Non-linear properties and yielding of enzymatic milk gels.

One of the first steps of cheese making is to suppress the colloidal stability of casein micelles by enzymatic hydrolysis and initiate milk gelation. Afterwards, the enzymatic milk gel is cut to promote syneresis and expulsion of the soluble phase of milk. Many studies have reported on the rheological properties of enzymatic milk gels at small strain, but they provide limited information on the ability of the gel to be cut and handled. In this study, we aim to characterize the non-linear properties and the yielding behavior of enzymatic milk gels during creep, fatigue and stress sweep tests. We evidence by both continuous and oscillatory shear tests that enzymatic milk gel displays irreversible and brittle-like failure, as reported for acid caseinate gels, but with additional dissipation during fracture opening. Before yielding, acid caseinate gels display strain-hardening only, while enzymatic milk gels also display strain-softening. By varying the gel aging time and the volume fraction of casein micelles, we are able to attribute the hardening to the network structure and the softening to local interactions between casein micelles. Our study highlights the crucial importance of the nanoscale organization of the casein micelles - or more generally of the building block of a gel - to retain the macroscopic nonlinear mechanical properties of the gel.

Ionic Strength Dependence of the Complex Coacervation between Lactoferrin and ß-Lactoglobulin.

Heteroprotein complex coacervation is an assembly formed by oppositely charged proteins in aqueous solution that leads to liquid-liquid phase separation. The ability of lactoferrin and ß-lactoglobulin to form complex coacervates at pH 5.5 under optimal protein stoichiometry has been studied in a previous work. The goal of the current study is to determine the influence of ionic strength on the complex coacervation between these two proteins using direct mixing and desalting protocols. The initial interaction between lactoferrin and ß-lactoglobulin and subsequent coacervation process were highly sensitive to the ionic strength. No microscopic phase separation was observed beyond a salt concentration of 20 mM. The coacervate yield decreased drastically with increasing added NaCl from 0 to 60 mM. The charge-screening effect induced by increasing the ionic strength is attributed to a decrease of interaction between the two oppositely charged proteins throughout a decrease in Debye length. Interestingly, as shown by isothermal titration calorimetry, a small concentration of NaCl around 2.5 mM promoted the binding energy between the two proteins. These results shed new light on the electrostatically driven mechanism governing the complex coacervation in heteroprotein systems.

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.

Mixed dairy and plant-based yogurt alternatives: Improving their physical and sensorial properties through formulation and lactic acid bacteria cocultures.

Food transition requires incorporating more plant-based ingredients in our diet, thus leading to the development of new plant-based products, such as yogurt alternatives (YAs). This study aimed at evaluating the impact of lactic acid bacteria (LAB) cocultures and formulation on the physico-chemical and sensory properties of YAs. YAs were made by emulsifying anhydrous milk fat (AMF) or coconut oil in milk and lupin protein suspensions. The starters used, in mono- and cocultures, were the strains Lactococcus lactis NCDO2125, Enteroccocus faecalis CIRM-BIA2412 and Lactiplantibacillus plantarum CIRM-BIA1524. Textural properties and metabolites of YAs were evaluated and their sensory properties compared using a sorting task. Some cocultures led to higher firmness, viscosity, and water holding capacity of YAs, compared to monocultures. AMF and a milk:lupin protein ratio of 67:33 gave firmer and more viscous YAs. YAs were sensorially discriminated on the basis of protein ratio and fat type, but not of starters. The cocultures exhibited more diverse functional outputs, such as texturing, production of flavour compounds, proteolysis, when the strains associated in coculture had distinct capacities. Appropriate associations of LAB and formulation offer interesting solutions to improve the perception of YAs, and ultimately, encourage their consumption.

Aroma-retention capacities of functional whey protein aggregates: Study of a strawberry aroma in solutions and in fat-free yogurts.

The aroma-retention capacity of functional whey protein aggregates (WPA) was compared to that of native whey protein isolate (WPI) in aqueous solutions and in fat-free yogurts. The retention of aroma compounds, constituting a model strawberry aroma, was evaluated by calculating gas-matrix partition coefficients using headspace gas chromatography (HS-GC). The retention capacity of WPA differed from the one of WPI for three out of seven aroma compounds detected in HS-GC. Incorporating WPA in fat-free yogurts tended to decrease the release of hydrophobic aroma compounds such as 2-nonanone or methyl-cinnamate. The magnitude of the differences between the partition coefficients of yogurts enriched in WPI or WPA was lower than in aqueous solutions, which is likely to be due to the higher complexity of the food matrix and potential interactions with other ingredients. Overall, the different aroma-retention capacities of native WPI and functional WPA are likely to lead to unbalanced aroma, especially in fat-free dairy products.

Major Role of Voluminosity in the Compressibility and Sol-Gel Transition of Casein Micelle Dispersions Concentrated at 7 °C and 20 °C.

The objective of this work is to bring new information about the influence of temperatures (7 °C and 20 °C) on the equation of state and sol-gel transition behavior of casein micelle dispersions. Casein micelle dispersions have been concentrated and equilibrated at different osmotic pressures using equilibrium dialysis at 7 °C and 20 °C. The osmotic stress technique measured the osmotic pressures of the dispersions over a wide range of concentrations. Rheological properties of concentrated dispersions were then characterized, respectively at 7 °C and at 20 °C. The essential result is that casein micelle dispersions are less compressible at 7 °C than at 20 °C and that concentration of sol-gel transition is lower at 7 °C than at 20 °C, with compressibility defined as the inverse to the resistance to the compression, and that is proportional to the cost to remove water from structure. From our interpretations, these two features were fully consistent with a release of soluble ß-casein and nanoclusters CaP and an increased casein micelle hydration and apparent voluminosity at 7 °C as compared with 20 °C.

The surface properties of milk fat globules govern their interactions with the caseins: Role of homogenization and pH probed by AFM force spectroscopy.

The surface of milk fat globules consists of a biological membrane rich in polar lipids and glycoproteins. However, high shear stress applied upon homogenization disrupts the membrane and leads to the adsorption of casein micelles, as the major protein fraction of milk. These changes in the interface properties could affect the interactions between native or homogenized milk fat globules and the surrounding protein matrix, at neutral pH and upon acidification. In this study, macroscale rheometry, microscopic observations, nanoscale AFM-based force spectroscopy and physico-chemical analysis were combined to examine the interfacial composition and structure of milk fat globules and to evaluate their interactions with casein micelles. We showed that the surface properties of milk fat globules (biological membrane vs. caseins) and pH govern their interactions with casein micelles. The adhesion between individual fat globules and casein micelles was higher upon homogenization, especially at acid pH where the work of adhesion increased from 3.3 x 10-18 to 14 x 10-18 J for native and homogenized fat globules, respectively. Consequently, casein-coated homogenized fat globules yield stiffer milk acid gels. These findings cast light on the importance of colloidal particle's surface properties and pH on their connectivity with the surrounding matrix, which modulates the bulk microstructure and rheological properties with potential functional consequences, such as milk lipid digestion.

Influence of casein on the formation of whey protein microparticles obtained by dry heating at an alkaline pH.

Dry heating (DH) at 100 °C for 36 h of a whey protein isolate powder conditioned at pH 9.5 leads to the formation of stable, large and porous whey protein microparticles (PMs), resulting from the crosslinking of proteins inside the powder. These PMs could be used as high-viscosity food ingredients. Casein, present as a contaminant in whey protein powders, has been shown to become incorporated into the PMs. In this study, we investigated the effect of adding increasing amounts of sodium caseinate to whey protein powders on the formation of PMs during DH at 100 °C for 36 h. In addition, we studied PM formation during DH of a micellar casein-enriched milk protein powder (Casmic). The browning index of the dry-heated powders, and the size and water content of the microparticles were also characterized. We confirmed that sodium caseinate was incorporated into the PMs. The highest PM D[4,3] values (270 µm) were observed for powders with around 40% caseinate. Powders without added caseinate displayed D[4,3] values of 150 µm. The yield of conversion of proteins into PMs increased from 0.6 to 0.8 g/g with caseinate addition, whereas the amount of water entrapped in the PMs decreased from around 30 to 20 g/g. PMs were also formed by DH of the Casmic powder, but these particles were smaller, with sizes of around 80 µm. In conclusion, our study shows that the process of DH at pH 9.5 could be applied to all milk proteins to obtain PMs with functional properties that could be used in the food industry.

Gastric Emptying and Dynamic In Vitro Digestion of Drinkable Yogurts: Effect of Viscosity and Composition.

Gastric emptying of food is mainly driven by the caloric concentration, the rheological properties of the chyme, and the physical state (liquid/solid) of food once in the stomach. The present work investigated: (1) The effect of the composition and the viscosity of drinkable yogurts on gastric emptying in pigs, and (2) the behavior of yogurts during dynamic in vitro digestion. Three isocaloric liquid yogurts were manufactured: Two enriched in protein and fiber showing either a low (LV) or high (HV) viscosity, one control enriched in sugar and starch (CT). They were labelled with 99mTc-sulfur colloid and given to pigs (n = 11) to determine gastric emptying pattern by gamma scintigraphy. Then dynamic in vitro digestion of the yogurts was done using the parameters of gastric emptying determined in vivo. Gastric emptying half-times were significantly longer for LV than CT, whereas HV exhibited an intermediate behavior. In vitro gastric digestion showed a quick hydrolysis of caseins, whereas whey proteins were more resistant in the stomach particularly for LV and HV. During the intestinal phase, both whey proteins and caseins were almost fully hydrolyzed. Viscosity was shown to affect the behavior of yogurt in the small intestine.

Dry heating of whey proteins leads to formation of microspheres with useful functional properties.

Modification of whey protein isolate (WPI) powders is used in the food industry to enhance the functional properties of WPI. We investigated the impact of severe dry heating (DH) at 100 °C for up to 36 h on an alkaline-treated (pH 9.5), spray dried (water activity of ~0.24) WPI powder. Dry heated powders and their reconstituted suspensions were analysed. DH for 0-6 h led to 47% loss of native proteins, increases in the levels of soluble aggregates (×2.2) and of advanced glycation end-products of the Maillard reaction (at least ×2.7) and to powder browning (at least ×3) with a 95% decrease in free lactose content. DH for at least 12 h led to a decrease in soluble aggregates with concomitant formation of large, stable and insoluble microparticles. These microparticles had a microsphere structure, contained 98% of water phase and were made of insoluble powder particles resulting from protein cross-links during DH. Microparticle size could be altered by varying the pH of the suspension: at pH 6.5, microsphere size was 3-5 times larger than powder particle size, but decreased as the suspension pH neared the isoelectric point. DH could be a useful method for producing functional protein ingredients as these microparticles had very high water retention properties and high viscosity values.

Dry heating of whey proteins.

Whey protein products are of widespread use as food ingredients because of their high nutritional, biological and functional properties. Whey proteins are important structural components in many foods as used in their native form, for example for their heat-induced gelation abilities. Furthermore, they also offer reliable functionalities when modified by heating processes as denatured or aggregated proteins. Heat treatment of whey proteins in a liquid state has received much attention in recent years. While dry heating of whey proteins, say heating whey proteins in the dry state, is frequently cited in the literature as a potential and efficient means to improve the functional properties of proteins, it has received very little attention. We report first on the dry heating of whey products as applied to promote glycation of whey proteins with a low denaturation, and second, to promote their denaturation and aggregation and on their consequences on the functional properties of whey proteins.

Lipid droplets coated with milk fat globule membrane fragments: Microstructure and functional properties as a function of pH.

Processed lipid droplets coated by milk fat globule membrane (MFGM) material are of primary interest to mimic the specific functions provided by the fat globules in milk and dairy products. The objectives were to investigate, as a function of pH, the properties and microstructure of MFGM-coated lipid droplets prepared with an ingredient rich in MFGM containing polar lipids and proteins. The samples were prepared in water and in milk ultrafiltrate. The combination of microscopy techniques, zeta potential and particle size measurements, and rheological determinations was used. We showed that all the components of the ingredient were highly sensitive to pH. Both the polar lipids and proteins contributed to the isoelectric point of the MFGM-rich ingredient at pI=4.2. Lipid droplets were coated with MFGM fragments both adsorbed at the surface of fat and protruding in the aqueous phase. Below pH5.5 the microstructure of the emulsions was affected by aggregation of the lipid droplets and formation of a gel. The emulsions prepared in water did not show coalescence upon 30days storage, while those prepared in milk ultrafiltrate showed coalescence for pH below 5.5. This study demonstrates that the MFGM-rich ingredient has excellent emulsifying properties and will contribute in the development of emulsions containing MFGM-coated lipid droplets for techno-functional, nutritional and health benefits (e.g. in infant formulas).

Immunomodulation properties of multi-species fermented milks.

Dairy propionibacteria (PAB) are used as a ripening starter in combination with Lactic acid bacteria (LAB) for dairy products such as Swiss-type cheese. LAB and PAB have also been studied for their probiotic properties but little is still known about their individual and/or synergistic beneficial effects within dairy matrices. In the context of a rising incidence of Inflammatory Bowel Diseases, it has become crucial to evaluate the immunomodulatory potential of bacteria ingested in large numbers via dairy products. We therefore selected different strains and combinations of technological LAB and PAB. We determined their immunomodulatory potential by IL-10 and IL-12 induction, in human peripheral blood mononuclear cells, on either single or mixed cultures, grown on laboratory medium or directly in milk. Milk was fermented with selected anti-inflammatory strains of LAB or PAB/LAB mixed cultures and the resulting bacterial fractions were also evaluated for these properties, together with starter viability and optimum technological aspects. The most promising fermented milks were evaluated in the context of TNBS- or DSS-induced colitis in mice. The improvement in inflammatory parameters evidenced an alleviation of colitis symptoms as a result of fermented milk consumption. This effect was clearly strain-dependent and modulated by growth within a fermented dairy product. These findings offer new tools and perspectives for the development of immunomodulatory fermented dairy products for targeted populations.

Casein Compared with Whey Proteins Affects the Organization of Dietary Fat during Digestion and Attenuates the Postprandial Triglyceride Response to a Mixed High-Fat Meal in Healthy, Overweight Men.

BACKGROUND: Postprandial lipemia is a risk factor for cardiovascular disease. The potential impacts of the type/nature of dietary protein on postprandial lipemia and associated dysregulations have been insufficiently investigated. OBJECTIVE: We investigated the postprandial effect of including in a high-fat meal some milk protein fractions that markedly differ in their physicochemical properties and composition [either casein (CAS), whey protein (WHE), or α-lactalbumin-enriched whey protein (LAC)]. METHODS: The protein fractions were incorporated as 15% energy in a high-fat meal in a 3-period, crossover postprandial study of 10 healthy overweight men with an elevated waist circumference (>94 cm). We measured postprandial changes in plasma lipids, amino acids, glucose, and oxidative stress markers, vascular function (using pulse contour analysis), and low-grade inflammation (using plasma markers). We also characterized in vitro the meal structures, including the size of the fat globule, and possible changes during digestion. RESULTS: The type of protein did not affect postprandial plasma glucose, amino acids, insulin, or nonesterified fatty acids, but, compared with WHE and LAC, which did not differ, CAS markedly reduced postprandial triglycerides (TGs), achieving a 22 ± 10% reduction in the 6-h area under the curve (P < 0.05). Similar trends were shown for plasma chylomicrons [apolipoprotein (apo)B-48; P < 0.05]. However, there were no significant differences between the meals regarding postprandial oxidative stress (plasma hydroperoxides and malondialdehyde), endothelial dysfunction (salbutamol-induced changes in pulse contour analysis), or low-grade inflammation. In vitro studies showed that when the pH of the meal decreased to stomach pH values, the reduction in the solubility of casein resulted in a phase separation between fat and protein, whereas the proteins in the other meals remained suspended with fat globules. CONCLUSION: In healthy overweight men, casein has specific physical interactions with fat that affect postprandial TGs, leading to the formation of fewer chylomicrons or an increase in chylomicron clearance. This trial was registered at clinicaltrials.gov as NCT00931151.

Acid and rennet gels exhibit strong differences in the kinetics of milk protein digestion and amino acid bioavailability.

This study aimed at determining the kinetics of milk protein digestion and amino acid absorption after ingestion by six multi-canulated mini-pigs of two gelled dairy matrices having the same composition, similar rheological and structural properties, but differing by their mode of coagulation (acidification/renneting). Duodenal, mid-jejunal effluents and plasma samples were collected at different times during 7h after meal ingestion. Ingestion of the acid gel induced a peak of caseins and ß-lactoglobulin in duodenal effluents after 20min of digestion and a peak of amino acids in the plasma after 60min. The rennet gel induced lower levels of both proteins in the duodenum (with no defined peak) as well as much lower levels of amino acids in the plasma than the acid gel. Plasma ghrelin concentrations suggested a potentially more satiating effect of the rennet gel compared to the acid gel. This study clearly evidences that the gelation process can significantly impact on the nutritive value of dairy products.

The heat treatment and the gelation are strong determinants of the kinetics of milk proteins digestion and of the peripheral availability of amino acids.

This study aimed to determine the kinetics of milk protein digestion and amino acid absorption after ingestion of four dairy matrices by six minipigs: unheated or heated skim milk and corresponding rennet gels. Digestive contents and plasma samples were collected over a 7 h-period after meal ingestion. Gelation of milk slowed down the outflow of the meal from the stomach and the subsequent absorption of amino acids, and decreased their bioavailability in peripheral blood. The gelled rennet matrices also led to low levels of milk proteins at the duodenum. Caseins and ß-lactoglobulin, respectively, were sensitive and resistant to hydrolysis in the stomach with the unheated matrices, but showed similar digestion with the heated matrices, with a heat-induced susceptibility to hydrolysis for ß-lactoglobulin. These results suggest a significant influence of the meal microstructure (resulting from heat treatment) and macrostructure (resulting from gelation process) on the different steps of milk proteins digestion.

Nisin quantification by ELISA allows the modeling of its apparent diffusion coefficient in model cheeses.

The diffusion of small solutes in cheese is of key importance for most enzymatic reactions involved in the ripening process. However, only a limited amount of data is available on salt diffusion and practically none on peptide diffusion. Nisin, a bacteriocin peptide, migrated in model cheeses made from ultrafiltered (UF) retentate. A profile concentration device and an enzyme-linked immunosorbent assay (ELISA), specifically developed for nisin quantification in cheese, were used to model the apparent diffusion coefficients for nisin according to Fick's law. This average coefficient was 49.5 µm(2)/s in UF cheese (n = 2). When 10% gelatin was added to the retentate, this value decreased to 34.4 µm(2)/s (n = 2). The two cheeses differed in their macrostructure (rheology) and microstructure (confocal microscopy). This study provides the first apparent diffusion coefficients for a peptide in cheese and supports the hypothesis that composition and structure influence the diffusion of small solutes such as peptides.

Characterization of heat-induced changes in skim milk using asymmetrical flow field-flow fractionation coupled with multiangle laser light scattering.

Separation and size measurement of protein particles are a relevant approach to monitor heat-induced changes in skim milk. Unfortunately, no method is currently available at low cost and without excessive preparation of the samples. Therefore, the present study aimed at evaluating the interest of asymmetrical flow field-flow fractionation (AFlFFF) coupled with multiangle laser light scattering (MALLS) for this purpose. Unheated and heated skim milk samples at pH 6.5 and 7.2 were prepared and comparatively analyzed using AFlFFF-MALLS, size exclusion chromatography (SEC-MALLS) and dynamic light scattering. The results showed that AFlFFF could evidence the conversion of the native whey proteins of unheated milk into heat-induced whey protein/κ-casein complexes in the serum phase of milk and possibly on the surface of the casein micelles. The pH-induced changes in the partition of the complexes between the serum and the micellar phases could also be observed. The results therefore showed the interest of AFlFFF-MALLS to monitor the heat-induced changes in particle sizes in skim milk and to separate the different protein components of unheated and heated skim milk.

Role of the heat-induced whey protein/kappa-casein complexes in the formation of acid milk gels: a kinetic study using rheology and confocal microscopy.

The effect of heat treatment of milk on the formation of acid gel was examined using confocal scanning laser microscopy and low-amplitude dynamic oscillation throughout acidification. Milk samples were reconstituted by mixing colloidal phase from unheated or preheated skim milk, labeled with rhodamine B isothiocyanate, with the aqueous phase from unheated or preheated milk, labeled with fluorescein isothiocyanate. Gels were made by acidification with glucono-delta-lactone. The presence of material from preheated milk, that is, either the colloidal or the aqueous phase or both, led to an increase in the gelation pH and in the final elastic modulus and to a more branched network with larger pores. During acidification, the heat-induced serum complexes and the casein micelles did not appear to form separated gels with time or in space. Moreover, the colocalization in the final network of serum heat-induced complexes and casein micelles is particularly well observed in the presence of an aqueous phase obtained from preheated milk. Finally, because the rheological and microstructural properties of acid gels containing either micelle-bound or serum heat-induced complexes were similar, it was suggested that the serum heat-induced complexes interacted with the casein micelles early in the course of acidification and that formation of the network did not differ significantly whether the heat-induced complexes were initially found in the aqueous phase of milk or bound to casein micelles.

Acid gelation properties of heated skim milk as a result of enzymatically induced changes in the micelle/serum distribution of the whey protein/kappa-casein aggregates.

Changes in the acid gelation properties of skim milk as a result of variations in the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates, induced by the combination of heat treatment and limited renneting, were investigated. No dramatic change in the zeta potential or the isoelectric point of the casein micelles was suggested, whether the aggregates were all attached to the casein micelle or not. Fluorescence intensity measurement using 8-anilino-1-naphthalenesulfonic acid (ANS) showed that the heat-induced aggregates were highly hydrophobic. Dynamic oscillation viscosimetry showed that acid gelation using glucono-delta-lactone (GDL) started at a higher pH value in prerenneted milk. However, no change in the gelation profile of skim milk could be related to the proportion of aggregates bound to the surface of the casein micelles. The results support the idea of an early interaction between the serum aggregates and the casein micelles on acidification.