Browsing by Author "Nag, Arup"

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    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, Arup
    According 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.
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    Studies on the stability of probiotic bacteria during long term storage : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Nag, Arup
    According 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. The primary objective of this project was to develop a novel stabilization technology for probiotic bacteria, through which a range of probiotic bacterial strains could potentially be delivered to the host through shelf stable dry and intermediate moisture foods. For preliminary experiments (reported in Chapter 4.0), Lactobacillus casei 431, a commercial strain from Chr Hansen, Denmark, was chosen as the experimental strain and milk powders (both skimmed and full-fat) were chosen as the principal supporting agent while stabilizing the bacterial cells. Stabilization efficiency in terms of long term ambient temperature storage viability was compared using freeze and fluidized bed drying techniques. Fluidized bed drying was able to retain 2.5 log cfu/g higher viability after 52 weeks of storage at 25 °C. A combination of fluidized bed drying and osmotic stress adaptation to the probiotic cells yielded further improvement of 0.83 log cfu/g higher viability compared to the unstressed cells. The findings were validated with other two lactobacilli and two bifidobacterium strains with probiotic characteristics and significant improvements in storage stability over freeze-dried samples were observed. Fortification of vitamin E in the stabilization matrix as an antioxidant improved the stability by 0.18 log cfu/g during 20 weeks storage period at 25 °C, whereas any similar benefit of fortifying inulin as a prebiotic was not observed. Incubation in simulated gastric fluid and intestinal fluid (in vitro) revealed that the L. casei 431 cells were better protected within the stabilized matrix than in the free form. The survival of the stabilized cells were 5.0 and 2.1 log cycles higher than free cells in gastric juice and bile salt solution respectively. Physical characterization of the probiotic ingredient showed very good flow-ability and solubility, with 470 Kg/m3 bulk density, water activity of 0.27 and agglomerated particles of 125.6 μm mean diameter. Thereafter, the project aimed to understand the underlying mechanism of the processes responsible for gradual decay in cell viability of another probiotic strain (Lactobacillus reuteri LR6) during long term storage at 37 °C (Chapter 5.0 onwards). Vacuum drying of sorbitol- or xylitol-coated Lactobacillus reuteri LR6 cells and fluidized bed drying of the same coated cells with different excipients were compared for the cell viability post drying. LR6 cells coated with xylitol and desiccated in unsupported form or together with skim milk powder as an excipient were found to be better protected when exposed to moderate as well as high drying temperatures. In Chapter 6.0, a closer examination of the protein and polypeptide components of the cell envelopes (amide regions) via Fourier transform infrared spectroscopy revealed different degrees of structural deformation in individual samples, which correlated well with the residual cell viability. It was also important to understand the underlying mechanisms responsible for the loss of viability of stabilized probiotic cells when stored at non-refrigerated temperatures. In Chapter 7.0, the stabilized Lactobacillus reuteri LR6 cells were stored at 37 °C and at two water activity (aw) levels. Superior storage stability was recorded in a lower aw environment, supported by a stronger glassy matrix when skim milk powder was used as the excipient. Fourier transform infrared spectroscopic examination of the cell envelopes revealed substantial dissimilarities between samples at the beginning and at the end of the storage period. In milk powder-based matrices, adjusting the aw to 0.30 resulted in a weaker or no glassy state whereas the same matrices had a high glass transition temperature at aw 0.11. This strong glassy matrix and low aw combination was found to enhance the bacterial stability at the storage temperature of 37 °C. During storage of the stabilized cells for 121 days at 37 °C, the measured Tg for all the samples was slightly lower than what was recorded at the beginning. Scanning electron microscopy revealed the formation of corrugated surfaces and blister-type deformations on the cell envelopes during the stabilization process whereas the freshly harvested cells were found to be with a smooth surface and undamaged membrane. Inspection of the cell bodies via transmission electron microscopy showed freshly harvested cells with normal shapes with no damage in the inner membrane structure. An almost intact but slightly waved outer membrane structure was observed. The findings emphasize the importance of protecting the integrity of the membrane of probiotic cells by using suitable protecting agents to enhance their stability during long term storage. The stabilized cell matrix samples were segregated into 4 groups based on the average particle diameter by passing through sieves of different mesh sizes. The degree of agglomeration had a very important role in offering physical protections to the LR6 cells during the desiccation process. The viable cell populations in the higher particle size groups (above 500μm and 1000μm) were between 9.5 to 9.9 log cfu/g whereas the same for the lower particle size (below 500μm but above 250μm) group was only 7.8 log cfu/g. The minimum viable cell concentration was recorded (7.3 log cfu/g) in the finer particles having less than 250μm diameter but having the maximum mass fraction. In case of stored samples, it was found that the bacterial cells adhered to the finest particles suffered the maximum loss in viability (41.4%) whereas the minimum loss (14.9%) was within the particles with average diameter above 500μm. In order to assess the effect of stabilization and storage (12 weeks, 37 °C) on the common probiotic attributes of the LR6 cells, an in vitro study on acid, bile salts tolerance and surface hydrophobicity was conducted. The results showed considerable reductions in cell viability for the desiccated as well as stored cells when incubated in simulated gastric (acid tolerance) and intestinal (bile salts tolerance) environments. A coating of xylitol over the cell bodies during desiccation was found to be marginally protective against these stresses. High aw storage was found to be more detrimental to the cells in terms of their ability to survive in the acid or bile environments. The cell surface hydrophobicity towards various hydrocarbons was also found to be adversely affected due to desiccation and non-refrigerated storage. Considerable degradation in hydrophobicity was found to be occurring in the cells stored at aw 0.30, a trend similar to the acid and bile resistance properties.