Peptide separation of commercial fermented milk during refrigerated storage

Milk is an important source of bioactive compounds. Many of these compounds are released during fermentation and refrigerated storage. The aim of this study was to determine the release of peptides by lactic acid bacteria in commercial fermented milk during refrigerated storage. The size and profile of peptides were analyzed by polyacrylamide gel electrophoresis and sizeexclusion HPLC. During electrophoresis, it was observed that the peptides were released from caseins, whereas β-lactoglobulin was the whey protein with the highest degradation. HPLC analysis confirmed the pattern of peptide formation observed in electrophoresis. Two fractions lower than 2 kDa with aromatic amino acids in their structure were separated. These results were consistent with those reported for structures of peptides with antihypertensive activity. Therefore, the presence of aromatic amino acids in the peptide fractions obtained increases the likelihood of finding peptides with such activity in refrigerated commercial fermented milk. In conclusion, during cold storage, peptides with different molecular weights are released and accumulated. This could be due to the action of proteinases and peptidases of the proteolytic system in lactic acid bacteria.

Lactose intolerance and cow's milk protein allergy

Abstract Adverse reactions to food intake have very diverse etiology and symptomatology. Regarding milk, its food allergy is presented as lactose intolerance, the sugar in milk, or allergy to milk protein. Despite having different symptomatology, confusions among allergic conditions to dairy and its mediators are common. Milk protein allergy originates from protein components present in milk, causing reactions to either the protein fractions in emulsion (caseins) or in whey (milk albumin). The allergic reaction is type IV mediated by T lymphocytes. The allergic reaction produces severe cellular damage and it triggers physical, mental and emotional symptomatology that may vary in time, intensity and severity. Lactose intolerance is originated by total or partial absence of the enzyme that digests this disaccharide. Lactose intolerance can be primary or congenital and secondary; the former being more rare and severe, the latter being more common. Lactase deficiency can be diagnosed by symptoms associated with cramping and diarrhea. Thus, the objective of this study was to conduct a review of available literature on cow’s milk protein allergy and lactose intolerance.

Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite

Protein, generally agreed to be the most satiating macronutrient, may differ in its effects on appetite depending on the protein source and variation in digestion and absorption. We investigated the effects of two milk protein types, casein and whey, on food intake and subjective ratings of hunger and fullness, and on postprandial metabolite and gastrointestinal hormone responses. Two studies were undertaken. The first study showed that energy intake from a buffet meal ad libitum was significantly less 90 min after a 1700 kJ liquid preload containing 48 g whey, compared with an equivalent casein preload (P<0.05). In the second study, the same whey preload led to a 28 % increase in postprandial plasma amino acid concentrations over 3 h compared with casein (incremental area under the curve (iAUC), P<0.05). Plasma cholecystokinin (CCK) was increased by 60 % (iAUC, P<0.005), glucagon-like peptide (GLP)-1 by 65 % (iAUC, P<0.05) and glucose-dependent insulinotropic polypeptide by 36 % (iAUC, P<0.01) following the whey preload compared with the casein. Gastric emptying was influenced by protein type as evidenced by differing plasma paracetamol profiles with the two preloads. Greater subjective satiety followed the whey test meal (P<0.05). These results implicate post-absorptive increases in plasma amino acids together with both CCK and GLP-1 as potential mediators of the increased satiety response to whey and emphasise the importance of considering the impact of protein type on the appetite response to a mixed meal.

Selective separation of the major whey proteins using ion exchange membranes

Synthetic microporous membranes with functional groups covalently attached were used to selectively separate beta-lactoglobulin, BSA, and alpha-lactalbumin from rennet whey. The selectivity and membrane performance of strong (quaternary ammonium) and weak (diethylamine) ion-exchange membranes were studied using breakthrough curves, measurement of binding capacity, and protein composition of the elution fraction to determine the binding behavior of each membrane. When the weak and strong anion exchange membranes were saturated with whey, they were both selective primarily for beta-lactoglobulin with less than 1% of the eluate consisting of alpha-lactalbumin or BSA. The binding capacity of a pure alpha-lactoglobulin solution was in excess of 1.5 mg/cm(2) of membrane. This binding capacity was reduced to approximately 1.2 mg/cm(2) when using a rennet whey solution (pH 6.4). This reduction in protein binding capacity can be explained by both the competitive effects of other whey proteins and the effect of ions present in whey. Using binary solution breakthrough curves and rennet whey breakthrough curves, it was shown that alpha-lactalbumin and BSA were displaced from the strong and weak anion exchange membranes by beta-lactoglobulin. Finally, the effect of ionic strength on the binding capacity of individual proteins for each membrane was determined by comparing model protein solutions in milk permeate (pH 6.4) and a 10 mM sodium phosphate buffer (pH 6.4). Binding capacities of beta-lactoglobulin, alpha-lactalbumin, and BSA in milk permeate were reduced by as much as 50%. This reduction in capacity coupled with the low binding capacity of current ion exchange membranes are 2 serious considerations for selectively separating complex and concentrated protein solutions.

A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g gluconodelta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.

Effect of milk fat concentration and gel firmness on syneresis during curd stirring in cheese-making

An experiment was undertaken to investigate the effect of milk fat level (0%, 2.5% and 5.0% w/w) and gel firmness level at cutting (5, 35 and 65 Pa) on indices of syneresis, while curd was undergoing stirring. The curd moisture content, yield of whey, fat in whey and casein fines in whey were measured at fixed intervals between 5 and 75 min after cutting the gel. The casein level in milk and clotting conditions was kept constant in all trials. The trials were carried out using recombined whole milk in an 11 L cheese vat. The fat level in milk had a large negative effect on the yield of whey. A clear effect of gel firmness on casein fines was observed. The best overall prediction, in terms of coefficient of determination, was for curd moisture content using milk fat concentration, time after gel cutting and set-to-cut time (R2 = 0.95).

Evaluation of an improved tracer method to monitor cheese curd syneresis at varying milk fat levels in a cheese vat

Previous studies have reported that cheese curd syneresis kinetics can be monitored by dilution of chemical tracers, such as Blue Dextran, in whey. The objective of this study was to evaluate an improved tracer method to monitor whey volumes expelled over time during syneresis. Two experiments with different ranges of milk fat (0-5% and 2.3-3.5%) were carried out in an 11 L double-O laboratory scale cheese vat. Tracer was added to the curd-whey mixture during the cutting phase of cheese making and samples were taken at 10 min intervals up to 75 min after cutting. The volume of whey expelled was measured gravimetrically and the dilution of tracer in the whey was measured by absorbance at 620 nm. The volumes of whey expelled were significantly reduced at higher milk fat levels. Whey yield was predicted with a SEP ranging from 3.2 to 6.3 g whey/100 mL of milk and a CV ranging from 2.03 to 2.7% at different milk fat levels.

Effect of dairy-based protein sources and temperature on growth, acidification and exopolysaccharide production of Bifidobacterium strains in skim milk

The aim of the present study was to find out the best growing conditions for exopolysaccharide (EPS) producing bifidobacteria, which improve their functionality in yoghurt-like products. Two Bifidobacterium strains were used in this study, Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205. In the first part of the study the effect of casein hydrolysate, lactalbumin hydrolysate, whey protein concentrate and whey protein isolate, added at 1.5% w/v in skim milk, was evaluated in terms of cell growth and EPS production; skim milk supplemented with yeast extract served as the control. Among the various nitrogen sources, casein hydrolysate (CH) showed the highest cell growth and EPS production for both strains after 18 h incubation and therefore it was selected for subsequent work. Based on fermentation experiments using different levels of CH (from 0.5 to 2.5% w/v) it was deduced that 1.5% (w/v) CH resulted in the highest EPS production, yielding 102 and 285 mg L− 1 for B. infantis NCIMB 702205 and B. longum subsp. infantis CCUG 52486, respectively. The influence of temperature on growth and EPS production of both strains was further evaluated at 25, 30, 37 and 42 °C for up to 48 h in milk supplemented with 1.5% (w/v) CH. The temperature had a significant effect on growth, acidification and EPS production. The maximum growth and EPS production were recorded at 37 °C for both strains, whereas no EPS production was observed at 25 °C. Lower EPS production for both strains were observed at 42 °C, which is the common temperature used in yoghurt manufacturing compared to that at 37 °C. The results showed that the culture conditions have a clear effect on the growth, acidification and EPS production, and more specifically, that skim milk supplemented with 1.5% (w/v) CH could be used as a substrate for the growth of EPS-producing bifidobacteria, at 37 °C for 24 h, resulting in the production of a low fat yoghurt-like product with improved functionality.

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterize the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilization of β-lactoglobulin and casein derived peptides (CDP) from whey. The adsorbed proteins were hydrolyzed in-situ which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC50 =287μg/mL) and (ii) from β-lactoglobulin (IC50=128μg/mL). IC50 is the concentration of inhibitor needed to inhibit ACE by half. The well known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterized in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC50).

Mozzarella-type curd made from buffalo, cows’ and ultrafiltered cows’ milk. 2. Physicochemical properties, curd yield and quality, casein fractions & micelles size

Buffalo milk contains (40–60 %) more protein, fat and calcium than cows’ milk. These constituents were enhanced by ultrafiltration (UF) of cows’ milk to give a product with similar levels to those found in the buffalo milk. Mozzarella-type curd was made from buffalo, cows’ and UF cows’ milk to compare the overall curd yield and quality. The curd yield on both dry and wet weight basis, curd moisture content and overall curd fat retention were found to be higher in the UF cows’ milk than for either the buffalo or the cows’ milk preparations. The minimum whey fat losses occurred in the UF cows’ curd when compared to the cows’ and the buffalo curd. The whey protein losses were found to be higher in the UF cows’ curd than those for the buffalo and the cows’ curds. The total mineral content of the curd was also higher in the UF cows’ milk than that found in either the buffalo or the cows’ milk. SEM micrographs showed that casein micelles sizes were different in the two different types of milk. Casein micelles were also observed to be deformed in the UF cows’ milk samples. UF cows’ milk contained higher amounts of both the αs1- and αs2-casein moieties than either the buffalo or the cows’ milk. Buffalo milk was found to contain a higher concentration of β-casein than either the UF cows’ or untreated cows’ milk samples. Gel strength was found to be higher in the resultant buffalo curd than for curds made from either native cows’ milk or those made from UF cows’ milk. The mineral distribution was also different in the three different types of bovine milk, measured by energy-dispersive X-ray (EDX) analysis. Differences in the curd quality observed between the buffalo and the cows’ milk appear to result from the differences in casein composition and overall micelle structure, rather than casein concentration alone.

Investigation of milk proteins binding to the oral mucosa

High protein dairy beverages are considered to be mouth drying. The drying sensation may be due to the product protein content; however the mechanism of this mouth drying is uncertain. This study investigated the potential adhesion of milk proteins to porcine oral mucosal tissues and their resistance to wash out with simulated saliva was monitored using fluorescence microscopy. Cadein was found to be more adhesive to porcine mucosa then lactogloubulin. Some investigation into the reason for this difference in mucoadhesion was conducted by thiol-content analysis, rheology and zeta-potential measurements. The higher viscosity of casein solution and smaller zeta-potential is believed to be responsible for its better retention on mucosal surfaces. These findings suggest that casein and whey protein are both capable of binding and eliciting mouth drying in high protein dairy beverages.

The impact of milk proteins and peptides on blood pressure and vascular function: a review of evidence from human intervention studies.

CVD are the leading cause of death worldwide. Hypertension, a major controllable risk factor of CVD, is intimately associated with vascular dysfunction, a defect which is also now recognised to be a major, modifiable risk factor for the development of CVD. The purpose of the present review was to critically evaluate the evidence for the effects of milk proteins and their associated peptides on blood pressure (BP) and vascular dysfunction. After a detailed literature search, the number of human trials evaluating the antihypertensive effects of casein-derived peptides (excluding isoleucine-proline-proline and valine-proline-proline) was found to be limited; the studies were preliminary with substantial methodological limitations. Likewise, the data from human trials that examined the effects of whey protein and peptides were also scarce and inconsistent. To date, only one study has conducted a comparative investigation on the relative effects of the two main intact milk proteins on BP and vascular function. While both milk proteins were shown to reduce BP, only whey protein improved measures of arterial stiffness. In contrast, a growing number of human trials have produced evidence to support beneficial effects of both milk proteins and peptides on vascular health. However, comparison of the relative outcomes from these trials is difficult owing to variation in the forms of assessment and measures of vascular function. In conclusion, there is an accumulating body of evidence to support positive effects of milk proteins in improving and/or maintaining cardiovascular health. However, the variable quality of the studies that produced this evidence, and the lack of robust, randomised controlled intervention trials, undermines the formulation of firm conclusions on the potential benefits of milk proteins and peptides on vascular health.

Potential sources of mouth drying in beverages fortified with dairy proteins: a comparison of casein and whey-rich ingredients

Oral nutritional supplement drinks (ONS) are beverages high in dairy proteins that are prescribed to individuals at risk of malnutrition. Consumption of ONS is poor in elderly care facilities, with patients commenting that the sensory attributes of these drinks reduce their enjoyment and willingness to consume. Mouth drying is an attribute of ONS found to build with repeated consumption, which may further limit liking of these products. This study investigated the sources of drying sensations by sequential profiling, with a trained sensory panel rating a range of model milk systems and ONS over repeated sips and during after-effects. Sequential profiling found that fortification of milk with both caseinate and whey protein concentrate significantly increased the perception of mouth drying over repeated consumption, increasing by between 35 and 85% over consumption of 40mL. Enrichment of ONS with either whey protein concentrate or milk protein concentrate to a total protein content of 8.7% (wt/wt) resulted in whey and casein levels of 4.3:4.4% and 1.7:7.0% respectively. The product higher in whey protein was substantially more mouth drying, implying that whey proteins may be the most important contributor to mouth drying in ONS. However, efforts to mask mouth drying of protein-fortified milk by increasing sweetness or fat level were unsuccessful at the levels tested. Increasing the viscosity of protein-fortified milk led to a small but significant reduction in mouth drying. However, this approach was not successful when tested within complete ONS. Further analysis is required into the mechanism of protein-derived mouth drying to mask negative sensations and improve the enjoyment and consumption of protein-rich ONS.

Short communication: estimation of the financial benefit of using Jersey milk at different inclusion rates for Cheddar cheese production using partial budgeting

Partial budgeting was used to estimate the net benefit of blending Jersey milk in Holstein-Friesian milk for Cheddar cheese production. Jersey milk increases Cheddar cheese yield. However, the cost of Jersey milk is also higher; thus, determining the balance of profitability is necessary, including consideration of seasonal effects. Input variables were based on a pilot plant experiment run from 2012 to 2013 and industry milk and cheese prices during this period. When Jersey milk was used at an increasing rate with Holstein-Friesian milk (25, 50, 75, and 100% Jersey milk), it resulted in an increase of average net profit of 3.41, 6.44, 8.57, and 11.18 pence per kilogram of milk, respectively, and this additional profit was constant throughout the year. Sensitivity analysis showed that the most influential input on additional profit was cheese yield, whereas cheese price and milk price had a small effect. The minimum increase in yield, which was necessary for the use of Jersey milk to be profitable, was 2.63, 7.28, 9.95, and 12.37% at 25, 50, 75, and 100% Jersey milk, respectively. Including Jersey milk did not affect the quantity of whey butter and powder produced. Althoug further research is needed to ascertain the amount of additional profit that would be found on a commercial scale, the results indicate that using Jersey milk for Cheddar cheese making would lead to an improvement in profit for the cheese makers, especially at higher inclusion rates.

Whey protein mouth drying influenced by thermal denaturation

Whey proteins are becoming an increasingly popular functional food ingredient. There are, however, sensory properties associated with whey protein beverages that may hinder the consumption of quantities sufficient to gain the desired nutritional benefits. One such property is mouth drying. The influence of protein structure on the mouthfeel properties of milk proteins has been previously reported. This paper investigates the effect of thermal denaturation of whey proteins on physicochemical properties (viscosity, particle size, zeta-potential, pH), and relates this to the observed sensory properties measured by qualitative descriptive analysis and sequential profiling. Mouthcoating, drying and chalky attributes built up over repeated consumption, with higher intensities for samples subjected to longer heating times (p < 0.05). Viscosity, pH, and zeta-potential were found to be similar for all samples, however particle size increased with longer heating times. As the pH of all samples was close to neutral, this implies that neither the precipitation of whey proteins at low pH, nor their acidity, as reported in previous literature, can be the drying mechanisms in this case. The increase in mouth drying with increased heating time suggests that protein denaturation is a contributing factor and a possible mucoadhesive mechanism is discussed.