Ultrasound viscous reduction effects on the proteolysis of soy protein isolate at a limited degree of hydrolysis: Changes in the functional characteristics and protein structure.

High-concentration soy protein isolate was subjected to ultrasonication for viscosity reduction to assist the process of limited enzymatic hydrolysis. Ultrasonication (20 kHz, 10 min, 160 W/L) effectively reduced the viscosity of soy protein isolate at a comparatively high concentration of 14 % (w/v) and promoted the limited enzymatic hydrolysis (controlled degree of hydrolysis of 12 %) with a higher peptide yield than that of the conventional method. The correlations between substrate viscosity and peptide yield, as well as the viscosities of the resulting hydrolysates, were studied. The findings revealed positive correlations between the viscosities of the substrate and hydrolysate, underscoring the potential impact of altering substrate viscosity on the final product. Furthermore, the utilization of ultrasonic viscosity reduction-assisted proteolysis has shown its capability to improve the functional and physicochemical properties, as well as the protein structure of the hydrolysate, while maintaining the same level of hydrolysis. It is worth noting that there were significant alterations in particle size (decrease), ß-sheet content (increase), ß-turn content (increase), and random coil content (increase). Interestingly, ultrasonication unexpectedly impeded the degradation of molecular mass in proteins during proteolysis, while increasing the hydrophobic properties of the hydrolysate. These findings aligned with the observed reduction in bitterness and improvement in emulsifying properties and water-holding capacity.

Experimental investigation into the implications of low-intensity magnetic field treatment on the structural and functional properties of rapeseed meal during biofermentation.

The functionality of rapeseed meal is limited, to acquire more utilization, the functional attributes were improved by altering its structural features using magnetic field-assisted solid fermentation. The magnetic treatment was performed every 24 h (specifically at 24, and 48 h), each treatment having a duration of 4 h. The magnetic intensity was set at 120 Gs, and the fermentation temperature 37 °C. Magnetic field-assisted solid fermentation resulted in higher surface hydrophobicity, fluorescence intensity, UV absorption, and sulfhydryl groups of rapeseed meal. Magnetic field treatment considerably enhanced solubility, antioxidant activity, emulsifying activity, and stability by 8.8, 19.5, 20.7, and 12.3 %, respectively. Magnetic field-assisted solid fermentation also altered rapeseed meal structure, as shown by scanning electron microscopy, atomic force microscopy, and Raman spectroscopy outcomes. Correlation analysis displayed positive interrelationships between functional characteristics, and surface hydrophobicity, ß-sheets, and polydispersity index.

Effect of carbonyl-amino condensation, non-covalent cross-linking and conformational changes induced by ultrasound-assisted Maillard reaction on lysinoalanine formation in silkworm pupa protein.

The inhibition of cross-linked lysinoalanine (LAL) formation in silkworm pupa protein isolates (SPPI) by Maillard reaction (using varying xylose concentration) and ultrasound treatment was studied. Results showed that sonicated SPPI was effectively grafted with high concentration of xylose (5 %), resulting in the lowest LAL content, which was 48.75 % and 30.64 % lower than the control and ultrasound-treated samples, respectively. Chemical bond analysis showed that the combined treatment destroyed the ionic bonds, intrachain (g-g-t), and interchain (g-g-g) disulfide bonds, but stimulated the polymerization of hydrogen and hydrophobic bonds between SPPI and xylose, and as well enhanced the net negative charge between SPPI/Xylose complexes. The particles of the complexes were more loose, dispersed and rough, and had a stronger hydrophilic microenvironment, accompanied by alterations in microscopic, secondary and tertiary structures. Ultrasound treatment induced the breakdown of the oxidative cross-linking in SPPI, and promoted the sulfhydryl group-dehydroalanine binding and the carbonyl-amino condensation of the protein and xylose, and thus inhibited the formation of cross-linked LAL. Furthermore, the physicochemical and structural parameters were highly interrelated with cross-linked LAL content (|r| > 0.9). The outcomes provided a novel avenue and theoretical basis for minimizing LAL formation in SPPI and improving the nutrition and safety of SPPI.

Effect of magnetic field-assisted fermentation on the in vitro protein digestibility and molecular structure of rapeseed meal.

BACKGROUND: There has been a significant growth in demand for plant-derived protein, and this has been accompanied by an increasing need for sustainable animal-feed options. The aim of this study was to investigate the effect of magnetic field-assisted solid fermentation (MSSF) on the in vitro protein digestibility (IVPD) and functional and structural characteristics of rapeseed meal (RSM) with a mutant strain of Bacillus subtilis. RESULTS: Our investigation demonstrated that the MSSF nitrogen release rate reached 86.3% after 96 h of fermentation. The soluble protein and peptide content in magnetic field feremented rapeseed meal reached 29.34 and 34.49 mg mL-1 after simulated gastric digestion, and the content of soluble protein and peptide in MF-FRSM reached 61.81 and 69.85 mg mL-1 after simulated gastrointestinal digestion, which significantly increased (p > 0.05) compared with the fermented rapeseed meal (FRSM). Studies of different microstructures - using scanning electron microscopy (SEM) and atomic force microscopy (AFM) - and protein secondary structures have shown that the decline in intermolecular or intramolecular cross-linking leads to the relative dispersion of proteins and improves the rate of nitrogen release. The smaller number of disulfide bonds and conformational alterations suggests that the IVPD of RSM was improved. CONCLUSIONS: Magnetic field-assisted solid fermentation can be applied to enhance the nutritional and protein digestibility of FRSM. © 2024 Society of Chemical Industry.

Comparison of prediction models for soy protein isolate hydrolysates bitterness built using sensory, spectrofluorometric and chromatographic data from varying enzymes and degree of hydrolysis.

The bitterness of soy protein isolate hydrolysates prepared using five proteases at varying degree of hydrolysis (DH) and its relation to physicochemical properties, i.e., surface hydrophobicity (H0), relative hydrophobicity (RH), and molecular weight (MW), were studied and developed for predictive modelling using machine learning. Bitter scores were collected from sensory analysis and assigned as the target, while the physicochemical properties were assigned as the features. The modelling involved data pre-processing with local outlier factor; model development with support vector machine, linear regression, adaptive boosting, and K-nearest neighbors algorithms; and performance evaluation by 10-fold stratified cross-validation. The results indicated that alcalase hydrolysates were the most bitter, followed by protamex, flavorzyme, papain, and bromelain. Distinctive correlation results were found among the physicochemical properties, influenced by the disparity of each protease. Among the features, the combination of RH-MW fitted various classification models and resulted in the best prediction performance.

Inhibition of cross-linked lysinoalanine formation in pH12.5-shifted silkworm pupa protein, and functionality thereof: Effect of ultrasonication and glycation.

We added three different carbohydrates (Xylose/Xyl, Maltose/Mal, and Sodium alginate/Sal) to pH12.5-shifted silkworm pupa protein isolates (SPPI), and examined the influence of multi-frequency ultrasound (US) on them, with reference to lysinoalanine (LAL) formation, changes in conformational characteristics and functionality. Results showed that, the LAL content of the glycoconjugates - SPPI-Xyl, SPPI-Mal, and SPPI-Sal decreased by 1.47, 1.39, and 1.54 times, respectively, compared with the control. Notably, ultrasonication further reduced the LAL content by 45.85 % and brought SPPI-Xyl highest graft degree (57.14 %). SPPI-Xyl and SPPI-Mal were polymerized by different non-covalent bonds, and SPPI-Sal were polymerized through ionic, hydrogen, and disulfide (covalent/non-covalent) bonds. Significant increase in turbidity, Maillard reaction products and the formation of new hydroxyl groups was detected in grafted SPPI (p < 0.05). US and glycation altered the structure and surface topography of SPPI, in which sugars with high molecular weight were more likely to aggregate with SPPI into enormous nanoparticles with high steric hindrance. Compared to control, the solubility at pH 7.0, emulsifying capacity and stability, and foaming capacity of SPPI-US-Xyl were respectively increased by 244.33 %, 86.5 %, 414.67 %, and 31.58 %. Thus, combined US and xylose-glycation could be an effective approach for minimizing LAL content and optimizing functionality of SPPI.

Hexagonal plate ultrasound pretreatment on the correlation between soy protein isolate structure and cholesterol-lowering activity of peptides, and protein's enzymolysis kinetics, thermodynamics.

In this study, a hexagonal plate ultrasound (HPU) pretreatment technology was employed to modify soy protein isolate (SPI) and enhance the hypocholesterolemic activity of enzymatic digests from SPI. Results demonstrated that under the condition of ultrasound power density of 40 W/L, the hypocholesterolemic activity of enzymatic digests from HPU-pretreated SPI (HPU-SPI) increased by 88.40 % compared to control group after gastrointestinal digestion. The sulfhydryl content of HPU-SPI increased by a maximum of 45.32 % compared to control group. Fourier transform infrared and scanning electron microscopy revealed that HPU pretreatment partially unfolded the SPI conformation, reduced the intermolecular interactions, and exposed the internal hydrophobic regions. Pearson correlation analysis showed that sulfhydryl groups (r = 0.860), disulfide bonds (r = -0.875) and random coil (r = 0.917) were strongly correlated with the cholesterol-lowering activity of soy protein hydrolysate (SPH), following a simulated gastrointestinal digestion. Finally, the effects of HPU pretreatment on enzymolysis kinetics and thermodynamics of the SPI enzymatic process showed that HPU pretreatment significantly reduced the Mie's constant, activation energy, activation enthalpy, activation entropy and Gibbs free energy. Overall, the study outcome suggested that HPU pretreatment could positively influence the hypocholesterolemic peptide activity, and thus, may be beneficial to the pharmaceutical/food industry.

Effect of pectin concentration on emulsifying properties of black soldier fly (Hermetia illucens) larvae albumin modified by pH-shifting and ultrasonication.

The effect of pectin concentration on the structural and emulsifying properties of black soldier fly larvae albumin (BSFLA) modified by pH-shifting (pH12) and ultrasound (US) was studied. The results (intrinsic fluorescence, surface hydrophobicity, Fourier transform infrared spectrum, and disulfide bonds) showed that modified BSFLA samples, especially pH12-US, were more likely to bind to pectin through hydrogen bonding, electrostatic interactions, and hydrophobic interactions due to the unfolding of BSFLA, the collapse of disulfide bonds and exposure of hydrophobic groups. Thus, a BSFLA-pectin complex with smaller particle size, more negative charges, and a relatively loose structure was formed. The emulsifying activity (EAI) and stability index (ESI) of pH12-US modified BSFLA were significantly enhanced by the addition of pectin, reaching the highest values (associated with 174.41 % and 643.22 % increase, respectively) at pectin concentration of 1.0 %. Furthermore, the interface modulus of the emulsion prepared by the modified BSFLA was mainly viscous, and had higher apparent viscosity, smaller particle size and droplet size, contributing to higher EAI and ESI. The study findings suggest the addition of pectin to pH12-US treated BSFLA could be used in industry to prepare BSFLA-pectin emulsion with exceptional/desirable properties.

Preparation of housefly (Musca domestica) larvae protein hydrolysates: Influence of dual-sweeping-frequency ultrasound-assisted enzymatic hydrolysis on yield, antioxidative activity, functional and structural attributes.

Dual-sweeping-frequency ultrasound (DSFU) was utilized in the preparation of polypeptides from housefly (Musca domestica) larvae protein (HLP). Results indicated that ultrasonication (20 ± 2/28 ± 2 kHz, 42 W/L, 25 min) significantly increased peptide yield and DPPH scavenging capacity by 8.25 % and 14.83 %, respectively. Solubility, foaming and emulsification properties of polypeptides were improved by 19.89 %, 33.33 % and 38.74 % over the control; along with notable reduction in particle size and increase in zeta potential. Tertiary structural changes of the sonicated hydrolysates were illustrated by UV and fluorescence spectra. FTIR showed that ultrasonication increased α-helix, ß-turn, and random coil by 38.23 %, 46.35 % and 16.36 %, respectively, but decreased ß-sheet by 48.03 %, indicating partial unfolding in HLP hydrolysate conformation and reduction in intermolecular interactions. The research results demonstrated that dual-sweeping-frequency ultrasonication has a great prospect in industry application for the purpose of improving enzymolysis efficiency and product quality for housefly larvae protein hydrolysates production.

Enhancing the growth of thermophilic Bacillus licheniformis YYC4 by low-intensity fixed-frequency continuous ultrasound.

The effect of low-intensity fixed-frequency continuous ultrasound (LIFFCU) on the growth of Bacillus licheniformis YYC4 was investigated. The changes in morphology and activity of the organism, contributing to the growth were also explored. Compared with the control, a significant increase (48.95%) in the biomass of B. licheniformis YYC4 (at the logarithmic metaphase) was observed following the LIFFCU (28 kHz, 1.5 h and 120 W (equivalent to power density of 40 W/L)) treatment. SEM images showed that ultrasonication caused sonoporation, resulting in increased membrane permeability, evidenced by increase in cellular membrane potential, electrical conductivity of the culture, extracellular protein and nucleic acid, and intracellular Ca2+ content. Furthermore, LIFFCU action remarkably increased the extracellular protease activity, volatile components of the culture medium, microbial metabolic activity, and spore germination of the strain. Therefore, LIFFCU could be used to efficiently promote the growth of targeted microorganisms.

Effect of dual-frequency thermosonication, food matrix, and germinants on Alicyclobacillus acidoterrestris spore germination.

The off-odors associated with spoilage of acidic beverages are linked to the germination and growth of Alicyclobacillus acidoterrestris (AAT) spores. As a consequence, we determined the influence of nutrients, non-nutrient germinants, dual-frequency thermosonication (DFTS), and food matrix on spore germination. AAT spores in orange juice (OJ), supplemented by L-alanine (L-ala), had the highest germination rate and lowest DPA content at 10 h of incubation. The formation of microscopic pores in cell membranes during DFTS caused irreversible damage in AAT spores in citrate buffer solution (CBS); however, it stimulated AAT spore germination in CBS containing L-ala. Hence, the germination potential was established in the order: L-ala > Calcium dipicolinate > asparagine, glucose, fructose, and potassium ion mixture (AGFK) > L-valine. The conductivity analysis indicated that membrane damage could be a key factor contributing to the artificial germination in CBS. AFM images revealed that after 2 h of adding L-ala, the protein content increased with increased germinated cells. TEM showed that membrane poration and coat detachment were the main pre-germination morphological changes detected after DFTS treatment. This study provides evidence that germination stimulated with DFTS might be an effective strategy for reducing A. acidoterrestris spores in fruit juices.

Mono-frequency ultrasonic-assisted thawing of frozen goose meat: Influence on thawing efficiency, product quality and microstructure.

This study aimed to investigate the influences of mono-ultrasound assisted thawing on the thawing efficiency, product quality and conformational characteristics of frozen goose meat. The thawing time, thawing loss, muscle quality, and microstructure of frozen goose meat were studied. The results displayed that ultrasonic-assisted thawing effectively reduced the thawing time by 45.37-57.58% compared with non-sonicated group, and significantly decreased the thawing loss. For the quality properties of goose meat tissue, ultrasound-assisted thawing with single-frequency of 50 kHz indicated a lower protein turbidity; meanwhile, hardness values were also significantly increased, and displayed a higher springiness, gumminess and chewiness of goose meat tissue. The microstructure analysis exhibited that the conformation of goose myofibrillar protein (MP) was modified following ultrasonic-assisted thawing, and became closer and more irregular. Therefore, ultrasound-assisted thawing treatments at 50 kHz mono-frequency (temperature 25℃) have a high potential application value in the thawing research of frozen goose meat, and lay a theoretical foundation for use in the meat process industries.

Synergistic effects of pH shift and heat treatment on solubility, physicochemical and structural properties, and lysinoalanine formation in silkworm pupa protein isolates.

The application of silkworm pupa protein isolates (SPPI) in food industry was limited because SPPI's solubility is poor and it contains a potential harmful component of lysinoalanine (LAL) which formed during protein extraction. In this study, combined treatments of pH shift and heating were performed to improve the solubility of SPPI and to reduce the content of LAL. The experimental results showed that the promoting effect on SPPI's solubility by alkaline pH shift + heat treatment was greater than that by acidic pH shift + heat. And an 8.62 times increase of solubility was observed after pH 12.5 + 80 â„ƒ treatment compared to the control SPPI sample which was extracted at pH 9.0 without pH shift treatment. Very strong positive correlation was found between alkali dosage and SPPI solubility (Pearson's correlation coefficient r = 0.938). SPPI with pH 12.5 shift treatment showed the highest thermal stability. Alkaline pH shift combined with heat treatment altered the micromorphology of SPPI and destroyed the disulfide bonds between macromolecular subunits (72 and 95 kDa), resulting in reduced particle size and increased zeta potential and free sulfhydryl content of the isolates. The fluorescence spectra analysis showed red shifts phenomena with pH increasing and fluorescence intensity increase with temperature increasing, implying the alterations in the tertiary structure of protein. Compared to the control SPPI sample, the amount of LAL reduced by 47.40 %, 50.36 % and 52.39 % using pH 12.5 + 70 â„ƒ, pH 12.5 + 80 â„ƒ and pH 12.5 + 90 â„ƒ treatment, respectively. These findings provide fundamental information for the development and application of SPPI in food industry.

Isolation and genomic characterization of Vmp-1 using Vibrio mimicus as the host: A novel virulent bacteriophage capable of cross-species lysis against three Vibrio spp.

Vibrio mimicus is a zoonotic pathogen that is widely distributed in aquatic habitats/environments (marine coastal water, estuaries, etc). The development of biocontrol agents for V. mimicus is imperative for the prevention and control of aquatic animal diseases and human food-borne infections. In this study, a broad-spectrum bacteriophage Vmp-1 was isolated from dealt aquatic product in a local market by double-layer agar plate method using V. mimicus CICC21613 as the host bacteria. Results indicated that Vmp-1, which belongs to the family Podoviridae, showed good pH tolerance (pH 3.0-12.0) and thermal stability (30-50 °C). The optimal multiplicity of infection (MOI) of Vmp-1 was 0.001 for a 20-min incubation and 100-min lysis period. Vmp-1 effectively controlled V. mimicus CICC21613 in LBS model (MOI = 0.0001, 0.001, 0.01, 0.1, 1) within 8 h. The full length of the Vmp-1 genome was 43,312 bp, with average GC content of 49.5%, and a total of 44 protein-coding regions. This study provides a novel phage strain that has the highest homology with vB_VpP_HA5 (GenBank: OK585159.1, 95.96%) for the development of biocontrol agents for V. mimicus.

Sorption Isotherms and Thermodynamic Characteristics of Gelatin Powder Extracted from Whitefish Skin: Mathematical Modeling Approach.

Moisture adsorption and desorption isotherms of gelatin extracted from whitefish skin powder (FSGP) at different temperatures across a wide range of water activity were determined along with their thermodynamic properties. Nine mathematical models were utilized for fitting the experimental data and simulating the adsorption and desorption behavior. The thermodynamic properties were determined and fitted to the experimental data. The results showed that Peleg and GAB models were the best fit for FSGP. The energies involved in the adsorption and desorption process of FSGP indicated a stronger dependence on equilibrium moisture content (Xe). When Xe decreased, there was a consistent trend of increasing thermodynamic properties. Both the moisture adsorption and desorption behaviors of FSGP were, therefore, non-spontaneous processes. Linear correlations between the changes in enthalpy and entropy for adsorption and desorption were observed, indicating the presence of enthalpy-entropy compensation for FSGP. For preserving FSGP quality, it should be stored with Xw ≤ 8 (gw/gdm, d.b.) at temperatures below 53 °C and an RH of 50% to avoid it becoming rubbery. These findings are crucial for providing insight into the optimal drying and storage conditions.

Application and potential of multifrequency ultrasound in juice industry: Comprehensive analysis of inactivation and germination of Alicyclobacillus acidoterrestris spores.

The majority of acidic fruits are perishable owing to their high-water activity, which promotes microbial activity, thus exhibiting metabolic functions that cause spoilage. Along with sanitary practices, several treatments are used during processing and/or storage to inhibit the development of undesirable bacteria. To overcome the challenges caused by mild heat treatment, juice manufacturers have recently increased their involvement in developing novel non-thermal processing procedures. Ultrasonication alone or in combination with other hurdle technologies may be used to pasteurize processed fruit juices. Multifrequency ultrasound has gained popularity due to the fact that mono-frequency ultrasound has less impact on bacterial inactivation and bioactive compound enhancement of fruit juice. Here, we present and discuss the fundamental information and technological knowledge of how spoilage bacteria, specifically Alicyclobacillus acidoterrestris, assemble resistant spores and inactivate and germinate dormant spores in response to nutrient germinants and physical treatments such as heat and ultrasound. To the authors' knowledge, no prior review of ultrasonic inactivation and germination of A. acidoterrestris in fruit juice exists. Therefore, this article aims to provide a review of previously published research on the inactivation and germination of A. acidoterrestris in fruit juice by ultrasound and heat.

Influence of Low-Intensity Ultrasound on ε-Polylysine Production: Intracellular ATP and Key Biosynthesis Enzymes during Streptomyces albulus Fermentation.

The effect of low-intensity sonication treatment on cell growth, ε-polylysine (ε-PL) yield and its biological mechanism were investigated, using a 3-L-jar fermenter coupled with an in situ ultrasonic slot with a Streptomyces albulus strain SAR 14-116. Under ultrasonic conditions (28 kHz, 0.37 W cm-2, 60 min), a high biomass of SAR 14-116 and concentration of ε-PL were realized (i.e., they increased by 14.92% and 28.45%, respectively) when compared with a control. Besides this, ultrasonication increased the mycelia viability and intracellular ATP as well as activities of key enzymes involved in the ε-PL biosynthesis pathway, resulting in an improvement in the production of ε-PL. Data on qRT-PCR revealed that ultrasonication also affected the gene expression of key enzymes in the ε-PL biosynthesis pathway, including ε-PL synthetase (PLS). These outcomes provided the basis for understanding the effects of ultrasound-assisted fermentation on the stimulation of metabolite production and fermentation procedure in a fermenter.

Recent Insight on Edible Insect Protein: Extraction, Functional Properties, Allergenicity, Bioactivity, and Applications.

Due to the recent increase in the human population and the associated shortage of protein resources, it is necessary to find new, sustainable, and natural protein resources from invertebrates (such as insects) and underutilized plants. In most cases, compared to plants (e.g., grains and legumes) and animals (e.g., fish, beef, chicken, lamb, and pork), insect proteins are high in quality in terms of their nutritional value, total protein content, and essential amino acid composition. This review evaluates the recent state of insects as an alternative protein source from production to application; more specifically, it introduces in detail the latest advances in the protein extraction process. As an alternative source of protein in food formulations, the functional characteristics of edible insect protein are comprehensively presented, and the risk of allergy associated with insect protein is also discussed. The biological activity of protein hydrolyzates from different species of insects (Bombyx mori, Hermetia illucens, Acheta domesticus, Tenebrio molitor) are also reviewed, and the hydrolysates (bioactive peptides) are found to have either antihypertensive, antioxidant, antidiabetic, and antimicrobial activity. Finally, the use of edible insect protein in various food applications is presented.

Pulsed multifrequency thermosonication induced sonoporation in Alicyclobacillus acidoterrestris spores and vegetative cells.

Alicyclobacillus acidoterrestris (AAT) was proposed as an index of pasteurization design for high-acid fruit products due to its spore resistance and repeated spoilage incidences in fruit juices. This study aimed to determine the effectiveness of pulsed multifrequency ultrasound to minimize AAT spores and vegetative cells in aqueous suspension. For this research, an investigation of the reactive oxygen species and antioxidant activity was performed to examine the effect of temperature and frequency on AAT spore inoculation. Total decreases in AAT bacteria were 5.99, 5.74 Log CFU/mL in vegetative cells for dual-frequency thermosonication (DFTS) and dual-frequency ultrasonication (DFUS), respectively, while 5.90 and 5.38 Log CFU/mL in spores for both DFUS and DFTS, respectively. The loss of the percentage of cells in ultrasound (US) and thermosonication (TS) treatments was inversely associated with the rate of O2-and H2O2 development. The fluorescence microscopy revealed a higher bactericidal efficacy of DFTS compared to the DFUS and control. Scanning Electron Microscopy (SEM) and Transmission electron microscopy (TEM) demonstrated ultra-structural modifications such as the interruption of cell walls by cavitation and pores in the membrane structure of the AAT bacteria induced by sonoporation. Several TS frequencies of 20/40/60, 20/40, and 20 kHz treated spores had a higher electrical conductivity than untreated ones, with an improvement of 7.94, 5.68, and 3.72 %, respectively. Fourier-transform infrared (FTIR) spectroscopy revealed major changes in the spectral region of membrane fatty acids and proteins of AAT. Simultaneously, AAT inactivation specific energy rate was significantly reduced using dual-frequency ultrasound, compared to mono-frequency thermosonication. The significant results of this work recommended pulsed DFUS as an alternative application to mono-frequency US in beverage industries.