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Item Metabolism of Caprine Milk Carbohydrates by Probiotic Bacteria and Caco-2:HT29⁻MTX Epithelial Co-Cultures and Their Impact on Intestinal Barrier Integrity(MDPI (Basel, Switzerland), 2018-07-23) Barnett AM; Roy NC; Cookson AL; McNabb WCThe development and maturation of the neonatal intestine is generally influenced by diet and commensal bacteria, the composition of which, in turn, can be influenced by the diet. Colonisation of the neonatal intestine by probiotic Lactobacillus strains can strengthen, preserve, and improve barrier integrity, and adherence of probiotics to the intestinal epithelium can be influenced by the available carbon sources. The goal of the present study was to examine the role of probiotic lactobacilli strains alone or together with a carbohydrate fraction (CF) from caprine milk on barrier integrity of a co-culture model of the small intestinal epithelium. Barrier integrity (as measured by trans epithelial electrical resistance (TEER)), was enhanced by three bacteria/CF combinations (Lactobacillus rhamnosus HN001, L. plantarum 299v, and L. casei Shirota) to a greater extent than CF or bacteria alone. Levels of occludin mRNA were increased for all treatments compared to untreated co-cultures, and L. plantarum 299v in combination with CF had increased mRNA levels of MUC4, MUC2 and MUC5AC mucins and MUC4 protein abundance. These results indicate that three out of the four probiotic bacteria tested, in combination with CF, were able to elicit a greater increase in barrier integrity of a co-culture model of the small intestinal epithelium compared to that for either component alone. This study provides additional insight into the individual or combined roles of microbe⁻diet interactions in the small intestine and their beneficial contribution to the intestinal barrier.Item Vital members in the gut microbiotas altered by two probiotic Bifidobacterium strains against liver damage in rats(BMC, 2020-06-05) Zha H; Fang D-Q; van der Reis A; Chang K; Yang L-Y; Xie J-J; Shi D; Xu Q-M; Li Y-T; Li L-JBACKGROUND: Probiotics are effective to rectify the imbalanced gut microbiota in the diseased cohorts. Two Bifidobacterium strains (LI09 and LI10) were found to alleviate D-galactosamine-induced liver damage (LD) in rats in our previous work. A series of bioinformatic and statistical analyses were performed to determine the vital bacteria in the gut microbiotas altered by the LI09 or LI10 in rats. RESULTS: Two groups of representative phylotypes could distinguish the gut microbiotas of LI09 or LI10 groups from the other groups. Among them, OTU170_Porphyromonadaceae acted as a gatekeeper in LI09 group, while OTU12_Bacteroides was determined with multiple correlations in the gut network of LI10 group. Multiple reduced OTUs associated with LC and increased OTUs associated with health were determined in LI09 or LI10 groups, among which, increased OTU51_Barnesiella and reduced OTU99_Barnesiella could be associated with the protective effects of both the two probiotics. The gut microbiotas in LI09, LI10 and positive control groups were clustered into three clusters, i.e., Cluster_1_Microbiota, Cluster_2_Microbiota and Cluster_3_Microbiota, by Partition Around Medoids clustering analysis. Cluster_2_Microbiota was determined at least dysbiotic status due to its greatest LD dysbiosis ratio, lowest levels of liver function variables and plasma cytokines compared with the two other clustered microbiotas, suggesting the treated rats in Cluster_2 were at better health status. CONCLUSION: Our findings suggest that OTU170_Porphyromonadaceae and OTU12_Bacteroides are vital in the gut microbiotas altered by LI09 and LI10. Characteristics of the LD cohorts treated by LI09 or LI10 at different gut microbial colonization states could help monitor the cohorts' health status.Item Streptococcus salivarius inhibits immune activation by periodontal disease pathogens(BioMed Central Ltd, 2021-05-07) MacDonald KW; Chanyi RM; Macklaim JM; Cadieux PA; Reid G; Burton JPBACKGROUND: Periodontal disease represents a major health concern. The administration of beneficial microbes has been increasing in popularity over efforts to manipulate the microbes using antimicrobial agents. This study determined the ability of Streptococcus salivarius to inhibit IL-6 and IL-8 production by gingival fibroblasts when activated by periodontal pathogens and their effect on the salivary microbiome. METHODS: Primary human gingival fibroblasts were challenged with Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum and a combination of all three. IL-6 and IL-8 cytokine release were measured. Using this same model, S. salivarius K12, M18 and different supernatant and whole-cell lysate fractions of S. salivarius K12 were administered to pathogen-induced fibroblasts. A patient study of healthy participants was also conducted to determine the effect S. salivarius K12 had on the native microbiome using 16S next generation sequence analysis. RESULTS: All pathogens tested induced a significant IL-6 and IL-8 response. S. salivarius K12 or M18, did not exhibit an increase in inflammatory cytokines. When either of the probiotic strains were co-administered with a pathogen, there were significant reductions in both IL-6 and IL-8 release. This effect was also observed when gingival fibroblasts were pre-treated with either S. salivarius K12 or M18 and then stimulated with the oral pathogens. Chewing gum containing S. salivarius K12 did not alter the salivary microbiome and did not increase inflammatory markers in the oral cavity. CONCLUSION: S. salivarius K12 and M18 prevented immune activation induced by periodontal disease pathogens. S. salivarius K12 did not alter the salivary microbiome or induce immune activation when administered as a chewing gum. These results warrant further study to determine if it may be an effective treatment in a model of periodontal disease.Item Time-to-conception and clinical pregnancy rate with a myo-inositol, probiotics, and micronutrient supplement: secondary outcomes of the NiPPeR randomized trial.(Elsevier B.V., 2023-05-26) Chan S-Y; Barton SJ; Loy SL; Chang HF; Titcombe P; Wong J-T; Ebreo M; Ong J; Tan KM; Nield H; El-Heis S; Kenealy T; Chong Y-S; Baker PN; Cutfield WS; Godfrey KM; NiPPeR Study GroupObjective To determine whether a combined myo-inositol, probiotics and micronutrient nutritional supplement impacts time-to-natural-conception and clinical pregnancy rates. Design Secondary outcomes of a double-blind randomized controlled trial. Setting Community recruitment. Patients Women aged 18 to 38 years planning to conceive in the United Kingdom, Singapore, and New Zealand, excluding those with diabetes mellitus or receiving fertility treatment. Intervention A standard (control) supplement (folic acid, iron, calcium, iodine, β-carotene), compared with an intervention additionally containing myo-inositol, probiotics, and other micronutrients (vitamins B2, B6, B12, D, zinc). Main Outcome Measures Number of days between randomization and estimated date of natural conception of a clinical pregnancy, as well as cumulative pregnancy rates at 3, 6, and 12 months. Results Of 1729 women randomized, 1437 (83%; intervention, n=736; control, n=701) provided data. Kaplan-Meier curves of conception were similar between intervention and control groups; the time at which 20% achieved natural conception was 90.5 days (95% confidence interval: 80.7, 103.5) in the intervention group compared with 92.0 days (76.0, 105.1) in the control group. Cox's proportional hazard ratios (HRs) comparing intervention against control for cumulative achievement of pregnancy (adjusted for site, ethnicity, age, body mass index, and gravidity) were similar at 3, 6, and 12 months. Among both study groups combined, overall time-to-conception lengthened with higher preconception body mass index, and was longer in non-White than in White women. Among women who were overweight the intervention shortened time-to-conception compared with control regardless of ethnicity (12-month HR=1.47 [1.07, 2.02], P=.016; 20% conceived by 84.5 vs. 117.0 days) and improved it to that comparable to nonoverweight/nonobese women (20% conceived by 82.1 days). In contrast, among women with obesity, time-to-conception was lengthened with intervention compared with control (12-month HR=0.69 [0.47, 1.00]; P=.053; 20% conceived by 132.7 vs. 108.5 days); an effect predominantly observed in non-White women with obesity. Conclusions Time-to-natural-conception and clinical pregnancy rates within a year were overall similar in women receiving the intervention supplement compared with control. Overweight women had a longer time-to-conception but there was suggestion that the supplement may shorten their time-to-conception to that comparable to the nonoverweight/nonobese women. Further studies are required to confirm this. Clinical Trial Registration Number clinicaltrials.gov (NCT02509988)Item 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, 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. 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.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 Pre- and pro-biotics may improve mineral absorption and retention in the growing male rat : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Nutritional Science at Massey University, Turitea, Palmerston North, New Zealand(Massey University, 2005) Fear, Alison LindsayProbiotics are bacteria, which reside in the large intestine and concur beneficial health effects on their host. Their abundance can be selectively-stimulated by prebiotics, such as fructo-oligosaccharide (FOS); prebiotics are oligosaccharides, which are not digested in the small intestine, but pass into the large intestine where they are fermented into short-chain fatty acids. Several studies have suggested that prebiotics may improve mineral absorption. This study aimed to determine the effects of pro- and pre-biotic supplementation on mineral absorption and bone quality in growing male rats. Sixty three-week old male Sprague-Dawley rats were randomised into five groups and fed either a high-calcium milk powder (HCMP) with or without a probiotic added (groups were subsequently named HCMP - and HCMP + respectively), or HCMP and vitamin K with or without the probiotic (HCMPK - and HCMPK +), or the HCMP with FOS replacing the sucrose in other diets, and the probiotic (the dietary group was named FOS). Animals were maintained on diets for 10 weeks. Balance studies were carried out during weeks 3 - 4 and 8 - 9 of the study. The earlier balance study suggested that dietary interventions may affect mineral absorption. The latter balance study, however, showed no discernable differences between groups. Several reasons were postulated for this. Active-absorption may have been down-regulated as a result of long-term supplementation, or an increased abundance of probiotics could cause an elevation of nutritional demands. Alternatively, supplementation may not prove beneficial once animals had passed their period of peak absorption. Bone resorption and formation did not appear to have been altered as a result of dietary intervention, when measured after 10-weeks. Bone mineral density and content, calcium, magnesium, zinc and ash contents and bone biomechanical testing also showed no significant differences between dietary groups. Further research is required to determine whether results obtained were due to long-term supplementation and / or due to the joint-supplementation of pre- and pro-biotics.Item Black tea water kefir beverage : a thesis submitted in partial fulfilment of the requirement for the degree of Master of Food Technology, Massey University, Albany, New Zealand(Massey University, 2017) Subardjo, Maria Vina KaniaFermented foods and beverages play an important role in the human diet as they provide essential nutrients as well as contribute towards prevention of diseases. Lactic acid bacteria and yeasts are a major group of microorganisms associated with fermented products. Some of the microorganisms, known as probiotics, confer health properties to human health. Thus, many different types of fermented foods and beverages containing probiotics are produced around the world to support wellness and health. In recent years, there has been increased interest in the development of fermented functional plant-based foods and beverages due to a surge in scientific research of the products. Further, there is evidence that probiotic microorganisms can grow well in plant-based substrates. Water kefir is a sparkling fermented beverage with an acidic, sweet, slightly alcoholic taste, and a yeasty flavour. Water kefir fermentation can be achieved by the inoculation of water kefir grains as a starter culture into a solution containing sugar. Kefir grains consist of a symbiotic starter culture of lactic acid bacteria (LAB) and yeasts contained in a polysaccharide matrix. Microorganisms present in kefir grains are recognized as probiotics. The majority of previous studies have focused on the isolation and identification of water kefir cultures responsible for fermentation. There is, therefore, scanty information on the fermentation of plant-based water kefir beverages. The main objective of this study was to develop fermented black tea beverage using water kefir grains as a starter culture. Fermentation of black tea infusions as single and mixed substrate with carrot juice using water kefir grains were investigated. Microflora of water kefir grains used consisted of symbiotic starter culture of lactic acid bacteria (Lactococcus spp. and Lactobacillus spp.) and a yeast (Saccharomyces cerevisiae). The study was conducted in three main phases. The first phase investigated the effect of sucrose concentration (5% and 10%) and fermentation temperature (25°C and 30°C) in black tea water kefir fermentation for 72 h. Meanwhile, the effect of added carrot juice (5%, 10%, and 15%) on kefir beverage during secondary fermentation (24 h) at 25°C was investigated in the second phase. The stability of the final black tea water kefir beverage formulation during storage (4°C) for four weeks was investigated in phase three. Samples of black tea water kefir beverages were subjected to various analyses during fermentation and storage (4°C) for 4 weeks: titratable acidity, total soluble solids (°Brix), colour, viable cell counts of constituent starter culture, sensory evaluation, sugars, organic acids, antioxidants, and pH was also measured. Results showed that fermentation temperature, sugar concentration, and carrot juice concentration contributed to the physico-chemical and microbiological characteristic as well as sensory properties of the product. In phases one and two, pH and total soluble solids (°Brix) decreased, while titratable acidity and cell counts of LAB and yeasts increased during fermentation of the products. LAB and yeasts were able to grow in black tea and addition of carrot juice into the beverages slightly increased their growth. The best fermentation conditions based on physico-chemical and sensory properties were kefir beverage containing sugar (10%) and carrot juice (10%) fermented at 25°C for 96 h. In phase three, the growth and survival of Lactococcus spp. and Lactobacillus spp. were low during storage of the product (4°C) while Saccharomyces cerevisiae maintained high cell numbers (7.03±0.07 log cfu/ml) at the end of storage (28 days). Results showed the possibility to produce low sugar water kefir beverage containing 0.08±0.01% (w/v) sucrose, 1.55±0.04% (w/v) glucose, and 2.93±0.20% (w/v) fructose. The fermented kefir beverage also contained 0.202±0.02% (w/v) lactic acid, 0.114±0.03% (w/v) acetic acid and some antioxidants (gallic acid, ECG, EGC, EGCG, theobromine and caffeine) which may be beneficial to human health. There was significant difference (p<0.05) in the colour (L*, a*, b*) of the fermented beverages during storage (4°C). Black tea water kefir beverage containing 10% sugar and 10% carrot juice fermented at 25°C for 96 h was well-liked by consumer sensory panellists.Item Stability of the probiotic Lactobacillus paracasei CRL 431 under different environmental conditions : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatu, New Zealand(Massey University, 2015) Poddar, DevastotraProbiotics are live microorganisms which provide health benefits to the host upon consumption. There is a wealth of information available on the health benefits associated with the consumption of probiotics. However, currently probiotic microorganisms are delivered mainly through refrigerated, short shelf-life products. When incorporated into ambient shelf-life products, the products generally fail to meet the regulatory criteria, which require probiotic bacteria to be viable in high numbers at the end of shelf-life. Storage temperature, oxygen and residual moisture content often result in loss of viability of probiotics during storage and distribution. A preliminary study was carried out to explore the effects of matrix composition (fat, protein and carbohydrate) on the probiotic bacterial (Lactobacillus paracasei CRL 431) viability, during fluidized bed drying and subsequent storage. The finding suggests that whole milk powder provided a superior protection to bacteria during fluidized bed drying and subsequent storage, compared to skim milk powder or milk protein isolate. Moreover, water activity of the powders during storage played a key role in determining the probiotic viability. The effects of drying techniques, moisture content and water activity on the storage stability of L. paracasei in a whole milk matrix were studied. Whole milk powder-bacteria mixtures were dried using spray drying, freeze drying or fluidized bed drying and stored at 25 ºC under controlled water activity ( 0.11 aw, 0.33 aw and 0.52 aw) for 105 days. At 0.11 aw, cell viability loss was minimal, while at 0.52 aw viability was lost in all powders within 22 days. At the intermediate 0.33 aw, there were marked differences among stored powders. Further, various analytical techniques (X-ray diffraction, FT-IR, Raman, NMR spectroscopy) were used to explore why and how structural differences in the matrix-bacteria mixtures, produced using different drying technologies, under different water activity storage conditions, influence bacterial viability. The results suggest that fluidized bed drying provided a better protection to the bacteria during storage, which was attributed to unique powder structure that reduced the absorption of water. The lower absorption of water resulted in the maintenance of a more rigid structure, which limited molecular mobility. Lactobacillus sp. is known to accumulate large amounts of inorganic manganese which apparently provides defense against oxidative damage by scavenging free radicals. The ability of L. paracasei to maintain viability during long term ambient storage may be enhanced by the ability of microorganism to accumulate manganese, which may act as free radical scavenger. To investigate this hypothesis, X-ray fluorescent microscopy (XFM) was employed to determine the changes in the elemental composition of L. paracasei during growth in MRS medium with or without manganese as a function of physiological growth state (early log vs. stationary phase). The results revealed that lower level of manganese accumulation occurred during the early log phase of bacterial growth compared with the stationary phase cells. The lower level of manganese accumulation was found to be related to the loss in bacterial viability during storage. Manganese has been known to possess pro- and anti-oxidant properties, and understanding of the changes in the manganese oxidation state was considered to provide some further insights into the bacterial death mechanisms. In view of the relatively high concentration of manganese in lactobacilli, it was of interest to better understand the oxidation state, coordination number and ligands of the manganese in the bacteria. It was possible to characterize the changes of manganese within bacteria using XANES. The results confirmed that manganese present within L. paracasei is in Mn(II) oxidation state and no changes in the manganese ligands could be observed during storage. In summary, the thesis provides a mechanistic insight into the ways to improve the stability of probiotics for application into ambient long shelf-life products. Future studies on tracking the genetic and proteomic aspects of the bacteria during storage might be useful for further understanding and process optimization.Item Identification and functional characterisation of a novel surface protein complex of Lactobacillus rhamnosus : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu Campus, New Zealand(Massey University, 2016) Wen, Wesley XingliProteins are the most diverse structures on bacterial surfaces; hence they are candidates for species- and strain-specific interactions of bacteria with the host, environment and other microorganisms. In probiotic bacteria, some surface and secreted proteins mediate interactions with the host and may consequently contribute to the health-promoting effects. However, a limited fraction of surface-associated proteins from probiotic bacteria have been functionally characterised to date. A secreted protein of Lactobacillus rhamnosus HN001, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like), was previously shown to bind to HN001 cells, however the nature of its ligand on the surface of the cells was unknown. In this study, a series of binding assays first demonstrated that SpcA binds to a cell wall anchored protein of HN001. Next, the SpcA-“docking” protein, named SpcB, was identified using phage display. SpcB is a 3275-residue cell-surface protein that has all the features of large glycosylated serine-rich adhesins/fibrils from Gram-positive bacteria, including the hallmark glycoprotein signal sequence motif KxYKxGKxW and the cell wall anchor motif LPxTG. The spcA and spcB genes are located in a gene cluster, spcBCDA, which is present in 94 out of 100 strains of L. rhamnosus species and some strains of L. casei and L. paracasei whose genome sequences have been determined, but was absent from other Lactobacillus clades. To confirm the role of SpcB as the SpcA anchor and investigate the roles of these two proteins in surface properties of probiotic L. rhamnosus strains HN001 and GG, stable double-crossover mutations of these two genes were constructed. Binding assays to L. rhamnosus mutant cells confirmed dependence on SpcB in both GG and HN001 strains. Comparison of the wild-type and mutant surface properties suggested that SpcB in GG interferes with biofilm formation and aggregation, while it might contribute to the protective effect against TNFa-mediated disruption of the polarised Caco-2 cell monolayer integrity. Deletion of HN001 spcB or spcA had no effect on functions other than the SpcA binding. Our findings indicate that the roles of a surface protein can vary considerably among the strains of a species, requiring functional data to validate the bioinformatics-based hypotheses.