Ultrasound-assisted processing: Science,technology and challenges for the plant-based protein industry

The present-day consumer is not only conscious of the relationship between food consumption and positive health, but also keen on environmental sustainability. Thus, the demand for plant-based proteins, which are associated with nutrition and environmental sustainability. However, the plant-based protein industry still demands urgent innovation due to the low yield and long extraction time linked with traditional extraction methods. Although ultrasound is an eco-innovative technique, there exist limited data regarding its impact with plant-based protein. In this paper, the scientific principles of ultrasonication with regards to its application in plant-based protein research were reviewed. After comparing the cavitational and shearing impacts of different ultrasonic parameters, the paper further reviewed its effects on extracted protein characteristics and techno-functional properties. Additionally, current technological challenges and future perspectives of ultrasonication for the plant-based protein industry were also discussed. In summary, this review does not only present the novelty and environmental sustainability of ultrasound as a plant-based protein assisted-extraction method, but also highlights on the correlation between protein source, structure and subsequent functional properties which are important crucial factors for maximum application of ultrasound in the growing plant-based protein market.     

Effect of ultrasound and enzymatic pre-treatment on yield and properties of banana juice

Effect of ultrasound and enzymatic pre-treatments with cellulase and pectinase on yield and properties of banana juice were investigated. A two-level full factorial design was employed. The factors selected were ultrasonication time (0 and 30 min), cellulase concentration (0 and 0.2%) and pectinase concentration (0 and 0.2%). The responses studied were yield, viscosity, clarity, total soluble solids (TSS) and pH. It was observed that pectinase was more effective in increasing the yield of juice compared to cellulase. Ultrasonic pre-treatment alone did not significantly increase the yield of juice. When ultrasound was combined with pre-treatment with both the enzymes maximum yield of 89.40% was obtained compared to 47.30% in the control. The viscosity of the juice decreased with addition of enzymes and with application of ultrasound. The clarity of the juice was not affected by cellulase treatment, but improved with pectinase treatment. Ultrasonication alone was found to be more effective than pectinase or cellulase treatment in improving the clarity of the juice. The TSS increased with enzymatic treatment, ultrasonication and their combination. pH was not affected by treatment type, but was found to be lower for the treated juices. Significant correlations were observed between the various responses.     

Ultrasonication promotes extraction of antioxidant peptides from oxhide gelatin by modifying collagen molecule structure

This study primarily explored the internal mechanism underlying the ultrasonication-induced release of antioxidant peptides. An oxhide gelatin solution was treated ultrasonically (power = 200, 300, and 400 W), followed by enzymatic hydrolysis and structural and morphological analysis. The results showed that ultrasonication increased not only the degree of hydrolysis (DH) and protein recovery rate of the oxhide gelatin but also the ABTS radical scavenging, DPPH radical scavenging, ferrous chelating, and ferric reducing activities of its hydrolysate. The oxhide gelatin hydrolysate treated with 300-W ultrasonication had the maximum antioxidant activities. Ultrasonication inhibited hydrogen bond formation, reduced the crosslinking between collagen molecules, transformed part of the folded structure into a helical structure, and lowered the thermal stability of collagen molecules. The micromorphological analysis revealed that ultrasonication caused the gelatin surface to become loose and develop cracks, and as the power of the ultrasonication increased, the repetition interval distance (dÅ) also increased. Moreover, ultrasonication improved the solubilization, surface hydrophobicity, and interface characteristics and increased the content of basic and aromatic amino acids in the hydrolysate. In conclusion, ultrasonication modifies the protein structure, which increases the enzyme’s accessibility to the peptide bonds and further enhances antioxidant peptide release. These findings provide new insights into the application of ultrasonication in the release of antioxidant peptides.     

Ultrasound for microalgal cell disruption and product extraction: A review

Microalgae are a promising feedstock for the production of biofuels, nutraceuticals, pharmaceuticals and cosmetics, due to their superior capability of converting solar energy and CO₂ into lipids, proteins, and other valuable bioactive compounds. To facilitate the release of these important biomolecules from microalgae, effective cell disruption is usually necessary, where the use of ultrasound has gained tremendous interests as an alternative to traditional methods. This review not only summarizes the mechanisms of and operation parameters affecting cell disruption, but also takes an insight into measuring techniques, synergistic integration with other disruption methods, and challenges of ultrasonication for microalgal biorefining. Optimal conditions including ultrasonic frequency, intensity, and duration, and liquid viscosity and sonochemical reactor are the key factors for maximizing the disruption and extraction efficiency. A combination of ultrasound with other disruption methods such as ozonation, microwave, homogenization, enzymatic lysis, and solvents facilitates cell disruption and release of target compounds, thus provides powerful solutions to commercial scale-up of ultrasound extraction for microalgal biorefining. It is concluded that ultrasonication is a sustainable “green” process, but more research and work are needed to upscale this process without sacrificing performance or consuming more energy.     

The promoted hydrolysis effect of cellulase with ultrasound treatment is reflected on the sonicated rather than native brown rice

Brown rice is nutritionally superior to polished white rice, as it maintains a large content of external bran that involves a series of bioactive compounds. However, the presence of bran also restricts water diffusion and results in adverse quality of brown rice. In this work, ultrasound conditions were optimized for cellulase to improve its hydrolysis effect on rice bran, and combinations of enzymatic and ultrasound treatment in different manners were conducted on brown rice, to improve the textural attributes. The results showed significant improvements in the catalytic activity and efficiency of cellulase after ultrasonication at the optimal intensity of 1.67 W cm⁻³ and duration of 30 min, with the conformational variation of cellulase observed from the fluorescence spectra and circular dichroism (CD). Despite the enhanced activity of ultrasonicated cellulase, it leaded to a similar rice surface morphology and a comparable amount of released glucose, and equivalent textural parameters of brown rice treated by native cellulase. However, for the pre-sonicated brown rice, the ultrasonicated cellulase showed a significantly higher hydrolysis capacity than the untreated enzyme, suggesting the important influence of ruptured bran surface on amplifying the hydrolysis effect of cellulase. Compared to the successive ultrasound stimulation on both cellulase and brown rice, ultrasound-assisted cellulase treatment on brown rice produced less glucose from rice bran, but induced similar textural properties of brown rice, possibly resulting from the simultaneously promoting effect of ultrasonication on cellulase and water diffusion. Ultimately, this study highlighted that the mild rice surface rupture is a crucial factor to display the promoted hydrolysis effect of ultrasonicated cellulase on brown rice. Ultrasound-assisted cellulase treatment potentially provides an effective strategy to improve the edible quality of brown rice.     

Inactivation of harmful Anabaena flos-aquae by ultrasound irradiation: Cell disruption mechanism and enhanced coagulation

Harmful algal blooms pose a potential threat to the safety of drinking water sources. Ultrasound is an effective method for algae removal. However, this method can lead to the release of algal organic matter and the effects and toxic mechanisms of ultrasound on Anabaena are still poorly understood. The destruction mechanism of Anabaena flos-aquae cells under different ultrasonic conditions, the safety of intracellular organic matter (IOM) release to water and the enhanced coagulation efficiency of ultrasound were studied. Results showed that high-frequency ultrasound was effective in breaking down algae cells. After 10 min ultrasonication at 20 kHz, 5 min at 740 kHz and 1 min at 1120 kHz, the algae cells were inactivated and algae growth was halted. Ultrasound radiation can lead to the release of IOM, primarily chlorophyll a and phycocyanin, followed by some tryptophan and humic substances, polysaccharides, and proteins. The sonicated ribosomes were considerably reduced, and the antioxidant system of cells was also damaged to some extent. The coagulation effect of algae cells was substantially improved after ultrasonication. Thus, the safety of algae cell removal could be improved by controlling the changes in physiological structure and IOM release of algae cells by adjusting the ultrasound parameters.     

Ultrasonic-assisted enzymatic extraction of sulfated polysaccharide from Skipjack tuna by-products

The effect of ultrasound pretreatment on extraction efficiency of sulfate polysaccharides (SPs) using alcalase from different by-products of Skipjack tuna including head, bone and skin was evaluated. Structural, functional, antioxidant and antibacterial properties of the recovered SPs using the ultrasound-enzyme and enzymatic method were also investigated. Ultrasound pretreatment significantly increased the extraction yield of SPs from all the three by-products compared with the conventional enzymatic method. All extracted SPs showed high antioxidant potential in terms of ABTS, DPPH and ferrous chelating activities where the ultrasound treatment enhanced antioxidant activities of the SPs. The SPs exerted strong inhibiting activity against various Gram-positive and Gram-negative bacteria. The ultrasound treatment remarkably increased antibacterial activity of the SPs against L. monocytogenes but its effect on other bacteria was dependent on the source of the SPs. Altogether, the results suggest that ultrasound pretreatment during enzymatic extraction of SPs from tuna by-products can be a promising approach to improve extraction yield but also bioactivity of the extracted polysaccharides.     

Ultrasonic extraction of plant materials--investigation of hemicellulose release from buckwheat hulls

Various one- and two-step extraction procedures with and without a short application of ultrasound at the beginning of the extraction were used to examine the effect of sonication on the extractibility of the hemicellulose components of buckwheat hulls. The polysaccharides recovered from the extracts were characterised by yield as well as composition determined by chemical methods and spectroscopic techniques. They comprised a complex of glucuronoxylan and co-extracted amylose-rich starch in various proportions contaminated with other cell wall components (protein, pectic polysaccharides). The hemicellulose fractions obtained by classical and ultrasound-assisted extraction exhibited significant immunomodulatory activities. The increased yield of ultrasonically extracted hemicelluloses, which have preserved their structural and molecular properties as well as immunological activity, confirmed the importance and great potential of ultrasound-assisted extraction of industrially important polysaccharides from different tissues of plant materials.     

Mechanisms underlying the effects of ultrasound-assisted alkaline extraction on the structural properties and in vitro digestibility of Tenebrio molitor larvae protein

Edible insects have been considered as a sustainable and novel protein source to replace animal-derived proteins. The present study aimed to extract Tenebrio molitor larvae proteins (TMP) using ultrasound-assisted alkaline extraction (UAE). Effects of different UAE times (10, 20, 30, 40, and 50 min) on the structural properties and in vitro digestibility of TMP were comparatively investigated with the traditional alkaline extraction method. The results revealed that ultrasonication could effectively alter the secondary/tertiary structures and thermal stability of TMP during UAE. The molecular unfolding and subsequent aggregation of TMP during UAE were mainly attributed to the formation of disulfide bonds and hydrophobic interactions. Moreover, TMP extracted by UAE had higher in vitro digestibility and digestion kinetics than those extracted without ultrasound, and the intermediate UAE time (30 min) was the optimal ultrasound parameter. However, longer UAE times (40 and 50 min) lowered the digestibility of TMP due to severe protein aggregation. The present work provides a potential strategy for the extraction of TMP with higher nutritional values.     

Conjoint application of ultrasonication and redox pair mediated free radical method enhances the functional and bioactive properties of camel whey-quercetin conjugates

Ultrasonication, redox-pair generated free radical method and their combination (Ultrasonication/redox-pair method) was used for production of camel whey-quercetin conjugates. FTIR and SDS-PAGE confirmed successful production of whey-quercetin conjugates using ultrasonication and ultrasonication/redox-pair method. FTIR suggested existence of covalent (appearance of new peak at 3399 cm⁻¹) and non-covalent linkages (shifting of peak at 3271 cm⁻¹, 1655 cm⁻¹ (amide I), 1534 cm⁻¹ and 1422 cm⁻¹ (Amide II)) in the whey-quercetin conjugates. Moreover, SDS-PAGE of conjugates produced by ultrasonication as well redox-pair method indicated shifting of protein bands slightly towards high molecular weight due to increase in the mass of proteins due to the binding of polyphenols. All conjugates showed improved techno-functional and bioactive properties in comparison to whey proteins. Conjugates produced through ultrasonication showed smaller particle size, improved solubility, emulsifying and foaming properties while conjugates produced through ultrasonication/redox-pair method depicted superior antioxidant properties in comparison to whey. Furthermore, conjugated samples showed higher inhibition of enzymatic markers involved in diabetes and obesity with highest potential recorded in conjugates produced using ultrasonication. Therefore, ultrasonication can be successfully used individually as well as in combination with redox-pair for production of whey-quercetin conjugates with enhanced bioactive and techno-functional properties.     

Ultrasonication as an innovative approach to tailor the apple seed proteins into nanosize: Effect on protein structural and functional properties

In this study, protein was extracted from the apple seed flour using alkali-acid precipitation method. The main objective of this study was to evaluate the impact of ultrasonication on structural and techno-functional properties of apple seed protein. Both native (N-protein) and ultra-sonicated protein (US-protein) were characterized for size, zeta potential, structure, protein pattern, crystallinity, thermal stability and functional properties. The results revealed that the hydrodynamic diameter of N-protein and US-protein was 1.2 µm and 484 nm while zeta potential was −11 and −19 mV, respectively. Fourier transform infrared-spectroscopy and X-ray diffraction analysis showed change in the conformational characteristics and functional groups of proteins after nano-reduction. SEM revealed change in the surface morphology of protein molecule upon ultrasonication. Differential scanning calorimetry showed decreased denaturation temperature for US-protein compared to N-protein . SDS-PAGE depicted no change in protein pattern upon ultrasonication. Ultrasonicated protein exhibited increased functional properties like emulsification, foaming, hydrophobicity and oil absorbing properties and hence can be efficiently used as functional ingredient in food and nutraceutical industry.     

Impact of ultrasound on extractability of native collagen from tuna by-product and its ultrastructure and physicochemical attributes

The effect of ultrasound treatment (300 W; 0, 5, 10, 15, 20 and 25 min) on the extractability of acid soluble collagen from yellowfin tuna skin and its structural, physicochemical and functional properties were investigated. Ultrasound treatments significantly increased collagen extraction yield from the tuna skin up to 2.7 times, compared to the conventional extraction with acetic acid. The level of proline, hydroxyproline and thermal stability of collagens increased by applying ultrasound while their native triple-stranded helical structure was well-preserved, as revealed by X-ray diffraction and FTIR spectroscopy. However, ultrasound treatment reduced the particle size of the collagens which increased their pH and salt induced solubility. The water holding capacity and the emulsifying properties of ultrasound treated collagens were also higher than those produced with the conventional method. Altogether, the results suggested that ultrasonication can be a promising assistant technology for improving native collagen extraction efficiency from tuna skin and its functionality but its duration should be carefully optimized.     

Ultrasound assisted enzymatic pre-treatment of high fat content dairy wastewater

This paper illustrates the application of ultrasound in a dairy waste water treatment for the removal of fat using enzyme as a catalyst. Lipase Z was used to perform the enzymatic pre-hydrolysis of a synthetic dairy wastewater containing around 2000 mg/L of fat content coupled with ultrasound irradiation. Different process parameters like effect of enzyme loading, temperature, ultrasound power, frequency, duty cycle and speed of agitation are optimized. The maximum hydrolysis of 78% is achieved at 0.2% enzyme loading (w/v), 30 °C temperature, 165 W of ultrasonication power at 25 kHz and 66% duty cycle. It was observed that the enzymatic pre-hydrolysis under the influence of ultrasound drastically reduces the reaction time from 24 h to 40 min as compared to conventional stirring with improved yield.     

Effects of ultrasound on Microcystis aeruginosa cell destruction and release of intracellular organic matter

Harmful algal blooms negatively impact ecosystems and threaten drinking water sources. One potential method to effectively counteract algal blooms is ultrasonication. However, ultrasonication can easily lead to the release of intracellular organic matter (IOM). The purpose of this study was to investigate the relationship between the destruction of algal cells and IOM release at different ultrasound frequencies. Microcystis aeruginosa cells were ultrasonicated at 20 kHz with an intensity of 0.038 W/mL, 740 kHz with an intensity of 0.113 W/mL, and 1120 kHz with an intensity of 0.108 W/mL. The IOM release was detected by fluorescence spectroscopy in addition to the more commonly used haemocytometry and optical density. After ultrasonication for 15 min, the removal rate of algal cells reached 10.5% at 20 kHz, 9.46% at 740 kHz, and 35.4% at 1120 kHz. The 20 kHz and 740 kHz ultrasound caused local damage to algal cells and then disrupted them, whereas the 1120 kHz ultrasound directly disrupted most algal cells. The extracellular organic matter (EOM), which was increased by ultrasonication, mainly consisted of protein-like compounds, chlorophyll, and a small amount of humic-like substances. Gas vacuoles had been destructed before the cells were broken, as indicated by the decrease of cell size and the wrinkles on the cell surface. Moreover, the removal of algae cells while upholding integrity is more conducive to the safety of the water environment.     

Enzymolysis kinetics and structural characteristics of rice protein with energy-gathered ultrasound and ultrasound assisted alkali pretreatments

This research investigated the structural characteristics and enzymolysis kinetics of rice protein which was pretreated by energy-gathered ultrasound and ultrasound assisted alkali. The structural characteristics of rice protein before and after the pretreatment were performed with surface hydrophobicity and Fourier transform infrared (FTIR). There was an increase in the intensity of fluorescence spectrum and changes in functional groups after the pretreatment on rice protein compared with the control (without ultrasound and ultrasound assisted alkali processed), thus significantly enhancing efficiency of the enzymatic hydrolysis. A simplified kinetic equation for the enzymolysis model with the impeded reaction of enzyme was deduced to successfully describe the enzymatic hydrolysis of rice protein by different pretreatments. The initial observed rate constants (K_in,0) as well as ineffective coefficients (k_imp) were proposed and obtained based on the experimental observation. The results showed that the parameter of k_in,0 increased after ultrasound and ultrasound assisted alkali pretreatments, which proved the effects of the pretreatments on the substrate enhancing the enzymolysis process and had relation to the structure changes of the pretreatments on the substrate. Furthermore, the applicability of the simplified model was demonstrated by the enzymatic hydrolysis process for other materials.     

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.     

Synergistic effect of ultrasonication and co-immobilized enzymes on tomato peels for lycopene extraction

In the present work, tomato peels were pre-treated using combination of ultrasound and enzyme co-immobilized amino-functionalized magnetic nanoparticles (AMNPs) for the efficient release of lycopene. To achieve maximum activity of enzymes in the co-immobilized form, optimization of several parameters were carried out. Moreover, the influence of ultrasound and enzyme co-immobilized magnetic nanoparticles on lycopene release was studied. Maximum lycopene release was obtained at 3% (w/w) enzyme co-immobilized AMNPs, pH 5.0, temperature of 50 °C, at 10 W ultrasound power and 20 min incubation time. After enzymatic pre-treatment, lycopene from the pre-treated mixture was extracted and separated using tri-solvent extraction method. Maximum recovery of lycopene using solvent extraction was obtained at 50 °C, 90 min of incubation time and agitation speed of 150 rpm. The presence of lycopene in the extract was confirmed by FT-IR, UV–vis spectroscopy and HPLC analysis. The co-immobilized bio-catalyst showed excellent reusability giving more than 50% lycopene yield even after 6th cycles of reuse.     

Different multi-scale structural features of oat resistant starch prepared by ultrasound combined enzymatic hydrolysis affect its digestive properties

In this research, oat resistant starch (ORS) was prepared by autoclaving-retrogradation cycle (ORS-A), enzymatic hydrolysis (ORS-B), and ultrasound combined enzymatic hydrolysis (ORS-C). Differences in their structural features, physicochemical properties and digestive properties were studied. Results of particle size distribution, XRD, DSC, FTIR, SEM and in vitro digestion showed that ORS-C was a B + C-crystal, and ORS-C had a larger particle size, the smallest span value, the highest relative crystallinity, the most ordered and stable double helix structure, the roughest surface shape and strongest digestion resistance compared to ORS-A and ORS-B. Correlation analysis revealed that the digestion resistance of ORS-C was strongly positively correlated with RS content, amylose content, relative crystallinity and absorption peak intensity ratio of 1047/1022 cm⁻¹ (R_1047/1022), and weakly positively correlated with average particle size. These results provided theoretical support for the application of ORS-C with strong digestion resistance prepared by ultrasound combined enzymatic hydrolysis in the low GI food application.     

Chemistry and morphology of dried‐up pollen suspension residues

Pollen grains are covered with lots of different biochemical compounds, like proteins, saccharides and lipids, which are only loosely attached to the pollen. Therefore, they can be separated from the pollen by suspending them in water. Since these compounds play a key role in many atmospheric processes (e.g. cloud condensation nucleation, ice nucleation, aerial allergen exposure), their separation and analyzing are of interest. The chemical composition of whole pollen grains is compared by both Raman and infrared spectroscopy with material that could be extracted from pollen with water. The dominant signals in the pollen grain Raman spectra are those from sporopollenin and carotenoids. These bands decrease in the washing water spectra, since sporopollenin is high molecular and thus is not extractable. The released material shows in turn a chemical composition that differs significantly between species, what is quite expected, since they differ even in the optical properties of their aqueous suspensions. The FTIR spectra show some additional bands to appear in comparison to the Raman spectra. Furthermore, we investigated the pollen rupturing and material release in the aqueous suspensions by drying them up and picturing the residues with a scanning electron microscope. We saw that corn pollen ejected loads of micrometer‐sized organelles, which are most likely starch granules. The more the pollen disrupted, the more the measured samples were covered with an amorphous film, which consists of the extracted pollen material, like lipids, sugars, and proteins – the same substances we detected by spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Inactivation of Enterobacter aerogenes in reconstituted skim milk by high- and low-frequency ultrasound

The inactivation of Enterobacter aerogenes in skim milk using low-frequency (20 kHz) and high-frequency (850 kHz) ultrasonication was investigated. It was found that low-frequency acoustic cavitation resulted in lethal damage to E. aerogenes. The bacteria were more sensitive to ultrasound in water than in reconstituted skim milk having different protein concentrations. However, high-frequency ultrasound was not able to inactivate E. aerogenes in milk even when powers as high as 50 W for 60 min were used. This study also showed that high-frequency ultrasonication had no influence on the viscosity and particle size of skim milk, whereas low-frequency ultrasonication resulted in the decrease in viscosity and particle size of milk. The decrease in particle size is believed to be due to the breakup of the fat globules, and possibly to the cleavage of the κ-casein present at the surface of the casein micelles. Whey proteins were also found to be slightly affected by low-frequency ultrasound, with the amounts of α-lactalbumin and β-lactoglobulin slightly decreasing.