Wool keratin - A novel dietary protein source: Nutritional value and toxicological assessment.

Keratin derived protein (KDP) was extracted from sheep wool using high pressure microwave technology and food acids and investigated for its potential as a novel dietary protein. The proximate composition, amino acid profile, element profile, in vitro cytotoxicity and digestibility of KDP were evaluated. Nutritive effects of KDP at 50% dietary supplementation were compared with a casein-based diet in a growing rat model for 95 days. Results indicate KDP to be rich in protein (86%), amino acid cysteine (8.8 g/100 g) and element selenium (0.29 µg/g). KDP was non-cytotoxic in vitro at ≤ 2 mg/mL concentration. There were no differences in the rat's weight gain compared to the control group (P > 0.05). Overall, the inclusion of the KDP in the diet was an effective substitute for casein protein at 50% and KDP has the potential to be used in the food industry as a novel dietary protein, free of fat and carbohydrate.

Effects of indirect indicators of udder health on nutrient recovery and cheese yield traits in goat milk.

In dairy goats, very little is known about the effect of the 2 most important indirect indicators of udder health [somatic cell count (SCC) and total bacterial count (TBC)] on milk composition and cheese yield, and no information is available regarding the effects of lactose levels, pH, and NaCl content on the recovery of nutrients in the curd, cheese yield traits, and daily cheese yields. Because large differences exist among dairy species, conclusions from the most studied species (i.e., bovine) cannot be drawn for all types of dairy-producing animals. The aims of this study were to quantify, using milk samples from 560 dairy goats, the contemporary effects of a pool of udder health indirect indicators (lactose level, pH, SCC, TBC, and NaCl content) on the recovery of nutrients in the curd (%REC), cheese yield (%CY), and daily cheese yields (dCY). Cheese-making traits were analyzed using a mixed model, with parity, days in milk (DIM), lactose level, pH, SCC, TBC, and NaCl content as fixed effects, and farm, breed, glass tube, and animal as random effects. Results indicated that high levels of milk lactose were associated with reduced total solids recovery in the curd and lower cheese yields, because of the lower milk fat and protein contents in samples rich in lactose. Higher pH correlated with higher recovery of nutrients in the curd and higher cheese yield traits. These results may be explained by the positive correlation between pH and milk fat, protein, and casein in goat milk. High SCC were associated with higher recovery of solids and energy in the curd but lower recovery of protein. The higher cheese yield obtained from milk with high SCC was due to both increased recovery of lactose in the curd and water retention. Bacterial count proved to be the least important factor affecting cheese-making traits, but it decreased daily cheese yields, suggesting that, even if below the legal limits, TBC should be considered in order to monitor flock management and avoid economic losses. The effect of NaCl content on milk composition was linked with lower recovery of all nutrients in the curd during cheese-making. In addition, high milk NaCl content led to reductions in fresh cheese yield and cheese solids. The indirect indicators of the present study significantly affected the cheese-making process. Such information should be considered, to adjust the milk-to-cheese economic value and the milk payment system.

Ultrafast Multiplexed-Allergen Detection through Advanced Fluidic Design and Monolithic Interferometric Silicon Chips.

A silicon-based miniaturized sensor chip combined with an advanced microfluidic module for the simultaneous, label-free immunochemical determination of four allergens, bovine milk protein, peanut protein, soy protein, and gliadin, is presented. The sensor chip consists of an array of 10 broad-band Mach-Zehnder interferometers (BB-MZIs) monolithically integrated on silicon, along with their respective broad-band light sources. The BB-MZIs were biofunctionalized with the targeted allergens and their responses during immunoreaction were monitored by multiplexing their transmission spectra through an external miniaturized spectrometer. The assay is performed by running mixtures of calibrators or samples with the antibodies against the four allergens followed by an antispecies specific antibodies solution. Employing a fluidic module of nearly one-dimensional geometry, that provided for uniform delivery of the reagents, CV values <6% were achieved for the responses of the 10 BB-MZIs, allowing for reliable multianalyte determinations. The analysis is completed in 6.5 min, and the detection limits were 0.04 µg/mL for bovine k-casein, 1.0 µg/mL for peanut protein, 0.80 µg/mL for soy protein, and 0.10 µg/mL for gliadin. The assays were accurate (recoveries 88-118%) and repeatable (intra- and interassay CVs <7% for all four allergens). Finally, the sensor was evaluated by analyzing samples from a cleaning in place system (CIP) of a dairy industry and the results obtained were in good agreement with those received by the respective ELISAs. The analytical characteristics of the sensor combined with the short analysis time and the small chip size make the proposed system an ideal tool for on-site multianalyte determinations.

Identification of bitter peptides in aged cheddar cheese.

The compounds responsible for the bitter taste of aged "sharp" Cheddar cheese were characterized. Sensory-guided fractionation techniques using gel permeation chromatography and multi-dimension semi-preparative reversed-phase high-performance liquid chromatography revealed the presence of multiple bitter compounds. The compounds with the highest perceived bitterness intensity were identified by tandem mass spectrometry de novo peptide sequencing as GPVRGPFPIIV, YQEPVLGPVRGPFPI, MPFPKYPVEP, MAPKHKEMPFPKYPVEPF, and APHGKEMPFPKYPVEPF; all originated from ß-casein. Subsequent quantitative liquid chromatography-tandem mass spectrometry analysis reported that the concentrations of GPVRGPFPIIV, YQEPVLGPVRGPFPI, and MPFPKYPVEP increased during maturation by 28.7-, 3.1-, and 1.8-fold, respectively. When directly compared to young "mild" Cheddar, APHGKEMPFPKYPVEPF was reported only in the sharp Cheddar cheese, whereas the concentration of MAPKHKEMPFPKYPVEPF did not change. Further taste re-engineering sensory experiments confirmed the importance of the identified peptides to the bitterness of sharp Cheddar. The bitter intensity of the aged "sharp" Cheddar model (mild Cheddar with equivalent concentrations of the five bitter peptides in the sharp sample) was rated as not significantly different from the authentic sharp Cheddar cheese. Among the five peptides, GPVRGPFPIIV was reported to be the main contributor to the bitterness intensity of sharp Cheddar. Furthermore, a difference from control sensory test also confirmed the significance of the bitter taste to the overall perception of aged Cheddar flavor. The sharp Cheddar model was reported to be significantly more similar to aged "sharp" Cheddar in comparison to the young "mild" Cheddar cheese sample.

Development of a method for the quantification of caseinate traces in Italian commercial white wines based on liquid chromatography-electrospray ionization-ion trap-mass spectrometry.

A method using the combination of size exclusion-solid phase extraction and ultrafiltration, followed by tryptic digestion and analysis of the protein digest by liquid chromatography-electrospray ionization-3D ion trap-mass spectrometry (LC-ESI-3D IT-MS), was developed for the detection and quantification of caseinate traces potentially resulting from fining processes in white wines. In particular, several tryptic peptides generated from the main proteins constituting caseinate (ß-, αS1-, and αS2-caseins) were used as markers of its presence in the wine matrices; among them, the ß-casein peptide GPFPIIV was found to be the best marker for quantification purposes. Method linearity and sensitivity were assessed on a series of Italian commercial white wines, first checked for the absence of any peptide signal attributable to caseins introduced during their production and subsequently spiked with increasing concentrations of caseinate, to provide samples for matrix-matched calibrations. Limits of detection ranging between 0.09 and 0.29 mg/L (S/N = 3), according to the wine, were achieved using a 10 mL sample volume and the MS signal of GPFPIIV as the response related to the caseinate concentration. Such levels are comparable or even lower than the one (0.25 mg/L) recently adopted as a threshold by European Union legislation concerning the indication of milk- and egg-derived fining agents on wine labels, that is, the most restrictive one among those currently proposed in the world.

Proportions of A1, A2, B and C ß-casein protein variants in retail milk in the UK.

The A1 variant protein of the ß-casein family has been implicated in various disease states although much evidence is weak or contradictory. The primary objective was to measure, for the first time, the proportions of the key ß-casein variant proteins in UK retail milk over the course of one year. In total, 55 samples of semi-skimmed milk were purchased from five supermarkets over the course of a year and the proportions of the A1, A2, B and C casein variant proteins were measured, using high resolution HPLC-MS. The results showed that ß-casein in UK retail milk comprises approximately 0.58, 0.31, 0.07 and 0.03 A2, A1, B and C protein variants, respectively. The proportion of A2 is higher than some early studies would predict although the reasons for this and any implications for health are unclear.

[Assessment of biological value of protein-peptide ingredients of functional drink in growing rats].

The abstract is devoted to research in vivo on the growing rats of biological value of protein hydrolyzate of meat-and-bone residues of poultry, fortified by defatted cow milk. Cow milk casein was chosen as a template. PER was determined by growing method, based on lab animals' growth rate assessment. True protein digestibility was determined by balance method. The indices were fixed individually for each animal and then the group mean value was calculated. Accessibility of protein hydrolyzate of meat-and-bone residues of poultry fortified by defatted cow milk equals 100%. PER is 1.25 lower in comparison with casein, which can be explained by a lower retention.

Development and application of a processing model for the Irish dairy industry.

A processing-sector model was developed that simulates (i) milk collection, (ii) standardization, and (iii) product manufacture. The model estimates the product yield, net milk value, and component values of milk based on milk quantity, composition, product portfolio, and product values. Product specifications of cheese, butter, skim and whole milk powders, liquid milk, and casein are met through milk separation followed by reconstitution in appropriate proportions. Excess cream or skim milk are used in other product manufacture. Volume-related costs, including milk collection, standardization, and processing costs, and product-related costs, including processing costs per tonne, packaging, storage, distribution, and marketing, are quantified. Operating costs, incurred irrespective of milk received and processing activities, are included in the model on a fixed-rate basis. The net milk value is estimated as sale value less total costs. The component values of fat and protein were estimated from net milk value using the marginal rate of technical substitution. Two product portfolio scenarios were examined: scenario 1 was representative of the Irish product mix in 2000, in which 27, 39, 13, and 21% of the milk pool was processed into cheese (€ 3,291.33/t), butter (€ 2,766.33/t), whole milk powder (€ 2,453.33/t), and skim milk powder (€ 2,017.00/t), respectively, and scenario 2 was representative of the 2008 product mix, in which 43, 30, 14, and 13% was processed into cheese, butter, whole milk powder, and skim milk powder, respectively, and sold at the same market prices. Within both scenarios 3 milk compositions were considered, which were representative of (i) typical Irish Holstein-Friesian, (ii) Jersey, and (iii) the New Zealand strain of Holstein-Friesian, each of which had differing milk constituents. The effect each milk composition had on product yield, processing costs, total revenue, component values of milk, and the net value of milk was examined. The value per liter of milk in scenario 1 was 24.8, 30.8, and 27.4 cents for Irish Holstein-Friesian, Jersey, and New Zealand strain of Holstein-Friesian milk, respectively. In scenario 2 the value per liter of milk was 26.1, 32.6, and 28.9 cents for Irish Holstein-Friesian, Jersey, and New Zealand strain of Holstein-Friesian milk, respectively.

Importance of casein micelle size and milk composition for milk gelation.

The economic output of the dairy industry is to a great extent dependent on the processing of milk into other milk-based products such as cheese. The yield and quality of cheese are dependent on both the composition and technological properties of milk. The objective of this study was to evaluate the importance and effects of casein (CN) micelle size and milk composition on milk gelation characteristics in order to evaluate the possibilities for enhancing gelation properties through breeding. Milk was collected on 4 sampling occasions at the farm level in winter and summer from dairy cows with high genetic merit, classified as elite dairy cows, of the Swedish Red and Swedish Holstein breeds. Comparisons were made with milk from a Swedish Red herd, a Swedish Holstein herd, and a Swedish dairy processor. Properties of CN micelles, such as their native and rennet-induced CN micelle size and their zeta-potential, were analyzed by photon correlation spectroscopy, and rennet-induced gelation characteristics, including gel strength, gelation time, and frequency sweeps, were determined. Milk parameters of the protein, lipid, and carbohydrate profiles as well as minerals were used to obtain correlations with native CN micelle size and gelation characteristics. Milk pH and protein, CN, and lactose contents were found to affect milk gelation. Smaller native CN micelles were shown to form stronger gels when poorly coagulating milk was excluded from the correlation analysis. In addition, milk pH correlated positively, whereas Mg and K correlated negatively with native CN micellar size. The milk from the elite dairy cows was shown to have good gelation characteristics. Furthermore, genetic progress in relation to CN micelle size was found for these cows as a correlated response to selection for the Swedish breeding objective if optimizing for milk gelation characteristics. The results indicate that selection for smaller native CN micelles and lower milk pH through breeding would enhance gelation properties and may thus improve the initial step in the processing of cheese.

HPLC-MS analysis of iodotyrosines produced by sample hydrolysis: A simple method for monitoring iodinated casein in feed premixes.

A new and simple HPLC-MS method was developed for monitoring iodinated casein in feed premixes. In this method, feed premixes were hydrolyzed, and the iodotyrosines thus released were analyzed. Sample pretreatment included precipitation of transition metals ions with Na(2)S, hydrolysis with sodium hydroxide, and cleaning up with an Oasis SAX cartridge. Gradient elution was carried out on a C(18) column with water (containing 0.1% formic acid) and acetonitrile. Ion detection was performed using ESI positive SIM at m/z 262, 308, 388, and 434. Iodinated casein levels were monitored by qualitative analysis of the iodotyrosines released upon sample hydrolysis and by quantifying the 3,5-diiodotyrosine released. The validation data demonstrated that the method was selective and sensitive (

A binary matrix for improved detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry.

Application of matrix-assisted laser-desorption/ionization mass spectrometry (MALDI MS) to analysis and characterization of phosphopeptides in peptide mixtures may have a limitation, because of the lower ionizing efficiency of phosphopeptides than nonphosphorylated peptides in MALDI MS. In this work, a binary matrix that consists of two conventional matrices of 3-hydroxypicolinic acid (3-HPA) and alpha-cyano-4-hydroxycinnamic acid (CCA) was tested for phosphopeptide analysis. 3-HPA and CCA were found to be hot matrices, and 3-HPA not as good as CCA and 2,5-dihydroxybenzoic acid (DHB) for peptide analysis. However, the presence of 3-HPA in the CCA solution with a volume ratio of 1:1 could significantly enhance ion signals for phosphopeptides in both positive-ion and negative-ion detection modes compared with the use of pure CCA or DHB, the most common phosphopeptide matrices. Higher signal intensities of phosphopeptides could be obtained with lower laser power using the binary matrix. Neutral loss of the phosphate group (-80 Da) and phosphoric acid (-98 Da) from the phosphorylated-residue-containing peptide ions with the binary matrix was decreased compared with CCA alone. In addition, since the crystal shape prepared with the binary matrix was more homogeneous than that prepared with DHB, searching for 'sweet' spots can be avoided. The sensitivity to detect singly or doubly phosphorylated peptides in peptide mixtures was higher than that obtained with pure CCA and as good as that obtained using DHB. We also used the binary matrix to detect the in-solution tryptic digest of the crude casein extracted from commercially available low fat milk sample, and found six phosphopeptides to match the digestion products of casein, based on mass-to-charge values and LIFT TOF-TOF spectra.

Valuation of milk composition and genotype in cheddar cheese production using an optimization model of cheese and whey production.

A mass balance optimization model was developed to determine the value of the kappa-casein genotype and milk composition in Cheddar cheese and whey production. Inputs were milk, nonfat dry milk, cream, condensed skim milk, and starter and salt. The products produced were Cheddar cheese, fat-reduced whey, cream, whey cream, casein fines, demineralized whey, 34% dried whey protein, 80% dried whey protein, lactose powder, and cow feed. The costs and prices used were based on market data from March 2004 and affected the results. Inputs were separated into components consisting of whey protein, ash, casein, fat, water, and lactose and were then distributed to products through specific constraints and retention equations. A unique 2-step optimization procedure was developed to ensure that the final composition of fat-reduced whey was correct. The model was evaluated for milk compositions ranging from 1.62 to 3.59% casein, 0.41 to 1.14% whey protein, 1.89 to 5.97% fat, and 4.06 to 5.64% lactose. The kappa casein genotype was represented by different retentions of milk components in Cheddar cheese and ranged from 0.715 to 0.7411 kg of casein in cheese/kg of casein in milk and from 0.7795 to 0.9210 kg of fat in cheese/kg of fat in milk. Milk composition had a greater effect on Cheddar cheese production and profit than did genotype. Cheese production was significantly different and ranged from 9,846 kg with a high-casein milk composition to 6,834 kg with a high-fat milk composition per 100,000 kg of milk. Profit (per 100,000 kg of milk) was significantly different, ranging from $70,586 for a high-fat milk composition to $16,490 for a low-fat milk composition. However, cheese production was not significantly different, and profit was significant only for the lowest profit ($40,602) with the kappa-casein genotype. Results from this model analysis showed that the optimization model is useful for determining costs and prices for cheese plant inputs and products, and that it can be used to evaluate the economic value of milk components to optimize cheese plant profits.

Two mathematical programming models of cheese manufacture.

The standardization problem faced by cheese makers is formulated as a nonlinear programming problem using the assumptions of the Van Slyke cheese yield formula. The objective function of the model is to minimize the net cost of producing a given quantity of cheese subject to a set of production constraints. An approximation of the standardization problem formulated as a linear programming problem is also presented. Two different approaches to finding a solution are provided. The model is implemented in Microsoft Excel and solved with the standard add-in solver available in that program. An example is provided to contrast the difference between the nonlinear programming and its linear approximation, and a second example is used to illustrate the yield implications of ultrafiltered milk protein products in Cheddar cheese production. Additionally, a method for pricing inputs using the sensitivity analysis generated by the solver is demonstrated.

A camel milk whey protein rich in half-cystine. Primary structure, assessment of variations, internal repeat patterns, and relationships with neurophysin and other active polypeptides.

The amino acid sequence of a recently isolated camel milk protein rich in half-cystine has been determined by peptide analyses. The 117-residue protein has 16 half-cystine residues, concluded to correspond to disulfide bridges and suggesting a tight conformation of the molecule. Comparisons of the structure with those of other proteins reveal several interesting relationships. The camel protein is clearly homologous with a previously reported rat whey phosphoprotein of possible importance for mammary gland growth regulation, and with a mouse protein of probable relationship to neurophysins. The camel, rat and mouse proteins may represent species variants from a rapidly evolving gene. Residue identities in pairwise comparisons are 40% for the camel/rat proteins and 33% for the camel/mouse proteins, with 38 positions conserved in all three forms. The camel protein also reveals an internal repeat pattern similar to that for the other two proteins. The homology between the three milk whey proteins has wide implications for further relationships. Thus, previously noticed similarities, involving either of the milk proteins, include limited similarities to casein phosphorylation sites for the camel protein, to neurophysins in repeat and half-cystine patterns for the mouse and rat proteins, and to an antiprotease for the rat protein. These similarities are reinforced by the camel protein structure and the recognition of the three whey proteins as related. Finally a few superficial similarities with the insulin family of peptides and with some other peptides of biological importance are noticed. Combined, the results relate the camel protein in a family of whey proteins, and extend suggestions of relationships with some binding proteins.

Effects of dietary nutrients and foraging costs on meal patterns of rats.

Rats were fed either a cereal-based or a purified casein-based diet in a foraging paradigm in which the costs of procurement and consumption were varied. The group offered the cereal-based diet consumed about 10% more calories than the group offered the casein-based diet, but both groups grew at the same rate. The intake of a control group offered a choice between the two diets was approximately 80% from the casein diet, and the growth of this group did not differ from that of the experimental groups. Variations in the cost of procurement and the cost of consumption affected the patterning of meals differentially for the two diets: changes in meal patterns tended to control the time and/or energy spent feeding. These results show that (1) meal patterns in the foraging paradigm are sensitive to subtle differences in diets, and (2) the amount of diet consumed (acceptance) and the choice between diets (preference) are determined by the economics of feeding and the nutritive quality of the foods, as well as by their palatability.