Comparison of different ultrasound assisted extraction techniques for pectin from tomato processing waste

Concept of waste to wealth is a hot topic with research ongoing globally to reduce carbon footprint. In an effort to follow up this cause present study focused on tomato industry waste specifically the peel of tomatoes for extraction of pectin. Pectin extraction was performed using five different extraction techniques (Ultrasound assisted extraction (UAE); microwave assisted extraction (MAE); ohmic heating assisted extraction (O_HAE); ultrasound assisted microwave extraction (UAME) and ultrasound assisted ohmic heating extraction (UAO_HE) at different power levels to study its extraction and degradation kinetics and in turn to optimize the extraction process. The extracted pectin yield ranged from 9.30% for O_HAE to 25.42% for MAE. Also, there was very less difference in the yield of MAE and UAME extracted pectin, but at the cost of major difference in degree of esterification 59.76 ± 0.70 and 73.33 ± 1.76%, respectively. In addition, all the pectin extracted under optimized conditions was having acceptable purity, [Galacturonic acid (GalA) content ranged from 675.8 ± 11.31 to 913.3 ± 20.50 g/kg of pectin]. FTIR analysis confirmed the presence of functional groups in the finger print region of identification for polysaccharide in all the extracted pectin. According to obtained results, UAME can be considered as better green extraction technology in terms of extraction yield as well as in quality of pectin compared to the other treatments used. Therefore, results suggest that UAME can be used as an efficient pectin extraction method from tomato processing waste.     

Influence of ultrasound on extraction of tannins from deoiled salseed cake

Deoiled salseed cake which contains 13–18% of tannins has extensive use in the production of poultry and cattle feed mixtures, where the tannin content is reduced to 4% or less. Studies have been carried out to remove tannins by the application of ultrasound as a function of frequency (43, 720 and 1000 kHz); intensity (2 to 9 W cm^?2); time (0 to 60 min); and by using different solvents. It has been observed that the percentage of extraction is greater at lower frequencies: 78% of tannins is extracted in a 40% aqueous acetone solvent using ultrasound in only 30 min compared to 20 h with conventional techniques.     

Extraction of phenolics and anthocyanins from purple eggplant peels by multi-frequency ultrasound: Effects of different extraction factors and optimization using uniform design

In this work, multi-frequency ultrasound (working modes for the single-, dual- and tri-frequency in simultaneous ways) was applied to extract bioactive compounds from purple eggplant peels. The single-factor experiments were performed by varying six independent variables. A six-level-five-factor uniform design (UD) was further employed to evaluate the interaction effects between different factors. It was found that extraction temperature and extraction time significantly affected the total phenolic content (TPC), whereas the total monomeric anthocyanins (TMA) was mainly influenced by ethanol concentration, extraction temperature and solid-liquid ratio. Based on partial least-squares (PLS) regression analysis, the optimal conditions for TPC extraction were: 53.6 % ethanol concentration, 0.336 mm particle size, 44.5 °C extraction temperature, 35.2 min extraction time, 1:43 g/mL solid-liquid ratio, and similar optimal conditions were also obtained for TMA. Furthermore, the TPC and TMA extraction were investigated by ultrasound in different frequencies and power levels. Compared with single-frequency (40 kHz) and dual-frequency ultrasound (25 + 40 kHz), the extraction yield of TPC and TMA with tri-frequency ultrasound (25 + 40 + 70 kHz) increased by 23.65 % and 18.76 % respectively, which suggested the use of multi-frequency ultrasound, especially tri-frequency ultrasound, is an efficient strategy to improve the TPC and TMA extracts in purple eggplant peels.     

Optimization of ultrasonic cell grinder extraction of anthocyanins from blueberry using response surface methodology

Ultrasonic cell grinder extraction (UCGE), using water as the solvent, was firstly applied to extract anthocyanins from blueberry. Extraction yield was related with four variables, including ratio of solution to solid, extraction power, buffer time, and extraction time. On the basis of response surface methodology (RSM), the optimal conditions were determined to be the ratio of solution to solid as 25:1 (mL/g), the extraction power as 1500 W, the buffer time as 3.0 s, and the extraction time as 40 min. The experimental yield of anthocyanins using UCGE was 2.89 mg/g higher than that of conventional ultrasound-assisted extraction (CUAE). This study indicated that UCGE was an innovative, efficient, and environment friendly method in ultrasonic extraction fields, and had a potential to effectively extract other bioactive constituents.     

Effect of fluid properties on ultrasound assisted liquid-liquid extraction in a microchannel

When immiscible liquids are subjected to an ultrasonic field, they form emulsions. This principle has been used to improve the mass transfer characteristics of a liquid-liquid extraction process in microreactor systems. The formation of emulsion and its characteristics are prominently dependent on the properties of the liquids used and this also holds true for emulsion brought about by ultrasound. This paper focuses on the properties of fluids that are reported to have an influence on the cavitation behaviour, namely viscosity, interfacial tension and vapour pressure. These properties were examined by changing the solvent of the organic phase in the hydrolysis of p-nitrophenyl acetate. The study is performed by comparing pairs of solvents that are different in one property but similar in the other two. The pairs selected are toluene – chlorobenzene for viscosity, toluene – methyl Isobutyl ketone for interfacial tension and methyl isobutyl ketone – 2-Methyl tetrahydrofuran for vapour pressure effects. A qualitative study was performed with a high-speed camera in flow to understand the emulsification initiation mechanisms and behaviours. These findings were further explored by performing the sonicated emulsion in a batch-sonicated reactor. The quantitative analysis of the fluid properties was evaluated and compared based on the relative percentage increase in yield upon sonication with respect to their individual silent conditions. The quantitative results were further supported by the quantification of the emulsion performed with an FBRM probe. The results indicate a two times improvement in yield with solvent of lower viscosity as 2 times more droplets were formed in the emulsion. Both the solvent systems with higher interfacial tension and vapour pressure had an improved yield of 1.4 times owing to larger number of droplets formed.     

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.     

Effect of combined ultrasonic and enzymatic extraction technique on the quality of noni (Morinda citrifolia L.) juice

In order to obtain noni juice with high yield and good quality, the effect of combined extraction technique of enzymatic treatment (EZ) and ultrasonication (US) on the overall quality of noni juice was investigated. Moreover, the extraction performance of the EZ-US combined extraction technique was compared with that of EZ-based extraction and the US-based extraction. Response surface methodology (RSM) was designed to optimize the parameters of ultrasonic treatment, by taking consideration of the extraction efficiency, quality parameters and bioactive ingredients of noni juice. The results indicated that combined ultrasonic and enzymatic treatment achieved a synergistic effect on promoting the quality of noni juice. The maximum juice yield of 67.95 % was obtained under ultrasonication for 10 min at 600 W after enzymatic treatment (EZU). In addition, EZU-treated juice exhibited the highest contents of total phenolic and flavonoid, which were 148.19 ± 2.53 mg gallic acid/100 mL and 47.19 ± 1.22 mg rutin/100 mL, respectively, thus contributing to better antioxidant activity. Moreover, the EZU treatment significantly reduced the particle size of noni juice, and improved its suspension stability and rheological properties. FTIR results indicated that the treatments did not bring major changes in the chemical structure and the functional groups of compounds in noni juice. Therefore, EZU treatment can be successfully applied to the extraction of noni juice with better nutritional properties and overall quality.     

Intensification of extraction of curcumin from Curcuma amada using ultrasound assisted approach: Effect of different operating parameters

Curcumin, a dietary phytochemical, has been extracted from rhizomes of Curcuma amada using ultrasound assisted extraction (UAE) and the results compared with the conventional extraction approach to establish the process intensification benefits. The effect of operating parameters such as type of solvent, extraction time, extraction temperature, solid to solvent ratio, particle size and ultrasonic power on the extraction yield have been investigated in details for the approach UAE. The maximum extraction yield as 72% was obtained in 1 h under optimized conditions of 35 °C temperature, solid to solvent ratio of 1:25, particle size of 0.09 mm, ultrasonic power of 250 W and ultrasound frequency of 22 kHz with ethanol as the solvent. The obtained yield was significantly higher as compared to the batch extraction where only about 62% yield was achieved in 8 h of treatment. Peleg’s model was used to describe the kinetics of UAE and the model showed a good agreement with the experimental results. Overall, ultrasound has been established to be a green process for extraction of curcumin with benefits of reduction in time as compared to batch extraction and the operating temperature as compared to Soxhlet extraction.     

Effect of ultrasound processing on anthocyanins and color of red grape juice

Grape juice samples were sonicated with processing variables of amplitude level (24.4–61.0 μm) and treatment time (0–10 min) at a constant frequency of 20 kHz and pulse durations of 5 s on and 5 s off. A full factorial experimental design with regression modeling was employed to investigate the main effects of amplitude level and treatment time on anthocyanins and color parameters. Significant effects of sonication on major anthocyanins cyanidin-3-O-glucosides (CA), malvanidin-3-O-glucosides (MA) and delphinidin-3-O-glucosides (DA), color values (L*, a*, b*) and color index (CI) were observed. Prediction models were found to be significant (p < 0.05) with low standard errors and high coefficients of determination (R²). Model predictions for critical quality parameters of anthocyanins (CA; MA; DA), color values (L*, a*, b*), TCD and CI inactivation were closely correlated to the experimental results obtained. Significant retention of anthocyanin content in grape juice was observed for CA (97.5 %); MA (48.2 %) and DA (80.9%) during sonication. CI and other color combinations (L*a*b*, L*a*/b* and L*b*/a*) were found to be strongly correlated with anthocyanin content. This study shows that sonication could be employed for as a preservation technique for fruit juice processing where anthocyanin retention is desired.     

Comparison of ultrasound and microwave assisted extraction of diosgenin from Trigonella foenum graceum seed

From the recent market trend, there is a huge demand for the bioactive compounds from various food matrices that could be capable enough to combat the emerging health effects in day-to-day life. Fenugreek is a well-known spice from ancient times for its medicinal and health benefits. In the present study, two methods of green extraction microwave (MAE) and ultrasound (UAE) assisted were studied in regard of extraction of fenugreek diosgenin. In this study, solvent type (acetone, ethanol, hexane and petroleum ether), solvent concentration (40, 60, 80 and 100%) and treatment time (1.5, 3.0, 4.5 and 6.0 min and 30, 40, 50 and 60 min for MAE and UAE method respectively) was varied to observe the effect of these parameters over extract yield and diosgenin content. The results of this study revealed that treatment time, type of solvent and its concentration and method adopted for extraction of diosgenin has significant effect. In relation with better yield extract and diosgenin content, the yield of fenugreek seed extract was 7.83% with MAE and 21.48% with UAE of fenugreek seed powder at 80% ethanol concentration at 6 and 60 min respectively. The content of diosgenin was observed in fenugreek seed powder extract was 35.50 mg/100 g in MAE and 40.37 mg/100 g in UAE with 80% ethanol concentration at 6 and 60 min respectively. The overall range of yield of fenugreek extract was varied from 1.04% to 32.48% and diosgenin content was 15.82 mg/100 g to 40.37 mg/100 g of fenugreek seed powder including both extraction methods. This study revealed that UAE would impose better ways for preparing fenugreek extract and observing diosgenin content from fenugreek seeds.     

Optimization of ultrasound-assisted extraction of anthocyanins in red raspberries and identification of anthocyanins in extract using high-performance liquid chromatography-mass spectrometry

Anthocyanins (Acys) are naturally occurring compounds that impart color to fruit, vegetables and plants. The extraction of Acys from red raspberry (Rubus idaeus L. var. Heritage) by ultrasound-assisted process (UAP) was studied. A central composite rotate design (CCRD) was used to obtain the optimal conditions of ultrasound-assisted extraction (UAE), and the effects of operating conditions, such as the ratio of solvents to materials, ultrasonic power and extraction time, on the extraction yield of Acys were studied through response surface methodology (RSM). The optimized conditions of UAE were as follows: ratio of solvents to materials was 4:1 (ml/g), extraction time was 200 s, and ultrasonic power was 400 W. Under these conditions 34.5 mg of Acys from 100 g of fresh fruits (T_Acy, expressed as cyanidin-3-glucoside), approximately 78.13% of the total red pigments, could be obtained by UAE. The Acys compositions of extracts were identified by high-performance liquid chromatography–mass spectrometry (HPLC–MS), 12 kinds of Acys had been detected and eight kinds of Acys were characterized. Result indicated that cyanidin-3-sophoroside, cyanidin-3-(2^G-glucosylrutinoside), cyanidin-3-sambubioside, cyanidin-3-rutinoside, cyanidin-3-xylosylrutinoside, cyanidin-3-(2^G-glucosylrutinoside), and cyanidin-3-rutinoside were main components in extracts. In addition, in comparison with the conventional solvent extraction, UAE is more efficient and rapid to extract Acys from red raspberry, due to the strong disruption of fruit tissue structure under ultrasonic acoustic cavitation, which had been observed with the scanning electron microscopy (SEM). However, the Acys compositions in extracts by both methods were similar, which were investigated using HPLC profile.     

Impact of ultrasonication on the aggregation structure and physicochemical characteristics of sweet potato starch

In order to understand the relationship between the aggregation structure and physicochemical characteristics of ultrasonicated sweet potato starches, sweet potato starch modified with different sonication time (15, 20, 25 and 30 min) was studied in this work for developing starch-based ingredients with tailored functionality. SEM, XRD, FTIR, Raman and DSC measurements were conducted to investigate the transformations of morphological features, crystalline structure, short-range ordered structure and ordered molecular structure of starch granules. Not only could pores and cracks be observed at the surface of starch, but also the structural disorganizations were more evidently induced with the treatment time increasing, especially for the degrees of crystallinity, short-range molecular orders and ordered molecular structures. And these disordering in the aggregation structure not only increased the swelling power and solubility but also declined the pasting temperature, peak viscosity, final and breakdown viscosity of gelatinized starch. In addition, the strengthened retrogradation and gel structures formed by the rearrangement and entanglement of starch polymer chains also demonstrated the structural disorganizations of starch granules during ultrasonic treatment. Therefore, the results of this study may provide further insight in understanding the structural basis of starch physicochemical properties.     

Combined effect of ultrasound,heat, and pressure on Escherichia coli O157:H7, polyphenol oxidase activity,and anthocyanins in blueberry (Vaccinium corymbosum) juice

The objective of this study was to evaluate the effect of different treatments—heat treatment (HT), sonication (SC), thermosonication (TS), manosonication (MS), manothermal (MT), and manothermosonication (MTS) on Escherichia coli O157:H7, polyphenol oxidase (PPO), and anthocyanin content in blueberry juice. First, samples were treated at different temperatures (30, 40, 50, 60, 70, and 80 °C) and power intensities (280, 420, 560, and 700 W) for 10 min. Subsequently, samples were treated using combinations of power intensity and mild temperature for 10 min. For further study, samples were treated using HT (80 °C), TS (40 °C, 560 W), MT (350 MPa, 40 °C), MS (560 W, 5 min/350 MPa), or MTS (560 W, 5 min, 40 °C/350 MPa, 40 °C) for 5, 10, 15, 20 min for each treatment, and the results compared between treatments. HT significantly reduced PPO activation (2.05% residual activity after only 5 min), and resulted in a 2.00-log reduction in E. coli O157:H7 and an 85.25% retention of anthocyanin. Escherichia coli O157:H7 was slightly inactivated by TS after 5 min (0.17-log reduction), while residual PPO activity was 23.36% and anthocyanin retention was 98.48%. However, Escherichia coli O157:H7 was rapidly inactivated by MTS (5.85-log reduction) after 5 min, while anthocyanin retention was 97.49% and residual PPO activity dropped to 10.91%. The destruction of E. coli cells as a result of these treatments were confirmed using SEM and TEM. Therefore, a combination of sonication, high pressure, and mild heat allows the safety of blueberry juice to be maintained without compromising the retention of desirable antioxidant compounds.     

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.     

超声场强化超临界CO2萃取除虫菊酯的初步研究

本文以除虫菊花中的除虫菊酯萃取为对象,研究用超临界CO2对其萃取时,施加超声场对萃取物降解及萃取速率的影响.结果表明,在实验范围内施加超声场(200kHz,200W电功率)没有引起超临界CO2流体"空化",从而没有使易分解组分除虫菊酯降解;施加超声场可以提高除虫菊酯的萃取速率,且对萃取速率的影响比用水为共溶剂强,在实验范围内,萃取优化工艺条件为萃取压力(20MPa);施加超声场(200kHz,200W电功率);萃取温度(40℃);添加共溶剂水(0.5ml水/g干花);CO2流量(2.0L·min-1).     

Influence of the ultrasound cavitation intensity on reduced graphene oxide functionalization

Graphene is a valuable and useful nanomaterial due to its exceptionally high tensile strength, electrical conductivity and transparency, as well as the ability to tune its materials properties via functionalization. One of the most important features needed to integrate functionalized graphene into products via scalable processing is the effectiveness of graphene dispersion in aqueous and organic solvents. In this study, we aimed to achieve the functionalization of reduced graphene oxide (rGO) by sonication in a one-step process using polyvinyl alcohol (PVA) as a model molecule to be bound to the rGO surface. We investigated the influence of the sonication energy on the efficacy of rGO functionalization. The correlation between the performance of the high-intensity ultrasonic horn and the synthesis of the PVA functionalized rGO was thoroughly investigated by TGA coupled with MS, and IR, Raman, XPS, Laser diffraction, and SEM analysis. The results show that the most soluble PVA-functionalized rGO is achieved at 50% of the ultrasonic horn amplitude. Analysis of cavitation dynamics revealed that in the near vicinity of the horn it is most aggressive at the highest amplitude (60%). This causes rGO flakes to break into smaller domains, which negatively affects the functionalization process. On the other hand, the maximum of the pressure pulsations far away from the horn is reached at 40% amplitude, as the pressure oscillations are attenuated significantly in the 2-phase flow region at higher amplitudes. These observations corelate well with the measured degree of functionalization, where the optimum functionalized rGO dispersion is reached at 50% horn amplitude, and generally imply that cavitation intensity must be carefully adjusted to achieve optimal rGO functionalization.     

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.     

Ultrasonication-assisted synthesis of alcohol-based deep eutectic solvents for extraction of active compounds from ginger

An ultrasonication-assisted synthesis of alcohol-based deep eutectic solvents (DESs) is described. Several DESs were synthesized simultaneously under the same conditions. The prepared DESs were used for the extraction of gingerols from ginger powder via ultrasonication-assisted extraction. Notably, some of the prepared DESs exhibited superior extraction performance than those in traditional organic solvents. The viscosity of the DESs, which was suggested to be typically lower than 100 mPa*s had a critical effect on extraction performance. However, the higher gingerol contents in the extracts did not translate to higher active antioxidant abilities. The extraction temperature was found to be a key determinant of the antioxidant capability of the extracted gingerols while the use of higher temperatures (>50 °C) induced degradation and loss of phenolic compounds during extraction. Response surface methodology was applied for determining the optimal extraction conditions to achieve maximum antioxidant capacity with suitable gingerol content. All compounds used for the preparation of the DESs in this study have been widely employed in cosmetic and pharmaceutical fields. Therefore, the extracts in these DES solutions can be considered for direct application development without further product isolation.     

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.     

An expeditious synthetic approach towards the synthesis of Bis-Schiff bases (aldazines) using ultrasound

Aldazines (Bis-Schiff bases) 1–24 were synthesized using aromatic aldehydes (heterocyclic and benzaldehydes) and hydrazine hydrate under reflux using conventional heating and/or via ultrasound irradiation using BiCl₃ as catalyst. Ultrasonication conditions with cat. BiCl₃ proved to be an effective, environmentally friendly synthetic procedure. This methodology is robust in the presence of electron donating and electron withdrawing groups affording desired products with high yields (>95%) in just a couple of minutes vs. hours using conventional heating.