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Wine and alcoholic fermentations are complex and fascinating ecosystems. Wine aroma is shaped by the wine’s chemical compositions, in which both microbes and grape constituents play crucial roles. Activities of the microbial community impact the sensory properties of the final product, therefore, the characterisation of microbial diversity is essential in understanding and predicting sensory properties of wine. Characterisation has been challenging with traditional approaches, where microbes are isolated and therefore analyzed outside from their natural environment. This causes a bias in the observed microbial composition structure. In addition, true community interactions cannot be studied using isolates. Furthermore, the multiplex ties between wine chemical and sensory compositions remain evasive due to their multivariate and nonlinear nature. Therefore, the sensorial outcome arising from different microbial communities has remained inconclusive.
In this thesis, microbial diversity during Riesling wine fermentations is investigated with the aim to understand the roles of microbial communities during fermentations and their links to sensory properties. With the advancement of high-throughput tools based ‘omic methods, such as next-generation sequencing (NGS) technologies, it is now possible to study microbial communities and their functions without isolation by culturing. This developing field and its potential to wine community is reviewed in Chapter 1. The standardisation of methods remains challenging in the field. DNA extraction is a key step in capturing the microbial diversity in samples for generating NGS data, therefore, DNA extraction methods are evaluated in Chapter 2. In Chapter 3, machine learning is utilized in guiding raw data mining generated by the untargeted GC-MS analysis. This step is crucial in order to take full advantages of the large scope of data generated by ‘omic methods. These lay a solid foundation for Chapters 4 and 5 where microbial community structures and their outputs - chemical and sensory compositions are studied by using approaches and tools based on multiple ‘omics methods.
The results of this thesis show first that by using novel statistical approaches, it is possible to extract meaningful information from heterogeneous biological, chemical and sensorial data. Secondly, results suggest that the variation in wine aroma, might be related
to microbial interactions taking place not only inside a single community, but also the
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interactions between communities, such as vineyard and winery communities. Therefore, the true sensory expression of terroir might be masked by the interaction between two microbial communities, although more work is needed to uncover this potential relationship. Such potential interaction mechanisms were uncovered between non- Saccharomyces yeast and bacteria in this work and unexpected novel bacterial growth was observed during alcohol fermentation. This suggests new layers in understanding of wine fermentations. In the future, multi-omic approaches could be applied to identify biological pathways leading to specific wine aroma as well as investigate the effects upon specific winemaking conditions. These results are relevant not just for the wine industry, but also to other industries where complex microbial networks are important. As such, the approaches presented in this thesis might find widely use in the food industry.
Carotenoids are organic lipophilic tetraterpenes ubiquitously present in Nature and found across the three domains of life (Archaea, Bacteria and Eukaryotes). Their structure is characterized by an extensive conjugated double-bond system, which serves as a light-absorbing chromophore, hence determining its colour, and enables carotenoids to absorb energy from other molecules and to act as antioxidant agents. Humans obtain carotenoids mainly via the consumption of fruits and vegetables, and to a smaller extent from other food sources such as fish and eggs. The concentration of carotenoids in the human plasma and tissues has been positively associated with a lower incidence of several chronic diseases including, cancer, diabetes, macular degeneration and cardiovascular conditions, likely due to their antioxidant properties. However, an important aspect of carotenoids, namely β- and α-carotene and β-cryptoxanthin, in human health and development, is their potential to be converted by the body into Vitamin A.
Yet, bioavailability of carotenoids is relatively low (< 30%) and dependent, among others, on dietary factors, such as amount and type of dietary lipids and the presence of dietary fibres. One dietary factor that has been found to negatively impact carotenoid bioaccessibility and cellular uptake in vitro is high concentrations of divalent cations during simulated gastro-intestinal digestion. Nevertheless, the mechanism of action of divalent cations remains unclear. The goal of this thesis was to better understand how divalent cations act during digestion and modulate carotenoid bioavailability. In vitro trials of simulated gastro-intestinal digestion and cellular uptake were run to investigate how varying concentrations of calcium, magnesium and zinc affected the bioaccessibility of both pure carotenoids and carotenoids from food matrices. In order to validate or refute results obtained in vitro, a randomized and double blinded placebo controlled cross-over postprandial trial (24 male participants) was carried out, testing the effect of 3 supplementary calcium doses (0 mg, 500 mg and 1000 mg) on the bioavailability of carotenoids from a spinach based meal. In vitro trials showed that addition of the divalent cations significantly decreased the bioaccessibility of both pure carotenoids (P < 0.001) and those from food matrices (P < 0.01). This effect was dependent on the type of mineral and its concentration. Strongest effects were seen for increasing concentrations of calcium followed by magnesium and zinc. The addition of divalent cations also altered the physico-chemical properties, i.e. viscosity and surface tension, of the digestas. However, the extent of this effect varied according to the type of matrix. The effects on bioaccessibility and physico-chemical properties were accompanied by variations of the zeta-potential of the particles in solution. Taken together, results from the in vitro trials strongly suggested that divalent cations were able to bind bile salts and other surfactant agents, affecting their solubility. The observed i) decrease in macroviscosity, ii) increase in surface tension, and the iii) reduction of the zeta-potential of the digesta, confirmed the removal of surfactant agents from the system, most likely due to precipitation as a result of the lower solubility of the mineral-surfactant complexes. As such, micellarization of carotenoids was hindered, explaining their reduced bioaccessibility. As for the human trial, results showed that there was no significant influence of supplementation with either 500 or 1000 mg of supplemental calcium (in form of carbonate) on the bioavailability of a spinach based meal, as measured by the area-under curve of carotenoid concentrations in the plasma-triacylglycerol rich fraction, suggesting that the in vitro results are not supported in such an in vivo scenario, which may be explained by the initial low bioaccessibility of spinach carotenoids and the dissolution kinetics of the calcium pills. Further investigations are necessary to understand how divalent cations act during in vivo digestion and potentially interact with lipophilic nutrients and food constituents.
