Extraction and Characterization of Bioactive Compounds from Diverse Marine Microalgae and Their Potential Antioxidant Activities.

This study compared free and bound phenolic compounds in various marine microalgae species. It assessed total phenolic content (TPC), total flavonoid content (TFC) and total condensed tannin content (TCT) and their antioxidant capacities using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⋅+ ) radical cation-based assay and ferric ion reducing antioxidant power assay. Liquid chromatography-mass spectrometry (LC-MS) was also employed to characterize the phenolic profiling. Results showed that free phenolic compounds ranged from 1.83-6.45 mg GAE/g d. w., while bound phenolic compounds ranged from 4.03-26.03 mg GAE/g d. w., indicating significant differences. These variations were consistent across assays, highlining unique profiles in different species. A total 10 phenolics were found in these seven microalgae, including 1 phenolic acid, 6 flavonoids, 1 other polyphenol and 2 lignans. 4'-O-methyl-(-)-epigallocatechin 7-O-glucuronide and chrysoeriol 7-O-glucoside in microalgae were firstly reported in microalgal samples. These findings have implications for future applications in industries.

Ultrasound-induced protein restructuring and ordered aggregation to form amyloid crystals.

Amyloid crystals, a form of ordered protein aggregates documented relatively recently, have not been studied as extensively as amyloid fibres. This study investigates the formation of amyloid crystals with low frequency ultrasound (20 kHz) using ß-lactoglobulin, as a model protein for amyloid synthesis. Acoustic cavitation generates localised zones of intense shear, with extreme heat and pressure that could potentially drive the formation of amyloid structures at ambient bulk fluid temperatures (20 ± 1 °C). Thioflavin T fluorescence and electron microscopy showed that low-frequency ultrasound at 20 W/cm3 input power induced ß-stacking to produce amyloid crystals in the mesoscopic size range, with a mean length of approximately 22 µm. FTIR spectroscopy indicated a shift towards increased intermolecular antiparallel ß-sheet content. An increase in sonication time (0-60 min) and input power (4-24 W/cm3) increased the mean crystal length, but this increase was not linearly proportional to sonication time and input power due to the delayed onset of crystal growth. We propose that acoustic cavitation causes protein unfolding and aggregation and imparts energy to aggregates to cross the torsion barrier, to achieve their lowest energy state as amyloid crystals. The study contributes to a further understanding of protein chemistry relating to the energy landscape of folding and aggregation. Ultrasound presents opportunities for practical applications of amyloid structures, presenting a more adaptable and scalable approach for synthesis.

The assimilation of glycerol into lipid acyl chains and associated carbon backbones of Nannochloropsis salina varies under nitrogen replete and deplete conditions.

Mixotrophic cultivation can increase microalgae productivity, yet the associated lipid metabolism remains mostly unknown. Stable isotope labeling was used to track assimilation of glycerol into the triacylglyceride (TAG) and membrane lipids of Nannochloropsis salina. In N-replete media, glycerol uptake and 13 C incorporation into acyl chains were, respectively, 6-fold and 12-fold higher than in N-deplete conditions. In N-replete cultures, 42% of the carbon in the consumed glycerol was assimilated into lipid acyl chains, mostly in membrane lipids rather than TAG. In N-deplete cultures, only 11% of the limited amount of consumed glycerol was fixed into lipid acyl chains. Labeled lipid-associated glycerol backbones were predominantly 13 C3 labeled, suggesting that intact glycerol molecules were directly esterified with fatty acids/polar head groups. However, the presence of singly and doubly labeled lipid-bound glycerol species suggested that some glycerol also went through the central carbon metabolism before forming glycerol-3-phosphate destined for lipid esterification. 13 C incorporation was higher in the saturated and monounsaturated than the polyunsaturated acyl chains of TAG, indicating the flux of carbon from glycerol went first to de novo fatty acid synthesis before acyl editing reactions. The results demonstrate that nitrogen availability influences both glycerol consumption and utilization for lipid synthesis in Nannochloropsis, providing novel insights for developing mixotrophic cultivation strategies.

A study of the effectiveness and energy efficiency of ultrasonic emulsification.

Three essential experimental parameters in the ultrasonic emulsification process, namely sonication time, acoustic amplitude and processing volume, were individually investigated, theoretically and experimentally, and correlated to the emulsion droplet sizes produced. The results showed that with a decrease in droplet size, two kinetic regions can be separately correlated prior to reaching a steady state droplet size: a fast size reduction region and a steady state transition region. In the fast size reduction region, the power input and sonication time could be correlated to the volume-mean diameter by a power-law relationship, with separate power-law indices of -1.4 and -1.1, respectively. A proportional relationship was found between droplet size and processing volume. The effectiveness and energy efficiency of droplet size reduction was compared between ultrasound and high-pressure homogenisation (HPH) based on both the effective power delivered to the emulsion and the total electric power consumed. Sonication could produce emulsions across a broad range of sizes, while high-pressure homogenisation was able to produce emulsions at the smaller end of the range. For ultrasonication, the energy efficiency was higher at increased power inputs due to more effective droplet breakage at high ultrasound intensities. For HPH the consumed energy efficiency was improved by operating at higher pressures for fewer passes. At the laboratory scale, the ultrasound system required less electrical power than HPH to produce an emulsion of comparable droplet size. The energy efficiency of HPH is greatly improved at large scale, which may also be true for larger scale ultrasonic reactors.

Computational models of populations of bacteria and lytic phage.

The use of phages to control and reduce numbers of unwanted bacteria can be traced back to the early 1900s, when phages were explored as a tool to treat infections before the wide scale use of antibiotics. Recently, phage therapy has received renewed interest as a method to treat multiresistant bacteria. Phages are also widely used in the food industry to prevent the growth of certain bacteria in foods, and are currently being explored as a tool for use in bioremediation and wastewater treatment. Despite the large body of biological research on phages, relatively little attention has been given to computational modeling of the population dynamics of phage and bacterial interactions. The earliest model was described by Campbell in the 1960s. Subsequent modifications to this model include partial or complete resistance, multiple phage binding sites, and spatial heterogeneity. This review provides a general introduction to modeling of the population dynamics of bacteria and phage. The review introduces the basic model and relevant concepts and evaluates more complex variations of the basic model published to date, including a model of disease epidemics caused by infectious bacteria. Finally, the shortcomings and potential ways to improve the models are discussed.

Nitrogen deprivation of microalgae: effect on cell size, cell wall thickness, cell strength, and resistance to mechanical disruption.

Nitrogen deprivation (N-deprivation) is a proven strategy for inducing triacylglyceride accumulation in microalgae. However, its effect on the physical properties of cells and subsequently on product recovery processes is relatively unknown. In this study, the effect of N-deprivation on the cell size, cell wall thickness, and mechanical strength of three microalgae was investigated. As determined by analysis of micrographs from transmission electron microscopy, the average cell size and cell wall thickness for N-deprived Nannochloropsis sp. and Chlorococcum sp. were ca. 25% greater than the N-replete cells, and 20 and 70% greater, respectively, for N-deprived Chlorella sp. The average Young's modulus of N-deprived Chlorococcum sp. cells was estimated using atomic force microscopy to be 775 kPa; 30% greater than the N-replete population. Although statistically significant, these microstructural changes did not appear to affect the overall susceptibility of cells to mechanical rupture by high pressure homogenisation. This is important as it suggests that subjecting these microalgae to nitrogen starvation to accumulate lipids does not adversely affect the recovery of intracellular lipids.

Impact of nutrient deficiency on biological sewage treatment - Perspectives towards urine source segregation.

Human urine contains 9 g/L of nitrogen (N) and 0.7 g/L of phosphorus (P). The recovery of N and P from urine helps close the nutrient loop and increase resource circularity in the sewage treatment sector. Urine contributes an average of 80 % N and 50 % P in sewage, whereby urine source segregation could reduce the burden of nutrient removal in sewage treatment plants (STPs) but result in N and P deficiency and unintended negative consequences. This review examines the potential impacts of N and P deficiency on the removal of organic carbon and nutrients, sludge characteristics and greenhouse gas emissions in activated sludge processes. The details of how these impacts affect the operation of STPs were also included. This review helps foresee operational challenges that established STPs may face when dealing with nutrient-deficient sewage in a future where source separation of urine is the norm. The findings indicate that the requirement of nitrification-denitrification and biological P removal processes could shrink at urine segregation above 80 % and 100 %, respectively. Organic carbon, N and biological P removal processes can be severely affected under full urine segregation. The decrease in solid retention time due to urine segregation increases treatment capacity up to 48 %. Sludge flocculation and settleability would deteriorate due to changes in extracellular polymeric substances and induce various forms of bulking. Beneficially, N deficiency reduces nitrous oxide emissions. These findings emphasise the importance of considering and preparing for impacts caused by urine source segregation-induced nutrient deficiency in sewage treatment processes.

A quantitative analysis of microalgal lipids for optimization of biodiesel and omega-3 production.

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC-MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega-3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products.

Ultrasound-enhanced interfacial adsorption and inactivation of soy trypsin inhibitors.

In this study, liquid-liquid interfacial protein adsorption was proposed as a means of inactivating soy trypsin inhibitors (TIs, including Kunitz (KTI) and Bowman-Birk inhibitor (BBI)). Hexane-water was first selected as a model system to compare three emulsification methods (hand shaking, rotor-stator and ultrasound mixing). Ultrasound could generate the smallest and least polydisperse emulsion droplets, resulting in highest interfacial adsorption amount of KTI and BBI as well as the highest inactivation percentage of TIs (p < 0.05). Therefore, ultrasound was selected to further explore the effect of the non-aqueous phase on interfacial adsorption and inactivation kinetics of TIs in a food emulsion system containing vegetable oil (VTO). The adsorption amounts of KTI and BBI in the VTO-aqueous emulsion increased by âˆ¼ 25 % compared to the hexane-aqueous emulsion. In addition, the adsorption amounts of KTI and BBI were rapidly increased as a function of sonication time, especially for the hexane-aqueous emulsion system. This result suggests that such inactivation of TIs could be implemented in continuous systems for large-scale processing. Finally, the pathways of interface-induced inactivation of BBI and KTI were investigated based on separate experiments on individual BBI and KTI systems. The results showed that the interface adsorption caused the changes in the secondary and tertiary structure of KTI that led to its activitation. However, BBI was quite stable at the liquid-liquid interface without significant conformational change. Overall, ultrasound-assisted interfacial adsorption can be considered a rapid and highly efficient method to inactivate KTI.

Kinetic and mechanistic study of ultrasonic inactivation of Kunitz (KTI) and Bowman-Birk (BBI) inhibitors in relation to process-relevant parameters.

In this study, quantitative monitoring of low-frequency (20 kHz) and high-frequency (355 kHz) ultrasound-induced inactivation of Kunitz (KTI) and Bowman-Birk inhibitor (BBI) using RP-HPLC was achieved, and its consistency with a traditional TI activity assay was verified. The effect of TI concentration, ultrasonic frequency, power density and pH on inactivation kinetics of KTI and BBI was explored. Results showed that the pseudo-first-order kinetic rate constants of KTI and BBI were decreased by over 60% when the initial TI concentration was increased from 100 mg/L to 1000 mg/L. Also, the amounts of inactivated KTI and BBI were increased by around 4-fold at the higher TI concentration of 1000 mg/L (20 kHz, 1.71 W/mL and pH 4). The colloidal environment and ultrasonic conditions influenced the secondary and tertiary structure and particle size of TIs in LF-induced inactivation. In comparison, the abovementioned conditions affected the oxidation of methionine and the conformational change of TIs in HF-induced inactivation.

The Impact of High-Intensity Ultrasound-Assisted Extraction on the Structural and Functional Properties of Hempseed Protein Isolate (HPI).

Hempseed protein has become a promising candidate as a future alternative protein source due to its high nutritional value. In the current study, hempseed protein isolate (HPI) was obtained using ultrasonic-assisted extraction with the aim to improve the functionality of HPI via protein structure modification. The solubility of HPI could be improved twofold under 20 kHz ultrasound processing compared to conventional alkaline extraction-isoelectric point precipitation. The protein solubility was gradually enhanced as the ultrasonic power improved, whereas excessive ultrasound intensity would cause a decline in protein solubility. Ultrasonic processing was found to have beneficial effects on the other functionalities of the extracted HPI, such as emulsifying and foaming properties. This improvement can be ascribed to the physical effects of acoustic cavitation that changed the secondary and tertiary structures of the protein to enhance surface hydrophobicity and decrease the particle size of the extracted protein aggregates. In addition, more available thiols were observed in US-treated samples, which could be another reason for improved functionality. However, the results of this study also revealed that prolonged high-power ultrasound exposure may eventually have a detrimental impact on HPI functional properties due to protein aggregation. Overall, this study suggests that high intensity ultrasound can enhance the functionality of HPI, which may ultimately improve its value in HPI-based food products.

Bioaccessibility and bioactivities of phenolic compounds from microalgae during in vitro digestion and colonic fermentation.

Microalgae are a developing novel source of carbohydrates, phenolic compounds, carotenoids and proteins. In this study, in vitro digestion and colonic fermentation were conducted to examine the total phenolic content and potential antioxidant activity of four microalgal species (Chlorella sp., Spirulina sp., Dunaliella sp., and Isochrysis sp.). The bioaccessibility of targeted phenolic compounds and the short-chain fatty acid (SCFA) production were also estimated. Particularly, Spirulina sp. exhibited the highest total phenolic content (TPC) and free radical scavenging (2,2'-diphenyl-1-picrylhydrazyl, DPPH) capacity after gastrointestinal digestion of 7.93 mg gallic acid equivalents (GAE) per g and 2.35 mg Trolox equivalents (TE) per g. Meanwhile, it had the highest total flavonoid content (TFC) of 1.07 quercetin equivalents (QE) per g after 8 h of colonic fermentation. Dunaliella sp. and Isochrysis sp. showed comparable ferric reducing antioxidant power (FRAP) of 4.96 and 4.45 mg QE per g after 4 h of faecal reaction, respectively. p-hydroxybenzoic and caffeic acid almost completely decomposed after the intestine and fermented in the colon with the gut microflora. In Dunaliella sp. and Isochrysis sp., these phenolic acids were found in the colonic fermented residual, probably due to the presence of dietary fibre and the interactions with other components. All four species reached the highest values of SCFA production after 16 h, except Spirulina sp., which displayed the most increased total SCFA production after 8 h of fermentation. It is proposed that Spirulina sp. could be more beneficial to gut health.

Evaporative concentration of skimmed milk: effect on casein micelle hydration, composition, and size.

Understanding the effect of evaporative concentration on casein micelle composition is of high importance for milk processing. Alterations to the hydration, composition and size of casein micelles were investigated in skimmed milk evaporated to concentrations of 12-45% total solids content. The size of casein micelles was determined by dynamic light scattering, and the water content and composition determined by analysis of supernatants and pellets obtained by ultracentrifugation. The mass balance and hydration results showed that during the evaporation process, while micelles were dehydrated, water was removed preferentially from the serum. The amount of soluble casein and calcium in the serum decreased as a function of increasing solids content, indicating a shift of these components to the micelles. The formation of a small proportion of micelle aggregates at high concentrations appeared dependent on the time kept at these concentrations. Upon redilution with water, casein micelles were immediately rehydrated and aggregates were broken up in a matter of minutes. Soluble calcium and pH returned to their original state over a number of hours; however, only a small percentage of original soluble casein returned to the serum over the 5h period investigated. These results showed that casein micelles are significantly affected by evaporative concentration and that the alterations are not completely and rapidly reversible.

Turbulence-induced formation of emulsion gels.

Emulsion gels have a wide range of applications. We report on a facile and versatile method to produce stable emulsion gels with tunable rheological properties. Gel formation is triggered by subjecting a mixture containing aqueous colloidal particle (CP) suspensions and water-immiscible liquids to intense turbulence, generated by low frequency (20 kHz) ultrasound or high-pressure homogenization. Through systematic investigations, requisite gel formation criteria are established with respect to both formulation and processing, including ratio/type of liquid pairs, CP properties, and turbulence conditions. Based on the emulsion microstructure and rheological properties, inter-droplet bridging and CP void-filling are proposed as universal stabilization mechanisms. These mechanisms are further linked to droplet-size scaling and sphere close-packing theory, distinctive from existing gel-conferring models. The study thereby provides the foundation for advancing the production of emulsion gels that can be tailored to a wide range of current and emerging applications in the formulation and processing of food, cosmetics or pharmaceutical gels, and in material science.

Mechanism of low-frequency and high-frequency ultrasound-induced inactivation of soy trypsin inhibitors.

In this study, the effect of ultrasonic frequency and power on the inactivation of soy trypsin inhibitors (TIs) was investigated to explore the ultrasound-induced inactivation mechanism. It was observed that 20 kHz and 355 kHz ultrasound have better inactivation efficiency than 1056 kHz. First-order rate constants for the inactivation process were obtained, which increased with increasing ultrasonic power at both 20 kHz and 355 kHz. For 20 kHz ultrasound, the formation of TI aggregates resulting from the physical effects of acoustic cavitation decreased the interactions between the active sites of TIs and trypsin, thus reducing the TI activity. For 355 kHz ultrasound, most of the methionine in the TIs was oxidised within 5 mins, resulting in a faster reduction of TI activity. Subsequent aggregation of TIs resulted in further TI inactivation. SDS-PAGE showed that neither disulphide bonds nor CC coupling were involved in the formation of aggregates.

Innovative Technologies for Extraction and Microencapsulation of Bioactives from Plant-Based Food Waste and their Applications in Functional Food Development.

The by-products generated from the processing of fruits and vegetables (F&V) largely are underutilized and discarded as organic waste. These organic wastes that include seeds, pulp, skin, rinds, etc., are potential sources of bioactive compounds that have health imparting benefits. The recovery of bioactive compounds from agro-waste by recycling them to generate functional food products is of increasing interest. However, the sensitivity of these compounds to external factors restricts their utility and bioavailability. In this regard, the current review analyses various emerging technologies for the extraction of bioactives from organic wastes. The review mainly aims to discuss the basic principle of extraction for extraction techniques viz. supercritical fluid extraction, subcritical water extraction, ultrasonic-assisted extraction, microwave-assisted extraction, and pulsed electric field extraction. It provides insights into the strengths of microencapsulation techniques adopted for protecting sensitive compounds. Additionally, it outlines the possible functional food products that could be developed by utilizing components of agricultural by-products. The valorization of wastes can be an effective driver for accomplishing food security goals.

Interplay between interfacial behaviour, cell structure and shear enables biphasic lipid extraction from whole diatom cells (Navicula sp.).

HYPOTHESIS: Bacillariophyceae (i.e., diatoms) are an important class of algae with potential use in the production of proteins and lipids including long-chain ω-3 polyunsaturated fatty acids. Biphasic extraction of microalgae lipids using water-immiscible solvents such as hexane, can avoid the excessive energy required to distil solvents from water, but generally requires energy-intensive rupture of the cells. The unique cell structure and surface chemistry of diatoms compared to other microalgae species might allow biphasic lipid extraction without prior cell rupture. EXPERIMENTS: The kinetics of biphasic lipid extraction from intact Navicula sp. cells was investigated during low-shear and high-shear mixing, and with prior or simultaneous application of ultrasound (20 kHz at 0.57 W/mL). Dynamic interfacial tension measurements and electron microscopic analysis were used to investigate lipid extraction in relation to interfacial behaviour and cell structure. RESULTS: High yields (>80%) of intracellular lipids were extracted from intact cells over the course of hours upon low-shear contacting with hexane. The cells associated with and stabilised the hexane-water interface, allowing hexane to infiltrate pores in the frustule component of the cell walls and access the intracellular lipids. It was shown that mucilaginous extracellular polymeric substances (EPS) bound to the cell walls acted as a barrier to solvent penetration into the cells. This EPS could be removed by prior ultrasonication. Biphasic extraction was greatly accelerated by shear applied by rotor-stator mixing or ultrasound. High-shear could remove mucilaginous EPS from the cell surfaces to facilitate direct contact of the cell surface with hexane and produced smaller emulsion droplets with increased surface area. The combination of high-shear in the presence of hexane resulted in the in-situ rupture of the cells, which greatly accelerated lipid extraction and allowed high yields of neutral lipid (>95%) to be recovered from freshly harvested cells within less than 5 min. The study demonstrated the ability of shear to enable simultaneous cell rupture and lipid extraction from a diatom alga based on its cell structure and interfacial behaviour.

Wastewater treatment using filamentous algae - A review.

Wastewater treatment using algae is a promising approach for efficient removal of contaminating nutrients and their conversion into useful products. Monocultures of filamentous algae provide easier harvesting compared to microalgae, and better control of biomass quality than polyculture systems such as algal turf scrubbers. In this review, recent research into wastewater treatment using freshwater filamentous algae is compiled and critically analysed. Focus is given to filamentous algae monocultures, with key relevant findings from microalgae and polyculture systems discussed and compared. The application of monocultures of filamentous algae is an emerging area of research. Gaps are identified in our understanding of key aspects important to large-scale system design, including criteria for species selection, influence of nutrient type and loading, inorganic carbon supply, algae-bacteria interactions, and parameters such as pond depth, mixing and harvesting regimes. This technology has much promise, however future research is needed to maximise productivity and wastewater treatment efficiency.

Effect of sonication, microwaves and high-pressure processing on ACE-inhibitory activity and antioxidant potential of Cheddar cheese during ripening.

Dairy processing provides acceptable safety and shelf-life to final products, and improves their bioactivity. The present study evaluated the potential of different milk processing techniques to improve the antioxidant and angiotensin-I converting enzyme (ACE)-inhibitory activity of Cheddar cheese, during ripening. Cheese was made from milk subjected to different pre-treatments (C = untreated control, US-1 = ultrasonication, specific energy = 23 J/g, 20 kHz frequency; US-2 = Ultrasonication specific energy = 41 J/g, 20 kHz; HPP = high-pressure processing, 400 MPa for 15 min, at temperature < 40 °C; MW = microwave, temperature<40 °C, specific energy = 86.5 J/g) and analysed after ripening for 0, 3, 6 and 9 months. The results showed that the rate of proteolysis during both cheese making and subsequent ripening was significantly affected by the pre-treatment. Antioxidant activity and ACE-inhibitory potential of cheeses made from pre-treated milk significantly increased (p < 0.05) in the following order: US-2 > HPP > US-1 > MW > C. These findings demonstrate the possibility of using ultrasound, microwaves or high-pressure processing as pre-treatments to improve the nutritional attributes of cheese.

Ultrasonic pretreatment of food waste to accelerate enzymatic hydrolysis for glucose production.

Recovering valuable materials from food waste by applying the concept of a bio-refinery is attracting considerable interest. To this effect, we investigated the possibility of enhancing the enzymatic hydrolysis of food waste using ultrasonic technology. The effect of pre-treating blended food waste with high-intensity ultrasound (20 kHz) on subsequent hydrolysis by glucoamylase was investigated as a function of sonication time and temperature. Particle sizing by laser diffraction, and imaging via scanning electronic microscopy showed that ultrasonic pre-treatment could reduce the particle size of the blended food waste significantly, resulting in a better interaction with the enzyme. As a consequence, the glucose yield of enzymatic hydrolysis was ∼10% higher for food waste pre-sonicated using the most intensive ultrasonication conditions studied (5 min sonication at a power of 0.8 W/mL at 20 °C) than for the untreated control. In addition, the time required to achieve high yields of glucose could be more than halved using ultrasonic pre-treatment. This could enable the hydrolysis reactor size or the enzyme usage to be reduced by more than 50%. Therefore, an ultrasound-assisted bioconversion process from food waste into a value-added product has been demonstrated.