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.

In the period from November 8 to 19, 2021, PhD student Ana Boban, mag. chem. stayed at the Universita Politecnica delle Marche, Ancona in Italy. The host and mentor at the training was Dr Vesna Milanović, collaborator on the project.

PhD student Ana acquired knowledge and practical skills in basic microbiological methods for isolation and identification of isolated non-Saccharomyces yeasts from the first phase of the project. During her stay, she improved on methods of oenological characterization of isolated yeasts, such as testing the resistance of isolates to different temperatures, concentrations of alcohol and sulfur (IV) oxide, and carried out testing of enzyme activity of yeasts related to the aroma of the variety using enzymatic methods. PhD student Ana Boban also learned about the PCR method used in the preparation of samples for molecular identification of isolated yeasts from our project.

A Fourier transform infrared spectroscopic (FTIR) device for the analysis of wine, must and must in fermentation, model Lyza 5000 Wine, with an included automatic sample changer, from the equipment manufacturer Anton Paar GmbH, was installed in the Oenology Laboratory at the Institute for Adriatic Crops and Karst Reclamation. The device was procured in the Open Public Procurement Procedure announced on June 8, 2021. On October 27 and 28, 2021, advanced training for working on the device was carried out for project collaborators. Next step is the validation of the FTIR analyzer on must and wine samples with reference OIV methods. The FTIR analyzer has the ability to measure a large number of chemical parameters important for wine quality. This device will significantly contribute to research on the fermentation activity of isolated autochthonous non-Saccharomyces yeasts due to automatic sample changer, very short analysis time and small sample volume.

The isolation of autochthonous yeasts from fresh grapes of the Maraština variety is in progress. Isolation is carried out in the Microbiological Laboratory by our colaborator Dr Vesna Milanović from the Polytechnic University of Marche in Ancona, Italy. The pictures show yeasts grown on a WL nutrient medium. The different morphology of the yeasts is visible. The samples were prepared for the following stages of work: purification and isolation of DNA, PCR analysis, and sequencing of PCR products.

September is an intense month for our team. We went to tour of vineyards in the area of Korčula, Kaštela, the hinterland of Zadar, and Šibenik with the aim of collecting samples of the Maraština variety for the isolation of non-Saccharomyces yeasts and the identification of grape metabolites. We sampled grapes from 10 commercial vineyards located in Dračevica, Prapatna, Kruševo, Kaštel Kambelovac, Smilčić, Polača, Stankovci, Vukšić and Oklaj and from the germplasm collection of autochthonous varieties at the Institute. We determined the parameters of grape maturity, collected data on location and viticultural practices. In the laboratory of the Institute, we separated the obtained samples for microbiological and chemical analyses. We set up spontaneous fermentations in laboratory conditions with grapes from all locations.