Study of the preparation of a stable foam

Author: Anh Nguyen

Supervisor: Marie Wahlgren, Lund University Assistant Supervisor: Lucia Casal Dujat, Aventure AB Examiner: Björn Bergenståhl, Lund University

Degree Project in Pharmaceutical Technology Department of Food Technology

Faculty of Engineering Lund University

Sweden, 2018

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Abstract On a daily basis, abdominal examinations with x-ray imaging are run at hospitals for diagnosis of abdominal diseases. To enable doctors to visualize organs and tissues on an x-ray image with colors based on the radiodensity, the patient needs to drink an oral contrast that fills up the digestive system. Current filling agents have a radiodensity similar to that of organs and tissues which makes the contrast between the agent and the organs low, and a challenge for the doctor to give a correct diagnosis.

The aim of this work was to scale down the production of a protein-based foam used as contrast agent, as the current process is not adapted to the time it takes for the patient to drink the full dose, leading to varying quality of the portions of foam swallowed. A production time of below 3 min was desired. Ultrasonication and scaling parameters of the current mixing method were evaluated for the mixing of powder ingredients in liquid (dispersion production). The foaming process was downscaled by evaluating equipment dimensions. Since the foam properties need to be unaffected by the downscaling, various analyses were conducted.

Results from the ultrasonication experiments showed that ultrasound enhances foaming properties and decreases the bubble size to 1/3 of that obtained with the original method. Downscaling the dispersion production and foaming 3 downscaled dispersions according to the original foaming process resulted in foams that fulfilled the product requirements. However, when the same dispersion was foamed alone, the obtained foam did not fulfill the requirements. Increasing the dispersion mixing time and decreasing the foaming jar diameter resulted in downscaled foams with similar properties as the original foams. In vitro digestive treatments and an x-ray showed that the downscaled foams were acceptable but need slight improvement to eliminate visible bubbles.

Downscaling the production resulted in foams indifferent from the original ones, but the time goal of 3 min production time was not accomplished since there seemed to be a minimum limit for the dispersion production essential for maintaining the foam properties. Increasing the foaming duration appeared to improve the stability of downscaled foams and needs to be further evaluated to eliminate the appearing visible bubbles.

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Populärvetenskaplig artikel

Skummigt kontrastmedel för buktomografi utan biverkningar Alla har vi sett i sjukhusserier en röntgenapparat med en cirkel som man åker in i, men vilka förberedelser krävs för att det ska bli bra bilder som kan hjälpa läkaren att ställa en korrekt diagnos? Visste du att man behöver dricka runt 1 L vätska innan för att fylla upp magen om man vill få en bra bild? I ett projekt har tillverkningen av ett skum som underlättar röntgenbesöket undersökts. Inför en röntgenundersökning behöver du alltså fylla upp magtarmkanalen med en vätska som gör det enklare att skilja olika organ åt på en röntgenbild. Oftast smakar den här vätskan rätt äckligt och inte nog med det, den kan göra att du får ont i magen efteråt och måste stanna kvar på sjukhuset någon timme. Hade det inte varit skönt att slippa den delen och bara gå hem istället?

Detta är fullt möjligt! Nu finns det nämligen ett drickbart skum som har samma effekt men gör att du slipper de obehagliga konsekvenserna, och som dessutom gör det enklare för läkaren att diagnosticera. Det finns i flera olika smaker och innehåller inga skumma ingredienser – faktiskt mestadels protein. Det enda kruxet är att du får runt ett skum som du dricker fördelat i tre glas under 45–60 min. Eftersom skummet förändras snabbt under den här tiden, blir röntgenbilderna kanske inte så bra som de hade kunnat om varje glas innehöll identiska skum.

I ett projekt har detta problem identifierats där man undersökte olika sätt att framställa skummet i mindre skala d.v.s. ett glas i taget. På så sätt får man ett färskt glas skum vid tre tillfällen precis innan man ska dricka det, och då blir det garanterat fina röntgenbilder. Först skulle steget där pulveringredienserna blandas i vatten göras i mindre skala och under mycket kortare tid än vanligtvis. Sedan vispades den här vätskan som vanligt för att bli ett skum. Det visade sig att volymen av vätskan spelar en stor roll i hur skummet blir, för när man blandade tre glas av den här vätskan och vispade till ett skum så uppfyllde det alla kraven. När man däremot vispade ett glas så fick man ett skum som inte uppfyllde några av kraven. Gjorde man exakt samma sak med ett glas av vätskan som blandats enligt den ursprungliga metoden så fick man ett skum som var bra enligt kraven. Därför bestämde man sig för att blanda pulvren under längre tid och byta ut burken som man vispade i mot en mycket mindre. Denna nya uppställning löste alla problemen och skummet var stabilt i rumstemperatur liknande skummet som producerats på ursprungligt vis.

Eftersom skummet ska färdas genom magtarmsystemet var det viktigt att det behöll dess struktur under dessa förhållanden. Det här kunde testas genom att simulera systemet i en in vitromodell innan man testar det i en människa. Detta innebär att man undersöker hur skummet förändras i ett värmeskåp inställt på 37 °C för att efterlikna kroppstemperaturen. Efter en stund tillsätter man en simulerad magsyra och efter ytterligare en stund simulerad bukspott. Därefter utvärderades skummet på hur bubbelstorleken och luftinnehållet påverkats. Man tog även röntgenbilder på skummet för att försäkra sig om att det uppfyllde alla krav.

Resultatet var att man fick fram en ny tillverkningsmetod av skummet anpassat till att producera ett glas i taget där tiden från ingredienser till färdig produkt kunde hållas rimligen kort, och det resulterade skummet var stabilt i rumstemperatur. Genom att öka vispningstiden skulle man kunna få ett skum där det inte dyker upp stora bubblor som det gjorde i in vitro testet och röntgenbilderna.

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Preface This report is the result of a master thesis of 20 weeks conducted during the spring semester 2018 at Aventure AB. The project is a concluding part of the engineering studies at the Faculty of Engineering, Lund University and equals 30 credits.

Acknowledgements I would first like to express my sincere gratitude to my supervisor, Dr. Lucia Casal Dujat at Aventure AB for her generous support and encouragement throughout this work. The door to her office was always open whenever I encountered a problem or had a question about my research or writing. She consistently allowed this thesis to be my own work but navigated me in the right direction. It has been a fantastic experience to work with this project under her supervision.

Professor Björn Bergenståhl and professor Marie Wahlgren of the Food Technology Department at Lund University are thanked for their support and expert advice in statistical analysis and the field of mixing operations and colloidal chemistry during the process of this work. I would also like to thank Dr. Ingvar Adnerhill and Dr. Thomas Fork at Skåne University Hospital for providing the opportunity to visit the hospital and sharing their knowledge in the clinical field. Without their passionate participation and input, this work could not have been successfully conducted.

I would also like to acknowledge Dr. Olof Böök at Aventure AB for offering me this great opportunity to conduct this work. The rest of the team at Aventure AB are also thanked for all the good moments together, always contributing to a pleasant working environment and sharing inspiration.

Finally, I must express my thankfulness to my family, friends and boyfriend for providing me with unfailing support and encouragement throughout my years of study and the process of researching and writing this thesis. This accomplishment would not have been possible without them. Thank you. Anh Nguyen Lund, June 2018

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Table of contents 1. INTRODUCTION ................................................................................................................................. 7

1.1. AIM ................................................................................................................................................ 7 2. THEORY ............................................................................................................................................... 7

2.1. PRODUCT REQUIREMENTS ................................................................................................................ 8 2.2. PRODUCT COMPOSITION .................................................................................................................. 8

2.2.1. Buffer salts and flavoring ........................................................................................................... 8 2.2.2. Egg white protein powder (EWP) ............................................................................................... 9 2.2.3. Xanthan gum.............................................................................................................................. 9

2.3. MIXING OF POWDER ........................................................................................................................ 9 2.3.1. Segregation................................................................................................................................ 9 2.3.2. Processing modes for mixing of powders .................................................................................. 10 2.3.3. Granulation ............................................................................................................................. 10

2.4. MIXING AND AGITATION OF LIQUIDS .............................................................................................. 10 2.4.1. Agitated vessels........................................................................................................................ 11

2.5. AGITATOR SELECTION AND SCALING .............................................................................................. 13 2.5.1. Scaling up ................................................................................................................................ 13 2.5.2. Scaling down ........................................................................................................................... 14

2.6. MIXING OF POWDER IN LIQUID ....................................................................................................... 14 2.6.1. Degree of suspension ............................................................................................................... 15 2.6.2. Colloidal systems ..................................................................................................................... 15

2.7. ULTRASOUND ............................................................................................................................... 15 2.7.1. Low power ultrasound.............................................................................................................. 16 2.7.2. High power ultrasound ............................................................................................................ 16

2.8. FOAMING OF EGG PROTEIN ............................................................................................................. 17 3. MATERIAL AND METHODS ........................................................................................................... 18

3.1. EXPERIMENTAL OUTLINE ............................................................................................................... 18 3.2. ORIGINAL METHOD ....................................................................................................................... 19

3.2.1. Mixing of dispersion ................................................................................................................ 19 3.2.2. Foaming .................................................................................................................................. 19

3.3. ULTRASOUND-ASSISTED DISPERSION PRODUCTION FOR 1/3 DOSE .................................................... 19 3.3.1. Ultrasound device A ................................................................................................................. 19 3.3.2. Ultrasound device B ................................................................................................................. 20

3.4. DOWNSCALING OF MIXING PROCESS FOR THE PRODUCTION OF 1/3 DOSE DISPERSION ........................ 20 3.5. DOWNSCALING OF FOAMING PROCESS FOR 1/3 DOSE ....................................................................... 20 3.6. EVALUATION OF FINAL PRODUCT ................................................................................................... 21 3.7. IN VITRO DIGESTIVE TREATMENT OF FOAM MADE WITH THE DOWNSCALED METHOD ......................... 21 3.8. STATISTICAL ANALYSIS ................................................................................................................. 21

4. RESULTS AND DISCUSSION ........................................................................................................... 22 4.1. ULTRASOUND-ASSISTED DISPERSION PRODUCTION FOR 1/3 DOSE .................................................... 22 4.2. DOWNSCALING OF DISPERSION PRODUCTION .................................................................................. 24 4.3. DOWNSCALING OF FOAMING PROCESS ............................................................................................ 27

4.3.1. Assessment of radiodensity of the downscaled foam in x-ray imaging ........................................ 31 4.3.2. In vitro digestive treatment of the downscaled foam .................................................................. 31

5. CONCLUSION .................................................................................................................................... 33 REFERENCES ............................................................................................................................................. 35 APPENDICES .............................................................................................................................................. 39

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1. Introduction An abdominal examination using x-ray images enables doctors to see organs, bones and blood vessels in the abdominal cavity, which aids in the diagnosis of abdominal diseases (Johns Hopkins Medicine, 2018).

Prior to a scan, the patient needs to drink an oral contrast agent that moves through the gastrointestinal tract and fills up the bowel. The contrast agent is necessary for the images to be interpreted, as tissues and contrast agent appear in different grayscale depending on the radiodensity measured in Hounsfield Units (HU). Since current oral contrast agents are displayed in a grayscale range too close to organs, tissues and body liquid they provide x-ray images with contrast that need to be improved for a better and easier diagnosis. Moreover, the current products are not pleasant to drink. Thus, a new product needs to be developed for a better diagnosis and patient acceptance.

A new protein-based oral contrast agent in the form of foam that fulfill the aimed properties is under study. However, since one dose is divided into 3 portions for the patient to slowly drink over a period of 45-60 minutes, and the foam properties change quickly during the first hour after production, the production needs to be adapted to 1/3 dose to eliminate the quality differences between each portion.

The production of the foam is done in two steps. The first process step is the dispersion production with mixing of powders in liquid. Since each powder ingredient is added separately and remains on the liquid surface, the mixing time is long. Until the foam is needed, the dispersion is stored in a fridge. In the second step, the dispersion is foamed. A possible method to investigate is the premixing of the powder ingredients and the downscaling of both the production steps without changing the product properties. If this is achievable, the cold storage of dispersions due to short shelf-life is eliminated. Additionally, the distribution of the dispersions will be eliminated and replaced by powder distribution that occupies less space. 1.1. Aim

The aim of this study was to study the theory of mixing and scaling and to adapt the foam production to the hospital setting by downscaling and evaluating the mixing of powder ingredients directly in water. The study was divided into three parts: a literature study on the mixing operations and the experimental work on mixing and foaming.

In the mixing part, the dispersion production was downscaled by evaluating mixing with ultrasonication and regular mixing. Different order of ingredient addition, different parameters and scaling of the equipment was evaluated. The obtained downscaled dispersion was foamed according to the original foaming method. In the second part, the foam production was downscaled by evaluating scaling parameters. Evaluation of the foams was performed by visual detection of drainage stability, and measuring bubble size, overrun and consistency. To study the structural changes of the product under gastrointestinal conditions, in vitro digestive treatments were conducted, and an x-ray was performed to verify the radiodensity.

2. Theory The downscaling of the foam production is required for adapting the process to the hospital setting where it is desirable to produce 1/3 amount of the total dose from a powder mix. A reasonable production time based on this has been decided to be below 3 minutes. The same formulation as the original foam is used for the downscaled foam and it is desired that the properties should be unaffected by the downscaling. Obtaining a homogeneous powder mix with no aggregates is of great importance, but since the powder mix in this study is prepared separately for each experiment and not stored, the focus will only be on the theoretical part of powder mixing.

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2.1. Product requirements

For the product to have a radiodensity below -300 HU, the air content, measured by the overrun, has to be controlled. The overrun is the relative increase of volume by inclusion of gas (van Aken, 2001): 𝑂𝑣𝑒𝑟𝑟𝑢𝑛(%) = ,-.,/

,/× 100 (1)

where mD is the weight of the dispersion and mF the weight of the foam. The overrun correlates with the air content and consistency, and a foam with a too high air content leads to a stiff product that is difficult to drink. The air content is the volumetric increase in % between dispersion and foam as expressed in Equation 2: 𝐴𝑖𝑟𝑐𝑜𝑛𝑡𝑒𝑛𝑡(%) = 8/.8-

8/× 100 (2)

where VF is the volume of foam and VD is the volume of dispersion.

The temperature and consistency of the foam must be in a range that is drinkable for the patient acceptance. The bubble size of the foam must also be within a certain range to avoid drainage and this drainage must not occur when the foam is stored in room temperature for at least 2 h. Foams with smaller bubbles decrease drainage, and a more uniform bubble size distribution gives more stable foams (Bisperink et al., 1992). Smaller bubbles also create more pressure which is beneficial for the distension of the intestines after intake. This is important for being able to visualize the crevices in the intestines.

Table 1 shows the parameters that are required to characterize to evaluate whether the foams fulfill the requirements or not for its application and use. Table 1. Parameters characterized for each foam.

Bubble size Consistency Overrun Air content

Radiodensity Temperature Foam height Free of drainage

Microscale range.

Affects the foam

stability and consistency.

For patient acceptance.

Product must flow easily for

easy intake.

Determined by weight. Crucial to assess air content, and thus contrast.

Determined by volume. Crucial to assess air content, and thus contrast.

Crucial to assess

contrast provided in

x-ray images.

Important for patient

acceptance and gut

behavior.

Helpful for effective air

content determination

in lab.

Important to assess from life-

time during

intake/use.

2.2. Product composition

To fulfill the product requirements, all the powder ingredients have been carefully selected and evaluated. In the following sections, each ingredient and its purpose are presented.

2.2.1. Buffer salts and flavoring K2HPO4 and NaH2PO4 are used for stabilizing and regulating the pH (∼7.3) of the product when passing the stomach and bowel. With a buffer, the product remains stable in the stomach despite the low pH and the enzymes in the gastric and duodenal fluids.

Flavoring for each dose has previously been evaluated to provide a pleasant taste that masks the egg flavor just enough without being too intense.

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2.2.2. Egg white protein powder (EWP) Egg powders are technical products with functional properties that cannot be found in liquid egg products, and in egg white powder these can be enhanced by dry heating. Egg white foaming properties are retained during mild treatments due to low protein denaturation. The super-high gel egg white powders have a pH around 6.5-8.5, and foaming powders are neutral or slightly acidic. (Lechevalier et al., 2011)

The functional properties, such as gelation and foam formation, of ovalbumin proteins in hen egg white make it useful as food ingredients (Mine, 1995). Gel formation by proteins occurs through ordinate polymerization of molecules which creates a three-dimensional network. For a viscous liquid, this transformation results in a viscous-elastic matrix (Hermansson, 1979). The ability of the proteins to form and stabilize foam by encapsulating and maintaining air is associated with its amphiphilic behavior (Du et al., 2002). Ovalbumin can be degraded by surface absorption, by heat, in films, through agitation, or by denaturant agents (Vadehra et al., 1973).

2.2.3. Xanthan gum Water-soluble polymers are extensively used as additive to improve foam stability. With

xanthan gum in a foam system, the aqueous phase increases in viscosity and the liquid drainage is slowed down. The stabilizing mechanisms of polymer-surfactant foam delay the liquid drainage of liquid films and enhance the adsorption behavior of the film surface. The polymer has the ability of reducing the surface tension and has high interfacial viscoelasticity. This is due to the larger adsorption quantity in the interface, and the strong electrostatic interactions. Consequently, liquid films are strengthened, and film permeability is reduced which makes polymer enhanced foam more resistant to external disturbance. However, in this study, the protein probably contributes to the stability more than the polymer which mainly enhances the rheological properties. Mixtures that have a larger shear capacity produce higher viscosity after foam formation and thus delay liquid drainage of the foam. (Pu et al., 2017)

With a suitable stabilizer, the size of gas bubbles in the foam is more uniformly distributed and thus are exposed to lower capillary pressure. A change of the microstructure appears with time when big bubbles become even bigger due to coalescence and small bubbles disappear from drainage. As a consequence of this, foam density is reduced. (Pu et al., 2017) 2.3. Mixing of powder

A critical stage that determines the quality of products of many industrial applications is the mixing of granular materials. The characteristics of the mixed materials, the mixing device type and the process conditions are parameters that affect the mixing operation (Cleary and Sinnott, 2008). The aim of mixing is to obtain a homogeneous distribution of each component of the mixture throughout the whole volume. However, granular mixtures can never be perfectly homogeneous similar to liquid and gaseous systems (Krolczyk, 2016).

2.3.1. Segregation Segregation (de-mixing) generally is an unwanted occurrence that is related to particulate materials-related unit operations, such as mixing (Tang and Puri, 2007). This is due to the different physicochemical properties of the components in one formulation. Different density, particle size, size distribution, shape, and surface texture of the components can lead to a non-uniform blend (Tang and Puri, 2007). This leads to weight variation of each component when the blend is divided into smaller portions. A mixture of granular materials with similar properties reaches a random state after mixing for a sufficiently long period. In contrast, a heterogeneous multi-element granular system cannot be described in the same way because of the significant differences in density, shape and size and this is a fundamental problem in

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mixing (Krolczyk, 2016). Knowledge of the interaction of shape, density and particle size on segregation is important. In a binary mixture, the smoother the surface of fine particles and the higher the density, the segregation potential is higher (Tang and Puri, 2007). However, segregation may not be the only reason for inhomogeneity – poor operation of equipment and in-process agglomeration can also have an effect (Oka et al., 2017). To minimize segregation, large particles should be avoided as well as matching of ingredients with very different properties. The strongest effect on segregation is caused by mixed particles with a significant variation in relative diameter (Krolczyk, 2016). Adding larger components first and then components of smaller size is an industrial practice that can reduce this effect. Mixing method and time can affect the mixing degree as it reaches a maximum and then becomes worse due to secondary segregation (Krolczyk, 2016).

2.3.2. Processing modes for mixing of powders Batch mode of processing characteristically does not give a sufficiently homogenous mixture when there are large differences in particle properties. In this process, powder ingredients are mixed in a large vessel that is tumbled for numerous hundred revolutions before the material is discharged. Ingredients with largely different properties are close to impossible to mix in batch systems and can even segregate maximally. Continuous processing of ingredients is an alternative to batch processing and implicates that ingredients are fed into a unit operation at a specific mass rate and the blend exits at the same rate. The material spends a fraction of time in a single unit operation compared to the time it spends in a batch mode unit operation. This lack of time and space leads to no dissociation of the material from dissimilar particles. Thus, the process conditions rather than the properties of the materials have a larger effect on the final state of the blend. (Oka et al., 2017)

2.3.3. Granulation Granulation is often used in pharmaceutical mixtures to bypass segregation. The most common approach is wet granulation in which a liquid binder is sprayed on the mixture. Mixing the wet mass creates a homogeneous powder granule that is dried and filled into capsules or compacted into tablets. Dry granulation is suitable for moisture sensitive ingredients and involves compaction of the dry powder mixture into ribbons which are milled to create granules that are filled into capsules or compressed. Several additional processing steps are required, and additional ingredients may be involved in granulation which makes the process lengthier, energy intensive and consequently more expensive. (Oka et al., 2017) 2.4. Mixing and agitation of liquids

Agitation and mixing of fluids is essential for many processing operations. Agitation is achieved by inducing motion of a material, often in a circular pattern inside a jar. When there are two initially separate phases that randomly distribute into each other, it is a mixing operation. A variety of operations employ mixing, differing extensively in the homogeneity of the mixed material. The purpose of agitation depends on the goal of the processing step, e.g. suspending solid particles, dispersing gas through the liquid or blending miscible liquids. One agitator can meet numerous purposes at the same time. (McCabe et al., 2005)

Mixing is a more difficult operation to study because the results of mixing studies are not often highly reproducible and depend on how mixing is defined by the experimenter. The criterion for mixing is often visual such as the visually observed uniformity of a suspension, or color changes. (McCabe et al., 2005)

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2.4.1. Agitated vessels Agitation of liquids is usually done in a cylindrical tank or vessel. The top can be open to the air but is more often closed. In many applications a standardized design with a rounded tank bottom, liquid depth equal to the tank diameter and an impeller mounted to an overhung motor driven shaft, is applicable. However, depending on the type of agitation problem, the proportions of the tank can vary widely. (McCabe et al., 2005)

2.4.1.1. Impellers The circulatory motion of liquid in the vessel is caused by the impeller. Axial-flow impellers generate currents parallel with the axis of the impeller shaft, and radial-flow impellers generate currents in a tangential or radial direction. For low- to moderate-viscosity liquids, the three main types of impeller are turbines, propellers, and high-efficiency impellers. (McCabe et al., 2005)

2.4.1.1.1. Turbines A simple straight-blade trubine drives the liquid radially and tangentially which generates currents that travel outward to the vessel wall and then upward or downward. This creates almost no vertical motion at the impeller. At 20-150 rpm, the impellers typically turn in process vessels. A disk turbine creates zones of high shear rate and is therefore useful for dispersing gas in a liquid. When the process requires good overall circulation, a pitched-blade turbine can provide axial flow in addition to the radial flow. (McCabe et al., 2005)

2.4.1.1.2. Propellers A propeller is a high-speed, axial-flow impeller for low-viscosity liquids. The liquid is forced downward and the flow currents continue until deflected by the bottom of the vessel. The liquid leaving the impeller is highly turbulent and pulls along stagnant liquid, and the propeller blades cut or shear the liquid. Propeller agitators are effective in large vessels because of the persistence of the flow currents. Small propellers turn at full motor speed i.e. 1150 or 1750 rpm, whereas larger ones turn at 400-800 rpm. (McCabe et al., 2005)

2.4.1.1.3. High-efficiency impellers To reduce the power required for a given flow rate and provide better mixing and uniform axial flow, variations of the pitched-blade turbine have been developed. These impellers are useful for mixing of low- or moderate-viscosity liquids. For dispersing gases and mixing of very viscous liquids, the high-efficiency impellers are not recommended.

2.4.1.2. Baffles Vessels equipped with four standard baffles generate a strong axial or radial flow depending on the type of stirrer and prevents circular motion of the liquid (Freudig et al., 1999). In partly baffled or unbaffled vessels, the circular motion causes centrifugal forces to appear which develops a vortex on the surface of the liquid. This is an advantage when submerging slowly sinking or floating particles because the vortex aids in transporting the powder away from the feed point towards the impeller, avoiding layer formation (Ciofalo et al., 1996). A higher maximum powder feed rate is possible for an unbaffled system Additionally, a vessel without baffles can improve the dispersion rate despite the much lower mixing power achieved compared to the baffled one (Freudig et al., 1999).

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2.4.1.3. Flow patterns The type of impeller, the size and proportions of the tank, baffles and impeller, and the

characteristics of the liquid – especially the viscosity, affect the way liquid moves in an agitated vessel. At any point in the tank the liquid velocity has three components, and variations in these affect the overall flow pattern in the tank. The first velocity component is radial, acting perpendicularly to the shaft of the impeller. The second one is longitudinal and acts in a direction parallel with the shaft. The third component is tangential, i.e. rotational, acting in a circular path around the shaft. The radial and tangential velocity components are in a horizontal plane whilst the longitudinal component is vertical, in the case of a vertical shaft. The longitudinal and radial components provide the necessary flow for the mixing operation. The tangential component is usually disadvantageous with a vertical and centrally located shaft in the tank. The tangential flow creates a vortex in the liquid. The swirling creates differences in various levels without providing longitudinal flow between them. Solid particles are thrown to the outside by centrifugal force and then move to the center at the bottom of the tank. The reverse of mixing – concentration – occurs instead because of the reduced relative velocity between the blades and the liquid, so limited power can be absorbed by the liquid. This is due to the circulatory flow of the liquid which is in the same direction as the impeller blades. At high impeller speeds, the vortex can be deep enough to reach the impeller and gas is drawn down into the charge, which is usually undesirable. (McCabe et al., 2005)

To prevent swirling and circulatory flow in small tanks, the impeller can be mounted off the center. In larger tanks the agitator can be mounted in the side of the tank with the shaft in a horizontal plane at an angle. Large tanks with vertical agitators benefit from the installment of baffles that reduce swirling. These impede rotational flow without interfering longitudinal or radial flow. (McCabe et al., 2005)

2.4.1.4. Circulation rates The volume of the fluid circulated by the impeller should be big enough to sweep out the

whole vessel in a reasonable time regardless of the agitation problem. In dispersion and mixing operations, turbulence in the moving stream is the most important factor and often controls the effectiveness. Turbulence is a result of large velocity gradients and well directed currents in the liquid. Both circulation and turbulence consume energy, and the selection of size and type of impeller has an influence. Generally, flow is promoted by large impellers at medium speed and turbulence is created by smaller impellers at high speed. (McCabe et al., 2005)

For a propeller or turbine agitator, the circulation rate and tip velocity are increased by increasing the impeller speed. However, the fluid velocity at a given position is not increased in the same proportion because the jet velocity decreases quickly with increasing distance from the impeller. (McCabe et al., 2005)

2.4.1.5. Power consumption To assess the power required to rotate a particular impeller at a given speed, correlations of power with the variables of the system are required. The variables are measurements of tank and impeller, the density 𝜌 and viscosity µ of the liquid, and the speed n. The impeller Reynolds number Re can be used to predict the flow in a vessel (McCabe et al., 2005): 𝑅𝑒 = <=>=?

@ (3)

where Ni is the impeller rotation speed (1/s) and Di is the impellor diameter. Low Reynolds numbers (Re < 10) yield viscous flow throughout the vessel and at high Reynolds numbers (Re > 104), there is turbulent flow everywhere. At intermediate Reynolds numbers, there is a transition region.

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The system geometry has influence on the power consumption, e.g. a decrease of the ratio of impeller diameter to tank diameter decreases the power number Np when using a flat-blade turbine and many baffles at high Reynolds numbers. The shape of the tank affects Np relatively little, but the circulation patterns are strongly affected. 2.5. Agitator selection and scaling

The power consumed, and the amount or degree of mixing is not necessarily correlated. In an unbaffled vessel, a low-viscosity liquid is swirled about, and the particles follow circular paths but mix little or not at all, so the energy supplied is not used for mixing. Mixing becomes rapid when baffles are added, and less energy is used for circulation because a larger fraction is used for mixing. A mixer that mixes with the smallest amount of power yet in the required time is the best one when mixing time is critical. (McCabe et al., 2005) On lab scale, a simple agitator vessel can be a beaker and a magnetic stirrer. Using an octagonal stirring bar with an integral pivot ring provides added turbulence and larger surface area than a smooth cylindrical stirring bar because of its interrupted profile (Bel-Art Products, 2018). Friction and chattering is reduced with the pivot ring because it minimizes the contact area between the bar and the vessel.

2.5.1. Scaling up When scaling up, provided that geometric similarity is maintained, lower stirrer speed is needed to suspend solids in larger tanks. The safe scale up rule is to keep the geometric similarity and constant power per unit volume. Ratios recommended are Da/Dt = 1/3, H/ Dt = 1 and E/ Dt = 1/4, in which Da is the agitator diameter, Dt is the tank diameter, H is the liquid height and E is the distance between the agitator and tank bottom (see Figure 1). For solids suspension, some prefer Da/Dt = 0.4. For a given power input, the optimum ratio of impeller to vessel diameter is an important scaleup factor. A strong influence on this ratio is the nature of the agitation problem. The impeller should be small relative to the vessel size for some purposes, and large for other purposes. Regardless of the operation, the smaller the impeller, the higher the impeller speed since the power input is kept constant. Operations that require high circulation rates rather than large velocity gradients, are suited with large, slow-moving impellers. For operations that demand large velocity gradients rather than high circulation rates, the best is to use small, high-speed impellers. A measure of mixing effectiveness for low-viscosity liquids is the amount of power consumed by the impeller per unit volume. The reasoning behind this is that more power gives more turbulence which means better mixing. (McCabe et al., 2005)

At constant power to volume unit, P/V, scale up leads to longer mixing time because of the decreased impeller speed. Maintaining a constant mixing time is mostly impractical due to the large amount of power required. An increase in mixing time in the larger tank is a necessary tradeoff in scale up of agitation equipment. (McCabe et al., 2005)

14

Figure 1. Measurements of vessel with turbine.

2.5.2. Scaling down When scaling down, the mixing time is predicted for a practical power input in the large vessel and then in a smaller tank tests are made at a stirrer speed that gives the same mixing time (McCabe et al., 2005). Mixing intensity depends to some extent on the amount of material that is agitated – more intense mixing is achieved in a smaller batch than a larger one with a given impeller (Dickey, 2015). 2.6. Mixing of powder in liquid

A common industrial process is the dispersion of powders in water or aqueous solutions performed in stirred vessels (Freudig et al., 1999; Hellborg et al., 2012). Some vessels and stirrers are specially designed for immersion and dispersion of powder. Despite the design, the powder can still be difficult to disperse due to lacking instant properties (Freudig et al., 1999). Instant properties are the physical properties of a powder associated with the four steps of dispersing it: wetting, submerging, dispersing, and dissolving (if soluble) (Schubert, 1993). Some occasional overlap of two steps can occur. Dispersion of insoluble powders in liquid is generally described in two steps: submersion followed by destruction of aggregates in the liquid (Freudig et al., 1999).

The wettability of a bulk powder is its capability to absorb a liquid and depends on the interaction between the surface and the liquid (Hellborg et al., 2012). Wetting of the particles is often the rate-controlling step and mixing can improve this since the surface of the stirred vessel is in continuous movement and causes movement in the powder (Freudig et al., 1999). As a result of mixing powder in liquid particles, the viscosity of the liquid can increase and slow down the wetting.

Particle falling through the liquid surface is the submerging step and is dependent on the density and the level of trapped air (Hellborg et al., 2012). It is rarely the rate-controlling step for large and heavy particles (Ortega-Rivas, 2008), but might be in a rapidly thickening system (Hellborg et al., 2012). Very fine particles that are hollow, resulting from spray-drying, have a small particle density that can cause problems in the sinking of the product into the liquid

15

(Freudig et al., 1999). Powders with “good” instant properties disintegrate quickly into its primary particles with little input of mechanical energy (Schubert, 1993).

Powder can be added in stages or through a charge port while the mixer is operating. The former allows closing of the vessel and establishing vacuum before turning on the mixer (Banaszek, 2015). Whether to add water to the powder ingredients or ingredients to water is also important to consider. Adding a more viscous material to a less viscous one is easier than the opposite (Dickey, 2015).

2.6.1. Degree of suspension The degree of suspended solids in an agitated tank depends on the process and is important to define in a design or scale up problem.

In a nearly complete suspension most of the solid is suspended. A few percent are in stationary fillets of solid at the edge of the tank bottom or other places. A small amount of stagnant solid may be acceptable in a feed tank to a processing unit if the solids do not cake or the fillets grow. (McCabe et al., 2005)

Complete particle motion indicates when all particles are suspended or moving along the bottom of the tank. The mass transfer coefficient for particles moving on the bottom is much lower than for suspended particles, which may affect the unit performance. (McCabe et al., 2005)

When all particles are suspended and do not stay on the bottom more than 1-2 s, the condition is a complete suspension. Usually, there will be a concentration gradient and a region of clear liquid near the top of the tank when this stage is just reached. Nevertheless, it has little effect on performance of the unit as a dissolver or a chemical reactor. Increasing the stirrer speed additionally does not increase the mass transfer coefficient much. (McCabe et al., 2005)

In a uniform suspension there is no clear liquid near the top of the tank and the suspension looks uniform. Vertical concentration gradients may still exist especially when the size distribution of the particles is wide. This condition is reached when the stirrer speed is substantially above that required for a complete suspension. (McCabe et al., 2005)

2.6.2. Colloidal systems Particles or molecules dispersed in a medium having a width between 1 nm and 1 µm at least in one direction are in a colloidal state. A system where particles of colloidal size are dispersed in a continuous phase of a different composition is defined as a colloidal dispersion. When the dispersed phase is solid, and the dispersion medium is a liquid, the system is called a sol. A hydrophobic colloidal dispersion is composed of an aqueous solution as the dispersion medium, and the colloid particles are repelled by water. (IUPAC, 2002) 2.7. Ultrasound

Ultrasound is sound waves with a frequency that surpasses the hearing of human ears (20 kHz). It is used in developing technologies to minimize processing and maximizing quality as well as ensure safety of foods. (Knorr et al., 2011)

Ultrasound can be divided into low and high energy based on the frequency range. Ultrasound with frequencies higher than 100 kHz is low energy (low power and intensity) and can be used for non-invasive analysis of food materials during processing and storage. At frequencies 20-500 kHz, ultrasound is classified as high energy (high power and intensity) and disrupt physical, mechanical or chemical properties of foods. This has a positive effect on processing, preservation and safety. (Awad et al., 2012)

Ultrasound probes and instruments are designed for applications in food science and technology as modifiers which uses high power ultrasound, and as sensors using low power ultrasound. Usually, equipment has to be custom designed for the varying demands of a specific

16

application. Probe geometry, design, characteristics and operation conditions are selected depending on the physicochemical and functional properties of the product to be analyzed or processed. (Awad et al., 2012)

2.7.1. Low power ultrasound Currently, low power ultrasound together with spectroscopy and nuclear magnetic resonance are the most popular ad widely used non-destructive analytical methods (Awad et al., 2012). Changes in ultrasound properties allow for the assessment of properties of opaque fluids through jar walls without sample contact. This enables measurements on-line and in labs with a robust and reasonably cheap equipment (Coupland, 2004).

Low power ultrasound for food analysis utilizes the changes in compressions and decompressions resulting from the propagation of sound as mechanical waves (Blitz, 1968, 1971). Attenuation and velocity of the sound waves changes, through absorption and/or scattering mechanisms, at interaction with matter (McClements, 2005). Ultrasonic velocity (v) can be determined by elasticity (E) and density (𝜌) of the medium according to Newton-Laplace equation (Blitz, 1968):

𝑣 = AB? (4)

The ultrasound velocity of the solid form is larger than that of the liquid form (Awad et al.,

2012). Determination of structure, composition, physical state and molecular processes using ultrasound velocity measurements is suitable because of the sensitivity to intermolecular interactions and molecular organization (Buckin et al., 2002).

Attenuation coefficient and acoustic impedance also correlate with physicochemical properties of materials. Absorption and scattering leads to energy loss in ultrasonic waves in compression and decompression which causes attenuation (Buckin, Kudryushov and O'Driscoll, 2002). Absorption impacts attenuation in homogeneous materials and scattering impacts in heterogeneous ones. Viscosity, wall material and viscosity are other parameters that affect attenuation (Povey, 1997). Acoustic impedance affects the reflection coefficient so different densities in a material will have different acoustic impedances. This results in reflections from the border of two materials with different acoustic impedances (Awad et al., 2012).

2.7.2. High power ultrasound Ultrasound conducted on biological material leads to compressions and decompressions of the medium particles which gives a large amount of energy. Ultrasound with frequency higher than 20 kHz has biochemical, mechanical and chemical effects which can be applied in processing of several food systems (Mason et al., 2011). Some of the mechanical effects that are utilized are degassing, extraction of flavors, and destruction of foams (Higaki et al., 2001). The biochemical and chemical effects are effective for preventing contamination of equipment surfaces, sterilizing equipment, and removal of bacterial biofilms (Baumann et al., 2009). Depending on the application, ultrasonic probes with various lengths, diameters and tip geometries, or sonication baths can be used for high power ultrasound (Awad et al., 2012).

Intensity, energy, pressure, velocity and temperature are the principal parameters affecting high power ultrasound. It can be described by (Patist and Bates, 2008): 𝑃D = 𝑃D,DE ⋅ 𝑠𝑖𝑛(2𝜋𝑓𝑡) (5)

17

The acoustic pressure Pa depends on time (t), frequency (f) and the maximum pressure amplitude of the wave (Muthukumaran et al., 2006). Pamax is dependent on the power input or the intensity (I) of the transducer: 𝐼 = 𝑃D,DE/2𝜌𝑣 (6) where 𝜌 is the density of the medium and v the sound velocity.

Acoustic streaming is the main mechanism at high frequencies or low intensities and is the motion and mixing in the fluid with no formation of bubbles (Leighton, 2007). A steady flow is generated by an energy gradient of the ultrasonic wave (Ito et al., 2002). Lower frequencies and higher intensities give rise to acoustic cavitation because of generation of bubbles and the growth and collapse of these. This results in the release of large amount of highly localized energy. The collapse of cavitation bubbles close to solid surfaces can be used for cleansing of surfaces from contaminants because of the formation of asymmetrical microjets (Suslick, 1989). Water molecules can be broken to form free radicals through cavitation, which induces crosslinking of proteins in aqueous medium and increases chemical reactions (Cavalieri et al., 2008).

Mixing can be achieved by movement of material between parts of a bulk liquid mass due to flow and diffusion that is generated by ultrasound. When using mechanically agitated contactors, the type and speed of impeller, dimensions of vessel and physico-chemical properties of the system affect the mixing time. In the case of acoustic cavitation using an ultrasonic horn, energy is dissipated by the vibrating horn surface, and mixing time depends on the horn position, tip dimensions and geometry of the vessel. Measuring the mixing time is a means for the characterization of batch mixing performance. (Vichare et al., 2001) 2.8. Foaming of egg protein

There are two foam classes, spherical and polyedric foams. Foam based on proteins consist of droplets of a thin and continuous film of protein molecules filled with air. A lamella containing fluid separates each bubble. Originally, air bubbles are spherical because of high internal pressure and a dense lamella. In the polyedric foam, liquid is distributed between lamellar channels where the films are situated. The destabilization strength causing air bubbles to become closer and take on polyedric shape is caused by the drainage of lamellar fluids. (Alleoni, 2006)

The formation of protein-based foam is based on a sequence of reactions. For triggering the process, an input of energy is required to create new surface area. To this interface soluble proteins can be transported and adsorbed to the air-water interface which lowers the critical surface tension. Rearrangement of polypeptides in the interface is based on polar mobility in which nonpolar parts lead to air particles and polar moieties are directed to water. This is the base of a continuous, cohesive film formed by non-covalent interactions of the polypeptides. Structural components and attractive electrostatic interactions enable intermolecular links to improve the foaming properties, whereas exceeding repulsive electrostatic interactions reduce the foaming capability. However, excess attractive electrostatic force might cause coagulation of the polypeptides. (Alleoni, 2006)

Foam formation and stabilization depend on the kind of protein and the condition of the solution. Proteins that are quickly opened and adsorbed in the air-water interface have better foaming abilities than those slowly adsorbed and with difficultly opened structures (Kinsella and Morr, 1984). Flexibility and structure of proteins also determine the interfacial behavior. However, since there are a lot of other factors that affect foam formation and many kinds of protein, this description is a generalization. The foaming properties depend on the scale of these strengths that keep the native protein structure in solution or in the interface (Adamson and

18

Gast, 1997). Film stability is improved by surface rheological properties in a point of the isoelectric point range of many proteins, but this is also where solubility is minimized (Phillips et al., 1994). The balance between ionic and hydrophobic components of a protein determines its foaming properties. Nonpolar residues contribute to the interactive forces at the hydrophobic interface by increasing the surface activity.

The mixing velocity, beater geometry and surface properties of the substances relates to the foam formation process (Phillips et al., 1990). The dynamic equilibrium between mechanical strength and bubble destruction reflects the maximum capability of air incorporation (Phillips et al., 1994). Foam stability is measured by the time needed for a certain amount liquid to be drained from the foam. The ability of the protein to reduce the surface tension between the protein solution and the air droplet is related to the ability of protein film formation (Alleoni, 2006). Low molecular weight and amphipathic molecules are structural characteristics that lead to fast foam formation of proteins (Kinsella, 1981).

For egg powder, foam stability and delay of film formation can be caused by electrostatic repulsion, and disulphide linkages reduce protein flexibility. Reduction of disulphide linkages between protein molecules improves film formation and foam stability (Alleoni, 2006). A stable foam of ovalbumin is built up from a network by non-covalent interactions and the formation of disulphide linkages in the air-water interface improves foam stability (Alleoni, 2006). Protein concentration, pH, temperature, other components, and physical-chemical properties affect the foaming features. A higher protein concentration yields more stable foams because of the formation of a thick lamellar film (Phillips et al., 1994).

Foaming properties are influenced by liquid pH, the optimal being 6.5. Dilution of the egg white up to 40 % does not modify foam stability. Salt can be added to improve foaming capacity, or sucrose to improve stability. However, salt reduces stability and sucrose reduces foaming capacity. Addition of hydrocolloids, such as xanthan, retains foaming properties but decreases foam volume.

3. Material and methods 3.1. Experimental outline

The study was divided into two main parts in which the first one consisted of scaling down the dispersion production. Since the original foaming protocol was adapted to 1 full dose of dispersion, 3 portions of downscaled dispersion were prepared separately with the same parameters and put together for foaming. Part of this consisted of testing an ultrasound equipment but since this test was run during a limited trail time, the results were limited and not as thoroughly presented. Focus was primarily on improving the mixing method with a beaker and a magnetic stirrer by evaluating scaling parameters. This method was based on the original method and the available equipment.

When optimal parameters for the dispersion production were determined, the study proceeded to scaling down the foaming process. At this stage of the product development, it was preferred to use the existing foaming equipment and only modify the position and movement of the blender. In case the downscaled method would not provide foams that fulfilled the product requirements, the dispersion production needed to be reevaluated (see Scheme 1). However, there was a possibility of changing parts of the foaming equipment if the existing one was not suitable for downscaling.

If time allowed and a downscaled foam that fulfilled the product requirements could be obtained, replacement of distilled water would be evaluated to reduce the distribution cost of distilled water.

19

Scheme 1. Control points for downscaling of foaming process and implementation of distilled water.

3.2. Original method 3.2.1. Mixing of dispersion

In the original method, the dispersion is prepared for 1 dose. The buffer is made from the buffer salts and distilled water and stored in the fridge. Egg white protein, stabilizer and flavoring are added to the buffer separately during mixing with a magnetic stirrer. This process takes around 30-35 min.

3.2.2. Foaming The foam is produced by foaming the dispersion with an electronic blender in a plastic jar. The blender is fixed to a clamp stand to control the highest and lowest limit positions that the blender reaches in the jar during the foaming. The foaming is carried out applying controlled cycles of up and down movements. 3.3. Ultrasound-assisted dispersion production for 1/3 dose

Dispersing of the powder ingredients in water was evaluated by using two different ultrasound devices with varying tip diameter, frequencies, and amplitudes. The aim was to decrease the 1/3 dose dispersion production time as much as possible, and possibly improve the properties of the final product.

3.3.1. Ultrasound device A An ultrasonic laboratory device with sonotrode (7 mm tip diameter) was used to study the dispersing of the powder ingredients in water. Powders for 1/3 dose were premixed and added

Foam height & homogeneity

Fulfilled

Consistency

Fulfilled

Bubble size & Overrun

Fulfilled

In vitro digestive tests

Stable

1st goal reached -Downscaling of

process

Replace distilled water with drinking

water

Repeat all previous steps

All steps passed

2nd goal reached -Implement

alternative water

Steps not passed -keep distilled water

Not stable

Repeat previous steps

Not fulfilled

Repeat previous steps

Not fulfilled

Change dispersing/foaming

method

Not fulfilled

Change dispersing/foaming

method

20

to a glass beaker with refrigerated distilled water for 1/3 dose, placed in a cooling jacket at 10 °C. A spatula was used to submerge the premixed powder prior to the ultrasonic treatment. The device was immersed in the bulk halfway to the bottom and as close to the surface as possible to incorporate the powders at the surface. The amplitude and ultrasonication time were varied and the temperature was measured before and after.

3.3.2. Ultrasound device B An industrial ultrasonic device (higher power than device A) with sonotrode equipped with a cooling jacket set to -2 °C was used for studying the dispersing of the premixed powders in a glass beaker with distilled water for 1/3 dose. After the powder was added to the water, a spatula was used for submerging the powder. In the next experiments, powders were predispersed by gradually adding distilled water to the premixed powder until covered and the rest of the distilled water was slowly added while mixing at high speed using a magnetic stirrer with polygonal magnetic bar for less than 30 seconds. The amplitude and ultrasonication time were varied and the temperature was measured before and after.

For foaming, three 1/3 doses ultrasound treated dispersions were mixed together and foaming was performed according to the original foaming protocol and foaming duration was varied. The bubble diameter, consistency and overrun were measured, as well as checking the stability after 6 h. 3.4. Downscaling of mixing process for the production of 1/3 dose dispersion

The downscaling of the dispersion production was performed by preparing 3 x 1/3 dose dispersions for each foam since the existing foaming equipment was used and requires the volume of 1 dose. A magnetic stirrer and a polygonal magnetic bar was used for mixing. Two glass beakers were evaluated for the mixing: 600 mL (i.d. = M) and 400 mL (i.d. = S). Powder ingredients were added to the beaker in the order of descending particle size as follows: Phosphate salt A, stabilizeer, egg white powder, Phosphate salt B, flavoring. The powders were mixed, distilled water for 1/3 dose was added in two controlled steps and the mixture was mixed under several stirrer speeds and mixing time to assess the influence of speed and time on the dispersion preparation. For foaming, three 1/3 doses of dispersions were transferred to a plastic jar and the original foaming protocol was followed but for a shorter time of 4 min 20 s. Stability tests were performed every hour for 3 hours if the foam fulfilled the initial requirements to proceed to this step (see Scheme 1). 3.5. Downscaling of foaming process for 1/3 dose

The same equipment as used for the original foaming process was used for foaming of 1/3 dose of dispersion prepared with the downscaled dispersing method. At constant foaming duration, lowest and highest position that the blender reaches in the jar during the foaming process, and the movement of the blender were varied. The initial tests were made with the total volume of dispersion for 1 dose to get an estimation of the limits, and several tests were performed to assess reproducibility. Then, dispersions made with the downscaled mixing method were used and tested for reproducibility. The foam height, consistency and homogeneity were the determining factors for repeating a set of parameters and proceeding to stability tests (see Scheme 1). In the stability tests, the overrun was only measured for 2 h due to insufficient volume. This was also evaluated in a smaller plastic jar. The minimum foam height to reach corresponding to 1/3 dose was 4.5 cm. The dispersion production was also modified in this step by means of mixing time and the replacement of the previously used polygonal stirrer bar with another one with less sides to optimize the mixing method.

21

Once a reproducible and acceptable foam was obtained, the radiodensity of the foam was determined in x-ray imaging. The time between production of the foam and the x-ray imaging was longer than desired and involved high temperature and movements due to travel conditions. 3.6. Evaluation of final product

The temperature, bubble size, consistency, overrun and foam height were measured right after foaming. Then, stability tests were done in which bubble size and overrun were measured and visual detection of drainage was performed, every hour for 3 h.

The foam volume (V) was determined using Equation 7, which was obtained by measuring the corresponding height (H) in the jar for volumes (207 – 360 mL) measured in a graduated cylinder.

V=82.2H–38.9 (7)

The consistency was calculated by taking an average of the shortest and longest distance that

the foam travelled in a Bostwick consistometer for 30 s. An optical microscope provided with a camera was used for studying the bubble size. A

small sample of the foam was pipetted with a Pasteur pipette onto an imaging chamber gasket, put on a microscope slide and inserted in the microscope for viewing. With a software, the bubble diameter was measured. The mean bubble size in a foam sample was determined by picking the three largest ones with at least 10 µm difference and selecting the smallest out of these as the representative size. This was done in three positions in the microscope slide and the mean of the three sizes was calculated.

The dispersion and foam weight were measured by filling a cup with the dispersion or foam and levelling with a spatula. The mean of three measurements was recorded for calculating the overrun. 3.7. In vitro digestive treatment of foam made with the downscaled method

Right after foaming, the overrun and bubble size were measured. Then, 250 mL foam was transferred to a 250 mL volumetric cylinder, covered with aluminum foil and incubated for 15 minutes at 37 °C in an incubator. This was repeated for another foam as control. The simulated gastric fluid was prepared by dissolving pepsin in HCl under stirring at 500 rpm and 30 °C to obtain an enzymatic activity of 37 U/mL in 250 mL foam. The solution was added dropwise on the surface of the foam. The foam was transferred to a beaker and mixed with a spatula and transferred back into the cylinder. The cylinder was incubated at 37 °C for 30 minutes. The simulated duodenal fluid was prepared by dissolving pancreatin in NaHCO3 under stirring at 500 rpm and 30 °C. Ursofalk suspension was added afterwards under the same conditions. Adding this solution to the 250 mL foam, an enzymatic activity of 10 U/mL should be obtained. The solution was added dropwise on the surface of the foam and the foam was transferred to a beaker and mixed with a spatula. The foam was transferred back to the cylinder and incubated for 30 minutes at 37 °C. All the steps were done for the control foam but without the addition of enzyme solutions. 3.8. Statistical analysis

Tests of significance are carried out within and between the groups with different parameters. This is to test that the method is reproducible and does not differ significantly from the original method. It also defines that the results are real (statistically significant) and not due to chance (Herkenhoff and Fogli, 2013). In order to compare the data, the data is plotted against time and the linear axes are converted to a logarithmic scale. Using the log transformed values the slope and standard error of a linear curve is obtained, using Excel’s Regression tool (Excel,

22

Microsoft®). Analyses of variance (ANOVA) are used for the slopes by taking the errors into account. The largest value that occurs by chance is the critical value of F (Fcrit). If the calculated F is larger than Fcrit the null hypothesis of equivalent group means is rejected, and the results are statistically significant. The probability that a value of F≥ Fcrit occurred by chance when there is no difference in the means, is given by the p-value. Statistically significant results give a p-value less than the chosen confidence level.

4. Results and discussion 4.1. Ultrasound-assisted dispersion production for 1/3 dose

After using the ultrasound A device for 165 s with amplitude 54 µm, powder agglomerates remained on the surface of the dispersion. Raising the amplitude to 90 µm did not solve this problem (see Table 2). It appears that the powder was not able to be completely dispersed, probably due to non-sufficient power.

Using the ultrasound B device, powder was completely dispersed. The temperature was initially too high due to the long ultrasonication duration but decreasing the time to 10 s resulted in lower temperature. Some agglomerates remained on the surface when the powder was submerged in water by stirring with a spatula. When a magnetic stirrer was used for submerging the powder, this problem was eliminated because of the vortex that pulled the powders into the core. Positioning the sonication tip close to the surface also resulted in better dispersed powders. Incorporation of water after the addition of the powders appeared to improve the submersion because this way the powder became wetted and did not have to fall through the water surface. The dispersions made with ultrasonication looked slightly clearer compared to those prepared with the original method. This could indicate that ultrasonication enhances incorporation of the powder ingredients.

23

Table 2. Parameters and results of the ultrasonic treatment tests on a powder dispersion. At 26 kHz and 20 kHz, the devices A and B were used, respectively.

Ultrasonic treatment parameters

Initial dispersion properties

Ultrasonic device

Dispersion volume (mL)

Ingredient order

Submerging method

Frequency (kHz)

Amplitude (µm)

Duration (s)

T (°C)

Powder on

surface

A

200

1

A

26

54 55 10 Yes 54 110 10 Yes 90 88 13 Yes

B

200 1 A 20 100 60 20 No 200

2

B

20

100 10 15 Little

200 10 12 No 200 10 10 No

Note. Ingredient order – 1: Water first. 2: Powder first Submerging method – A: Spatula. B: Magnetic stirrer.

The consistent results confirm the repeatability of using ultrasound device B for 10 s at frequency 20 kHz and amplitude 100 µm. Employing a higher frequency does not lead to better dispersion of the powders. Even though the difference is small between the frequencies employed (20 and 26 kHz), a lower frequency can enhance the properties of food proteins such as solubility and foaming ability. This is an advantage since the dispersion is processed into a foam.

In the beginning of the foaming process, the dispersion appeared to be less viscous than normal and thus more turbulence was created. Despite this, the consistency and the overrun were similar to those obtained with the original method even though the bubble diameter was smaller (see Table 3). This indicates that ultrasound enhances the stability of the bubbles by decreasing the size of them. A good foam appearance was achieved before the foaming process was completed to T1. Decreasing the foaming duration to T2 did not affect the foam properties which is an indication of the ultrasound treatment of the dispersion increasing the foaming ability. The bubble size in both foams was smaller than the 100-140 µm obtained with the original method. The consistency and overrun values were within the acceptable limits which indicates that the foam properties are maintained. The foams were stable for a minimum of 6 h.

24

Table 3. Foaming parameters and properties of foams prepared from 3 x 200 mL dispersions treated with ultrasound, 5 minutes after foaming.

Foaming parameters

Initial foam properties

Free of

drainage 6 h

Foaming duration

Jar size Dispersion volume (mL)

Bubble size (µm)

Consistency (cm)

Overrun (%)

Homogeneity

T1 Original 600 30 ✓ ✓ ✓ ✓ T2 Original 600 24 ✓ ✓ ✓ ✓

Note. Stability: ✓ means no drainage. Consistency, overrun and homogeneity: ✓ means within acceptable range. 4.2. Downscaling of dispersion production

The initial bubble size, overrun and consistency of foams prepared from downscaled dispersions were similar to foams prepared according to the original method (see Table 5). This indicates that the foaming ability was not affected by the short mixing time. The mixing time of the downscaled process is at a maximum 13 % of that in the original method whilst the volume is 33 % of the original. From this rough calculation, only taking the volumes into account and disregarding the geometry, the mixing time is vastly reduced and so the quality could be affected. Taking the geometry into account, the increase of the ratio Da/Dt by keeping Da constant and decreasing Dt, compensated for the mixing time reduction.

However, reducing the mixing time primarily decreased the viscosity of the dispersion. This was probably due to powders not being completely dispersed, which could be observed as a few particles remaining on the bottom of the beaker. The dispersing of the powders may have been completed in the foaming step, contributing to the same degree of mixing in the end and resulting in a similar initial bubble size. The important fact is that no agglomerates were present on the surface after mixing and that the foams were stable for at least 2 h.

The dispersing mixing time did not affect the consistency of the foams despite the fact that the dispersions were less viscous, and powders not being completely dispersed especially when mixing for only 30 s. There was no difference between the overrun of the foams made with the downscaled process compared to the original foams. There seems to be no relationship between foam consistency and overrun with mixing time. However, the overrun and consistency depend mainly on the foam height because of the amount of air incorporated. Increased foam height gives lower consistency and larger overrun.

Splashing of the liquid at start and a thicker foam layer in the end was observed in the beaker M, but not in the beaker S. In the original method, splashing does not occur which lead to the decision to continue the experiments only with the beaker with inner diameter 7.5 cm. The high stirrer speed and smaller dimensions of the beaker created a deeper vortex than in the original method. Due to this, air was incorporated which resulted in a layer of foam on the dispersion surface. To circumvent this, stirrer speed and mixing time could be decreased but with the risk of not obtaining the same degree of dispersed particles. The foam layer may affect the foaming process negatively due to the foam acting as a lid, making it more difficult to incorporate air at the start. It also remained on top of the blender throughout the foaming process and was difficult to homogenize. When the blender was removed, this inhomogeneous foam got into the bulk foam and affects the homogeneity. It might seem to be of benefit to create foam already before the foaming process, enabling a decrease of the foaming duration, but since it is such a small amount that rather causes problems the foam formation during mixing should be avoided. For all mixing times, bubbles were present in the dispersion bulk which should also be avoided.

25

Tabl

e 4.

Dow

nsca

led

disp

ersi

ng p

aram

eter

s an

d pr

oper

ties

of fo

ams

mad

e fr

om 3

dow

nsca

led

disp

ersi

ons

5 m

inut

es a

fter

foam

ing

and

valu

es o

btai

ned

with

the

orig

inal

met

hod

as w

ell a

s re

com

men

ded

valu

es. T

wo

diffe

rent

bea

kers

wer

e ev

alua

ted

for t

he d

ispe

rsio

n pr

oduc

tion

(siz

es M

and

S) t

oget

her

with

a m

agne

tic s

tirre

r an

d a

mag

netic

bar

(App

endi

x B)

.

Lite

ratu

re v

alue

s (m

ixin

g)

Dow

nsca

led

foam

ing

para

met

ers

(com

pare

d to

ori

gina

l val

ues)

In

itial

dow

nsca

led

foam

pro

pert

ies

c

Bea

ker

size

D

a/Dt

H/D

ta St

irrer

sp

eed

b (rp

m)

Mix

ing

time

(min

)

Jar

size

H

/Dta

Foam

ing

dura

tion

Ble

nder

sp

eed

Bub

ble

size

(µm

) C

onsi

sten

cy

(cm

) O

verru

n Fo

am

heig

ht

T (°

C)

Hom

ogen

eity

- 1/

3 1

- -

O

rigi

nal d

ispe

rsin

g pa

ram

eter

s co

mpa

red

to

liter

atur

e va

lues

O

rigin

al

Hig

her

Low

er

630-

1000

30

– 3

5

Dow

nsca

led

disp

ersi

ng

para

met

ers

(com

pare

d to

or

igin

al v

alue

s)

Dow

nsca

led

disp

ersi

ng

para

met

ers

M

↑

18 %

↑

12%

1000

3

0%

0%

↓

13%

0%

128

✓ ✓

- 18

✓

1000

2

126

✓ ✓

✓ 16

✓

1000

1

124

✓ ✓

✓ 18

✓

1000

0.

5 13

0 ±

0.7

✓ ✓

✓ 14

± 0

.0a

✓

S

↑ 34

%

↑ 28

%

780

3

0%

0%

↓

13%

0%

126

± 4.

7 ✓

✓ ✓

18 ±

1.8

✓

780

2 13

4 ±

0.4

✓ ✓

✓ 17

± 0

.6

✓ 78

0 1

128

± 7.

5 ✓

✓ ✓

16 ±

1.4

✓

780

0.5

137

± 8.

4 ✓

✓ ✓

16 ±

3.5

✓

1000

0.

5 12

5 ✓

✓ ✓

18.0

✓

Not

e. M

eans

± S

D a

re s

how

n fo

r exp

erim

ents

don

e in

trip

licat

es. M

eans

with

subs

crip

t a w

ithin

row

s are

sign

ifica

ntly

diff

eren

t fro

m th

e va

lues

obt

aine

d w

ith th

e or

igin

al m

etho

d.

a H/D

t in

whi

ch H

is th

e di

sper

sion

hei

ght b

efor

e fo

amin

g

b App

roxi

mat

e sp

eed.

See

App

endi

x fo

r con

vers

ion

tabl

e.

c Con

sist

ency

, ove

rrun

, foa

m h

eigh

t and

hom

ogen

eity

: ✓ m

eans

w

ithin

acc

epta

ble

rang

e.

26

The value of Da/Dt for the original dispersing setup is the closest to the recommended value 1/3. H/Dt for the beaker S is the closest to the literature value of 1. Since the production is scaled down, these values are not as important to consider as would if the process were to be scaled up. When scaling up, it may be impractical and expensive to employ an impeller that is larger than 1/3 of the tank diameter. In this study, the downscaling is done to a dimension in which stirrer bars larger than 1/3 of the beaker diameter are highly available and easy to employ. Since the power consumption is decreased when scaling down, financial issues are not a matter either.

The bubble size of all foams made from the downscaled dispersions was below the critical limit of 300 µm for at least 3 h after foaming (see Table 5). The bubble size growth follows the same pattern having the highest growth rate during the first hour. The overrun was relatively stable for at least 3h with small fluctuations with time but remaining within the acceptable range. increment Table 5. Changes in bubble size and overrun of foams made from 3 downscaled dispersions and stored at room temperature.

Downscaled dispersing

parameters

Acceptable bubble size range: 100 - 300 µm

Overrun after foaming a

Free of drainage

3 h b

Changes in bubble size (µm) after foaming

Mixing time (min)

Beaker size

Initial After 3 h Initial After 3 h

3 S

126 ± 4.7 269 ± 13 ✓ ✓ ✓ 2 134 ± 0.4 267 ± 18 ✓ ✓ ✓ 1 128 ± 7.5 277 ± 2.5 ✓ ✓ ✓

0.5 137 ± 8.4 268 ± 2.3 ✓ ✓ ✓ Note. Means ± SD with subscript a are significantly not within the acceptable range. a Overrun: ✓ means within acceptable range. b Free of drainage: ✓ means no drainage.

There is no significant difference in the slopes of the bubble size graphs of foams made with 3 downscaled dispersions compared to that of the original foam (see Figure 2). This indicates that the downscaled dispersing method with shorter mixing times between 30 s and 3 min does not give significantly different foams when they are foamed in one jar with the original foaming method. Based on these results, any of the mixing times are suitable for producing downscaled dispersions that can be used for foaming. Proceeding to the downscaling of the foaming process, it was decided that 1 min mixing for the production of dispersions is suitable for the reduction of the process time without risking a too large amount of remaining powders in the bottom as was observed in the dispersions prepared with 30 s mixing time.

27

Figure 2. Slopes of bubble size development graphs obtained from regression of logarithmically converted values. Data from foams prepared with the original foaming method but with 3 downscaled dispersions prepared with different mixing times. Average value ± SE for the original method and values ± SE for each replicate A, B and C of the foams.

4.3. Downscaling of foaming process Using the original foaming jar for foaming of 1/3 dose dispersion, made according to the original method, resulted in foams that fulfilled the product requirements. Foaming of the downscaled dispersions made with 1 min mixing time did not result in acceptable foams, despite the same foaming parameters (see Table 6). The viscosity difference between the original dispersions and the downscaled ones was most likely the reason. The degree of dispersed particles in the downscaled dispersions seemed to be lower than in the original ones due to the short mixing time and thus the viscosity was lower. This indicates that the downscaled dispersions were more sensitive to variations than the original ones. Due to this, the dispersion production had to be reevaluated as well as the dimensions of the foaming equipment. Increasing the dispersion mixing time whilst decreasing the stirrer speed and replacing the octagonal stirrer with a triangular one resulted in dispersions with no air bubbles inside the bulk and an easily removed foam layer on the surface. However, excessively increasing the dispersion mixing time resulted in very viscous dispersions which decreased the foaming ability.

Increasing the dispersion mixing time to 5 min and decreasing the stirrer speed to 480 rpm resulted in dispersions with a viscosity more similar to those prepared with the original method. For the foaming, replacing the original foaming jar with a smaller jar resulted in downscaled foams that fulfilled the product requirements. With this change, the ratio H/Dt increased and became closer to the H/Dt value in the original foaming method. The increase of Da/Dt because of the change to a smaller foaming jar at constant Da = 3.5 cm (blender diameter), seemed to not affect the process to a large extent or was balanced by the increase of H/Dt. This could indicate that for foaming, the ratio H/Dt is more important to consider than Da/Dt.

All foams produced in the smaller foaming jar had a temperature within the acceptable range. They had a slightly higher consistency than the original foams, but this is not an issue because then they are even easier to drink. As long as the overrun is in the acceptable range, the air content is in the acceptable range and the product has the correct radiodensity. As seen in Table 7, the downscaled foams had an acceptable overrun. The initial bubble size of the downscaled foams was larger than the upper acceptable limit, but this could be improved by increasing the foaming duration. Increasing foaming duration leads to decreased consistency, so there is a limit for the increase. However, since the consistency of the downscaled foams was above the upper limit, there is a margin for increasing the foaming duration without risking decreasing

1,8

1,85

1,9

1,95

2

2,05

2,1

Slop

e of

bub

ble

size

gra

phSlope of bubble size graphs of foams made from downscaled

dispersions

Original 3 min (A) 3 min (B) 3 min (C) 2 min (A) 2 min (B) 2 min (C)

1 min (A) 1 min (B) 1 min (C) 30 s (A) 30 s (B) 30 s (C)

28

the consistency to the lower limit. A single trial where foaming duration was increased with 0.3 % points resulted in a foam that fulfilled all criteria and had a smaller initial bubble size. This shows that only a small change is needed but needs to be further evaluated.

29

Ta

ble

6. S

etup

par

amet

ers

for

the

dow

nsca

led

met

hod

and

foam

pro

pert

ies

5 m

in a

fter

foam

ing.

L

itera

ture

val

ues

(mix

ing)

D

owns

cale

d fo

amin

g pa

ram

eter

s (c

hang

es

com

pare

d to

ori

gina

l val

ues)

D

esir

ed in

itial

foam

pro

pert

ies

for

dow

nsca

led

foam

ing

met

hod

Bea

ker

size

D

a/Dt

H/D

ta St

irrer

sp

eed

b (rp

m)

Mix

ing

time

(min

)

Jar s

ize

H/D

tb D

a/Dt

Ble

nder

sp

eed

Foam

ing

dura

tion

Bubble size (µm)

Consistency (cm)

Overrun (%)

Foam height (cm)

Temperature (°C)

Homogeneity

O

rigin

al

1/3

1 -

-

10

0-14

0 C

O

F

15

-21

✓ O

rigi

nal d

ispe

rsin

g pa

ram

eter

s a (c

ompa

red

to li

tera

ture

val

ues)

Ori

gina

l di

sper

sing

pa

ram

eter

s

In

itial

foam

pro

pert

ies

of d

owns

cale

d fo

amin

g m

etho

d

- H

ighe

r Lo

wer

63

0 –

1000

30

– 3

5

Dow

nsca

led

disp

ersi

ng

para

met

ers

(cha

nges

co

mpa

red

to o

rigi

nal

valu

es)

Dow

nsca

led

disp

ersi

ng

para

met

ers

ND

1 N

D1

ND

1 N

D1

ND

1 O

rigin

al

↓ 52

%

0%

0%

↓ 80

%

148

± 20

✓

✓ ✓

18

✓

S ↑

34%

↑

28%

78

0 1

Orig

inal

↓

52%

0%

0%

↓

80%

N

D2

< C

ND

2 ✓

ND

2 -

S ↑

34%

↑

28%

78

0 6

Orig

inal

↓

52%

0%

0%

↓

80%

N

D2

< C

ND

2 ✓

ND

2 -

S ↑

34%

↑

28%

48

0 5

Smal

ler

(↓ 1

7%)

↓ 54

%

↑ 21

%

0%

↓ 80

%

152

± 3.

1 a

> C

✓ ✓

19 ±

1.

2 ✓

S ↑

34%

↑

28%

48

0 5

Smal

ler

(↓ 1

7%)

↓ 54

%

↑ 21

%

0%

↓ 77

%

128

> C

✓ ✓

18

✓

Not

e. M

eans

± S

D fo

r exp

erim

ents

don

e in

dup

licat

es/tr

iplic

ates

. Mea

ns w

ith su

bscr

ipt a

are

sign

ifica

ntly

diff

eren

t fro

m th

e de

sire

d va

lues

or v

alue

s obt

aine

d w

ith th

e or

igin

al m

etho

d.

a H/D

t in w

hich

H is

the

disp

ersi

on h

eigh

t bef

ore

foam

ing.

b A

ppro

xim

ate

spee

d. S

ee A

ppen

dix

for a

con

vers

ion

tabl

e.

d Con

sist

ency

, ove

rrun

, foa

m h

eigh

t and

hom

ogen

eity

: ✓ m

eans

with

in a

ccep

tabl

e ra

nge.

N

D1:

Not

det

erm

ined

bec

ause

the

disp

ersi

on w

as m

anuf

actu

red

in th

e sm

all-s

cale

pro

duct

ion.

N

D2:

Not

det

erm

ined

due

to h

omog

enei

ty re

quire

men

t not

fulfi

lled.

30

The bubble size of foams produced from downscaled dispersions was below the critical limit of 300 µm for at least 2 h after foaming (see Table 7). As previously, the overrun was relatively stable for at least 2 h with small fluctuations with time but remaining within the acceptable range. Table 7. Changes in bubble size and overrun of foams made from downscaled dispersions with the downscaled foaming method and stored at room temperature.

Downscaled foaming parameters compared

to original values

Acceptable bubble size range: 100 - 300 µm

Changes in %overrun after

foaming a

Free of drainage 2 h b

Changes in bubble size (µm) after

foaming Foaming duration

(s)

Jar size Initial After 2 h Initial After 2 h

↓ 80% Smaller 152 ± 3.1 265 ± 9.1 ✓ ✓ ✓ ↓ 77% 128 259 ✓ ✓ ✓

Note. Means ± SD for experiments performed in duplicates or triplicates. Values with subscript a are significantly not within the acceptable range. a Overrun: ✓ means within acceptable range. b Free of drainage: ✓ means no drainage.

One of the slopes of the bubble size development graphs of the foams produced with the downscaled foaming method (80% shorter foaming duration compared to original) is similar to the one for the original foaming method (see Figure 3). The values of the replicates overlap which shows that the method is reproducible. However, only one of the triplicates overlap with the original method which indicates that improvements are necessary. Foaming duration 77% shorter compared to the original resulted in a foam with bubble size growing at a lower rate similar with the original method, showing a lower slope. However, no secure decisions can be made about this matter until further experiments are performed with this foaming duration.

Figure 3. Slopes of bubble size development graphs obtained from regression of logarithmically converted values. Data from foams prepared with the downscaled dispersing and foaming method. Average value ± SE for the original method and values ± SE for each replicate A, B and C of the downscaled foams.

1,851,9

1,952

2,052,1

2,15

Slop

e of

bub

ble

size

gra

ph

Slope of bubble size graphs for foams made with downscaled foaming method

OriginalDispersion 5 min - Foaming 80% lower (A)

Dispersion 5 min - Foaming 80% lower (B)Dispersion 5 min - Foaming 80% lower (C)Dispersion 5 min - Foaming 77% lower

31

4.3.1. Assessment of radiodensity of the downscaled foam in x-ray imaging Two foams produced with the downscaled foaming method were tested with x-ray imaging. The images showed that both foams had an acceptable radiodensity to provide acceptable contrast. A small difference in radiodensity in the foams was found which was probably due to small differences in the foaming process. The dispersion for Foam 2 had foam on the surface that could not be completely removed before foaming which could have contributed to the difference. In the x-ray images using a window setting, to assess homogeneity, both foams showed some visible bubbles in the bulk, but Foam 1 was considered acceptable since there were few bubbles that would interfere in the diagnosis. The reason for the occurrence of these bubbles could be due to the transport and time delay between production and x-ray. In the window setting used for diagnosis, the x-ray images showed both foams in black with no visible bubbles. This window setting is used for diagnosis so therefore the current state of the foams was considered acceptable for diagnosis purposes. However, for the foam to become an industrial product the x-ray image must not show any visible bubbles using the window setting for homogeneity assessment. Due to the transport under non-ideal conditions, the scan showed the stressed state of the foams which should be taken into consideration. since in the future situation, there will be no transportation before product intake.

4.3.2. In vitro digestive treatment of the downscaled foam The stability of foams, produced with the downscaled foaming method, under gastric and duodenal conditions was assessed by performing in vitro digestive treatments as described in section 3.5. The digestion was conducted in duplicates with controls (only incubation and same mechanical stress, no enzymatic digestion) and the overrun and bubble size after treatment were taken as the response factors.

As shown in Table 8, the overrun significantly increased after the in vitro digestive treatment. The overrun in the control also significantly increased, which shows that the temperature had an effect. It was observed that the foam volume increased even after the short incubation before the enzymatic treatment started, which could explain this change in overrun. However, the overrun of the foams remained within the acceptable overrun range of 78-112 %. The bubble size also significantly increased in both the in vitro digested foams and the control foams. No significant difference is found in bubble size between the in vitro digested foams and the control foams after treatment. The bubble size after in vitro digestive treatment is similar to that of foams produced with the original full-scale method and stored in room temperature for 3 h. This indicates that the temperature affects the foam since the same bubble size as for a foam produced with the original method and stored in room temperature, is reached after a shorter time at higher temperature. Nevertheless, the bubble size in all foams remained within the acceptable range of 100-300 µm in the studied timeframe. None of the foams showed drainage during and after treatment but visible bubbles were observed in both the in vitro digested foams and the control foams. This indicates that the foams were stable in the sense of drainage, but the foaming production needs to be improved in order to avoid a large bubble size distribution after in vitro digestive treatment.

32

Table 8. Overrun and bubble size of foams produced with the downscaled foaming method before and after in vitro digestive treatment. The same mechanical stress was conducted on the control foams as the in vitro digested foams but without the addition of enzymes. Overrun for foams produced with the original method right after and 3 h after foaming for comparison.

Foam type

Overrun (%) a Bubble size (µm) Free of drainage after treatment Before

treatment After

treatment Before

treatment After

treatment Enzymes

added OE1a OE2b 128 ± 1.6 238 ± 8.7 ✓

Control OC1a OC2b 131 ± 4.2 251 ± 11.5 ✓ a Different subscripts within rows are significantly different.

33

5. Conclusion The focus of this work was on the optimization and downscaling of the whole process of the production of a foam used as a filling agent for x-ray abdominal examination. The first part of the study focused on the downscaling of the dispersion production. A small study on ultrasonic treatment for dispersion production was conducted to assess whether it would be of benefit to integrate an ultrasonic device into the process. The main focus of this part was on studying the effects of ingredient addition, equipment, mixing time and stirrer speed in the original dispersion production. The second part of the study was focused on the downscaling of the foaming process using the downscaled dispersion and evaluating the effects of dimensions of the equipment and movement of the blender. In vitro digestive treatments were conducted on the downscaled foams to assess the stability in gastric and duodenal conditions and x-ray imaging tests were performed to confirm the correct radiodensity and homogeneity.

It was discovered that using ultrasound for dispersing the powder ingredients mainly enhanced the foam properties, such as decreasing the bubble diameter while maintaining the consistency, overrun and stability. For the purpose of decreasing the time for dispersing powder, ultrasonication is a promising method but could possibly be skipped since it was observed that the predispersing step using a magnetic stirrer could be efficient enough by its own when time is increased. This increase of time is worth foregoing giving the price of an ultrasonic device. An important observation during this part of the study was that the order of ingredient addition plays an essential role, especially the order of powders and water addition. Not only this, but also the stirrer speed and the ratio of material dimensions affected the predispersing step which was useful information to bring into the next part where the original dispersion production was evaluated for downscaling. Nevertheless, the ultrasonic device could eventually be taken into consideration.

Downscaling the dispersion production and using the downscaled dispersions prepared with different mixing times in the original full-scale foaming process, produced foams that were similar to foams made in the original process. No significant effects of mixing times below the required were found in the dispersion appearance. However, it was not possible to produce a foam that fulfilled the product requirements from a downscaled dispersion using the original foaming equipment. Even after the dispersion production was reevaluated using other mixing times, this could not be accomplished. Replacing the original foaming jar with a smaller one, using a polygonal stirrer bar with less sides than the previously one and increasing the mixing time of the dispersion production to 5 min solved the problem. The need of increasing the dispersion mixing time to 5 min indicates that there is an optimal degree of dispersed powder ingredients that is essential for producing a good foam. Increasing the mixing time increased the viscosity of the dispersion which was beneficial to a certain extent. As viscosity became higher than that of the original dispersion, not only the long production time was nonbeneficial but also the foaming properties decreased. It was also found an important dependence on dimension relation of all material used during the production process.

The downscaling of the process seemed to influence the properties of the foam under gastrointestinal conditions. This was shown in the in vitro digestive treatments where the temperature had an effect on the overrun and bubble size as well as increased the formation of visible bubbles. The x-ray images confirmed the presence of the visible bubbles but also that the foams produced with the downscaled method fulfilled the radiodensity criterion for acceptable contrast. For diagnosis purposes, the downscaled foam in the current state was satisfactory but for a better improvement the visible bubbles need to be eliminated.

The aim of decreasing the total production time to below 3 minutes was not fulfilled as the

optimal dispersion production already takes 5 min. The decrease in patient waiting time can be minimized by starting the dispersion production shortly before use during the conversation with

34

the doctor. This would reduce the active patient waiting time to only the duration of foaming. However, the original method produces 1 full dose of foam at once which leads to a decrease of foam quality with time due to the time it takes for the patient to drink the 3 portions. The original method requires a separate facility for the production of dispersions and a distribution system that has a larger impact on the environment. Since the dispersions are stored in fridges for a long time, a large amount of energy is required, and the shelf-life has to be taken into account. With production of both dispersion and foam at the hospital, these problems are eliminated. In the distribution of the premixed powders, less space is required than in the distribution of dispersions and the quality of the final product can be more controlled. Since the long-term goal is to distribute premixed powder ingredients in the amount required for 1/3 dose per package, the shelf-life of the mixture needs to be evaluated. The problem with premixing and storing them in the same jar is the possible interaction between the ingredients leading to a decrease in quality.

The current downscaled process shows results with promising possibilities for improvements of the current foam quality. Further evaluation of the foaming duration and movement of the blender during foaming is important as this can improve the stability of the downscaled foam. Since the consistency was higher in the downscaled foams than obtained with the original method, there is a margin for increasing the foaming duration to increase homogeneity and stability without risking a decrease of consistency below the lower limit. The evaluation of interaction between the different powder ingredients to determine whether it would be necessary to separate certain ingredients is another important further investigation.

35

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Appendices

A. Bubble size and overrun development

0 50 100 150 200

Ove

rrun

(%)

Time after foaming (min)

Overrun development of foams made from downscaled dispersions

Original 3 min 2 min

1 min 30 s Lower limit

Upper limit

0

50

100

150

200

250

300

350

0 50 100 150 200

Bub

ble

diam

eter

(µm

)

Time after foaming (min)

Bubble size development of foams from downscaled dispersions

Original 3 min 2 min

1 min 30 s Critical limit

40

B. Conversion table for magnetic stirrer The speed was obtained in approximate values by the supplier of the magnetic stirrer and the g values are calculated from (Thermo Scientific, 2009): g = (1.118 × 10-5) R S2 Steps Speed [rpm] g 0 0 0 1 6 0,0020124 2 128 0,9158656 3 163 1,4852071 4 216 2,6080704 5 274 4,1967484 6 365 7,4472775 7 482 12,9869116 8 627 21,9759111 9 781 34,0968199 10 996 55,4536944 11 1229 84,4336519

100

1000

1 10 100 1000Bub

ble

diam

eter

(µm

)

Log time after foaming (min)

Bubble development of downscaled dispersions after foaming according to the original method

Original (A) Original (B) Original (C) 3 min (A) 3 min (B)

3 min (C) 2 min (A) 2 min (B) 2 min (C) 1 min (A)

1 min (B) 1 min (C) 30 s (A) 30 s (B) 30 s (C)

0

200

400

600

800

1000

1200

1400

0 2 4 6 8 10 12

Spee

d [rp

m]

Steps

RCT basic Speed