Lydia Ong

Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells

Amino acid-based core cross-linked star (CCS) polymers (poly(L-lysine)(arm)poly(L-cystine)(core)) with peripheral allyl functionalities were synthesized by sequential ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides (NCAs) via the arm-first approach, using N-(trimethylsilyl)allylamine as the initiator. Subsequent functionalization with a poly(ethylene glycol) (PEG)-folic acid conjugate via thiol-ene click chemistry afforded poly(PEG-b-L-lysine)(arm)poly(L-cystine)(core) stars with outer PEG coronas decorated with folic acid targeting moieties. Similarly, a control was prepared without folic acid, using just PEG. A fluorophore was used to track both star polymers incubated with breast cancer cells (MDA-MB-231) in vitro. Confocal microscopy and flow cytometry revealed that the stars could be internalized into the cells, and higher cell internalization was observed when folic acid moieties were present. Cytotoxicity studies indicate that both stars are nontoxic to MDA-MB-231 cells at concentrations of up to 50 μg/mL. These results make this amino acid-based star polymer an attractive candidate in targeted drug delivery applications including chemotherapy.

The effect of milk processing on the microstructure of the milk fat globule and rennet induced gel observed using confocal laser scanning microscopy.

Confocal laser scanning microscopy (CLSM) was successfully used to observe the effect of milk processing on the size and the morphology of the milk fat globule in raw milk, raw ultrafiltered milk, and standardized and pasteurized milk prepared for cheese manufacture (cheese-milk) and commercial pasteurized and homogenized milk. Fat globule size distributions for the milk preparations were analyzed using both image analysis and light scattering and both measurements produced similar data trends. Changes to the native milk fat globule membrane (MFGM) were tracked using a MFGM specific fluorescent stain that allowed MFGM proteins and adsorbed proteins to be differentiated on the fat globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the identity of native MFGM proteins isolated from the surface of fat globules within raw, UF retentate, and cheese-milk preparations, whereas only casein was detected on the surface of fat globules in homogenized milk. The microstructure, porosity, and gel strength of the rennet induced gel made from raw milk and cheese-milk was also found to be comparable and significantly different to that made from homogenized milk. Our results highlight the potential use of CLSM as a tool to observe the structural details of the fat globule and associated membrane close to its native environment.

Probiotic cheddar cheese: influence of ripening temperatures on proteolysis and sensory characteristics of cheddar cheeses.

Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI B94, Lactobacillus casei 279, Lb. casei LAFTI L26, Lb. acidophilus 4962, or Lb. acidophilus LAFTI L10 were used as an adjunct in the production of Cheddar cheeses, which were ripened at 4 and 8 degrees C for 24 wk. Effects of ripening temperatures and probiotic adjuncts on proteolysis and sensory evaluation of the cheeses were examined. Higher ripening temperature increased the level of proteolysis in the cheeses. Product of proteolysis and organic acids released during ripening were shown to be important for the flavor of Cheddar cheeses. There were positive and significant correlations between the levels of soluble nitrogen, lactic, acetic, and butyric acids, percentage hydrolysis of alpha(s1)-CN and beta-CN to the scores of cheddary flavor (P < 0.05). Scores for sour-acid and vinegary flavors were higher in cheeses with the addition of Bifidobacterium sp. or Lb. casei 279 ripened at 8 degrees C. The scores were positively and significantly correlated to the level of lactic, acetic, and free amino acids in the cheeses (P < 0.05). The results show that both 4 and 8 degrees C have potential for use in the ripening of probiotic Cheddar cheeses.

Microstructure and Composition of Full Fat Cheddar Cheese Made with Ultrafiltered Milk Retentate

Milk protein is often standardised prior to cheese-making using low concentration factor ultrafiltration retentate (LCUFR) but the effect of LCUFR addition on the microstructure of full fat gel, curd and Cheddar cheese is not known. In this work, Cheddar cheeses were made from cheese-milk with or without LCUFR addition using a protein concentration of 3.7%–5.8% w/w. The fat lost to sweet whey was higher in cheese made from cheese-milk without LCUFR or from cheese-milk with 5.8% w/w protein. At 5.8% w/w protein concentration, the porosity of the gel increased significantly and the fat globules within the gel and curd tended to pool together, which possibly contributed to the higher fat loss in the sweet whey. The microstructure of cheese from cheese-milk with a higher protein concentration was more compact, consistent with the increased hardness, although the cohesiveness was lower. These results highlight the potential use of LCUFR for the standardization of protein concentration in cheese-milk to 4%–5% w/w (equivalent to a casein to total protein ratio of 77%–79% w/w) to increase yield. Beyond this concentration, significant changes in the gel microstructure, cheese texture and fat loss were observed.

Nano-mechanical properties of clay-armoured emulsion droplets

There has been a growing interest in understanding the stabilization mechanism of particle-armoured emulsion droplets over the last few decades due to their importance in many everyday products. Here, the mechanical properties of clay-armoured emulsion droplets were investigated using laser scanning confocal microscopy with an in situatomic force microscopy measurement. This combination allows the visualization of droplet shape as a function of applied force. The emulsion droplets were found to be mechanically robust, stable against coalescence during drop collisions and able to recover from large deformations without disintegration. A Hookean constitutive law was used to extract the surface Youngs modulus of the clay-armoured droplets as a function of a range of solution conditions. The clay-armoured droplets were relatively insensitive to changes in solution ionic strength and pH. However, in the presence of cationic surfactants, the surface Youngs modulus decreases and shows significant reduction well above the critical micelle concentration. These changes are most likely due to desorption of clay platelets from the oil–water interface after charge neutralisation and the eventual solubilisation of the oil droplet. The elasticity measurements in this study should help illuminate the impact of the clay-armoured droplets on macroscopic properties of emulsions including rheological properties and emulsion stability.

Effect of calcium chloride addition and draining pH on the microstructure and texture of full fat Cheddar cheese during ripening.

Calcium chloride is commonly added to cheese-milk to improve coagulum formation and to increase cheese yield but high concentrations of calcium ions can have adverse effects. In this study, confocal laser scanning microscopy and cryo-scanning electron microscopy were coupled with textural and chemical analyses to observe microstructural and biochemical changes that occur in cheese during ripening when calcium chloride is added or the draining pH altered. For the cheese prepared with no additional calcium at a draining pH of 6.0, the cheese porosity increased with ripening time and the number of protein vertices in the microscopy images declined, indicative of protein solubilisation. As the amount of CaCl2 added was increased, however, these changes became less significant. Our findings show that calcium chloride addition can be used, together with a lower draining pH, to alter the manufacturing process without significantly impacting on the quality of the mature cheese.

The dynamics of the biological membrane surrounding the buffalo milk fat globule investigated as a function of temperature.

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.

Chapter 33 – Cheddar Cheese and Related Dry-Salted Cheese Varieties

This chapter describes the manufacture of Cheddar cheese and related dry-salted cheese varieties. Curd formation, whey separation, cheddaring, milling, salting, and pressing are described in detail, including an illustration of how the microstructure of this cheese develops. The chapter also explains the main factors that determine Cheddar cheese quality including the chemical composition, texture, and flavour of the cheese. It summarizes the role of lipolysis, proteolysis, starter, nonstarter lactic acid bacteria, or adjunct cultures in the development of Cheddar flavor during ripening. Although there is no universal standard for measuring Cheddar quality, different grading and assessments of Cheddar cheese are also presented including sensory evaluation and the use of instrumental analysis to measure key aspects of Cheddar cheese ripening.

Microstructure and physicochemical properties reveal differences between high moisture buffalo and bovine Mozzarella cheeses

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.

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy

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.