Sofia Nikolaevna Cherviak

Engineering wine yeasts to combine efficient extracellular polysaccharide degradation with reduced urea accumulation may support improvements in winemaking. This study aimed to replace the arginase-encoding CAR1 locus in the genome of the triploid industrial wine yeast Saccharomyces cerevisiae I-328 with an expression cassette for the endopolygalacturonase gene PGU1, placed under the control of the PGK1 gene promoter and terminator. CRISPR/Cas9-mediated genome editing was performed using an acetamidase-selectable plasmid followed by phenotypic screening and molecular genetic verification. Whole genome sequencing confirmed the replacement of all three CAR1 alleles by the PGU1 expression cassettes. The modified strain I-328-Y21 showed enhanced degradation of polygalacturonic acid relative to the parental strain. Consistently, RT-qPCR analysis showed that the transcription level of the PGU1 gene in strain I-328-Y21 was about 300 times higher than in I-328. Disruption of arginase—the enzyme that generates urea from arginine—reduced urea content in the growth medium by about ninefold. As ethyl carbamate is formed from ethanol and yeast-excreted urea, this strain could help lower ethyl carbamate in wine. Fermentation performance and other technological characteristics remained unaffected by the genome editing. The new strain, in which the arginase gene is replaced by an endopolygalacturonase expression cassette, is a promising candidate for winemaking.

Engineering wine yeasts to combine efficient extracellular polysaccharide degradation with reduced accumulation of ethyl carbamate, a group 2A carcinogen, may support improvements in winemaking. This study aimed to replace the arginase-encoding CAR1 locus in the genome of the triploid industrial wine yeast Saccharomyces cerevisiae I-328 with an expression cassette for the endopolygalacturonase gene PGU1, placed under the control of the PGK1 gene promoter and terminator. CRISPR/Cas9-mediated genome editing was performed using an acetamidase-selectable plasmid followed by phenotypic screening and molecular genetic verification. Whole genome sequencing confirmed the replacement of all three CAR1 alleles by the PGU1 expression cassettes. The modified strain I328-Y21 showed enhanced degradation of polygalacturonic acid relative to the parental strain. Consistently, RT-qPCR analysis showed that the transcription level of the PGU1 gene in strain I328-Y21 was about 300 times higher than in I-328. Inactivation of arginase, which forms urea through the breakdown of arginine, resulted in about nine-fold decrease in the content of urea in the growth medium. Since ethyl carbamate is formed as a result of the interaction between ethanol and urea excreted by yeast cells, this strain could be used to reduce its content in wine. Winemaking experiments show that genome editing did not affect fermentation performance and other technological characteristics. The new strain with the replacement of the arginase gene with the endopolygalacturonase expression cassette is promising for use in winemaking.

For the production of high-quality sparkling wines it is necessary to use pure yeast culture of Saccharomyces cerevisiae species, which is able to ferment sugars at elevated CO ₂ pressure and is characterized by cold and alcohol resistance. Technological properties of the yeast should be preserved regardless of the storage conditions of the culture. The aim of the research was to study the physicochemical and organoleptic properties of sparkling wines produced from white Muscat grapes using yeast strains ‘Sevastopolskaya 23’ (I-525) and ‘Leningradskaya’ (I-307) stored in the collection of microorganisms of winemaking "Magarach" by subcultivation and cryopreservation. The study used generally accepted methods of microbiology and enochemistry of wine. As a result of the studies, no significant influence of the storage conditions of the yeast cultures on their morphological and cultural characteristics was revealed. The strains retained their cell shape and size, unchanged sediment character, strain phenotype, ability to form a ring and spores. No significant differences were found in the process of must fermentation at 26 ± 1 °С using the studied strains. At the same time, fermentation with the strain I-525 was smooth, and with the strain I-307 — stepwise. Stable resistance of the studied yeast strains to changes in some abiotic factors was found. Fermentation of must at a temperature of 15–18 °C with the strain I-525 was much faster (5–21 days) than with I-307. The sparkling wine samples obtained with the yeast strain I-307 were characterized by higher CO ₂ pressure (by 9–21 %) and content of bound forms of CO ₂ (by 18.5–20.3 %), as well as by the value of the coefficient of sparkling properties (K>100) and better foam stability (more than 60 s). The method of storing yeast cultures by cryopreservation ensures the preservation of their basic morphological, cultural and technological properties at low labor costs, which makes it advisable to use it for long-term storage of microorganisms.

In the context of climate change, it is of utmost importance to study the transformation of carbohydrate-acid and phenolic complexes of grapes as they reach technical and phenolic ripeness, the discrepancy between which is increasing against the background of climate change. Studies in this direction are relevant, and in future they will open the way to predict the response of a grape plant to abiotic environmental factors, the formation of quality indicators of raw materials and finished products. The studies conducted allowed revealing the effect of various factors on the formation of phenolic complex of red grapes. It was shown that the total anthocyanin potential in the studied grape samples varied from 735 to 1976 mg/dm³ and was characterized as average → excellent. The dependence of the percentage of extracted anthocyanins on the mass concentration of sugars in grapes, expressed by a cuspidal function, was established. The maximum degree of anthocyanin extractability was observed at grape sugar content of 19–23 g/100 cm³, and amounted to 53–65%. The positive effect of the cold night index on the total anthocyanin potential of grapes was confirmed (r = –0.58). At the same time, a direct dependence (r = 0.75) was observed between the degree of anthocyanin extraction from grapes and the cold night index. The authors established a decrease in the content of phenolic substances in must after pressing whole berries as the value of the glucoacidimetric indicator increased (r = –0.70), as well as a decrease in the mass concentration of phenolic substances after 4 hours of infusion (r = –0.59). From 82.7 to 96.3% of all phenolic substances in grapes were represented by flavan3-ols and anthocyanins. The predominant anthocyanins in grapes were malvidin3-Oglucoside and malvidin3-Ocoumaroylglucoside. The mass concentration of malvidin3-Oglucoside ranged from 580 to 1224 mg/kg or 47.4–81.3% of all grape anthocyanins. The proportion of malvidin3-Ocoumaroylglucoside amounted to 9.3–23.8% of anthocyanin complex components.

In winemaking, malolactic fermentation (MLF), which converts L-malic acid to L-lactic acid, is often applied after the alcoholic fermentation stage to improve the sensory properties of the wine and its microbiological stability. MLF is usually performed by lactic acid bacteria, which, however, are sensitive to the conditions of alcoholic fermentation. Therefore, the development of wine yeast strains capable of both alcoholic fermentation and MLF is an important task. Using genome editing, we engineered a modified variant of the triploid wine yeast strain Saccharomyces cerevisiae I-328, in which the CAR1 arginase gene was replaced by the malate permease gene from Schizosaccharomyces pombe and the malolactic enzyme gene from Oenococcus oeni. Genome-wide transcriptional profiling confirmed the expression of the introduced genes and revealed a limited effect of the modification on global gene expression. Winemaking experiments show that genome editing did not affect fermentation activity and ethanol production, while use of the modified strain resulted in a tenfold reduction in malate content with simultaneous formation of lactate. The resulting wines had a softer and more harmonious taste compared to wine obtained using the parental strain. Inactivation of arginase, which forms urea and L-ornithine through the breakdown of arginine, also resulted in a twofold decrease in the content of urea and the carcinogenic ethyl carbamate in wine. Thus, the new strain with the replacement of the arginase gene with the MLF gene cassette is promising for use in winemaking.

In winemaking, malolactic fermentation (MLF), which converts L-malic acid to L-lactic acid, is often applied after the alcoholic fermentation stage to improve the sensory properties of the wine and its microbiological stability. MLF is usually implemented by lactic acid bacteria, which, however, are sensitive to the conditions of alcoholic fermentation. Therefore, the development of a wine yeast strains capable of both alcoholic fermentation and MLF is an important task. Using genome editing, we engineered a modified variant of the triploid wine yeast strain Saccharomyces cerevisiae I-328, in which the CAR1 arginase gene was replaced with the malate permease gene from Schizosaccharomyces pombe and the malolactic enzyme gene from Oenococcus oeni. Genome-wide transcriptional profiling confirmed the expression of the introduced genes and revealed a limited effect of the modification on global gene expression. Winemaking experiments show that genome editing did not affect the fermentation activity and the production of ethanol, while the use of the modified strain ensured a tenfold reduction in malate content with the simultaneous formation of lactate. The resulting wines had a softer and more harmonious taste compared to the wine obtained using the parental strain. Inactivation of arginase, which forms urea and L-ornithine through the breakdown of arginine, also led to a twofold decrease in the content of urea and the carcinogenic ethyl carbamate in wine. Thus, the new strain with the replacement of the arginase gene with the MLF gene cassette is promising for use in winemaking.

There is no uniform test or “black box method” to establish definitely the authenticity of wine samples. Methods and types of adulteration are constantly being modernized, requesting to improve and develop analytical methods of increasing the efficiency of identification and detection of counterfeits. The goal of the research is to create a system for diagnostics of prohibited process technologies and additives during the wine identification. The studies were carried out in 2016-2023. The objects of research are: wines; model systems obtained by introducing of permitted and prohibited additives. A total of 3.500 samples were examined. Enochemical indicators based on the principles of high-performance and gas-liquid chromatography, potentiometry and colorimetry were identified in the objects. The developed classifying system covers the range of detection of prohibited additives and a number of process technologies: coloring agents of different origin, flavoring agents, organic acids, water, glycerol; sugar- containing substances of non-grape origin, additives of non-grape raw materials. Each element of the system can be used to diagnose several types of counterfeits. The system is open for seeding and spreading. Methods of analysis of classifying indicators are quite simple and available for industrial laboratories of various levels.

Heat and moisture supply are key climatic parameters to determine the possibility and efficiency of growing crops including grapes. Climate change causes a significant uncertainty not only in potential adaptive capabilities of grape plants, but also in the prospects of viticulture development in general. This problem assumes a greater importance in the context of global climate change, since this process can lead to a shift in the areas of high-quality viticulture and winemaking. Studying the patterns of influence of climatic factors on the productivity of vineyards and quality characteristics of raw materials, as well as final products will allow optimizing the efficiency of using natural resources, and reducing the negative impact of climate change on agricultural production. The goal of the work is to study the effect of climatic factors on biochemical and physicochemical indicators of red grape varieties. Materials and methods. The objects of the study were red grape varieties ‘Bastardo Magarachskiy’ and ‘Cabernet Sauvignon’, growing in different viticulture and winemaking region of Crimea. As climatic indices characterizing the heat and moisture supply of the territory, we used the total amount of precipitation from the beginning of growing season until harvest, the total amount of precipitation for the last month before harvest, hydrothermal coefficient of Selyaninov, as well as the sum of active temperatures above 10 °C (∑ Т°С10), Huglin heliothermal index (HI), Winkler index (WI), average air temperature from the growing season beginning until harvest (tgr), average air temperature for the last month before harvest (tmonth), cool night index (CI). To assess the carbohydrate-acid complex of grapes, the following indicators were used: mass concentration of sugars, titratable acids and malic acid. Results and conclusion. Distribution of climatic factors characterizing different viticulture and winemaking regions of Crimea, as well as their effect on the quality characteristics of raw materials were assessed. It was established that the Mountain Valley Coastal and Mountain Valley viticulture and winemaking regions were characterized by higher values of Winkler and Huglin indices (by 354-435 and 97-124 units, respectively), than the Crimean Western Coastal Piedmont region and the city of Sevastopol. A similar tendency was observed for the indicators tmonth, tgr and CI. A linear dependence of the mass concentration of sugars in a berry on the level of heat supply in the region during growing season was established (rwi = 0.73). A correlation was estimated between Pgr and the mass concentration of malic acid in a berry (r = 0.88-0.9). Based on the meteorological data, the patterns established will allow oenologists to estimate quality indicators of further harvest, and plan in advance technological aspects of grape processing in order to obtain high-quality wine products.

The study of scientific problem, to which this research is devoted, is due to planetary climate change, manifested in an increase in the average annual air temperature, rapid changes in winter and summer temperatures, and a lack of precipitation. For today, there is a serious problem of adapting plants to climate change due to an increase in water deficiency in many regions of the world. Climate change creates significant uncertainty in the potential adaptability of grape plants, as well as in the prospects for viticulture development in general. The study and generalization of territorial distribution of climatic factors that characterize a particular region, as well as their effect on the quality characteristics of raw materials and finished products, are attractive from the point of better understanding and forecasting the potential impact on agricultural systems. The obtained data consumption will allow improving the forecast of climate change consequences in the medium term, and in relation to particular terroirs. The goal is to study the effect of climatic factors that characterize the moisture supply of the territory on the quality indicators of grapes. Materials and methods. The objects of the study were grapes of white and red cultivars, different in agroecological aspect, and growing in micro regions of Crimea. To identify the effect of factors characterizing the moisture supply of the territory on the quality indicators of raw materials for winemaking products, the amount of precipitation from the beginning of growing season to the harvesting, the amount of precipitation for the last month before harvest, and hydrothermal coefficient of Selyaninov were selected. The assessment of grapes was carried out according to the indicators of carbohydrate-acid complex (mass concentration of sugars, titratable acids, pH value, profile of organic acids), as well as the glucoacidimetric indicator revealing the degree of grape ripeness. Results and conclusion. Climatic factors characterizing the moisture supply of viticultural and winemaking regions of Crimea were assessed. The studies carried out made it possible to reveal variations in the concentration of carbohydrate-acid complex components depending on the changing ability of environmental factors. A significant precipitation deficiency was observed on the Peninsula. According to the value of hydrothermal coefficient of Selyaninov (did not exceed 1), most of the territory was classified as an arid or very arid zone. A correlation between the hydrothermal coefficient and mass concentration of grape sugars (r=-0.63) was established for red grapevine cultivars. The data obtained indicate that an increase in the amount of precipitation during growing season contributes to a moderation of sugar accumulation in grapes. Similar pattern was not established for white grapevine cultivars. There was a direct correlation of the mass concentration of malic acid on the amount of precipitation in the last month before harvest (r=0.78).

A grape plant is capable to adapt, grow and produce crops in different environmental conditions. At the same time, agro-climatic conditions of grape growing area have a significant effect on the product quality. Environmental climatic conditions of Crimea contribute to cultivating grapes from very early to late ripening varieties. Diverse range of abiotic conditions allows choosing an optimal terroir for the formation of necessary grape conditions depending on the application target. Significant spatial climate variability within the region makes it possible to evaluate the impact of environmental factors on biosynthetic and accumulative properties of grapes. The purpose is to study changes in the profile of carbohydrate-acid complex of wine grape varieties under the influence of agro-ecological growing conditions. Materials and methods. Study objects were white and red grape varieties growing in various viticulture and winemaking regions of Crimea. A research for grape plant response to the variability of environmental factors basing on a long-term comparative study of chemical composition of its fruits, determining the varietal biochemical identity and technological properties in a particular region, was carried out. Results and conclusion. As a research result, the cumulative effect of agro-ecological factors on the accumulation of components of carbohydrate-acid grape complex was established, and in particular years it was possible to identify the dependence between their quantitative content and individual environmental factors. Accumulation of the mass concentration of sugars in grape berry directly correlated with the Huglin index value (r = 0.7). With regard to the mass concentration of titratable acids, an inverse correlation was revealed with the Winkler index value (r = -0.59). A comparative analysis of the results shows that one grape variety grown under different climatic conditions may show different ability to synthesize the components of carbohydrate-acid complexes, determining its suitability for various output production. There is a direct correlation of the mass concentration of sugars on the average air temperature for the last month before harvest (r=0.70) within the variety. Виноградное растение способно адаптироваться, произрастать и давать урожаи в разнообразных природных условиях. В то же время агроклиматические условия территории возделывания винограда оказывают существенное влияние на качество продукта. Природно-климатические условия Крыма способствуют возделыванию винограда от очень ранних до поздних сортов, а широкое разнообразие абиотических условий позволяет подобрать оптимальный терруар для формирования необходимых кондиций винограда в зависимости от целей его применения. Значительная пространственная изменчивость климата в пределах региона позволяет оценить воздействие факторов среды на биосинтетические и аккумуляционные свойства винограда. Цель – изучение изменения профиля углеводно-кислотного комплекса винограда технических сортов под воздействием агроэкологических условий его произрастания. Материалы и методы. Объектом исследования являлся виноград белых и красных сортов, произрастающий в различных виноградо-винодельческих районах Крыма. Проведено исследование реакции виноградного растения на вариабельность экологических факторов на основе многолетнего сравнительного изучения химического состава его плодов, обусловливающих биохимическую индивидуальность и технологические свойства сорта в конкретной местности. Результаты и заключение. В результате проведенных исследований установлено совокупное влияние агроэкологических факторов на накопление компонентов углеводно-кислотного комплекса винограда, а в отдельные годы удалось выявить зависимости их количественного содержания от отдельных фактора среды. Установлена прямая зависимость накопления массовой концентрации сахаров в виноградной ягоде от величины индекса Хуглина (r = 0,7). В отношении массовой концентрации титруемых кислот от значения индекса Уинклера выявлена обратная зависимость (r = -0,59). Сравнительный анализ результатов, показывает, что один и тот же сорт винограда, выращенный в неодинаковых климатических условиях, может проявлять различную способность к синтезу компонентов...