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Item Enhancing antioxidant property of instant coffee by microencapsulation via spray drying(Editorial Universitat Politècnica de València, 2019-01-18) Sakawulan D; Archer R; Borompichaichartkul C; Cárcel JA; Clemente G; García-Pérez JV; Mulet A; Rosselló CThis study is aimed to improve the antioxidant property of instant coffee by using microencapsulation technique and spray drying. Concentrated coffee extract was mixed with Konjac glucomannan hydrolysate (KGMH) and Maltodextrin (MD). The mixture of coating material and coffee extract was then spray dried at 160 - 180 °C inlet air temperature and at 85-90 °C outlet air temperature. KGMH can preserve retention of phenolic compounds, DPPH scavenging activity and antioxidant activity of FRAP (p<0.05 of instant coffee better than other treatment.Item Development of microemulsion delivery systems for bioactive compounds : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Auckland, New Zealand(Massey University, 2020) Yuan, QuanMany bioactive compounds for health benefits are not readily stable against degradation and their solubility is also very low. As a result, a delivery system is required to encapsulate and protect bioactive compounds for their food applications. Emulsion is one of the delivery systems which has been studied by many researchers. But emulsion tends to destabilize during storage and its opaque optical properties makes it difficult for its use and incorporation into clear foods or beverages without affecting their original appearance. Therefore, microemulsion, which is known to be transparent, has been investigated to some extent to encapsulate and deliver bioactive compounds as a potential delivery system. The objective of this research was to fabricate oil-in-water (O/W) microemulsions which might be utilised as the delivery system for bioactive compounds. This thesis is mainly composed of two sections. The first section was to produce microemulsions via emulsion dilution method and water titration method as well as to study the characteristics of these microemulsions. Beta-carotene was a type of bioactive compound used in the second section to study the effect of beta-carotene on the formation and properties of microemulsion which was fabricated using the same methods described above. At first, emulsion dilution method was employed to fabricate microemulsions with different types and concentrations of oils, such as peanut oil, fractionated coconut oil, isopropyl myristate (IPM), lemon oil and Capmul 708G, and also with different surfactants (Tween 20, 40, 60 and 80). It was found that peanut oil and fractionated coconut oil could not be utilised to form microemulsions by this method, whereas IPM and lemon oil had the ability to fabricate microemulsions. When 1% Tween 80 was introduced as the surfactant and dilution medium, microemulsion could be formed when the concentration of IPM was less than 0.1% and that of lemon oil was less than 0.2%. Among the different types of Tween surfactants, Tween 80 was the most efficient when its solution containing Tween micelles was used as a dilution medium compared to the other Tween surfactants because more lemon oil could be incorporated into the Tween 80 micelles with an increase in Tween 80 concentration. In the following study, a water titration method was employed to create ternary or pseudo phase diagrams which indicated the ability to fabricate microemulsions of a mixture system. Various types of oils (Captex 100, Capmul PG-8, Capmul PG-12, Capmul PG-2L, lemon oil, Capmul MCM C8, Capmul 708G and Captex 355) and surfactants (Tween 80, Tween 20, Span 80 and Kolliphor EL) were used in this study. Absolute ethanol and propylene glycol (PG) were also incorporated as cosurfactant and cosolvent, respectively. It is concluded that all these oils and surfactants could be utilised by the water titration method to produce microemulsions, however, their ability to form microemulsions were different. Capmul 708G, which is a monoglyceride, was the most efficient in terms of producing microemulsions compared to diglyceride and triglyceride. Tween 20 and Kolliphor had the similar emulsifying properties compared to Tween 80 whereas Span 80 was not efficient. Both absolute ethanol and PG could assist the formation of microemulsions when they were introduced into the mixture system of oil, surfactant and water. In the following study, microemulsions containing 0.1% and 0.4% lemon oil and an emulsion containing 1.5% lemon oil (larger oil droplets), which were fabricated by the emulsion dilution method, were chosen to incorporate beta-carotene as a lipophilic model bioactive compound into lemon oil in order to study its impact on the formation and properties of the resulting microemulsion and emulsion systems. The encapsulation of beta-carotene into 0.1% and 0.4% lemon oil caused a significant increase in the particle size of the O/W microemulsions, but the particle size was still within the size range of microemulsion. As a result, the beta-carotene-loaded microemulsions containing 0.1 and 0.4% lemon oil were visually clear in appearance. However, the incorporation of beta-carotene did not increase and alter the particle size of the emulsion containing 1.5% lemon oil. The microemulsion sample containing 0.1% lemon oil and the emulsion containing 1.5% lemon oil were stored at 25 °C without exposed to oxygen and light for one month. While, the microemulsion containing 0.4% lemon oil was selected and placed at three different temperatures (4, 25 and 37 °C) for 1 month: at 4 and 37 °C without exposure to both oxygen and light and at 25 °C, four different environmental conditions (i.e. with oxygen/light, with oxygen and without light, without oxygen and with light, without oxygen/light). The results showed that the rate of beta-carotene degradation was lower in all these three samples when compared to the beta-carotene present in a hexane solution without encapsulation. Higher temperature accelerated the degradation rate of beta-carotene. As a consequence, the 0.4% lemon oil microemulsion at 4 °C exhibited the slowest degradation rate of beta-carotene. Next, the microemulsions fabricated by the water titration method were selected to encapsulate beta-carotene to study the encapsulation capacity of these microemulsion systems as well as their ability to protect beta-carotene against oxidative degradation during storage. Capmul 708G, Tween 80, Milli-Q water and PG mixture system were chosen to fabricate microemulsions and two formulations (L910 and L990) were prepared to incorporate beta-carotene. L910 was comprised of 81% Capmul 708G, 9% Tween 80, 5% water and 5% PG, whereas L990 contained 9% Capmul 708G, 1% Tween 80, 45% water and 45% PG. It was able to see clearly from this experiment that the L910 system could incorporate more beta-carotene than L990. Both L910 and L990 could reduce the degradation rate of beta-carotene when loaded into them compared to their presence in hexane solutions without encapsulation. Similar to the previous experiment as described above, when the beta-carotene incorporated microemulsions were placed at 4 °C and away from oxygen and light, beta-carotene had the highest retention rate after storage for 1 month. Furthermore, beta-carotene degradation rate in L910 was slower than that in L990, indicating L910 was more effective than L990 in terms of incorporating and protecting beta-carotene. It is shown clearly from the present study that microemulsions could be formed via the water titration and emulsion dilution methods. The type and concentration of oil phase and surfactant had a significant influence on the determination of whether a mixture system could form a microemulsion as well as the properties of the formed microemulsion. The microemulsions produced by these two different methods could be utilised to encapsulate beta-carotene as the incorporation of beta-carotene did not have a significant influence on the properties of the original microemulsions. Moreover, microemulsions provided the stability and protection to beta-carotene against oxidative degradation that could be caused by oxygen, light and temperature during storage, which might be possible to be applied to some liquid foods and beverages.Item Complexation between whey protein and octenyl succinic anhydride modified starch: a novel approach for encapsulation of lipophilic bioactive compounds : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand(Massey University, 2018) Wu, DanProteins and polysaccharides are frequently used in food industry, and their interactions in food systems could affect the properties of food products such as the texture and stability. Therefore, the knowledge of the interactions between these two macromolecules is of great significance for food manufacturers. The aim of this study was to investigate the complexation process between whey protein isolate (WPI) and octenyl succinic anhydride (OSA)-modified starch and explore the application of their interactions in the encapsulation of lipophilic bioactive compounds. The formation of complexes between WPI and OSA-modified starch was investigated as a function of pH (7-3), the heat treatment of WPI, and the concentration ratio of WPI and OSA-modified starch (1:1, 1:10 and 1:20). The complexation process was evaluated by the determinations of the absorbance, particle size and ζ-potential of the mixtures, which were determined by spectrophotometer and dynamic light scattering. It was found that the OSA-modified starch was more likely to interact with heated WPI (HWPI, 90°C for 20 min) rather than non-heated WPI (NWPI). The optimum condition for the formation of insoluble coacervates was at ratio of 1:10 and pH 4.5, which was driven by both electrostatic and hydrophobic interactions. The structure of the complexes formed under the optimum condition could be affected by different molecular characteristics of OSA-modified starch including molecular weight (Mw) and degrees of substitution (DS) value. It was found that OSA-modified starch with higher Mw was difficult to form a dense precipitation phase with HWPI due to its higher viscosity restricting the movement of any particles present. Stable soluble complexes could be formed between HWPI and OSA-modified starch with higher DS value under the same condition, which may be attributed to the stronger steric hindrance of OSA-modified starch with higher DS values. It seems that the complexation between HWPI and OSA-modified starch was induced by electrostatic interactions, while the structural properties of the complexes were determined by hydrophobic interactions. The soluble complexes between HWPI and OSA-modified starch with a DS value of 4.29 ± 0.11% formed at ratio of 1:10 and pH 4.5 were applied to encapsulate β-carotene, which was used as a model of lipophilic bioactive compounds in this study. The apparent aqueous solubility of β-carotene was enormously improved (264.05±72.53 μg/g) after encapsulation in the soluble complexes. No significant differences were observed under transmission electron microscopy (TEM) and scanning electron microscope (SEM) between the soluble complexes before and after encapsulation of β-carotene whether in a liquid or a powdered form. Results of Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC) indicated that the β-carotene was in an amorphous form loaded inside the soluble complexes, which suggested that the molecules of β-carotene evenly distribute within the complex particles by hydrophobic force. In addition, the β-carotene-loaded freeze-dried soluble complexes showed good redispersion behaviour and a high retention rate of the loaded β-carotene (89.75%), which indicated that the β-carotene-loaded soluble complexes could be successfully converted into a powdered form. The accelerated stability study showed that these soluble complexes could effectively protect the loaded β-carotene at pH 4.5 during storage, especially after 7 days of storage. This indicated the potential of using the soluble complexes between HWPI and OSA-modified starch to protect lipophilic bioactive compounds for long-term storage under low pH conditions. This study may be beneficial for the potential using the soluble complexes between HWPI and OSA-modified starch as delivery systems for lipophilic bioactive compounds in commercial applications.Item Particle coating using foams and bubbles : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemical and Bioprocess Engineering at Massey University, Palmerston North, New Zealand(Massey University, 2017) Singh, ShaktiThis thesis investigates powder coating using foams or bubbles. The work initially started on foams. Wettability studies first showed that foams can be used to coat powders. Research then focussed on the fundamental unit of foams, the bubble. An experimental apparatus was designed and built to perform particle-bubble impact studies in air. Bubble solutions comprised of water, hydroxypropyl methylcellulose (HPMC) and sodium dodecyl sulphate (SDS). Four distinct physical behaviours occur when a particle impacts a bubble: (i) particle capture, (ii) particle slide-off, (iii) bubble burst and (iv) bubble self-healing. The rate processes that occur during particle-bubble impact are; (i), surface area creation by bubble film stretching; (ii), delivery of surface active molecules to the newly created surface; and (iii), stress dissipation as the film is stretched. The ability of the solutions to do (ii) and (iii) are highly complex relying on the thermodynamic equilibrium of the solutions and the local perturbations in the near surface region. Therefore, establishing quantitative boundaries of behaviour is a difficult exercise. It is proposed that, for solutions above the cac or cmc, (critical aggregate concentration, critical micelle concentration) where self-healing occurs, the rate of (ii) > rate of (i) and the rate of (iii) > rate of (i). For solutions below the cac, where bursting occurs, the opposite is true, the rate of (ii) < rate of (i) and the rate of (iii) < rate of (i). Intermediate behaviours such as slide-off of capture are within the range of self-healing behaviours, but where the energy of the particle is insufficient to penetrate the bubble. These behaviours are explained by complexation theory. For SDS concentration ≥ cac and cmc, small aggregates of SDS and HPMC locally supply surfactant to the surface of the stretching bubble film. This maintains low surface tension stress and self-healing results. For SDS concentrations < cac, self-healing occurs because the complexation is a HPMC-SDS sea containing SDS islands. The HPMC-SDS sea structure is sufficiently interlinked to simply stretch with the film, while the SDS islands de-aggregate quickly in the near surface region to supply the newly created surface with surfactant. Here the supply rate is faster than the stretching and so the new surface area is populated with SDS molecules. In contrast bursting occurs when the complexation is HPMC-SDS islands in a SDS sea. Here, the rapid film extension is so fast that the islands of HPMC-SDS become isolated and the film loses structural homogeneity. Furthermore, the rate of new surface creation is too fast for diffusion of SDS molecules from the bulk ‘sea’ to the newly created surface. This results in both an inhomogeneous structure and local increases in surface tension, causing both stress concentration in the film and the Marangoni effect. Extensional viscosity measurements, conducted in collaboration with Monash University, Australia, produced three behaviours as solutions were thinned: bead-on-string, blob and long-lived filaments. Solutions which produced long lived filaments here correspond to those that self-healed during particle impact (when the impact velocity was sufficient). It is proposed that this long-lived filament behaviour is due to the SDS concentration being > cmc, where the SDS micelles act like ‘ball-bearings’ between the extending HPMC chains. Coatings were characterised by SEM and gravimetric measurement. Cross-sectional imaging of the soft particle that penetrated self-healing bubbles were found to have a continuous coating layer around the particle. Surface topography of bubble coated particles were compared with classical droplet coated single particles from the literature. Bubble coated particles were found to be smoother than the droplet coated particle. The knowledge gained here was used to suggest how an industrial-scale particle coater using bubbles may be designed.Item Stabilisation of dried Lactobacillus rhamnosus against temperature-related storage stresses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatū, New Zealand(Massey University, 2019) Priour, SarahIn the past few years, research has established a link between gut health and overall health and wellbeing. A diverse microbiome is a major step towards a healthy gut. Probiotics could help by improving the gut microbiome diversity and thus, are being added to a wide range of food products. However, maintaining them in a viable state within these food products is a considerable challenge. In order to increase the shelf-life of probiotics, numerous encapsulation systems have been developed to help protect them. Techniques such as emulsification, coacervation, or drying methods have all been employed with varying levels of success. While the final encapsulated bacteria may have enhanced protection and stability, a range of stresses are imposed on the bacterial cells during the actual encapsulation process, including mechanical, physical and chemical. Drying is the technique that confers the most protection to the probiotics, potentially stabilising them for up to several years. However, water plays a structural role and upon its removal, forces appear between cell components leading to the denaturation of proteins or the phase transition of the phospholipids membrane. Thus, bacterial cells need to be dried in the presence protectants that can prevent detrimental events from occurring and damaging the cells. It is thought that there are three main mechanisms by which protectants will confer superior stability. Firstly, the protective matrix can form a glassy system preventing further chemical reactions from happening, and thus protecting the bacteria. Secondly, if protectants are introduced for a period prior to drying, they can interact with the cellular biomolecules, replacing the structural role of the water, and maintaining the biomolecules in their native state when the water is removed from the system. Finally, the protectants can increase the free energy of water, maintaining it in the vicinity of the biomolecules, so that when the water is removed, the biomolecules are still hydrated and in their native state. Therefore, it is obvious that the role of protectants during the drying step is critical. The question that has remained largely unanswered, however, is how long and under what conditions should the protectants be introduced, and what type of protectants work best? Once the probiotics are successfully dehydrated, storage stresses may impair their stability on the shelf. Among these stresses, high temperatures of the surrounding environment is one that has been well documented to be detrimental to the cells and generally leads to a rapid drop in shelf stability. These temperatures can be experienced not only during the life of the product on the supermarket shelves, but also during transport of these consumables around the globe. The effect of changes in temperature on bacterial cell viability is an area which has not been explored in great depth, and the impact that encapsulation may have on the viability under these conditions even less so. Once again, like in the case of the protectants, the materials used to encapsulate the bacteria will be critical to final stability. Materials such as ‘phase change materials’ (PCM), which can absorb and release heat over different temperature ranges could be the key to protecting bacteria under extreme conditions. The aim of this thesis was thus to stabilise a model probiotic: Lactobacillus rhamnosus HN001 to high temperatures occurring during storage and transport. In order to do so, the study was separated into four principal research questions. Firstly, can a pre-drying step (for example the uptake of protectants) help the stability/viability of the bacteria during storage? Secondly, what are the best protectants for long-term storage of Lb. rhamnosus HN001, and why? Thirdly, is it possible that combinations of the most suitable protectants act in synergy, bringing increased storage stability compared to either protectant on its own? Finally, can the inclusion of PCM in the encapsulation matrix give extra protection to the cells during storage? This question would be of particular significance when examining the effect of the fluctuating temperatures experienced during the transport of the probiotics. The first study, therefore, consisted of establishing a protocol to prepare the cells for drying, by finding the early stationary phase where cells are known to be most stable to stress, and then optimising the exposure of the cells to potentially protective solutions of glucose and sucrose at 4 and 20°C. The uptake of the solutes was explored using HPLC, before drying the cells and evaluating the effect that their uptake had on the shelf-life stability of freeze-dried cells. In order to try and understand any interactions between the intracellular biomolecules and the protectants, the Nano DSC was used. Results showed that when cells were exposed to glucose at 20°C, metabolisation took place, and the longer the exposure, the lower the stability of the cells after drying and over storage. Overall, the study revealed that cells exposed to sucrose at 20°C for 4 hours presented best stability indicating that both the type of protectant, and exposure settings are critical to a successful outcome. The results from the Nano DSC showed that sucrose interacted with some of the cell biomolecules, rendering them more stable. The exposure temperature for the rest of the experiments was thus set at 4°C to avoid metabolisation, and the time was set at one hour so that exposure settings would be adapted for both sugars. In the second part of the study, a range of nine protectants (glucose, fructose, galactose, sucrose, lactose, trehalose, betaine, monosodium glutamate (MSG) and sorbitol) were compared for their ability to stabilise freeze-dried Lb. rhamnosus at 30°C for 6 months. Inulin was used as a carrier. The impact of galactose, sucrose, betaine, MSG and sorbitol was studied using a Nano DSC to again try and establish links between biomolecule interaction and stability during storage. Interestingly, MSG led to the best stability overall with a cell loss of 0.19 /month, even though it had the highest water activity of all the samples following freeze-drying. This is contradictory to general thought on how water activity affects bacterial cell stability, with higher water activity generally resulting in increased cell death over time. It was shown, using the Nano DSC, that MSG interacted with most of the cell biomolecules rendering them more stable. MSG was thus selected for further study. Three additional protectants were selected (galactose, sucrose and sorbitol) to look for potential synergistic effects with MSG in terms of protecting the bacteria during storage. The study followed a mixture design of experiment (DoE) in order to obtain an optimal protective matrix. The powder structure was also studied at this point by microscopy along with analysis using the DSC to try and comprehend the importance of the powder structure on the stability of the dried cells. Multivariate analysis was used to link all factors and their relative impact on the cell death rate together. Interestingly, it was found that neither a high glass transition temperature (Tg) nor a low water activity helped to stabilise the bacteria. Instead, the amount of MSG was clearly shown to improve the shelf-life, and a synergy was found between sorbitol and MSG. Microscopy showed that this powder led to a unique structure that most likely collapsed during drying resulting in the shrinkage of the cake and the loss of the porous structure, thus lowering the exposure of the bacteria to oxygen. In addition, a small amount of the sorbitol present in the matrix seemed to help in stabilising additional biomolecules as shown by the Nano DSC. The slowest death rate results obtained were 0.04 /month when MSG alone was mixed with inulin, but the model predicted an even lower death rate due to the synergy occurring between MSG and sorbitol. Finally, this optimised stabilisation matrix was used to study the impact of further protection, in the form of an encapsulate containing a PCM, on the stability of the bacteria. Powders with two different structures were compared using freeze-drying and spray drying techniques. The viability of the resulting powders was assessed during two separate storage studies designed to test the cells against fluctuating temperatures (20 to 50°C) and at constant temperature (35°C). The results showed that PCM appeared to have little impact on the overall stability of the powder. However, it was confirmed that a dense and smooth powder structure helped to maintain the bacteria in a viable state for a longer time than a more porous structure. This was most likely due to the lower surface-area ratio decreasing the exposure with the environment and preventing detrimental reaction such as oxidation. The bacteria in the optimised stabilisation matrix had the best stability, with a death rate of 0.07 /month at 35°C and 0.18 /month under fluctuating temperature from 20 to 50°C. In conclusion, it was found that the interaction of the protectants with cells is of paramount importance in maintaining the cells in a dried, viable state for longer periods at elevated temperatures. In addition, the structure of the powder should also be considered as one of the main mechanisms for protecting the bacteria, as it has a substantial impact on the shelf-life of the powder. Conversely, in this body of work it was shown that a high glass temperature did not enhance, or indeed help to maintain cell viability as has been suggested by many previous studies. A dense structure is, however, believed to protect the bacteria through preventing exchanges with the environment, especially with oxygen. If future work is to be done, it should follow the oxidation of the cells during storage and link it with measures of the powder porosity to gain further insight into the impact of the structure on oxidation stress.Item Design and characterisation of polyhydroxyalkanoate bead-alginate hybrid materials : a dissertation presented in partial fulfilment of the requirements of the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand(Massey University, 2018) Ogura, KampachiroEncapsulation is a technique to entrap a material of interest within a carrier material, and it has been used in a wide range of industries such as biopharmaceutical and biotechnological companies. Among biopolymers used for the carrier material, alginate has been widely used due to its attractive properties of biodegradability, biocompatibility and ease of gelation under mild conditions. In this study, a novel hybrid micro- or millisphere composed of functionalised polyhydroxyalkanoate (PHA) beads and alginate was fabricated through ionotropic gelation methods. The selected functional proteins displayed on PHA beads were the IgG binding domain (ZZ) of Protein A from Staphylococcus aureus, and organophosphate hydrolase (OpdA). The effect of alginate encapsulation on their functionality was assessed. In addition, alginate millispheres encapsulating PHA beads were assessed for their ability to remove lipophilic compounds from an aqueous solution. This utilised the hydrophobic polyester core of the PHA beads and was assessed by using the lipophilic dye, Nile red, as the reporter. The results of IgG binding assays using PHA beads encapsulated in alginate microspheres showed no significant difference compared with negative controls, and no elution of bound IgG was observed. However, the alginate encapsulation enhanced IgG binding to the PhaC derived proteins on PHA beads within alginate microspheres. In contrast, alginate encapsulation limited the activity of PHA beads displaying OpdA when compared to free PHA beads. The qualitative data obtained from phase contrast and fluorescence microscopy suggested that the enzyme displayed on PHA beads was active within alginate microspheres. The lipophilic dye removal by PHA beads encapsulated in alginate millispheres was influenced by different parameters used in the millisphere preparation. The adsorption kinetics aligned with a pseudo second-order kinetic model, and the equilibrium adsorption data was in agreement with the Freundlich isotherm model. This study has provided preliminary data for fabrication of a hybrid material of functionalised PHA beads with alginate. The development of combinations of highly functional PHA beads with other biomaterials is expected to expand the application field of PHA beads.Item Design and development of a modified spouted bed coater for the micro-encapsulation of powders : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Chemical Technology, Massey University(Massey University, 2003) Bishop, Peter AndrewA modified spouted bed coater was designed and constructed for the micro-encapsulation of solid particles. The coating of small particles with a polymer film can alter physical factors such as taste and release rate. These properties are particularly important in the field of pharmacology as the nature of the coating can be changed to prolong or target drug release based on physiological conditions such as pH and time. The spouted bed coater was modified to induce gas and particle recirculation through a draft tube containing a venturi to increase droplet and particle mixing, while a high velocity gas jet and large diameter draft tube promotes the recirculation of gas and solid within the apparatus. The effectiveness of the design was tested in terms of gas and solid mass flows through the draft tube using a venturi within the draft tube and an induction detector to measure the mass flow. To determine the effectiveness of the coater design in terms of coalescence and the influence of operational variables, a factorial experiment was conducted. The result of this experiment showed that the coalescence of particles was dominated by the relative humidity in the apparatus which was unable to be directly related to the operational variables. The capacity to micro-encapsulate particles was demonstrated by coating fine table salt with an acrylic polymer Eudragit NE 40D in combination with bentonite clay as a free flow agent or glident. The results of this trial showed the distribution of polymer/clay and the reduction in dissolution rate as a function of particle size.Item Development of a microencapsulation technique for probiotic bacteria Lactobacillus casei 431 using a protein-polysaccharide complex : a thesis presented in partial fulfillment of the requirements of the degree of Masters of Technology in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2011) Nag, ArupAccording to the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), probiotics are defined as ‘‘live microorganisms which when administered in adequate amounts confer a health benefit for the host’’ (FAO/WHO, 2001). Lactobacilli and Bifidobacteria are two major group of organisms considered to have probiotic properties. Probiotic bacteria are accepted universally for conferring beneficial effects to human gut health. However, the successful delivery of these bacteria to the human intestine via a proper food matrix is challenging because the stresses encountered by the probiotics during processing, storage and gastric transition cause major loss of viability. The primary objective of this study was to develop a novel protection system using a complexation product of dairy protein and a bacterial exo-polysaccharide which should be able to protect the probiotic bacteria during their gastric transit and also release them under suitable conditions in the intestine. Lactobacillus casei 431, a commercial strain from Chr Hansen, Denmark, was chosen as the experimental strain and the protein-polysaccharide complex was made up of sodium caseinate and gellan gum. Gelation of the sodium caseinate and gellan gum mixture was achieved by a gradual decrease of pH with slow hydrolysis of glucono-delta-lactone (GDL) and Lactobacillus casei 431 cells were successfully entrapped into this gel matrix. An intermediate step of forming a water-in-oil emulsion was involved in this process for producing micron level gel particles. The appropriate combination of ingredients, based on final elastic modulus to attain adequate gel strength, was finally decided as 10% (w/w) sodium caseinate, 0.25% (w/w) gellan gum and 2.5% (w/w) GDL. This combination resulted in a very fine and uniform capsule size distribution and up to 89% encapsulation efficiency was achieved. The gelled microcapsules were freeze dried to obtain better shelf stability and easy handling properties. The particles obtained had diameters ranging from 40 to 1100 μm for wet and 46 to 631 μm for freeze dried microcapsules. The mean diameters (D32) of wet and freeze dried microcapsules were found as 287 and 152 μm, respectively. II Scanning electron microscopic examination of the freeze dried particles showed irregular surfaces and the presence of numorous pores. Tolerance of free and encapsulated bacterial cells in simulated gastric juice at pH 2.0 was tested in an in vitro model and the results showed better survivability of encapsulated cells in both wet and dry microcapsules as compared to the free cells. The log CFU reduction figures after a 2 hour incubation period, were 4.56 for free cells, 3.03 for cells inside wet capsules and 2.28 for cells protected inside freeze dried particles. Incubation of free and encapsulated cells in the presence of 1% (w/v) bile extract for 8 hours showed 2.51 log CFU/gm reductions for free cells with almost no detrimental effect on wet microencapsulated cells and 2.44 log CFU reductions for freeze dried cells. Further research work was undertaken to improve the post freeze drying survivability of the L. casei 431 cells by including cryoprotective solutes in both the culture growth and the drying media. Trehalose and lactose were chosen as cryoprotecting agents. Compared to an average 1.70 log CFU reduction in case of control (no cryoprotectant) samples, trehalose and lactose containing samples both showed a much better survival rate; only 0.84 and 0.37 log CFU/gm reduction respectively, in cell population, were recorded. A membrane coating over the produced microcapsules was applied and the properties of such coated samples were checked separately. The coating process aided in the post drying survivability and only 0.53 and 0.13 log CFU/gm reductions were recorded for trehalose and lactose supplemented samples, respectively. The presence of cryoprotecting compounds proved to be beneficial against the simulated gastric environment and the membrane coating gave additional improvement in this regard. During the gastric fluid incubation tests, cryoprotected samples (freeze dried) containing trehalose and lactose shown a higher survival of 3.13 log CFU/gm and 2.04 log CFU/gm respectively, compared to cells in free form. Improvements offered by the membrane coating were recorded as an additional 0.23 log CFU/gm and 0.66 log CFU/gm higher survival for trehalose and lactose respectively. The same trend was observed for bile salt tolerance also. Cryoprotected samples (freeze dried) containing trehalose and lactose showed a higher survival of 0.41 log CFU/gm and 0.84 log CFU/gm respectively, compared to cells in free form. Additionally, the membrane coating process contributed III towards further improvement in viability of 0.25 log CFU/gm and 0.26 log CFU/gm for trehalose and lactose respectively. Overall, lactose has been found to be a marginally better protectant of cells than trehalose against freeze drying, acid and bile salt stresses. The membrane coating process helped in forming a very smooth surface morphology devoid of any visible pores. Perhaps the presence of a membrane coating was responsible for this better protective nature of coated microcapsules. But as a drawback, this coating process resulted into higher particle mean diameters, both for wet and freeze dried beads. Storage of freeze dried samples at 37°C proved to be more detrimental to the entrapped cells than at 4°C. But the results obtained were better compared to the situation where no protective compounds were used. It was found that lactose and trehalose helped in maintaining high levels of viable cell populations during the storage period but the cell degradation rate was positively correlated with the storage temperature. Therefore, it can be concluded that a low pH sodium caseinate-gellan gum gel matrix can offer adaptation and protection to the probiotic cells before encountering a high acid stomach environment and therefore can be utilized as an effective microencapsulation technique. The survibility of the L. casei 431 cells could be further improved during freeze drying as well as gastrointestinal transit by incorporation of protectants, viz., lactose or trehalose and applying a membrane coating of gellan gum. High acid food preparations such as, yogurt and fruit juice could be the probable applications for the current findings.