Grape and Wine Metabolites: Biotechnological Approaches to Improve Wine Quality

Grape metabolites can be affected by many extrinsic and intrinsic factors, such as grape variety, ripening stage, growing regions, vineyard management practices, and edaphoclimatic conditions. However, there is still much about the in vivo formation of grape metabolites that need to be investigated. The winemaking process also can create distinct wines. Nowadays, wine fermentations are driven mostly by single-strain inoculations, allowing greater control of fermentation. Pure cultures of selected yeast strains, mostly Saccharomyces cerevisiae, are added to grape must, leading to more predictable outcomes and decreasing the risk of spoilage. Besides yeasts, lactic acid bacteria also play an important role, in the final wine quality. Thus, this chapter attempts to present an overview of grape berry physiology and metabolome to provide a deep understanding of the primary and secondary metabolites accumulated in the grape berries and their potential impact in wine quality. In addition, biotechnological approaches for wine quality practiced during wine alcoholic and malolactic fermentation will also be discussed.

Recent Developments in Wine Tartaric Stabilization

Tartrate precipitation is still a relevant subject in Enology, being one of the most common problems of wine physical-chemical instability. Potassium bitartrate and calcium tartrate precipitations are undesirable phenomena which can occur in bottled wines, especially when these are stored at low temperatures. The occurrence of tartrate salt crystals (potassium hydrogen tartrate – KHT and calcium tartrate – CaT) in bottles has severe consequences in the final aspect of the wine and therefore on the consumer’s acceptance, making tartrate wine stabilization virtually mandatory before bottling. Currently, several solutions to prevent this haze are available: subtractive methods including the conventional cold treatments that promote the cristalization of KHT, removal of potassium and calcium ions either by electrodialysis or ion exchange resins; and additive methods such as the addition of carboxymethylcellulose, mannoproteins or metatartaric acid. For monitoring the KHT stability, several analytical methods have been developed based on conductivity evaluation, namely the mini-contact test and the saturation temperature measurements (TS). These methods will also be revisited, aiming to raise awareness of their utility as tools in quality control of wines. This review addresses tartrate precipitation subject and the most recent preventive solutions available, pointing out the advantages and drawbacks of each one, and its impact on the final characteristics of the wine.

Evaluation of the evolution of the anthocyanins profile in red wine grape varieties in Alentejo

The flavonoids (including anthocyanins) are wine compounds with important anti-oxidant activity, protecting the cells against oxidative processes, preventing cardiovascular and neurodegenerative diseases, cancer, among others (Antoniolli et al. 2015; Castañeda-Ovando et al. 2009; Hosu et al. 2014; Huang et al. 2009; Kong et al. 2003). Anthocyanins in grapes at harvest are determinant to red wine quality and their development in the grape must be characterised in order to determine the most suitable date for the harvest. Thus the aim of this research is the evaluation of anthocyanins composition in two red wine grape varieties from véraison continuing through ripening. Anthocyanins were quantified by high resolution liquid chromatography (HPLC-DAD). Additionally, the total phenols content were quantified by UV-Vis Spectrometry. The anthocyanins’ profile evolution may be dependent on the variety and ripening phase. During ripening grape samples have shown an increase of coumaryl derivatives. This information may lead us to understand the anthocyanins biosynthesis pathway in different grape varieties. The development of anthocyanins from the véraison seems to follow a pattern that coincides with the increasing accumulation of soluble sugars.

Nitrate and potassium concentration in fertigated soil cultivated with wine vines.

In the semiarid region of Brazil the use of irrigation systems for applying fertilizers in horticulture is the primary means for incorporating nutrients in the soil. However, this technique still requires its use in wine vines to be assessed. In view of this, this study aimed to assess nitrate and potassium concentrations in soil fertigated with nitrogen and potassium fertilizers in 3 wine grape growing cycles. A field experiment was conducted with ?Syrah? wine grapes, in Petrolina, Pernambuco, Brazil; it assessed five nitrogen doses (0, 15, 30, 60 and 120 kg ha-1) and five K2O doses (0, 15, 30, 60 and 120 kg ha-1) applied by drip irrigation system with two emitters per plant, with a flow rate of 4 L h-1. The experimental design used was the factorial split-plot, making up 13 combinations arranged in 4 randomized blocks. Soil solution samples were collected weekly with the aid of porous cup extractors for all treatments and at depths of 0.4 and 0.6 m by determining nitrate and potassium concentrations and electrical conductivity. Increased levels of both nutrients in the irrigation water increased the availability of nitrate and potassium in the soil solution. The highest nitrate and potassium concentrations were found in the second growing cycle at both depths studied.

Aromatic stability of Syrah and Petit Verdot tropical wines from Brazil.

The production of fine wines in the Sub-middle of the São Francisco River Valley, Northeast of Brazil, is relatively recent, about twenty-five years ago. This region presents different characteristics, with a tropical semiarid climate, in a flat landscape. Presenting high annual average temperature, solar radiation and water in abundance for irrigation, it?s possible the scaling the grape harvests for winemaking throughout the year, allowing to obtain until two harvests per year. Several factors may affect the aromatic compounds in wines, such as viticulture practices, climatic conditions, cultivars and winemaking process. This study aimed to evaluate the aromatic stability of Syrah and Petit Verdot tropical wines elaborated in two different periods in the year. The grapes were harvested in the first and second semesters of 2009, in June and November. The wines were elaborated and then, they were bottled and analyzed in triplicate, thirty days and one year after bottling, by gas chromatography with ionization detector flame (GC-FID), to evaluate the profile and the stability of the aroma compounds. Principal component analysis was applied to discriminate between wine samples and to find the compounds responsible by the variability. The results showed that Syrah and Petit Verdot tropical wines presented different responses, for stability of higher alcohols, esters and carboxylic acids.

Influence of the vintage, clone and rootstock on the chemical characteristics of Syrah tropical wines from Brazil.

In the Northeast of Brazil, vines can produce twice a year, because annual average temperature is 26ºC, with high solar radiation and water availability for irrigation. Many cultivars have been tested according to their adaptation to the climate and soil, and the main variety used for red wines is Syrah. This work aimed to evaluate five clones of Syrah, grafted on two rootstocks, in two harvests of the second semester of 2009 and 2010, according to the chemical analyses of the wines.The clones evaluated were 100, 174, 300, 470 and 525, the rootstocks were Paulsen 1103 and IAC 313 (Golia x Vitis caribeae). Grapes were harvested in November 2009 and 2010 and the yield was evaluated. Climate characteristics of each harvest was determined and correlated to the results. Wines were elaborated in glass tanks of 20 L, with alcoholic fermentation at 25ºC for seven days, then wines were pressed and malolactic fermentation was carried out at 18ºC for 20 days. The following parameters were analyzed: alcohol content, dry extract, total anthocyanins, total phenolic index. High performance liquid chromatography was used to determine tartaric, malic, lactic and citric organic acids. Results showed that wines presented different concentrations of classical analyses, phenolics and organic acids according to the harvest date, rootstocks and clones. Principal component analysis was applied on data and clusters with wine samples were formed, explaining the variability, and results are discussed.

The development of the viticulture for a high quality tropical wine production in the world.

2011

High altitude wine in Santa Catarina, Brazil: a collective brand.

2010

Development of methodology for the determination of organic acids in must, grape juice and wine by high performance liquid chromatography (HPLC).

2012

Detection of ochratoxin A in tropical wine and grape juice from Brazil.

Ochratoxin A (OTA) is the main mycotoxin found in grapes, wines and grape juices and is considered one of the most harmful contaminants to human health. In this study, samples of tropical wines and grape juices from different grape varieties grown in Brazil were analysed for their OTA content by high-performance liquid chromatography. The detection and quantification limits for OTA were 0.01 and 0.03 ?g L?1 respectively. OTA was detected in 13 (38.24%) of the samples analysed, with concentrations ranging from <0.03 to 0.62 micron g L-1. OTA was not detected in any of the grape juice samples. Most of the red wine samples proved to be contaminated with OTA (75%), while only one white wine sample was contaminated. However, the OTA levels detected in all samples were well below the maximum tolerable limit (2 micron g L-1) in wine and grape juice established by the European Community and Brazilian legislature. The results of this study indicate a low risk of exposure to OTA by consumption of tropical wines and grape juices from Brazil.

SELinux policy management framework for HIS

Health Information Systems (HIS) make extensive use of Information and Communication Technologies (ICT). The use of ICT aids in improving the quality and efficiency of healthcare services by making healthcare information available at the point of care (Goldstein, Groen, Ponkshe, and Wine, 2007). The increasing availability of healthcare data presents security and privacy issues which have not yet been fully addressed (Liu, Caelli, May, and Croll, 2008a). Healthcare organisations have to comply with the security and privacy requirements stated in laws, regulations and ethical standards, while managing healthcare information. Protecting the security and privacy of healthcare information is a very complex task (Liu, May, Caelli and Croll, 2008b). In order to simplify the complexity of providing security and privacy in HIS, appropriate information security services and mechanisms have to be implemented. Solutions at the application layer have already been implemented in HIS such as those existing in healthcare web services (Weaver et al., 2003). In addition, Discretionary Access Control (DAC) is the most commonly implemented access control model to restrict access to resources at the OS layer (Liu, Caelli, May, Croll and Henricksen, 2007a). Nevertheless, the combination of application security mechanisms and DAC at the OS layer has been stated to be insufficient in satisfying security requirements in computer systems (Loscocco et al., 1998). This thesis investigates the feasibility of implementing Security Enhanced Linux (SELinux) to enforce a Role-Based Access Control (RBAC) policy to help protect resources at the Operating System (OS) layer. SELinux provides Mandatory Access Control (MAC) mechanisms at the OS layer. These mechanisms can contain the damage from compromised applications and restrict access to resources according to the security policy implemented. The main contribution of this research is to provide a modern framework to implement and manage SELinux in HIS. The proposed framework introduces SELinux Profiles to restrict access permissions over the system resources to authorised users. The feasibility of using SELinux profiles in HIS was demonstrated through the creation of a prototype, which was submitted to various attack scenarios. The prototype was also subjected to testing during emergency scenarios, where changes to the security policies had to be made on the spot. Attack scenarios were based on vulnerabilities common at the application layer. SELinux demonstrated that it could effectively contain attacks at the application layer and provide adequate flexibility during emergency situations. However, even with the use of current tools, the development of SELinux policies can be very complex. Further research has to be made in order to simplify the management of SELinux policies and access permissions. In addition, SELinux related technologies, such as the Policy Management Server by Tresys Technologies, need to be researched in order to provide solutions at different layers of protection.

Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings