In the period from September 26 to September 30, project collaborator Dr Vesna Milanović held a research workshop on yeast growth kinetics at the Institute for Adriatic Crops. During the workshop, we worked with selected non-Saccharomyces yeasts taken from a collection of autochthonous yeasts at the Institute. Yeasts were isolated from Maraština grapes during the first year of the project, identified and tested for basic oenological characteristics. During the workshop, we carried out analyses related to the determination and monitoring of yeast growth kinetics using several different techniques, such as seeding and counting live yeasts on solid nutrient media, preparation of yeast inoculum for inoculation into sterile Maraština, and including other important steps related to growth and yeast development. Acquired microbiological skills and knowledge are of great use for the methodology of monitoring alcoholic fermentation experiments.

As part of the research on the WINE AROMAS project, the first thesis entitled “Isolation and identification of non-Saccharomyces yeasts from grapes of the Maraština variety” was defended.

On December 5 2021, student Pierpaolo Nocchi successfully defended his diploma thesis at the Università Politecnica della Marche, Dipartimento di Scienze Agrarie Alimentari e Ambientali, Ancona, Italy under the supervision of a project associate, Dr Vesne Milanović, with the commentary of Dr Federica Cardinali, and the mentorship of Dr Cristiane Garofalo. These are also the first results from the project presented in the diploma thesis.

Student Giacomo Migliorini defended his graduation bachelor thesis entitled Oenological characterization of Saccharomyces cerevisiae strains isolated from Maraština grapes at the Polytechnic University of Marche, Department of Agricultural, Food and Environmental Sciences, Ancona, Italy under the mentorship of Dr Vesna Milanović. This is the second diploma thesis defended from the research results of our collaborators on the WINE AROMAS project.

In the journal, Food Research International (I.F.= 7,425, Q1) a scientific paper is published on the topic of molecular identification and characterization of autochthonous microbiota identified for the first time from the Maraština variety.

Milanović, V., Cardinali, F., Ferrocino, I., Boban, A., Franciosa, I., Gajdoš Kljusurić, J., Mucalo, A., Osimani, A., Aquilanti, L., Garofalo, C., Budić-Leto, I. Croatian white grape variety Maraština: first taste of its indigenous mycobiota. Food Research International 162, 111917, 2022 (I.F.=7.425, Q1).

The paper is available at the link: https://doi.org/10.1016/j.foodres.2022.111917

Ana Boban, mag.chem. is currently staying (May 12 – June 12, 2022) at the Research and Innovation Center – Fondazione Edmund Mach, San Michele all’Adige in Italy, as part of a Short-Term Scientific Missions scholarship (STSM) within the COST Action CA 17111 INTEGRAPE; “Data integration to maximize the power of OMICs for grapevine improvement”.

During the scholarship, Ana Boban acquires knowledge and skills in the field of metabolomics, related to the profiling of aromas and lipids from grapes and wine under the mentorship of Dr Urska Vrhovsek, who is part of the COST Action CA 17111 working group, together with Dr Budić-Leto.

We thank COST Action 17111 Integrape for recognition and financial support!

In the period from April 20 to May 4 2022, as part of training on a project in the field of metabolomics, Ana Boban, mag.chem., stayed at the Research and Innovation Center – Fondazione Edmund Mach (FEM) in San Michele all’Adige in Italy. The host and mentor at the training was Dr Urska Vrhovsek, head of the Metabolomics Unit and our collaborator on the project.

Ana Boban acquired knowledge and gained practical experience in profiling aromas and lipids from grapes and wine. She became familiar with advanced analytical instruments and methods. She actively participated in aroma profiling with a targeted metabolic approach using SPE-GC-QqQ-MS/MS and a untargeted approach with SPME-GCxGC-TOF-MS in samples of must and experimental wines of the Maraština variety from our project. In addition, the PhD student worked on lipid profiling with a targeted metabolic approach using UPLC/QqQ-MS/MS in grape skin samples of the Maraština variety from different growing areas. The analyzes included sample preparation, familiarization with the way the instrument works, chromatogram analysis, and identification and quantification of numerous chemical compounds.

Collaborator on the project Prof Jasenka Gajdoš Kljusurić presented the results related to the application of NIR spectroscopy in establishing the protected mark of origin of Maraština wine, at the meeting as part of COST Action CA 19145 “European Network for assuring food integrity using non-destructive spectral sensors“. The meeting was held from May 10 to 12, 2022 in Izola, Slovenia. Prof Gajdoš Kljusurić participated in the meeting as the Croatian representative and member of the Management Committee – Management Committee COST Action 19145.

The first scientific paper on the project is published in the journal Foods (IF= 5.561, Q1) about application of NIR spectroscopy in determining the physicochemical parameters of experimental wines of the Maraština variety from the Dalmatia subregion

Gajdoš Kljusurić, J., Boban, A., Mucalo, A., Budić-Leto, I. Novel Application of NIR Spectroscopy for Non-Destructive Determination of ‘Maraština’ Wine Parameters. Foods, 2022, 11(8), 1172. The complete paper is available at the link: https://www.mdpi.com/2304-8158/11/8/1172/htm

Following the planned activities of the project, after microbiological and molecular identification, the oenological characterization of all isolates was completed. The results of the resistance of non-Saccharomyces yeasts to different concentrations of SO₂ and ethanol were obtained, the capacity of H₂S production was determined, and the ability to grow at different temperatures, the production of acetic acid and the activity of hydroxycinnamic acid decarboxylase (HCDC) were tested. The photographs show samples of the results of H₂S production capacity (Figure 1), resistance of isolates to different concentrations of SO₂ (Figure 2) and positive for HCDC (Figure 3).

The molecular identification of autochthonous non-Saccharomyces yeasts, isolated for the first time from the Maraština variety, has been completed. Identification was performed by sequencing PCR products and BLAST (Basic Local Alignment Search Tool) analysis of the ITS1-5.8S-ITS2 region. The photos in the gallery show the growth of representatives of all 27 different types of non-Saccharomyces yeasts isolated from grapes of the Maraština variety on differential nutrient medium WL (Wallerstein Laboratory). The isolated yeasts will be multiplate at the Universita Politecnica delle Marche, Ancona, Italy and store in the first established collection of indigenous yeast at the Institute of Adriatic Crops.

Gallery: Isolated and molecularly characterized autochthonous yeasts on differential nutrient medium WL.