Evaluation of Extraction Techniques for Recovery of Microalgal Lipids under Different Growth Conditions.

Microalgal lipids contain a wide array of liposoluble bioactive compounds, but lipid extraction remains a critical limitation for their commercial use. An accelerated solvent extraction (ASE) was used to extract lipids from Chlamydomonas reinhardtii, Arthrospira platensis (Spirulina), and Chlorella vulgaris grown under either standard or nitrogen depletion conditions. Under standard growth conditions, ASE using methanol:chloroform (2:1), methyl tert-butyl ether (MTBE):methanol:water, and ethanol at 100 °C resulted in the highest recovery of total lipids (352 ± 30, 410 ± 32, and 127 ± 15 mg/g biomass from C. reinhardtii, C. vulgaris, and A. platensis, respectively). Similarly, the highest total lipid and triacylglycerols (TAGs) recovery from biomass cultivated under nitrogen depletion conditions was found at 100 °C using methanol:chloroform, for C. reinhardtii (total, 550 ± 21; TAG, 205 ± 2 mg/g biomass) and for C. vulgaris (total, 612 ± 29 mg/g; TAG, 253 ± 7 mg/g biomass). ASE with MTBE:methanol:water at 100 °C yielded similar TAG recovery for C. reinhardtii (159 ± 6 mg/g) and C. vulgaris (200 ± 4 mg/g). Thus, MTBE:methanol:water is suggested as an alternative substitute to replace hazardous solvent mixtures for TAGs extraction with a much lower environmental impact. The extracted microalgal TAGs were rich in palmitic (C16:0), stearic (C18:0), oleic (C18:1,9), linoleic (C18:2n6), and α-linolenic (C18:3n3) acids. Under nitrogen depletion conditions, increased palmitic acid (C16:0) recovery up to 2-fold was recorded from the biomasses of C. reinhardtii and C. vulgaris. This study demonstrates a clear linkage between the extraction conditions applied and total lipid and TAG recovery.

Octenyl succinic anhydride-modified amyloid protein fibrils demonstrate enhanced ice recrystallization inhibition activity and dispersibility.

Octenyl succinic anhydride (OSA) modification of amyloid proteins fibrils (APFs) was employed to improve dispersibility and ice recrystallization inhibition activity. OSA mainly reacted with the amino groups of APFs without significantly changing morphology. OSA-modified APFs (OAPFs) had lower pI, carried more negative charges, and were more hydrophobic. OSA-modification showed a pH-dependent effect on the dispersibility of fibrils. At pH 7.0, OSA-modification improved dispersibility and inhibited heat-induced gelation of fibrils at weakened electrostatic repulsion. OAPFs were more prone to aggregation with lower dispersity at acidic pH values and demonstrated stronger IRI activity than unmodified fibrils at pH 7.0. Our findings indicate OSA-modification favors the industrial application of APFs as an ice recrystallization inhibitor with enhanced dispersibility.

Role of Ca2+ in the pepsin-induced coagulation and in the dynamic in vitro gastric digestion behavior of casein micelles.

The effect of Ca2+ on pepsin-induced hydrolysis of κ-casein and subsequent coagulation of casein micelles was studied in a micellar casein (MC) solution at pH ≈ 6.0 at 37 °C without stirring. An NaCl-supplemented MC solution was used as a positive control to assess the effect of increased ionic strength after CaCl2 addition. Quantitative determination of the released para-κ-casein during the reaction using reverse-phase high-performance liquid chromatography showed that specific hydrolysis of κ-casein by pepsin was little affected by the addition of either CaCl2 or NaCl. However, rheological properties and microstructures of curds induced by pepsin hydrolysis depended markedly on the addition of salts. Addition of CaCl2 up to 17.5 mM facilitated coagulation, with decreases in coagulation time and critical hydrolysis degree, and increases in firming rate and maximum storage modulus (G'max); further addition of CaCl2 (22.5 mM) resulted in a lower G'max. Increased ionic strength to 52.5 mM by adding NaCl retarded the coagulation and resulted in a looser curd structure. In a human gastric simulator, MC, without the addition of CaCl2, did not coagulate until the pH decreased to ≈5.0 after ≈50 min of digestion. Addition of CaCl2 facilitated coagulation of casein micelles and resulted in more cohesive curds with dense structures during digestion, which slowed the emptying rate of caseins. At the same CaCl2 concentration, a sample with higher ionic strength coagulated more slowly. This study provides further understanding on the effect of divalent (Ca2+) ions and ionic strength on the coagulation of casein micelles and the digestion behavior of milk.

Cholesterol stabilization of phospholipid vesicles against bile-induced solubilization.

Sphingomyelin (SM) and cholesterol complex to form functional liquid-ordered (Lo) domains. It has been suggested that the detergent resistance of these domains plays a key role during gastrointestinal digestion of the milk fat globule membrane (MFGM), which is rich in both SM and cholesterol. Small-angle X-ray scattering was employed to determine the structural alterations that occur when milk sphingomyelin (MSM)/cholesterol, egg sphingomyelin (ESM)/cholesterol, soy phosphatidylcholine (SPC)/cholesterol, and milk fat globule membrane (MFGM) phospholipid/cholesterol model bilayer systems were incubated with bovine bile under physiological conditions. The persistence of diffraction peaks was indicative of multilamellar vesicles of MSM with cholesterol concentrations > 20 % mol, and also for ESM with or without cholesterol. The complexation of ESM with cholesterol is therefore capable of inhibiting the resulting vesicles from disruption by bile at lower cholesterol concentrations than MSM/cholesterol. After subtraction of background scattering by large aggregates in the bile, a Guinier fitting was used to determine changes in the radii of gyration (Rgs) over time for the biliary mixed micelles after mixing the vesicle dispersions with bile. Swelling of the micelles by phospholipid solubilization from vesicles was a function of cholesterol concentration, with less swelling of the micelles occurring as the cholesterol concentration was increased. With 40% mol cholesterol, the Rgs of the bile micelles mixed with MSM/cholesterol, ESM/cholesterol, and MFGM phospholipid/cholesterol were equal to the control (PIPES buffer + bovine bile), indicating negligible swelling of the biliary mixed micelles.

Variation in milk fat globule size and composition: A source of bioactives for human health.

Milk fat globules (MFGs) are secreted from the mammalian gland and are composed of a triacylglycerol core surrounded by a triple membrane structure, the milk fat globule membrane (MFGM). The MFGM contains complex lipids and proteins reported to have nutritional, immunological, neurological and digestive functions. Human and ruminant milk are shown to share a similar MFG structure but with different size, profile and abundance of protein and polar lipids. This review summarizes the reported data on human, bovine, caprine and ovine MFG composition and concentration of bioactive components in different MFG-size fractions. A comprehensive understanding of compositional variations between milk from different species and MFG size fractions may help promote various milk sources as targeted supplements to improve human development and health. MFG size and MFGM composition are species-specific and affected by lactation, diet and breed (or maternal origin). Purification and enrichment methods for some bioactive proteins and lipids present in the MFGM have yet to be established or are not scaled sufficiently to be used to supplement human diets. To overcome this problem, MFG size selection through fractionation or herd selection may provide a convenient way to pre-enrich the MFG fraction with specific protein and lipid components to fulfill human dietary and health requirements.

Pepsin-induced coagulation of casein micelles: Effect of whey proteins and heat treatment.

The effect of whey proteins and heat treatment (90 °C, 5 min) on pepsin-induced hydrolysis of κ-casein, and subsequent coagulation of casein micelles, was investigated at pH 6.3 and 6.0 using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. Whey proteins did not affect the hydrolysis of κ-casein but retarded the coagulation process. Heat treatment did not affect the hydrolysis kinetics in whey protein (WP)-free samples, but slightly impaired the hydrolysis rate in WP-containing samples. The coagulation process of WP-free samples was little affected by heat-treatment. However, compared with unheated WP-contained sample at the same pH, the coagulation process of the heated sample was retarded at pH 6.3 but enhanced at pH 6.0. The curd in heated samples with smaller pores had higher water holding capacity. This knowledge provides further understanding on the role of whey proteins and heat treatment on the coagulation mechanisms of milk under gastric conditions.

Kinetics of pepsin-induced hydrolysis and the coagulation of milk proteins.

Hydrolysis-induced coagulation of casein micelles by pepsin occurs during the digestion of milk. In this study, the effect of pH (6.7-5.3) and pepsin concentration (0.110-2.75 U/mL) on the hydrolysis of κ-casein and the coagulation of the casein micelles in bovine skim milk was investigated at 37°C using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. The hydrolysis of κ-casein followed a combined kinetic model of first-order hydrolysis and putative pepsin denaturation. The hydrolysis rate increased with increasing pepsin concentration at a given pH, was pH dependent, and reached a maximum at pH ∼6.0. Both the increase in pepsin concentration and decrease in pH resulted in a shorter coagulation time. The extent of κ-casein hydrolysis required for coagulation was independent of the pepsin concentration at a given pH and, because of the lower electrostatic repulsion between para-casein micelles at lower pH, decreased markedly from ∼73% to ∼33% when pH decreased from 6.3 to 5.3. In addition, the rheological properties and the microstructures of the coagulum were markedly affected by the pH and the pepsin concentration. The knowledge obtained from this study provides further understanding on the mechanism of milk coagulation, occurring at the initial stage of transiting into gastric conditions with high pH and low pepsin concentration.

Cholesterol-phospholipid interactions resist the detergent effect of bovine bile.

Sphingomyelin (SM) and cholesterol complexation gives rise to detergent-resistant liquid-ordered domains. The persistence of these domains and subsequent mixed micelle formation was examined in the presence of bile under physiological digestive in vitro conditions for vesicles comprising either SM/cholesterol, porcine brain phosphatidylcholine (BPC)/cholesterol, or soy phosphatidylcholine (SPC)/cholesterol bilayers, the latter two systems having no liquid-ordered domains. Micellization of these digested phospholipid multilamellar vesicle systems was confirmed by transmission electron microscopy. Bovine bile was found to consist of large multilamellar sheets which subsumed phospholipid vesicles to form aggregated superstructures. Budding off from these superstructures were vesicle-to-micelle transition intermediates: unilamellar vesicles and cylindrical micelles. The presence of cholesterol (60/40 phospholipid/cholesterol mol/mol) delayed the initial rapid onset of digestion, but not for BPC and SPC vesicle systems. Acyl chain order/disorder before and after vesicle-to-micelle transition of all three phospholipid/cholesterol systems was examined using Raman spectroscopy. The addition of bovine bile to both PC/cholesterol vesicle systems reduced the overall ratio of acyl chain disorder to order. In SM/cholesterol vesicles with ≤ 20% mol cholesterol, only the lateral inter-acyl chain packing was reduced, whereas for SM/cholesterol vesicles with ≥ 30% mol cholesterol, a higher proportion of gauche-to-trans isomerization was apparent, demonstrating that SM/cholesterol complexes modify the acyl chain structure of micelles.

Addition of milk to tea infusions: Helpful or harmful? Evidence from in vitro and in vivo studies on antioxidant properties.

Tea consumption is practised as a tradition, and has shown potential to improve human health. Maximal uptake of tea antioxidants and milk proteins without a negative impact on tea flavor is highly desired by consumers. There is a conflicting evidence of the effect of milk addition to tea on antioxidant activity. Differences in the type of tea, the composition, type and amount of milk, preparation method of tea-milk infusions, the assays used to measure antioxidant activity, and sampling size likely account for different findings. Interactions between tea polyphenols and milk proteins, especially between catechins and caseins, could account for a decrease in antioxidant activity, although other mechanisms are also possible, given the similar effects between soy and bovine milk. The role of milk fat globules and the milk fat globule membrane surface is also important when considering interactions and loss of polyphenolic antioxidant activity, which has not been addressed in the literature.

Molecular interactions between green tea catechins and cheese fat studied by solid-state nuclear magnetic resonance spectroscopy.

Molecular integrations between green tea catechins and milk fat globules in a cheese matrix were investigated using solid-state magic angle spinning nuclear magnetic resonance spectroscopy. Full-fat cheeses were manufactured containing free catechin or free green tea extract (GTE), and liposomal encapsulated catechin or liposomal encapsulated GTE. Molecular mobility of the carbon species in the cheeses was measured by a wide-line separation technique. The (1)H evolution frequency profile of the (13)C peak at 16ppm obtained for the control cheese and cheeses containing encapsulated polyphenols (catechin or GTE) were similar, however, the spectrum was narrower for cheeses containing free polyphenols. Differences in spectral width indicates changes in the molecular mobility of --CH3- or -C-C-PO4- species through hydrophobic and/or cation-π associations between green tea catechins and cheese fat components. However, the similar spectral profile suggests that encapsulation protects cheese fat from interaction with catechins.

Effects of (+)-Catechin on the Composition, Phenolic Content and Antioxidant Activity of Full-Fat Cheese during Ripening and Recovery of (+)-Catechin after Simulated In Vitro Digestion.

(+)-Catechin, the representative catechin in green tea, was incorporated into a full-fat cheese (at 125-500 ppm) followed by ripening for 90 days at 8 °C and digesting for six hours. Determination of pH, proximate composition, total phenolic content (TPC) and antioxidant activity (AA) after manufacture and ripening demonstrated that the addition of (+)-catechin significantly (p ≤ 0.05) decreased the pH of both whey and curd during cheese manufacturing and ripening with no significant (p > 0.05) effect on the moisture, protein and fat contents. (+)-Catechin increased TPC, as well as AA, though the increase was not proportional with increasing the concentration of added (+)-catechin. About 57%-69% of (+)-catechin was retained in the cheese curd, whereas about 19%-39% (depending on the concentration) was recovered from the cheese digesta. Transmission electron micrographs showed that the ripened control cheese had a homogeneous pattern of milk fat globules with regular spacing entrapped in a homogenous structure of casein proteins, whereas the addition of (+)-catechin disrupted this homogenous structure. The apparent interaction between (+)-catechin and cheese fat globules was confirmed by Fourier transform infrared spectroscopy. These associations should be taken into account when incorporating antioxidants, such as (+)-catechin, to create functional dairy products, such as cheese.

A novel functional full-fat hard cheese containing liposomal nanoencapsulated green tea catechins: manufacture and recovery following simulated digestion.

(+)-Catechin or green tea extract were encapsulated in soy lecithin nanoliposomes and incorporated into a full-fat cheese, then ripened at 8 °C for 90 days. Cheese samples were subjected to simulated gastrointestinal digestion to measure total phenolic content (TPC) and antioxidant activity of the cheese digesta, and to determine the catechin recovery after digestion by high performance liquid chromatography (HPLC). Addition of catechin or green tea extract significantly (P ≤ 0.05) increased TPC and antioxidant activity (measured by ferric reducing antioxidant power and oxygen radical absorbance capacity) of the full-fat cheese without affecting pH or proximate composition. HPLC analysis confirmed retention of encapsulated catechins in the cheese curd; however, individual catechins were recovered in differing amounts (15-52%) from cheese digesta after 6 h of digestion. Transmission electron microscopy and Fourier transform infrared spectroscopy provided evidence for association of nanoliposomes with the surface of milk fat globules inside the cheese matrix. The study shows the potential for using cheese as a delivery vehicle for green tea antioxidants.

The behaviour of green tea catechins in a full-fat milk system under conditions mimicking the cheesemaking process.

Due to their well-known health benefits, green tea catechins have received recent attention as natural additives in foods such as dairy products. However, they may present some irreversible associations with milk components (e.g. protein and milk fat globules). To investigate the behaviour of two important green tea catechins, (+)-catechin (C) and (-)-epigallocatechin gallate (EGCG), in a standard whole milk system under the conditions of cheesemaking, 250 and 500 ppm of each catechin were added to whole milk (3.3% fat). Although both C and EGCG at either concentration increased both total phenolic content and total antioxidant capacity of the subnatants obtained from the milk system, there was a less linear increase when the concentration of the catechins was doubled, whereas C or EGCG were recovered (measured by HPLC) differently. Overall, these results suggest a degree of associations between green tea catechins with milk proteins and milk fat.

Thermal properties of milk fat, xanthine oxidase, caseins and whey proteins in pulsed electric field-treated bovine whole milk.

Thermodynamics of milk components (milk fat, xanthine oxidase, caseins and whey proteins) in pulsed electric field (PEF)-treated milk were compared with thermally treated milk (63 °C for 30 min and 73 °C for 15s). PEF treatments were applied at 20 or 26 kV cm(-1) for 34 µs with or without pre-heating of milk (55 °C for 24s), using bipolar square wave pulses in a continuous mode of operation. PEF treatments did not affect the final temperatures of fat melting (Tmelting) or xanthine oxidase denaturation (Tdenaturation), whereas thermal treatments increased both the Tmelting of milk fat and the Tdenaturation for xanthine oxidase by 2-3 °C. Xanthine oxidase denaturation was ∼13% less after PEF treatments compared with the thermal treatments. The enthalpy change (ΔH of denaturation) of whey proteins decreased in the treated-milk, and denaturation increased with the treatment intensity. New endothermic peaks in the calorimetric thermograms of treated milk revealed the formation of complexes due to interactions between MFGM (milk fat globule membrane) proteins and skim milk proteins. Evidence for the adsorption of complexes onto the MFGM surface was obtained from the increase in surface hydrophobicity of proteins, revealing the presence of unfolded hydrophobic regions.

Interactions between milk fat globules and green tea catechins.

The determination of putative chemical interactions between the milk fat globule membrane and green tea catechins provided useful information about the role of milk fat globules (MFGs) in high-fat dairy systems, such as cheese, and containing bioactive compounds, such as tea catechins. Catechins from green tea (125-1,000 ppm), including (+)-catechin, (-)-epigallocatechin gallate, and green tea extract were added to washed MFGs to examine possible interactions. The addition of catechins gave a significant change in the size and ζ-potential of MFGs. The recovery of different catechins from the milk fat globule suspensions was found to vary, suggesting selective association with the milk fat globule membranes. The interactions were further investigated using transmission electron microscopy and Fourier transform infra-red spectroscopy. It is suggested that catechins are localised in association with milk fat globule membrane domains as they contain both hydrophobic and hydrophilic moieties with potential points of molecular interaction.

Formulation of oil-in-water ß-carotene microemulsions: effect of oil type and fatty acid chain length.

The impact of oil type and fatty acid chain length on the development of food-grade microemulsions for the entrapment of ß-carotene was investigated. The microemulsion region of a ternary phase diagram containing short chain monoglycerides was larger than for di- and triglycerides when Tween 80 was used as surfactant. The cytotoxicity of microemulsions composed of a 30% monoglyceride oil, 20% Tween 80 and 50% aqueous buffer were evaluated using an in vitro cell culture model (human epithelial colorectal adenocarcinoma, Caco-2). The cytotoxicity test showed that the viability of Caco-2 cells against ß-carotene microemulsions at concentrations of 0.03125% (v/v) was higher than 90%. This study suggests that short chain monoglycerides could be used with Tween 80 to prepare transparent ß-carotene-encapsulated O/W microemulsions in the particle size range of 12-100 nm.

Lateral lipid organization of the bovine milk fat globule membrane is revealed by washing processes.

Evidence for the asymmetric distribution of phospholipids in the milk fat globule membrane (MFGM) was obtained by applying 3 washing processes using aqueous solutions with different degrees of stringency (mild, intermediate, and intensive) to milk fat globule (MFG) surfaces in simulated milk ultrafiltrate buffer. We detected no change in the amount of cholesterol after the mild washing process; however, intensive washing yielded a relative enrichment of surface cholesterol with concomitant damage to the outer bilayer of the MFGM. This finding supports the hypothesis of repartitioning of cholesterol on MFG surfaces during mechanical treatments. An updated model system of lipid organization was developed according to the results of relative depletion of individual phospholipids, as analyzed by HPLC.

Phospholipid Architecture of the Bovine Milk Fat Globule Membrane Using Giant Unilamellar Vesicles as a Model.

Giant unilamellar vesicles (GUVs) were constructed using an electroformation technique to mimic the morphology of the native milk fat globule membrane (MFGM) for the purpose of structural investigation. Bovine milk derived phospholipids were selected to manufacture GUVs which were characterized by confocal laser scanning microscopy after fluorescent staining. Circular nonfluorescent dark regions were observed in a 3/7 (mol/mol) surface mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine. Linear shaped dark lipid domains were found in GUVs containing sphingomyelin (SM) in the absence of cholesterol. The dark regions were interpreted as a gel phase formed by a high gel-liquid phase transition temperature (Tm) of DPPC and SM. This study provides a strategy for investigating the lipid structural organization within the native MFGM using a model lipid bilayer system and reveals that a SM and cholesterol association network is not the only requirement for nonfluorescent lipid domain formation and that PE is preferably located in the inner leaflet of the phospholipid bilayer.

Delivery of green tea catechin and epigallocatechin gallate in liposomes incorporated into low-fat hard cheese.

The encapsulation of green tea catechin and epigallocatechin gallate (EGCG) in soy lecithin liposomes was examined at four concentrations (0%, 0.125%, 0.25% and 0.5% w/v), and inclusion in cheese at 0% and 0.25% w/v. The empty capsules had a mean diameter of 133nm and significantly (p<0.05) increased with the addition of catechin or EGCG. Electron microscopy revealed the lamellae and central core of the liposomes. Addition of antioxidants gave a significant (p<0.05) increase in the size of liposomes. Liposomes had surface potentials of -42.4 to -46.1mV with no significant difference between treatments, suggesting stable liposome systems. High efficiency (>70%) and yield (∼80%) were achieved from the incorporation of catechin or EGCG inside the liposome structure. Addition of either antioxidant increased the liposome phase transition temperature (>50°C). Nanocapsules containing these antioxidants were effectively retained within a low-fat hard cheese, presenting a simple and effective delivery vesicle for antioxidants.