The physicochemical and rheological properties of yoghurt made from unstandardised unhomogenised buffalo milk were investigated during fermentation and 28 days of storage and compared to the properties of yoghurt made from homogenised fortified bovine milk. A number of differences observed in the gel network can be linked to differences in milk composition. The microstructure of buffalo yoghurt, as assessed by confocal laser scanning microscopy (CLSM) and cryo scanning electron microscopy (cryo-SEM), was interrupted by large fat globules and featured more serum pores. These fat globules have a lower surface area and bind less protein than the homogenised fat globules in bovine milk. These microstructural differences likely lead to the higher syneresis observed for buffalo yoghurt with an increase from 17.4 % (w/w) to 19.7 % (w/w) in the weight of whey generated at days 1 and 28 of the storage. The higher concentration of total calcium in buffalo milk resulted in the release of more ionic calcium during fermentation. Gelation was also slower but the strength of the two gels was similar due to similar protein and total solids concentrations. Buffalo yoghurt was more viscous, less able to recover from deformation and less Newtonian than bovine yoghurt with a thixotropy of 3,035 Pa.s−1 measured for buffalo yoghurt at the end of the storage, at least four times higher than the thixotropy of bovine yoghurt. While the titratable acidity, lactose consumption and changes in organic acid concentrations were similar, differences were recorded in the viability of probiotic bacteria with a lower viability of Lactobacillus acidophilus of 5.17 log (CFU/g) recorded for buffalo yoghurt at day 28 of the storage. Our results show that factors other than the total solids content and protein concentration of milk affect the structural properties of yoghurt. They also illustrate the physicochemical reasons why buffalo and bovine yoghurt are reported to have different sensory properties and provide insight into how compositional changes can be used to alter the microstructure and properties of dairy products.

The properties of buffalo and bovine milk differ and the procedures developed to make bovine yoghurt may require optimisation for the production of buffalo yoghurt. This study aimed to apply cryo-scanning electron microscopy and confocal laser scanning microscopy to determine the optimal temperature for processing buffalo yoghurt. Milk was fermented at three different temperatures (37, 40 and 43 °C), stored for 28 days and the yoghurt microstructure, physicochemical and rheological properties assessed. Yoghurt fermented at 37 °C had a compact microstructure and the probiotic Lactobacillus acidophilus La-5 was more viable on storage. In contrast, yoghurt produced from a faster fermentation at 43 °C was firmer with a more porous microstructure that exhibited a higher degree of syneresis. The rheological properties during storage including the thixotropy, consistency coefficient and flow behaviour index were not significantly affected by temperature nor were the concentration of lactose, ionic calcium or titratable acidity. This study shows how changes to processing can be used to alter the microstructure of buffalo products and suggests that a decrease in fermentation temperature could be used to improve the quality of buffalo yoghurt.

Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pre-treatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Super-resolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments.

Cutting and cooking settings have a strong effect on curd particle features, whey syneresis and cheese properties. In the present study, the impact of curd grain size and cooking temperature on the microstructure, texture and composition of cheese, whey losses and cheese yield was studied with specific focus on sheep milk. Cooking temperature especially affected cheese microstructure, texture and composition, while cutting process was largely responsible for fat losses in the whey. Additionally, cheese yield increased with a bigger curd grain size and lower cooking temperatures. Higher cooking temperatures reduced the moisture content of the curd grains and cheese and lead to cheeses with reduced porosity and more free fat in their structure, resulting in harder and chewier cheeses. Interactions between the microstructural arrangement of fat and textural parameters were also observed. These results contribute with new data on the relationships between curd grain size and cooking conditions on the microstructure and physico-chemical properties of cheese. In addition, reducing the compound losses in whey would have a direct effect on the improvement of processing, cheese quality and yield, and the ulterior by product management.

The biological membrane surrounding fat globules in milk (the MFGM) is poorly understood, despite its importance in digestion and in determining the properties of fat globules. In this study, in situ structural investigations of buffalo MFGM were performed as a function of temperature (4–60 °C), using confocal microscopy. We demonstrate that temperature and rate of temperature change affected the lipid domains formed in the MFGM with the lateral segregation (i) of high Tm lipids and cholesterol in a Lo phase for both T < Tm and T > Tm and (ii) of high Tm lipids in a gel phase for T < Tm. Rapid cooling favours nucleation, while slow cooling favours growth, leading to the formation of small and large lipid domains, respectively. Changes in the interfacial properties of the MFGM, as a function of temperature, could modulate the functions of fat globules during processing and digestion.

Mozzarella cheese is a classical dairy product but most research to date has focused on low moisture products. In this study, the microstructure and physicochemical properties of both laboratory and commercially produced high moisture buffalo Mozzarella cheeses were investigated and compared to high moisture bovine products. Buffalo and bovine Mozzarella cheeses were found to significantly differ in their microstructure, chemical composition, organic acid and proteolytic profiles but had similar hardness and meltability. The buffalo cheeses exhibited a significantly higher ratio of fat to protein and a microstructure containing larger fat patches and a less dense protein network. Liquid chromatography mass spectrometry detected the presence of only β-casein variant A2 and a single β-lactoglobulin variant in buffalo products compared to the presence of both β-casein variants A1 and A2 and β-lactoglobulin variants A and B in bovine cheese. These differences arise from the different milk composition and processing conditions. The differences in microstructure and physicochemical properties observed here offer a new approach to identify the sources of milk used in commercial cheese products.

Milk fat globules and their surrounding biological membrane (the MFGM) are not well understood despite the importance of these milk components in human nutrition and the role of fat globules in determining the properties of dairy products. The objectives of this study were to investigate these unique colloidal assemblies and the microstructure of the MFGM in buffalo milk, which is the second largest global source of dairy products. In-situ structural investigations were performed at room temperature using confocal microscopy with multiple fluorescent probes (Nile Red, Rh-DOPE, the lectin WGA-488). Microscopic observations showed cytoplasmic crescents around fat globules and the heterogeneous distribution of glycosylated molecules and polar lipids with the occurrence of lipid domains. The lipid domains in the buffalo MFGM appear to form by the segregation of lipids with a high phase transition temperature (e.g. sphingomyelin and saturated phosphatidylcholine molecular species) and cholesterol resulting in a gel phase or a Lo phase forming circular domains. The structure of the buffalo MFGM results from a non-random mixing of components, consistent with observations for other species. Structural heterogeneities of the MFGM could affect the processability of buffalo fat globules and the bioavailability of milk lipids.

The properties of casein micelles are known to be affected by modifications to the environment, such as variations in pH or the addition of salts, yet the scientific literature typically considers the effects of one factor at a time, while in industrial processes, several modifications are performed simultaneously. The aim of this study was to assess the impact of multifactorial environmental modifications on the colloidal, structural and rennet coagulation properties of casein micelles in a simplified model system. A key finding was that dense regions (~20 nm in size) could be released from the casein micelle. The addition of NaCl and CaCl2 had opposing effects, i.e. enhancing or limiting this micellar disruption, respectively. A decrease in pH had the strongest impact on the mineral balance, causing the colloidal CaP to solubilize and the micelle to swell. The rennet clotting time was impacted by variations in pH and NaCl content. Interestingly, a consideration of all three levels of casein micelle structure and their interactions was needed to explain variations in the firmness of rennet gels. This study illustrates the complex interplay of factors affecting micellar structure and improves our understanding of how micelles can be manipulated to control their properties.

Elevated temperatures have been widely studied as a route to accelerate cheese ripening and decrease energy and storage requirements but the impact of temperature on the underlying microstructure of the cheese during prolonged periods of ripening is poorly understood. In this study, Cheddar cheese was matured at four different ripening temperatures (8 °C, 15 °C, 20 °C or a combination of 8 °C and 15 °C) and the impact on cheese microstructure assessed using confocal laser scanning microscopy, cryo scanning electron microscopy and quantitative image analysis of 3D images. An increase in ripening temperature was shown to alter the microstructure of the cheese protein network after only a few weeks of ripening. Incubation at 20 °C significantly reduced branching within the protein network, leading to thicker protein strands and larger pores after 33 days. These structural changes coincided with increased proteolysis, consistent with solubilisation of the protein network; they also led to a softer, less chewy and less cohesive cheese. While the concentration of biogenic amines tryptamine and tyramine were observed to increase with ripening temperature, the concentrations were generally low, confirming that biogenic amines do not represent a health concern under the conditions examined. This study illustrates how 3D image analysis can be used to observe and quantify the effect of process changes on cheese structure, assisting our understanding of the link between structure and function in Cheddar cheese.

Natural creaming of raw milk is the first step in production of Grana Padano and Parmigiano Reggiano Protected Denomination of Origin cheeses. This process decreases the fat content and plays an important role in the removal of clostridia species that may cause late-blowing defects in ripened cheeses. Partial coalescence of fat globules—that may influence fat behavior in cheese making and affect the microstructure of fat in the final cheese product—was observed at creaming temperatures higher than 22°C by confocal laser scanning microscopy. The widespread practice of heating of milk at 37°C before creaming at 8°C resulted in important changes in the size distribution of fat globules in raw milk, potentially altering the ability of fat to entrap clostridia spores. We investigated the role of immunoglobulin classes in both the clustering of fat globules and the agglutination of Clostridium tyrobutyricum to fat globules during creaming. Immunogold labeling and transmission electron microscopy showed that IgA and IgM but not IgG were involved in both clustering and agglutination. Both vegetative cells and spores were clearly shown to agglutinate to fat droplets, a process that was suppressed by thermal denaturation of the immunoglobulins. The debacterization of raw milk through natural creaming was improved by the addition of purified immunoglobulins. Overall, these findings provide not only a better understanding of the phenomena occurring during the natural creaming but also practical insights into how the process of creaming may be optimized in cheese production plants.

Increasing frataxin protein levels through gene therapy is envisaged to improve therapeutic outcomes for patients with Friedreich's ataxia (FRDA). A non-viral strategy that uses submicrometer-sized multilayered particles to deliver frataxin-encoding plasmid DNA affords up to 27 000-fold increase in frataxin gene expression within 2 days in vitro in a stem cell-derived neuronal model of FRDA.

Multifunctional and biodegradable nanostructured hybrid interfaces based on biopolymers are potentially useful in many applications in catalysis, bioanalytical sensing and nanomedicine. Herein, we report the engineering of multifunctional hybrid films by assembling adhesive biological nanoparticles composed of lipoate-conjugated phytoglycogen (L-PG). These nano building blocks possess adhesive properties, arising from their amphiphilic nature, and reactive functional disulfide groups. The assembly of L-PG on surfaces enabled the rapid and conformal deposition of a thin film on substrates of varying chemical composition and wettability. The L-PG films showed negligible cytotoxicity and moderate stability under different conditions but displayed enzyme-mediated degradability. In addition, metal nanoparticles were embedded into the L-PG layers to build up multilayered hybrid films. Specifically, gold and silver nanoparticle-loaded L-PG multilayered films with catalytic and surface-enhanced Raman scattering properties were prepared. Finally, we highlight the versatility of the present approach to engineer multifaceted interfaces for catalysis and sensing applications.