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-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.

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.

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.

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.

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.

A method for measuring investigative journalism in local newspapers.

Major changes to the operation of local newsrooms-ownership restructuring, layoffs, and a reorientation away from print advertising-have become commonplace in the last few decades. However, there have been few systematic attempts to characterize the impact of these changes on the types of reporting that local newsrooms produce. In this paper, we propose a method to measure the investigative content of news articles based on article text and influence on subsequent articles. We use our method to examine over-time and cross-sectional patterns in news production by local newspapers in the United States over the past decade. We find surprising stability in the quantity of investigative articles produced over most of the time period examined, but a notable decline in the last 2 y of the decade, corresponding to a recent wave of newsroom layoffs.

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.

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.

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.

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.

Thermally coupled dark-anoxia incubation: A platform technology to induce auto-fermentation and thus cell-wall thinning in both nitrogen-replete and nitrogen-deplete Nannochloropsis slurries.

Nitrogen-deprived Nannochloropsis cells invested their fixed carbon into the accumulation of triacylglycerol and cell wall cellulose (thickness of N-replete cell walls = 27.8 ±â€¯5.8, N-deplete cell walls = 51.0 ±â€¯10.2 nm). In this study, the effect of nitrogen depletion on the ability of the cells to weaken their own cell walls via autolysis was investigated. Autolytic cell wall thinning was achieved in both N-replete and N-deplete biomass by incubating highly concentrated slurries in darkness at 38 °C. The incubation forced cells to anaerobically ferment their intracellular cellulose and resulted in 30-40% reduction in cell wall thickness for both biomass types. This wall depletion weakened the cells and increased the extent of cell rupture by mechanical force (from 42 to 78% for N-replete biomass, from 36 to 62% for N-deplete biomass). Importantly, autolysis did not adversely impact the amino acid content of protein-rich N-replete biomass or the fatty acid content of lipid-rich N-deplete biomass.

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.

Functionalised dairy streams: Tailoring protein functionality using sonication and heating.

Ultrasound can be used to modify the functional interactions between casein and whey proteins in dairy systems. This study reports on ongoing developments in understanding the effect of ultrasound and heating on milk proteins in systems with modified casein-whey protein ratios (97:3, 80:20 and 50:50), prepared from milk protein concentrates that were fractionated by microfiltration, based on protein size. Heating of concentrated casein streams (9% w/w) at 80.0 °C for up to 9 min resulted in reduced gelation functionality and increased viscosity, even in the absence of added whey proteins. 20 kHz ultrasonication at 20.8 W calorimetric power for 1 min was able to break protein aggregates formed during heating, resulting in improved gelation and reduced viscosity. Interestingly, when heated whey protein was recombined with unheated casein the gelation properties were similar to unheated controls. In contrast, when heat treated casein streams were recombined with unheated whey protein, the gel forming functionality was reduced. This study therefore shows that using specific combinations of heat and/or ultrasound, fractionated dairy streams can be tailored for specific functional outcomes.

Emulsifying properties of ruptured microalgae cells: Barriers to lipid extraction or promising biosurfactants?

A systematic investigation of the emulsifying properties of ruptured algae cells was performed for the first time. The slurry of ruptured algae cells was separated into different biomass fractions, namely the cell debris, the delipidated debris, the serum, and the lipid. The interfacial interactions of these biomass fractions with a nonpolar solvent (e.g. hexane or hexadecane) were characterized using pendant drop tensiometry and interfacial shear rheology. The stability of the different emulsions (formed by the different biomass fractions) was tested using analytical centrifugation. The extracted lipid was an excellent surfactant that reduced the interfacial tension, however, it was not effective at stabilizing the emulsions. The protein-rich serum produced a strong interfacial film that stabilized the emulsions against coalescence during centrifugation. The cell debris stabilized the emulsions to a lesser extent by adsorbing to the droplet surface, presumably via interactions with hydrophobic extracellular polymeric substances (EPS). However, neither the serum nor the cell debris were very effective surfactants, and required the presence of the lipid fraction to produce small emulsion droplets. When present together, the components exhibited competitive interfacial adsorption, which influenced emulsion stability. In particular, the interruption of the protein film by the presence of lipid or cell debris reduced the stability of the emulsions. This study provides a new mechanistic understanding of emulsification during wet lipid extraction from microalgae that will be useful for determining strategies to improve solvent recovery. The results also suggest potential for developing effective bioemulsifiers or biosurfactants from fractionated microalgae biomass for commercial application.