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.     

Rhenium oxide nanoparticles – Sonochemical synthesis and integration on anode powders for solid oxide fuel cells

Rhenium oxide nanoparticles have been prepared using ultrasonication at 20 kHz. Samples characterization was committed via SEM-EDX, TEM, XRD, and Raman spectroscopy. Various experimental parameters were examined, including precursor/substrate amounts, ultrasonication intensity, and type of solvent used. Insights to the agglomeration of the prepared nanoparticles depending on the preparation parameters are given. As ultrasonic source we used either an ultrasonic probe by Sonics & Materials Inc. (20 kHz, 750 W net output) or a Bandelin SONOPULS HD 3200 ultrasound generator (20 kHz, 200 W net output) at intensities between 30 and 100 W/cm². The rhenium oxide nanoparticles haven been decorated on state-of-the-art anode materials (NiO/GDC) for solid oxide fuel cells (SOFCs) in order to prepare catalytically more active anode powders. These experiments revealed that ultrasonication intensity and solvents used are able to affect final nanoparticles size distribution and morphology. At the same time, ratio of precursor and substrate compounds amounts as well as ultrasonication intensity and duration were all found to affect the decoration loading extend of nanoformations on substrate powders. The results showing the influence of the above-mentioned parameters allowed for the quantification of the effects on the loading and the preferable sites of the decoration.     

An investigation of mechanisms for the enhanced coagulation removal of Microcystis aeruginosa by low-frequency ultrasound under different ultrasound energy densities

There is a lack of studies elaborating the differences in mechanisms of low-frequency ultrasound-enhanced coagulation for algae removal among different ultrasound energy densities, which are essential to optimizing the economy of the ultrasound technology for practical application. The performance and mechanisms of low-frequency ultrasound (29.4 kHz, horn type, maximum output amplitude = 10 μm) -coagulation process in removing a typical species of cyanobacteria, Microcystis aeruginosa, at different ultrasound energy densities were studied based on a set of comprehensive characterization approaches. The turbidity removal ratio of coagulation (with polymeric aluminum salt coagulant at a dosage of 4 mg Al/L) was considerably increased from 44.1% to 59.7%, 67.0%, and 74.9% with 30 s of ultrasonic pretreatment at energy densities of 0.6, 1.11, and 2.22 J/mL, respectively, indicating that low-frequency ultrasound-coagulation is a potential alternative to effectively control unexpected blooms of M. aeruginosa. However, the energy density of ultrasound should be deliberately considered because a high energy density (≥18 J/mL) results in a significant release of algal organic matter, which may threaten water quality security. The specific mechanisms for the enhanced coagulation removal by low-frequency ultrasonic pretreatment under different energy densities can be summarized as the reduction of cell activity (energy density ≥ 0.6 J/mL), the slight release of negatively charged algal organic matter from cells (energy density ≥ 1.11 J/mL), and the aggregation of M. aeruginosa cells (energy density ≥ 1.11 J/mL). This study provides new insights for the ongoing study of ultrasonic pretreatment for the removal of algae via coagulation.     

Effects of ultrasonic irradiation on crystallization kinetics,morphological and structural properties of zeolite FAU

In this work, NaX zeolite was synthesized and the effect of ultrasound irradiation on reaction kinetics, morphological and structural properties was investigated. Ultrasound was applied, by using a plate transducer (91.8 kHz), for the first time during the crystallization of zeolite NaX, at high temperature, varying the irradiation moment and its duration. Furthermore, ultrasound was applied after the crystallization by a horn-type transducer (20–24 kHz) at low temperature. The effects of irradiated volume (100–300 mL), sonication time (2–10 min) and ultrasound power (10–200 W) were studied with a power intensity up to 100 W/cm². It was found that the application of ultrasound during the first hour of crystallization resulted in 20% reduction of reaction time compared to a standard crystallization. Ultrasound can also reduce the agglomeration degree of the final powder by combining high power and long sonication time. After 5 min sonication time at 0.3 W/mL, the tapped density of the powder was increased by 10%, from 0.37 to 0.41 g/mL. Finally, by scanning electron microscopy (SEM) it was demonstrated that ultrasound can disrupt the agglomerates without affecting the morphology of individual crystals.     

Application of high-frequency ultrasound standing waves for the recovery of lipids from high-fat dairy effluent

Effect of high-frequency ultrasonication was examined on wastewater of a cheese manufacturing plant. Tests were carried out at two frequencies (500 kHz and 1 MHz) and two temperatures (22 and 40 °C). Samples were subjected to different energy densities; 7.5, 30.2, 60.5 and 121.0 J/mL at 500 kHz and 7.9, 31.7, 63.4 and 126.8 J/mL at 1 MHz to observe the creaming and recovery of lipid. These energy densities correspond to 30, 120, 240 and 480 s of sonication. Sonication was performed using a single plate transducer and reflector system at 40 W to create standing wave to coalesce and flocculate lipid globules. Recovery was higher at 40 °C after 480 s of sonication at both frequencies (77% at 500 kHz and 75% at 1 MHz). The lowest recovery of 47% was observed at 500 kHz and 22 °C at all applied energy densities. Changes in particle size and turbidity in the bottom aliquot indicated that high-frequency ultrasound caused coagulation and aggregation and settling of colloidal particles. Increase in particle size was observed to be highest at 1 MHz, 40 °C and 480 s of sonication. These results confirm that high-frequency ultrasound standing wave technology can be used to recover lipid from high-lipid dairy wastewater including that from cheese manufacturing.     

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.     

Effects of microfiltration combined with ultrasonication on shelf life and bioactive protein of skim milk

To extend the shelf life and retain bioactive proteins in milk, this study utilized microfiltration (MF) combined with ultrasonication to treat skim milk and investigated its efficiency in removing bacteria and retaining bioactive proteins compared with HTST pasteurization and microfiltration alone. Results showed that microfiltration combined with ultrasonication at 1296 J/mL could completely remove the bacteria in skim milk. Ultrasonication further extended the shelf life (4 °C) of microfiltered skim milk, which could reach at least 40 days when MF was combined with ˃1296 J/mL ultrasonication. In addition, ELISA showed that HTST pasteurization significantly decreased the levels of IgG by ~30%, IgA by ~ 50%, IgM by ~60%, and lactoferrin by ~40%, whereas the activity of the enzymes lactoperoxidase and xanthine oxidase were also decreased by ~ 20%. Compared with HTST, MF alone or combined with ultrasonication retained these bioactive proteins to a larger degree. On the other hand, proteomics indicated both damage to casein micelle and fat globule structures in milk when ultrasonication at >1296 J/mL was applied, as shown by increases in caseins and milk fat globular proteins. Simultaneously, this ultrasound intensity also decreased levels of bioactive proteins, such as complement factors. Taken together, this study provided new insights that may help to implement this novel combination of non-thermal technologies for the dairy industry aimed at improving milk quality and functionality.     

Ultrasound stimulated production of a fibrinolytic enzyme

The present study is aimed at enhanced production of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672 under ultrasonic stimulation. Various process parameters viz; irradiation at different growth phases, ultrasonication power, irradiation duration, duty cycle and multiple irradiation were studied for enhancement of fibrinolytic enzyme productivity. The optimum conditions were found as follows, irradiation of ultrasonic waves to fermentation broth at 12 h of growth phase with 25 kHz frequency, 160 W ultrasound power, 20% duty cycle for 5 min. The productivity of fibrinolytic enzyme was increased 1.82-fold from 110 to 201 U/mL compared with the non sonicated control fermentation. Drop in glucose concentration from 0.41% to 0.12% w/v in ultrasonicated batch implies that, ultrasonication increases the cell permeability, improves substrate intake and progresses metabolism of microbial cell. Microscopic images before and after ultrasonic stimulation clearly signifies the impact of duty cycle on decreasing biomass concentration. However, environmental scanning electron micrograph does not show any cell lysis at optimum ultrasonic irradiation. Offshoots of our results will contribute to fulfill the demand of enhancement of microbial therapeutic enzyme productivity, through ultrasonication stimulation.     

Variation of dissolved organic nitrogen concentration during the ultrasonic pretreatment to Microcystis aeruginosa

Algae cells were the main sources of dissolved organic nitrogen (DON) in raw water with plenty of algae, and ultrasonic pretreatment was one of the algae-controlling methods through the damage of algae cells. However, the variation of DON concentration during the ultrasonic treatment process was not confirmed. Variation of DON concentration during the processes of low frequency ultrasound treatment of Microcystis aeruginosa was investigated. In addition, the effect of sonication on the metabolite concentration, algae cellar activity and the subsequent coagulation performance were discussed. The results showed that after a long duration of ultrasonic (60 s), nearly 90% of the algal cells were damaged and the maximum concentration of DON attained more than 3 mg/L. In order to control the leakage extent of DON, the sonication time should be less than 30 s with power intensity of more than 1.0 W/cm³. In the mean time, ultrasonic treatment could inhibit the reactivation and the proliferation of algal, keep the algae cell wall integrity and enhance coagulation effectively under the same condition. However, ultrasound frequency had little effect on DON at the frequency range used in this study (20–150 kHz).     

Lysinoalanine formation and conformational characteristics of rice dreg protein isolates by multi-frequency countercurrent S-type sonochemical action

The influences of multi-frequency countercurrent S-type ultrasound (MFSU), with various frequency modes, on lysinoalanine (LAL) formation and conformational characteristics of rice dreg protein isolates (RDPI) were investigated. The ultrasonic operating mode with dual-frequency combination (20/40 kHz) indicated lower LAL content and higher protein dissolution rate of RDPI compared with that of other ultrasound operating modes. Under the dual-frequency ultrasound mode of 20/40 kHz, acoustic power density of 60 W/L, time of 20 min, and temperature of 35 °C, the relative reduction rate of LAL of RDPI reached the highest with its value of 26.95%, and the protein dissolution rate was 71.87%. The changes in chemical interactions between protein molecules indicated that hydrophobic interactions and disulfide bonds played a considerable role in the formation of LAL of RDPI, especially the reduction of g-g-g and g-g-t disulfide bond. Alterations in microstructure showed that ultrasonication loosened the protein structure and created more uniform protein fragments of RDPI. In conclusion, using MFSU in treating RDPI was an efficacious avenue for minimizing LAL content and modifying the conformational characteristics of RDPI.     

Study of continuous flow ultrasonication to improve total phenolic content and antioxidant activity in sorghum flour and its comparison with batch ultrasonication

Ultrasonic technology was applied to release the phenolics bound with starch and protein matrix in order to enhance total phenolic content (TPC) and antioxidant activity (AA) of the sorghum flour. Both the continuous flow and batch ultrasonication were implied with independent variables such as flour to water ratio (FWR), ultrasonication intensity (UI), and ultrasonication time (UT) with an additional variable as flow rate (FR) in continuous flow ultrasonication. All the process variables showed a significant effect on the corresponding ultrasonication process. The optimal conditions for the continuous flow ultrasonication were a FWR of 10% w/v, an UI of 20 W/cm², an UT of 130 s, and 15 ml/s FR which produced a maximum values of 70.9 mg GAE/100 g dry matter (d.m.) for TPC and 143.9 µmol TE/100 g d.m. for AA. Regarding the batch ultrasonication, the maximum values were 65.6 mg GAE/100 g d.m. and 141.0 µmol TE/100 g d.m. for TPC and AA, respectively at optimum conditions of 10% w/v FWR, 30 W/cm² UI, and 200 s UT. When comparing with the batch ultrasonication, the continuous flow process saved 35% time and 33% of energy consumption to obtain comparatively higher TPC and AA of the sorghum flour. Ultrasonication improved free phenolic acid content by releasing bound phenolics in the sorghum flour. Impact of various process parameters on specific energy was analyzed during both the processes, and influence of energy on TPC and AA of the sorghum flour was also observed for the batch and continuous flow ultrasonication.     

Dual-frequency multi-angle ultrasonic processing technology and its real-time monitoring on physicochemical properties of raw soymilk and soybean protein

To improve the soybean protein content (SPC), flavor and quality of soymilk, the effects of dual-frequency ultrasound at different angles (40 + 20 kHz 0°, 40 + 20 kHz 30°, 40 + 20 kHz 45°) on physicochemical properties and soybean protein (SP) structure of raw soymilk were mainly studied and compared with the conventional single-frequency (40 kHz, 20 kHz) ultrasound. Furthermore, the intensity of the ultrasonic field in real-time was monitored via the oscilloscope and spectrum analyzer. The results showed that 40 + 20 kHz 45° treatment significantly increased SPC. The ultrasonic field intensity of 40 + 20 kHz 0° treatment was the largest (8.727 × 10⁴ W/m²) and its distribution was the most uniform. The emulsifying stability of SP reached the peak value (233.80 min), and SP also had the largest particle size and excellent thermal stability. The protein solubility of 40 + 20 kHz 30° treatment attained peak value of 87.09%. 20 kHz treatment significantly affected the flavor of okara. The whiteness and brightness of raw soymilk treated with 40 kHz were the highest and the system was stable. Hence, the action mode of ultrasonic technology can be deeply explored and the feasibility for improving the quality of soymilk can be achieved.     

Effect of ultrasound on the flocculation-sedimentation and thickening of unclassified tailings

In back-fill mining, how to rapidly increase the concentration of tailings is an important problem facing mining engineers. In this paper, the effects of ultrasound frequency (17–25 kHz), power (50–100 W), duration (5–20 min) and start time (3–12 min) on the final underflow concentration (FUC) of unclassified tailings (UTs) were investigated. The flocculation-sedimentation and thickening of tailings were compared with and without ultrasound application. The response surface method was applied to analyze the primary and secondary relationships and interactive relationships between the various ultrasound operating parameters and the FUC, and the optimal conditions were determined. In addition, Environmental Scanning Electron Microscope (E-SEM) was used to analyze the structural changes of underflow aggregates and clearly demonstrated a denser underflow after ultrasound treatment. The results indicated that ultrasound can significantly improve the underflow concentration of the UTs slurry. The frequency and power are the most important influencing factors. The best conditions for ultrasound application are a frequency of 20.4 kHz, power of 90 W, duration of 6.2 min and start time at 15.0 min. The FUC reached 71.75% after several minutes of sonication, which is 4.31% higher than the FUC of free flocculation. The results of E-SEM analysis showed significant differences among the microstructures of settled tailings (STs) after free flocculation and 17 and 20 kHz ultrasound treatment. Ultrasound at a frequency of 20 kHz has a more effective mechanical vibration and cavitation action and can therefore effectively break the polymer flocculant chains into shorter chains and promote the compaction of tailings and the release of water. The size distributions of the flocs before and after sonication also support this conclusion.     

Does power ultrasound affect hydrocarbon Ionomers?

The effect of low-frequency high-power ultrasound on hydrocarbon-based ionomers, cation exchange sulfonated phenylated polyphenylene (sPPB-H⁺) and anion exchange hexamethyl-p-terphenyl poly(benzimidazolium) (HMT-PMBI), was studied. Ionomer solutions were subjected to ultrasonication at fixed ultrasonic frequencies (f = 26 and 42 kHz) and acoustic power (P_acous = 2.1 – 10.6 W) in a laboratory-grade ultrasonication bath, and a probe ultrasonicator; both commonly employed in catalyst ink preparation in research laboratory scale. Power ultrasound reduced the polymer solution viscosity of both hydrocarbon-based ionomers. The molecular weight of sPPB-H⁺ decreased with irradiation time. Changes in viscosity and molecular weight were exacerbated when ultrasonicated in an ice bath; but reduced when the solutions contained carbon black, as typically used in Pt/C-based catalyst inks. Spectroscopic analyses revealed no measurable changes in polymer structure upon ultrasonication, except for very high doses, where evidence for free-radical induced degradation was observed. Ionomers subjected to ultrasound were used to prepare catalyst layers and membrane electrode assemblies (MEA)s. Despite the changes in the ionomer described above, no significant differences in electrochemical performance were found between MEAs prepared with ionomers pre-subjected to ultrasound and those that were not, suggesting that fuel cell performance is tolerant to ionomers subjected to ultrasound.     

Ultrasound assisted cyanotoxin extraction for nematode inhibition in soil

Root-knot nematodes are one of the plant damaging nematodes in agriculture causing a projected annual yield loss of ∼12 % (∼$160 billion) worldwide. Conventional solutions to control these plant-parasitic nematodes involve chemical nematicides. To reduce the use of harmful chemicals, microalgal extracts can be used as greener alternatives for nematode management. Microalgae produce valuable metabolites, including cyanotoxins which can aid in nematode suppression.In this study, two microalgae species, Trichormus variabilis and Nostoc punctiforme, were treated with ultrasound for intensified recovery of secondary metabolites. Ultrasound results in cell wall disruption of the microalgal species, thus resulting in enhanced release of secondary metabolites. Microalgal biomass was treated with an ultrasound probe at 50 % amplitude, 20 kHz frequency, using water as the extraction medium, for 5–30 min. The extraction efficiency was determined in terms of the total chlorophyll (Chl) content of the extract. Microscopic images of the treated cells were also investigated to gain insight into the effect of the ultrasonication time on the cell morphology. Our results suggest that ultrasonication resulted in the intensified release of secondary metabolites, as established through the total chlorophyll content of the ultrasonicated microalgal samples as well as the microscopic images of the ruptured cells. The best extraction for Trichormus variabilis was achieved with 15 min extraction time where the Total Chl content increased by 29 times (compared to the non-ultrasonicated sample), and for the Nostoc punctiforme, 30 min extraction time gave the highest metabolite recovery of 6.4 times higher than the non-ultrasonicated sample.Ultrasonicated algal extracts were then tested for their nematicidal potential against root-knot nematode, Meloidogyne hapla, in infested field soil samples. Experimental study was conducted using different concentrations of each microalga, Trichormus sp. and Nostoc sp., individually, as well as in combination. The nematode count for the treated soil was compared with that of the control (untreated soil). Ultrasonicated microalgal extracts showed 66% to 100% inhibition on root-knot nematodes in the soil samples tested.     

Inactivation of foodborne pathogens based on synergistic effects of ultrasound and natural compounds during fresh produce washing

Ultrasound has potential to be used for disinfection, and its antimicrobial effectiveness can be enhanced in presence of natural compounds. In this study, we compared the antimicrobial effects of ultrasound at 20 kHz (US 20 kHz) or 1 MHz (US 1 MHz) in combination with carvacrol, citral, cinnamic acid, geraniol, gallic acid, lactic acid, or limonene against E. coli K12 and Listeria innocua at a constant power density in water. Compared to the cumulative effect of the individual treatments, the combined treatment of US 1 MHz and 10 mM citral generated >1.5 log CFU/mL additional inactivation of E. coli K12. Similarly, combined treatments of US 1 MHz and 2 mM carvacrol (30 min), US 20 kHz and 2 mM carvacrol, 10 mM citral, or 5 mM geraniol (15 min) generated >0.5–2.0 log CFU/mL additional inactivation in L. innocua. The synergistic effect of citral, as a presentative compound, and US 20 kHz treatment was determined to be a result of enhanced dispersion of insoluble citral droplets in combination with physical impact on bacterial membrane structures, whereas the inactivation by US 1 MHz was likely due to generation of oxidative stress within the bacteria. Combined ultrasound and citral treatments improved the bacterial inactivation in simulated wash water in presence of organic matter or during washing of inoculated blueberries but only additive antimicrobial effects were observed. Findings in this study will be useful to enhance fresh produce safety and shelf-life and design other alternative ultrasound based sanitation processes.     

Effect of high-intensity ultrasound on the technofunctional properties and structure of jackfruit (Artocarpus heterophyllus) seed protein isolate

The influence of high-intensity ultrasound (HIU) on the technofunctional properties and structure of jackfruit seed protein isolate (JSPI) was investigated. Protein solutions (10%, w/v) were sonicated for 15 min at 20 kHz to the following levels of power output: 200, 400, and 600 W (pulse duration: on-time, 5 s; off-time 1 s). Compared with untreated JSPI, HIU at 200 W and 400 W improved the oil holding capacity (OHC) and emulsifying capacity (EC), but the emulsifying activity (EA) and emulsion stability (ES) increased at 400 W and 600 W. The foaming capacity (FC) increased after all HIU treatments, as opposed to the water holding capacity (WHC), least gelation concentration (LGC), and foaming stability (FS), which all decreased except at pH 4 for FS. Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) showed changes in the molecular weight of protein fractions after HIU treatment. Scanning electron microscopy (SEM) demonstrated that HIU disrupted the microstructure of JSPI, exhibiting larger aggregates. Surface hydrophobicity and protein solubility of the JSPI dispersions were enhanced after ultrasonication, which increased the destruction of internal hydrophobic interactions of protein molecules and accelerated the molecular motion of proteins to cause protein aggregation. These changes in the technofunctional and structural properties of JSPI could meet the complex needs of manufactured food products.     

Ultrasound driven conformational and physicochemical changes of soy protein hydrolysates

The effect of ultrasound on the conformational and physicochemical properties of soy protein isolate hydrolysates (SPHs) was investigated. SPHs were prepared at hydrolysis times of 20 min, 60 min, and 180 min, then treated with ultrasound for 10 min, 20 min, and 30 min at a frequency of 20 kHz and output powers of 150 W and 450 W. The structural properties and antioxidant capacities of the aqueous layer of SPHs (ASPHs) after sonication were evaluated by Fourier-transform infrared spectroscopy (FTIR), intrinsic fluorescence, DPPH radical scavenging activity assays, and microscopy observations. Results obtained showed that ultrasound treatment significantly disrupted the peptide aggregates formed during protein hydrolysis. The protein solubility was significantly increased after sonication (by up to 18.33%), as did the percentage of proteins with MW < 1 kDa in ASPHs. The antioxidant capacity of ASPHs also increased, as measured by DPPH assay. FTIR analysis of ASPHs indicated that the protein secondary structures were different, with an increase in β-sheet and a decrease in α-helix and β-turn. Furthermore, the changes in fluorescence spectra of ASPHs showed the transition of protein tertiary structure with a greater exposure of Trp residues in the side chains. Scanning electron microscope (SEM) and atomic force microscope (AFM) observations of the morphological structure of ASPHs further confirmed the significant effect of sonication on disrupting peptide aggregates. In conclusion, ultrasound can be used as an efficient treatment to promote the solubility of protein hydrolysates.     

超声处理黑豆蛋白结构变化的拉曼光谱分析

探究了拉曼光谱应用于黑豆蛋白结构变化研究的可行性,研究了黑豆蛋白溶液在低频超声处理在不同超声强度、不同处理时间下的结构变化,并进行了热力学特性分析。低、中超声处理强度下TD的降低表明蛋白质分子的内部疏水作用被破坏,使黑豆蛋白不稳定的聚集体解聚为小分子可溶性聚集物,而在高超声处理强度下TD的增高表明聚集体重聚。拉曼光谱分析表明超声处理下除了E样品(300 W, 24 min)所有黑豆蛋白均发生了α-螺旋结构含量降低和β-折叠结构含量增高。聚集体的聚合/解聚导致黑豆蛋白二级结构的重组,尤其是β-折叠。超声处理使拉曼光谱在760 cm-1的色氨酸归属谱线强度降低表明超声处理使黑豆蛋白发生了部分的解折叠。超声处理下酪氨酸归属谱线强度变化不显著,表明超声处理并未显著改变黑豆蛋白酪氨酸的微环境。1 450 cm-1拉曼归属谱带随着超声处理强度和时间的增加而增大,但随着功率及处理时间的进一步增大此值有所降低。在超声处理下聚集体的形成使二硫键的g-g-t构型转变为t-g-t构型。尽管黑豆蛋白聚集体重组的机理仍有待研究,但拉曼光谱是一种研究超声处理黑豆蛋白结构变化的可行方法,也可为蛋白质结构研究提供一种新的研究思路。     

Ultrasound enhanced butyric acid-lauric acid designer lipid synthesis: Based on artificial neural network and changes in enzymatic structure

Ultrasound is a green technology for intensifying enzymatic reactions. In this study, an ultrasonic water bath with equipment parameters of 28 kHz, 1750.1 W/m², 60% duty cycle was used to assist the synthesis of butyric acid-lauric acid designer lipid (BLDL), which was catalyzed by Lipozyme 435. A convincing three-layer feed-forward artificial neural network (ANN) model was established (R² = 0.949, RMSE = 4.759, ADD = 7.329) to accurately predict the optimal parameters combination, which was described as 13.72 mL reaction volume, 15.49% enzyme loading, 0.253 substrate molar ratio (tributyrin/lauric acid), 56.58 °C reaction temperature and 120 min reaction time. The ultrasonic assistance increased actual butyric acid conversion rate by 11.38%, and also enhanced the consumption rate of tributyrin and lauric acid during the reaction. Meanwhile, the esterification activity of Lipozyme 435 was enhanced and its effectiveness up to 6 cycles. Structurally, ultrasound assistance significantly disrupted the secondary structure of the Lipozyme 435: reduced the content of α-helices, increased the content of β-sheet and β-turn. In addition, sonication caused an increase in crevice and micro-damage on the surface of the immobilized enzyme. In conclusion, low-intensity ultrasound at 28 kHz improved the synthesis efficiency of BLDL, which was scientifically predicted by ANN model, and the change of enzyme structure may be the vital reason for ultrasound enhanced reaction. However, the effect of ultrasound on immobilized enzymes’ activity needs to be further explored.