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Coagulation properties of milk : association with milk protein composition and genetic polymorphism /Hallén, Elin, January 2008 (has links) (PDF)
Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2008. / Härtill 5 uppsatser.
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Effect of heat denaturation of bovine milk beta-lactoglobulin on its epithelial transport and allergenicityRytkönen, J. (Jani) 06 June 2006 (has links)
Abstract
Beta-lactoglobulin (β-lg) is the main whey protein in bovine milk. It belongs to the lipocalin protein family, and it is one of the main milk allergens. Resistance to hydrolysis is a particular feature of β-lg making it possible that β-lg reaches the small intestine in its native form. Heat treatments during milk processing may change the native structure of bovine β-lg and change its intestinal transport properties. Heat induced conformational alterations may also expose new antigenic sites. However, there have been no previous studies on the effects of heat treatment on the transport of β-lg or on its sensitizing properties.
Cow's milk allergy is one of the most important food allergies affecting about 2.4% of infants. Milk proteins, including β-lg, in breast milk substitute formulas are often the earliest foreign antigens in the diet of newborns. According to the hygiene hypothesis, natural infections and vaccinations may modify the immunological balance and decrease the risk of allergy.
Isoelectric precipitations followed by anion exchange and gel filtration were used to purify bovine milk β-lg in its native form. Transport of native and heat-denatured β-lg was compared in two in vitro cell models, Caco-2 and M-cells. Sensitization properties of native and heat-denatured β-lg were studied with an animal model using Hooded-Lister rats. Effects of BCG vaccination in combination with the native β-lg were also studied. Effects of different sensitizations were assessed by antibody levels in serum and inflammation locally in the gastrointestinal tract.
Heat denaturation of β-lg made its transport slower in both enterocytes and M-cells. M-cells were more effective transporters of both native and heat-denatured β-lg than caco-2 cells. Animals generated higher levels of IgE when sensitized with native β-lg, but heat-denatured β-lg induced a more intense inflammatory cell reaction in the gastrointestinal tract. Vaccination with BCG decreased serum IgE concentration and modified the predominant site of the inflammatory cell response in intestine.
The results indicate that, heat denaturation of β-lg and BCG vaccination, change both the systemic and the mucosal response to bovine milk β-lg. The reasons for this remain speculative. The effect of BCG vaccination is consistent with the hygiene hypothesis. The observed alteration of transport properties could be one mechanism by which heat denaturation modifies the allergenic properties of this protein, but additional studies are necessary to assess whether other mechanisms, such as exposure of new antigenic determinants are also relevant.
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Selective cation-exchange adsorption of the two major whey proteinsEl-Sayed, Mayyada January 2010 (has links)
Whey is a by-product of cheese manufacture, containing a mixture of proteins of commercial value, each having unique attributes for nutritional, biological and food ingredient applications. A tremendous amount of whey, normally treated as a waste product, is produced worldwide each year. This work describes the cation-exchange adsorption of the two major whey proteins, alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) with the purpose of optimising a process for isolating them from whey. Adsorption of pure BLG and ALA was studied onto SP Sepharose FF using 0.1M acetate buffer. Batch experiments were carried out at various pH values for ALA and BLG, and the relevant Langmuir isotherm parameters, dissociation constant, Kd, and maximum binding capacity, qm, were determined. The optimum pH for separation was chosen to be pH 3.7. At pH 3.7, both Kd and qm pertaining to ALA were found to have higher numerical values than those of BLG, implying different characteristics of adsorption of the two proteins on this adsorbent. The Kd for the former protein was almost four times larger than the latter, while qm was 1.3 times higher. Packed-bed column adsorption was performed using a 1-ml column at pH 3.7, flow rate 1 ml/min and initial concentration of 3 mg/ml for BLG and 1.5 mg/ml for ALA both in 0.1M sodium acetate buffer. The t1/2 for the resulting ALA breakthrough was 75% longer than its BLG counterpart. The above results suggest the possibility of the occurrence of competitive adsorption between the proteins when adsorbed simultaneously. In traditional batch uptake experiments, the kinetic rate constants of ALA and BLG in both the single- and two-component systems were determined using the simple kinetic model. The values so obtained implied that BLG was adsorbed faster than ALA. In the confocal laser scanning microscopy experiments, the different behaviour of ALA and BLG in the single-component system with regard to their penetration within the adsorbent beads suggested that the two proteins have different transport mechanisms governing their adsorption. The two-component system results showed that ALA was able to displace BLG in spite of the lower affinity of the former protein to the adsorbent. The packed-bed adsorption and elution of a mixture of ALA and BLG were then investigated under the above conditions but using a 5-ml column. BLG breakthrough occurred first, and its concentration in the outlet exceeded its feed value by 1.6 fold before declining to the feed value, followed by the breakthrough of ALA. ALA displaced and eluted all the BLG from the column in a pure form. Pure ALA could then be eluted with good recovery. The single- and two-component breakthrough curves for ALA and BLG were simulated by the simple kinetic model using the isotherm parameters, but the overshoot phenomenon could only be predicted after correcting these parameters. The evidence of the competitive nature of adsorption observed in binary mixtures was used to develop a facile separation procedure for the two proteins from aqueous solutions of whey concentrate powders. A novel consecutive two-stage separation process was developed to separate ALA and BLG from whey concentrate mixtures. Almost all the BLG in the feed was recovered, with 78% being recovered at 95% purity and a further 20% at 86% purity. In addition, 67% of ALA was recovered, 48% at 54% purity and 19% at 60% purity. The correction factors employed for the pure binary mixture were used to simulate the breakthrough curves of the two proteins in experiments conducted with whey concentrate in each of the two stages of the novel separation process, and there was agreement between the experimental and theoretical results.
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A study of protein aggregation processes using Dynamic Light Scattering : Validation of the technique and experimental trial with an active pharmaceutical ingredientArnroth, Cornelia January 2020 (has links)
Protein pharmaceuticals is one of the fastest growing class of therapeutics today. However, they pose a lot of challenges in production lines due to their poor stability. Protein aggregation is one of the most common results of protein instability and is a risk factor regarding the quality of therapeutics. This master thesis at RISE focused on validating the techniques Dynamic Light Scattering (DLS) and multi angle DLS (MADLS) with respect to detection of aggregation. The model protein B-lactoglobulin was used to assess the robustness and accuracy of DLS. A comparison between two instruments from Malvern, Zetasizer Nano (2006) and Zetasizer Ultra (2018) was done with respect to DLS. It was determined that they were in many ways equivalent, but the newer model Ultra was favourable due to reduced noise and its ability to detect a lower concentration of aggregates. MADLS produced more precise results which is reflected in narrower distributions and has a higher sensitivity than DLS with regards to separating particles near in size. Both techniques proved sensitive enough to differentiate between aggregates and native protein. Experimental trials were performed with an active pharmaceutical ingredient, API. The experimental trials with the API aimed to investigate what conditions and surface-interfaces that might pose a risk for aggregation. Despite efforts put in creating an environment where aggregation could be monitored, aggregation could not be established. Measurements with the API generated less reliable results due to noisy data and a lack of reproducibility between individual measurements.
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Designing Novel Emulsion Performance by Controlled Hetero-Aggregation of Mixed Biopolymer SystemsMao, Yingyi 01 September 2013 (has links)
The increase in obesity and overweight in many countries has led to an upsurge of interest in the development of reduced fat food products. However, the development of these products is challenging because of the many roles that fat droplets normally plays in these food products, including contributing to flavor, texture, appearance, and bioactivity. The goal of this research was to develop novel reduced-fat emulsions based on hetero-aggregation of oppositely charged food-grade colloidal particles or polymers.
Initially, lactoferrin (LF) and β-lactoglobulin (β-Lg) were selected as emulsifiers to form protein-coated fat droplets (d43 ≈ 0.38 μm) with opposite charges at neutral pH: pKaβ-Lg ≈ 5 < pH 7 < pKaLF ≈ 8.5. Droplet aggregation occurred when these two emulsions were mixed together due to electrostatic attraction. The structural organization of the droplets in these mixed emulsions depended on the positive-to-negative particle ratio, particle concentration, pH, ionic strength, and temperature. The nature of the structures formed influenced the rheology, stability, and appearance of the mixed emulsions, which enabled some control over emulsion functionality. The largest microclusters were formed at particle ratios of 40% LF-coated and 60% β-Lg-coated fat droplets, which led to mixed emulsions with the highest apparent viscosity or gel strength. At low total particle concentrations (0.1%), there was a relatively large distance between microclusters and the mixed emulsions were fluid. At high particle concentrations (>20%), a three-dimensional network of aggregated droplets formed that led to gel-like or paste-like properties. The influence of environmental stresses on the physicochemical stability of the microclusters formed by hetero-aggregation was investigated: pH (2-9); ionic strength (0-400 mM NaCl); and temperature (30-90 ºC). Large microclusters were obtained at pH 7 (d43 ≈ 10 μm) with the absence of salt at room temperature. More acidic (< pH 6) or alkaline (> pH 8.5) solutions resulted in smaller aggregates by minimizing the electrostatic attraction between the protein-coated fat droplets. Microclusters dissociated upon addition of intermediate levels of salt, which was attributed to screening of attractive electrostatic interactions. Heating the microclusters above the thermal denaturation temperature of the proteins led to an increase in gel-strength, which was attributed to increased hydrophobic attraction.
The influence of hetero-aggregation of lipid droplets on their potential biological fate was studied using a simulated gastrointestinal tract (GIT). Results showed that the mixed emulsions had high viscosity in the simulated oral environment but exhibited similar rheological properties and particle characteristics as single-protein emulsions in the simulated gastric and small intestinal tract regions. The mixed emulsions also had similar lipid digestion rates in the simulated small intestine as single-protein emulsions suggesting that they could be used as delivery systems for bioactive lipophilic compounds in reduced fat food products.
The possibility of using more practical food ingredients to promote heteroaggregation system was also examined. Whey protein isolate (positive) and modified starch (negative) were selected as building blocks due to their opposite charges at pH 3.5. The largest aggregates and highest viscosities occurred at a particle ratio of 70% MS and 30% WPI, which was attributed to strong electrostatic attraction between the oppositely charged droplets. Particle aggregation and viscosity decreased when the pH was changed to reduce the electrostatic attraction between the droplets.
Finally, the influence of interfacial properties on the chemical stability of bioactive components in emulsion-based delivery systems containing mixed proteins was studied. Lactoferrin (LF: pI ≈ 8) and β-lactoglobulin (β-Lg: pI ≈ 5) were selected to engineer the interfacial properties. Interfaces with different structures were formed: LF only; β-Lg only; LF-β-Lg (laminated); β-Lg -LF (laminated); β-Lg /LF (mixed). The influence of pH, ionic strength, and temperature on the physical stability of β-caroteneenriched emulsions was then investigated. LF- emulsions were stable to the pH change from 2 to 9 but the aggregation was occurred in intermediate pH for other emulsions. β- Lg- emulsions aggregated at low salt concentration (≥ 50mM NaCl), however other emulsions were stable (0 - 300mM NaCl). β-Lg /LF (mixed) emulsions were unstable to heating (≥ 60 ºC), but all other emulsions were stable (30 to 90 ºC). Color fading due to β-carotene degradation occurred relatively quickly in β-Lg-emulsions (37 ºC), but was considerably lower in all other emulsions, which was attributed to the ability of LF to bind iron or interact with β-carotene.
Overall, this study shows that hetero-aggregation may be a viable method of creating novel structures and rheological properties that could be used in the food industry.
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Atividade antioxidante da vanilina e do ácido vanílico e o efeito da complexação por proteínas do soro do leite na desativação de radicais e ferrilmioglobina em condições simulando o trato gastrointestinal / Antioxidant activity of vanillin and vanillic acid and the effect of complexation by milk whey proteins in the deactivation of radicals and ferrylmyoglobin under conditions simulating the gastrointestinal tractLibardi, Silvia Helena 23 July 2010 (has links)
O presente trabalho procurou investigar influência da presença de proteínas do soro do leite na atividade antioxidante da vanilina e ácido vanílico frente ao radical DPPH• e a espécie de ferro hipervalente ferrilmioglobina MbFe(IV)=O em meio simulando o trato gastrointestinal. A constante de associação (KA) entre a vanilina e a β-lactoglobulina (BLG) foi determinada utilizando-se as técnicas de espectroscopia de emissão molecular (KA = 400 ± 12·102 L·mol-1) e microcalorimétria (KA = 5,6±0,3·102 L·mol-1) ambas em tampão fosfato com CH+ = 10-7,4 mol·L-1 e força iônica 0,32 (NaCl). Para a interação entre a vanilina e albumina de soro bovino (BSA) encontrou-se o valor de 340 ± 13·102 L·mol-1 em meio de tampão fosfato com CH+ = 10-6,4 mol·L-1 e força iônica 0,32 (NaCl), obtido por espectroscopia de emissão molecular. Constatou-se pela técnica de microcalorimetria que a complexação possui caráter exotérmico e as contribuições de interações hidrofóbicas para a complexação são fracas. A reatividade da vanilina e ácido vanílico com o radical DPPH• foi investigada em meio de emulsão aquosa Tween-20® com CH+ = 10-2,0 mol·L-1. Os resultados obtidos demonstraram que a vanilina não pode ser considerada um bom antioxidante frente ao DPPH• (k298 = 1,42±0,04·10-1 L·mol-1·s-1), no entanto, o ácido vanílico apresentou maior reatividade frente ao radical DPPH• (k298 = 17,1±0,3 ·10-1 L·mol-1·s-1). A presença das proteínas BLG e BSA nas reações de redução do radical DPPH• pela vanilina e ácido vanílico conduziu a um efeito antagônico na constante de velocidade de reação. Os parâmetros termodinâmicos do estado de transição da reação com DPPH• apresentaram valores relativamente altos de entalpia de ativação e moderados valores de entropia de ativação: ΔH‡298 = 34,0 ± 0,3 kJmol-1 para a vanilina e 46,2 ± 0,1 kJmol-1 no complexo com BSA e 51,0 ± 0,6 kJ·mol-1 no complexo com BLG, valores negativos de entropia ΔS‡298 = -147,4 ± 0,9 J·mol-1·K-1, -105,3 ± 0,5 J·mol-1·K-1 e -90 ± 2 J·mol-1·K-1 respectivamente. Os valores de entalpia e entropia de ativação encontrados para o ácido vanílico foram: ΔH‡298= 19,6 ± 0,2 kJ·mol-1, 10,2 ± 0,03 kJ·mol-1 e 37,6 ± 0,3 kJ·mol-1 para os complexos com BSA e BLG respectivamente e valores negativos de entropia ΔS‡298=-174 ± 0,5 J·mol-1·K-1, -206,0 ± 0,1 J·mol-1·K-1 e -116 ± 1 J·mol-1·K-1. A partir destes valores de entalpia e entropia de ativação o mecanismo de redução do radical DPPH• foi atribuído a um processo de abstração de átomo de hidrogênio (HAT/PCET). A reação de desativação da espécie MbFe(IV)=O pela vanilina apresentou constante de velocidade de k298 = 57±1 L·mol-1·s-1 sendo superior quando comparada ao ácido vanílico k298 = 15±1 L·mol-1·s-1, fato este atribuído as cargas totais, negativa, do redutor e da proteína nas presentes condições experimentais. Observa-se um efeito antagônico da complexão da vanilina pelas proteínas na atividade antioxidante frente à ferrilmioglobina, onde o efeito reduziu, mas não impediu a reação de transferência de elétrons por esfera-externa à longa distância. Em contrapartida, a presença das proteínas BLG e BSA não influenciaram a reatividade do ácido vanílico frente à espécie MbFe(IV)=O. Os parâmetros de ativação encontrados para a reação de redução da MbFe(IV)=O com a vanilina apresentaram valores de ΔH‡298 = 58,8 ± 0,3 kJmol-1 e ΔS‡298 = -14 ± 1 J·mol-1·K-1, ΔH‡298 = 45,5 ± 0,3 kJ·mol-1 e ΔS‡298 = -60 ± 1 J ·mol-1·K-1, ΔH‡298 = 68,6 ± 0,4 kJ·mol-1 e ΔS‡298 = 17 ± 1 J ·mol-1·K-1 para vanilina \"livre\", complexo com BSA, e complexo com BLG respectivamente. Para a redução com ácido vanílico foram determinados os seguintes valores de entalpia e entropia de ativação: ΔH‡298 = 41,8 ± 0,2 kJ·mol-1 e ΔS‡298 = -82,4 ± 0,7 J·mol-1·K-1, ΔH‡298 = 37,7 ± 0,3 kJ·mol-1 e ΔS‡298 = -96 ± 1,0 J·mol-1·K-1, ΔH‡298 = 53,5 ± 0,2 kJ·mol-1 e ΔS‡298 = -44 ± 1,0 J·mol-1·K-1 para vanilina \"livre\", complexo com BSA, e complexo com BLG respectivamente. / The present study evaluate the influence of the presence of whey proteins in the antioxidant activity of vanillin and vanillic acid towards the DPPH• radical species and the hypervalent iron species ferrylmyoglobin, MbFe(IV)=O under conditions simulating the gastrointestinal tract. The association constant (KA) between vanillin and β- lactoglobulin (BLG) was obtained using molecular emission spectroscopy (KA = 400 ± 12·102 L·mol-1) and microcalorimetric titration (KA = 5.6±0.3·102 L·mol-1) both in phosphate buffer CH + = 10-7.4 mol·L -1 and ionic strength 0.32 (NaCl). For the interaction between vanillin and bovine serum albumin (BSA) it was founded value of 340 ± 13·102 L·mol-1 in phosphate buffer with CH+ = 10-6,4 mol·L-1 and ionic strength 0.32 (NaCl), as obtained by molecular emission spectroscopy. It was founded by microcalorimetry tritation that the complexation has a exothermic character and the contributions of hydrophobic interactions for complexation are weak. The reactivity of vanillin and vanillic acid toward DPPH• radical was studied in aqueous emulsion using Tween-20® with CH + = 10-2.0 mol·L-1. The results show that vanillin can not be considered a good antioxidant (k298 = 1.42±0.04·10-1 L·mol-1·s-1), however vanillic acid show higher reactivity than vanillin towards the radical DPPH• (k298 = 17.1±0.3·10-1 L·mol-1·s-1). The presence of the proteins BLG and BSA in the reduction reactions of the DPPH• radical by vanillin and vanillic acid led to an antagonic effect in the reaction rate constant. The thermodynamic parameters for the transition state of the reaction with DPPH• showed relatively high values of enthalpy of activation and moderately negative entropy of activation: ΔH‡298= 34.0 ± 0.3 kJmol-1 for vanillin and 46.2 ± 0.1 kJmol-1 for complex with BSA and 51.0 ± 0.6 kJ·mol-1 for complex with BLG, negatives values of entropy ΔS‡298 = -147.4 ± 0.9 J·mol-1·K-1, -105.3 ± 0.5 J·mol-1·K-1 and -90 ± 2 J·mol-1·K-1 respectively. The values of enthalpy and entropy of activation found for vanillic acid were: ΔH‡298 = 19.6 ± 0.2 kJ·mol-1, 10.2 ± 0.03 kJ·mol-1 and 37.6 ± 0.3 kJ·mol-1 for BSA and BLG respectively and negative values of entropy ΔS‡298 = -174 ± 0.5 J·mol-1·K-1, -206.0 ± 0.1 J·mol-1·K-1 and -116 ± 1 J·mol-1·K-1. From these values of enthalpy and entropy of activation the mechanism of radical DPPH• reduction was assigned to a process of hydrogen atom transfer (HAT/PCET). The deactivation reaction of the MbFe(IV)=O species by vanillin shown rate constant of k298 = 57±1 L·mol-1·s-1, which it is higher than vanillic acid k298 = 15±1 L·mol-1·s-1. This fact is assigned to the total negative charges of the reductor and the protein under the experimental conditions. It is observed an antagonistic effect of the complexation of vanillin by proteins in the antioxidant activity, in which the effect diminish, but not avoid the long range electron transfer by out-sphere reaction. On the other hand, the presence of BLG and BSA do not affect the reactivity of vanillic acid towards the MbFe(IV)=O species. The activation parameters found for the reduction of MbFe(IV)=O by vanillin revealed values of ΔH‡298 = 58.8 ± 0.3 kJmol-1 and ΔS‡298 = -14 ± 1 J·mol-1·K-1, ΔH‡298 = 45.5 ± 0.3 kJ·mol-1 e ΔS‡298 = -60 ± 1 J ·mol-1·K-1, ΔH‡298 = 68.6 ± 0.4 kJ·mol-1 and ΔS‡298 = 17 ± 1 J ·mol-1·K-1 for free vanillin, complex with BSA and complex with BLG respectively. For the reduction by vanillic acid it were with the following values of enthalpy and entropy of activation: ΔH‡298 = 41.8 ± 0.2 kJ·mol-1 and ΔS‡298 = -82.4 ± 0.7 J·mol-1·K-1, ΔH‡298 = 37.7 ± 0.3 kJ·mol-1 and ΔS‡298 = -96 ± 1.0 J·mol-1·K-1, ΔH‡298 = 53.5 ± 0.2 kJ·mol-1 and ΔS‡298 = -44 ± 1.0 J·mol-1·K-1 for free vanillin, complex with BSA and complex with BLG respectively.
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Variantes genéticas de beta-lactoglobulina em vacas leiteiras e características físico-químicas e de composição do leite / Beta-lactoglobulina polymorphism in dairy cows and milk composition and physico-chemical characteristicsBotaro, Bruno Garcia 09 February 2007 (has links)
O presente estudo teve como objetivo avaliar a associação entre o polimorfismo da β-lactoglobulina e as características físico-químicas (pH, acidez e crioscopia), de composição (gordura, sólidos totais, uréia, proteína bruta, proteína verdadeira, nitrogênio não-protéico e caseína), e de estabilidade do leite. Para tanto, 11 rebanhos leiteiros foram selecionados, 5 da raça Holandesa e 6 da raça Girolanda, dos quais foram coletadas 4 amostras de leite de 164 vacas da raça Holandesa e 74 da raça Girolanda, sendo duas coletas realizadas na estação das secas e 2 na estação das chuvas. Cada amostra foi submetida à análise de composição e de características físico-químicas. Para a identificação do genótipo para β-lactoglobulina, foram coletadas amostras de sangue de cada vaca, as quais foram submetidas à reação de polimerase em cadeia (PCR), determinando-se as freqüências alélicas e genotípicas dos animais. A estabilidade do leite foi avaliada pelo teste de estabilidade ao etanol, nas seguintes concentrações alcoólicas: 70, 76, 80 e 84ºGL. As freqüências genotípicas foram 0,28, 0,30 e 0,41 para os genótipos AA, AB e BB, respectivamente. A freqüência do alelo B foi maior que do alelo A, 0,52 e 0,47, para a raça Holandesa, e 0,58 e 0,41, para a raça Girolanda, respectivamente. Não houve efeito do polimorfismo da β-lactoglobulina (AA, AB e BB), entre os animais das raças, avaliadas sobre as propriedades físico-químicas e a composição do leite. Observou-se efeito de raça (Holandesa e Girolanda, respectivamente) sobre a acidez titulável (16,16 e 17,07°D) e pH (6,78 e 6,75), e de composição do leite quanto as variáveis gordura (3,31 e 3,20%), NUL (16,62 e 14,45mg/dL) e PB (3,13 e 3,04%). Houve efeito da estação (chuvosa e seca, respectivamente) sobre as características físico-químicas de acidez titulável (16,62 e 16,34°D), pH (6,76 e 6,79) e crioscopia (-0,5411 e -0,5376°H), e de composição do leite quanto as variáveis lactose (4,34 e 4,50%), sólidos totais (11,65 e 11,90%), LogCCS (2,44 e 2,34), PB (3,08 e 3,14%), PV (2,84 e 2,91%), caseína (2,01 e 2,13%) e relação caseína:proteína verdadeira (0,70 e 0,72). Verificou-se também efeito da raça e estação do ano sobre a estabilidade do leite, sendo que o leite foi mais instável para raça Girolanda e durante a estação seca, mas não se observou efeito do polimorfismo da β-lactoglobulina sobre esta característica. / The objective of this study was to evaluate the association between beta-lactogobulin polymorphism and physico-chemical characteristics, composition (fat, total solids, urea, crude protein, true protein, non protein nitrogen and casein), and stability of milk. For this aim, 11 dairy herds were selected, six of them composed of crossbred Holstein-Zebu (H-Z) cows and five from Holstein cows. Milk samples were taken four times (twice in dry season and twice in rainy season), from 278 Holstein and 156 crossbred Holstein-Zebu cows. Individual milk samples were analyzed for milk composition and physico-chemical properties. For β-lactoglobulin polymorphism analysis, 10 mL of blood samples were ollected from each cow and then submitted to polymerase chain reaction (PCR). Following β-lactoglobulin protein variants detection, genotype and allele frequencies for the 11 herds were analyzed. Heat stability of milk was determined by the alcohol-induced precipitation test, using the following ethanol concentrations 70, 76, 80 and 84ºGL. The genotype frequencies were 0.28, 0.30 and 0.41 for AA, AB and BB, respectively. Allele B frequency was higher than A, 0.52 and 0.47, for Holstein cows, 0.58 and 0.41, for Holstein-Zebu, respectively. Genetic variants of β-lactoglobulin (AA, AB and BB) had no effect on physico-chemical (acidity, pH and crioscopy), and compositional characteristics (fat, total solids, urea, crude protein, non-protein nitrogen, true protein and casein percentages), either among milk from Holstein cows, or from crossbred Holstein-Zebu. Breed effect for Holstein and H-Z on titrable acidity (16,16 and 17,07°D, respectively), pH (6,78 and 6,75, respectively), fat (3,31e 3,20%, respectively), milk urea nitrogen (16,62 e 14,45mg/dL, respectively) and crude protein (3,13 e 3,04%, respectively) could be observed. Effect of seasonality between rainy and dry seasons was also observed on physico-chemical variables of titrable acidity (16,62 and 16,34°D, respectively), pH (6,76 and 6,79, respectively) and freezing point (-0,5411and -0,5376°H, respectively), and on composition characteristics of lactose (4,34 and 4,50%, respectively), total solids (11,65 and 11,90%, respectively), LogCCS (2,44 and 2,34, respectively), crude protein (3,08 and 3,14%, respectively), true protein (2,84 and 2,91%, respectively), casein content (2,01 and 2,13%, respectively) and casein:true protein ratio (0,70 and 0,72, respectively). Effect of breed and seasonality on milk ethanol stability test was observed. Holstein-Zebu milk was ethanol-unstable on dry season. No effect of β-lactoglobulin on milk stability was observed.
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Kvalitativní ukazatele mléka původní valašky / Qualitative indexes of milk of ewes the Original ValachianPEŠINOVÁ, Petra January 2010 (has links)
The aim of study was to evaluate milk efficiency of sheep Original Valachian (OV). Observation took place in the period 2006-2009 and were involved 123 ovine. Samples were taken from the morning milking during the months April to August (method ET). Milking proces was realised mechanically. In 84 sheep were known genotypes AA (n = 13), AB (n = 18), BB (n = 53). After evaluation of essential components of ewe{\crq}s milk from flock of lambing ewes OV during lactation were found these average values: Fat (F) 4,90 g.100g-1, crude protein (CP) 5,94 g.100g-1, casein (CAS) 4,40 g.100g-1, serum protein (SP) 1,18 g.100g-1, lactose (L) 5,07 g.100g-1, dry matter (DM) 16,45 g.100g-1, solid not fat (SNF) 11,63 g.100g-1 and utilizable dry matter (UDM) 10,85 g.100g-1. Average daily milk yield of OV was 0,70 l. Effect of stage of lactation was provable on all the indicators in the level of significance 0,001. It was evidenced a statistically significant effect of the control year on the content of SNF (P{<}0,05), on the L (P{<}0,01) and on all other components (P{<}0,001). At comparing milk production OV according to the genetic polymorphism of {$\beta$}-lactoglobulin have been identified probably significant differences in milk yield (P{<}0,05). The highest daily milk yield reached genotype AB (0,76 l.day-1) {>} BB (0,68 l.day-1) {>} AA (0,66 l.day-1). In AB genotype was found the lowest levels of these essential components F, CP, CAS, DM, SNF and UDM. Highly significant effect of genotype was found on content of L (P{<}0,001). The highest content of L was confirmed by genotype BB (5,13 g.100g-1) {>} AB (5,08 g.100g-1) {>} AA (4,91 g.100g-1). Less significant effect was found on SNF (P{<}0,1). The highest average content of SNF was found in genotype BB (11,72 g.100g-1) {>} AA (11,62 g.100g-1) {>} AB (11,54 g.100g-1). In genotype AA was found the highest values of F, CP, CAS, SP, DM. Both genotypes AA, BB showed the same content of DM (16,32 g.100g-1).
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Atividade antioxidante da vanilina e do ácido vanílico e o efeito da complexação por proteínas do soro do leite na desativação de radicais e ferrilmioglobina em condições simulando o trato gastrointestinal / Antioxidant activity of vanillin and vanillic acid and the effect of complexation by milk whey proteins in the deactivation of radicals and ferrylmyoglobin under conditions simulating the gastrointestinal tractSilvia Helena Libardi 23 July 2010 (has links)
O presente trabalho procurou investigar influência da presença de proteínas do soro do leite na atividade antioxidante da vanilina e ácido vanílico frente ao radical DPPH• e a espécie de ferro hipervalente ferrilmioglobina MbFe(IV)=O em meio simulando o trato gastrointestinal. A constante de associação (KA) entre a vanilina e a β-lactoglobulina (BLG) foi determinada utilizando-se as técnicas de espectroscopia de emissão molecular (KA = 400 ± 12·102 L·mol-1) e microcalorimétria (KA = 5,6±0,3·102 L·mol-1) ambas em tampão fosfato com CH+ = 10-7,4 mol·L-1 e força iônica 0,32 (NaCl). Para a interação entre a vanilina e albumina de soro bovino (BSA) encontrou-se o valor de 340 ± 13·102 L·mol-1 em meio de tampão fosfato com CH+ = 10-6,4 mol·L-1 e força iônica 0,32 (NaCl), obtido por espectroscopia de emissão molecular. Constatou-se pela técnica de microcalorimetria que a complexação possui caráter exotérmico e as contribuições de interações hidrofóbicas para a complexação são fracas. A reatividade da vanilina e ácido vanílico com o radical DPPH• foi investigada em meio de emulsão aquosa Tween-20® com CH+ = 10-2,0 mol·L-1. Os resultados obtidos demonstraram que a vanilina não pode ser considerada um bom antioxidante frente ao DPPH• (k298 = 1,42±0,04·10-1 L·mol-1·s-1), no entanto, o ácido vanílico apresentou maior reatividade frente ao radical DPPH• (k298 = 17,1±0,3 ·10-1 L·mol-1·s-1). A presença das proteínas BLG e BSA nas reações de redução do radical DPPH• pela vanilina e ácido vanílico conduziu a um efeito antagônico na constante de velocidade de reação. Os parâmetros termodinâmicos do estado de transição da reação com DPPH• apresentaram valores relativamente altos de entalpia de ativação e moderados valores de entropia de ativação: ΔH‡298 = 34,0 ± 0,3 kJmol-1 para a vanilina e 46,2 ± 0,1 kJmol-1 no complexo com BSA e 51,0 ± 0,6 kJ·mol-1 no complexo com BLG, valores negativos de entropia ΔS‡298 = -147,4 ± 0,9 J·mol-1·K-1, -105,3 ± 0,5 J·mol-1·K-1 e -90 ± 2 J·mol-1·K-1 respectivamente. Os valores de entalpia e entropia de ativação encontrados para o ácido vanílico foram: ΔH‡298= 19,6 ± 0,2 kJ·mol-1, 10,2 ± 0,03 kJ·mol-1 e 37,6 ± 0,3 kJ·mol-1 para os complexos com BSA e BLG respectivamente e valores negativos de entropia ΔS‡298=-174 ± 0,5 J·mol-1·K-1, -206,0 ± 0,1 J·mol-1·K-1 e -116 ± 1 J·mol-1·K-1. A partir destes valores de entalpia e entropia de ativação o mecanismo de redução do radical DPPH• foi atribuído a um processo de abstração de átomo de hidrogênio (HAT/PCET). A reação de desativação da espécie MbFe(IV)=O pela vanilina apresentou constante de velocidade de k298 = 57±1 L·mol-1·s-1 sendo superior quando comparada ao ácido vanílico k298 = 15±1 L·mol-1·s-1, fato este atribuído as cargas totais, negativa, do redutor e da proteína nas presentes condições experimentais. Observa-se um efeito antagônico da complexão da vanilina pelas proteínas na atividade antioxidante frente à ferrilmioglobina, onde o efeito reduziu, mas não impediu a reação de transferência de elétrons por esfera-externa à longa distância. Em contrapartida, a presença das proteínas BLG e BSA não influenciaram a reatividade do ácido vanílico frente à espécie MbFe(IV)=O. Os parâmetros de ativação encontrados para a reação de redução da MbFe(IV)=O com a vanilina apresentaram valores de ΔH‡298 = 58,8 ± 0,3 kJmol-1 e ΔS‡298 = -14 ± 1 J·mol-1·K-1, ΔH‡298 = 45,5 ± 0,3 kJ·mol-1 e ΔS‡298 = -60 ± 1 J ·mol-1·K-1, ΔH‡298 = 68,6 ± 0,4 kJ·mol-1 e ΔS‡298 = 17 ± 1 J ·mol-1·K-1 para vanilina \"livre\", complexo com BSA, e complexo com BLG respectivamente. Para a redução com ácido vanílico foram determinados os seguintes valores de entalpia e entropia de ativação: ΔH‡298 = 41,8 ± 0,2 kJ·mol-1 e ΔS‡298 = -82,4 ± 0,7 J·mol-1·K-1, ΔH‡298 = 37,7 ± 0,3 kJ·mol-1 e ΔS‡298 = -96 ± 1,0 J·mol-1·K-1, ΔH‡298 = 53,5 ± 0,2 kJ·mol-1 e ΔS‡298 = -44 ± 1,0 J·mol-1·K-1 para vanilina \"livre\", complexo com BSA, e complexo com BLG respectivamente. / The present study evaluate the influence of the presence of whey proteins in the antioxidant activity of vanillin and vanillic acid towards the DPPH• radical species and the hypervalent iron species ferrylmyoglobin, MbFe(IV)=O under conditions simulating the gastrointestinal tract. The association constant (KA) between vanillin and β- lactoglobulin (BLG) was obtained using molecular emission spectroscopy (KA = 400 ± 12·102 L·mol-1) and microcalorimetric titration (KA = 5.6±0.3·102 L·mol-1) both in phosphate buffer CH + = 10-7.4 mol·L -1 and ionic strength 0.32 (NaCl). For the interaction between vanillin and bovine serum albumin (BSA) it was founded value of 340 ± 13·102 L·mol-1 in phosphate buffer with CH+ = 10-6,4 mol·L-1 and ionic strength 0.32 (NaCl), as obtained by molecular emission spectroscopy. It was founded by microcalorimetry tritation that the complexation has a exothermic character and the contributions of hydrophobic interactions for complexation are weak. The reactivity of vanillin and vanillic acid toward DPPH• radical was studied in aqueous emulsion using Tween-20® with CH + = 10-2.0 mol·L-1. The results show that vanillin can not be considered a good antioxidant (k298 = 1.42±0.04·10-1 L·mol-1·s-1), however vanillic acid show higher reactivity than vanillin towards the radical DPPH• (k298 = 17.1±0.3·10-1 L·mol-1·s-1). The presence of the proteins BLG and BSA in the reduction reactions of the DPPH• radical by vanillin and vanillic acid led to an antagonic effect in the reaction rate constant. The thermodynamic parameters for the transition state of the reaction with DPPH• showed relatively high values of enthalpy of activation and moderately negative entropy of activation: ΔH‡298= 34.0 ± 0.3 kJmol-1 for vanillin and 46.2 ± 0.1 kJmol-1 for complex with BSA and 51.0 ± 0.6 kJ·mol-1 for complex with BLG, negatives values of entropy ΔS‡298 = -147.4 ± 0.9 J·mol-1·K-1, -105.3 ± 0.5 J·mol-1·K-1 and -90 ± 2 J·mol-1·K-1 respectively. The values of enthalpy and entropy of activation found for vanillic acid were: ΔH‡298 = 19.6 ± 0.2 kJ·mol-1, 10.2 ± 0.03 kJ·mol-1 and 37.6 ± 0.3 kJ·mol-1 for BSA and BLG respectively and negative values of entropy ΔS‡298 = -174 ± 0.5 J·mol-1·K-1, -206.0 ± 0.1 J·mol-1·K-1 and -116 ± 1 J·mol-1·K-1. From these values of enthalpy and entropy of activation the mechanism of radical DPPH• reduction was assigned to a process of hydrogen atom transfer (HAT/PCET). The deactivation reaction of the MbFe(IV)=O species by vanillin shown rate constant of k298 = 57±1 L·mol-1·s-1, which it is higher than vanillic acid k298 = 15±1 L·mol-1·s-1. This fact is assigned to the total negative charges of the reductor and the protein under the experimental conditions. It is observed an antagonistic effect of the complexation of vanillin by proteins in the antioxidant activity, in which the effect diminish, but not avoid the long range electron transfer by out-sphere reaction. On the other hand, the presence of BLG and BSA do not affect the reactivity of vanillic acid towards the MbFe(IV)=O species. The activation parameters found for the reduction of MbFe(IV)=O by vanillin revealed values of ΔH‡298 = 58.8 ± 0.3 kJmol-1 and ΔS‡298 = -14 ± 1 J·mol-1·K-1, ΔH‡298 = 45.5 ± 0.3 kJ·mol-1 e ΔS‡298 = -60 ± 1 J ·mol-1·K-1, ΔH‡298 = 68.6 ± 0.4 kJ·mol-1 and ΔS‡298 = 17 ± 1 J ·mol-1·K-1 for free vanillin, complex with BSA and complex with BLG respectively. For the reduction by vanillic acid it were with the following values of enthalpy and entropy of activation: ΔH‡298 = 41.8 ± 0.2 kJ·mol-1 and ΔS‡298 = -82.4 ± 0.7 J·mol-1·K-1, ΔH‡298 = 37.7 ± 0.3 kJ·mol-1 and ΔS‡298 = -96 ± 1.0 J·mol-1·K-1, ΔH‡298 = 53.5 ± 0.2 kJ·mol-1 and ΔS‡298 = -44 ± 1.0 J·mol-1·K-1 for free vanillin, complex with BSA and complex with BLG respectively.
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Variantes genéticas de beta-lactoglobulina em vacas leiteiras e características físico-químicas e de composição do leite / Beta-lactoglobulina polymorphism in dairy cows and milk composition and physico-chemical characteristicsBruno Garcia Botaro 09 February 2007 (has links)
O presente estudo teve como objetivo avaliar a associação entre o polimorfismo da β-lactoglobulina e as características físico-químicas (pH, acidez e crioscopia), de composição (gordura, sólidos totais, uréia, proteína bruta, proteína verdadeira, nitrogênio não-protéico e caseína), e de estabilidade do leite. Para tanto, 11 rebanhos leiteiros foram selecionados, 5 da raça Holandesa e 6 da raça Girolanda, dos quais foram coletadas 4 amostras de leite de 164 vacas da raça Holandesa e 74 da raça Girolanda, sendo duas coletas realizadas na estação das secas e 2 na estação das chuvas. Cada amostra foi submetida à análise de composição e de características físico-químicas. Para a identificação do genótipo para β-lactoglobulina, foram coletadas amostras de sangue de cada vaca, as quais foram submetidas à reação de polimerase em cadeia (PCR), determinando-se as freqüências alélicas e genotípicas dos animais. A estabilidade do leite foi avaliada pelo teste de estabilidade ao etanol, nas seguintes concentrações alcoólicas: 70, 76, 80 e 84ºGL. As freqüências genotípicas foram 0,28, 0,30 e 0,41 para os genótipos AA, AB e BB, respectivamente. A freqüência do alelo B foi maior que do alelo A, 0,52 e 0,47, para a raça Holandesa, e 0,58 e 0,41, para a raça Girolanda, respectivamente. Não houve efeito do polimorfismo da β-lactoglobulina (AA, AB e BB), entre os animais das raças, avaliadas sobre as propriedades físico-químicas e a composição do leite. Observou-se efeito de raça (Holandesa e Girolanda, respectivamente) sobre a acidez titulável (16,16 e 17,07°D) e pH (6,78 e 6,75), e de composição do leite quanto as variáveis gordura (3,31 e 3,20%), NUL (16,62 e 14,45mg/dL) e PB (3,13 e 3,04%). Houve efeito da estação (chuvosa e seca, respectivamente) sobre as características físico-químicas de acidez titulável (16,62 e 16,34°D), pH (6,76 e 6,79) e crioscopia (-0,5411 e -0,5376°H), e de composição do leite quanto as variáveis lactose (4,34 e 4,50%), sólidos totais (11,65 e 11,90%), LogCCS (2,44 e 2,34), PB (3,08 e 3,14%), PV (2,84 e 2,91%), caseína (2,01 e 2,13%) e relação caseína:proteína verdadeira (0,70 e 0,72). Verificou-se também efeito da raça e estação do ano sobre a estabilidade do leite, sendo que o leite foi mais instável para raça Girolanda e durante a estação seca, mas não se observou efeito do polimorfismo da β-lactoglobulina sobre esta característica. / The objective of this study was to evaluate the association between beta-lactogobulin polymorphism and physico-chemical characteristics, composition (fat, total solids, urea, crude protein, true protein, non protein nitrogen and casein), and stability of milk. For this aim, 11 dairy herds were selected, six of them composed of crossbred Holstein-Zebu (H-Z) cows and five from Holstein cows. Milk samples were taken four times (twice in dry season and twice in rainy season), from 278 Holstein and 156 crossbred Holstein-Zebu cows. Individual milk samples were analyzed for milk composition and physico-chemical properties. For β-lactoglobulin polymorphism analysis, 10 mL of blood samples were ollected from each cow and then submitted to polymerase chain reaction (PCR). Following β-lactoglobulin protein variants detection, genotype and allele frequencies for the 11 herds were analyzed. Heat stability of milk was determined by the alcohol-induced precipitation test, using the following ethanol concentrations 70, 76, 80 and 84ºGL. The genotype frequencies were 0.28, 0.30 and 0.41 for AA, AB and BB, respectively. Allele B frequency was higher than A, 0.52 and 0.47, for Holstein cows, 0.58 and 0.41, for Holstein-Zebu, respectively. Genetic variants of β-lactoglobulin (AA, AB and BB) had no effect on physico-chemical (acidity, pH and crioscopy), and compositional characteristics (fat, total solids, urea, crude protein, non-protein nitrogen, true protein and casein percentages), either among milk from Holstein cows, or from crossbred Holstein-Zebu. Breed effect for Holstein and H-Z on titrable acidity (16,16 and 17,07°D, respectively), pH (6,78 and 6,75, respectively), fat (3,31e 3,20%, respectively), milk urea nitrogen (16,62 e 14,45mg/dL, respectively) and crude protein (3,13 e 3,04%, respectively) could be observed. Effect of seasonality between rainy and dry seasons was also observed on physico-chemical variables of titrable acidity (16,62 and 16,34°D, respectively), pH (6,76 and 6,79, respectively) and freezing point (-0,5411and -0,5376°H, respectively), and on composition characteristics of lactose (4,34 and 4,50%, respectively), total solids (11,65 and 11,90%, respectively), LogCCS (2,44 and 2,34, respectively), crude protein (3,08 and 3,14%, respectively), true protein (2,84 and 2,91%, respectively), casein content (2,01 and 2,13%, respectively) and casein:true protein ratio (0,70 and 0,72, respectively). Effect of breed and seasonality on milk ethanol stability test was observed. Holstein-Zebu milk was ethanol-unstable on dry season. No effect of β-lactoglobulin on milk stability was observed.
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