Precipitation Of Casein Research Articles

Comparative Studies on Physico-chemical and Sensory Attributes of Natural and Artificial Whey Protein Beverages

Whey, the liquid remaining after the precipitation of milk casein was used and there physicochemical properties were measured. The aim of the study was to utilise the valuable nutrients present in whey by preparing a ready-to-serve modified whey beverage. Two types of beverages were prepared i.e., natural whey beverage using Himsagar mango and other one was artificial beverage using artificial colours and flavours. Shelf life of these beverages were studied (0-11th day) and compared on the basis of acidity, viscosity, ph and organoleptic properties. Student t-test was applied here and found that the natural whey beverage was curdled on the 7th day but the artificial whey beverage remained normal till 11th day of the study. From this observation, it was concluded that the natural whey beverage tasted better initially but curdled on the 7th day and in case of artificial whey beverage, though the taste of the beverage initially was not acceptable but gradually it improved its taste on storage and it remained in good condition till the 11th day. Keywords: Whey beverage, Channa, Paneer, Casein, Serum albumin, β-lactoglobulin. Cite this Article Debasree Ghosh, Deep Biswas. Comparative Studies on Physico-chemical and Sensory Attributes of Natural and Artificial Whey Protein Beverages. Research & Reviews: Journal of Food Science & Technology. 2020; 9(1): 1–6p.

Valorization of cheese whey using microbial fermentations.

Cheese whey (CW), the liquid resulting from the precipitation and removal of milk casein during cheese-making, and the second cheese whey (SCW) derived from the production of cottage and ricotta cheeses are the main byproducts of dairy industry. The major constituent of CW and SCW is lactose, contributing to the high BOD and COD content. Because of this, CW and SCW are high-polluting agents and their disposal is still a problem for the dairy sector. CW and SCW, however, also consist of lipids, proteins, and minerals, making them useful for production of various compounds. In this paper, microbial processes useful to promote the bioremediation of CW and SCW are discussed, and an overview on the main whey-derived products is provided. Special focus was paid to the production of health-promoting whey drinks, vinegar, and biopolymers, which may be exploited as value-added products in different segments of food and pharmaceutical industries.

Physicochemical properties of skim milk powder dispersions after acidification to pH 2.4–3.0 and heating

Application of skim milk powder (SMP) in acidified beverages is limited due to precipitation and sedimentation of caseins. In this study, 5% w/v SMP dispersion was acidified to pH 2.4–3.0 using citric acid and heated at different conditions. Lowering pH resulted in dissolution of colloidal calcium phosphate from casein micelles, corresponding to lowered turbidity and reduced hydrodynamic diameter of dispersions. When heated for 10 min at different temperatures, 70 °C was the most effective to produce transparent dispersions; whereas a shorter heating time between 2 and 60 min was desired at 90 °C. The decreased turbidity after heating was attributed to weakened hydrophobic interaction between casein molecules based on fluorescence spectrometry and ion analysis. Scanning transmission electron microscopy showed the altered structure and decreased dimension of casein micelles after acidification and heating. The current study may expand the use of SMP for manufacturing new types of protein drinks.

IMPROVEMENT OF THE METHOD OF COMPARATIVE STUDY OF MILK WHEY PROTEINS ENZYMATIC HYDROLYSIS

Milk whey proteins are valueble nutritional ingredients with a number of health-beneficial properties. Whey proteins are also a source of bioactive peptides that can be released in the process of proteins enzymatic hydrolysis. In this connection, there often is a need to compare their proteolytic action on milk whey proteins. It is important to take into account the specificities of the composition and properties of milk whey proteins. The aim of the research was to improve the method of comparative study of milk whey proteins enzymatic hydrolysis. Casein and whey were obtained from fresh cow skimmed milk. The whey was separated by centrifugation after casein precipitation at the isoelectric point. The following enzyme preparations were used in the research: neutral protease, papain, trypsin, chymotrypsin and pancreatin. To select β-LG, gel filtration of the milk whey on the chromatographic column with Sephadex G-150 (Pharmacia) was used. The homogeneity of the received β-LG preparation was analyzed by express electrophoresis in the polyacrylamide gel plates (PAG). The preparation of general casein was isolated by repeated precipitation at the isoelectric point. The fractional composition of the casein substrate was analyzed by electrophoresis in the anode system of homogeneous PAG in the presence of urea. Quantitative treatment of electrophoregrams of the β-LG preparation was performed using the imread reading function. Determination of proteolytic activity of enzyme preparations was carried out according to the method of V. F. Selemenev [6]. In the course of the research, it was determined, that for the research of proteolysis under conditions of identical total proteolytic activity, the concentration of neutral protease should be increased by 1.02 times, papain – by 4.2 times, trypsin – by 2.8 times, pancreatin – by 2.12 times as compared to chymotrypsin. As a result, it has been shown that the use of β-lactoglobulininstead of serum albumin in spectrophotometric determinations allows obtaining more accurate values of the concentrations of whey protein and proteolytic products. In determining the ratio of enzyme : substrate it is advisable to take into account the general proteolytic activity of various enzyme preparations in comparative studies of whey proteins proteolysis with various enzyme preparations. These will simplify the methodology and reduce the time for objective evaluation of enzymatic preparations for proteolysis of milk whey proteins. In some cases, considering the specificity of proteases it could increase the yield of biologically active peptides.

Chemical fractionation of caseins by differential precipitation: influence of pH, calcium addition, protein concentration and temperature on the depletion in α‐ and β‐caseins

SummaryThe impact of pH, calcium and casein concentrations, and temperature on the efficiency of the differential precipitation of caseins by calcium affinity was investigated, using native phosphocaseinate as starting material. We adapted one of the most recent methods for the separation of caseins that is based on the addition of calcium at alkaline pH. Increasing the pH to 11 disturbed the micellar structure by enhancing electrostatic repulsion of caseins, leading to a marked viscosity increase and a significant particle size decrease, indicating casein micelle disruption. This pH‐driven increase in negative charges enhanced the affinity of individual caseins for calcium, proportionally to the number of phosphate groups carried by each casein. The addition of calcium first led to a progressive increase in the proportion of precipitated caseins, before reaching a plateau. Hence, an optimal calcium/casein molar ratio of about 40 was evidenced to optimise casein precipitation (and fractionation), leading to significant depletion of α‐casein (around 80%) and, in a lesser extent, β‐casein (around 65%) and κ‐casein (around 55%). This method led to relative proportions of caseins significantly differing from the starting material: 31% α‐casein, 45% β‐casein and 24% κ‐casein.

Detection of buffalo milk adulteration with cow milk by capillary electrophoresis analysis

The addition of cow milk during the production of buffalo mozzarella is a common fraud in dairy industries because of the lower price and greater availability of cow milk throughout the year. The aim of this study was to develop a new, rapid, and robust capillary electrophoresis method for detecting and quantifying cow milk in buffalo milk by exploiting cow α-lactalbumin as a marker of adulteration. In particular, a linear calibration curve was generated, using a training set of calibrators consisting of 7 series of 17 buffalo/bovine whey mixtures, obtained after casein precipitation, with increasing percentages of cow whey. The capillary electrophoresis method showed high linearity (R2 = 0.968), repeatability [relative standard deviation (RSD) = 2.11, 3.02, 4.38, and 1.18%, respectively for 5, 10, 20, and 50% of buffalo/bovine whey mixtures], and intermediate precision (RSD = 2.18, 2.49, 5.09, and 3.19%, respectively, for 5, 10, 20, and 50% buffalo/bovine whey mixtures). Moreover, the minimum amount of detectable fraudulent cow milk was 1%, and the limit of quantification was 3.1%.

OBTAINING OF Β-LACTOGLOBULIN BY GEL FILTRATION OF COW MILK WHEY

Milk whey proteins carry out a number of important biological functions and also they are precursors of many biologically active peptides (antihypertensive peptides, antagonists of opioid receptors, regulators of intestinal motility, immunomodulatory, anti-microbial and anti-cancer peptides, appetite regulators and so on.). An important stage in natural bioactive peptides obtaining from milk whey proteins is the isolation of homogeneous proteins-precursors. Considering the significant difference in the molecular masses of whey proteins, a promising method for their selection is gel filtration. The purpose of the research was the fractionation of bioactive peptides precursors from milk whey using gel filtration on Sephadex G-150. The whey was obtained from fresh skimmed milk after isoelectric precipitation of casein. Gel filtration was carried out on the columns from a liquid chromatography kit by the “Reanal” company. The fractional composition and the degree of homogeneity of milk whey proteins were determined by disc-electrophoresis in the plates of a polyacrylamide gel. A repeated gel filtration of fractions from the chromatographic peaks, separated into sections, was performed to increase the fractionation efficiency. While choosing a dextran gel for gel filtration of precursors of biologically active peptides from milk whey proteins, we have taken into account the range of their molecular weights (from 10000 to 150000 Da), the ability to form supramolecular structures (β-LG), as well as the previously obtained results of gel filtration. As a result, it was shown that repeated gel filtration of milk whey on Sephadex G-150 allows efficiently fractionate the proteins-precursors of bioactive peptides. The range of peptides and proteins molecular weights that can be fractionated on this Sephadex is from 5000 to 300 000 Da. The usage of repeated gel filtration on Sephadex G-150 with the chromatogram separation into sectors allows to effectively fractionate proteins-precursors of bioactive peptides from milk whey. In particular, homogeneous β-lactoglobulin (degree of homogeneity > 95 %) and partially purified α-lactalbumin, as well as a group of immunoglobulins and a proteose-peptone fraction were obtained.

Short communication: Multi-component interactions causing solidification during industrial-scale manufacture of pre-crystallized acid whey powders

Acid whey (AW) is the liquid co-product arising from acid-induced precipitation of casein from skim milk. Further processing of AW is often challenging due to its high mineral content, which can promote aggregation of whey proteins, which contributes to high viscosity of the liquid concentrate during subsequent lactose crystallization and drying steps. This study focuses on mineral precipitation, protein aggregation, and lactose crystallization in liquid AW concentrates (∼55% total solids), and on the microstructure of the final powders from 2 independent industrial-scale trials. These AW concentrates were observed to solidify either during processing or during storage (24 h) of pre-crystallized concentrate. The more rapid solidification in the former was associated with a greater extent of lactose crystallization and a higher ash-to-protein ratio in that concentrate. Confocal laser scanning microscopy analysis indicated the presence of a loose network of protein aggregates (≤10 µm) and lactose crystals (100-300 µm) distributed throughout the solidified AW concentrate. Mineral-based precipitate was also evident, using scanning electron microscopy, at the surface of AW powder particles, indicating the formation of insoluble calcium phosphate during processing. These results provide new information on the composition- and process-dependent physicochemical changes that are useful in designing and optimizing processes for AW.

Improving Fractionation of Human Milk Proteins through Calcium Phosphate Coprecipitation and Their Rapid Characterization by Capillary Electrophoresis.

This work describes a simple sample pretreatment method for the fractionation of human milk proteins (into their two main groups, whey and caseins) prior to their analysis. The protein-extraction protocol is based on the addition of calcium phosphate to nonadjusted pH human milk. The combination of calcium ions with phosphate results in an effective coprecipitation of caseins. To assess the suitability of this fractionation protocol, the protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), LC-MS/MS, and capillary electrophoresis (CE) analysis. The results evidence a significant decrease in contamination of casein fraction with whey proteins and vice versa compared with the conventional isoelectric precipitation of caseins. In addition, CE fraction collection coupled to LC-MS/MS (off-line coupling) has been successfully applied to the identification of minor proteins in this complex matrix. The methodology presented here constitutes a promising tool to enlarge the knowledge of human milk proteome.

Production of calcium- and magnesium-enriched caseins and caseinates by an ecofriendly technology

Finding new environmentally friendly ways of producing proteins has never been of such critical public interest, both to meet consumers' needs and to preserve the environment. Milk proteins are among the most attractive protein types due to their high nutritional value and attractive functional properties. In this work, the separation of caseins by conventional chemical acidification was compared with electrodialysis with bipolar membrane coupled to an ultrafiltration module (EDBM-UF), a green process that allows the precipitation of caseins by H+ generated in situ by the bipolar membrane and, simultaneously, the production of a separated NaOH stream from OH- electrogenerated by the bipolar membrane. Caseinate production using this NaOH stream by-product and the quantity of NaOH needed to produce caseinates from both methods were also investigated. Hence, the purity and composition of caseins and caseinates were compared in terms of protein, ash, and lactose contents as well as mineral composition. The results showed for the first time that caseinates can be produced by solubilizing caseins with NaOH stream from the EDBM process. Furthermore, the caseins and caseinates produced by EDBM-UF were equivalent in terms of lactose and protein contents to their respective caseins and caseinates that were chemically produced but presented slightly lower sodium content and 3 to 4 times higher magnesium and calcium contents. The fact that calcium and magnesium are likely bound to milk caseins would ensure their favorable absorbability. These caseins or caseinates from the new EDBM-UF process could be suitable as an improved protein-based calcium or magnesium supplement, both for their enhanced nutritional quality and because they are produced by a "green" process.

Підбір умов для препаративного електрофорезу протеїнів сироватки молока у нативних умовах

Протеїни сироватки молока є попередниками більше сотні біоактивних пептидів. Ці пептиди можуть утворюватися в процесі травлення у шлунково-кишковому тракті, а також за дії протеаз молочнокислих бактерій і молокозгортальних препаратів. Серед них знайдено інгібітори ангіотензин-перетворювального ензиму, пептиди з опіоїдною та бактерицидною дією, імуномодуляторні та гіпохолестеролемічні пептиди, пептиди, які впливають на моторику кишечника, апетит та інші. Біоактивні пептиди з протеїнів сироватки молока становлять значний інтерес для створення функціональних продуктів або продуктів з профілактичними властивостями. Створення таких продуктів передбачає виділення біоактивних пептидів певної біологічної дії. Враховуючи специфіку їх розміщення у первинній структурі доцільним є використання для їх отримання гомогенних протеїнів-попередників з сироватки молока. Одним з методів для одностадійного розділення протеїнів сироватки молока є диск-електрофорез в нативній системі ПААГ для лужних і нейтральних протеїнів. Метою даної роботи було встановити параметри препаративного варіанту диск-електрофорезу для виділення гомогенних протеїнів сироватки молока. Дослідження проводили на модифікованому апараті Стадієра. Протеїни сироватки молока виділяли осадженням казеїнів в ізоелектричній точці. Осад казеїнів відділяли центрифугуванням. Для ідентифікації протеїнів сироватки молока використовували гомогенні фракції α–лактальбуміну(α-LA) і β-лактоглобуліну(β-LG), які отримували шляхом повторної гель-фільтрації на сефадексі G-100(fine). Гомогенність і фракційний склад протеїнів сироватки молока визначали аналітичним диск-електрофорезом у пластинах поліакриламідного гелю. Концентрацію протеїнів у зразках визначали спектрофотометрично (спектрофотометр СФ-46) при довжині хвилі λ = 280 нм.
 Отримані результати доводять доцільність використання пластинок ПААГ для препаративного фракціонування протеїнів сироватки молока. Встановлено допустиму тривалість і умови ідентифікації протеїнових фракцій. Показано, що вихід протеїнів у складі гомогенних фракцій становить 69,2%. При цьому співвідношення між кількістю отриманих фракцій (β-LG, α-LA, BSA і ІG) близьке до їх розподілу у сироватці молока.

Isolation of human milk whey proteins by solid phase extraction with a polymeric material modified with gold nanoparticles

This work describes a method for the isolation of human milk whey proteins by solid-phase extraction (SPE) with a polymeric material modified with gold nanoparticles. Human serum albumin, α-lactalbumin, lactoferrin and lysozyme were selected as target proteins to establish the performance of SPE support. Several experimental variables (pH and ionic strength) that affect the SPE protocol were investigated to achieve the maximum extraction efficiency. Under optimal conditions, the SPE sorbent gave excellent recoveries, and offered a high permeability and reusability (>20 times). The feasibility of this methodology was successfully demonstrated by isolating the target proteins in a milk whey extract and from a direct dilution of human milk samples (without isoelectric precipitation of caseins).

A Zymogram technique for preliminary screening and characterization of feruloyl esterases.

We report here, for the first time, a zymogram technique designed for rapid screening of feruloyl esterase using ethyl ferulate as enzyme substrate and casein precipitation as enzyme activity indicator.

Effect of calcium on the aggregation behaviour of caseinates

Insights into the structure of caseinate aggregates have been obtained from small angle X-ray scattering (SAXS) with complementary measurements obtained by turbidity and dynamic light scattering (DLS). The caseinate precipitation method produced a cold insoluble and cold soluble caseinate (CN) fraction. The caseinates were then recombined in ratios of these fractions from 0% to 100% cold soluble CN (3.3% w/w total protein), and calcium was added up to 22mM. This allowed individual structural features due to calcium and/or protein to be identified as the relative concentrations of each component were varied in a controlled fashion. SAXS analysis indicated the presence of scattering objects on three different length scales in the system: a feature from the largest objects which was ascribed to the overall aggregate size; a small feature at q>0.1Å−1 (where q is the magnitude of the scattering vector) corresponding to protein inhomogeneities with a radius of gyration of 1.9–5.9nm, and a mid-range feature at q=0.02Å−1 which corresponds to a protein cluster interaction distance of 22–28nm. DLS and turbidity measurements showed that the overall aggregate size and turbidity increased with increasing calcium and cold insoluble CN concentration. The hydrodynamic diameter for the cold insoluble CN increased from 190nm to 295nm with 0mM and 22mM calcium, respectively. In contrast, a cold soluble CN with 22mM calcium had a diameter of 140nm. Collectively this is consistent with calcium binding to the phosphoserine clusters via ionic bonding, leading to particle growth; the higher ratio of cold insoluble CN allowed the aggregates to grow to larger sizes, which is likely due to different structuring of κ-CN around the aggregates.

Detection of the Presence of Reconstituted Milk in Raw Milk and in Pasteurized Milk Using Synchronous Fluorescence Spectroscopy

The increasing demand for dairy products has led to the adulteration of milk with reconstituted milk. This may be a problem for unsuspecting consumers. In this study, synchronous fluorescence spectroscopy was used to detect the adulteration of raw milk and pasteurized milk by the addition of reconstituted milk. Milk samples were prepared by the precipitation of casein with a pH 4.6 buffer solution to separate the whey protein. Synchronous fluorescence spectra were then captured over an excitation wavelength between 200 and 500 nm at a wavelength interval (Δλ) of 75 nm. The raw data of the spectra were then subjected to a second derivative, and the ratio of the peaks of characteristic Maillard reaction products and tryptophan (2nDFM/T) were used to detect the adulteration of raw and pasteurized milk with reconstituted milk. This method is unaffected by variations in environmental temperature. The results indicate that the detection of reconstituted milk in raw and pasteurized milk can be achieved with an accuracy of 86.7 and 90%, while linear equations could be used to estimate the content range of reconstituted milk roughly according to the fluorescence data.

Casein precipitation by acid and rennet coagulation of buttermilk: Impact of pH and temperature on the isolation of milk fat globule membrane proteins

Milk fat globule membrane (MFGM) proteins make up only 2–3% of the total protein content in buttermilk. Prior isolation of buttermilk MFGM material is therefore required for further investigation of its functional properties and application in food systems. Rennet induced coagulation has been applied to remove caseins, but 30–70% of MFGM proteins are entrapped in the casein curd under classical cheese making conditions. Therefore, factors influencing the rennet as well as acid induced coagulation of buttermilk with regard to a reduction of MFGM protein losses into the casein curd, i.e., pH, temperature and pre-heat treatment of buttermilk, were examined. Complete casein removal from buttermilk was reached using both coagulation methods, whereas retention of MFGM proteins in the serum phase was higher during renneting. Rennet induced coagulation at higher pH and lower temperatures resulted in weaker gel structures, which increased the yield of MFGM proteins in the buttermilk whey.

Hybrid bipolar membrane electrodialysis/ultrafiltration technology assisted by a pulsed electric field for casein production

Ecofriendly EDBM-UF-PEF technology demonstrates good performance in the isoelectric casein precipitation process due to elimination of EDBM stack clogging and a decrease in membrane scaling.

Improvement of a sustainable hybrid technology for caseins isoelectric precipitation (electrodialysis with bipolar membrane/ultrafiltration) by mitigation of scaling on cation-exchange membrane

Abstract Electroacidification of milk by electrodialysis with bipolar membranes (EDBM) can be performed to produce isoelectric precipitated casein. In spite of advantages of EDBM, a problem of fouling hampers the industrial application of this technology. There are two types of fouling occurring during milk electroacidification such as protein fouling inside the EDBM stack and scaling on cation-exchange membrane (CEM). Recent studies demonstrated that protein fouling can be avoided by coupling an EDBM module with an ultrafiltration (UF) module. The present study aims the mitigation of scaling on the CEM. In order to attain this goal, two approaches were tested: 1) addition of KCl in the milk reservoir and 2) use of a UF membrane allowing higher permeate flux and consequently higher flow rates of solutions in the EDBM stack. Results of this study demonstrate the reasonableness of both approaches resulting in the significant decrease of CEM scaling (more than 30%). Industrial Relevance Caseins are the major milk proteins largely produced in the world (around 330 000 t per year) and widely applied in the food and pharmaceuticals industries. There are several relevant features of proposed method (electrodialysis with bipolar membranes coupled with ultrafiltration (EDBM-UF)) for the casein production such as 1) absence of the hazardous reagents; 2) high purity of caseins due to the demineralization during EDBM; 3) no waste generation; 4) the base stream from EDBM module (co-product) can be used for the formation of caseinates. The present work demonstrates the significant decrease of scaling on the cation-exchange membrane (more than 30%), which is the major problem of the EDBM-UF process. Indeed, during milk acidification, there is a liberation of minerals (Ca 2 + and Mg 2 + ), which form the membrane deposit. This deposit negatively affects the process performance and leads to the membrane replacement. The cost of ion-exchange membranes in ED module comprises a major part of the total cost of EDBM-UF process. Thus, control and prevention of the membrane deposit will allow to increase a lifetime of ion-exchange membranes and to decrease a price of EDBM-UF treatment.

Preparation and characterisation of a milk polar lipids enriched ingredient from fresh industrial liquid butter serum: Combination of physico-chemical modifications and technological treatments

Milk polar lipids (PLs) are poorly utilised as ingredients for human consumption, despite their interesting nutritional and functional properties. The objective of this study was to valorise by-products by developing a technological process able to provide a milk PL-enriched ingredient from industrial fresh liquid butter serum. The process comprised the following successive steps: skimming, heat treatment, acid precipitation of caseins, concentration and purification of whey butter serum by ultrafiltration and diafiltration. The proposed process yields a recovery of 62% of PLs present in the initial butter serum. The final ingredient contained 31.5%, 26% and 34% of PLs, proteins and triacylglycerols (on a dry matter basis). The identified proteins were caseins, whey proteins and proteins from the milk fat globule membrane. This process will allow the preparation of a milk PL-rich ingredient for food applications.

A DIGITAL TOOL FOR CASEIN ISOLECTRIC POINT DETERMINATION

Introduction: Casein is a milk protein that has the same number of positive and negative charges in a pH around 4.7. This characteristic is called the isoelectric point (pI). The pI varies from protein to protein and it depends on the charges of the lateral chain of the constituent aminoacids. At this pH value the protein has its minimum solubility since the net charge is zero and the repulsion between molecules is decreased. Furthermore, the electrostatic interaction occurs between the protein molecules. Thus, they form clumps which tend to precipitate. On the other hand, when they are placed in a solution whose pH is above or below of its pI, the protein molecules have respectively a negative or positive net charge, with strong repulsion between themselves and great interaction with the solvent (water). Objective: This learning object presents a simulation of a laboratory practice for the determination of casein isoelectric point. Materials and Methods: Cartoons were planned in order to show the methodology procedures and biochemical fundamentals. Animations were developed with the aid of the Adobe ® Flash 8 software associated with logic programming. Results: The simulation consists of six steps that reproduce the activities performed in the laboratory. Among them, the user can observe different degrees of turbidity and / or precipitation of casein in solutions with different pH values, he can measure these values with paper indicator strips and then determine the pI of this protein. Conclusions: This learning object was tested by students of Biochemistry I, Pharmacy course since 2012/2. The navigation features, design, interaction, interactivity were considered excellent by 80% of students indicating that this object can be used as an interesting tool to assist the teaching and learning of basic biochemistry. Available at:www.ufrgs.br/gcoeb/PontoIsoleletricoDaCaseina/PontoIsoleletricoDaCaseina.swf