Action Of Rennin Research Articles

Sensory indicators of suluguni cheese when using enzyme preparations of different origins in the technology of soft cheeses

Cheeses are food products obtained by concentration and biotransformation of the main components of milk under the influence of physicochemical factors of rennet enzyme preparations and microorganisms. Suluguni occupies an important place among cheeses in Ukraine. Suluguni cheese has been known for a long time, it was first mentioned in the book of the Pope's personal chef Bartolomeo Scipri. Industrial production of the product was established at the end of the 18th century. The main operation in the production of rennet cheese is the enzymatic coagulation of milk under the action of chymosin, resulting in the formation of a milk clot with a large part of casein and whey. In the body of animals, chymosin, similar to cheese-making technology, coagulates milk at the beginning of its digestion. Thus, the processing of milk in the process of cheese production corresponds to natural physiological processes. III groups of milk samples (n = 5) were formed to conduct the experiment. In the control group of samples, rennet enzyme of microbial origin was used for curdling milk. In the 1st experimental group of samples, an enzyme preparation from the rennet of dairy calves, extracted according to the method of Yu. Ya. Svyridenko, was used. In the II experimental group, an enzyme preparation was used, which was extracted from the rennet of dairy calves according to the method of S. V. Merzlova. The duration of protein coagulation during the production of “Suluguni” cheese using a microbial enzyme preparation is 23 min., when using enzymes of the first research group – 27 min. and when using rennet enzymes of the II research group – 33 min. In view of the output of ready-made Suluguni cheese with a commercial purpose to obtain more profit, it is advisable to use an enzyme of microbial origin. According to sensory analysis, samples using calf rennet enzyme extracted according to the method of S. V. Merzlova (I research group of samples) were characterized by pronounced cheesy, sour-milk, without extraneous tastes and smells, taste and smell characteristic of soft fresh cheese; the surface is clean without mechanical damage, elastic; the consistency of the smear is gentle, moderately dense; the color is white, uniform throughout the mass. Samples of the II experimental group were characterized by the worst organoleptic indicators.

MILK CLOTTING ACTIVITY OF RECOMBINANT CAMEL CHYMOSIN

One of the earliest biotechnological applications of enzymes is cheese production. The high specific milk-clotting activity of chymosin gives it an advantage over other proteases in the cheese industry. The action of chymosin is based on hydrolysis of the bond between the amino acid residues Phe105 and Met106 in the κ-casein molecule, which causes destabilisation of casein micelles, coagulation of milk and its separation into a clot and a whey. A recombinant chymosin of the camel Camelus Bactrianus was obtained using the yeast producer strain Pichia pastoris GS115/pGAPZαA/ProchymCB. The recombinant chymosin was purified by ion exchange chromatography. The milk-clotting activity of recombinant camelian chymosin against mare's milk was studied as a function of pH and temperature. It was found that the optimum value at which the maximum activity of recombinant camel's chymosin with respect to mare's milk is observed is pH 4.0 and the optimum temperature is 70°C. The activity of recombinant camel chymosin on mare, cow and goat milk was 123, 9605 and 4650 units/mg, respectively. Recombinant camel chymosin was shown to have high coagulation activity, sufficient to produce curd and cheese. The yield of cheese clot from 6 l of goat milk was 936 grams. Recombinant camel chymosin has prospects for use in cow's and goat's milk processing and cheese production technologies.

Glycomacropeptide in PKU-Does It Live Up to Its Potential?

The use of casein glycomacropeptide (CGMP) as a protein substitute in phenylketonuria (PKU) has grown in popularity. CGMP is derived from κ casein and is a sialic-rich glycophosphopeptide, formed by the action of chymosin during the production of cheese. It comprises 20–25% of total protein in whey products and has key biomodulatory properties. In PKU, the amino acid sequence of CGMP has been adapted by adding the amino acids histidine, leucine, methionine, tyrosine and tryptophan naturally low in CGMP. The use of CGMP compared to mono amino acids (L-AAs) as a protein substitute in the treatment of PKU promises several potential clinical benefits, although any advantage is supported only by evidence from non-PKU conditions or PKU animal models. This review examines if there is sufficient evidence to support the bioactive properties of CGMP leading to physiological benefits when compared to L-AAs in PKU, with a focus on blood phenylalanine control and stability, body composition, growth, bone density, breath odour and palatability.

Proteolysis in Irish farmhouse Camembert cheese during ripening.

Proteolysis in an Irish farmhouse Camembert cheese was studied during 10weeks of ripening. Urea-polyacrylamide gel electrophoresis of pH 4.6-insoluble fractions of cheese showed the degradation of caseins, initially due to the action of chymosin and plasmin and later due to Penicillium camemberti proteinases. Proteolytic specificities of Penicillium camemberti proteinases on the caseins in milk hydrolysates were determined and 64, 6, 28, and 2 cleavage sites were identified in αs1 -, αs2 -, β-, and κ-casein, respectively. Proteolysis in cheese was studied and peptides produced were determined and compared to the cleavage specificities of Penicillium camemberti proteinases. Regions most susceptible to proteolysis were 1-40, 79-114, and 168-199 in αs1 -casein; 42-79 and 97-116 in αs2 -casein; 40-57, 101-125, 143-189, and 165-209 in β-casein; and 31-81 and 124-137 in κ-casein. The present study describes in detail the proteolytic action of proteinases from Penicillium camemberti in Camembert cheese during ripening. PRACTICAL APPLICATIONS: Camembert cheese is a major international cheese variety, made in many countries around the world. The ripening of the cheese involves many biochemical changes and this study provides new information on peptides produced during ripening. Penicillium camemberti is an important mold used in the production of this type of cheese and detailed information is provided on the action of its enzymes on the caseins. Data reported in this study furthers the understanding of the ripening of Camembert cheese.

Study of milk coagulation induced by chymosin using atomic force microscopy

Caseins (αs1-, αs2, β-, and κ-caseins) form the major protein fraction of milk, irrespective of their origins. They are able to form well-ordered colloidal structures in association with colloidal calcium phosphate, named casein micelles. Chymosin-mediated milk coagulation takes place through loss of casein micelles’ stability by hydrolyzing κ-caseins. This process is critical for the quality of cheese and other milk derivatives. Therefore, many microscopy techniques have been used to understand the structural aspects underlying the integrity of casein micelles during chymosin action. However, these technologies can be costly and laborious. In this study, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to study milk coagulation by chymosin. Following 15 min of chymosin action, the AFM images showed the start of the formation of casein micelle aggregates. After 30–45 min, the micelles continued aggregating, forming structures such as bunches of grapes. Finally, after 45–60 min, these structures formed large clusters of casein micelles. After 60 min, the zeta potential did not drop to zero when the milk clotted, but still had a negative value, suggesting that the κ-caseins on the micelle surface were not totally hydrolyzed. The results corroborate those previously described and suggest this tool as an alternative to study the milk-clotting process at the ultrastructural level induced by proteases.

Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese

We applied capillary electrophoresis, liquid chromatography coupled with tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to determine the composition of water-insoluble and water-soluble proteinaceous fractions of the cheese and to study in detail the degradation of caseins during 8 mo of ripening of Estonian high-temperature cooked hard cheese Old Saare. The application of high-resolution and high-accuracy MS/MS enabled identification of more than 3,000 small peptides, representing a fairly full casein peptidome containing peptides of 4 to 25 AA in length: 1,049 from β-casein (CN), 944 from αS1-CN, 813 from αS2-CN, and 234 from κ-CN. The majority of β-CN- and αS1-CN-derived peptides originated from the N-terminal parts of the molecule, f6-93 and f1-124, respectively; peptides from αS2-CN arose predominantly from the C-terminal end f100-162. At the beginning of ripening, we found a relatively high amount of peptides originating from the glycomacropeptide part of κ-CN, whereas peptides from para-κ-CN prevailed during the later stages of ripening of the cheese. The cleavage patterns of β-CN, αS2-CN, as well as αS1-CN, showed that primary proteolysis was started mainly by plasmin, although a low proteolytic activity of chymosin was also evident. Based on the analysis of cleavage sites, we observed a significant participation of proteolytic enzymes, including amino- and carboxypeptidases, of both mesophilic and thermophilic starter bacteria in further hydrolysis of oligopeptides during the ripening. Several new phosphopeptides were detected in the result of MS/MS data analysis. The profiles of the estimated concentrations of phosphopeptides revealed that those originating from β-CN and αS1-CN accumulated during cheese maturation. In contrast, we did not notice any generation of phosphopeptides from the highly phosphorylated part of αS2-CN, f25-80, presumably due to the inaccessibility of this region to the action of plasmin and chymosin. The analysis of cleavage sites and the combination of principal component and clustering analyses provided a characterization of the complex dynamics of formation and degradation of peptides during cheese maturation. We made an attempt to obtain a comprehensive picture of proteolysis during Old Saare cheese ripening on the basis of the detailed peptidomic data, including also the less abundant peptides determined by MS/MS, and complemented by the data on intact caseins and free AA and reported the results in the paper.

Glycomacropeptide Reduces Intestinal Epithelial Cell Barrier Dysfunction and Adhesion of Entero-Hemorrhagic and Entero-Pathogenic Escherichia coli in Vitro.

In recent years, the potential of glycosylated food components to positively influence health has received considerable attention. Milk is a rich source of biologically active glycoconjugates which are associated with antimicrobial, immunomodulatory, anti-adhesion, anti-inflammatory and prebiotic properties. Glycomacropeptide (GMP) is the C-terminal portion of kappa-casein that is released from whey during cheese-making by the action of chymosin. Many of the biological properties associated with GMP, such as anti-adhesion, have been linked with the carbohydrate portion of the protein. In this study, we investigated the ability of GMP to inhibit the adhesion of a variety of pathogenic Escherichia coli strains to HT-29 and Caco-2 intestinal cell lines, given the importance of E. coli in causing bacterial gastroenteritis. GMP significantly reduced pathogen adhesion, albeit with a high degree of species specificity toward enteropathogenic E. coli (EPEC) strains O125:H32 and O111:H2 and enterohemorrhagic E. coli (EHEC) strain 12900 O157:H7. The anti-adhesive effect resulted from the interaction of GMP with the E. coli cells and was also dependent on GMP concentration. Pre-incubation of intestinal Caco-2 cells with GMP reduced pathogen translocation as represented by a decrease in transepithelial electrical resistance (TEER). Thus, GMP is an effective in-vitro inhibitor of adhesion and epithelial injury caused by E. coli and may have potential as a biofunctional ingredient in foods to improve gastrointestinal health.

The inhibitory roles of native whey protein on the rennet gelation of bovine milk

Rennet gelation is used to produce many types of cheese. The effect of native whey protein on rennet gelation kinetics was investigated. Milks with a wide range of whey protein:casein (WP:CN) ratios (with standardised casein concentrations) were made from powders produced by microfiltration. Measurements of casein macro peptide release showed that native whey protein inhibited the enzymatic action of chymosin, which delayed the onset and reduced the subsequent rate of gelation. Experiments in which increased chymosin concentrations compensated for the inhibition, demonstrated that other factors also contributed to the reduced gelation rate. Neither an increase in viscosity nor a reduction in soluble calcium was responsible, leading to the conclusion that in addition to inhibiting chymosin, native whey proteins present a physical barrier to para-casein aggregation. This study demonstrates and explains how casein-enriched retentates from microfiltration gel faster than regular cheese milk that contains higher amounts of native whey protein.

Glycomacropeptide Attenuates Inflammation, Pruritus, and Th2 Response Associated with Atopic Dermatitis Induced by 2,4-Dinitrochlorobenzene in Rat.

Atopic dermatitis (AD) is one of the most common skin diseases, whose incidence is increasing in industrialized countries. The epicutaneous application of a hapten, such as 2,4-dinitrochlorobenzene (DNCB), evokes an experimental murine AD-like reaction. Glycomacropeptide (GMP) is a dairy bioactive peptide derived from hydrolysis of κ-casein by chymosin action. It has anti-inflammatory, prebiotic, and immunomodulatory effects. The present study was aimed to investigate the effect of GMP administration on DNCB-induced AD in rats. The severity of inflammatory process, pruritus, production of cytokines, and total immunoglobulin E (IgE) content were measured, and the histopathological features were analyzed. GMP reduced the intensity of inflammatory process and edema of DNCB-induced dermatitis, with a significant decrease in eosinophils recruitment and mast cells hyperplasia. In addition GMP suppressed the serum levels of total IgE and IL-4, IL-5, and IL-13 expression in AD-lesions. Besides, the levels of IL-10 were significantly increased. Remarkably, GMP administration before AD-induction abolished pruritus in dermatitis-like reactions in the rats. Taken together, these results indicate that GMP has an inhibitory effect on AD by downregulating Th2 dominant immune response, suggesting GMP as a potential effective alternative therapy for the prevention and management of AD.

Lactobacillus rhamnosus GG encapsulation by spray-drying: Milk proteins clotting control to produce innovative matrices

A well-known probiotic strain, L. rhamnosus GG, was encapsulated by spray-drying in milk water-insoluble matrices upon reconstitution in hot water by exploiting and controlling the clotting reaction of milk proteins during the process. The feed solution, composed of probiotic bacteria and milk proteins, was or not subjected to the action of chymosin, a proteolytic enzyme. To optimize microencapsulation efficiency, different outlet air temperatures were tested (55, 70 and 85 °C). After spray-drying, small microparticles were recovered for further characterization. All drying conditions led to excellent bacterial survival rates (<0.5 log reduction) whereas only the highest outlet air temperature allowed the production of microparticles with acceptable moisture contents (<7%) to ensure storage stability. Finally, enzymatic cleavage of milk proteins by chymosin before atomization led to matrices presenting innovative functionalities when microparticles are reconstituted with water: rehydration or dispersion in cold (8 °C) or warm (40 °C) water, respectively.

Study of proteolysis in river buffalo mozzarella cheese using a proteomics approach

The guarantee of the origin and quality of raw material is essential for the protection and valorization of Campana buffalo mozzarella cheese. The risk of utilization of semifinished products and stored milk in substitution for fresh milk is increasing, due to the continuous desire to reduce production costs. A proteomics approach and electrophoresis survey of retail mozzarella cheeses indicated different rates of proteolysis in the production of dairy industries. The use of fresh milk and correct cheesemaking protocol yielded only γ-caseins, which are derived from β-casein by plasmin, and para-κ-casein, which is derived from κ-casein by chymosin. The detection of abnormal hydrolysis resulting in β- and αS1-casein fragments, identified by mass spectrometry, indicates the use of stored milk or stored and pressed curd, or the reuse of unsold mozzarella cheese, to produce mozzarella. The formation of γ-caseins and other fragments during a long storage of raw materials at room or refrigeration temperature was ascribed to plasmin (endogenous milk enzyme), whereas formation of αS1-casein fragments, mainly αS1-I6P- and αS1-I7P-casein during the storage of curd was ascribed to the action of chymosin (exogenous enzyme) from rennet. Sodium dodecyl sulfate-PAGE and alkaline urea-PAGE permitted us to evaluate the freshness of the raw materials used in the manufacturing of buffalo mozzarella cheese and to reveal possible inappropriate preservation.

A Sialic Acid Assay in Isolation and Purification of Bovine .&amp;#954;-Casein Glycomacropeptide: A Review

Sialic acid is a carbohydrate moiety of k-casein glycomacropeptide (GMP), which is a 64 amino acid residue C-terminal sialylated phosphorylated glycopeptide released from k-casein by the action of chymosin during cheese making. GMP lacks aromatic amino acids including phenylalanine, tyrosine, and tryptophan. Because of its unique amino acid composition and various biological activities, GMP is thought to be a potential ingredient for dietetic foods (e.g., a food for PKU patients) and pharmaceuticals. Thus, increased attention has been given to the development of techniques to purify GMP. In this review, techniques of GMP purification described in patents and scientific research papers were introduced. A sialic acid assay is the important method to track GMP isolation and purification processes, for which the thiobarbituric acid reaction with 1-propanol as a chromophore extracting solvent is an inexpensive, practical and specific technique. Sephacryl S-200 gel filtration chromatography, cellulose acetate electrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis are the major techniques to identify sialic acid specific to GMP. Sephacryl S-200 chromatography and cellulose acetate electrophoresis are also used to detect GMP sialic acid in whey pearmeate and whey added commercial margarine samples. Future research includes development of an economical industrial scale method to produce high purity GMP.

Detection of αs1-I casein in mozzarella Fiordilatte: A possible tool to reveal the use of stored curd in cheesemaking

The assessment of the origin and quality of raw materials is pivotal for the protection and valorization of typical dairy products. Italian high moisture mozzarella cheese, also called “Fiordilatte”, is manufactured by stretching in hot water the curd, which can be produced inside the dairy (fresh curd) or purchased as semi-finished product from specialized companies (pre-made stored curd). The employment of stored curd allows cheese manufacturers to obtain economic profit thanks to the reduction of the production costs. The use of the semi-finished product has not to be mentioned in the label according to the actual EU regulation, and this has given rise to competition between local milk farmers and curd-producing companies causing misinformation among the consumers, and unfair competition to the disadvantage of the traditional dairies. In the present research, a proteomic approach was used to investigate “Fiordilatte” produced with or without the employment of stored curd. The results obtained allowed us to identify a molecular marker which is present in very small amounts in cheese made from fresh curd and at high levels in cheese made from stored curd. The marker is αs1-I casein (fragment 24–199), the main product of primary proteolysis formed by the action of chymosin on αs1 casein. It can be easily detected by electrophoresis in the presence of urea (urea-PAGE) and is measured by image analysis. Even though the kinetic of formation of this proteolytic product has been well known for several decades, in mozzarella it has a peculiar behavior compared to other cheeses. As a matter of fact, it originates during the first part of cheesemaking, and has a very slow increase during storage of the cheese, due to the denaturation of the clotting enzyme by the hot water stretching process. The results obtained demonstrated that the quantification of αs1-I fragment should be recommended to guarantee the production of “Fiordilatte” obtained from fresh milk under normal conditions, and could be the basis for the development of a method able to fully protect the traditional cheesemaking procedure.

Chemical and functional properties of glycomacropeptide (GMP) and its role in the detection of cheese whey adulteration in milk: a review

Glycomacropeptide (GMP) is a C-terminal part (f 106–169) of kappa-casein which is released in whey during cheese making by the action of chymosin. GMP being a biologically active component has gained much attention in the past decade. It also has unique chemical and functional properties. Many of the biological properties have been ascribed to the carbohydrate moieties attached to the peptide. The unique set of amino acids in GMP makes it a sought-after ingredient with nutraceutical properties. Besides its biological activity, GMP has several interesting techno-functional properties such as wide pH range solubility, emulsifying properties as well as foaming abilities which are shown to be promising for applications in food and nutrition industry. These properties of GMP have given new dimension for the profitable utilization of cheese whey to the dairy industry. A number of protocols for isolation of GMP from cheese whey have been reported. Moreover, its role in detection of sweet/rennet whey adulteration in milk and milk products has also attracted attention of various researchers, and many GMP-specific analytical methods have been proposed. This review discusses the chemico-functional properties of GMP and its role in the detection methods for checking cheese or sweet whey adulteration in milk. Recent concepts used in the isolation of GMP from cheese whey have also been discussed.

Interfacial and foaming interactions between casein glycomacropeptide (CMP) and propylene glycol alginate

Proteins and polysaccharides are widely used in food formulation. While most of the proteins are surface active, only few polysaccharides can adsorb at the air–water interface; this is the case of propylene glycol alginates (PGA). It is known that casein glycomacropeptide (CMP), a bioactive polypeptide derived from κ-casein by the action of chymosin, presents a great foaming capacity but provides unstable foams. So, the objective of this work was to analyze the impact of mixing CMP and a commercial variety of PGA, Kelcoloid O (KO), on the interfacial and foaming properties at pH 7.0. It was determined the surface pressure isotherm, the dynamics of adsorption and the foaming properties for CMP, KO and the mixed system CMP–KO.CMP dominated the surface pressure of CMP–KO mixed system. The presence of KO synergistically improved the viscoelastic properties of surface film. The foaming capacity of CMP was altered by KO. KO foam presented a higher stability than CMP foam and it controlled the stability against drainage and the initial collapse in the mixed foam.

The rennet coagulation mechanisms of a concentrated casein suspension as observed by PFG-NMR diffusion measurements

Pulsed field gradient-nuclear magnetic resonance (PFG-NMR) was used to monitor the diffusion of caseins throughout the rennet coagulation of a highly concentrated casein suspension. Two types of casein species were distinguished according to their diffusing properties and attributed to the dissolved casein fraction, i.e. dissociated caseins and caseinomacropeptides (CMP), and casein particles, respectively. The NMR signal intensity coming from the dissolved casein fraction, which is related to the number of protons it contains, increased all along the experiment whereas the opposite tendency was observed for casein particles. This was explained by the increasing amount of CMP in the whey and the reciprocal loss of protons contained by casein particles, and used to quantitatively estimate the kinetics of the chymosin action. The diffusion of the dissolved casein fraction remained nearly constant during the coagulation process whereas, as revealed by rheometry measurements, the diffusion of casein particles was very sensitive to the structural reorganization of the sample. It decreased by about 35% during the sol–gel transition and increased in similar proportions during the succeeding rise in gel porosity. Our results also provide different types of information on the respective behaviors of dissociated caseins and casein particles during the course of the process, the most remarkable one being that all the casein particles did not aggregate during the sol–gel transition of our sample. This strongly suggests that the rennet gelation of a concentrated dairy solution may be better visualized by the formation of a network backbone during the sol–gel transition which is thereafter reinforced upon further incorporation of casein particles.

From humans to giraffes: The evolution of hypertension and hormones

From humans to giraffes: The evolution of hypertension and hormones

Casein glycomacropeptide pH-driven self-assembly and gelation upon heating

Casein glycomacropeptide (CMP) found in cheese whey is a C-terminal hydrophilic glycopeptide released from κ-casein by the action of chymosin during cheese making. In a previous work a self-assembly model for CMP at room temperature was proposed, involving a first step of hydrophobic assembly followed by a second step of electrostatic interactions which occurs below pH 4.5. The objective of the present work was to study, by dynamic light scattering (DLS), the effect of heating (35–85°C) on the pH-driven CMP self-assembly and its impact on the dynamics of CMP gelation. The concentration of CMP was 3%w/w for DLS and 12%w/w for rheological measurements. The solutions at pH 4.5 and 6.5 did not show any change in the particle size distributions upon heating. In contrast the solutions at pH lower than 4.5 that showed electrostatic self-assembly at room temperature were affected by heating. The mean diameter of assembled CMP increased by decreasing pH. For all solutions with pH lower than 4.5, the particle size did not change on cooling, suggesting that the assembled CMP forms formed during heating were stable. The gel point determined as G′–G″ crossover, occurred in all systems at 70°C, but at different times. The rate of self-assembly determined by DLS as well as the rate of gelation increased with increasing temperature and decreasing pH from 4 to 2. Increasing temperature and decreasing pH, the first step of CMP self-assembly by hydrophobic interactions is speed out. All the self-assembled structures and the gels formed at different temperatures were pH-reversible but did not revert to the initial size (monomer) but to associated forms that correspond mainly to CMP dimers.

A study on bovine kappa-casein aggregation after the enzymatic action of chymosin

Kappa-casein (κ-CN) is a milk phospho-glycoprotein that plays an important role in the electrostatic and steric stabilization of casein micelles suspensions. κ-CN characteristics such as its aminoacidic composition and its high denaturalization temperature make this protein an excellent nutrient. The enzymatic destabilization of κ-CN is the basis of cheese and yoghurt manufacture. In this paper, modifications on the κ-CN aggregation process by hydrolysis with chymosin derived from the initial protein state and the presence of sucrose and lactose were studied. Our study shows that, during its self-association, κ-CN opens its conformation by raising the exposition of hydrophobic regions to the medium. Hence, the initial polymerization or association state of κ-CN affects on the aggregation stage after the enzymatic action of chymosin. The inhibition of aggregation when the protein was previously associated suggests that the region which participates in the aggregation of p-κ-CN particles, would be the one involved in the self-association during the heating of κ-CN samples. Sucrose and lactose also affect the aggregation of proteolized particles of κ-CN. Furthermore, the nature and the concentration of the added sugars had significant influence on the protein aggregation process.

Effects of Acidification with and without Rennet on a Concentrated Casein System: A Kinetic NMR Probe Diffusion Study

The self-diffusion coefficients of 620 and 96 750 g/mol poly(ethylene glycol) (PEG) were measured by pulsed field gradient (PFG)-NMR throughout the coagulation processes of a concentrated casein suspension induced by acidification alone and with the concomitant action of chymosin. The diffusion of water molecules was also investigated during the acidification process. In all experiments, the diffusion coefficient of the molecule studied was modified nonlinearly, and the precise moments at which its evolution changed corresponded to key stages of both coagulation processes. These variations were not directly related to sol−gel transitions as revealed by rheological measurements but were caused by other types of structural changes in the sample whatever its viscoelastic state (solution or gel). The diffusion of the large PEG was very sensitive to variations in the size of the casein particles and the casein aggregates constituting the network before and after gelation, respectively. In contrast, the diffusi...