Biomedical Science
Faculty of Health and Society Malmö University
SE-205 06 Malmö Sweden
Master programme in Biomedical Surface Science http://edu.mah.se/en/Program/VABSE
2020-06-03
Master degree thesis, 30 ECTS Examensarbete, 30 hp
Rheological Behaviour of Probiotic Bacteria
Dispersed in Maltodextrin and Sucrose
Solutions
Nousin Akter
SUPERVISOR: Dr. Vitaly Kocherbitov
Professor, Department of Biomedical Science, Faculty of Health and Society,
2 ABSTRACT
Probiotic bacteria are live microorganisms, which manifest health benefits in humans. The goal of this work was to characterize rheological properties of probiotic bacteria (Lactobacillus reuteri) formulation in which maltodextrin and sucrose used as excipients. To fulfil the goal, first, thermal, structural and rheological properties of maltodextrin, sucrose solutions and probiotic bacteria slurry were investigated. Probiotic bacteria formulations were prepared by adding probiotic bacteria slurry to maltodextrin and sucrose solutions at different mass fractions. Finally, rheological properties of probiotic bacteria formulations were evaluated. From TGA, the water content of PB slurry including intracellular water found 81%. In DLS, three different types of aggregations of maltodextrin were observed and characteristic size of Probiotic bacteria found 1μm. The optical microscopy results indicate that at the liquid - air interface and in dehydrated state the bacteria are birefringent and arranged in an ordered fashion resembling a nematic phase. Most of the MD, SU and MD+SU solutions show Newtonian behaviour. MD and SU solutions show strong increase of viscosity with increasing concentration. This dependence can be described by using the Spurlin–Martin–Tennent’s model. The viscosities of MD+SU solutions increase with increasing proportion of maltodextrin. The oscillation data of MD and MD+SU solutions can be described by Maxwell model. The viscosities of MD, SU, MD+SU mixed solutions decrease with increasing temperature. This temperature dependency can be described by Arrhenius model of viscosity. At very high concentrations of MD, a deviation from this behaviour is observed. The probiotic bacteria slurry shows shear thinning behaviour at low shear stress and Newtonian behaviour at higher stresses. All probiotic formulations in which probiotic bacteria dispersed in maltodextrin and sucrose solutions show Newtonian behaviour. The viscosities of maltodextrin solutions and MD+SU mixed solutions decrease by addition of probiotic bacteria whereas mixed effects of probiotic bacteria addition on the viscosity of sucrose solutions were observed. The viscosity of probiotic bacteria slurry decreases with increasing temperature, although deviations from this behavior are seen at certain conditions.
Keywords: Lactobacillus reuteri, Maltodextrin, Probiotic bacteria, Rheological properties, Sucrose.
This master thesis has been defended on June 3rd, 2020 at the Faculty of Health and Society, Malmö University.
Opponent: Dr. Christopher Garvey Examiner: Professor Sergey Shleev
Biomedical Science,
Faculty of Health and Society, Malmö University
3
ABBREVIATIONS LIST
MD - Maltodextrin SU - Sucrose
PB – Probiotic Bacteria
TGA – Thermogravimetric Analysis DLS – Dynamic Light Scattering
4 TABLE OF CONTENTS
Page
ABSTRACT 2
1. INTRODUCTION 6
2. MATERIALS AND METHODS 11
2.1. Materials 11
2.2. Methods 13
2.2.1. Thermo-gravimetrical analysis 13
2.2.2. Dynamic light scattering 13
2.2.3. Microscopic Study 14
2.2.4. Rheometry 14
3. RESULTS 16
3.1. Thermo-gravimetrical analysis 16
3.1.1. Maltodextrin powder 16
3.1.2. Probiotic bacteria slurry 17
3.2. Dynamic light scattering of maltodextrin and probiotic
solution 18
3.3. Microscopic study of probiotic bacteria slurry 20
3.4. Viscometry 23
3.4.1. Viscosity of sucrose and maltodextrin solutions 23 3.4.2. Temperature dependency of viscosity of sucrose
and maltodextrin solutions 25
3.4.3. Viscosity of probiotic bacteria slurry 26 3.4.4. Temperature dependency of viscosity of probiotic
bacteria slurry 26
3.4.5. Viscosity of probiotic bacteria slurry with sucrose
and maltodextrin solution 27
5
3.5.1. Amplitude and frequency sweep of sucrose and
maltodextrin solutions 30
3.5.2. Amplitude and frequency sweep of probiotic
bacteria slurry 31
4. DISCUSSION 32
4.1 Water content and thermal degradation of materials 32 4.2. Structural properties of maltodextrin solution and probiotic
bacteria slurry 33
4.3. Rheological behaviour of sucrose, maltodextrin solutions and
probiotic bacteria slurry. 34
4.3.1. Sucrose and maltodextrin solutions 34 4.3.2. Effect of probiotic bacteria addition on viscosity of
sucrose and maltodextrin solutions 38
4.3.3. Temperature dependency of viscosity of sucrose,
maltodextrin solutions and probiotic bacteria slurry 40
5. CONCLUSIONS 41
ACKNOWLEDGEMENTS 43
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1. INTRODUCTION
1.1.Probiotic Bacteria
Probiotics are live microorganisms which promoted with claims that they provide health benefits when consumed. Now-a-days, researchers are claiming that probiotics can stabilize gut microbiota, make nutrients more bioavailable to the human body and stimulate the immune system [Qinghui, 2018].
The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) define probiotics as ‘live microorganisms which, when administered in adequate amounts, confer a health benefit to the host’ [Schlundt, 2012]. From some studies, it is concluded that probiotics can stabilize gut microbiota, make nutrients more bioavailable to the human body and stimulate the immune system [Chen, 2016]. According to Fuller (1989), ‘probiotics are biopreparations containing living cells or metabolites of stabilised autochthonous micro-organisms that optimise the colonisation and composition of the gut microflora in both animals and humans and stimulate digestive processes and immunity’ [Fuller, 1989]. One of the most scientifically well-documented probiotic bacteria is Lactobacillus reuteri. These bacteria can be found in different body sites of human including the gastrointestinal tract, urinary tract, skin, and breast milk. By meeting with the acidic environment of the stomach, many probiotics die, while Lactobacillus reuteri can survive throughout the entire GI tract and beyond. These bacteria can colonize in the intestine and vagina, decrease incidence of bacterial vaginosis, urinary tract infections. [Hekmat, 2009; Gardiner, 2002]. The approximate length of Lactobacallus reuteri is 1 μm (Fig.1).
Fig.1: SEM image of Lactobacillus reuteri [Dishisha, 2013]
Lactobacillus reuteri has been used in 105 clinical studies in children, from newborns up to 18 years old, and 98 studies in adults and results have been published in 180 papers in scientific journals till January 2020 [biogaia.com]. Lactobacillus reuteri help babies to digest breast milk and solids foods. They inhibit the colonization of pathogenic microbes and remodel the commensal microbiota composition in the host without killing beneficial gut bacteria by its antimicrobial activity. L. reuteri can secrete sufficient amounts of reuterin which is a potent antimicrobial compound [Casas, 2000]. Lactobacillus reuteri also can reduce the production of pro-inflammatory cytokines while promoting regulatory T cell development and function [Qinghui, 2018].
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Due to the modern-day lifestyle of human with increased urbanization and altered eating habits, an imbalance of their microbiota is resulted. Especially, the decrease of the amount of Lactobacillus reuteri in human body in the past decades is correlated with an increase of inflammatory diseases over the same period. Therefore, direct supplementation or probiotic modulation of Lactobacillus reuteri may be an attractive preventive and/or therapeutic avenue against inflammatory diseases and can be used to strengthen the intestinal barrier of gut microbiota [Qinghui, 2018].
1.2. Formulations of probiotics and excipients
Probiotic bacteria formulation products marketed as tablet, capsules, liquid drops, fermented milk, yogurt, chewing gum etc [Govender, 2014]. To prepare a formulation, excipients as bulking agents, fillers, diluents, stabilizer etc are needed.
Recently few authors claimed that maltodextrin (MD) is a good excipient for a probiotic product. Sucrose is also used extensively in pharmaceutical industries as excipient.[Samedi, 2019; Carpenter, 1987].
Maltodextrin
Maltodextrins are maltooligosaccharides with a degree of polymerization ranging from three to nine and often act as flavour enhancers, fat replacers and bulking agents in dairy product. Maltodextrins are classified by DE (dextrose equivalent) and have a DE between 3 and 20. Dextrose equivalent value is a measure of the reducing sugar content. Maltodextrin is produced from vegetable starch by partial hydrolysis and is usually found as a white hygroscopic spray-dried powder. They are starch hydrolysates and saccharide polymers consisting of a-D-glucose units bounded by (1-4) glycosidic linkages (primarily) as well as by (1-6) linkages (Fig. 2), having long carbohydrate chains along with 2-3% glucose and 5-7% maltose. [Wang, 2000].
Fig.2: Structure of maltodextrin, x=1/6 linkages and y=1/4 linkages [Ninni, 2005] The empirical formula of maltodextrin is C6nH(10n+2)O(5n+1) and the molar mass is typically
between 504.5 g/mol (n = 3) and 2774.7 g/mol (n = 17). Maltodextrins from different sources may have different properties. They are soluble in cold water, non-sweet, flavourless and slightly soluble to almost insoluble in alcohol. They have found wide application in food industry as bodying agents, coatings, carrier for flavours, fragrances and oils coatings [Wang, 2000].
8 Sucrose
Sucrose is common sugar and a disaccharide. Its molecule is composed of two monosaccharides: glucose and fructose (Fig.3). Sucrose is produced naturally in plants, from which table sugar is refined. It has the molecular formula C12H22O11 and molar mass
is 342.3 g/mol. Sucrose is a nonreducing disaccharide, widely used as an ingredient and preserving agent in the food and pharmaceutical industries. It is used to stabilize labile biomolecules in aqueous solutions as cryo- preservatives. [Carpenter, 1987].
Fig.3: Structure of sucrose [Amchra, 2018]
Effect of MD and SU on probiotic bacteria
Recently very few authors performed the research on maltodextrin (MD) as excipient with a probiotic and they found maltodextrin as a good excipient and preservation agent. Samedi et al. [Samedi, 2019] improved the viability of bacteria in the low pH gastro-intestinal tract by using maltodextrin and arrowroot composite encapsulation. Bomba et al. [Bomba, 2002] also found that maltodextrin can potentiate the probiotic effect in the small intestine. Batawy et al. [Batawy, 2018] observed that maltodextrin could enhance the viability of probiotic strains and sensory properties of final product during the storage period.
Sucrose is widely applied as a protectant in the freeze-drying of lactic acid bacteria. The protective effect of sucrose is attributed to stabilization of proteins and membranes during the cellular dehydration associated with freeze-drying. Sucrose also can keep membranes in a fluid state in the absence of water [Li, 2010]. Giulio et al. [Giulio, 2005]also reported the cryo-protective effect of sucrose on the survival rate of different strains of lactic acid bacteria, after freezing or freeze-drying procedures. Akin et al [Akin, 2005] reported that sugar improved physical and sensory properties of products having probiotic bacteria. Addition of sucrose is an important factor that influences the acceptance of fermented milks from the sensory point of view [Maganha, 2013].
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Oldenhof et al [Oldenhof, 2005] evaluated the effect of sucrose, maltodextrin and skim milk on survival of Lactobacillus Bulgaricus after drying. They found that survival rates of Lactobacillus Bulgaricus after drying can be improved using sucrose and maltodextrin as excipients. They also reported that sucrose interacts directly with lipids and proteins and maltodextrin strengthen the glassy matrix as an osmotically inactive bulking compound. 1.3. Rheological behaviour of probiotic bacteria, maltodextrin and sucrose aqueous
solution
There is no detailed information on rheological behaviour of the probiotic bacteria in previous literature. Researchers evaluated rheological properties of suspension of other bacteria like Escherichia coli, Bacillus subtilis etc and they reported about some interesting phenomena. They found that the viscosity of the bacteria suspension can be lower than the viscosity of the suspending fluid. Which means after bacteria addition in the suspension, viscosity decreased [Gachelin, 2013; Martinez, 2020; Sokolov, 2009]. This phenomenon is clearly opposite to expected behaviour. For example, according to Einstein equation, viscosity of solution or dispersion increases with increasing concentration:
𝜂𝑟 = 1 + 2.5𝜑...(1)
Where, ηr is the relative viscosity of solution and φ is the volume fraction of solute. This
equation is expected to be valid at low volume fractions, i.e. φ<0.01[Willenbacher, 2013]. Even higher viscosity increase is expected at higher concentrations. The exact mechanism causing this anomalous rheological behaviour of bacteria still unknown.
Previous research reported in literature was mainly focused on rheological properties of products containing probiotic bacteria. There is no information about the effects of probiotic bacteria addition on rheological properties of these products.
Many authors studied rheological properties of maltodextrin, where they characterized mainly maltodextrin gel [Reuther, 1983]. Although Dokic et al. [Dokic, 1998] and Avaltroni et al. [Avaltroni, 2004] evaluated rheological properties of maltodextrin solutions but the rheological data for aqueous solutions of maltodextrins are still rather scarce. According to Dokic et al. and Avaltroni et al, aqueous solutions of maltodextrins show Newtonian behaviour.
The one of the most rheologically studied sugars is sucrose. Several authors have measured the viscosity of aqueous sucrose solutions for over 100 years. Many authors have reported viscosity data of aqueous sucrose solutions over a wide range of temperatures and concentrations. Therefore, those aqueous solutions are being used as standard solutions for viscosity calibration. Sucrose solutions show Newtonian behaviour and follow the Arrhenius model of the temperature dependence of viscosity [Longinotti, 2008].Sillick et al [Sillick, 2009] measured the rheological properties of mixed solutions of maltodextrin and sucrose and found near-Newtonian behaviour throughout the measurable regime.
10 1.4. Importance of rheological characterisation of probiotic bacteria formulation
with excipients
The pressure, temperature, concentration and time dependency of rheological properties of a pharmaceutical solution should be known before going to manufacture the final product from that solution.It is very important to set the processing parameters in the production processes like freeze-drying, spray drying, extrusion or encapsulation, etc.
Especially rheological behaviour of probiotic bacteria solution or dispersion can be complex. According to previous literature, after addition of bacteria in a solution, viscosity of the sample may decrease. This viscosity reduction can be a crucial element when manufacturing the final product containing probiotic bacteria. Moreover, bacteria must be handled very carefully in the solution, otherwise they will die.
Excipients like maltodextrin and sucrose could affect negatively on the final probiotic bacteria product during formulation and storage. If these are crystallized in final product at different conditions, then patient cannot get desired amount of probiotic from the product. Therefore, before manufacturing the MD/SU-probiotic product, behaviour of the formulation solution or dispersion should be rheologically investigated.
1.5. Rheological glossary
Rheology is the study of flow and deformation of materials. Viscosity, storage modulus, loss modulus, phase angle etc against an external force are the rheological properties of a material. If an external force or stress applied on a liquid, the layers of liquid will be displaced relative to each other. This displacement of liquid layer is known as strain. Therefore, rheology is also considered to be the study of stress-strain relationships in materials. [Mezger, 2006].
Viscosity- The resistance of a fluid to flow. In shear deformation viscosity is the ratio of applied shear stress (σ) to resulting shear rate (γ). Symbol- η, units: Pa.s.
𝜂 =𝜎 𝛾̇
If viscosity of a material is independent from shear stress, this material shows Newtonian behaviour. But if viscosity decreases against continuously changing shear stress, it is called shear thinning behaviour and if viscosity increases against continuously changing shear stress, it is called shear thickening behaviour [Mezger, 2006].
Viscoelasticity- It is rheological property of a material which shows both viscous and elastic characteristics when undergoing deformation. Viscoelasticity of a material can be described by storage and loss modulus of a material. Storage and loss modulus can be obtained from an oscillatory shear of material against shear stress or frequency [Barnes, 2000].
Storage modulus is a measure of the energy stored in a material in which a deformation (for example sinusoidal oscillatory shear) has been imposed. Put simply, storage modulus can
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be thought of that proportion of the total rigidity (the complex modulus) of a material that is attributable to elastic deformation. Symbol G', typically reported in Pascals (Pa) [Mezger, 2006].
Loss modulus is a measure of the energy dissipated in a material in which a deformation (for example sinusoidal oscillatory shear) has been imposed. Loss modulus can be thought of that proportion of the total rigidity (the complex modulus) of a material that is attributable to viscous flow, rather than elastic deformation. Symbol G”, typically reported in Pascals (Pa) [Mezger, 2006].
1.6.Aim
The aim of this work was to make a rheological characterization of maltodextrin, sucrose aqueous solutions, probiotic bacteria and evaluate the rheological effect of probiotic bacteria addition on those solutions at various conditions like different temperatures, against continuously changing and steady shear stresses. Thermal behavior and structure of probiotic bacteria formulation components also will be characterized.
2. MATERIALS AND METHODS
2.1. Materials
Sucrose
Crystalline sucrose supplied by Sigma-Aldrich Co. (St. Louis, USA). Maltodextrin
White maltodextrin powder with dextrose equivalent value 8 named Glucidex 9 supplied by Roquette Frères (Lestrem, France).
Probiotic bacteria
Freeze dried Lactobacillus reuteri (DSM 20016) with sucrose supplied by BioGaia AB (Lund, Sweden).
Preparation of samples: Maltodextrin solutions:
At first, thermogravimetrical analysis of maltodextrin powder has done three times and found 4.87% water content in MD powder. Maltodextrin solutions were prepared by dissolving maltodextrin powder in MQ water at room temparature. 10%, 20%, 30%, 40%, 50%, 55%, 60% (w/w) solutions were prepared. Therefore, amount of maltodextrin powder needed to making solution and solution concentration were calculated by following manner:-
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C = (mMD)/ms = (mp * XMD)/ms
Where, Concentration of Maltodextrin solutuion= C Mass of Maltodextrin powder = mp
Mass of water in Maltodextrin powder = mw
Mass fraction of Maltodextrin in Maltodextrin powder = XMD = 𝑚𝑝
𝑚𝑝+𝑚𝑤
Actual mass of Maltodextrin = mMD = mp * XMD
Mass of Maltodextrin solution with water = ms
All the concentrations were prepared weight per weight (w/w). To dissolve maltodextrin and obtain a transparent solution, mixture of maltodextrin powder and water was stirred vigorously right after mixing until the solution become transparent.
Sucrose solutions:
Sucrose solutions were prepared by dissolving crystalline sucrose in MQ water at room temperature. 10%, 20%, 30%, 40%, 50%, 55% (w/w) solutions were prepared.
Maltodextrin + Sucrose solutions:
Two sucrose and maltodextrin mixed solutions were prepared. The sucrose to maltodextrin mass ratio in those mixed solutions were 75:25 and 50:50. At first, required amounts of sucrose and maltodextrin were mixed and after then, required amount of MQ water was added. To prepare transparent solution, mixture of solute and water were stirred vigorously right after mixing. 10%, 20%, 30%, 40%, 50%, 55% (w/w) of both types of solutions were prepared.
Maltodextrin + Sucrose dispersions with probiotic bacteria:
The freeze-dried probiotic bacteria with sucrose were washed five times to remove sucrose and culture media salt. After that, the probiotic bacteria slurry was obtained and added to sucrose and maltodextrin solutions. For probiotic bacteria addition. 50% sucrose, 50% maltodextrin and 25%sucrose + 25% maltodextrin solutions and water were chosen. The bacteria slurry was mixed in five different mass ratios with all types of solutions. The mass fractions of the probiotic bacteria slurry in the mixed dispersions were 0, 0.33, 0.5, 0.66 and 1. True Mass fraction of water in dispersion were calculated by following manner:-
True Xwater = Xwater + (XPB Slurry * 0.8136)
Where, mass fraction of added water in dispersion = Xwater
Mass fraction of PB Slurry= XPB Slurry
As water content in PB slurry determined 81.36% (from TGA) So, mass fraction of water in PB Slurry= (XPB Slurry * 0.8136)
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Table.1.a: Composition of probiotic bacteria and maltodextrin or sucrose dispersion Mass ratio of PB slurry
and 50% MD or SU solution XPB Slurry XMD or XSU of solution Xwater True Xwater 0:1 0 0.5 0.5 0.5 1:2 0.33 0.33 0.37 0.64 1:1 0.5 0.25 0.25 0.6568 2:1 0.66 0.165 0.175 0.712 1:0 1 0 0 0.8136
Table.1.b: Composition of probiotic bacteria and MD+SU dispersion Mass ratio of PB slurry
and 50% MD+SU solution XPB Slurry XMD of solution XSU of solution Xwater True Xwater 0:1 0 0.25 0.25 0.5 0.5 1:2 0.33 0.165 0.165 0.37 0.64 1:1 0.5 0.125 0.125 0.25 0.6568 2:1 0.66 0.0825 0.0825 0.175 0.712 1:0 1 0 0 0 0.8136
2.2.
Methods
2.2.1. Thermo-gravimetrical analysisThermo-gravimetrical analysis was performed using a TGA Q500 (thermogravimetric analyser) from TA Instruments. It has a maximum sample weight of 1 g, a precision of ±0.01%, sensitivity of 0.1µg and a temperature range of ambient to 1000ºC.). The maltodextrin powder, sucrose, probiotic bacteria samples were heated in a platinum pan from 25°C to 1000°C (for maltodextrin up to 3000C), under a nitrogen atmosphere at a heating rate of 10°C/min. Thermograms of all samples were processed using the software TA Universal Analysis. The onset of degradation temperature was obtained from the intersection of the horizontal baseline and tangent line of the curve representing degradation of material.
2.2.2. Dynamic light scattering
Maltodextrin particle and probiotic bacteria size distributions in solutions were measured using Zetasizer Nano ZS analyzer from Malvern Instruments. Particle size measurements were performed using 1 mL disposable cuvettes filled with maltodextrin solutions of different concentrations and 1% (w/w) freeze-dried probiotic bacteria+ sucrose solution in
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MQ water. The refractive index used for maltodextrin solution was 1.67. and for bacteria, that was 1.45 (refractive index of protein and liposome).
2.2.3. Microscopic Study
An optical microscope (Nikon Optiphot Epi-Fluorescence microscope equipped with polarizing filters, phase contrast and a digital camera) was employed to examine probiotic bacteria slurry. That slurry placed directly on glass slides and their zoomed appearance through microscope and cross polarisation filter were captured as electronic images.
2.2.4. Rheometry
Viscosity curves of freshly prepared maltodextrin, sucrose and bacteria solutions were obtained using Bohlin rotational rheometer CVO (Malvern, Worcestershire, United Kingdom) against continuously changing shear stresses and against steady shear stresses. Different ranges of linear shear stress were used for different concentrations. The measured viscosities were used to analyse the concentration and temperature dependent behaviour of the solutions. Both parallel-plate (diameter 20 mm, gap 150 μm) and cone-plate geometry (diameter 40 mm and, gap 150 μm) were used e.To prevent water evaporation, samples were covered by a plastic cover with some water droplets.
Oscillatory curves were obtained also. To determine the shear stress dependent behaviour of the solutions, amplitude sweeps were carried out against continuously changing shear stress changes with constant frequency 0.1 Hz. To determine, the frequency dependent behaviour of the solutions, frequency sweeps were carried out against continuously changing frequency changes with constant shear stresses 2 and 5 Pa.
All measurements were performed in triplicate samples. 2.2.4.1. Effect of inertia in viscosity measurement
Rheometer fundamentally measures torque or force, angular displacement and angular velocity of material under external force. Rheometer measures the torque and the deflection angle of the measuring chuck. This means, in a viscosity measurement, the rheometer pre-sets a certain current that correlates to a defined torque. The sample provides resistance or a reset torque to the setting and the resulting deflection angle is then measured very precisely by the rheometer’s encoder. The speed is calculated from the deflection angle and the time. The rheological parameter shear stress is calculated from the torque in relation to the shearing surface. The same is true for the shear rate, which correlates with the speed. [anton-paar.com]. Due to rotation of chuck, artifact might be risen because of inertia.
During viscosity measurement against continuously changing shear stresses in this work, unexpected changes of viscosity were observed (Fig 4). In Bohlin rotational rheometer CVO, there are three options of inertia management during viscosity measurement. Those are- a) no inertial corrections, b) active inertia control, c) inertial compensation.
At first, viscosity measurements were done with no inertia corrections. But after getting strange viscosity curves, viscosity measurements were done with active inertia control. With
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this active inertia control, again similar result obtained. Finally, inertial compensation option was used and expected results was obtained (Fig 4).
Fig.4: Comparison among three ways of viscosity measurements of 30% MD solution (Scan rate 2 Pa/min, 200C)
In Fig.4, the viscosity curve obtained from viscosity measurement against continuously changing shear stresses using three options of inertia control were plotted and difference in result can be observed. As Newtonian curve obtained only with inertial compensation, therefore, for further measurement of viscosity, inertial compensation option was used. 2.2.4.2. Statistical analysis
Descriptive statistical analyses for calculating the mean viscosity and the standard deviation of the mean were performed using Microsoft Excel software (Microsoft Office 365). All obtained results were expressed as the mean ± standard deviation (±SD).
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3. RESULTS
3.1. Thermo-gravimetrical analysis
Thermo-gravimetrical analysis involves the measurement of change of sample mass with change of temperature.
3.1.1. Maltodextrin powder
Maltodextrin is a hygroscopic material. To determine water content in the maltodextrin powder, thermogravimetrical analysis of maltodextrin powder was done.
Fig.5a: Comparison between 3 different thermogravimetrical analysis of Maltodextrin powder (same source) at 100C/min scan rate
Fig.5b: Comparison between derivative thermogravimetric curves of Maltodextrin powder (same source) at 100C/min scan rate
In Fig.5, three TGA curves of maltodextrin powder were obtained and water content in maltodextrin powder determined from each curve. The values found 4.72%, 4.70% and 4.68% respectively. The onset degradation temperature also determined from each curve. The temperatures are 227.90C, 237.30C and 233.80C respectively and associated with mass
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loss of about 94%. Plateau of all curves can be observed in between 1300C to 2000C temperature range.
3.1.2. Probiotic bacteria
Probiotic bacteria were freeze dried with sucrose. To determine water content in freeze dried probiotic bacteria and amorphous sucrose, thermogravimetrical analysis of those were done. To remove sucrose, freeze dried probiotic bacteria washed five times. After removing culture medium and sucrose, to determine water content in probiotic bacteria slurry, TGA was done (measured by Ekaterina Bogdanova, PhD student, MAU.)
Fig.6a: TGA data on freeze-dried probiotic bacteria with sucrose, amorphous sucrose and probiotic bacteria slurry at 100C/min scan rate
Fig.6b: Derivative TGA curves of freeze-dried probiotic bacteria with sucrose, amorphous sucrose and probiotic bacteria slurry at 100C/min scan rate
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In Fig.6, three TGA curves of freeze-dried probiotic bacteria with sucrose, amorphous sucrose and probiotic bacteria slurry were obtained and water content values found 1.95%, 2.12% and 81.36% respectively. The onset degradation temperature also determined from TGA curves of probiotic bacteria slurry and amorphous sucrose and found 214.20C, mass
loss 98% and 244.70C, mass loss 17.90% respectively. Plateau of sugar TGA curve can be observed in between 800C to 2100C temperature range and in TGA curve of probiotic bacteria slurry, plateau can be observed in between 1800C to 2400C temperature range. 3.2.
Dynamic light scattering of maltodextrin solution
To determine the size distribution of maltodextrin particles in solution, maltodextrin solutions of different concentrations were subjected to dynamic light scattering.
Fig.7a: The size distribution of maltodextrin particles in solution of different concentrations (1%, 0.5%, 1.8%, 4.6%, w/w) from dynamic light scattering analysis
Fig.7b: The size distribution of maltodextrin particles in solution of different concentrations (18.4%, 9.2%, 4.6% w/w) from dynamic light scattering analysis
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In Fig.7a and Fig.7b, comparison between size distribution profiles of maltodextrin in solutions of different concentrations (0.5%, 1%, 1.8%, 4.6%, 9.2% and 18.4%, w/w) can be observed. The average size measured for 0.5%, 1%, 1.8%, 4.6%, 9.2% and 18.4% maltodextrin solutions found 12.79 nm, 13.01 nm, 13.26 nm, 14.28 nm, 16.64 nm and 19.65 respectively. In the DLS curves of lower concentrated MD solutions, only one peak appeared. But in the DLS curve of 9.2% (w/w) solution, two peaks have appeared at approximately 10 nm and between 80-100 nm. In the DLS curve of 18.4% (w/w) solution, three peaks have appeared at approximately 10 nm, between 80-100 nm and 1000 nm. To determine the size distribution profile of probiotic bacteria with sucrose in solution, that also was subjected to dynamic light scattering (measured by Ekaterina Bogdanova, PhD student, MAU.)
Fig.8: The size distribution of freeze-dried probiotic bacteria with sucrose in solution (1% w/w) from dynamic light scattering analysis
In Fig.8, size distribution profile of freeze-dried probiotic bacteria with sucrose in 1% solution can be observed. The average particles size and polydispersity index found 1373 nm, and 0.22 respectively.
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3.3. Microscopic study of probiotic bacteria slurry
The probiotic bacteria slurry was examined by microscope. It was also examined through the cross-polarizer filter.
Fig.9: Probiotic bacteria slurry under microscope
In Fig.9. probiotic bacteria slurry and interface between probiotic bacteria slurry and air bubble can be easily observed. The dark dots represent bacteria.
Fig.10a: Probiotic bacteria slurry under microscope through cross polarization filter
Probiotic bacteria
Air bubble
Probiotic bacteria
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In Fig.10a. birefringence of probiotic bacteria slurry and interface between probiotic bacteria slurry and air bubble through the cross-polarizer filter can be observed. Fig, 10b represents zoomed picture of Fig.10a.
Fig.10b: Probiotic bacteria slurry under microscope through cross polarization filter The probiotic bacteria slurry was diluted and examined by microscope.
Fig.11: Dilute Probiotic bacteria slurry under microscope
Probiotic bacteria
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In Fig.11, separated bacteria in diluted probiotic bacteria slurry can be easily observed.
Fig.12: Dried probiotic bacteria slurry under microscope through cross-polarisation filter (without cover)
In Fig.12, aggregations of bacteria can be observed in dried probiotic bacteria slurry examined under microscope through cross-polarisation filter.
Probiotic bacteria Air bubble
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3.4.
Viscometry
3.4.1. Viscosity of Sucrose and Maltodextrin solution
Viscosity curves of maltodextrin, sucrose and two sucrose+maltodextrin (different mass ratio of solute) solutions of different concentrations against continuously changing shear stresses at 200C obtained and plotted in Fig.13. In this figure, comparison among viscosity curves of all concentrations also done. Different ranges of linear shear stresses and different steady shears were used for viscosity measurement of different concentrations of different solutions.
Fig.13: Viscosity comparison between of SU, MD and two SU+MD (different mass ratio of solute) solutions of different concentrations against continuously changing shear
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Average viscosity of sucrose, maltodextrin and sucrose+maltodextrin solutions of different concentrations were determined and plotted against concentrations in Fig.14 and found increases of viscosity with increasing concentrations. The average viscosity values can be found in Table 2 and it can be observed that average viscosity increases with increasing concentration of the excipients.
Fig.14: Average Viscosity comparison between of SU, MD and two SU+MD (different mass ratio of solute) solutions of different concentrations at 200C. Determined viscosity
values of sucrose also compared with values found in literature. [Telis, 2007] The average viscosities of the solutions are determined from viscosity values measured against continuously changing shear stresses. From Fig.14, it can be seen that, except 10-30% sugar solutions, 10-20% sucrose+maltodextrin (mass ratio of solute -75:25) and 10% sucrose+maltodextrin (mass ratio of solute -50:50), all solutions are Newtonian. That’s why, to determine average viscosities of 10-30% sugar solutions, 10-20% sucrose+maltodextrin (mass ratio of solute -75:25) and 10% sucrose+maltodextrin (mass ratio of solute -50:50), viscositiy values measured at steady shear stresses were considered.
1 10 100 1000 10000 10% 20% 30% 40% 50% 55% Vi sco si ty , m P as Concentration, % (w/w)
25
Table.2: Average viscosities of SU, MD and two SU+MD (different mass ratio of solute) solutions of different concentrations at 200C. Uncertainties are standard deviations.
Mass percentage
Viscosity (mPas) of the solutions
MD SU (Literature) SU SU+MD (mass ratio of solute -75:25) SU+MD (mass ratio of solute -50:50) 10% 2.84 ± 0.32 (0-1.5 Pa, 33 values) 1.26 1.58 ± 0.51 (0-1 Pa, 55 values) 1.74 ± 0.49 (0-1 Pa, 58 values) 1.97 ± 0.51 (0-1 Pa, 98 values) 20% 8.22 ± 0.30 (0-3.5 Pa, 82 values) 1.96 2.02 ± 0.53 (0-1 Pa, 95 values) 3.03 ± 0.42 (0-1.5 Pa, 33 values) 4.44 ± 0.31 (0-2 Pa, 48 values) 30% 35.00 ± 1.66 (0-14 Pa, 344 values) 3.21 3.72 ± 0.49 (0-1.5 Pa, 95 values) 6.52 ± 0.51 (0-1.5 Pa, 98 values) 10.46 ± 0.97 (0-4.5 Pa, 107 values) 40% 176.16 ± 2.19 (0-69 Pa, 1716 values) 6.2 5.47 ± 0.37 (0-2 Pa, 95 values) 13.91 ± 0.64 (0-2.6 Pa, 138 values) 36.49 ± 0.59 (0-15 Pa, 358 values) 50% 1278.11 ± 21.09 (0-400 Pa, 9997 values) 15.04 14.14 ± 1 (0-5.5 Pa, 223 values) 47.06 ± 2.45 (0-19 Pa, 458 values) 139.34 ± 2.37 (0-55 Pa, 1363 values) 55% 5375.67 ± 190.69 (0-450 Pa, 17986 values) N/A 29.39 ± 0.74 (0-6.5 Pa, 278 values) 108.96 ± 3.09 (0-22 Pa, 998 values) 392.21 ± 5.93 (0-65 Pa, 2798 values)
3.4.2. Temperature dependency of viscosity of sucrose and maltodextrin solution Viscosity curve of 50% (w/w) sucrose, maltodextrin and sucrose+maltodextrin solutions were obtained against continuously changing shear stresses at different temperatures, plotted in Fig.15 and found decreases in viscosity with increasing temperature.
26
Fig.15: Comparison between viscosity curves of 50% (w/w) SU, MD and two SU+MD (different mass ratioof solute) solutions against continuously changing shear stresses at
three different temperatures. 3.4.3. Viscosity of Probiotic bacteria slurry
Viscosity curve of probiotic bacteria slurry against continuously changing shear stresses was obtained at 200C and plotted in Fig.16. Probiotic bacteria slurry shows shear thinning
behaviour against continuously changing shear stresses.
Fig.16: Viscosity curve of probiotic bacteria slurry against continuously changing shear stresses at 200C
27 3.4.4. Temperature dependency of Viscosity of Probiotic bacteria slurry
Viscosity curves of probiotic bacteria slurry at different temperatures are plotted in Fig.17a and Fig.17b. In Fig.17a, for each viscosity measurement, different samples from PB slurry was used and found decreases in viscosity with increasing temperature. Again, In Fig.17b, for each viscosity measurement, same sample was used and found different results from Fig.17a. In this Fig 17b, viscosity at 250C is lower than viscosity at 200C but viscosity at 300C is higher than viscosity at 200C.
Fig.17a: Comparison between viscosity curves of probiotic bacteria slurry against continuously changing shear stresses at three different temperatures (for each temperature,
different sample used)
Fig.17b: Comparison between viscosity curves of probiotic bacteria slurry against continuously changing shear stresses at three different temperatures (for all temperatures
same sample used) Up scan
Up scan
Back scan
28 3.4.5. Viscosity of Probiotic bacteria slurry with sucrose and maltodextrin solution The probiotic bacteria dispersions were prepared by mixing probiotic bacteria slurry with 50% sucrose, maltodextrin and sucrose+maltodextrin solutions. The mass fraction of probiotic bacteria slurry in the dispersions were 0, 0.33, 0.5, 0.66 and 1. Those dispersions were subjected to continuously changing shear stresses to observed effect of probiotic bacteria addition on viscosity of sucrose and maltodextrin solutions. The average viscosities of the mixed dispersions were also determined. To calculate average viscosity of probiotic bacteria slurry, viscosities of Newtonian part of the curve were considered.
Fig.18: Comparison between viscosity curves of probiotic bacteria with 50% (w/w) SU, MD and SU+MD dispersions against continuously changing shear stress at 200C. The mass fraction of probiotic bacteria slurry in the mixed dispersions were 0, 0.33, 0.5, 0.66
and 1.
In Fig.18, viscosity curves of probiotic bacteria with 50% (w/w) sucrose, maltodextrin and sucrose+maltodextrin dispersions against continuously changing shear stress at 200C were plotted and Newtonian behaviour observed.
29
In Fig.19, average viscosities of probiotic bacteria with 50% (w/w) sucrose, maltodextrin and sucrose+maltodextrin dispersions were plotted against mass fraction of probiotic bacteria slurry and values can be found in Table 3.
Fig.19: Effect of Probiotic bacteria slurry addition on viscosity of 50% (w/w) SU, MD and SU+MD solutions at 200C. The mass fraction of probiotic bacteria slurry in the mixed
dispersions were 0, 0.33, 0.5, 0.66 and 1.
Table.3: Average viscosity of probiotic bacteria with 50% (w/w) SU, MD and SU+MD dispersions at 200C. Uncertainties are standard deviations.
Mass fraction of PB slurry
Viscosity (mPas) of mixed solutions 50% SU solution 50% MD solution 25% SU + 25% MD solution Water 0 14.14 ± 1 1278.11 ± 21.09 139.34 ± 2.37 1 0.33 10.31 ± 1.21 (0-2.5 Pa, 37 values) 283.40 ± 7.62 (0-50 Pa, 996 values) 46.82 ± 1.71 (0-20 Pa, 396 values) 2.32 ± 3.83 (0-2 Pa, 98 values) 0.5 10.89 ± 0.85 (0-2.5 Pa, 37 values) 171.98 ± 4.40 (0-50 Pa, 996 values) 44.73 ± 2.58 (0-20 Pa, 396 values) 3.20 ± 0.64 (0-2 Pa, 98 values) 0.66 17.23 ± 3.39 (0-2.5 Pa, 37 values) 125.31 ± 5.03 (0-50 Pa, 996 values) 44.97 ± 2.96 (0-20 Pa, 396 values) 5.54 ± 1.71 (0-2 Pa, 98 values) 1 61.56 ± 1.76 (0-20 Pa, 197 values) 57.67 ± 3.67 (0-20 Pa, 303 values) 57.67 ± 3.67 (0-20 Pa, 303 values) 57.67 ± 3.67 (0-20 Pa, 303 values) 1 10 100 1000 0 0,33 0,5 0,66 1
Vis
cos
it
y,
mP
as
Mass fraction of PB slurry
30
3.5. Oscillation test
3.5.1. Amplitude and frequency sweep of sucrose and maltodextrin solutions
To investigate the rheological behaviour of 50% sucrose, maltodextrin and sucrose+maltodextrin solutions against oscillation, those solutions were subjected to amplitude sweep at frequency of 0.1 Hz and frequency sweep at 2 Pa and plotted in Fig.20a and Fig.20b.
Fig.20a: Amplitude sweep of 50% (w/w) SU, MD and SU+MD (different mass ratioof solute) solutions at 0.1 Hz and 200C. All G’’ curves are plotted as blue circles and indicated by sample name in blue words. G’ curves are plotted as circle also and indicated
31
Fig.20b: Frequency sweep of 50% (w/w) MD and SU+MD (mass ratioof solute-50:50) solutions at 2 Pa and 200C.
3.5.2. Amplitude and frequency sweep of Probiotic Bacteria slurry
To investigate the rheological behaviour of probiotic bacteria slurry against oscillation, these solutions were subjected to amplitude sweep at frequency of 0.1 Hz and frequency sweep at 2 Pa and plotted in Fig.21a and Fig.21b.
32
33
4. DISCUSSION
4.1. Water content and thermal degradation of materials
In the TGA curves of maltodextrin (Fig.5), the first thermal event was observed in the temperature range of 50–1500C, associated with evaporation of water and corresponding to approximately 4.7% (average of three values from three curves) of mass loss. The second thermal event started at the temperature of approximately 2320C. This thermal event attributed to the thermal decomposition of long molecular chains of maltodextrin and depolymerization of the macromolecules. This onset decomposition temperature of maltodextrin is 400C and 260C higher than the temperature reported by Zenaida et al. and Castro et al. respectively [Zenaida, 2015; Castro, 2016]. As the onset decomposition temperature depends on dextrose equivalent value and molecular weight distribution of maltodextrin [Avaltroni, 2004], Another reason might be the difference in algorithms for determination of onset temperature.
In the TGA curve of amorphous sucrose (Fig.6), at 1000C, 2.12% of mass loss which can be considered as evaporation of water. The second thermal event started at the temperature of 2140C which indicates starting thermal degradation of sucrose. This onset decomposition temperature of amorphous sucrose is 340C higher than the degradation temperature of crystalline sucrose found in literature. [Hurtta, 2004].
To know thermal response of probiotic bacteria only, TGA of probiotic bacteria should be done. To do that, freeze dried probiotic bacteria+sucrose was washed five times. After removing culture medium and sucrose, the probiotic bacteria slurry subjected to TGA. In TGA curve of probiotic bacteria slurry (Fig.6), from the plateau of the curve after 1000C, mass loss was determined 81.36% which can be considered as evaporation of water. As an accepted standard value for dry-matter content of bacterial cells is approximately 20% [Luria, 1960; Bratbak, 1984] so, it can be concluded that the evaporated water is the water content of the slurry plus intracellular water of bacteria. Bratbak et al also claimed that the dry-matter content of bacteria may be more than twice as high as generally assumed [Bratbak, 1984]. After evaporating of water, thermal degradation of probiotic bacteria started at 2450C.
In TGA curve of freeze-dried probiotic bacteria with sucrose (Fig.6), mass loss at 1000C
was 1.95% which can be considered as evaporation of water. In this curve, gradual mass loss can be observed after 1000C. To understand this curve, TGA curves of freeze-dried probiotic bacteria with sucrose, amorphous sucrose and probiotic bacteria slurry were compared in Fig.6.
From Fig.6, in TGA curve of sucrose, the onset degradation temperature is found at 2140C and 2450C in the TGA curve of washed PB slurry. This confirms that there is no sucrose in
the washed PB slurry. Moreover, it can be concluded that after five times washing, that culture media substances were also removed. So, 2450C represents the starting of thermal degradation of probiotic bacteria only. Therefore, in the TGA curve of freeze-dried probiotic bacteria with sucrose, from 2450C to 10000C, the gradual mass loss at this temperature range might be attributed to loss of culture media substances with bacteria.
34
4.2. Structural properties of maltodextrin solution and probiotic bacteria
dispersion
The size distribution profile of maltodextrin in solution was determined by dynamic light scattering of maltodextrin solutions of different concentrations (0.5%-18.4% w/w). It can be observed from Fig.7, there are broader size distribution curves with one peak appeared at approximately 10 nm for 0.5%-4.6% (w/w) maltodextrin and average size found approximately 12-15 nm.
As we know, length of glucose molecular unit is approximately 1 nm. The dextrose equivalent gives an indication of the average degree of polymerization (DP) for starch sugars. The relation between DE and DP described by following equation: -
DE × DP = 120.
So, fully extended chain length of maltodextrin having DE value 8 is about 15 nm.
Therefore, the only peak of DLS curves of maltodextrin solutions of different concentrations (0.5%, 1%, 1.8%, 4.6%) represents maltodextrin molecules and perhaps very small aggregates of these molecules (a small shoulder on the left side of the peak at the lowest concentration may correspond to non-aggregated molecules). The DLS curve of maltodextrin solutions of 9.2% concentration also has one peak at 10 nm and another peak at 80-100 nm. This 2nd peak represents aggregation of maltodextrin molecules. Again, the DLS curve of maltodextrin solutions of 18.4% concentration has three peaks: at 10 nm, at 80-100 nm and at 1000 nm. Therefore, the 3rd peak might represent maltodextrin crystalline particles. Although 18.4% MD solution was transparent but from DLS curve, it can be concluded that there is a two-phase system in MD solution of this concentration. Carlstedt et al also reported about observation of crystallization of maltodextrin at this concentration [Carlstedt, 2014].
So, there are different types of aggregates of maltodextrin formed in maltodextrin solution and the size of the aggregates also become greater than aggregate present in lower concentration solution. Therefore, it can be concluded that, aggregation tendency of maltodextrin molecules in solution increases with increasing concentrations.
Reuther et al proposed a structural model of Maltodextrin gel. Maltodextrin molecules might follow this model to aggregate in solution, (Fig 22)
35
In the dynamic light scattering analysis of freeze-dried probiotic bacteria with sucrose solution, (Fig.8) one peak in DLS curve can be observed and the average diameter of particles is 1.373 µm. The length of Lactobacillus Reuteri found at literature is approximately 1 µm. [Dishisha, 2013]. The polydispersity index found greater than 0.05, (if polydispersity index found less than 0.05, sample is monodispersed). This result suggests that some aggregation of bacteria may take place, which also can be observed in Fig 11. Probiotic bacteria slurry was also examined by microscope and the dark dots in Fig.9 represent bacteria. It seems, that slurry is highly concentrated. Same slurry was also examined through cross-polarisation filter (Fig.10a) and found to be birefringent at the interface between liquid and air. Birefringence is the ability of substances to change the polarization state of light. Fig.10b is zoomed picture of Fig.10a. In the zoomed Fig.10b, it can be observed birefringent bacteria are highly concentrated and, at the interface, are arranged in an ordered fashion resembling a nematic phase. Diluted bacteria slurry also examined by microscope and the separated bacteria and some very small aggregations of bacteria can be observed in Fig.11. Those aggregations of bacteria can be observed in Fig.12 also, which figure represents picture of dried probiotic bacteria slurry examined under microscope through cross-polarisation filter.
4.3. Rheological behavior of sucrose, maltodextrin solutions and probiotic
bacteria slurry
4.3.1. Sucrose and Maltodextrin solutions Observation of phase behaviour:
Freshly prepared maltodextrin and sucrose solutions of different concentrations have different physical states: 10-40% fresh Maltodextrin solutions were transparent, 50% solution was opaque viscous solution, 55% solution was turbid viscous solution,, 60% solution was very thick, turbid paste and too sticky to measure. Precipitation time of MD solutions of different concentrations were different: 10% solution was remained transparent after seven days, , 20% solution started to precipitate after 2 days, 30% solution started to precipitate after 1 day while 40% solution needed only two hours to precipitate. 50% solution became thick turbid paste after 6 hours while 55% solution became thick turbid paste within 1 hour.
Sucrose and sucrose+maltodextrin (75:25) solutions remain transparent while 40%-55% sucrose+maltodextrin (50:50) solutions started to precipitate after one day.
Table.4: Physical states of freshly prepared solutions Concentration of solution (w/w) MD SU SU+MD (mass ratio of solute -75:25) SU+MD (mass ratio of solute -50:50) 10% Transparent Transparent Transparent Transparent 20% Transparent Transparent Transparent Transparent 30% Transparent Transparent Transparent Transparent
36
40% Transparent Transparent Transparent Transparent 50% Opaque viscous
solution Transparent Transparent Transparent 55% Turbid viscous
solution Transparent Transparent
Opaque viscous solution 60% Thick, turbid and
sticky paste N/A N/A N/A
Rheological behaviour of Sucrose and Maltodextrin: -
Viscosities of sucrose and maltodextrin solutions of 10-55% (w/w) were measured at 200C and found that viscosity increases with increasing concentration for both sucrose and maltodextrin solutions (Fig.14). In MD solutions, the presence of increasing maltodextrin or sucrose molecules or particles in solutions perturb the normal liquid flow more and hinder the mobility of polymer chain segments in solution. As a result, viscosity of MD and SU solutions increases with increasing concentration.
For all concentrations of maltodextrin solutions, viscosity vs shear stress flow curves were Newtonian (Fig.13). Dokic et al. and Avaltroni et al also reported about this behaviour of maltodextrin solutions [Dokic, 1998; Avaltroni, 2004]. For sucrose solutions, at higher concentrations (40%-55%), viscosity vs shear stress flow curves were Newtonian. For lower sugar concentrations like 10%, 20% and 30%, viscosity vs shear stress flow curves showed some deviations from Newtonian behaviour. From the literature, it is known that sugar solutions show Newtonian behaviour. Therefore, in viscosity measurement for lower concentrations of sugar solution against continuously changing shear stresses, a machine artifact (for example related to inertia, see methods section 2.2.4.1.) might affect the results. The average viscosity values of sucrose solutions which determined in this work, are in a good agreement (Fig.14) with values reported by Telis et al [Telis, 2007].
The viscosity of a aqueous solution depends on solute concentration, temperature and pressure which are the thermodynamic state parameters. In literature, many equations relating those parameters with viscosity were derived by the researchers. Einstein first pointed out the relation between viscosity with volume fraction of solute at Eq.1 [Longinotti, 2008]. In Eq.1, Einstein considered solution as infinitely dilute solution and the solutes were non-interacting hard spheres. As, application of Einstein equation is limited to dilute solution only, for concentrated solution, Krieger and Dougherty proposed another equation of viscosity to observe the concentration dependence of the viscosity, [Willenbacher, 2013].
𝜂𝑟 = (1 − 𝜑 𝜑𝑚𝑎𝑥))
−2.5𝜑𝑚𝑎𝑥...(2)
Where, φmax is the maximum packing fraction or the volume fraction at which the zero-shear
viscosity diverges. In this equation, solutes are also considered as spheres.
From Eq.1 and Eq.2, it also can be observed that viscosity of solution or dispersion increases with increasing volume fraction of solute.
37
In this work, maltodextrin particles in concentrated solution can be assumed as solid spheres. It is however difficult to measure the volume fraction of maltodextrin particles in solution. Therefore, to describe viscosity data of both maltodextrin and sucrose, Spurlin, Martin, and Tennent’s equation [Avaltroni, 2004] are considered. In this empirical equation, no assumption about type and shape of aggregates is used and solution concentration is considered instead of volume fraction of particles. Avaltroni et al. also reported that, viscosity changing with concentrations of maltodextrin solutions follows this equation [Avaltroni, 2004]. The empirical Spurlin, Martin, and Tennent’s equation (1946) is-
𝑙𝑛𝜂𝑠𝑝
𝐶 = ln([𝜂]0) + 𝑘[𝜂]0𝐶...(3)
Where, ηsp is the specific viscosity, [η]0 is the intrinsic viscosity and c is the concentration
in g/dl and K is constant. The formula of specific viscosity is:- 𝜂𝑠𝑝 = 𝜂
𝜂0− 1 Where, η0 is the viscosity of pure solvent.
To fit the obtained viscosity data in Spurlin, Martin, and Tennent’s model, specific viscosities of MD and SU solutions were divided by concentration and logarithmic value of quotient was plotted against concentrations (in g/dl) in Fig. 23.
Fig.23: Plot of Spurlin–Martin–Tennent’s model for maltodextrin (blue) and sucrose (orange) solutions
From Fig.23, it can be concluded that the Spurlin–Martin–Tennent’s model is readily applicable to maltodextrin solutions of different concentrations. This model is applicable for sucrose solutions also although viscosity of some sucrose solutions deviated from this model. The inaccuracy in measured viscosity values might be a reason for that.
y = 0,0503x - 2,4133 y = 0,0141x - 3,3802 y = 0,0321x - 2,8132 -4 -3 -2 -1 0 1 2 3 4 0 20 40 60 80 100 120 140 ln ( ηsp/C) C, g/dl MD SU MD+SU (50:50)
38
After characterising MD and SU solutions, MD+SU solutions also characterized. For that, two sucrose and maltodextrin mixed solutions were also prepared. The sucrose to maltodextrin mass ratio in those mixed solutions were 75:25 and 50:50. Viscosity of both solutions of 10-55% (w/w) were measured also against continuously changing shear stresses at 200C and found that viscosity increases with increasing concentration of solute in solutions (Fig.14). Except 10-20% sucrose+maltodextrin (mass ratio of solutes -75:25) and 10% sucrose+maltodextrin (mass ratio of solutes -50:50), where some deviations were observed, for all concentrations of both solutions, viscosity curves were Newtonian (Fig.13).
From the four types of solutions, it also can be observed that viscosity of the maltodextrin solutions had the highest value and viscosity of the sucrose solutions had the lowest value (Fig.14). From Fig.14, it also can be concluded that with increasing concentration of maltodextrin in solute, viscosity of mixed solutions increases. This is explained by the fact that maltodextrin is a polymer and might have internally connected chain network while sucrose is disaccharide [Chronakis, 1998; Longinotti, 2008]. Therefore, it is more difficult to move the maltodextrin polymer chains against applied shear stresses. As a result, maltodextrin solutions show higher viscosity than sucrose.
This behaviour also can be observed from the Fig.20a; the results of amplitude sweeps of 50% of all four types of solutions. The viscous modulus curves of all four types of solutions were Newtonian and 50% maltodextrin viscous modulus curve have highest values against continuously changing shear stresses and 50% sucrose viscous modulus curve have lowest values.
From the Fig.20b, the results of frequency sweeps of 50% maltodextrin solution and 25% sugar + 25% maltodextrin solution, it can be observed that both solutions are frequency dependent. To describe oscillatory data obtained from frequency sweeps, the Maxwell model was considered. The Maxwell model is- [Barnes, 2000].
𝐺′= 𝐺(𝜔𝜏)2
1+(𝜔𝜏)2...(4)
𝐺′′ = 𝜂𝜔
1+(𝑤𝜏)2...(5)
Where, G’’ is the viscous modulus, G’ is the elastic modulus, ω if frequency in rad/s, 𝜏 is the relaxation time. 𝐺 =𝜂⁄ 𝜏
If 𝜏 is assumed very small, then
𝐺′′ ≈ 𝜂𝜔
Or, log 𝐺′′ ≈ log 𝜂 + log 𝜔...(7) Since the shear modulus, 𝐺 =𝜂⁄ then, from Eq.4, 𝜏
𝐺′ ≈𝜂 𝜏(𝜔𝜏)
2
39
To check applicability of Maxwell model for obtained oscillation data, logarithmic values of obtained G’’ plotted against logarithmic values frequency (rad/s) in Fig.24.
Fig.24: Plot of Maxwell model for 50% MD and 25%MD+25%SU solutions
From Fig.24, viscosity of 50% MD and 25%MD+25%SU determined and found 2176.2 mPas and 149.4 mPas respectively. Those values are little bit higher from measured viscosities of those two solutions (Table 2). Nonetheless it might be concluded that the measured oscillation data is in good agreement with Maxwell model. The relaxation time τ calculated from Fig.25 and found 0.007 s and 0.011s for 50% MD and 25%MD+25%SU respectively.
From both Fig.20a and Fig.20b, the oscillation behaviour of all four types of solutions were dominated by the loss (or viscous) modulus, G’’. Therefore, it can be concluded that the systems in all four types of solutions have a little internal network and are easily disturbed. Therefore, all solutions behave like a liquid, not a gel.
4.3.2. Effect of Probiotic bacteria addition on viscosity of sucrose and maltodextrin solutions
Viscosity of probiotic bacteria slurry was measured against continuously changing shear stresses at 200C. The probiotic bacteria slurry show shear thinning behaviour at low shear
stress but after 10 Pa, it shows Newtonian behaviour (Fig.16). With application of shear stress, bacteria in the slurry, might be arranged in a nematic order, and become easy to move. As a result, viscosity decreases with increasing shear stresses. To determine average viscosity of probiotic bacteria slurry, viscosities of Newtonian part of the curve were considered.
This shear thinning behaviour against continuously changing shear stresses also can be observed from the Fig.21a; the results of amplitude sweeps of probiotic bacteria slurry. The viscous modulus values decrease with increasing shear stresses.
From the Fig.21b, the results of frequency sweep probiotic bacteria slurry, it can be observed that probiotic bacteria slurry is frequency dependent.
40
From both Fig.21a and Fig.21b, the oscillation behaviour of probiotic bacteria slurry was dominated by the loss (or viscous) modulus, G’’. Therefore, it can be concluded that the system in probiotic bacteria slurry has a little internal network and is easily disturbed. Therefore, probiotic bacteria slurry behaves like a liquid, not a gel.
To check applicability of Maxwell model for obtained oscillation data of PB slurry, logarithmic values of obtained G’’ plotted against logarithmic values frequency (rad/s) in Fig.25.
Fig.25: Plot of Maxwell model for probiotic bacteria slurry
From Fig.25, viscosity of probiotic bacteria slurry determined and found 57.7 mPas. This value in excellent agreement with the measured viscosity of PB slurry (Table 3). Therefore, it might be concluded that the measured oscillation data is in good agreement with Maxwell model. The relaxation time τ calculated from Fig.25 and found 0.06 s.
The probiotic bacteria were added to sucrose and maltodextrin solutions. For probiotic bacteria addition. 50% sucrose, 50% maltodextrin and 25%sucrose + 25% maltodextrin solutions and water were chosen. The bacteria slurry was added in five different mass fractions in all types of solutions. The mass fractions of the probiotic bacteria slurry in the mixed dispersions were 0, 0.33, 0.5, 0.66 and 1.
Viscosities of those bacteria mixed dispersions were measured against continuously changing shear stresses at 200C. In case of 50% sucrose dispersion, viscosity first decreased
and then increased with increasing mass fraction of probiotic bacteria slurry. But in case of 50% maltodextrin solution + bacteria dispersion and 25%sucrose + 25% maltodextrin with bacteria dispersions, viscosity is decreasing with increasing mass fraction of probiotic bacteria slurry (Fig.19). For all bacteria mixed dispersions, viscosity vs shear stress flow curves were Newtonian. (Fig.18). Viscosity of probiotic bacteria slurry with water is increasing with increasing mass fraction of probiotic bacteria slurry (Fig.19).
Relative viscosities of PB dispersions were calculated. Unlike Fig.19, relative viscosity increase with increasing PB slurry concentration in dispersion. This behaviour is observed
41
much stronger than Einstein equation (Eq.1) and can be discribed in term of Krieger and Dougherty equation (Eq.2).
To evaluate Eq.2, volume fraction of probiotic bacteria need to be known. Since the volume fraction of PB is not accurately known, relative viscosities of the dispersions are plotted against mass fraction of PB slurry in PB dispersion in Fig.26. The course of the curves becomes more similar and continuous increase of relative viscosities as a function of PB concentration is observed.
Fig.26: Relative viscosities of PB dispersions
4.3.3. Temperature dependence of Viscosities of sucrose, maltodextrin solutions and probiotic bacteria slurry
The viscosity curves of 50% sucrose, 50% maltodextrin, 37.5% sucrose+12.5% maltodexrin and 25% sucrose+25% maltodextrin solutions at three different temperatures against continuously changing shear stresses were obtained (Fig.15) and found that, the viscosity changes with the temperature according to an Arrhenius relationship.
From Arrhenius equation of viscosity
𝜂 = 𝐴 𝑒𝑥𝑝(𝐸𝑎𝑅𝑇)...(8)
or, ln 𝜂 = 𝐸𝑎
𝑅𝑇+ ln 𝐴 ...(9)
where Ea is activation energy, A is constant, and R is gas constant.
Therefore, log η ∝ 𝟏/𝑻, i.e., log of viscosity is inversely proportional to temperature. On the molecular level - at higher temperatures, the molecular movement of the maltodextrin polymer is easier due to the decrease of the inter-chain interaction and with
1
10
100
0
0,33
0,5
0,66
1
η
r
Mass fraction of PB slurry
42
increasing temperature, solubility of sucrose increases. Therefore, viscosity of sucrose solutions decreases with increasing temperature [Charles, 1912].
By using Eq.9, activation energies for all four types of 50% solutions were calculated and values can be found in Table 5.
Table.5: Activation energy of 50% solutions.
Sample Ea (KJ/mol)
50% MD 38.59
50% SU 28.88
50% SU (Literature value) 33.05 [Telis, 2007]
25% MD + 25% SU 31.53
12.5% MD + 37.5% SU 29.69
From, Table.5. it can be observed that 50% Maltodextrin solution has highest activation energy. This also correlates with the fact that this solution has the highest viscosity also among all four types of solutions.
The viscosity curves of probiotic bacteria slurry at three different temperatures against continuously shear stresses were obtained also (Fig 17). In Fig.17a, viscosity measurement was done by using different samples and in Fig.17b, viscosity measurement was done by using same sample.
From Fig.17a, the viscosity decreases with the increasing temperature according to the Arrhenius relationship. It can be an effect of temperature dependence of solvent (water). Another reason might be increasing cell membrane fluidity with increasing temperature. Membrane fluidity refers to the viscosity of the lipid bilayer of a cell membrane. When lipids are heated up, they acquire thermal energy and energetic lipids move around more, making the membrane more fluid by arranging and rearranging randomly. [Gennis, 1989] However, when the same sample was used at all three temperatures, viscosity at 300C was higher than viscosity of 200C. It might be associated with growth of bacteria with increasing temperature when using same sample of PB slurry for viscosity measurement. Valik et al also reported about the positive effect of temperature on bacteria growth [Valik, 2013]. In 300C, PB also may absorb water from surrounding making the dispersion more concentrated. Finally drying of PB slurry also may occurred. With bacteria growth and drying at 300C, PB slurry became more concentrated, movement of bacteria became harder and viscosity increased.
