Enzyme-assisted extraction of bioactives from plants

Demand for new and novel natural compounds has intensified the development of plant-derived compounds known as bioactives that either promote health or are toxic when ingested. Enhanced release of these bioactives from plant cells by cell disruption and extraction through the cell wall can be optimized using enzyme preparations either alone or in mixtures. However, the biotechnological application of enzymes is not currently exploited to its maximum potential within the food industry. Here, we discuss the use of environmentally friendly enzyme-assisted extraction of bioactive compounds from plant sources, particularly for food and nutraceutical purposes. In particular, we discuss an enzyme-assisted extraction of stevioside from Stevia rebaudiana, as an example of a process of potential value to the food industry.

The determination of bioactives using multidimensional separation and detection systems

This project was focused on the development of platform technology for the separation and detection of complex samples. The samples investigated included roasted coffee beans, opiate alkaloids and amino acids.

Plant bioactive determinations using flow analysis, chromatography with chemiluminescence detection

The development of rapid analytical methodologies for the determination of plant bioactives including antioxidants, amino acids and opium alkaloids was investigated using novel flow analysis and liquid chromatographic techniques. These methodologies were applied to the wine, food and pharmaceutical industries.

Setting the foundation for molecular nuts and bolts

The thesis outlines effective methods of designing and constructing chiral nano-porous materials. These materials may facilitate cheaper and large-scale production of chiral molecules, such as pharmaceuticals and bioactives. Computer models of target materials correlated well with laboratory experiments and effective synthetic strategies for manufacture were developed.

Identification and functional characterization of a novel monotreme - specific antibacterial protein expressed during lactation

Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother’s body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.

Transcriptomic characterisation of pigeon ‘milk’ production and its effect on young

Pigeon milk is fed to young pigeons by both their parents. This thesis described the genetic basis of pigeon milk production in the crop and also its effect on young chickens. This revealed that pigeon milk has functional similarities to mammalian milk, and bioactives with future potential in poultry production were identified.

Omega-3 fatty acid production from enzyme saccharified hemp hydrolysate using a novel marine thraustochytrid strain

In this work, a newly isolated marine thraustochytrid strain, Schizochytrium sp. DT3, was used for omega-3 fatty acid production by growing on lignocellulose biomass obtained from local hemp hurd (Cannabis sativa) biomass. Prior to enzymatic hydrolysis, hemp was pretreated with sodium hydroxide to open the biomass structure for the production of sugar hydrolysate. The thraustochytrid strain was able to grow on the sugar hydrolysate and accumulated polyunsaturated fatty acids (PUFAs). At the lowest carbon concentration of 2%, the PUFAs productivity was 71% in glucose and 59% in the sugars hydrolysate, as a percentage of total fatty acids. Saturated fatty acids (SFAs) levels were highest at about 49% of TFA using 6% glucose as the carbon source. SFAs of 41% were produced using 2% of SH. This study demonstrates that SH produced from lignocellulose biomass is a potentially useful carbon source for the production of omega-3 fatty acids in thraustochytrids, as demonstrated using the new strain, Schizochytrium sp. DT3.

Role of marsupial tammar wallaby milk in lung maturation of pouch young

Background: Marsupials such as the tammar wallaby (M.Eugenii) have a short gestation (29.3 days) and at birth the altricial young resembles a fetus, and the major development occurs postnatally while the young remains in the mother's pouch. The essential functional factors for the maturation of the neonate are provided by the milk which changes in composition progressively throughout lactation (300 days). Morphologically the lungs of tammar pouch young are immature at birth and the majority of their development occurs during the first 100 days of lactation. Results: In this study mouse embryonic lungs (E-12) were cultured in media with tammar skim milk collected at key time points of lactation to identify factors involved in regulating postnatal lung maturation. Remarkably the embryonic lungs showed increased branching morphogenesis and this effect was restricted to milk collected at specific time points between approximately day 40 to 100 lactation. Further analysis to assess lung development showed a significant increase in the expression of marker genes Sp-C, Sp-B, Wnt-7b, BMP4 and Id2 in lung cultures incubated with milk collected at day 60. Similarly, day 60 milk specifically stimulated proliferation and elongation of lung mesenchymal cells that invaded matrigel. In addition, this milk stimulated proliferation of lung epithelium cells on matrigel, and the cells formed 3-dimensional acini with an extended lumen. Conclusions: This study has clearly demonstrated that tammar wallaby milk collected at specific times in early lactation contains bioactives that may have a significant role in lung maturation of pouch young.

The inhibitory effect of stevioside on bacillus cereus growth in milk: validation and its response surface optimization

Stevioside, a glycoside present in the leaves of Stevia rebaudiana Bertoni, offers therapeutic benefits such as anti-hyperglycemic, anti-hypertensive, antiinflammatory, anti-tumor, diuretic and immune influencing properties. In this work antimicrobial activity of stevioside against Bacillus cereus, a major source of milk contamination was investigated. The isolate was confirmed by various biochemical and 16S rRNA gene sequencing. The effect of temperature, incubation time and concentration of stevioside was optimized from a central composite response surface design. The standard plate count (SPC) of pasteurized milk was drastically reduced in comparison to toned and fresh milk. The optimal temperature, incubation time and stevioside concentration were observed to be 60.23°C, 21 h, and 275 μg/ mL respectively. The synergism of stevioside with the external factors (temperature and time) against B. cereus was observed. Our studies showed that addition of stevioside in fresh as well as pasteurised milk would control growth of B. cereus in milk.

Bioactive functions of milk proteins: a comparative genomics approach

The composition of milk includes factors required to provide appropriate nutrition for the growth of the neonate. However, it is now clear that milk has many functions and comprises bioactive molecules that play a central role in regulating developmental processes in the young while providing a protective function for both the suckled young and the mammary gland during the lactation cycle. Identifying these bioactives and their physiological function in eutherians can be difficult and requires extensive screening of milk components that may function to improve well-being and options for prevention and treatment of disease. New animal models with unique reproductive strategies are now becoming increasingly relevant to search for these factors.

Exploring omega-3 fatty acids, enzymes and biodiesel producing thraustochytrids from Australian and Indian marine biodiversity

The marine environment harbours a vast diversity of microorganisms, many of which are unique, and have potential to produce commercially useful materials. Therefore, marine biodiversity from Australian and Indian habitat has been explored to produce novel bioactives, and enzymes. Among these, thraustochytrids collected from Indian habitats were shown to be rich in saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), together constituting 51-76 % of total fatty acids (TFA). Indian and Australian thraustochytrids occupy separate positions in the dendrogram, showing significant differences exist in the fatty acid profiles in these two sets of thraustochytrid strains. In general, Australian strains had a higher docosahexaenoic acid (DHA) content than Indian strains with DHA at 17-31 % of TFA. A range of enzyme activities were observed in the strains, with Australian strains showing overall higher levels of enzyme activity, with the exception of one Indian strain (DBTIOC-1). Comparative analysis of the fatty acid profile of 34 strains revealed that Indian thraustochytrids are more suitable for biodiesel production since these strains have higher fatty acids content for biodiesel (FAB, 76 %) production than Australian thraustochytrids, while the Australian strains are more suitable for omega-3 (40 %) production.

Bead milling for lipid recovery from thraustochytrid cells and selective hydrolysis of Schizochytrium DT3 oil using lipase

Marine microalgae present a renewable alternative source for sustainable production of omega-3 fatty acids, as compared to conventional sources such as krill oil and fish oil. In this study, we optimised a method for lipid extraction from marine thraustochytrids using a bead mill and enzymatic concentration of omega-3 fatty acids from the thraustochytrid oil. The optimised lipid extraction conditions were, bead size 0.4-0.6μm, 4500rpm, 4min of processing time at 5g biomass concentration. The maximum lipid yield (% dry weight basis) achieved at optimum conditions were 40.5% for Schizochytrium sp. S31 (ATCC) and 49.4% for Schizochytrium sp. DT3 (in-house isolate). DT3 oil contained 39.8% docosahexaenoic acid (DHA) as a percentage of lipid, a higher DHA percentage than S31. Partial hydrolysis of DT3 oil using Candida rugosa lipase was performed to enrich omega-3 polyunsaturated fatty acids (PUFAs) in the glyceride portion. Total omega-3 fatty acid content was increased to 88.7%.

Comparative activities of milk components in reversing chronic colitis

Inflammatory bowel disease (IBD) is a poorly understood chronic immune disorder for which there is no medical cure. Milk and colostrum are rich sources of bioactives with immunomodulatory properties. Here we compared the therapeutic effects of oral delivery of bovine milk-derived iron-saturated lactoferrin (Fe-bLF), angiogenin, osteopontin (OPN), colostrum whey protein, Modulen IBD (Nestle Healthsciences, Rhodes, Australia), and cis-9,trans-11 conjugated linoleic acid (CLA)-enriched milk fat in a mouse model of dextran sulfate-induced colitis. The CLA-enriched milk fat significantly increased mouse body weights after 24d of treatment, reduced epithelium damage, and downregulated the expression of proinflammatory cytokines and nitrous oxide. Modulen IBD most effectively decreased the clinical score at d 12, and Modulen IBD and OPN most effectively lowered the inflammatory score. Myeloperoxidase activity that denotes neutrophil infiltration was significantly lower in mice fed Modulen IBD, OPN, angiogenin, and Fe-bLF. A significant decrease in the numbers of T cells, natural killer cells, dendritic cells, and a significant decrease in cytokine expression were observed in mice fed the treatment diets compared with dextran sulfate administered mice. The Fe-bLF, CLA-enriched milk fat, and Modulen IBD inhibited intestinal angiogenesis. In summary, each of the milk components attenuated IBD in mice, but with differing effectiveness against specific disease parameters.