Optimization of Thermal Pretreatment Conditions for the Separation of Native α-Lactalbumin from Whey Protein Concentrates by Means of Selective Denaturation of β-Lactoglobulin

ABSTRACT: In this study a method to obtain native α-lactalbumin with a high degree of purity of 98% (m/m) and recovery of 75% (m/m) by selective denaturation of β-lactoglobulin was developed. To achieve this goal, the thermal pretreatment of whey protein concentrate was optimized varying the composition of the liquid whey protein concentrate in terms of total protein, lactose and calcium content, and pH value. The kinetics of the thermal denaturation of α-la and β-lg were then investigated at predetermined optimal composition (protein content 5 to 20 g/L, lactose content 0.5 g/L, calcium content 0.55 g/L, and pH 7.5). Using the activation energies and reaction rate constants obtained, lines of equal effects for targeted denaturation degrees of α-la and β-lg were calculated. Depending on total protein content, an area of optimal heating temperature/time conditions was identified for each protein concentration level.     

Essential oil composition of commercial black tea (Camellia sinensis)

The essential oil components of different tea brands were investigated by gas chromatography. The oil yields of dried tea samples were ranged from 0.09% to 0.63%. Twenty-five compounds from Supreme and Lipton Yellow Label tea brands representing 98.0% and 88.0% of the Camellia sinensis oil were identified, respectively. The main ones were β-pinene (51.2%) and α-pinene (30.2%). Nineteen components from Tapal tea brand representing 76.7% of the C. sinensis oil were determined with high contents of muurol-5-en-4-a-ol (10.5%) and muurol-5-en-4-b-ol (31.3%). Fifteen components from Deer and Diana tea brands were identified, accounting for 83.3% and 78.2% of the oil containing α-cadinol and β-pinene. Seventeen components from non-branded teas were determined with high contents of muurol-5-en-4-a-ol and muurol-5-en-4-b-ol. Twenty-one compounds from non-branded Bangladeshi Shezan and Indian teas were also identified. All oils consisted of monoterpenic hydrocarbons, oxygenated monoterpenes and sesquiterpenes.     

Selective Solubilization of α- Lactalbumin and β-Lactoglobulin into Reversed Micelles from Their Mixtures

Phase transfer experiments were performed, involving contact between an aqueous 1:1 solution of α-lactalbumin and β-lactoglobulin and an AOT-in-isooctane reversed micellar phase. The resulting extraction and separation of the two proteins were analyzed as functions of pH, ionic strength and total protein concentration using SDS-PAGE, and compared with extractions from pure solutions. At low protein concentrations, the extent of reversed micellar solubilization of the two pure proteins predicted well the extraction from mixtures. However, at higher protein concentrations β-lactoglobulin appeared to be excluded from the micellar droplets. Because of the significantly different partitioning behavior of the two proteins, reversed micellar extraction from an initially equal weight mixture led to an effective separation of the proteins.     

Expression of recombinant wild-type and mutant β-Lactoglobulins in the yeast Pichia pastoris

Summary We have produced β-lactoglobulin gene constructs in the pGAPZαA vector for over expression of recombinant wild-type and a putative monomeric mutant protein in Pichia pastoris . Levels of expression of at least 150 mg L⁻¹ of the wild-type were achieved in shake flask culture. Ion exchange chromatography showed that the expressed recombinant wild-type β-Lg eluted as a heterogeneous mixture of species, some of which may be owing to small variations in the N-terminus and some to ligand binding. The putative monomeric β-lactoglobulin construct gave an expressed protein that was recognized by anti bovine β-lactoglobulin antibody, but we do not know yet if it forms monomers rather than dimers.     

Quantitative X-Ray Diffraction Determination of α-Lactose Monohydrate and β-Lactose in Chocolate

ABSTRACT:  Lactose is a constituent of milk chocolate. During processing and cooling, lactose may precipitate as α-lactose monohydrate and β-lactose. The presence of α-lactose monohydrate has a deleterious effect on the quality of milk chocolate. A quantitative X-ray diffraction method for determination of α-lactose monohydrate and β-lactose in chocolate is described. The α-lactose monohydrate signal at 19.9°2θ with Cu-Kα X-rays is a cubic function of concentration. The β-lactose signal at 20.9°2θ is a linear function of concentration. α-Lactose monohydrate is detectible at about 0.1 weight% and can be quantified at >0.5 weight%. β-Lactose is detectible at about 1 weight% and can be quantified at >3 weight%. About 10 min is required to prepare and run a sample.
Practical Application: The crystalline form of lactose affects the quality of chocolate. A rapid method for quantifying crystalline forms of lactose in chocolate is described. The method can be used for quality control and for improving chocolate quality.     

Surface active molecules: preparation and properties of long chain nα-acyl-l-α-amino-ω-guanidine alkyl acid derivatives

A new route for the synthesis of long chain N ^α-acyl- l -α-amino-ω-guamdine alkyl acid derivatives, with cationic or amphoteric character has been established. The general formula of these compounds is shown below.
A physico-chemical and antimicrobial study of these products as a function of the alkyl ester or sodium salt (R), the straight chain length of the fatty acid residue ( x ) and the number of carbons between the ω-guanidine and ω -carboxyl group ( n ) has been investigated.
The water solubility, surface tension, critical micelle concentration (c.m.c.) and minimum inhibitory concentration (MIC) against Gram-positive and Gram-negative bacteria (including Pseudomonas) has been determined.
Dicyclohexylcarbodiimide has been used to condense fatty acids and α -amino-ω-guanidine alkyl acids. In these conditions protection of the ω-guanidine group is not necessary. The main characteristic of this synthetic procedure is the use of very mild experimental conditions (temperature, pH) to form the amide linkage which leads to pure optical compounds in high yield in the absence of electrolytes.
The results show that some structural modifications, particularly the protection of the carboxyl group, promote variations of the surfactant and antimicrobial properties. Only those molecules with the blocked carboxyl group (cationic molecules, where R = Me, Et or Pr) showed a good surfactant and antimicrobial activity. When the carboxyl group was unprotected (amphoteric molecules, where R = Na⁺) the resulting compounds were inactive.     

Heat-induced gelation of whey protein at high pH studied by combined UV spectroscopy and refractive index measurement after size exclusion chromatography and by in-situ dynamic light scattering

Summary The interactions between β-lactoglobulin and α-lactalbumin involved in gelation at 67.5 °C at high pH and low salt concentration were studied by size exclusion chromatography, followed by UV and refractive index measurements, and by in-situ dynamic light scattering. This was achieved by choosing whey protein samples with different proportions of the two proteins. The ratio of absorbance at 280 nm to the refractive index was used to demonstrate that α-lactalbumin was incorporated in aggregates and gels and drastically changed the properties of the gel, making them much more turbid than the transparent gels formed by β-lactoglobulin alone at the same pH and ionic strength. At a ratio of 1:2 for α-lactalbumin relative to β-lactoglobulin in the samples, the gel consisted of a 1:1 mixture of the two proteins. The aggregates present after 10 min of heating at 67.5 °C had molar mass of about 6.10⁶ g/mol and a radius of gyration of about 40 nm. After gel formation the field autocorrelation function could be described as a power law over many decades of lag time for all samples, demonstrating selfsimilarity of the gel structure. The only exception to this was for the gel with high content of α-lactalbumin which showed an oscillatory behaviour of the autocorrelation function. Significant amounts of glycosylated caseino-macro-peptide were observed in many of the samples at the position of β-lactoglobulin. However it did not affect gelation as it remains in solution.     

Rheological properties of commercial whey protein samples from the MADGELAS survey

Summary Thermally induced gelation of commercial whey protein samples from the Molecular Basis of the Aggregation, Denaturation, Gelation and Surface Activity of Whey Proteins ( MADGELAS ) survey has been studied in order to develop a current status report on their rheological properties. Solutions of 10% protein (w/v) were prepared in distilled water, 200 mm NaCl or 10 mm CaCl₂ at neutral pH. Small-scale deformation of the samples was measured by dynamic oscillatory rheometry using a Bohlin CS Rheometer. Large-scale deformation at penetration mode was measured using a Texture Analyser (Stable Micro Systems, Surrey, UK). For solutions containing salts, there was a general trend for gel point to decrease and G' values to increase, the effect being more marked in the presence of NaCl. Similarly, force values at failure also tended to increase in the presence of salts. Results obtained with samples of similar protein composition dissolved in water were highly scattered, these differences being reduced in the presence of added salts.     

Simultaneous determination of ketoacids and dicarbonyl compounds, key Maillard intermediates on the generation of aged wine aroma

ABSTRACT:  The aim of this work was the simultaneous determination of both ketoacids and dicarbonyl compounds in wine. To detect ketoacid compounds in wine, a method based on the quinoxaline derivatives by the reaction with diaminobenzene, currently employed to detect α-dicarbonyl compounds, was developed. The quinoxaline derivatives were detected by RP-HPLC with UV detection, which allows the determination of the major dicarbonyl compounds in wine: glyoxal, methylglyoxal, diacetyl and pentane-2,6-dione, and the quinoxaline/quinoxalinol derivatives of α-keto-γ-(methylthio)butyric acid and β-phenylpyruvic acid (intermediate ketoacid compounds of methional and phenylacetaldehyde) were simultaneously detected by a fluorescence detector. The identification was performed by comparison with standards and also by using LC-MSMS. The levels found in 15 wines analyzed (white wines, Madeira wines, and Port wines) diverge according to the type and the age of the wine. The ketoacid compounds ranged from 0.2 to 5.7 mg/L for α-keto-γ-(methylthio)butyric acid and 0.1 to 9.6 mg/L for β-phenylpyruvic acid. The quantities observed for dicarbonyl compounds were similar to those already reported.     

α-Tocopherol, β-Carotene and Ascorbic Acid as Antioxidants in Stored Poultry Muscle

Broilers were fed α-tocopherol or β-carotene for 3 wk or L-ascorbic acid for 24 hr prior to slaughter. α-Tocopherol maintained the redness of unheated meat stored for 8 wk (—20°C). Values for thiobarbituric acid reactive substances in ground, stored meat showed that L-ascorbic acid produced results similar to the control while a-tocopherol produced results lower than the control. Panelists rated meat with added α-tocopherol as different from the control for smell and flavor. β-Carotene was a pro-oxidant compared to the control and other additives. Meat from broilers fed β-carotene had lower α-tocopherol content than the control. Vitamin A in livers of birds fed β-carotene was 64% higher than that of the control.     

Quenching Mechanisms and Kinetics of α-Tocopherol and β-Carotene on the Photosensitizing Effect of Synthetic Food Colorant FD&C Red No. 3

ABSTRACT: Effects of FD&C Red No. 40, Red No. 3, Yellow No. 5, Yellow No. 6, Green No. 3, Blue No. 1 and Blue No. 2 on 0.03M soybean oil oxidation in acetone at 25 °C under light were studied by measuring headspace oxygen depletion. As Red No. 3 increased from 0 to 5, 20, 100 and 200 ppm, the headspace oxygen was reduced by 2 to 70, 73, 77 and 77%, respectively, for 4 h. Only Red No. 3 acted as a photosensitizer to produce singlet oxygen in the oil. The quenching rates of α-tocopherol and β-carotene for the singlet oxygen by Red No.3 were 4.1 × 10⁷ M⁻¹s⁻¹ and 7.3 × 10⁹ M⁻¹s⁻¹, respectively. When β-carotene was below 1.86 × 10⁻⁶ M, β-carotene quenched singlet oxygen, but it quenched both singlet oxygen and Red No. 3 at or above 3.72 × 10⁻⁶ M. However, α-tocopherol quenched singlet oxygen only.     

Oxymyoglobin and Lipid Oxidation in Phosphatidylcholine Liposomes Retarded by α-tocopherol and β-carotene

An n-3 fatty acid-constructed oxymyoglobin liposome model was used to study the effects of 10 μM α-tocopherol and/or various concentrations of β-carotene on oxymyoglobin and lipid oxidation. Oxidation was delayed by α-tocopherol or β-carotene treatments alone (p < 0.05). β-Carotene at 1.5 μM and 2μM caused greater delay in oxymyoglobin oxidation than α-tocopherol (p < 0.05). For lipid oxidation inhibition, 2 μM β-carotene was similar in effect to α-tocopherol. With α-tocopherol plus 1.5 or 2 μM β-carotene, greater oxidation delaying effect resulted for both oxymyoglobin and lipid oxidations than with 1.5 or 2 μM β-carotene treatment alone (p < 0.05).     

Effects of Combined Treatment of High Hydrostatic Pressure and Mild Heat on the Quality of Carrot Juice

ABSTRACT: This study was performed to investigate the effects of the combined treatment of high pressure and heat on the quality of carrot juice, and to optimize the process condition. About 95% of food quality-related enzymes were lost at 400 MPa and 70 °C, for 10 min, while α- and β-carotene were relatively stable at the combined process. The optimum process condition was estimated at 395 to 445 MPa, 70 °C, for 8 to 11 min. These results indicate that the combined treatment of high pressure and mild heat could be used as an effective process for production of high-quality carrot juice.     

Effects and prooxidant mechanisms of oxidized alpha-tocopherol on the oxidative stability of soybean oil

ABSTRACT:  α-Tocopherol was oxidized in methanol containing methylene blue for 30 h under light. The effects of 0, 250, 500, 1000, and 1500 ppm of oxidized α-tocopherol on the oxidative stability of purified soybean oil in the dark at 55 °C were studied by measuring the peroxide values and headspace oxygen contents in sample bottles. As the concentrations of oxidized α-tocopherol increased, the peroxide values increased and the headspace oxygen contents decreased during the 6 d of storage. The oxidized α-tocopherol compounds acted as prooxidant on the peroxide values and headspace oxygen contents of purified soybean oil. Tukey's test showed that oxidized α-tocopherol had a significant effect on the peroxide value and headspace oxygen disappearance of oil at P < 0.05. The prooxidant mechanisms of oxidized α-tocopherol may be due to α-tocopherol peroxy radical, α-tocopherol oxy radical, hydroxy radical, and singlet oxygen formed from the α-tocopherol. The oxidized α-tocopherol containing polar and nonpolar groups in the same molecule may reduce the surface tension of oil to increase the transfer of headspace oxygen to oil and accelerate the oil oxidation.     

利用枯草芽孢杆菌发酵生产环磷腺苷的工业化试验

为了实现环磷腺苷的发酵生产,对一株枯草芽孢杆菌Bacillus.subtilis jsim-1277在摇瓶、自控罐、20 M3发酵罐上的发酵条件进行了试验。结果表明,最适宜发酵条件是p H 6.7,且添加K2HPO41.0 g/d L,Na F 0.05 g/d L;在摇瓶、自控罐、大型发酵罐产苷分别为7.17、6.01、7.10 g/L。     

Lack of desquamation - the Achilles heel of the reconstructed epidermis

The use of human skin equivalents for screening tests aiming to assess repetitive application of various test agents is hampered by the lack of desquamation in vitro. The present study was undertaken to examine whether the desquamation can be induced by various treatments including mechanical stress, application of various agents that should decrease the surface pH and calcium level, activate the enzymes involved in desquamation process or UV irradiation. In addition, the effect of alpha-hydroxyacids, known to enhance desquamation and to improve the stratum corneum barrier function in vivo, was examined as well. Human epidermis reconstructed on de-epidermized dermis or on fibroblast-populated collagen matrices during a 2-week culture at the air-liquid interface underwent various treatments during an additional 3-week period. The effects of treatments were evaluated on the basis of tissue morphology and lipid composition. The results of the present study revealed that cell shedding could only be induced by a mild repetitive mechanical treatment. The lack of desquamation, under most in vitro conditions, has a practical consequence, since it may hamper the use of reconstructed epidermis for various screening studies aiming to examine the repetitive exposure to topical agents or UV irradiation. The gradual thickening of the stratum corneum will lead to its higher resistance to the environmental stimuli and in this way affect the outcome of the tests. Furthermore, from the results obtained in the present study, it became evident that one should be careful in selecting endpoints when, for example, the effects of agents known to modulate melanogenesis are examined.     

Influence of Dietary Supplementation with α-Tocopheryl Acetate and Canthaxanthin on Cholesterol Oxidation in ω3 and ω6 Fatty Acid-enriched Spray-dried Eggs

ABSTRACT: The effect of feeding laying hens linseed oil or sunflower oil, with and without α-tocopheryl acetate and/or canthaxanthin, was evaluated on cholesterol oxidation in spray-dried whole egg at various storage periods. Storage of spray-dried eggs at room temperature in the dark resulted in an increase in cholesterol oxidation products from 18.1 μg/g, after spray drying, to 39.3 μg/g, at 12 mo of storage. No differences were found with either dietary oil or canthaxanthin supplementation. However, α-tocopheryl acetate supplementation resulted in a lower formation of cholesterol oxidation products during storage. No synergistic effect between α-tocopherol and canthaxanthin was detected.