The adsorption of chymosin and lysozyme onto emulsion droplets and their association with casein

The proteolytic action of proteases present in cheese plays a major role in the ripening of cheese. These proteases originate from the rennet, the starter cultures and from the milk itself. The proteolysis in cheese results in the degradation of the casein proteins into smaller peptides and free amino acids, which act as flavour precursors. The ripening of cheese under conditioned storage is time consuming and costly. Addition of specific enzymes to the cheese milk is one of several options to accelerate ripening. A major problem then is that hardly any of these proteases end up in the cheese and most disappear with the whey stream. Entrapment of bacteria and milk fat globules into the casein matrix of the curd is due to their particle character. Immobilisation of proteases onto particles would thus result in retention of these proteins in the curd. Ideally, for reasons of acceptance, these particles should originate from the milk itself or at least be edible.In this study, in a more general approach, soya oil emulsion droplets and casein micelles, being protein aggregates, were tested as the carrier system. Chymosin and lysozyme were taken as the enzymes to be immobilized, because of their relevance to the dairy industry and because they are scientifically well-known. Moreover, their biochemical divergence make them suitable models for study.The literature provides several studies on adsorption of proteins onto interfaces. Few of these proteins are enzymes. In cases where lysozyme was studied, it was included because of its extraordinary properties as a protein and not because of its enzymatic activity. Apart from (phospho)lipases there is scarcely any literature that describes activity of enzymes adsorbed onto the oil/water or air/water interfaces. Proteins tend strongly to accumulate in interfaces and for that reason are said to be very surface active. This adsorption is accompanied by a conformational change of the three-dimensional structure of the protein that results in some unfolding, ranging from almost full stretching of the peptide chain to a more conserved conformation. Hydrophobic residues or patches of the protein, mostly buried inside the molecule, will tend to position themselves next to or even protrude partly into, the hydrophobic phase of an oil/water interface.The extent of conformational change depends on the conformational stability of the protein, which, in turn, depends on pH, temperature, ionic strength etc. Furthermore, the extent of unfolding will be dependent on the surface area available and on the time scale, and hence, on protein concentration. In a static condition, adsorption at the interface will be diffusion driven, whereas during emulsification the time of adsorption will be determined by convection and will be very much shorter. Consequently, conformational changes of proteins during emulsification should be smaller because full surface coverage may be reached before unfolding can occur. In the case of enzymes being adsorbed the emulsification process would therefore offer an opportunity to retain activity.The relation between the surface pressureΠi.e. the extent of surface tension decrease and the amount of protein adsorbed per unit surface area availableΓ, provides a possibility to relate the size of the adsorbed protein molecules, and thereby the extent of unfolding, to the surface load. As mentioned earlier the extent of unfolding should be less at a higherΓvalue. It has been calculated that lysozyme, an enzyme of high conformational stability, hardly unfolds at the air/water interface, even at low surface coverage. For the oil/water interface, however, a considerable increase in the radius of the protein molecules was observed at low surface load. At surface coverage of &gt; 1.5 mg.m -2, the radius remained more or less constant, indicating that substantial unfolding did not occur. Despite this rigidity, the enzyme had lost all of its enzymatic activity in situ and it even remained inactive after desorption.Apparently, conformational changes in the enzyme molecule do not necessarily become manifest in a larger size for the molecule. Chymosin, being an enzyme of smaller conformational stability, naturally lost all of its activity due to adsorption onto the oil/water interface. In experiments with the enzymes coadsorbed simultaneously with bovine serum albumin, or the one after the other, there was no retention of in situ activity. Chymosin also proved to be inactivated at the expanding air/water interface due to air incorporation, if this occurred e.g. during homogenization.During the cheese-making process enzymes like chymosin and lysozyme are retained in the curd. This retention must be due to association of the enzymes with the casein from milk. In order to adsorb the enzymes with retention of activity, the various casein fractions were used to make and stabilize a soya-oil emulsion, and the enzyme was subsequently allowed to associate with the casein. The extent of association of lysozyme with the casein fractions was in the orderα s -casein &gt;β-casein &gt;κ-casein. Only for theκ-casein stabilized emulsion, was lysozyme association dependent on pH within the range of pH 5.2 - 6.4 (greater for a lower pH). Furthermore, the association with the caseins was not dependent on temperature, indicating that hydrophobic interactions were not predominant. The same trends were found with the various caseins in solution, albeit that association withκ-casein hardly occurred. It should be kept in mind that casein adsorbed at an interface will expose other amino acid residues compared to its behaviour when free in solution. For that reason the association behaviour in the two systems may differ.Because the association varies between caseins the extent of association with lysozyme depended on the composition of the casein micelles (aggregates of many casein molecules and calcium phosphate, as occurring especially in milk). As expected, casein micelles containing a higher proportion ofκ-casein associated less with lysozyme. It was found that lysozyme did not lose activity due to association with casein adsorbed on soya oil droplets or free in solution. However, lysozyme activity was markedly reduced when the enzyme was associated with casein micelles. In this system lysozyme also associated with casein in the interior of the casein micelle. The apparent loss of activity was most probably due to internal diffusion limitation. The difference of association for the various systems was also reflected in the free equilibrium concentration at which the surface excess plateau value was reached. In the system of adsorbed caseins this value was reached at a free lysozyme concentration of about 3 _M, whereas for the micellar system this value was about 100 times higher.The association of chymosin with casein has been studied in the same three systems of casein adsorbed onto soya-oil emulsion droplets, caseins in solution and caseins aggregated in casein micelles. It appears that chymosin only associated with adsorbedκ-casein and not with adsorbedαs- orβ-casein. Preceding the association, the caseinomacropeptide part ofκ-casein is split off, followed immediately by the aggregation of the soya-oil emulsion droplets containing the remaining para-κ-casein. This coagulation behaviour is identical to the renneting of milk during the cheese-making process. The association characteristics for chymosin are also comparable. The association was strongly dependent on pH and ionic strength, and on chymosin and casein concentration. Theκ-casein stabilized emulsion has proven to be a good model system for studying chymosin retention in curd. The chymosin associated with para-κ-casein was shown to be still active on addedκ-casein or on a fluorescent small hexapeptide substrate. Consequently, the active centre of the enzyme is presumably not involved in the association with casein.The association of chymosin with caseins free in solution has also been studied. Only in a solution containingκ-casein will addition of chymosin result in protein flocculation and precipitation. This flocculation is due to splitting off the caseino-macropeptide part ofκ-casein and the consecutive aggregation of the fairly hydrophobic and almost electrically neutral para-κ-casein molecules. The precipitated protein fraction also contains associated chymosin, to an extent depending on conditions like pH, ionic strength and casein and chymosin concentrations. In this system time and temperature also affected the extent of chymosin association. The association decreased with increased contact time and was stronger at higher temperatures.The protein content in the supernatant after centrifugation increased not only due to dissociation of chymosin but also due to the presence of casein fragments. Apparently, the dissociation of chymosin was related to its proteolytic action. The dissociation rate increased with decreasing pH where chymosin becomes more active and less specific. The dissociation also increased with temperature for a given time of contact. However, when extrapolated to a contact time of t = 0 (i.e. when dissociation due to proteolysis has not occurred yet) the association was observed to be somewhat stronger for a higher temperature. The effect of temperature on the proteolysis-dependent dissociation, apparently was stronger than its effect on the increase of the association. Since chymosin association depends on mutual association of caseins (see below), it will also depend on the temperature dependence of the latter. Dissociation of chymosin was not found in the system of caseins adsorbed onto emulsion droplets.The addition of small amounts ofαs- orβ-casein strongly decreased the extent of association of chymosin with para-κ-casein. This effect was stronger forαs-casein than forβ-casein. It was also found that the extent of chymosin association (moles of chymosin per mole of para-κ-casein) was larger when the system was diluted or, in other words, when the casein concentration was reduced. Both phenomena can be explained by assuming that competitive association occurs between the caseins and chymosin for interaction with a para-κ-casein molecule. Chymosin is only able to associate with a para-κ-casein molecule when that is not associated with other casein molecules. Thermodynamically speaking, the extent of association of chymosin is determined by the association constants that exist between all caseins under conditions as in the system. These association constants vary with pH, ionic strength, casein concentration and temperature.The model of competitive association is further developed and applied to the association of chymosin with casein micelles of various composition. It follows that chymosin will associate less with casein micelles composed ofαs- andκ-casein than with micelles composed ofβ- andκ-caseins. Again, this behaviour can be explained by competitive association, since different association constants exist for the caseins and chymosin for association with para-κ-casein. The relations for association and dissociation found in this casein micelle system are comparable with those found with caseins in solution. The kinetic model for competitive association is only a crude approximation. It does not provide possibilities of calculating all the association constants occurring in milk from the relations found from retention of chymosin in curd.</p

The European Food Safety Authority scientific opinion on a risk profile related to production and consumption of insects as food and feed

The increased attention to the use of farmed insects as a novel protein source has raised the question of the safety of insects as human food and as animal feed. This was the background for the European Union (EU) Commission to mandate the European Food Safety Authority (EFSA) to conduct a review of the current knowledge about biological, chemical and environmental risks associated with production and consumption of insects. National authorities in some EU member states (Belgium, the Netherlands and France) have conducted national assessments (ANSES, 2015; FASFC, 2014; NVWA, 2014). However, in the EU, existing regulations constitute legal barriers for marketing insects for human consumption and as protein in animal feed for food producing animals

Imaging features and differential diagnoses of non-neoplastic diffuse mediastinal diseases.

Acute or chronic non-neoplastic diffuse mediastinal diseases have multiple causes, degrees of severity, and a wide range of management. Some situations require emergency care while others do not need specific treatment. Although the diagnosis may be suspected on chest X-ray, it is mainly based on CT. A delayed recognition is not uncommonly observed. Some findings may prompt the radiologist to look for specific associated injuries or lesions.This pictorial review will successively describe the various non-neoplastic causes of diffuse mediastinal diseases with their typical findings and major differentials.First, pneumomediastinum that can be provoked by extra- or intra-thoracic triggers requires the knowledge of patient's history or recent occurrences. Absence of any usual etiological factor should raise suspicion of cocaine inhalation in young individuals.Next, acute mediastinitis may be related to post-operative complications, esophageal perforation, or contiguous spread of odontogenic or retropharyngeal infections. The former diagnosis is not an easy task in the early stage, owing to the similarities of imaging findings with those of normal post-operative appearance during the first 2-3 weeks.Finally, fibrosing mediastinitis that is linked to an excessive fibrotic reaction in the mediastinum with variable compromise of mediastinal structures, in particular vascular and airway ones. Differential diagnosis includes tumoral and inflammatory infiltrations of the mediastinum

Dysrhythmias in patients with a complete atrioventricular septal defect: From surgery to early adulthood

Contains fulltext : 203679.pdf (publisher's version ) (Open Access

A Single Laser System for Ground-State Cooling of 25-Mg+

We present a single solid-state laser system to cool, coherently manipulate and detect $^{25}$Mg$^+$ ions. Coherent manipulation is accomplished by coupling two hyperfine ground state levels using a pair of far-detuned Raman laser beams. Resonant light for Doppler cooling and detection is derived from the same laser source by means of an electro-optic modulator, generating a sideband which is resonant with the atomic transition. We demonstrate ground-state cooling of one of the vibrational modes of the ion in the trap using resolved-sideband cooling. The cooling performance is studied and discussed by observing the temporal evolution of Raman-stimulated sideband transitions. The setup is a major simplification over existing state-of-the-art systems, typically involving up to three separate laser sources

Transthoracic 3D echocardiographic left heart chamber quantification in patients with bicuspid aortic valve disease

Integration of volumetric heart chamber quantification by 3D echocardiography into clinical practice has been hampered by several factors which a new fully automated algorithm (Left Heart Model, (LHM)) may help overcome. This study therefore aims to evaluate the feasibility and accuracy of the LHM software in quantifying left atrial and left ventricular volumes and left ventricular ejection fraction in a cohort of patients with a bicuspid aortic valve. Patients with a bicuspid aortic valve were prospectively included. All patients underwent 2D and 3D transthoracic echocardiography and computed tomography. Left atrial and ventricular volumes were obtained using t

Male–female differences in quality of life and coping style in patients with Marfan syndrome and hereditary thoracic aortic diseases

Hereditary thoracic aortic diseases (HTAD) such as Marfan syndrome (MFS) affect multiple organ systems and provide a risk of acute aortic dissection, which causes lifelong uncertainties. Although health-related quality of life (HRQOL) was found to be reduced in HTAD patients, no studies have evaluated male–female-specific aspects of HRQOL and coping in this population. This study aims to evaluate HRQOL in HTAD patients compared to the general population; assess male–female differences in HRQOL and factors associated with HRQOL; evaluate coping styles in male and female HTAD patients and identify factors associated with acceptance. All consecutive adult patients who visited the specialized HTAD outpatient clinic between 2013 and 2018 were asked to complete three HRQOL questionnaires: the Short Form 36 (SF-36), the Hospital Anxiety and Depression Scale (HADS), and the Nijmegen Clinical Screening Instrument (NCSI). In total, 142 patients were included (mean age 42.1 years, 65 females, 123 MFS). Compared to the general population, HTAD patients scored significantly lower on multiple SF-36 sub-domains (males: General Health 54.5 ± 18.8 vs. 71.6 ± 20.6, p <.001; Vitality 58.3 ± 20.4 vs. 71.9 ± 18.3, p <.001; females: Physical Functioning 67.5 ± 23.8 vs. 80.4 ± 24.2, p =.003; Role Physical 58.3 ± 45.1 vs. 73.8 ± 38.5, p =.047; General Health 49.4 ± 24.3 vs. 69.9 ± 20.6, p <.001; Social Functioning 73.5 ± 22.0 vs. 82.0 ± 23.5, p =.027). Females scored significantly lower than males on the SF-36 physical component score (41.6 [IQR 35.5–53.1] vs. 49.3 [IQR 42.3–54.6], p =.035). Males scored significantly higher on the coping style denial than females (2.75 [IQR 2.00–3.25] vs. 2.25 [IQR 1.75–3.25], p =.018). High scores on acceptance were found in 38 (26.8%) of HTAD patients, and these patients showed significantly better scores on the NCSI, SF-36, and HADS, except on NCSI Satisfaction Relationships and SF-36 Physical Functioning and Mental Health. Acceptance was associated with more medication use (beta blocker use, p =.008; angiotensin receptor blocker use, p =.003) and less hypertension (p =.001). In patients with MFS, employment was strongly associated with better scores on the NCSI. In conclusion, HTAD patients showed subnormal HRQOL, especially females. Interestingly, in both males and females factors such as employment, coping style, and disease acceptance seem more important for HRQOL than disease-related factors. This highlights the importance of genetic counseling and guidance for HTAD patients, and offers valuable leads for HRQOL improvement

Efficacy and feasibility of stereotactic radiotherapy after folfirinox in patients with locally advanced pancreatic cancer (LAPC-1 trial)

Background: We conducted a multicentre phase II trial to investigate feasibility and antitumor activity of sequential FOLFIRINOX and Stereotactic Body Radiotherapy (SBRT) in patients with locally advanced pancreatic cancer (LAPC), (LAPC-1 trial). Methods: Patients with biopsy-proven LAPC treated in four hospitals in the Netherlands between December 2014 and June 2017. Patients received 8 cycles of FOLFIRINOX followed by SBRT (5 fractions/8 Gy) if no tumour progression after the FOLFIRINOX treatment was observed. Primary outcome was 1-year overall survival (OS). Secondary outcomes were median OS, 1-year progression-free survival (PFS), treatment-related toxicity, and resection rate. The study is registered with ClinicalTrials.gov, NCT02292745, and is completed. Findings: Fifty patients were included. Nineteen (38%) patients did not receive all 8 cycles of FOLFIRINOX, due to toxicity (n = 12), disease progression (n = 6), or patients’ preference (n = 1). Thirty-nine (78%) patients received the SBRT treatment. The 1-year OS and PFS were 64% (95% CI: 50%-76%) and 3

Health-related quality of life and lived experiences in males and females with thoracic aortic disease and their partners

Objective Thoracic aortic disease (TAD) may have substantial impact on health-related quality of life (HRQOL). We described HRQOL in patients with TAD, cardiovascular screening participants and their partners; identified factors associated with HRQOL; and explored lived experiences and feelings of anxiety or depression using a mixed methods design. Methods For this cross-sectional study, all consecutive patients visiting the TAD outpatient clinic (2017–2019) at our centre were asked to complete three questionnaires: the Short Form 36 (SF-36), the Hospital Anxiety and Depression Scale (HADS) and the Rotterdam Disease Specific Questionnaire (RDSQ). A subsamp