Volatilome Analysis and Evolution in the Headspace of Packed Refrigerated Fish.

Fresh fish is a perishable food in which chemical (namely oxidation) and microbiological degradation result in undesirable odor. Non-processed fish (i.e., raw fish) is increasingly commercialized in packaging systems which are convenient for its retailing and/or which can promote an extension of its shelf-life. Compared to fish sent to its retail unpackaged, fish packaging results in a modification of the gaseous composition of the atmosphere surrounding it. These modifications of atmosphere composition may affect both chemical and microbiological degradation pathways of fish constituents and thereby the volatile organic compounds produced. In addition to monitoring Total Volatile Basic Nitrogen (TVB-N), which is a common indicator to estimate non-processed fish freshness, analytical techniques such as gas chromatography coupled to mass spectrometry or techniques referred to as "electronic nose" allow either the identification of the entire set of these volatile compounds (the volatilome) and/or to selectively monitor some of them, respectively. Interestingly, monitoring these volatile organic compounds along fish storage might allow the identification of early-stage markers of fish alteration. In this context, to provide relevant information for the identification of volatile markers of non-processed packaged fish quality evolution during its storage, the following items have been successively reviewed: (1) inner atmosphere gaseous composition and evolution as a function of fish packaging systems; (2) fish constituents degradation pathways and analytical methods to monitor fish degradation with a focus on volatilome analysis; and (3) the effect of different factors affecting fish preservation (temperature, inner atmosphere composition, application of hurdle technology) on volatilome composition.

Staphylococcus aureus membrane-damaging activities of four phenolics.

The membrane-damaging activities of four phenolics chosen for their bactericidal activity against Staphylococcus aureus CNRZ3 were investigated: 5,7-dihydroxy-4-phenylcoumarin (DHPC), 5,8-dihydroxy-1,4-naphthoquinone (DHNQ), epigallocatechin gallate (EGCG) and isobutyl 4-hydroxybenzoate (IBHB). Staphylococcus aureus CNRZ3 cells, as well as model liposomes mimicking its membrane phospholipids composition, were treated with each phenolic at its minimal bactericidal concentration. Membrane integrity, intracellular pH and intracellular esterase activity were examined by flow cytometric analysis of S. aureus cells stained with propidium iodide and SYTO® 9, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, and 5(6)-carboxyfluorescein diacetate, respectively. While intracellular pH was affected by the foyr phenolics, only DHNQ and to a lesser extent EGCG, caused a loss of membrane integrity. Flow cytometric analysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and DPPC/POPG (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoglycerol) liposomes stained with Coumarin 6 (which penetrates the lipid bilayer) or 5-N(octadecanoyl)-amino-fluorescein (which binds to the liposome shell) suggested that only EGCG and DHNQ penetrated the bilayer of phospholipids of liposomes. Taken together, these findings support the hypothesis that EGCG and DHNQ bactericidal activity results from their accumulation in the phospholipid bilayer of S. aureus cells membrane causing its disruption.

[Start to collaborate with partner patients†: an evidence?] / Entrer en collaboration avec des patients partenaires†: une évidence†?

The societal, political and institutional context is today favorable for the establishment of a partnership between patient and healthgivers. Despite the tangible benefits, the perception of partners ambivalent attitudes reinforces the importance of the construction for this collaboration. This article describes this collaborative approach born out of the transformation of a bariatric surgery preparation educational program. In this context, the implementation strategy is the founding stage to explore the needs of partners. This highlights the need to secure the healthgivers regarding power issues, as well as to question the skills required for patient partners. The definition of the partnership model by the partners provides answers.

Le contexte sociétal, politique et institutionnel est aujourd'hui favorable à la mise en place d'un partenariat entre patients et soignants. Malgré des bénéfices tangibles, la perception d'une ambivalence des partenaires renforce l'importance de la construction de cette collaboration. Cet article décrit cette démarche collaborative, née dans le contexte de la transformation d'un programme éducatif de préparation à la chirurgie bariatrique. Dans la stratégie d'implémentation, l'exploration contextuelle des besoins des partenaires constitue l'étape fondatrice. Elle met en lumière un besoin de sécurisation des soignants quant à des enjeux de pouvoir, et questionne sur les compétences requises des patients partenaires. La définition du modèle de partenariat par les partenaires apporte des réponses.

Potential of Incorporation of Antimicrobial Plant Phenolics Into Polyolefin-Based Food Contact Materials to Produce Active Packaging by Melt-Blending: Proof of Concept With Isobutyl-4-Hydroxybenzoate.

There is an increasing interest for active food packaging incorporated with natural antimicrobial agents rather than synthetic preservatives. However, most of plastics for direct contact with food are made of polyolefins, usually processed by extrusion, injection, or blow-molding methods while most of natural antimicrobial molecules are thermolabile compounds (e.g., essential oils). Therefore, addition of plant phenolics (with low volatility) to different polyolefins might be promising to design active controlled release packaging processed by usual plastic compounding and used for direct contact with food products. Therefore, up to 2% (wt/wt) of isobutyl-4-hydroxybenzoate (IBHB) was mixed with 3 polyolefins: EVA poly(ethylene-co-vinyl acetate), LLDPE (Linear Low Density Polyethylene), and PP (PolyPropylene) by melt-blending from 75 to 170°C and then pelletized in order to prepare heat-pressed films. IBHB was chosen as an antibacterial phenolic active model molecule against Staphylococcus aureus to challenge the entire processing. Antibacterial activity of films against S. aureus (procedure adapted from ISO 22196 standard) were 4, 6, and 1 decimal reductions in 24 h for EVA, LLDPE, and PP films, respectively, demonstrating the preservation of the antibacterial activity after melt processing. For food contact materials, the efficacy of antimicrobial packaging depends on the release of the antimicrobial molecules. Therefore, the three types of films were placed at 23°C in 95% (v/v) ethanol and the release rates of IBHB were monitored: 101 ± 1%, 32 ± 7%, and 72 ± 9% at apparent equilibrium for EVA, LLDPE, and PP films, respectively. The apparent diffusion coefficients of IBHB in EVA and PP films were 2.8 ± 0.3 × 10-12 and 4.0 ± 1.0 × 10-16 m2s-1. For LLDPE films, IBHB crystals were observed on the surface of films by SEM (Scanning Electron Microscopy): this blooming effect was due the partial incompatibility of IBHB in LLDPE and its fast diffusion out of the polymer matrix onto the film surface. In conclusion, none of these three materials was suitable for a relevant controlled release packaging targeting the preservation of fresh food, but a combination of two of them is promising by the design of a multilayer packaging: the release could result from permeation through an inner PE layer combined with an EVA one acting as a reservoir.

Homogeneous acylation of Cellulose diacetate: Towards bioplastics with tuneable thermal and water transport properties.

In this study, we report a simple, non-degrading and efficient homogeneous acylation of cellulose diacetate (CDA) by using a large panel of commercially available acylating aliphatic moieties, differing in their structure (fatty, ramified, bulky, cycloaliphatic, aromatic, more or less spaced from the cellulose backbone), in view of generating a library of well-defined cellulose mixed esters with enhanced thermoplasticity. As reflected by a lowering of the glass temperature (Tg), the covalent grafting confers an improved mobility to the cellulose chains, by disrupting the initial H-bonds. In particular, it appears that the gain in free volume is tailored by the substituent structure and that acylating reagents consisting in a terminal bulky moieties spaced from CDA chains by a linear chain efficiently separate macromolecular chains without generating detrimental stiffening interactions (low Tg around 125 °C). Moreover, free-standing films easily prepared by solvent casting exhibit relevant water transport properties, which are closely dictated and tuned by the water solubility of the cellulose mixed ester.

The impact of chronic disease management on primary care doctors in Switzerland: a qualitative study.

BACKGROUND: Patient-centeredness and therapeutic relationship are widely explored as a means to address the challenge of chronic disease and multi-morbidity management, however research focusing on the perspective of doctors is still rare. In this study, we aimed to explore the impact of the patient's chronic disease(s) on their healthcare provider. METHODS: A qualitative approach was taken using semi-structured interviews with general practitioners working in outpatient clinics either in individual practices or in a hospital setting in Geneva, Switzerland. Codes were developed through an iterative process and using grounded theory an inductive coding scheme was performed to identify the key themes. Throughout the analysis process the research team reviewed the analysis and refined the coding scheme. RESULTS: Twenty interviews, 10 in each practice type, allowed for saturation to be reached. The following themes relevant to the impact of managing chronic diseases emerge around the issue of feeling powerless as a doctor; facing the patient's socio-economic context; guidelines versus the reality of the patient; time; and taking on the patient's burden. Primary care practitioners face an emotional burden linked with their powerlessness and work conditions, but also with the empathetic bond with their patients and their circumstances. Doctors seem poorly prepared for this emotional strain. The health system is also not facilitating this with time constraints and guidelines unsuitable for the patient's reality. CONCLUSIONS: Chronic disease and multi-morbidity management is a challenge for healthcare providers. This has its roots in patient characteristics, the overall health system and healthcare providers themselves. Structural changes need to be implemented at different levels: medical education; health systems; adapted guidelines; leading to an overall environment that favors the development of the therapeutic relationship.

[How to support obese patients in a long-term process of change?]. / Comment accompagner les patients obèses dans un processus de changement à long-terme?

It is illusory to think losing weight effectively by acting only on diet or physical activity. To lose weight satisfactorily and to maintain that weight loss, we should move more on lifestyle changes, namely changes in behavior on several axes. Through concrete examples of an obese patient wanting to lose weight, we will see what skills he must acquire to achieve its objective and what tools the therapist can use to help.

Active packaging with antifungal activities.

There have been many reviews concerned with antimicrobial food packaging, and with the use of antifungal compounds, but none provided an exhaustive picture of the applications of active packaging to control fungal spoilage. Very recently, many studies have been done in these fields, therefore it is timely to review this topic. This article examines the effects of essential oils, preservatives, natural products, chemical fungicides, nanoparticles coated to different films, and chitosan in vitro on the growth of moulds, but also in vivo on the mould free shelf-life of bread, cheese, and fresh fruits and vegetables. A short section is also dedicated to yeasts. All the applications are described from a microbiological point of view, and these were sorted depending on the name of the species. Methods and results obtained are discussed. Essential oils and preservatives were ranked by increased efficacy on mould growth. For all the tested molecules, Penicillium species were shown more sensitive than Aspergillus species. However, comparison between the results was difficult because it appeared that the efficiency of active packaging depended greatly on the environmental factors of food such as water activity, pH, temperature, NaCl concentration, the nature, the size, and the mode of application of the films, in addition to the fact that the amount of released antifungal compounds was not constant with time.

Nisin as a Food Preservative: Part 2: Antimicrobial Polymer Materials Containing Nisin.

Nisin is the only bacteriocin approved as a food preservative because of its antibacterial effectiveness and its negligible toxicity for humans. Typical problems encountered when nisin is directly added to foods are mainly fat adsorption leading to activity loss, heterogeneous distribution in the food matrix, inactivation by proteolytic enzymes, and emergence of resistance in normally sensitive bacteria strains. To overcome these problems, nisin can be immobilized in solid matrices that must act as diffusional barriers and allow controlling its release rate. This strategy allows maintaining a just sufficient nisin concentration at the food surface. The design of such antimicrobial materials must consider both bacterial growth kinetics but also nisin release kinetics. In this review, nisin incorporation in polymer-based materials will be discussed and special emphasis will be on the applications and properties of antimicrobial food packaging containing this bacteriocin.

Nisin as a Food Preservative: Part 1: Physicochemical Properties, Antimicrobial Activity, and Main Uses.

Nisin is a natural preservative for many food products. This bacteriocin is mainly used in dairy and meat products. Nisin inhibits pathogenic food borne bacteria such as Listeria monocytogenes and many other Gram-positive food spoilage microorganisms. Nisin can be used alone or in combination with other preservatives or also with several physical treatments. This paper reviews physicochemical and biological properties of nisin, the main factors affecting its antimicrobial effectiveness, and its food applications as an additive directly incorporated into food matrices.

An integrated approach to bovine oocyte quality: from phenotype to genes.

In cattle, early embryonic failure plays a major role in the limitation of reproductive performance and is influenced by genetic effects. Suboptimal oocyte quality, including an inadequate store of maternal factors, is suspected to contribute to this phenomenon. In the present study, 13 Montbeliarde cows were phenotyped on oocyte quality, based on their ability to produce viable embryos after in vitro maturation, fertilisation and culture for 7 days. This discriminated two groups of animals, exhibiting developmental rates below 18.8% or above 40.9% (relative to cleaved embryos). Using microarrays, transcriptomic profiles were compared between oocytes collected in vivo from these two groups of animals. The difference in oocyte development potential was associated with changes in transcripts from 60 genes in immature oocytes and 135 genes in mature oocytes (following Bonferroni 5% correction). Of these, 16 and 32 genes were located in previously identified fertility quantitative trait loci. A subset of differential genes was investigated on distinct samples by reverse transcription-quantitative polymerase chain reaction. For SLC25A16, PPP1R14C, ROBO1, AMDHD1 and MEAF6 transcripts, differential expression was confirmed between high and low oocyte potential animals. Further sequencing and searches for polymorphisms will pave the way for implementing their use in genomic selection.

Influence of some formulation and process parameters on the stability of lysozyme incorporated in corn flour- or corn starch-based extruded materials prepared by melt blending processing.

In order to obtain an antimicrobial biodegradable material, corn flour was extruded with 1% of lysozyme. Since the limited stability of natural preservatives such as lysozyme is a common bottleneck to the elaboration of active biomaterials by melt blending processes, the influence of formulation and of extrusion processing temperature on its residual enzymatic activity was investigated. To assess the contribution of process parameters such as temperature, shear stress and of related formulation parameters such as glycerol and moisture contents, the stability of lysozyme following its extrusion or its thermoforming with plasticized corn starch or thermal treatments in aqueous glycerol solutions was also studied. Increasing glycerol content from 25% to 30% significantly limited inactivation of lysozyme during extrusion, while increasing initial moisture content of the mixture from 14.5% to 28.5% had the opposite effect. These observations open the possibility to prepare active materials retaining more than 60±7% of initial lysozyme activity.

Metabolomic prediction of pregnancy viability in superovulated cattle embryos and recipients with fourier transform infrared spectroscopy.

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared spectroscopy (FTIR) metabolomics to identify spectral models predictive of pregnancy outcome. Embryos collected on Day 6 from superovulated cows in 2 countries were individually cultured in synthetic oviduct fluid medium with BSA for 24 h before embryo transfer. Spent CM, blank controls, and plasma samples (Day 0 and Day 7) were evaluated using FTIR. The spectra obtained were analyzed. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the ROC curve (AUC). Endpoints considered were Day 60 pregnancy and birth. High AUC was obtained for Day 60 pregnancy in CM within individual laboratories (France AUC = 0.751 ± 0.039, Spain AUC = 0.718 ± 0.024), while cumulative data decreased the AUC (AUC = 0.604 ± 0.029). Predictions for CM at birth were lower than Day 60 pregnancy. Predictions with plasma at birth improved cumulative over individual results (Day 0: France AUC = 0.690 ± 0.044; Spain AUC < 0.55; cumulative AUC = 0.747 ± 0.032). Plasma generally predicted pregnancy and birth better than CM. These first results show that FTIR metabolomics could allow the identification of embryos and recipients with improved pregnancy viability, which may contribute to increasing the efficiency of selection schemes based on ET.

Polybutylene succinate adipate/starch blends: a morphological study for the design of controlled release films.

Films made of plasticized starch (PLS)/poly(butylene succinate co-butylene adipate) (PBSA) blends were prepared by thermomechanical processing varying the PBSA proportions in blends to obtain biphasic materials with distinct morphologies. These morphologies were characterized by selective extraction of each phase, microscopic observations, and selective water/oxygen permeation properties. These experiments allowed identifying the blend compositions corresponding to the beginning of partial continuity (cluster partial percolation) until total continuity of each phases. This property was related to the controlled release of model molecule (fluorescein) previously dispersed in the PLS and revealed that its release depended on the tortuosity of the PLS phase tailored by the polymer blends composition and by the limited swelling of the PLS when entrapped in the PBSA phase. Future applications will focus on food preservatives dispersed in PBSA-PLS blends to obtain active antimicrobial packaging put in direct contact with intermediate to high moisture foods.

Specific expression patterns and cell distribution of ancient and modern PAG in bovine placenta during pregnancy.

Pregnancy-associated glycoproteins (PAGs) constitute a multigenic family of aspartic proteinases expressed in the trophoblast of the ruminant placenta. In Bos taurus, this family comprises 21 members segregated into ancient and modern phylogenetic groups. Ancient PAGs have been reported to be synthesized throughout the trophoblastic cell layer whereas modern PAGs are produced by binucleate cells of cotyledons. The aim of this study was to investigate modern and ancient PAGs during gestation in cotyledonary and intercotyledonary tissues. To obtain convincing and innovative results despite the high sequence identity shared between PAGs, we designed specific tools such as amplification primers and antibodies. Using real-time RT-PCR, we described the transcript expression of 16 bovine PAGs. Overall, PAGs are characterized by an increase in their expression during gestation. However, we demonstrated a segregation of modern PAGs in cotyledons and of ancient PAGs in the intercotyledonary chorion, except for the ancient PAG2 expressed in cotyledons. By raising specific antibodies against the modern PAG1 and ancient PAG11 and PAG2, we established the expression kinetics of the proteins using western blotting. Immunohistochemistry showed that PAGs were produced by specific cellular populations: PAG1 by binucleate cells in the whole trophoblastic layer, PAG11 was localized in binucleate cells of the intercotyledonary trophoblast and the chorionic plate of the cotyledon, while PAG2 was produced in mononucleate cells of the internal villi of the cotyledon. These results revealed a highly specific regulation of PAG expression and cell localization as a function of their phylogenetic status, suggesting distinct biological functions within placental tissues.

In vitro maturation of oocytes alters gene expression and signaling pathways in bovine cumulus cells.

In vitro maturation (IVM) of immature oocytes is widely used in assisted reproduction technologies in cattle, and is increasingly used to treat human infertility. The development competence of IVM oocytes, however, is lower than preovulatory, in vivo-matured oocytes. During maturation, cumulus cells (CC) are metabolically coupled with an oocyte and support the acquisition of its developmental potential. Our objective was to identify genes and pathways that were affected by IVM in bovine CC. Microarray transcriptomic analysis of CC enclosing in vitro- or in vivo-mature oocytes revealed 472 differentially expressed genes, including 28% related to apoptosis, correlating with twofold higher cell death after IVM than in vivo, as detected by TUNEL. Genes overexpressed after IVM were significantly enriched in functions involved in cell movement, focal adhesion, extracellular matrix function, and TGF-beta signaling, whereas under-expressed genes were enriched in regulating gene expression, energy metabolism, stress response, and MAP kinases pathway functions. Differential expression of 15 genes, including PAG11 (increased) and TXNIP (decreased), which were never detected in CC before, was validated by real-time RT-PCR. Moreover, protein quantification confirmed the lower abundance of glutathione S-transferase A1 and prostaglandin G/H synthase 2, and the higher abundance of hyaluronan synthase 2 and SMAD4, a member of TGF-beta pathway, in CC after IVM. Phosphorylation levels of SMAD2, MAPK3/1, and MAPK14, but not MAPK8, were higher after IVM that in vivo. In conclusion, IVM provokes the hyper-activation of TGF-beta and MAPK signaling components, modifies gene expression, leads to increased apoptosis in CC, and thus affects oocyte quality.

Specific enzymatic tailoring of wheat arabinoxylan reveals the role of substitution on xylan film properties.

To increase understanding of the applicability of agro biomass by-products as biodegradable film formers, the effect of wheat arabinoxylan (WAX) fine structure on film properties was studied by applying specific enzyme modifications. WAX was selectively modified to mimic the natural variations of different arabinoxylans, particularly the degree of mono and disubstitution of α-L-arabinofuranosyl (Araf) units in ß-D-xylopyranosyl (Xylp) backbone residues. The resulting modified WAX samples had similar arabinose-to-xylose (Ara/Xyl) ratios, but they differed in the number of unsubstituted Xylp units. The substitution of WAX was found to affect, in particular, tensile strength, crystallinity, and oxygen permeability properties of the films, as statistically significant decreases in tensile strength and oxygen permeability took place after WAX de-branching. An increase in the number of unsubstituted Xylp units decreased the temperature of relaxation of small-scale molecular motions of WAX (ß-relaxation) and increased the degree of crystallinity of the films.

Expression of enzymes involved in the synthesis of prostaglandin E2 in bovine in vitro-produced embryos.

Prostaglandin E2 (PGE2) may play a major role in embryo development and the establishment of pregnancy in cattle. The biosynthesis of PGE2 implies the sequential transformation of arachidonic acid to PGH2 by cyclooxygenases (COXs), then the conversion of PGH2 to PGE2 by prostaglandin E synthases (PGESs). Quantitative RT-PCR was used to examine the expression of COX-1, COX-2, microsomal PGES-1 (mPGES-1), microsomal PGES-2 (mPGES-2) and cytosolic PGES (cPGES) mRNAs in day 7 in vitro-produced (IVP) embryos from oocytes collected by ovum pick-up in Holstein heifers. Transcripts for COX-2 and mPGES-1 were detected in all embryos, whereas transcripts for COX-1 and mPGES-2 were not detected and cPGESs were at the limit of detection in 40% of embryos. Levels of COX-2 and mPGES-1 mRNAs were significantly higher in blastocysts and expanded blastocysts than in morulae and early blastocysts. Furthermore, excellent-quality embryos (grade 1) displayed higher levels of both COX-2 and mPGES-1 than did embryos of good and medium qualities (grades 2-3). Our results suggest that bovine IVP embryos at the morula and blastocyst stages use exclusively the COX-2/mPGES-1 pathway for PGE2 biosynthesis, and that PGE2 is potentially involved in blastocyst expansion and developmental competence.

Reproductive technologies and genomic selection in cattle.

The recent development of genomic selection induces dramatic changes in the way genetic selection schemes are to be conducted. This review describes the new context and corresponding needs for genomic based selection schemes and how reproductive technologies can be used to meet those needs. Information brought by reproductive physiology will provide new markers and new improved phenotypes that will increase the efficiency of selection schemes for reproductive traits. In this context, the value of the reproductive techniques including assisted embryo based reproductive technologies (Multiple Ovaluation Embryo Transfer and Ovum pick up associated to in vitro Fertilization) is also revisited. The interest of embryo typing is discussed. The recent results obtained with this emerging technology which are compatible with the use of the last generation of chips for genotype analysis may lead to very promising applications for the breeding industry. The combined use of several embryo based reproductive technologies will probably be more important in the near future to satisfy the needs of genomic selection for increasing the number of candidates and to preserve at the same time genetic variability.

Thymosins ß-4 and ß-10 are expressed in bovine ovarian follicles and upregulated in cumulus cells during meiotic maturation.

ß-Thymosins are small proteins that regulate the actin cytoskeleton and are involved in cell motility, differentiation, the induction of metalloproteinases, in anti-inflammatory processes and tumourigenesis. However, their roles in the ovary have not yet been elucidated. Using transcriptomics and real time reverse transcription-polymerase chain reaction validation, the present study demonstrates that thymosin ß-4 (TMSB4) and thymosin ß-10 (TMSB10) are upregulated in bovine cumulus cells (CCs) during in vitro maturation of cumulus-oocyte complexes (COCs) in parallel with an increase in mRNA expression of HAS2, COX2 and PGR genes. Using immunocytochemistry, both proteins were found to be localised mainly in granulosa cells, CCs and oocytes, in both the cytoplasm and nucleus, as well as being colocalised with F-actin stress fibres in CCs. Using different maturation mediums, we showed that the expression of TMSB10, but not TMSB4, was positively correlated with COC expansion and progesterone secretion and negatively correlated with apoptosis. Immunofluorescence, coupled with terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL), demonstrated the absence of TMSB4 and/or TMSB10 in apoptotic cells. TMSB10 expression was higher in COCs matured in vivo than in vitro, and differences related to the age of the animal were observed. TMSB4 and/or TMSB10 expression was unchanged, whereas HAS2 overexpressed in CCs from oocytes that developed to the blastocyst stage in vitro compared with those that did not. Thus, TMSB4 and/or TMSB10 ovarian expression patterns suggest that these two thymosins may be involved in cumulus modifications during maturation.