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Microbial transglutaminase (mTGase) is an acyltransferase that predominantly catalyses the formation of covalent cross-links between protein-bound glutamine and lysine residues, referred to as isopeptide bonds. This typically results in protein polymerisation. The enzymatic polymerisation of caseins, the major protein fraction in milk, has been studied for decades because of its potential to modify physical properties of fermented dairy products. It was suggested that cross-linked caseins form denser gel networks, resulting in higher gel stiffness and increased water holding capacity. However, other studies indicated that there is an optimal cross-linking degree and that prolonged incubation with mTGase results in converse effects. The aim of this research was to elucidate the mechanisms that cause these alterations of the gelation properties. Using non-micellar casein preparations at 27 g·kg-1 protein as model systems, structure-function-interrelationships were studied by molecular characterisation in combination with rheological studies of acid-induced gels. The results suggested that casein molecules self-associate in aqueous solutions and that cross-linking occurs predominantly within distinct casein particles. These cross-links contributed directly to the stiffness of acid-induced gels as indicated by an increased maximum storage modulus. However, in the presence of ions, introduced either prior to or after cross-linking, the highest value was shifted to shorter incubation times. This was attributed to an increased inflexibility of the casein particles with ongoing internal cross-linking, which made them incapable of conformational changes to compensate for the screening of attractive electrostatic interactions through other non-covalent interactions. The findings provide important information for the direct application of mTGase in milk as well as on the utilisation of cross-linked caseinates as additives in food processing. Further studies should be conducted

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.

The digestion, absorption, uptake and bioavailability of a rosemary supercritical fluid extract encapsulated in oil in water emulsion were studied. Two emulsions with opposite surface charge were prepared, containing 7% canola oil, and either 2% lactoferrin or whey protein isolate. When absorption and uptake of carnosic acid and carnosol were followed on Caco-2 cell monolayers, there were no differences with protein type. However, when co-cultures of HT-29 MTX were employed, the presence of mucus caused a higher retention of carnosic acid in the apical layer for lactoferrin emulsions. The immune activity of the bioavailable fractions collected from cell absorption experiments was tested ex vivo on murine splenocytes. Although transport through the intestinal barrier models was low, the bioavailable fractions showed a significant effect on splenocytes proliferation. These results demonstrated the potential of using rosemary supercritical extract through protein stabilized oil in water emulsions, as a food with immunomodulatory functionality.

Rosmarinus officinalis L. is an aromatic plant with components known to confer health-promoting properties. The present study aimed to enhance the bioavailability of the main compounds, carnosic acid and carnosol, by encapsulation. The rosemary extracts were obtained by supercritical fluid extraction, and then different delivery systems were tested, namely oil emulsion droplets and casein micelles, native protein particles present in milk. Soybean oil in water emulsions and casein dispersions were digested in vitro with simulated gastric and intestinal fluids, and the bioavailability of the extract was tested on Caco-2 cell monolayers. The apical and basolateral compartments and the cell lysates were further analyzed for carnosic acid and carnosol content by means of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS). The encapsulation in both casein micelles and emulsion droplets significantly protected the rosemary extract from degradation during digestion, resulting in 81 and 97 % recovery of carnosic acid, respectively. This high protection rate prompted an increased bioavailability of carnosic acid and carnosol in Caco-2 monolayers, with up to 10 % carnosic acid recovered in the bioavailable fraction. Encapsulation in delivery systems like casein micelles or emulsion droplets opens new possibilities for successful incorporation of rosemary extracts in healthy food formulations with increased functionality.

Non-thermal processing methods, such as pulsed electric field (PEF) and tangential-flow microfiltration (TFMF), are emerging processing technologies that can minimize the deleterious effects of high temperature short time (HTST) pasteurization on quality attributes of skim milk. The present study investigates the impact of PEF and TFMF, alone or in combination, on color and volatile compounds in skim milk. PEF was applied at 28 or 40 kV/cm for 1122 to 2805 µs, while microfiltration (MF) was conducted using membranes with three pore sizes (lab-scale 0.65 and 1.2 µm TFMF, and pilot-scale 1.4 µm MF). HTST control treatments were applied at 75 or 95 °C for 20 and 45 s, respectively. Noticeable color changes were observed with the 0.65 µm TFMF treatment. No significant color changes were observed in PEF-treated, 1.2 µm TFMF-treated, HTST-treated, and 1.4 µm MF-treated skim milk (p ≥ 0.05) but the total color difference indicated better color retention with non-thermal preservation. The latter did not affect raw skim milk volatiles significantly after single or combined processing (p ≥ 0.05), but HTST caused considerable changes in their composition, including ketones, free fatty acids, hydrocarbons, and sulfur compounds (p < 0.05). The findings indicate that for the particular thermal and non-thermal treatments selected for this study, better retention of skim milk color and flavor components were obtained for the non-thermal treatments.

Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future. © 2014 the Partner Organisations.

The production of highly aerated foods remains a challenge that requires skill to obtain desired appearance and texture. Since foams are fragile and inherently unstable, evaluation of structure changes with time requires a delicate approach. Non-invasive but informative evaluation of changes in aerated food properties is a desired goal to be achieved in this thesis. I studied the aging of egg white foams of different void fraction using two noninvasive techniques - ultrasonic spectroscopy and electrical resistivity - with a view to understanding aging mechanisms that would affect the quality of the final product. To help in interpretation of the results, a commercial high void fraction foam, Gillette (Sensitive), was also analyzed. As a support technique for ultrasound results I used image analysis in order quantitatively evaluate the size of bubbles in the foams. Large increases in attenuation were observed with increasing aging time and frequency. Results were modeled using an effective medium theory originated by Foldy (1945) in order to understand the factors governing foam lifetime and texture. From electrical resistance measurements, liquid fraction changes in an egg white foam column were evaluated quantitatively and qualitatively by using a generalized free drainage model. Although egg-white foams were more prone to changes due to drainage, I observed that for all foams the ultrasonic scaling parameter alfa*lambda/freq was proportional to the square of average bubble size (indicative of a diffusively driven aging process due to disproportionation of bubbles). Slopes of alfa*lambda/freq versus aging time were 6*10-8 and 11*10-8 for egg white foams of void fraction 0.65 and 0.78, respectively, indicating that disproportionation progressed approximately twice as fast in the high void fraction foam. The slopes of alfa*lambda/freq versus aging time were similar for both Gillette foam void fractions (0.93 and 0.91) at a value of 1.5*10–8 s-1, attributable to a low