Unravelling the Influence of Composition and Heat Treatment on Key Characteristics of Dairy Protein Powders Using a Multifactorial Approach.

The purpose of this study was to improve understanding of the structural and functional property changes that milk-protein concentrates undergo during production, particularly how the manufacturing route (heat treatment position and intensity), standardization (in osmosed water or ultrafiltrate permeate) and formulation (casein:whey protein (Cas:WP) ratio) influence the physico-chemical characteristics-hygroscopicity, particle size, sphericity, density and evolution of browning during storage. To obtain a comprehensive understanding of the parameters responsible for the distinctive characteristics of different powders, a multifactorial approach was adopted. Hygroscopicity depended mainly on the standardizing solution and to a lesser extent the Cas:WP ratio. The particle size of the heat-treated casein-dominant powders was up to 5 µm higher than for those that had had no heat treatment regardless of the standardizing solution, which also had no influence on the sphericity of the powder particles. The density of the powders increased up to 800 kg·m-3 with a reduced proportion of casein, and lactose and whey proteins participated in browning reactions during storage at 13 °C. In increasing order, the modality of heat treatment, the standardizing solution and the Cas:WP protein ratio influenced the key characteristics. This work is relevant for industrial applications to increase control over the functionalities of powdered products.

Heat treatment of milk protein concentrates affects enzymatic coagulation properties.

Dairy ingredients with highly concentrated protein contents are high added value products with expanding market. The manufacture of such ingredients includes a succession of unit operations of which heat treatment is a key step to guarantee the microbial safety, that induces major changes in protein structures and thus ingredients functionalities. However, due to an incomplete understanding of phenomena taking place at high protein concentrations, shedding light on their mechanisms is a scientific challenge as well as an industrial need. In this study, the influence of heat treatment (74 °C/ 30 s) of highly concentrated milk protein systems (up to 20 % w/w) on protein denaturation/aggregation and enzymatic coagulation properties was studied using an original semi-industrial approach. 10 % w/w protein solutions constituted with whey protein and casein micelles at milk ratio, standardized in osmosed water or ultrafiltration permeate were used. These protein solutions were processed in different ways prior the manufacture of powders: heat treatment of the 10 % w/w protein solution before vacuum evaporation, heat treatment of the 20 % w/w protein solution after vacuum concentration, two consecutive heat treatments before and after vacuum evaporation. A fourth powder was prepared from unheated 10 % w/w protein solution. An increase in protein concentration led to a higher heat-induced protein denaturation. This phenomenon was reduced when increasing the lactose content. The effect of heat treatment on the extent of protein denaturation was not cumulative. At high protein concentration, interactions between κ-casein and whey protein were modified compared to milk, as mainly micelle-bound aggregates were formed at pH about 6.7. This phenomenon was enhanced at low ionic strength and lactose content. Our study showed that the enzymatic coagulation properties of reconstituted protein powders could be correlated with their physico-chemical compositions. An increase in protein denaturation disrupted the gel reorganization and led to the formation of weaker gels but did not interfere on the micelles aggregation phase and the early gelation. On the contrary, an increase in ionic strength and lactose content led to higher gel time.

The adhesion of homogenized fat globules to proteins is increased by milk heat treatment and acidic pH: Quantitative insights provided by AFM force spectroscopy.

The rheological properties and microstructure of dairy gels involve the connectivity between milk fat globules (MFG) and casein micelles that is affected by technological processes such as milk homogenization and heat treatment. The underlying mechanisms require further quantification of the interactions at the nanoscale level to be fully understood and controlled. In this study, we examined the adhesion of homogenized MFG to milk proteins and evaluated the role of ultra-high temperature (UHT) heat treatment and pH. The combination of physico-chemical analysis, rheology and microscopy observations at different scale levels associated to atomic force microscopy (AFM) force spectroscopy were used. AFM experiments performed at the particle scale level showed that adhesion of individual homogenized MFG to milk proteins (1) is increased upon acidification at pH 4.5: 1.4 fold for unheated samples and 3.5 fold for UHT samples, and (2) is enhanced by about 1.7 fold at pH 4.5 after UHT heat treatment of milk, from 176 pN to 296 pN, thanks to highly-reactive heat-denatured whey proteins located at the surface of MFG and caseins. The increased inter-particle adhesion forces accounted for more connected structures and stiffer UHT milk acid gels, compared to unheated-milk gels. Using a multiscale approach, this study showed that heat treatment of milk markedly affected the interactions occurring at the particle's surface level with consequences on the bulk structural and rheological properties of acid gels. Such findings will be useful for manufacturers to modulate the texture of fermented dairy products through the tailoring of heat-induced complexation of proteins and the connectivity of homogenized MFG with the protein network. This work will also contribute in a better understanding of the impact of process-induced changes on the digestibility and metabolic fate of proteins and lipids.

Anxiety and Psycho-Physiological Stress Response to Competitive Sport Exercise.

Introduction: Sport is recognized as beneficial for health. In certain situation of practice, it nevertheless appears likely to induce a stress response. Anxiety is a stress response-modulating factor. Our objective is to characterize the role of anxiety in the stress response induced by a selective physical exercise. Method: Sixty-three young male military conducted a selective sporting running event (a 8-km commando-walk) and were recorded the day before, the day of the race, and the day after. The variables were psychometric [personality questionnaires, coping and anxious/stress state, and physiological (nocturnal heart rate variability and actigraphy)]. The subjects were classified, using scores on anxiety questionnaires at baseline, into two groups according to their anxious (G ANX) or non-anxious (G N-ANX). Results: Before the race, the G ANX was characterized by a lower level of self-esteem, higher scores in dysfunctional coping and a greater perceived stress compared to the G N-ANX. Compared to G N-ANX, the stress response to the exercise was higher in G ANX: G ANX exhibited (Selye, 1950) in immediate post-exercise, greater level in activation markers, and mental fatigue associated with a same level of physical fatigue and (Kim et al., 2018) in nocturnal post-exercise, an increase in sympathetic activation associated with a higher sleep fragmentation. Conclusion: A competition selection sport exercise causes a stress response, particularly for anxious subjects. Anxious status could be involved in the risk of emergence of overtraining in sport practice. These results must be taken into account when sport practice is used for anxiety management.

Ser2 from Serratia liquefaciens L53: A new heat stable protease able to destabilize UHT milk during its storage.

The heat-stable protease Ser2 is secreted by the species Serratia liquefaciens, a psychrotrophic bacteria frequently found in raw milk. To understand the physicochemical modifications of casein micelles induced by Ser2 and to confirm its implication in UHT milk destabilization, the enzyme was purified and added to microfiltered raw milk before UHT treatment. UHT milk destabilization was investigated during 90days of storage. A visual destabilization appeared after 8days of storage with the presence of sediment. Zeta potential increase and formation of aggregates were observed during the storage. Using tandem mass spectrometry, numerous released peptides from the four caseins were identified at the end of storage. Caseins were hydrolyzed in the preferential order ß->αs1->κ->αs2. No specific peptidic hydrolysed bond was detected. The present study confirmed that the presence of the protease Ser2 in raw milk can be one of the main causes of UHT milk destabilization.

Quantitative and qualitative variability of the caseinolytic potential of different strains of Pseudomonas fluorescens: implications for the stability of casein micelles of UHT milks during their storage.

Pseudomonas fluorescens grows at low temperature and produces thermo-resistant protease(s) that can destabilize UHT (Ultra High Temperature) milk during its storage. The consequences of contamination of microfiltered milk with 9 strains of P. fluorescens on the stability of the corresponding UHT milk during storage had been investigated in this study. The strains were classified in two groups according to their ability to destabilize UHT milk. For the group of highly destabilizing strains, sedimentations of UHT milks, low values to phosphate test and the presence of aggregates were observed. Zeta potential and hydration of casein micelles decreased, whereas non casein nitrogen (NCN) and non protein nitrogen (NPN) contents increased. The analyses of NCN fraction by liquid chromatography coupled to mass spectrometry indicated that the different casein molecules were hydrolyzed in a similar way for the destabilizing strains suggesting that the same enzyme was implicated. For the group of slightly or not destabilizing strains no visual and biochemical alteration were found. This study showed that destabilization of UHT milk by P. fluorescens was highly variable and strain-dependent.

COMMD1-mediated ubiquitination regulates CFTR trafficking.

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.

Tina wooden vat biofilm: a safe and highly efficient lactic acid bacteria delivering system in PDO Ragusano cheese making.

In the Sicilian PDO Ragusano cheese making, raw milk is placed in a wooden vat called a Tina. As no starter is added, lactic acid is produced by milk flora and flora released from the Tina biofilm. The aim of this work was to assess the safety and efficiency of this natural inoculation system. From 15 Tinas' biofilms, bacteria total counts varied from 10(3) to 10(6) CFU/cm(2), with the predominance of thermophilic lactic acid bacteria. Low counts of yeasts and moulds were found in a few Tinas. Salmonella, Listeria monocytogenes, Escherichia coli O157:H7 were totally absent, as assessed by conventional plating and the Bax detection system after enrichment, highlighting the safety of the system. From four Tinas out of the 15, micropieces of wood were observed by confocal and scanning electron microscopy. The biofilm entrapped in a matrix covered almost entirely the surface of the wood. Polysaccharides were detected in the four Tinas. In three of the latter, cocci were predominant in the ecosystem whereas in the other one, cocci, bacilli, yeasts and moulds were observed. Fifty litres of microfiltrated milk (<10 CFU/mL) were poured in the four Tinas for 10 min of contact. Enumeration of lactic acid bacteria, yeasts and enterococci were performed in the milk after contact. Depending on the Tina, from 5.10(4) to 10(6) CFU/mL of Streptococcus thermophilus were released into the milk, and from 10(4) to 10(5) CFU/mL of thermophilic lactobacilli. Spontaneous acidification after contact confirmed the high efficiency of biofilm lactic acid bacteria delivery.

CSN5 binds to misfolded CFTR and promotes its degradation.

Cystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The aim of this study was to find cellular proteins interacting with CFTR and regulating its processing. We have used a genetic screen in yeast to identify such proteins and identified CSN5 that interacted with the third cytoplasmic loop of CFTR. CSN5 is the 5th component of the COP9 signalosome, a complex of eight subunits that shares significant homologies to the lid subcomplex of the 26S proteasome and controls the stability of many proteins. The present study shows that CSN5 associates with the core-glycosylated form of CFTR and suggests that this association targets misfolded CFTR to the degradative pathway. Identifying CSN5 as a new component of the degradative pathway is an important step towards the goal of unraveling the sorting between misfolded and correctly folded CFTR proteins.

pEg6, a spire family member, is a maternal gene encoding a vegetally localized mRNA in Xenopus embryos.

BACKGROUND INFORMATION: In Xenopus, during oocyte maturation and the segmentation period, cell cycle progression is independent of new transcription, but requires de novo translation. This suggests that the completion of oocyte maturation and then the rapid cell division period is controlled exclusively at a post-transcriptional level by specific gene products. To isolate these maternal genes, a differential screening of a Xenopus egg cDNA library was performed. Several cDNAs were isolated which correspond to mRNA polyadenylated in eggs and deadenylated in embryos, and these constitute the founders members of the Eg family of mRNAs. RESULTS: We report here the characterization of Eg6 mRNA as a novel maternal gene expressed in Xenopus egg until gastrula stage. The Eg6 transcript is initially concentrated in the vegetal cytoplasm of the egg, and later the distribution of the transcript marks the posterior vegetal end of developing embryos. pEg6 is a multidomain protein with a kinase non-catalytic C-lobe domain of unknown function, a cluster of four WH2 (Wiskott-Aldrich syndrome protein homology 2) domains and a modified FYVE zinc-finger motif. The amino acid sequence of pEg6 is related to PEM-5 (posterior end mark-5), from an ascidian maternal mRNA, and spire, a Drosophila protein required to establish dorsal-ventral and anterior-posterior axes of polarity and recently described as an actin nucleation factor. In Xenopus and Schizosaccharomyces pombe cells pEg6 expression induces filamentous actin clusters and is associated with vesicular structure. CONCLUSION: These data suggest that pEg6 acts as a vegetally localized factor contributing to the actin nucleation process during Xenopus early development.

Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis.

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth.

A first locus for isolated autosomal recessive optic atrophy (ROA1) maps to chromosome 8q.

In contrast to the frequent dominant optic atrophies (DOAs) in which the neuropathy is usually an isolated event, isolated recessive optic atrophies (ROAs) are very uncommon and have been described as severe congenital or early infantile conditions. To date, two loci for isolated DOA have been mapped, of which one was ascribed to mutations in the OPA1 gene. Conversely, no isolated autosomal ROA locus had previously been localised. Here, we report a large multiplex consanguineous family of French origin affected with an early onset but slowly progressive form of isolated OA. A genome-wide search for homozygosity allowed the localisation of the disease-causing gene to chromosome 8q21-q22 (Zmax of 3.41 at theta=0 for D8S270), in a 12 Mb interval flanked by markers D8S1702 and D8S1794. This localisation excludes allelism of the disease with both isolated DOAs, on one hand, or all known syndromic forms of ROA, on the other hand, supporting the mapping of a first gene for isolated autosomal ROA (ROA1) on the long arm of chromosome 8.