Mario Malacarne

Evolution of Yeast Populations during Different Biodynamic Winemaking Processes

This work was performed to evaluate the evolution of indigenous yeasts during wine productions carried out following the principles of biodynamic agriculture. Five trials were designed with different technological interventions consisting of the addition of nitrogen (in the form of ammonium salt), thiamine salt, oxygen, and pied de cuvee at varying concentrations. Yeasts were estimated by haemocytometer chamber and plate counts and identified by sequencing of the D1/D2 domain of the 26S rRNA gene. The isolates identified as Saccharomyces cerevisiae were found to dominate must fermentations and were genetically differentiated by interdelta sequence analysis (ISA). Several non-Saccharomyces species, in particular Hanseniaspora spp. and Candida spp., were found at subdominant levels during must fermentation. The trial added with both nitrogen and thiamine (NTV) showed the highest fermentation rate and microbial richness. The internal surfaces of the cellar equipment were characterised by a certain yeast biodiversity and hosted the species found during winemaking; the wooden surfaces represented the primary source of inoculation of a strain of S. cerevisiae found dominant in all winemaking trials.

Free and glycosylated simple phenol profiling in Apulian Italian wines

Free simple phenols have a significant role in defining the sensory and nutritional characteristics of wines, affecting the organoleptic profile and having positive effects on health, but glycosidically bound phenols can also be hydrolysed during the winemaking process, releasing the corresponding volatile compounds and making a possible contribution to the final sensory profile. In this work, application of on-line SPE liquid chromatography-high resolution mass spectrometry, operating in negative polarity with heated electrospray, allowed to detect over eighty free and glycosylated simple phenols in Primitivo di Manduria and Negroamaro wines. Sixty-one phenols, four of which phenolic glucosidic precursors, were quantified as having quantification limits ranging from 0.001 to 0.1μgmL(-1), calibration R(2) of 0.99 for over 92% of compounds, and precision (R.S.D.%) always lower than 12%. Twenty-four simple phenolic precursors were tentatively identified as hexoside, pentoside and hexoside-hexoside derivatives, on the basis of accurate mass, isotopic pattern and MS/MS fragmentation.

Verifying the botanical authenticity of commercial tannins through sugars and simple phenols profiles.

Commercial tannins from several botanical sources and with different chemical and technological characteristics are used in the food and winemaking industries. Different ways to check their botanical authenticity have been studied in the last few years, through investigation of different analytical parameters. This work proposes a new, effective approach based on the quantification of 6 carbohydrates, 7 polyalcohols, and 55 phenols. 87 tannins from 12 different botanical sources were analysed following a very simple sample preparation procedure. Using Forward Stepwise Discriminant Analysis, 3 statistical models were created based on sugars content, phenols concentration and combination of the two classes of compounds for the 8 most abundant categories (i.e. oak, grape seed, grape skin, gall, chestnut, quebracho, tea and acacia). The last approach provided good results in attributing tannins to the correct botanical origin. Validation, repeated 3 times on subsets of 10% of samples, confirmed the reliability of this model.

Simultaneous yeast–bacteria inoculum. A feasible solution for the management of oenological fermentation in red must with low nitrogen content

The simultaneous inoculum of yeasts and bacteria is a feasible solution for improving fermentation in wines with a harsh chemical composition, capable of inhibiting microbial activity. Considering the risk of wine spoilage due to lactic bacteria, co-inoculum is suggested in white wines with a low pH. However, climate change has also caused problems in achieving malolactic fermentation in red wines, due to the high concentration of ethanol and the low nutrient content. In this work, 5 pairs of commercial oenological starters were tested in simultaneous fermentation, using 4 red musts with a low nitrogen content, and compared with a traditional winemaking process. The simultaneous inoculum caused a slowdown in the activity of yeasts, although no problems in the accomplishment of alcoholic fermentations were observed. More reliable malolactic fermentation was performed in the co-inoculum trials, while, in traditional winemaking, some failures in the degradation of malic acid were observed. Microbiological analyses agreed with these observations. No differences were found in yeast density during alcoholic fermentation, demonstrating the absence of negative interaction between the yeast and the bacteria. However, simultaneous fermentation is not without risks; the highest increases of acetic acid were noted in the co-inoculum trials. The addition of yeast and bacteria to must with a serious lack of nutrients would appear to be a promising alternative to traditional fermentation; however, careful control of the chemical composition of must is mandatory to obtain reliable microbiological activity in the first stages of winemaking.

Botanical origin characterisation of tannins using infrared spectroscopy

Different approaches to analysing the botanical origin of tannins have been proposed in the last fifteen years, but are generally time consuming and require the use of advanced instrumentation. This study aims to suggest an effective, easy, rapid and cheap method based on the acquisition of FT-IR spectra of 3g/L hydroalcoholic tannin solutions, overcoming possible disadvantages due to sample or particle size inhomogeneity. 114 commercial powder tannins from 7 different botanical sources (oak, chestnut, gall, quebracho, tea, grape skin and grape seed) were collected and the FT-IR spectra were acquired in the region 926-5011cm-1. Partial Least Squares regression, Discriminant Analysis and Artificial Neural Networks were applied to FT-IR spectra to investigate the possibility of differentiating the 7 botanical origins. The best results were obtained using Discriminant Analysis, with 95% correct re-classification, and 97% grouping of grape skin and seed in a single source.

Exploitation of simultaneous alcoholic and malolactic fermentation of Incrocio Manzoni, a traditional Italian white wine

Malolactic fermentation (MLF) is a key feature in the production of high-quality wines. Its evolution is not always guaranteed, especially in white wine, due to certain limiting factors (low pH, sulphur dioxide, low temperature) acting against malolactic bacteria. The inoculation of grape must with bacteria is an alternative approach to the management of oenological fermentation, favouring the survival of bacteria due to the absence of ethanol or sulphur dioxide – toxic compounds made by yeasts in the first stages of winemaking. We compared the activity of two strains of Oenococcus oeni during MLF in wines made from an emerging white grape variety, native to north-eastern Italy, namely Incrocio Manzoni. Different winemaking protocols were assayed, comparing sequential or simultaneous inoculation of microbial starters. The monitoring of bacterial viability through fermentations and a comprehensive characterisation of the volatile profile of the wines were achieved by advanced analytical approaches, flow cytometry and GC-MS respectively. According to the preliminary hypothesis, the chemical composition of the grape must was characterised by high acidity, which represented a serious barrier to bacterial development. Simultaneous inoculation of the two O. oeni strains ensured a regular evolution of MLF. Some differences were highlighted, both in terms of fermentation kinetics and the aromatic profile of the wines obtained, in relation to the strain of lactic bacteria. The work provides an exhaustive overview of the opportunities and risks related to different wine fermentation approaches in order to enhance the quality of white wines made from “new” or “local” wine grapes.