Effect of pH on the gelation properties of skim milk gels made from plant coagulants and chymosin

The effect of three milk pH values, 6.0, 6.3 and 6.7, on gelation properties was monitored by small amplitude oscillatory rheology as well as a large deformation (yield) test for gels induced by the plant coagulants, Cynara cardunculus L. and Cynara humilis L., and chymosin. Gel microstructure was studied using confocal scanning laser microscopy. For each coagulant, a decrease in pH of milk resulted in a decrease in gelation time (tg), and an increase in the rate of increase in storage modulus (G'). At pH 6.0 and 6.3, plant coagulant-induced gels reached a maximum value in G' (G'max) followed by a decrease in G' values during the rest of the experiment, reflecting higher proteolytic activity of plant coagulants towards caseins as determined by SDS-PAGE. Gels produced at pH 6.0 and 6.3, exhibited a minimum in loss tangent (tan delta) followed by slight increase in tan delta during gel aging, that may have been associated with faster rearrangements of the gel network structure. In gels aged for approximately 6 h, the values for G', tan delta at low frequency (0.006 Hz) and yield stress were higher for chymosin than for plant-induced gels. For gels with the same pH value, no major differences were observed in microstructure between coagulants. Gels made at low pH values (6.3 and 6.0) appeared to have a denser or more interconnected structure than gels made at pH 6.7. Our results suggest that, at a low pH, the type of coagulant used in gelation is likely to have a considerably impact on gel/cheese structure.     

Effect of controlled kappa-casein hydrolysis on rheological properties of acid milk gels

An experimental method based on the controlled chymosin-induced kappa-casein hydrolysis of milk was proposed to modify micellar reactivity. Milk samples with a degree of kappa-casein hydrolysis of 19, 35, and 51% were obtained. The physicochemical properties of partially converted casein micelles were determined. The net negative charge of casein micelles was reduced with increasing degree of kappa-casein hydrolysis and a small but significant decrease in hydrodynamic diameter and micellar hydration were noted. Dynamic low amplitude oscillatory rheology was used to monitor the rheological properties of acid milk gels (GDL) made with partially chymosin-hydrolyzed milks in comparison with those of strictly acid and rennet gels. An increase in the gelation pH value was observed with increasing the degree of kappa-casein hydrolysis. The moduli values (G' and G') reached 2 h after the point of gel were, for all degrees of hydrolysis tested, significantly higher than those of strictly rennet and acid gels. Comparison of changes in delta G'/delta t with time indicated differences in gel formation that could be related to the increased values of G' obtained for acid gel made with chymosin-treated milk. At a given time after gelation (2 h), increasing the degree of kappa-casein hydrolysis in milk led also to an increase in the loss tangent and the serum holding capacity of acid milk gels suggesting a correlation between these two parameters.     

Acid and rennet gelation properties of sheep,goat, and cow milks: Effects of processing and seasonal variation

Gelation is an important functional property of milk that enables the manufacture of various dairy products. This study investigated the acid (with glucono-δ-lactone) and rennet gelation properties of differently processed sheep, goat, and cow milks using small-amplitude oscillatory rheological tests. The impacts of ruminant species, milk processing (homogenization and heat treatments), seasonality, and their interactions were studied. Acid gelation properties were improved (higher gelation pH, shorter gelation time, and higher storage modulus (G′) by intense heat treatment (95°C for 5 min) to comparable extents for sheep and cow milks, both better than those for goat milk. Goat milk produced weak acid gels with low G′ (<100 Pa) despite improvements induced by heat treatments. Seasonality had a marked impact on the acid gelation properties of sheep milk. The acid gels of late-season sheep milk had a lower gelation pH, no maximum in tan δ following gel formation, and 70% lower G′ values than those from other seasons. We propose the potential key role of a critical acid gelation pH that induces structural rearrangements in determining the viscoelastic properties of the final gels. For rennet-induced gelation, compared with cow milk, the processing treatments of the goat and sheep milks had much smaller impacts on their gelation properties. Intense heat treatment (95°C for 5 min) prolonged the rennet gelation time of homogenized cow milk by 8.6 min (74% increase) and reduced the G′ of the rennet gels by 81 Pa (85% decrease). For sheep and goat milks, the same treatment altered the rennet gelation time by only less than 3 min and the G′ of the rennet gels by less than 14 Pa. This difference may have been caused by the different physicochemical properties of the milks, such as differences in their colloidal stability, proportion of serum-phase caseins, and ionic calcium concentration. The seasonal variations in the gelation properties (both acid and rennet induced) of goat milk could be explained by the minor variation in its protein and fat contents. This study provides new perspectives and understandings of milk gelation by demonstrating the interactive effects among ruminant species, processing, and seasonality.     

Effect of heat treatment on the rennet coagulation properties of recombined high total solids milk made from milk protein concentrate powder

The effects of heat treatment on the small and large deformation rheological properties of rennet gels made from recombined high total solids milk made from milk protein concentrate were studied. Heat treatment of recombined milk resulted in a slower rate of increase in the storage modulus (G') of rennet induced gels, a reduction in the gelation time and a decrease in the yield force required to fracture gels. The extent of whey protein denaturation (as a result of heat treatment) was related to the decrease in the G' value of gels as well as the yield force.     

Rheological Properties and Microstructure of Acid Milk Gels as Affected by Fat Content and Heat Treatment

Gels were made from recombined milks containing 0, 1.5 or 3.5% fat that were heated at 75, 80 or 90°C for 30 min, followed by acidification with glucono-8-lactone at 30°C. The rheological and microstructural properties of acid gels were investigated using dynamic low-amplitude oscillatory rheology and confocal scanning laser microscopy (CSLM). Heating milks, at temperatures ≥80°C, increased the storage moduli (G′) and decreased the gelation time. Recombined milks containing high fat (3.5%) had higher G′ than gels made from low-fat or skim milk. Milk heat treatment resulted in gels with a cross-linked microstructure. Recombined fat globules appeared to be embedded in the protein matrix.     

Effect of insoluble calcium concentration on rennet coagulation properties of milk

Rennet-induced gels were made from milk acidified to various pH values or milk at pH 6.0 that had added EDTA. The objective was to examine the effect of removing insoluble Ca (INS Ca) from casein micelles (CM) on rennet gelation properties. For the pH trial, diluted lactic acid was added to reconstituted skim milk to decrease the pH to 6.4, 6.0, 5.8, 5.6, and 5.4. For the EDTA trial, EDTA was slowly added (0, 2, 4, and 6 mM) to reconstituted skim milk, and the final pH values were subsequently adjusted to pH 6.0. Dynamic low amplitude oscillatory rheology was used to monitor gel development. The Ca content of CM and rennet wheys made from these milks was measured using inductively coupled plasma spectroscopy. The INS Ca content of milk was altered by the acidification pH values or level of EDTA added. In all samples, the storage modulus (G′) exhibited a maximum (GM), with a decrease in G′ during longer aging times. Gels made at pH 6.4 had higher GM compared with gels made at pH 6.7 probably due to the reduction in electrostatic repulsion, whereas the INS Ca content only slightly decreased. The highest GM value of gels was observed at pH 6.4 and the GM value decreased with decreasing pH from 6.4 to 5.4. This was due to an excessive loss of INS Ca from CM. There was a decrease in GM with the increase in the concentration of added EDTA, which was probably due to the loss of colloidal calcium phosphate, which weakens the integrity of CM. Loss tangent (LT) values at GM increased with a reduction in milk pH and the addition of EDTA to milk. Rennet gels at the point of the GM were subjected to constant low shearing to fracture the gels. With a reduction in INS Ca content, the yield stress decreased, whereas LT values increased indicating a weaker, more flexible casein network. Microstructure of rennet-induced gels near the GM point and 2 to 10 h after this point was studied using fluorescence microscopy. At GM, gels made from milk acidified to pH 6.4 exhibited more branched, interconnected networks, whereas strands and clusters became larger with a reduction in milk pH to 5.4. Gels made from milk with EDTA added had more finely dispersed protein clusters compared with gels made from milk with no EDTA added. These microscopic observations supported the effect of loss of INS Ca on GM and LT. There was a decrease in apparent interconnectivity between strands in gel microstructure during aging, which agreed with the decrease in G′ after GM. It can be concluded that low levels of solubilization of INS Ca and the decrease in milk pH resulted in an increase in GM. With greater losses of INS Ca there was excessive reduction in cross-linking within CM, which resulted in weaker, more flexible rennet gels. This complex behavior cannot be explained by adhesive hard sphere models for CM or rennet gels made from these CM.     

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 β-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 β-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.     

Casein gelation under simultaneous action of transglutaminase and glucono-delta-lactone

Casein solutions (5% w/v) were treated with microbial transglutaminase (MTG) and glucono-delta-lactone (GDL) under varying conditions in order to obtain gels. Storage modulus (G') and gelation time of the gels were measured by oscillation rheometry, while protein cross-linking was determined by gel permeation chromatography. The addition of only GDL to milk resulted in very weak gels, while MTG on its own was not able to create gel networks. Simultaneous action of both ingredients led to gels, the firmness of which was linearly related to the added amount of MTG, but passed through a maximum with rising GDL concentrations. Using chromatographical analysis, increasing G' values were interrelated with the formation of MTG-induced oligomers. The gelation time was directly proportional to the GDL concentration but not influenced by the addition of MTG within the studied range of concentration.     

Protein interactions in heat-treated milk and effect on rennet coagulation

Skim milk was heated at different pH values to cause differential association of whey proteins (WP) with the casein micelles. All of these milk treatments coagulated poorly with rennet. To understand this in more detail, the casein micelles from heated milk were redispersed in unheated serum or unheated micelles were suspended in the sera from heat-treated milk. Systems containing micelles from milk heated at pH 6.7 and 7.1 were marginally better than the heated milk, but that from milk heated at pH 6.3 was not. The sera from milk heated at pH 6.7 and 7.1 impaired the clotting of native micelles but that from the pH 6.3 milk did not. Native casein micelles were suspended in permeates or dialyzed (against unheated milk) sera from heat-treated milk. Permeate systems free of WP/κ-casein complexes produced significantly stronger rennet gels; as did dialyzed systems. The impaired rennet clotting of heat-treated milk was attributed to a synergistic effect of the casein micelles with their heat-modified surfaces, the soluble serum WP/κ-casein complexes, and other dialyzable serum components.     

Capsule formation by nonropy starter cultures affects the viscoelastic properties of yogurt during structure formation

The aim of this work was to study the structure formation of yogurt made with cultures containing ropy Lactobacillus delbrueckii ssp. bulgaricus (R), capsule-forming nonropy Streptococcus thermophilus (CNR), and noncapsule-forming nonropy cultures (NCNR). Similar gelation profiles were shown for milk fermented by ropy and nonropy lactic cultures. The gelation point occurred at lower pH values in milk fermented with R or NCNR compared to that of milk fermented with CNR culture. Differences between capsule forming ropy and nonropy cultures were observed in the aggregation behavior of the caseins. Gels made with R culture had the highest maximum in loss tangent (tan delta). However, this maximum occurred at the highest pH value when CNR culture was used. The earlier gelation of milk fermented by the encapsulated nonropy strain of Strep. thermophilus resulted in increased structure rearrangement as the pH dropped, interfering with the formation of a more compact structure.     

Gelation properties of partially renneted milk

Acid- and rennet-induced gelation properties of milk with modified casein micelles, produced by partial renneting at 4^oC for 15 min, followed by inactivation of enzymes by heat at 60^oC/3 min (referred as low heat treatment milk) and 85^oC/30 min (high heat treatment milk), were investigated to provide a mechanistic understanding of gel formation from partially renneted milk. Acidification of low heat treatment milk gave firmer gel quality, this was reflected by its high elastic modulus (G′) and hardness. In addition, the high heating condition for enzyme inactivation of high heat treatment milk alone increased the elastic modulus of both the control and renneted milk samples. Gel development of the two milk types (low heat treatment and high heat treatment milks) was different. In contrast with acid gelation, rennet-induced gelation of partially pre-rennet treated milk had no impact on the elastic modulus of low heat treatment milk and the rennet gels were very weak. Similarly, the addition of rennet to pre-rennet treated high heat treatment milk did not produce “true gels,” most likely due to the effect of the heat treatment on impairing the rennet coagulation. The findings in this study confirmed that pre-rennet treated milk had positive effects on the end-product acid gels of low heat treatment and high heat treatment milk.     

The relationship between rheological parameters and whey separation in milk gels

The relation between whey separation of rennet-induced gels and rheological properties of those gels is reasonably well understood. A low fracture stress and a high value for the loss tangent at low frequencies have been correlated with a tendency to exhibit syneresis in rennet gels. In contrast, little is known about the relationship between mechanical properties of gels and whey separation in acid-induced milk gels, such as yoghurt, although this continues to be a major defect. In recent work, it has been found that conditions such as high milk heat treatment, fast rates of acidification and high incubation temperatures all gave high levels of whey separation compared with gels made from unheated milk that were incubated at low temperatures and where the rate of acidification was slow (i.e. when bacterial cultures were used instead of the acidogen, glucono-δ-lactone). The tendency to exhibit whey separation in acid gels made from heated milk was related to a low fracture strain and an increase in the loss tangent (observed even at high frequencies) during the gelation process (a high value indicates conditions favouring relaxation of bonds). Excessive rearrangements of particles in the gel network before and during gelation were implicated as being responsible for whey separation and rheological conditions that appeared to indicate this defect are described. It was also concluded that techniques that measure the spontaneous formation of surface whey should be distinguished from those that measure the expression of whey from networks under pressure as the latter tests only measure gel rigidity.     

Native vs. damaged milk fat globules: membrane properties affect the viscoelasticity of milk gels

The storage modulus G' of rennet and acid milk gels filled with milk fat globules was measured as a function of the fat globule surface composition (native milk fat globule membrane, caseins and whey proteins, or a mixture of the three due to mechanical treatments) and surface area (i.e., the fat globule size). By different technological procedures, it was possible to obtain fat globules of constant surface composition but various sizes, and vice-versa, which had never been done. For both rennet and acid gels, a critical fraction of the fat globule surface covered by caseins and whey proteins was identified (approximately 40%), beyond which G' increased. Below this threshold, the gel viscoelasticity was unaffected by mechanical treatments. When the diameter of native milk fat globules decreased, the G' of rennet gels increased slightly, whereas that of acid gels decreased sharply. For both types of gels, G' increased when the diameter of partially disrupted fat globules decreased. For recombined globules completely covered with caseins and few whey proteins, G' increased with fat globule surface area for rennet gels whereas it decreased for acid gels. With the help of confocal microscopy and in the light of general structural differences between rennet and acid gels, a mechanism is proposed for the effect of fat globule damage and diameter on G', depending on the interactions the globules can undergo with the casein network.     

Serum Protein Aggregates in the High-Heated Milk and Their Gelation Properties in Rennet-Induced Milk Gel

The influence of different heat treatments on the protein aggregates and changes in gelation properties of rennet-induced milk gels were investigated. In the heated milk, a visible difference in milk serum proteins was found resulting from the formation of protein aggregates. Meanwhile, the size of protein aggregates increased from 25 to 170 nm with increasing the intensity of heat treatment. Furthermore, the differences in textural variables of rennet gels were found among the heat treatments using the principal component analysis. The water holding capacity and cheese curd yield of rennet gels obtained from the heated milk were significantly greater than those of raw milk (p < 0.05). It was also found heat treatments above 80°C could endow rennet-induced milk gels with novel textural properties.     

Combined milk gel generated with a novel coagulating enzyme by Virgibacillus sp. SK37, a moderately halophilic bacterium

The hydrolysis of milk proteins by the recombinant AprX‐SK37 protease and the changes in the rheological properties of the milk gel generated with AprX‐SK37 and glucono‐δ‐lactone (GDL) were investigated. The AprX‐SK37 and rennet selectively hydrolysed κ‐casein to yield a 16‐kDa band, while subtilisin hydrolysed all of the casein components. Milk treated only with AprX‐SK37 formed softer gel. Storage modulus (G′) values of the combined gels increased with GDL concentrations up to 7 g/L. High tan δ was observed in the combined gel at 8.75 g/L GDL alongside syneresis. AprX‐SK37 is a promising milk‐clotting enzyme when combined with an optimal GDL concentration.     

Influence of denaturation and aggregation of β-lactoglobulin on rennet coagulation properties of skim milk and ultrafiltered milk

Skim milk and ultrafiltered milk were heated at temperatures in the range 80–140 °C for 4s in a UHT plate heat exchanger. The extent of denaturation of β-lactoglobulin and its association with the casein micelles increased with increase in heating temperature; the extent of association was markedly less than the amount that denatured. There was no noticeable difference in the extent of denaturation and association of β-lactoglobulin between skim milk and the corresponding ultrafiltered milk. The coagulation properties of renneted heated milks were measured by dynamic oscillation rheometry. Gelation times and storage modulus (G′) could be related to the extent of denaturation of β-lactoglobulin and its association with the casein micelles. Denaturation of β-lactoglobulin up to 60% had no effect on the gelation time, but additional denaturation markedly increased the gelation times. In contrast, G' decreased gradually with increase in the extent of denaturation of β-lactoglobulin and its association with casein micelles. The effects of β-lactoglobulin denaturation and association on coagulation properties were less pronounced in ultrafiltered milk. G' values for ultrafiltered milk were markedly higher than the corresponding skim milks under all heating conditions.     

EFFECT of SALT, SUGAR and MONOSODIUM GLUTAMATE ON the VISCOELASTIC PROPERTIES of FISH CRACKER ("KEROPOK") GEL

Rheological and microscopic studies were carried out on the fish cracker ('keropok') gels. A hard gel is very important for the production of uniform, round, and thin slices of dried 'keropok' and easier for mechanical slicing. the addition of 2% salt in the 'keropok' gel increased the storage modulus (G'), and reduced loss tangent (tan ×= G/G'). an increase in the fish content in 'keropok' also increased the G' and reduced tan γ. Gels having higher values of storage modulus G' and lower tan × values, indicative of a strong elastic network, showed well crosslinked fish muscle bundles in the microscopy studies. In the absence of salt, the fish protein appeared to be in lumps and this has resulted in low storage modulus G' and higher tan × values. Lower the temperature of the gel to 5C will result in 5–6 fold increase in the G' value.     

Use of cold ultrafiltered retentates for standardization of milks for pizza cheese: Impact on yield and functionality

Pizza cheese was manufactured from two types of Ultrafiltration (UF)-fortified milks: high solids (UFHS; 15.2% TS) and medium solids (UFMS; 13.5%). Cheese milks were obtained by blending cold processed UF retentate with partially skimmed milk and UF (skim milk) retentate. Cheese functionality was assessed using oscillatory rheology and by baking on a pizza. Gels made from UF-fortified milks had similar clotting times and they clotted faster than control milk. Shear stress values of gels from UF-fortified milks were higher than control. Fat recoveries in the cheeses increased in the order UFHS<control<UFMS. Nitrogen recoveries were lower in control than UF-fortified cheeses. During heating loss tangent curves shifted higher during the first month of ripening and the temperature for the maximum loss tangent decreased. Crossover temperature also decreased during ripening. Trichloroacetic acid-soluble nitrogen levels were similar in all cheeses. Standardization of cheese milk with cold UF retentates increased yield without adversely affecting functionality.     

Effect of increasing the colloidal calcium phosphate of milk on the texture and microstructure of yogurt

The effect of increasing the colloidal calcium phosphate (CCP) content on the physical, rheological, and microstructural properties of yogurt was investigated. The CCP content of heated (85°C for 30 min) milk was increased by increasing the pH by the addition of alkali (NaOH). Alkalized milk was dialyzed against pasteurized skim milk at approximately 4°C for 72 h to attempt to restore the original pH and soluble Ca content. By adjustment of the milk to pH values 7.45, 8.84, 10.06, and 10.73, the CCP content was increased to approximately 107, 116, 123, and 128%, respectively, relative to the concentration in heated milk. During fermentation of milk, the storage modulus (G′) and loss tangent values of yogurts were measured using dynamic oscillatory rheology. Large deformation rheological properties were also measured. The microstructure of yogurt was observed using fluorescence microscopy, and whey separation was determined. Acid-base titration was used to evaluate changes in the CCP content in milk. Total Ca and casein-bound Ca increased with an increase in the pH value of alkalization. During acidification, elevated buffering occurred in milk between pH values 6.7 to 5.2 with an increase in the pH of alkalization. When acidified milk was titrated with alkali, elevated buffering occurred in milk between pH values 5.6 to 6.4 with an increase in the pH of alkalization. The high residual pH of milk after dialysis could be responsible for the decreased contents of soluble Ca in these milks. The pH of gelation was higher in all dialyzed samples compared with the heated control milk, and the gelation pH was higher with an increase in CCP content. The sample with highest CCP content (128%) exhibited gelation at very high pH (6.3), which could be due to alkali-induced CN micellar disruption. The G′ values at pH 4.6 were similar in gels with CCP levels up to 116%; at higher CCP levels, the G′ values at pH 4.6 greatly decreased. Loss tangent values at pH 5.1 were similar in all samples except in gels with a CCP level of 128%. For dialyzed milk, the whey separation levels were similar in gels made from milk with up to 107% CCP but increased at higher CCP levels. Microstructure of yogurt gels made from milk with 100 to 107% CCP was similar but very large clusters were observed in gels made from milk with higher CCP levels. By dialyzing heated milk against pasteurized milk, we may have retained some heat-induced Ca phosphate on micelles that normally dissolves on cooling because, during dialysis, pasteurized milk provided soluble Ca ions to the heated milk system. Yogurt texture was significantly affected by increasing the casein-bound Ca (and total Ca) content of milk as well as by the alkalization procedure involved in that approach.