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At present in the Russian Federation fortified food products have gained extensive promotion as the newest and promising direction in the food industry. Almost every dairy processing enterprise has a line of fortified products in its product range. Vitamins, antioxidants, vitamin-like substances, flavoring components, micro and macro elements, phospholipids, prebiotics, probiotics, essential amino acids, polyunsaturated fatty acids can be used as enriching ingredients. On the other hand, the problem of targeted and economically profitable use of secondary milk processing resources is a significant problem. Production of cottage cheese, cheese and casein produces a liquid by-product, which is one of the largest additional sources of dietary protein and lactose as an important source of carbohydrates. The aim of the research is to develop whey fermented milk drink. The objectives of the research are to determine the effect of Jerusalem artichoke syrup on the organoleptic characteristics of the developed fermented milk drink; to optimize empirically the recipe composition of the developed fermented milk drink; to select experimentally the species composition of the starter micro flora of the developed fermented milk drink. The objects of the research are milk serum, Jerusalem artichoke syrup, ground ginger and cinnamon, starter culture on pure cultures of lactic acid microorganisms consisting of Str. thermophilus strains, viscous strains and Lbm. Bulgaricus and B. bifidum 791 in a ratio of 2:1:1. It can be concluded that the use of flavored fillers in the technology of whey fermented milk drink makes it possible to obtain a new product with competitive characteristics.

The objective of this research is to determine whether intramammary antibiotics with complementary acupuncture can reduce bovine mammary inflammation due to subclinical mastitis. Lactating cows were selected based on milk with a somatic cell count (SCC) greater than 500,000 cells/ml. Pre- and post-treatment milk samples were collected to determine SCC, aerobic bacterial content, milk ion conductivity, total protein, lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAGase) concentrations. Milk serum was prepared from milk samples by double centrifugation. Concentrations of LDH and NAGase were determined using commercial enzyme-linked immunosorbent assays. Cows being treated with intramammary antibiotics were separated by random assignment to the acupuncture group (n = 10) and a no-acupuncture (control) group (n = 9). Both the acupuncture and control group were restrained for 30 min in a head catch 12 hr apart for a total of four times. For front quarters affected by subclinical mastitis, the acupuncture points used were spleen (SP) 12, SP 17, SP 18, SP 21, stomach (ST) 18 and conception vessel (CV) 12. For rear quarters affected by subclinical mastitis, the acupuncture points used were bladder (BL) 30, BL 30-1, BL 49, kidney (KI) 10, conception vessel (CV) 2 and CV 3. All parameters were compared using a Student t test. Significance was defined as p < .05. Compared to control cows, complementary acupuncture treatment reduced NAGase enzymatic activity in quarters of cows with subclinical mastitis. The reduction in NAGase suggests that complementary acupuncture treatment may be associated with healing of the damaged mammary epithelial cells, which are the primary source of NAGase activity in milk serum.

During whey powder production, the feed is subjected to several heat treatments which can cause lactosylation of proteins. In this study, lactosylation of whey proteins was evaluated in spray-dried powders before and after storage by varying the native protein fraction as well as the serum protein/lactose ratio in the powders. The lactosylation of native α-lactalbumin and β-lactoglobulin in the powders before storage was not affected to a large extent by the protein denaturation or if the feed had been heat treated in a high or low lactose environment. After storage (relative humidity of 23.5%, 30 °C, 25 days), the kinetic of lactosylation tended to increase with increasing native protein fraction and bulk protein content in the powders. An explanation could be that proteins dissolved in the lactose glassy structure might have a lower reactivity, while proteins present in the protein glassy structure with dissolved lactose may display higher lactosylation reactivity. (Less)

Orientador: Cesar Costapinto Santana Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica Resumo: O crescente interesse e aplicações dos produtos biotecnológicos vem aumentando o desenvolvimento de novos processos de recuperação e purificação de proteínas. O soro de leite bovino, obtido da manufatura da caseína para a produção de queijo, é em sua maioria descartado em mananciais de água, causando sérios problemas ambientais devido a sua alta demanda biológica de oxigênio (DBO). As proteínas presentes no lactosoro apresentam um excelente valor nutritivo e farmacológico, porém, o seu uso no enriquecimento de produtos alimentícios é limitado devido a baixa concentração destas proteínas. As principais proteínas do lactosoro são: ~-Iactoglobulinas, a-Iactalbumina, albuminas de soro bovino, imunoglobulinas, lactoperoxidase, lactoferrina, lisozima e outras proteínas de menor proporção, que apresentam um alto valor agregado. A a-Iactalbumina atua no organismo estimulando os agentes do sistema imunológico por proporcionar a elevação de glutationa em vários órgãos e no sangue, resultando em benefícios para pacientes portadores de doenças degenerativas como os males de Parkinson e Alzheimer, câncer e AIDS. Neste trabalho foi estudado o processo de separação da a-Iactalbumina, através de técnicas cromatográficas empregando a metodologia de leito fixo e expandido. O leito expandido possibilita a redução nos custos do processo de purificação, eliminando etapas de separação necessárias quando o extrato apresenta material em suspensão, que é o caso dos lactosoros. Nos ensaios realizados foram estudados as melhores condições de adsorção da a-Iactalbumina visando a sua purificação empregando adsorventes de troca iônica e de interação hidrofóbica. Também foram realizados ensaios em sistemas de tanque agitados para a determinação das isotermas e cinéticas de adsorção. Neste trabalho obteve-se a a-Iactalbumina com uma pureza acima de 80% e apresentando um fator de purificação de 5 vezes utilizando as resinas de interação hidrofóbica com única etapa de purificação Abstract: The interest and applications of biotechnology products has been increasing the development of new recovery and purification processes for proteins. The bovine milk serum, obtained from casein manufacture for cheese production, is mostly rejected into watercourse, causing problems to the environment due to its high biological oxygen demand (BOD). The proteins of milk serum have excellent nutritious and pharmaceutical value, h oweve r, its application for protein enrichment of food products is limited due to its low content in the milk serum. The main proteins of milk serum are: ~-Iactoglobulins, a-Iactalbumine, bovine serum albumine, immunoglobulins, lactoperoxidase, lactoferrin, lisozime and other lower content proteins which have a high aggregate value. The a-Iactalbumine acts in the human organism by stimulating the agents of the immunologycal system due to increasing on glutathione levei in several organs and blood, resulting in benefits for patients of some diseases like Parkinson and Alzheimer's iII, cancer and AIDS. It was studied in this work the separation processes of a-Iactalbumine, by chromatographic techniques making use of fixed and expanded bed methods. The expanded bed enables cost reduction on purification process by reducing separation steps used for removing suspended solids, as in case of milk serum extracts. In our experiments were studied the adsorption conditions of alactalbumine aiming at its purification by using ionic exchange and hydrophobic interaction adsorbents. Other experiments were accomplished at stirred tank systems for the determination of isotherms and adsorption kinetics. It was obtained, in this work, an a-Iactalbumine purity higher than 80%, with a five fold purification factor by using the hydrophobic interaction resins in a single purification ste Mestrado Desenvolvimento de Processos Biotecnológicos Mestre em Engenharia Química

O presente trabalho teve como objetivo desenvolver uma bebida lactea fermentada desenvolvida com soro de leite, extrato de erva-mate e estevia e avaliar as caracteristicas fisico-ligadas, microbiologicas e sensoriais do produto. Primeiramente, nas formulacoes da bebida, variou-se a concentracao de soro / leite (25/75; 50/50 e 75/25) e de extrato de erva-mate (0,1; 0,25 e 0,35%, m / v) e mantendo-se fixas como regras de stevia (0,75%, m / v), fermento lacteo (3,5%, m / v), sorbato de potassio (0,1%, m / v) e cacau em po (1%, m / v). As formulacoes foram caracterizadas fisico-quimicamente quanto ao pH, acidez, lactose, proteina, gordura, minerais totais, cor (L, a * eb *), sinerese, microbiologica e aceitabilidade. Os resultados das analises fisico-aglomerados demostram uma variacao entre as devedor, a qual pode estar relacionada com a quantidade de soro / leite, extrato de erva-mate e stevia adicionadas. A partir dos resultados da aceitabilidade das 7 formulacoes, realizou-se novos ensaios com a combinacao fixa de soro / leite (50/50), extrato de erva-mate (0,25%, m / v) fermento lacteo (3,5 %, m / v), sorbato de potassio (0,1%, m / v) e cacau em po (1%, m / v) e variacao na concentracao de estevia (0,625; 0,750; 0,875%, p / v) , realizando-se novamente analises fisicas-compostas, microbiologicas e sensorial. A composto com 50/50% de soro / leite, 0,25% de extrato de erva-mate, 3,5% de fermento lacteo, 0,1% de sorbato de potassio, 1% de cacau em po, 0,25 % de extrato de erva-mate e 0,650% de estevia apresentada um indice de aceitacao de 81,57%. Este indice de aceitacao demostra que uma bebida lactea fermentada carregada com adicao de soro de leite

Volatile sulfur compounds in ultra-pasteurized (UP) milk are the major contributors to sulfur/burnt and eggy flavors, and these flavors are disliked by consumers. Previous research has established distinct differences in flavor profiles of fluid milk processed by high temperature, short time pasteurization (HTST) and UP by direct steam injection (DSI-UP) or indirect heat (IND-UP). An understanding of the contribution of the individual milk proteins to sulfur off-flavors would clarify the source of sulfur flavors in UP milks. The objective of this study was to determine the source of volatile sulfur compounds in fluid milk with a specific focus on the comparison of heat treatment effects on milks by HTST and UP. Formulated skim milks (FSM) were manufactured by blending micellar casein concentrate and serum protein isolate (SPI). Three different caseins as a percentage of true protein (FSM95, FSM80, and FSM60) were formulated to determine the source of sulfur/burnt and eggy flavors. Freshly processed micellar casein concentrate or SPI were blended to achieve a true protein content of about 3.2%. Raw skim milk served as a control. Skim milk and FSM were pasteurized at 78°C for 15 s (HTST) or 140°C for 2.3 s by IND-UP or DSI-UP. The experiment was replicated twice. Sensory properties of milks and FSM were documented by descriptive sensory analysis. Volatile sulfur compounds in milks and FSM were evaluated using solid-phase microextraction followed by gas chromatography-triple quadrupole mass spectrometry combined with a sulfur selective flame photometric detector. The FSM with higher SPI as a percent of true protein had higher sensory sulfur/burnt and eggy flavors along with elevated concentrations of hydrogen sulfide and carbon disulfide compared with skim milk or FSM with lower proportions of SPI. Sulfur compounds including dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, dimethyl sulfoxide, and methional were not associated with sulfur/burnt and eggy flavors, which suggests that these compounds may not specifically contribute to the sulfur/burnt and eggy off-flavors of UP milks. Sensory panelists found higher overall aroma impact, and cooked, sulfur/burnt, and eggy flavors for DSI-UP, followed by IND-UP and HTST. The combination of sensory and instrumental methods used in the current study effectively identified that milk serum proteins are the source of sulfur compounds in milk, and further confirmed the contribution of hydrogen sulfide and carbon disulfide to eggy and sulfur/burnt flavors, respectively.

Horns are living tissue and cows can use their horns for thermoregulatory purposes. We investigated the effect of the presence of horns on the metabolome of milk serum and lipidome of milk fat, to assess the physiological effect of dehorning. Milk sampling took place at low ambient temperatures of −6 to 2°C. Horned and dehorned cows were kept in a mixed herd of Holstein Friesian and Brown Swiss cows. The hypothesis was that horned cows needed to increase their metabolism to compensate for additional heat loss through the presence of their horns. No differences were observed in milk yield, milk solids, and somatic cell counts between horned and dehorned cows. For the milk metabolome, horned cows showed an upregulation of several glucogenic AA that could be transformed into glucose for energy supply and a downregulation of sugar intermediates and γ-glutamylcysteine compared with dehorned cows. The fatty acid (FA) composition in horned cows showed a shift toward decreased odd medium-chain FA (C7:0, C9:0, and C11:0) and increased cis-vaccenic acid (C18:1n-7 cis-11) and stearidonic acid (C18:4n-3). The changes in milk composition related to additional heat loss in horned cows indicate a competition in C3 metabolism for glucose synthesis and de novo FA synthesis under cold stress.

Orientador: Aloisio Jose Antunes Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: As proteínas da clara do ovo de galinha têm sido extensivamente usadas como ingredientes em alimentos processados, devido a suas propriedades funcionais, tais como gelatinização e formação de espuma. Durante o armazenamento, podem ocorrer mudanças nas características físicas, microbiológicas, químicas e funcionais dos ovos, que dependem das condições de armazenamento. As vantagens de utilizar filmes e coberturas comestíveis podem ser justificadas pela manutenção das propriedades funcionais dos alimentos, através da diminuição da perda de umidade e da diminuição do transporte de gases (02 e CO2). A aplicação de cobertura em ovos com casca reduz a perda de peso e mantém a qualidade interna do produto. Os objetivos foram quantificar a perda de peso, os valores da unidade Haugh, o pH e os teores de s-ovalbumina do albume, além de determinar alterações na estabilidade da espuma da clara, no perfil de textura e na umidade espremível de géis da clara de ovos frescos e de ovos com e sem cobertura à base de concentrado protéico de soro de leite, armazenados a 25°C, por 3,7, 10, 14,21 e 28 dias. Durante todo o período de estocagem houve decréscimo dos valores de unidade Haugh e perda de peso, tanto para os ovos com cobertura como para os ovos sem cobertura. Já o pH do albume aumentou. A variação dos valores nos ovos com cobertura foi menor do que para os ovos sem cobertura. Os valores da unidade Haugh para ovos com cobertura foram semelhantes aos de ovos armazenados sob refrigeração, encontrados em referências bibliográficas, no mesmo período de armazenamento. O volume de líquido drenado foi maior (P < 0,01) em ovos sem cobertura do que em ovos com cobertura armazenados a 25°C, em todos os períodos de armazenamento. A diferença no terceiro dia de armazenamento foi de 59% entre os ovos com cobertura e ovos sem cobertura, e no vigésimo oitavo dia foi 202%. Durante o período de armazenamento, ocorreu um aumento do pH e do volume do líquido drenado nos ovos sem cobertura. O teor de s-ovalbumina foi maior em ovos sem cobertura, comparados aos ovos com cobertura, em todos os períodos de armazenamento (P

The culture of rice has great potential to constitute the production systems of annual perennial crops, mainly in small properties. This allows this system to integrate with other activities such as the dairy industry, taking advantage of the milk by-product, reducing the environmental liability for disposal in the environment. Therefore, the objective was to evaluate the growth and productivity of rice grains under different doses of acid milk serum (0 m3 ha-1, 5 m3 ha-1, 10 m3 ha-1, 20 m3 ha-1, 50 m3 ha-1, 100 m3 ha-1, and 150 m3 ha-1), in soil applications with monitoring of fertility and electrical conductivity levels. The rice was sown in the UFLA experimental field, using the BRSMG Esmeralda lineage. Seven different doses were used, divided into two applications via soil. No cover fertilization was carried out. Yield and crop growth variables were determined by monitoring the nutrient content in the soil profile and the sodification and salinization capacity of the residue. It was found that the increase in serum doses increased the electrical conductivity and the percentage of exchangeable sodium by 1.75 and 7.75 compared to the control, but did not cause the process of sodification and salinization of the soil. As acid milk serum is a significant source of potassium, its use in the soil did not promote significant grain yield. It was concluded that the acid milk serum in the doses used did not cause sodification or salinization of the soil. This residue is the safe dose that can be used as potassium and nitrogen. The acid milk serum in the tested conditions did not increase the growth of the culture.

The turbidity of milk prohibits the use of optical density measurements for strain characterizations. This often limits research to laboratory media. Here, we cleared milk through centrifugation to remove insoluble milk solids. This resulted in a clear liquid phase, termed milk serum, in which optical density measurements can be used to track microbial growth until a pH of 5.2 is reached. At pH 5.2 coagulation of the soluble protein occurs, making the medium opaque again. We found that behavior in milk serum was predictive of that in milk for 39 Lactococcus lactis (R2 = 0.81) and to a lesser extent for 42 Lactiplantibacillus plantarum (formerly Lactobacillus plantarum; R2 = 0.49) strains. Hence, milk serum can be used as an optically clear alternative to milk for comparison of microbial growth and metabolic characteristics. Characterization of the growth rate, specific acidification rate for optical density at a wavelength of 600 nm, and the amount of acid produced per unit of biomass for all these strains in milk serum, showed that almost all strains could grow in milk, with higher specific acidification and growth rates of Lc. lactis strains compared with Lb. plantarum strains. Nondairy Lc. lactis isolates had a lower growth and specific acidification rate than dairy isolates. The amount of acid produced per unit biomass was relatively high and similar for Lc. lactis dairy and nondairy isolates, as opposed to Lb. plantarum isolates. Lactococcus lactis ssp. lactis showed slightly lower growth and acidification rates when compared with ssp. cremoris. For Lc. lactis strains a doubling of the specific acidification rate occurred with a doubling of the maximum growth rate. This relation was not found for Lb. plantarum strains, where the acidification rate remained relatively constant across 39 strains with growth rates ranging from 0.2 h−1 to 0.3 h−1. We conclude that milk serum is a valuable alternative to milk for high-throughput strain characterization during milk fermentation.

The use of antibiotics to prevent bovine mastitis is responsible for the emergence and selection of resistant strains. Lactic acid bacteria (LAB) could be introduced into animal feed as an alternative prevention method that would bypass the risk of resistance development. In previous research, we demonstrated that two probiotic LAB strains isolated from bovine milk were capable of stimulating the production of antibodies and the host’s immune cellular response in the udder. The present study aimed to elucidate whether the antibodies of animals inoculated with these strains were able to increase phagocytosis by neutrophils and inhibit the growth of different mastitis-causing pathogens. Moreover, the effect of LAB on the expression of pro-inflammatory cytokines was assessed. Ten animals were inoculated intramammarily with 106 cells of the two strains at dry-off. The blood serum was tested for its ability to opsonize bovine mastitis pathogens, the in vitro bactericidal activity of bovine blood and milk against these pathogens was determined, and cytokine mRNA expression was quantified in milk somatic cells. The inoculated animals did not show abnormal signs of sensitivity to the LAB. Their blood serum significantly enhanced the phagocytosis of Staphylococcus spp. and the LAB. Escherichia coli and Streptococcus uberis were inhibited by the milk serum but not the blood serum, whereas Staphylococcus aureus and Staphylococcus haemolyticus were inhibited by both. In regard to cytokine expression, interleukin (IL)-1β increased markedly for up to 4 h post-inoculation, and an increase in IL-8 was observed 4, 12 and 24 h after inoculation. Tumour necrosis factor-α mRNA increased 1 and 2 h after inoculation and a significant difference was registered at 6 h for interferon-γ. This rapid immunomodulatory response shows that inoculating animals with LAB at dry-off, when they are especially susceptible, could be a useful strategy for the prevention of bovine mastitis.

Human milk contains, among others, a range of biofunctional components, like serum proteins and human milk oligosaccharides (HMOs). This PhD thesis provides information on the variability in serum proteins, serum protein N-glycans and HMOs in milk of individual mothers during lactation, and offers insights on the degradation of proteins using a newly developed infant (0–3 months) in vitro digestion model.The 15 most abundant HMOs cover >95% of the total HMO content. The mother’s secretor (Se) and Lewis (Le) histo-blood groups are related to specific fucosylated HMOs. Based on the total neutral fucosylated HMO concentrations in both Chinese and Dutch human milk, for the first time, Se+Le+ subgroups were identified. Variation among mothers was found in the composition of serum proteins in both colostrum and mature milk, although the group of immune-active proteins, enzymes, and transport proteins were the most abundant for all mothers. These three protein groups encompass many of the 15 most abundant proteins, covering >95% of the total protein concentrations, in both the Chinese and Dutch milk serum proteome. The serum protein N-glycan composition in human milk from 2 different lactational periods was investigated. Analysis showed that neutral fucosylated and nonfucosylated N-glycans dominated the human milk serum glycoproteome. Based on the levels of the individual N-glycans, for the first time, a clear difference can be observed between the milk of secretor and nonsecretor mothers. It also indicated that specific fucosylated N-glycans can be synthesized in the gland of nonsecretor mothers and even in much higher concentrations compared to secretor mothers. In an in vitro infant digestion model, the total milk protein content decreased from the start to the end of infant in vitro digestion with large variation between mothers, especially in the gastric phase (remaining between 25–80%). After intestinal digestion, still some undigested proteins could be found, ranging from 0.5% to 4.2% of the initial protein content, although no differences could be observed between colostrum and mature milk. More than 40 serum proteins could be detected after intestinal digestion. Overall, caseins are digested more than most serum proteins during digestion. Especially immune-active serum proteins, which are digested to a lower extent, might protect infants from pathogens.

The aim of this work is to study the ability of whey flavor precursors to form new volatile components under model conditions. The formation of new aromas is considered in the aspect of a two-stage process - enzymatic cleavage of the precursors and the subsequent Maillard reaction. The properties of whey components by their ability to form or modify aromatic descriptors at the stages of enzymatic cleavage of taste precursors are considered. It is proved that targeted enzymatic reactions with whey components lead to the formation of sweet, meat, cheese flavors at various stages of whey protein breakdown after interaction with carbohydrates. The influence of the depth of proteolysis on the subsequent formation of aroma from precursors is considered. It has been established that the breakdown products of milk sugar in whey do not play a significant role in the formation of aroma. Unlike proteolytic enzymes, the use of lactase (β-galactosidase) did not lead to a change in the aromatic characteristics of the modified serum. An increase in the concentration of lactulose, as a potential precursor of aroma in the sugar-amine reactions of serum, gives only a sweetish tint and does not significantly affect the change in aroma. The processes of aroma formation in modified whey have been consistently investigated, and the concept of flavored food foam based on it has been tested. It was shown that the addition of sodium chloride 5-10% to the lower residue after rectification of fermented milk whey significantly increases the stability of the aroma of the final product. It was found that the specific aroma of whey, limiting its use in food, can be modified by using new approaches to reactions with aroma precursors. We consider the fixation of the obtained aromas “soup mushroom”, “broth”, “cheese” on an oil basis for subsequent concentration and use in various products.

Infant formula is used as a supplement for newborns. Although bovine milk-based infant formulas dominate the market, caprine milk-based infant formula has attracted increasing attention because of its lower allergenicity. This study compared the digestive peptidome of bovine and caprine milk serum proteins by using in vitro infant simulating conditions. The result showed that the degradation pattern of milk proteins was similar, whereas the digestive rates of milk proteins differed between bovine and caprine milks. Several proteins, such as α-lactalbumin (LALBA), β-lactoglobulin (LGB), serum amyloid A protein (SAA1), glycosylation-dependent cell adhesion molecule 1 (GLYCAM1), and lactotransferrin (LTF), released more peptides during digestion of caprine milk serum than during digestion of bovine milk serum; however, more peptides derived from αS1-casein (CSN1S1) were found in bovine digesta. In addition, antimicrobial-related peptides were mostly only found in caprine intestinal digesta. The results of this study may be useful in understanding the digestion characteristics of milk serum proteins and providing guidance on the improvement of infant formula.

Our previous study identified peaks in the 31 P nuclear magnetic resonance ( 31 P NMR) spectra of skim milk, denoting the interaction of different phosphate species such as inorganic and casein-associated phosphate during the separation of colloidal and serum phases of skim milk by microfiltration (MF) and diafiltration (DF). In the current study, we investigated the same samples generated by the aforementioned separation using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy analysis. The results confirmed that the technique was not only capable of differentiating between the mineral equilibrium of the casein phosphate nanocluster (CPN) and milk serum, but also complemented the application of 31 P NMR. An ATR-FTIR broad band in the region of 1,055 to 1,036 cm −1 and a specific band at 1,076 cm −1 were identified as sensitive to the repartitioning of different phosphate species in milk in accordance with the 31 P NMR signals representing casein-associated phosphate and inorganic phosphate in the serum. A third ATR-FTIR signal at 1,034 cm −1 in milk, representing precipitated inorganic calcium phosphate, had not previously been detected by 31 P NMR. Thus, the results indicate that a combination of ATR-FTIR and 31 P NMR spectroscopies may be optimally used to follow mineral and protein phase changes in milk during membrane processing.

The article discusses the best options for the preparation of blood and milk samples for the purpose of diagnosing retroviral enzootic bovine leukosis in cattle and assessing the origin of dairy products from sick or healthy animals. The effectiveness of using samples in the native state and after their processing with the removal of red blood cells, their membranes, the fat component and casein were analyzed. It has been established that when performing screening analysis it can be carried out using whole blood and milk samples. This is especially possible in the case of diagnosis of the disease at the stage of its progressive development. The use of such samples significantly reduces analysis time and makes it simpler. But if it is necessary to carry out data verification and detection of cattle retroviral leukemia virus already in the early periods, then, in accordance with the results obtained by us, samples prepared in the form of blood serum or milk serum should be used. The latter should also be used to accurately assess the origin of milk, especially when it is necessary to control its quality when collecting from several animals, among which may be sick individuals, as well as in the early stages of the development of the pathological process.

This study investigated milk serum protein concentrate/lactose systems with varying ratios and how the morphology of the spray-dried particles of these systems could be described by the surface properties of the feed as well as the protein surface coverage of the particles. An extrapolation of the surface pressure of the feed to 0.3 s, the approximate time for molecular diffusion in an atomised droplet in the spray-dryer, showed a relationship with the particle morphology. At low protein concentrations (

In present study, 198, 169, 213 and 128 proteins were identified and quantified in human, cow, goat and yak milk serum respectively by using proteomics techniques. Large variations were observed between human and ruminant milk proteins. Human milk contained higher concentration of mucosal immune response, complement proteins and regulators. The concentration of bactericidal proteins were relatively higher in ruminants milk. Human milk exclusively expressed proteins important for delivery or utilization of nutrients. Peptidase inhibitors prevent the bioactive proteins/peptides in human milk from gastral-intestinal digestion. Protein composition among ruminants milk was similar but with variations. Goat milk contained high level of complement proteins but low level of corresponding regulators. In addition, the acute phase proteins were significantly higher in goat milk. Of note, osteopontin in yak milk was enriched, which could offer an alternative source for producing osteopontin and revealed possible nutritional value of yak milk.

This study investigated the effects of heating skim milk with soluble calcium salts, calcium chloride, calcium lactate, calcium gluconate and calcium lactobionate, on the physico-chemical and rheological properties of milk. Regardless of the type of salt added, the amount of casein in milk serum decreased and the amount of calcium in centrifuge sediment increased along with the serum calcium. The amount of calcium salt required for gelation and the gel firmness (G') varied depending on the salt. The aCa2+ order in milk was calcium chloride>calcium lactate>calcium gluconate>calcium lactobionate. With the same amount of added calcium salt, the final G' of milk gel followed the same order. The findings from this study suggest that the different association constants and formation of an intermediate calcium complex with hydroxycarboylate groups affected the calcium ion activity, which may influence the gelation properties in milk.

This study aims to evaluate the in vivo efficacy in terms of elasticity, hydration and anti-wrinkle enhancement of a new cosmetic cream formulation: Apollinea anti-ageing facial cream. The latter is O/W (oil/water) emulsion with high volume percentages of snail secretion filtrate and donkey milk serum in its composition. The investigation has been performed on (twelve) healthy subjects: 8 (eight) females, and 4 (four) males, all presenting evident skin wrinkles in typical facial areas (e.g. forehead and eye contour). The subjects had to apply about 2 grams of Apollinea anti-ageing facial cream every day. The efficacy of the new cosmetic formulation was assessed before and after the cosmetic treatment, precisely 2 hours, to assess the short-term efficacy, and 40 days after, this to evaluate the product long-term efficacy. Elasticity enhancements have been tested through measurements of the viscoelastic properties of the skin, whereas the hydration parameter through measurements of the skin and subcutaneous tissues dielectric constant. All the latter measurements have been performed with specific instrumentation, as discussed more in detail in the following. To evaluate the anti-wrinkles efficacy instead, we proceeded with digital camera imaging and image analysis processed through ImageJ software. The new Apollinea facial cream cosmetic formulation, based on donkey milk whey and snail secretion filtrate from Helix Aspersa Muller, showed significant enhancement effects on skin elasticity and hydration as well as an important skin-wrinkles-reduction.

One of the main factors determining physicochemical and structural properties of the dry permeate of spray drying is lactose which is present in permeate as a basic phase. If lactose is in the β-form in a dry powder, the powder easily absorbs moisture from the atmosphere during storage, causing agglutination, clumping, and caking of the product. Prevention of negative consequences associated with increased hygroscopicity, is ensured by the directed crystallization of lactose in a condensed product before spray drying. The purpose of this investigation was to study the crystallization process of lactose in permeate under industrial conditions when using various seed materials. Results of the studies showed that the type of a crystal forming agentdoes not affect the size and shape of lactose crystals and the level of lactose crystallization. Seed material is of critical significance as it will ensure both a high level of crystallization and homogenous crystals which, in general, leads to an increase in melting temperature, due to a decrease in the proportion of amorphous lactose, as well as improves the drying process.

Whey (milk serum) is produced by the dairy industry during the manufacture of cheese. In addition to being a valuable source of functional and nutritional proteins, whey also presents almost all the lactose from the original whole milk. However, many industries still consider the whey as an effluent, which can cause serious environmental problems when not properly treated. Therefore, it is important to develop alternatives for the adequate use of whey. The lactose obtained from whey permeate can be converted into lactulose, a prebiotic which may be metabolized in the intestine by probiotic bacteria, such as Lactobacillus sp. and Bifidobacterium sp., through enzymatic isomerization or by using alkaline catalysts, with minimal secondary reactions and high yield. This manuscript provides information about various techniques used to produce lactulose, its purification and analysis, as well as its mechanisms of action and alternative applications in food products and medicines.

Milk whey proteins are precursors of more than a hundred bioactive peptides. These peptides may be formed in the digestive process in the gastrointestinal tract, as well as under the influence of lactic acid bacteria proteases and milk-clotting preparations. Among them, inhibitors of angiotensin-converting enzyme, opioid and bactericidal peptides, immunomodulatory and hypocholesterolemic peptides, peptides that have an effect on intestinal motility, appetite, and others have been found. Bioactive peptides from whey proteins are of considerable interest for the production of functional products or products with prophylactic properties. The creation of such products includes the obtaining of bioactive peptides of define biological action. Taking into account the specifics of their placement in the primary structure, it is advisable to use homogeneous protein precursors from milk whey to obtain them. One of the methods for one-stage separation of whey proteins is disc-electrophoresis in the native PAGE system for alkaline and neutral proteins. The purpose of this work was to determine the parameters of the preparative variant of disc-electrophoresis for the selection of homogeneous whey proteins. The research was performed on a modified apparatus of Stadier type. Proteins of milk whey were isolated by the precipitation of caseins at an isoelectric point. The precipitate of caseins was separated by centrifugation. Homogenous α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) fractions were used to identify milk whey proteins, which were obtained by double gel-filtration on Sephadex G-100 (fine). Homogeneity and fractional composition of milk serum proteins were determined by analytical disс-electrophoresis in plates of polyacrylamide gel. The concentration of proteins in the samples was determined spectrophotometrically (spectrophotometer SF-46) at a wavelength λ = 280 nm.The obtained results prove the feasibility of using PAGЕ plates for the preparative fractionation of serum milk proteins. The allowable duration and conditions for identification of protein fractions are established. It was shown that the yield of proteins in the composition of homogeneous fractions was 69.2%. In this case, the correlation between the number of obtained fractions (β-LG, α-LA, BSA and IG) is close to their distribution in milk serum.

Proteins represent one of milk's macroconstituents (along with water, lipids, and carbohydrates), accounting for ~ 3.0%–3.5% of the total composition. In bovine milk, there are two main protein families, namely, casein and whey protein. Caseins are milk-specific proteins that exist as colloidal aggregates dispersed in milk serum. The bovine milk protein system is casein dominant, which enables large quantities of calcium to be delivered to the neonate. The natural function of milk is to supply the neonate of the species with its complete nutritional requirements for a period of time postpartum. Milk proteins (and their peptides) provide essential amino acids and amino groups for the biosynthesis of nonessential amino acids and, when in excess, also supply energy. They also provide many of milk's physiological functions, performed by immunoglobulins, enzymes, enzyme inhibitors, growth factors, hormones, and antibacterial agents. The development of advanced fractionation and processing techniques has led to the production of many milk-derived ingredients that have different functional properties and applications.

This study mainly investigated changes in milk serum proteins by simulating the industrial processing of whole milk powder using a label-free proteomic approach. A total of 391 proteins were identified, 89 of which were quantified in all groups. Compared with raw milk, the milk subjected to the heating pasteurization process showed significantly decreased the serum protein profile, whereas that subjected to vacuum concentration and spray-drying showed minimal changes. The LC-MS/MS results were further confirmed by determining the activity of bioactive xanthine oxidase and retention of lactoferrin and immunoglobulins. The data showed that 70%-85% of lactoferrin and immunoglobulins were retained in vacuum-concentrated or spray-dried milk but were hardly identifiable in heat-pasteurized milk or whole milk powders. These findings indicate the need for improving the current milk powder-manufacturing techniques to allow the retention of active milk proteins.

Bovine milk extracellular vesicles (EVs) attract research interest as carriers of biologically active cargo including miRNA from donor to recipient cells to facilitate intercellular communication. Since toxicity of edible milk seems to be negligible, milk EVs are applicable to use for therapeutics in human medicine. Casein separation is an important step in obtaining pure EVs from milk, and recent studies reported that adding hydrochloric acid (HCl) and acetic acid (AA) to milk accelerates casein aggregation and precipitation to facilitate EV isolation and purification; however, the effects of acidification on EVs remain unclear. In this study, we evaluated the acidification effects on milk-derived EVs with that by standard ultracentrifugation (UC). We separated casein from milk by either UC method or treatment with HCl or AA, followed by evaluation of EVs in milk serum (whey) by transmission electron microcopy (TEM), spectrophotometry, and tunable resistive pulse sensing analysis to determine EVs morphology, protein concentration, and EVs size and concentration, respectively. Moreover, we used anti-CD9, -CD63, -CD81, -MFG-E8, -HSP70, and -Alix antibodies for the detection of EVs surface and internal marker proteins by western blot (WB). Morphological features of EVs were spherical shape and similar structure was observed in isolated EVs by TEM. However, some of the EVs isolated by HCl and AA had shown rough surface. Although protein concentration was higher in whey obtained by UC, EV concentration was significantly higher in whey following acid treatment. Moreover, although all of the targeted EVs-marker-proteins were detected by WB, HCl- or AA-treatments partially degraded CD9 and CD81. These findings indicated that acid treatment successfully separated casein from milk to allow efficient EV isolation and purification but resulted in partial degradation of EV-surface proteins. Our results suggest that following acid treatment, appropriate EV-surface-marker antibodies should be used for accurate assess the obtained EVs for downstream applications. This study describes the acidification effects on EVs isolated from bovine milk for the first time.

We investigated the applicability of cryo-electron tomography as a method to quantify changes in the major constituents of casein micelles (i.e., casein proteins, putative colloidal calcium phosphate nanoclusters, and serum-filled voids and channels) in response to their environment. Skim milk diluted 20-fold in milk serum was used for this study. Tomograms were generated for multiple casein micelles at 2 different pH values (6.7 and 6.0) and pixel intensity thresholds were identified for each constituent. The volume of each constituent was determined using these thresholds and expressed as a fraction of micelle volume. At the given dilution, a significant decrease in the volume fractions of casein proteins (∼37%) and putative colloidal calcium phosphate nanoclusters (∼67%) was observed with the reduction of pH from 6.7 to 6.0. Assessment of casein micelle fraction obtained by ultracentrifugation of corresponding skim milk samples produced comparable results. When using such an approach, the imaging conditions, denoising methods, and thresholding approaches used can all affect the precision of the measurements, but the overall trends in constituent volumes are able to be tracked. The primary advantage of using cryo-electron tomography is that analysis can be done at the level of individual micelles, within a 3-dimensional morphological context. This workflow paves the way for high-throughput exploration of milk micelles and how their environment shapes their composition and structure.

Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low-density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%) were added to control extender. Vitrification was performed in 0.25-ml straws directly plunged into liquid nitrogen. Total motility (TM, %) and progressive motility (PM, %) were analysed by CASA, and plasma membrane (IMS, %) and acrosome membrane integrity (AIS, %) were assessed under epifluorescence microscopy. Post-warmed sperm parameters were compared between treatments by ANOVA. Results were expressed as mean ± SEM. In both experiments, the minimum concentration of LDL and Pronexcell obtained significantly higher values (p < 0.01) than the control extender for TM (L1 = 52.95 ± 4.4; P1 = 58.99 ± 4.6; C = 30.88 ± 3.0), PM (L1 = 36.79 ± 5.5; P1 = 47.25 ± 4.3; C = 19.20 ± 2.4), IMS (L1 = 68.88 ± 3.6; P1 = 47.25 ± 4.3; C = 52.81 ± 2.6) and AIS (L1 = 45.88 ± 3.6; P1 = 47.25 ± 4.3; C = 26.00 ± 2.1). No differences in sperm parameters were found among different concentrations of LDL or Pronexcell. In conclusion, the addition of 0.25% LDL and 1% Pronexcell to the vitrification extender is recommended to improve the quality of stallion sperm after vitrification.

Superoxide dismutase activity was shown to be present in bovine milk serum and was quantified by measuring the capacity of retentate from dialyzed milk serum to inhibit reduction of cytochrome c by xanthine-xanthine oxidase-generated superoxide anion. One unit of enzyme was defined as the quantity of superoxide dismutase which inhibits cytochrome c reduction by 20%. By this definition 19,500 units of enzyme were present per liter of retentate from dialyzed milk serum. This amount is equivalent to about 2.4 mg of purified bovine erythrocyte superoxide dismutase per liter. Polyacrylamide gel electrophoresis of a partially-purified superoxide dismutase from acid whey, followed by staining for enzymic activity, confirmed the presence of the enzyme in milk serum which was identical in electrophoretic properties to those of bovine erythrocyte copper-zinc superoxide dismutase. Pasteurization at 63 C for 30 min did not decrease superoxide dismutase activity in milk serum. Heating of purified bovine erythrocyte-superoxide dismutase at 100 C for 1 min resulted in almost complete loss of enzymic activity, whereas the partially-purified superoxide dismutase from acid whey still retained 40% of the original activity under these conditions. Bovine milk superoxide dismutase may be an important naturally-occurring antioxidant for increasing oxidative stability of milk and other dairy products.

The objective of this study was to evaluate the effectiveness of microfiltration (MF) of skim milk, alone or combined with ultraviolet-C (UVC) treatment, in extending the shelf life and retaining the native bioactive milk serum proteins, compared with high-temperature short-time (HTST) pasteurization. The vegetative bacterial load was reduced by ~3.5 log when applying M alone, by ~5 log when combining MF with UVC, while it was reduced by only ~2 log when applying HTST pasteurization. MF with UVC resulted in near complete elimination of vegetative bacteria, including coliforms. The refrigerated shelf life of skim milk was extended with increasing UVC dosage, as least for 40 days after MF combined with a UVC dosage at 39.3 mJ/cm2. An increasing UVC dosage would further reduce the bacterial load without obvious damage of bioactive milk serum proteins, such as lactoferrin, immunoglobulins, and lactoperoxidase. An LC-MS/MS based proteomics study also showed that HTST reduced the abundance of several native low-abundant milk serum proteins significantly, while MF, alone or in combination with UVC, did not change the proteome, even with higher UVC dosages. The results of this study can be used to facilitate the development of non-thermal treatments, and combinations therefore, for improving milk quality.

Skimmed milk was fractionated via a cascade system: Graphic plotting of microfiltration (MF); ultrafiltration (UF); nanofiltration (NF); and reverse osmosis (RO). The buffering curves of each fraction were studied over the pH range 4–7. Depending on their composition, the individual permeate streams showed different buffering capacity values and pH ranges where the buffering occurred. The concentration of active buffering substances in the permeates decreased (in mmol/L) from ~26.6 (MF) to ~17.4 (UF) to 1.39 (NF) to 0.07 (RO). Contributions to the total buffering capacity for MF permeate, which represents the serum phase of milk, were ~37% from whey proteins and ~63% from milk salts (especially citrates, phosphates and carbonates) including lactose and water.

Through the use of protein-free milk serum (PFMS) in comparison with deionised water (DW) or deionised water with lactose (DWL), the respective impact of lactose and milk salts on surface activity and foaming properties of selected individual milk proteins and relevant milk protein mixtures was evaluated. Against expectation, the lactose-dependent increase in solution viscosity did not impede protein diffusion and surface adsorption. Instead of molecular flexibility, protein interaction potential (inherent or milk salt-induced) correlated with foamability. Lactose and milk salts contributed to increased foam stabilities, which was also reflected in time-dependent evolution of bubble sizes as well as reduced drainage exponents. Generally, maxima in foamability and foam stability resulted when PFMS was used, which indicates the significance of pronounced protein–protein interactions as well as high packing densities at the air/water interface. The main contribution of lactose to foam volume gain and stabilisation was attributed to the reduction of coalescence.

The influence of different heat treatments on the protein aggregates and changes in gelation properties of rennet-induced milk gels were investigated. In the heated milk, a visible difference in milk serum proteins was found resulting from the formation of protein aggregates. Meanwhile, the size of protein aggregates increased from 25 to 170 nm with increasing the intensity of heat treatment. Furthermore, the differences in textural variables of rennet gels were found among the heat treatments using the principal component analysis. The water holding capacity and cheese curd yield of rennet gels obtained from the heated milk were significantly greater than those of raw milk (p < 0.05). It was also found heat treatments above 80°C could endow rennet-induced milk gels with novel textural properties.

To keep their functional properties, it is crucial that protein aggregates maintain their structure after spray drying and that the powders can be fully rehydrated. In this study, microgels and fractal aggregates were prepared by heating a mixture of milk serum protein concentrate and lactose (40/60; %, w/w) at 85 °C for 15 min by varying the pH. Various fractions of native proteins were added to the systems prior to spray drying. This study showed that microgels and fractal aggregates kept their structure after spray drying and reconstitution. The particle morphology could be correlated to the stiffness of the interface of the feed droplet. The forced imbibition rate showed a negative correlation with increasing amount of aggregated proteins in the powders that seems to be a result of denatured/aggregated proteins present at the surface. These findings are of importance for the formulation of spray-dried powders with improved rehydration characteristics.

Milk serum contains many immune-active proteins that are sensitive to heat treatment. This study compared the effects of thermal (63 °C, 30 min; 72 °C, 15 s; 85 °C, 5 min) and non-thermal (ultraviolet-C, UV-C; thermo-ultrasonication, TUS) treatments on bovine milk serum proteins by using label-free LC-MS/MS-based proteomics. UV-C (4500 J/L) and TUS (60 W, 6 min) treatments achieved a 5log microbial reduction as determined by plate counting. Proteomics showed that e.g., complement proteins, xanthine dehydrogenase/oxidase, and fatty acid-binding protein decreased significantly (p˂0.05, |fold change|˃1) after thermal treatments, and almost no lactoferrin, immunoglobulin, and lactoperoxidase was retained after heating at 85 °C for 5 min, whereas these proteins were mostly retained after non-thermal treatments. Most of these heat-sensitive proteins were located in membrane and extracellular regions and were involved in cellular and metabolic processes, response to stimulus, binding, immune process and catalytic functions. Finally, part of the proteomics results were verified by ELISA. This study thus provided insights for the development of optimized thermal and novel non-thermal treatments for dairy processing. Industrial relevance As alternatives to thermal processing technique, UV-C and ultrasonication showed a great potential in the processing of milk. This study not only showed that UV-C and ultrasonication were able to largely reduce the microbial load of raw milk, but also better retained the immune-related milk serum proteins than thermal processing, especially for the UV-C treatment. ELISA assays also demonstrated that the LC-MS/MS based proteomics technology used in this study was a robust method for quantifying damage to the milk serum proteome upon processing. Taken together, this study provided insights for development of optimized thermal and novel non-thermal techniques for dairy processing.

Churning is a common method to separate milk fat globule membrane (MFGM) from milk fat globules. In this work, we qualitatively and relative quantitatively analyzed the MFGM components obtained by churning cream for different time using a label-free proteomic approach. The result showed that 794 MFGM proteins were identified in all groups, of which 51, 14, 60 and 44 unique proteins were contained after churning cream for 2 min, 4 min, 6 min and 8 min, respectively. The sample churned for 6 min possessed the largest portion of total and unique proteins compared with other three groups. The relative abundances of proteins butyrophilin and xanthine dehydrogenase/oxidase, proteins cluster of differentiation 36 and fatty acid binding protein had the same change tendency during the continuous agitation process. Some milk serum proteins also were detected in the buttermilk. The analysis of protein profiles of MFGM at different churning time had an important guidance for enriching a certain MFGM protein.

For microbial safety, thermal processing is applied during caprine milk processing, which affects the stability of proteins, specially the whey proteins. Therefore, this study evaluated the changes of glycated serum proteome in caprine milk products using a robust ion mobility time-of-flight proteomic technique, without glycated peptides enrichment. In total, 302 proteins were identified in whey and 41 lactosylation sites from 16 proteins. The number of identified whey proteins decreased from 202 to 194, 178 and 186 in pasteurised milk (PM), spray-dried milk (SDM) and ultra-high temperature (UHT) milk serum, respectively, as the heating intensity increased. Nevertheless, lactosylation sites increased 16 in raw milk to 20 in PM, 25 in UHT and 27 in SDM serum. Moreover, major whey proteins contained the highest number of lactosylation sites as expected. The results of this study may contribute to measurement of the quality of caprine milk after industrial processing.

Whey powder rehydration is expected to be closely linked to both denaturation and lactosylation of the proteins. This study investigated the relation between the forced imbibition rate of spray-dried milk serum protein/lactose powders and the particle morphology and how it is related to the insoluble and lactosylated protein fraction, respectively. Despite extensive variation in protein denaturation, aggregation and lactosylation, only comparably small effects on the forced imbibition rate of the powders and the particle morphology could be observed. A possible explanation for this rather limited effect on the rehydration properties and particle morphology might be that the surface composition of the powder particles is rather similar and dominated by native proteins. These insights have relevance for the formulation of whey powders with improved rehydration properties.

Laccases (E.C. 1.10.3.2) are multicopper oxidases of great importance in the industry due to their non-specificity and high oxidative potential. Laccases are useful to bleach synthetic dyes, oxidize phenolic compounds and degrade pesticides, among others. Hence, the objective of this work was to optimize low cost culture media for recombinant (rPOXA 1B) laccase production from Pleurotus ostreatus in Pichia pastoris. To this end, low cost nitrogen sources were studied, such as malt extract, isolated soy protein and milk serum. Following, two central composite designs (CCD) were performed. In CCD-1 different concentrations of glucose USP (0–13.35 gL-1), protein isolated soy protein (5–25 gL-1), malt extract (3.5–17.5 gL-1) and (NH4)2SO4 (1.3–6.5 gL-1) were evaluated. In CCD-2 only different concentrations of glucose USP (7.9–22 gL-1) and isolated soy protein (15.9–44.9 gL-1) were evaluated. CCD-2 results led to a One Factor Experimental design (OFED) to evaluate higher isolated soy protein (20–80 gL-1) concentrations. In all designs, (CCD-1, CCD-2 and OFED) CuSO4 (0.16 gL-1) and chloramphenicol (0.1 gL-1) concentrations remained unchanged. For the OFED after sequential statistical optimization, an enzyme activity of 12,877.3 ± 481.2 UL−1 at 168 h was observed. rPOXA 1B activity increased 30.54 % in comparison with CCD-2 results. Final composition of optimized media was: 20 gL-1 glucose USP, 50 gL-1 isolated soy protein 90 % (w/w), 11.74 gL-1 malt extract, and 4.91 gL-1 (NH4)2SO4. With this culture media, it was possible to reduce culture media costs by 89.84 % in comparison with improved culture media previously described by our group.

Milk processing affects the molecular and supramolecular characteristics of milk. While many of these changes have been studied extensively, the effect of homogenisation on volatile components and lipids at different pressures and temperatures is still poorly understood. The aim of this study was to determine the effect of homogenisation pressure (0, 150, 250 and 350 bar) at different temperatures (45 °C, 60 °C and 80 °C) on the content of individual milk volatile components, phospholipids and triacylglycerols. The results demonstrated the importance of temperature and pressure (P > 0.001) on the formation of 2-heptanone. Temperature was the most important factor for significant (P > 0.05) loss of the milk fat globule membrane lipid phosphatidyletholamine (PE) from the milk serum. Moreover, a significant increase (P > 0.001) in the level of all triacylglycerols groups in the serum phase of homogenised milk was observed, independent of the temperature of the homogenisation process.

We report the use of a method to determine the refractive index of copper(II) serum (RICS) in milk as a tool to detect the fraudulent addition of water. This practice is highly profitable, unlawful, and difficult to deter. The method was optimized and validated and is simple, fast and robust. The optimized method yielded statistically equivalent results compared to the reference method with an accuracy of 0.4% and quadrupled analytical throughput. Trueness, precision (repeatability and intermediate precision) and ruggedness are determined to be satisfactory at a 95.45% confidence level. The expanded uncertainty of the measurement was ±0.38°Zeiss at the 95.45% confidence level (k=3.30), corresponding to 1.03% of the minimum measurement expected in adequate samples (>37.00°Zeiss).

The anti-penicillin G was conjugated to avidin-peroxidase and biotin to obtain immunogen and competitor which were then used to develop a competitive immunosensor assay for the detection of penicillin G and other β-lactam antibiotics, with Kaff values of the order of 10(8) M(-1). The new immunosensor appears to afford a number of advantages in terms of sensitivity, possibility of "in situ" analysis, but especially of simplicity and lower costs, compared with other existing devices, or different chemical instrumental methods reported in the literature and used for the analysis of β-lactam compounds. Satisfactory results were found in the analysis of real matrixes and good recoveries were obtained by applying the standard addition method to spiked milk, urine, serum and drug samples. The new device uses an amperometric electrode for hydrogen peroxide as transducer, the BSA-penicillin G immobilized on polymeric membrane overlapping the amperometric transducer and the peroxidase enzyme as marker. It proved to be highly sensitive, inexpensive and easily reproducible; LOD was of the order of 10(-11)M. Lastly, the new immunosensor displayed low selectivity versus the entire class of β-lactam antibiotics and higher selectivity toward other classes of non-β-lactam antibiotics.