Increasing Performance of Spiral-Wound Modules (SWMs) by Improving Stability against Axial Pressure Drop and Utilising Pulsed Flow.

Spacer-induced flow shadows and limited mechanical stability due to module construction and geometry are the main obstacles to improving the filtration performance and cleanability of microfiltration spiral-wound membranes (SWMs), applied to milk protein fractionation in this study. The goal of this study was first to improve filtration performance and cleanability by utilising pulsed flow in a modified pilot-scale filtration plant. The second goal was to enhance membrane stability against module deformation by flow-induced friction in the axial direction ("membrane telescoping"). This was accomplished by stabilising membrane layers, including spacers, at the membrane inlet by glue connections. Pulsed flow characteristics similar to those reported in previous lab-scale studies could be achieved by establishing an on/off bypass around the membrane module, thus enabling a high-frequency flow variation. Pulsed flow significantly increased filtration performance (target protein mass flow into the permeate increased by 26%) and cleaning success (protein removal increased by 28%). Furthermore, adding feed-side glue connections increased the mechanical membrane stability in terms of allowed volume throughput by ≥100% compared to unmodified modules, thus allowing operation with higher axial pressure drops, flow velocities and pulsation amplitudes.

The Role of Maltodextrin Concentration in Maintaining Storage Stability of Dried Fruit Foams Texturized Using Plant Protein-Polysaccharide Blends.

Hydrocolloids are widely used in food processing because of their texture-forming abilities, which help to preserve the quality of sensitive compounds, e.g., in dried fruit foams, which have recently emerged in healthier alternative snacks. Our aim was to investigate the protective role of maltodextrin in improving the storage stability of fruit foams. This study evaluated the effect of maltodextrin concentrations on the stability of the following quality parameters: anthocyanins, ascorbic acid, color, texture, and sensory perception of dried foamed raspberry pulp during storage. This study compared three concentrations (5%, 15%, and 30% w/w) of maltodextrin in mixtures, evaluating their effect on the stability of these parameters over a 12-week storage period. The foam samples were stored at 37 °C to accelerate chemical reactions under vacuum packaging conditions which excluded oxygen. The addition of 30% maltodextrin to the raspberry pulp blend resulted in the highest retentions in all compounds tested, i.e., 74% for ascorbic acid and 87% for anthocyanins. Color and texture were similarly preserved. Adding 30% maltodextrin to the mixture did not negatively influence the acceptability of sensory perception. Maltodextrin thus represents an effective protective agent for preserving nutritional and sensory qualities for a longer storage period. Hence, using MD together with potato protein was optimal for enhancing the storage stability of fruit foam, which is important for the food industry.

Protein-protein interactions explain the temperature-dependent viscoelastic changes occurring in colloidal protein gels.

Denaturation of protein solutions can be induced by higher temperatures and the presence of non-polar organic solutions. The denatured proteins form aggregates and gels through protein interactions occurring between their amino acid side chains. Depending on the involved side chains, the denaturation conditions lead to different gel properties. As model systems, a variety of food proteins were gelled through different mechanisms to cover a whole range of protein-protein interactions. Especially the temperature dependence of the viscoelastic properties in a simple rheometer method was found to be very different. These differences could be explained by the different thermodynamic properties of the involved protein-protein interactions. Electrostatic interactions were shown to weaken the resulting gel upon temperature increase whereas entropically driven interactions such as hydrophobic or covalent links were strengthened with increased temperatures. A proposed model explaining these results can be used to assess protein interactions in hydrogels in a non-invasive way and could also have applications to describe the temperature behavior of other hydrogels.

New technical concept for alternating tangential flow filtration in biotechnological cell separation processes.

Robust cell retention devices are key to successful cell culture perfusion. Currently, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) are most commonly used for this purpose. TFF, however, suffers from poor fouling mitigation, which leads to high filtration resistance and product retention, and ATF suffers from long residence times and cell accumulation. In this work, we propose a filtration system for alternating tangential flow filtration, which takes full advantage of the fouling mitigation effects of alternating flow and reduces cell accumulation. We have tested this novel setup in direct comparison with the XCell ATF® as well as TFF with a model feed comprising yeast cells and bovine serum albumin as protein at harsh permeate to feed flow conditions. We found that by avoiding the dead-end design of a diaphragm pump, the proposed filtration system exhibited a reduced filtration resistance by approximately 20% to 30% (depending on feed rate and permeate flow rate). A further improvement of the novel setup was reached by optimization of phase durations and flow control, which resulted in a fourfold extension of process duration until hollow fiber flow channel blockage occurred. Thus, the proposed concept appears to be superior to current cell retention devices in perfusion technology.

Comparison of one-step with two-step production of Bacillus atrophaeus spores for use as bioindicators.

The production method of spores significantly influences the resistance of spores used as bioindicators (BI) in the validation of sterilization of packaging material surfaces in aseptic food manufacturing. Therefore, the standardization of the spore production method represents an important and desirable goal in industrial BI production to ensure reliable validation test results. Previously, we recommended a two-step production approach for submerged spore production, in which the cultivation phase to obtain high cell mass was separate from the sporulation phase. In this work, a one-step manufacturing process was investigated to reduce production complexity and facilitate standardization of spore production. It was found that one-step BI production is technically possible but at the expense of spore yield. The two-step manufacturing process can realize almost 10-fold higher spore yields.

Determination of Compressibility and Relaxation Behavior of Yeast Cell Sediments by Analytical Centrifugation and Comparison with Deposit Formation on Membrane Surfaces.

Separation of cells from produced biomolecules is a challenging task in many biotechnological downstream operations due to deposit formation of the retained cells, affecting permeation of the target product. Compression and relaxation behavior of cell deposits formed during filtration are important factors affecting operational performance. The determination of these factors by flux or pressure stepping experiments is time- and labor-intensive. In this work, we propose a screening method by analytical centrifugation, which is capable of detecting small differences in compression and relaxation behavior induced by milieu changes, using a model system comprised of washed and unwashed yeast cells in the presence or absence of bovine serum albumin as a model target protein. The main effects observed were firstly the impact of pH value, affecting interaction of bovine serum albumin and yeast cells especially close to the isoelectric point, and secondly the effect of washing the yeast cells prior to analysis, where the presence of extracellular polymeric substances led to higher compressibility of the deposited cells. By comparing and validating the obtained results with dead-end filtration trials, the stabilizing role of bovine serum albumin in deposits formed at low pH values due to interactions with the yeast cells was underlined.

Concentration, purification and quantification of milk protein residues following cleaning processes using a combination of SPE and RP-HPLC.

Detection and quantification of milk protein residues can be of utmost importance for validation of cleaning process efficiency in removing even traces of residues as well as quality assurance and product safety. However, currently available assays cannot provide a combination of high sensitivity and a simultaneous quantification of the individual milk proteins. Furthermore, a low protein-to-protein-variability and high compatibility with other reagents such as residual cleaning agents (e.g. surfactants) cannot be ensured. Therefore, a new method was developed comprised of a pre-concentration of proteins by solid-phase extraction and optimisation of the sensitivity of an existing reversed-phase high performance liquid chromatography method for the separate quantification of bovine milk proteins κ-Casein, αS2-Casein, αS1-Casein, ß-Casein, α-Lactalbumin, and ß-Lactoglobulin. Hereby, solid-phase extraction enables robust and reproducible purification and concentration of protein residues with a high protein recovery rate and flexible adjustment of concentration factors. The increased sensitivity of the reversed-phase high performance liquid chromatography method was achieved by changes in the measurement wavelength and guanidine buffer concentration. This new method enables reproducible concentration, purification and quantification of protein concentrations below 7 ng mL-1 and thus can be used to detect milk protein residues in highly diluted aqueous systems.•Concentration, purification and quantification of milk protein residues with a high recovery rate of proteins (> 94%) and high reproducibility (coefficient of variation (CV) < 3.0%)•Flexible adjustment of sample volumes allows the utilisation of high concentration factors (≤ 500) without compromising the recovery rate of proteins (recovery rate of proteins decreases by 2.74% per 100 CF).

Heat-induced aggregation kinetics of potato protein - Investigated by chromatography, calorimetry, and light scattering.

In this study, the heat-induced aggregation behavior of patatin rich potato protein isolate (PPI) was investigated by reversed-phase high-pressure liquid chromatography (RP-HPLC), differential scanning calorimetry (DSC), and dynamic light scattering. It could be shown that aggregation already occurs at low temperatures, despite low degrees of unfolding. The unfolding temperature, determined by DSC, coincided with a change in the reaction kinetics, which is determined by the unfolding step below a critical temperature up to the point, where the proteins are completely unfolded. The reaction rate k as a function of the absolute temperature T is then determined by diffusion of unfolded proteins forming aggregates. This change can be visualized in the Arrhenius diagram by a change of the slope of the relationship k âˆ¼ 1/T. A change in pH from 7 to 6 shifted the critical temperature towards higher values and resulted in larger aggregate sizes, due to reduced electrostatic repulsion.

Transcriptome and fatty-acid signatures of adipocyte hypertrophy and its non-invasive MR-based characterization in human adipose tissue.

BACKGROUND: The adipocyte-hypertrophy associated remodeling of fat cell function is considered causal for the development of metabolic disorders. A better understanding of transcriptome and fatty acid (FA) related alterations with adipocyte hypertrophy combined with less-invasive strategies for the detection of the latter can help to increase the prognostic and diagnostic value of adipocyte size and FA composition as markers for metabolic disease. METHODS: To clarify adipocyte-hypertrophy associated transcriptomic alterations, fat cell size was related to RNA-Seq data from white adipose tissue and size-separated adipocytes. The relationship between adipocyte size and adipose tissue FA composition as measured by GC-MS was investigated. MR spectroscopy (MRS) methods for clinical scanning were developed to characterize adipocyte size and FA composition in a fast and non-invasive manner. FINDINGS: With enlarged adipocyte size, substantial transcriptomic alterations of genes involved in mitochondrial function and FA metabolism were observed. Investigations of these two mechanisms revealed a reciprocal relationship between adipocyte size and estimated thermogenic adipocyte content as well as depot-specific correlations of adipocyte size and FA composition. MRS on a clinical scanner was suitable for the in-parallel assessment of adipose morphology and FA composition. INTERPRETATION: The current study provides a comprehensive overview of the adipocyte-hypertrophy associated transcriptomic and FA landscape in both subcutaneous and visceral adipose tissue. MRS represents a promising technique to translate the observed mechanistic, structural and functional changes in WAT with adipocyte hypertrophy into a clinical context for an improved phenotyping of WAT in the context of metabolic diseases. FUNDING: Competence network for obesity (FKZ 42201GI1128), ERC (No 677661, ProFatMRI; No 875488, FatVirtualBiopsy), Else Kröner-Fresenius-Foundation.

Critical assessment of methods for measurement of temperature profiles and heat load history in microwave heating processes-A review.

Limitations of microwave processing due to inhomogeneities of power input and energy absorption have been widely described. Over- and underheated product areas influence reproducibility, product quality, and possibly safety. Although a broad range of methods is available for temperature measurement and evaluation of time/temperature effects, none of them is sufficiently able to detect temperature differences and thermally induced effects within the product caused by inhomogeneous heating. The purpose of this review is to critically assess different methods of temperature measurement for their suitability for different microwave applications, namely metallic temperature sensors, thermal imaging, pyrometer measurement, fiber optic sensors, microwave radiometry, magnetic resonance imaging, liquid crystal thermography, thermal paper, and biological and chemical time-temperature indicators. These methods are evaluated according to their advantages and limitations, method characteristics, and potential interference with the electric field. Special attention is given to spatial resolution, accuracy, handling, and purpose of measurement, that is, development work or online production control. Differences of methods and examples of practical application and failure in microwave-assisted food processing are discussed with a special focus on microwave pasteurization and microwave-assisted drying. Based on this assessment, it is suggested that infrared cameras for measuring temperature distribution at the product surface and partially inside the product in combination with a chemical time/temperature indicator (e.g., Maillard reaction, generating heat-induced color variations, depending on local energy absorption) appear to be the most appropriate system for future practical application in microwave food process control, microwave system development, and product design. Reliable detection of inhomogeneous heating is a prerequisite to counteracte inhomogeneity by a targeted adjustment of process and product parameters in microwave applications.

Sensomics-Assisted Aroma Decoding of Pea Protein Isolates (Pisum sativum L.).

The aroma of pea protein (Pisum sativum L.) was decrypted for knowledge-based flavor optimization of new food products containing pea protein. Sensomics helped to determine several volatiles via ultra-high performance liquid chromatography tandem mass spectrometry and 3-nitrophenylhydrazine derivatization. Among the investigated volatiles, representatives of aldehydes, ketones, and acids were reported in literature as especially important in pea and pea-related matrices. After validation of the method and quantitation of the corresponding analytes, sensory reconstitution as well as omission studies of a selected pea protein were performed and revealed nine odor-active compounds as key food odorants (3-methylbutanal, hexanal, acetaldehyde, (E,E)-2,4-nonadienal, (E)-2-octenal, benzaldehyde, heptanal, 2-methylbutanal, and nonanoic acid). Interestingly, eight out of nine compounds belonged to the chemical class of aldehydes. Statistical heatmap and cluster analysis of all odor activity values of different pea proteins confirmed the obtained sensory results and generalize these nine key food odorants in other pea proteins. The knowledge of key components gained shows potential for simplifying industrial flavor optimization of pea protein-based food.

Preservation by lyophilization of a human intestinal microbiota: influence of the cultivation pH on the drying outcome and re-establishment ability.

Faecal microbiota transplantation is an emerging medical concept for the treatment of gastrointestinal diseases. This concept, however, has disadvantages as low storability of stool and intensive donor screening. A solution to overcome these problems would be the preservation of an in vitro microbiota through freeze-drying. However, the influence of the entire preservation process, including cultivation and lyophilization, has not been assessed so far. In this study, the influences of the process steps cultivation, drying and re-cultivation were determined with cell count, production of metabolites, microbial composition and diversity in the system as evaluation criteria. All pH conditions resulted in stable, culturable communities after re-cultivation. Cell count, richness, diversity and microbial composition were affected by freeze-drying, but these effects were reversible and vanished during re-cultivation. Hence, the re-cultivated system did not differ from the system before drying. The metabolism, measured by short-chain fatty acids as indicators, showed slight changes due to natural dynamics. Consequently, the cultivation prior to drying was identified to have more influence than the drying itself on the preservation process and therefore the biggest potential for optimization. Hence, the highest similarity with the initial stool sample was obtained with pH 6.0 - 6.5 during cultivation.

Effect of Vertical and Horizontal Sample Orientations on Uniformity of Microwave Heating Produced by Magnetron and Solid-State Generators.

In this study, the effect of different horizontal and vertical orientations of a model sample (cuboid gellan gel samples containing Maillard reactants) on microwave heat processing was investigated in the solid-state and magnetron microwave systems. To achieve this target, seven orientations inside both microwave cavities were defined. Two of the investigated sample orientations were in a vertical position with and without turntable rotation, and five in a horizontal position. Furthermore, samples at horizontal orientations were put at an angle position without turntable rotation. To analyze the microwave heating patterns, infrared (IR) pictures and photographs of the gellan gel samples were taken after processing to document IR-based thermal and Maillard color changes, respectively. Three main factors for improvement of the heating homogeneity were identified: first, processing samples in the solid-state microwave system; second, position variation of the sample by turntable activated; and third, horizontal orientation. In addition, it was observed that placing the gellan gel samples in a vertical position in the magnetron microwave system resulted in considerably more absorbed power and a more uniform microwave heat processing compared to other horizontal orientations in this system. This indicated a non-uniform microwave field distribution. The results of this study can also confirm the importance of designing suitable food packaging: a vertical shape for more microwave energy absorbance and thus, more energy efficiency, and a horizontal shape for more uniform microwave heat processing.

Molecular Analytical Assessment of Thermally Precipitated α-Lactalbumin after Resolubilization.

Selective thermal precipitation followed by a mechanical separation step is a well described method for fractionation of the main whey proteins, α-lactalbumin (α-la) and ß-lactoglobulin (ß-lg). By choosing appropriate environmental conditions the thermal precipitation of either α-la or ß-lg can be induced. Whereas ß-lg irreversibly aggregates, the precipitated α-la can be resolubilized by a subsequent adjustment of the solution's pH and the ionic composition. This study reports on the analytical characterization of resolubilized α-la compared to its native counterpart as a reference in order to assess whether the resolubilized α-la can be considered close to 'native'. Turbidity and quantification by RP-HPLC of the resolubilized α-la solutions were used as a measure of solubility in aqueous environment. RP-HPLC was also applied to determine the elution time as a measure for protein's hydrophobicity. DSC measurement was performed to determine the denaturation peak temperature of resolubilized α-la. FTIR spectroscopy provided insights in the secondary structure. The refolding of α-la achieved best results using pH 8.0 and a 3-fold stoichiometric amount of Ca2+ per α-la molecule. The results showed that the mechanism of aggregation induced by gentle thermal treatment under acidic conditions with subsequent mechanical separation is reversible to a certain extent, however, the exact native conformation was not restored.

Submerged Bioreactor Production of Geobacillus stearothermophilus ATCC 7953 Spores for Use as Bioindicators to Validate Hydrogen Peroxide Inactivation Processes.

In the food and pharmaceutical industries, evaluating the sterilization performance preceding aseptic production processes is of central importance. In the case of hydrogen peroxide sterilization of solid surfaces, bioindicators (BI) consisting of spores of Bacillus atrophaeus or Geobacillus stearothermophilus are used to validate the effectiveness and efficiency of the inactivation procedure. Commercial production of G. stearothermophilus is commonly performed on agar plates, where cultivation and sporulation conditions are not well-defined. Therefore, the produced BI can vary in their resistance, which in turn creates unacceptable uncertainties in the evaluation of aseptic processes. Submerged production in the bioreactor would allow more control over sporulation conditions, while reducing production time, resistance variability, and avoidance of false-positive or false-negative test results. In addition, submerged production of G. stearothermophilus so far was a challenge to achieve sufficiently high spore concentrations for BI production. This study reports on the development of a method for submerged production of G. stearothermophilus spores (pH 7.0, 57 °C, 30% pO2) that can achieve 1.6 × 107 spores/mL with a resistance against 35% H2O2 at 25 °C of D25°C,35% H2O2 = 73 s. This resistance ranks within the range of commercially available BI, making the results directly transferable to industrial applications.

Cold-Renneted Milk Powders for Cheese Production: Impact of Casein/Whey Protein Ratio and Heat on the Gelling Behavior of Reconstituted Rennet Gels and on the Survival Rate of Integrated Lactic Acid Bacteria.

The idea was to develop powders for fresh/hard cheese or quark production comprising milk proteins in optimal composition and functional properties for manufacturing each of those cheese types. The aim was to avoid whey protein drainage by their prior removal or by their heat-induced structural integration in the curd. The pre-renneted powders already contain additives such as starter cultures and calcium chloride to instantaneously form homogeneous curds upon reconstitution. The impact of the casein/whey protein ratio (86:14 by ultrafiltration and 98:2 by microfiltration) and upfront heat treatment (80 °C/30 min) on the gelling behavior of reconstituted rennet gels and on the survival rate of integrated Lactobacillus paracasei ssp. paracasei F19 was investigated. The assessment criteria for the rennet gelation were curd firming rate, gel strength, and whey drainage. Furthermore, the amount of integrated whey proteins and the resulting cheese yield were evaluated. It could be shown that heating had a positive effect on the viable cell count of the bacteria after spray drying and on the gelation behavior of the reconstituted ultrafiltration concentrates. The curd firming rate and the gel strength could be increased to higher values than the reconstituted microfiltration concentrate at 25% total solids.

Quantification of protein-protein interactions in highly denatured whey and potato protein gels.

Understanding the stabilizing protein interactions in protein gels is of high importance for food- and biotechnology. Protein interactions in protein gels can help to predict hardness, deformability and other gel parameters. Currently there are two types methods used. One is to use protein interaction blocking agents and the other is to dissolve the gel in different buffer systems, which cleave the interactions. The first method alters the gelling mechanism, which is why the second method is the preferred one. However, currently published methods are often only suitable for specific gel systems as for example weakly bound protein gels. In this paper, a method is introduced, which is suitable for highly denatured whey and plant protein.•Suitable for strongly cross-linked whey protein and plant protein gels•Stronger buffer system to ensure cleavage of all protein interactions•More reproducible and simplified crushing of the gel without the introduction of uncontrolled shear stress excessively affecting the analysis of chemical bonds.

Effect of pentasodium triphosphate concentration on physicochemical properties, microstructure, and formation of casein fibrils in model processed cheese.

The effects of varying the concentration of pentasodium triphosphate (PP) emulsifying salt [0, 0.6, 1.2, 1.5, and 1.8%, plus 0.9% of a mixture of citric acid (CA) and disodium phosphate (DSP) to adjust cheese pH to 5.85] on rheological, textural, physicochemical, and microstructural properties were studied in a processed cheese model system containing ~20% micellar casein concentrate, ~20% sunflower oil, and ~59% water. Special emphasis was placed on the unique casein fibrils recently described in a comparable processed cheese model system. Our results show that during processing (90°C, 17.37 rpm over 270 min) the apparent viscosity increased more and faster for formulations containing higher concentrations of PP, in analogy to the so-called creaming reaction, a general thickening of the molten cheese mass with prolonged processing. We found that 1.2% PP (plus 0.9% CA-DSP) appeared to be the threshold for the creaming reaction to take place. With increasing PP concentrations, cheese hardness increased in a sigmoidal fashion, and insoluble (protein-bound) calcium concentration decreased exponentially. Light micrographs of samples taken at the end of processing indicated initially large and dense casein aggregates within the matrix that disappeared with higher levels of PP, in parallel with the development of a finer emulsion. With transmission electron microscopy analysis on the same samples, the highly complex restructuring of the casein matrix was evident; casein fibrils had formed de novo at the periphery of the loosening casein aggregates. With higher levels of PP, amorphous areas were observed in place of the dense casein aggregates that appeared progressively void of protein, whereas fibril concentration increased throughout the rest of the matrix. Fibrils progressively attached to the surface of fat globules, thereby emulsifying them. Reverse-phase HPLC analysis of insoluble and soluble fractions indicated κ-casein to be the most likely constituent of the newly formed fibrils. The results of this study suggest that PP induced a concentration-dependent dissociation of caseins (through increased calcium chelation) and further led to their spatial separation. In essence, their chaperone activity was hindered, which resulted in amorphous aggregation on the one hand and fibril formation on the other.

Stability of Foams in Vacuum Drying Processes. Effects of Interactions between Sugars, Proteins, and Surfactants on Foam Stability and Dried Foam Properties.

The hypothesis was that saccharides mediate interactions between surface-active components and that this will have an impact on foam decay during the drying process. Static light scattering was performed to determine changes in interactions between the foam stabilizer on a molecular level. Furthermore, pendant drop and oscillating drop measurements were performed to examine the surface tension and surface rheology. Foams were dried in conventional dryers as well as microwave-supported vacuum dryers. Final foam properties were determined. It was shown that the addition of sugars, often added as protective substances for sensitive organic molecules, resulted in lower repulsion between different types of surface-active components, namely polysorbate 80 and ß-lactoglobulin (ß-lg). Differences in impact of the types of sugars and between different types of surfactant, protein, and small molecules were observed influencing the foam decay behavior. The interfacial properties of polysorbate 80 and ß-lg were influenced by the type of the used sugars. The surface elasticity of protein stabilized surfaces was higher compared to that of polysorbate stabilized systems. Protein stabilized systems remained more stable compared to polysorbate systems, which was also affected by the used saccharide. Overall, a correlation between molecular interactions and foam decay behavior was found.

Correlation between Physico-Chemical Characteristics of Particulated ß-Lactoglobulin and Its Behavior at Air/Water and Oil/Water Interfaces.

It is widely accepted that protein-based particles can efficiently stabilize foams and emulsions. However, it is not fully elucidated which particle properties are decisive for the stabilization of air/water and oil/water interfaces. To unravel this correlation, selected properties of nano-sized soluble ß-lactoglobulin particles were changed one at a time. Therefore, particles of (1) variable size but similar zeta potential and degree of cross-linking and (2) similar size but different further properties were produced by heat treatment under a specific combination of pH value and NaCl concentration and then analyzed for their interfacial behavior as well as foaming and emulsifying properties. On the one hand, it was found that the initial phase of protein adsorption at both the air/water and the oil/water interface was mainly influenced by the zeta potential, independent of the particle size. On the other hand, foam stability as resolved from the time-dependent evolution of mean bubble area negatively correlated with disulfide cross-linking, whereas emulsion stability in terms of oil droplet flocculation showed a positive correlation with disulfide cross-linking. In addition, flocculation was more pronounced for larger particles. Concluding from this, foam and emulsion stability are not linked to the same particle properties and, thus, explanatory approaches cannot be used interchangeably.