INTRODUCTIONThe accumulation of wine production waste has anadverse effect on the environmental situation in grapegrowingregions. Grape pomace is a key winemakingby-product that can be used as an additional rawmaterial [1].Grape pomace is rich in biologically valuablecomponents, including polyphenols, pectin substances,and microelements [2, 3]. About 10–15% of this byproductis used as a biofertilizer to improve the soilstructure [4]. Grape pomace can also be a source ofdietary fiber, natural food colors, grape alcohol, tartaricacid, as well as extracts and concentrates [5–8]. Grapeseeds are used to extract grape oil, which is widely usedin cosmetology [9, 10]. Therefore, there is an urgent needfor effective methods to process grape pomace.Pomace can be sweet, fermented, and alcoholized,depending on the technology of grape processing.Sweet pomace is obtained by pressing the grapesafter the juice has separated. Such pomace containsmicroorganisms and major components of grape berries,including sugars. Sweet pomace is usually derived fromwhite grapes during the production of table wines and wine base for sparkling wines and champagne, as wellas from red grapes processed like white grapes (withoutmaceration or fermentation).Fermented pomace results from pressing thefermented grapes during red table wine production. Itcontains ethanol (a product of natural fermentation ofgrape sugars), organic acids, phenolic, nitrogenous andpectin substances, aromatic components of wine base, aswell as wine yeast and malolactic fermentation bacteriaused for fermentation and acidity reduction.Alcoholized pomace is produced through pressingfermented and alcoholized grapes in the production ofliqueur wines, especially Kagor (fortified dessert wine)and Muscat wines. The last 15–20 years have seen asignificant decrease in these wines due to a need to usegrape alcohols in their production. Alcoholized pomacecontains ethanol, sugars, and other components ofgrapes and wine base, including yeast. According to theRussian Ministry of Agriculture, alcoholized pomaceaccounts for 2.0–3.4% of total pomace, depending on thevolume of liqueur wine production.Various types of pomace differ in their componentsand microflora. Pomace can rapidly deteriorate duringstorage due to a combination of nutrients (sugars,nitrogenous compounds, organic acids, vitamins,etc.) and air exposed natural grape microflora (sweetpomace), as well as wine yeast and malolactic bacteria(fermented and alcoholized pomace). As a result,pomace becomes moldy, alcohol turns into acetic acid,and tartaric acid compounds get destroyed by bacteria.Therefore, pomace needs to be processedimmediately after its separation. However, sometimesit has to be stored for a certain time before processing(e.g., in the production of dietary fiber, powders,enocolorants, extracts, etc.). In this case, pomace mustbe stored under appropriate conditions, dependingon the amount, type, and the physiological state of itsmicroflora.Grape pomace is usually stored on specialsites, covered with tarpaulin or other material, ifany. However, its surface and inside contain molds(Aspergillus, Penicilium, Rhizopus nigricans, Cladosprium,Fusarium, Alternarium, Mucor, Botritis,and Oospora), yeasts (Saccharomyces and Torula),bacteria (Bacillus stearotermophilus, Bacillus sudtilis,and Staphylococcus aureus), and many othersmicroorganisms [11–14]. In this regard, the assessmentof its microbiological state is an important part ofpomace disposal, which depends on grape processingtechnology and storage conditions.Our aim was to study the influence of storageconditions on the microflora of white and red grapepomace treated with different methods.STUDY OBJECTS AND METHODSSampling and preparation for microbiologicalresearch. We studied fresh and one-month storedpomace from Vitis vinifera grapes produced inKrasnodar Krai (Russia), namely: sweet (Chardonnay,Riesling, Sauvignon Blanc, Traminer Rose, and PinotNoir), fermented (Cabernet Sauvignon, Merlot, andSaperavi), and alcoholized (Traminer Rose, CabernetSauvignon, and Saperavi). The pomace came fromthe production of white and red table and liqueurwines. Some grape processing technologies usedpectoproteolytic enzyme preparations – TrenolinBlanc and Trenolin Rouge (Erbsloeh Geisenheim AG,Germany) – in optimal manufacturer-recommendedamounts. The storage temperature varied from 14°C(at night) to 26°C (at daytime). An average sample wasobtained by mixing equal amounts of samples takenfrom the surface of the pomace mass. The sampleswere taken from a depth of 0.5 and 1.0 m, placed inglass flasks, filled with distilled water, and incubated at22–25°C for two days.Isolation of microorganisms. The samples wereinoculated and passed on yeast-peptone agar containing10 g yeast extract, 20 g peptone, 20 g agar-agar, and20 g glucose per 1 L of water (Research Center forPharmacotherapy, Russia). The elective test wasperformed on Lysine Medium Base (Himedia, India).Those isolates that were incapable of growing on theelective medium were considered as belonging to thegenus Saccharomyces.We also used solid nutrient media, such as grapejuice agar (2%) alcoholized with ethanol (14% alcohol) –to identify saccharomycetes, and OFS-agar (ErbsloehGeisenheim AG, Germany) – to quantify yeast, moldfungi, as well as lactic and acetic acid bacteria.Chloramphenicol (50 mg/L) was introduced intothe media to improve yeast growth and suppressbacterial growth. Yeast colonies were cultivated at24 ± 2°C for 6–7 days. Some of them were inoculated onselective solid nutrient media. During the cultivation, wemonitored the presence of other genera yeast, includingSaccharomyces, Pichia, Hansenula, and Hanseniaspora.The colonies were microscoped to identifysaccharomycetes and other microorganisms based ontheir cultural and morphological characteristics [13, 15].Generic identification of the isolates was based on theirmorphological and biochemical characteristics.Physical and chemical parameters. Thepomace was extracted with hot water (65–70°C) at ahydromodule of 1:5. The extracts were analyzed todetermine:– organic acids: by capillary electrophoresis StateStandard 52841-2007. Wine production. Determinationof organic acids by capillary electrophoresis method.Moscow: Standartinform; 2008. 7 p.;– ethyl alcohol: according to State Standard 32095-2013. The alcohol production and raw material for itproducing. Method of ethyl alcohol determination;– phenolic substances: by the Folin-Ciocalteu colorimetricmethod [16];– anthocyanins: by the colorimetric method [16];– polysaccharides: by the phenol sulfur method [16]; and– volatile impurities: by gas-liquid chromatography(Crystal 5000, nitrogen carrier gas, flame ionization  detector, PEG-based HP-FFAP column, 50 m, 0.32 mm,dosing device).Pomace treatment before storage. To studythe effect of storage conditions on microbiologicalparameters, the pomace samples were treated using thefollowing methods:– drying at 60–65°C to constant weight in a laboratorydrying oven with forced air convection (AB UMEGAGROUP,Lithuania);– drying at 60–65°C to constant weight in a drying ovenwith infrared radiation (Radiozavod, Russia);– exposing to sulfur dioxide (sulfitation) introduced as aconcentrated solution (at least 1g SO2/kg pomace); and– treating with sodium metabisulfite introduced in tabletform into the lower part of pomace (when decomposed,it produces sulfur dioxide that evenly spreads throughoutthe pomace).RESULTS AND DISCUSSIONMicrobiological studies of fresh and stored grapepomace. We compared the microbiological indicatorsfor fresh and one-month stored pomace samples fromvarious grape varieties obtained by different methods(Table 1). As we can see, fresh sweet pomace had asignificantly smaller amount of micromycetes (includingyeast fungi) and bacteria than fermented pomace. Thiswas because the fermented samples contained wineyeast, which is used for alcoholic fermentation, andlactic acid-reducing bacteria, which are often introducedat the final stage of fermentation. The smallest amountof microorganisms was found in the alcoholized pomace,which is associated with the inhibitory effect of ethylalcohol.We found that the pomace microflora includedmicroorganisms of various classes, species, and genera.Their metabolic interactions involved the transferof metabolites between partners, a producer and ametabolizer. For example, yeast converted residualsugars to ethyl alcohol that was consumed by acetic acidbacteria to produce acetaldehyde and acetic acid. Lacticacid bacteria and yeast have a symbiotic relationship.Yeast stimulates growth in lactic acid bacteria, fortifiesfoods with vitamins, as well as ferments lactose andother sugars to produce antibiotic substances actingagainst pathogenic microorganisms.With changes in environmental conditions,some microorganisms can suspend the processes ofreproduction and fermentation of other species. Somelactic acid bacteria, mainly rod-shaped (a threat towine production), can act antagonistically and destroyyeast cells, for example, in nitrogen-depleted media(pH < 6) [17]. Yeast and acetic acid bacteria stimulategrowth in lactic acid bacteria. Thus, some biochemicalprocesses that occur during storage can significantlychange the chemical composition of grape pomaceand make it unsuitable for production. In particular,pomace microorganisms destroy organic acids andpolysaccharides, basic components of dietary fiber.Moreover, they consume vitamins and vitamin-likesubstances, leading to a significant decrease in bioactivecomponents, so important for the production of extractsand concentrates.The Chardonnay samples can be used to show acorrelation between the method of pressing and thenumber of microorganisms (Table 1). Different pressingequipment produces pomace that varies in moisture. TheBusher Vaslin press (France) provided a higher degreeof pressing and, therefore, a higher mechanical effecton grapes (fresh, fermented or alcoholized) comparedto other presses, resulting in less active microorganismsand fewer colonies.The use of enzyme preparations to produce sweetand fermented pomace led to a decomposition ofmany high-molecular grape components (proteins,polysaccharides, complex compounds) into lowmolecularsubstances easily assimilated by themicroflora. The fermentation increased the concentrationof sugars and nitrogenous substances, stimulating thegrowth of micromycetes and bacteria cells on nutrientmedia.Storing the pomace samples in tarpaulin-coveredcement pits with air access activated microorganismcells, leading to their significant increase, especiallybacteria, in all the experiments.Figure 1 shows colonies of microorganisms in thepomace samples stored for one month in an open area.They were isolated by inoculation on solid nutrientmedia. The average pomace sample contained yeast of the Saccharomyces cerevisiae genus, characteristic ofwine production. Its colonies varied in shape (round,with or without septa, radial or feathery, some with awell-defined inner ring), appearance (shiny or matte, dryor wet, smooth or wrinkled, with smooth or deformededges), surface relief, and thickness. Such a varietywas due to their belonging to different species [12, 14,18–20].Growing on the pomace surface, Candidamycoderma consumes extractives and releases volatilecompounds that give the pomace a pungent taste andunpleasant odor, making it unsuitable for furtherprocessing [12, 14]. Moreover, its enzyme systems canbreak down high-molecular compounds (includingpectin substances), significantly reducing the value ofthe pomace as a secondary raw material.Almost all the samples contained filmy yeasts of theCandida, Pichia, Hansenula, Hanseniaspora/Kloeckera,аnd Torulaspora genera, with their greatest amount infresh pomace of white grape varieties and the smallestamount in alcoholized pomace. Noteworthily, yeasts ofthe Brettanomyces and Schizosaccharomyces genera,which are always present on grape berries, were low inour samples, under 0.7–1.0% [21]. Yeasts of the Pichiaand Hansenula genera were under 1.2%, dependingon the technology of pomace production. The growthof these microorganisms in our pomace samplessignificantly changed their aroma, giving them the tonesof fermentation, ethyl acetate, and sour milk.Debaryomyces yeasts, which we identified in theaverage pomace sample, have a poor ability to absorbsugars, metabolize tartaric, lactic, and citric acids intoesters, synthesize extracellular enzymes, and decomposetoxins [22, 23]. They make the pomace unsuitable forfurther processing.Molds were clearly visible on the pomace surface(3.5–6.4%), namely Mucor, Aspergillus niger, andPenicillium. They are highly undesirable since they canproduce mycotoxins and compounds with unpleasantodors and tastes [24, 25].Prevailing bacteria included acetic acid bacteria(mainly Acetobacter aceti) and lactic acid bacteria(including Lactobacillus plantarum, Pediococcus, andLeuconostoc) amounting to 6–9%, with their greatestincrease in sweet pomace during storage.The greatest growth in microorganisms was inthe sweet pomace samples during storage: yeast cellsconverted sugars to ethanol, which was then used byacetic acid bacteria to synthesize acetic acid. Lactic acidbacteria were especially frequent in fermented pomace.We found that microorganism growth was much greaterin white grape pomace compared to red grape pomace,which is rich in phenolic compounds with antiseptic andantibacterial action [26–28].Microflora also increased in alcoholized pomace,despite 7–10% ethyl alcohol, although not as much asin the other types of samples. With acetic and lacticacid fermentation, alcoholized pomace (e.g. Cabernet-Sauvignon) still retained grape-wine tones in its aroma.Thus, we found that red grape pomace did betterduring storage than white pomace due to the presence ofpolyphenols with antiseptic effects. Alcoholized pomaceshowed the smallest growth in micromycetes.Physicochemical parameters of fresh and onemonthstored pomace extracts. Changes in thephysicochemical parameters of the Traminer Rosepomace extracts (sweet and fermented) are presentedin Table 2.The chemical composition of the extracts (Table 2)showed that microorganism growth in the stored pomacesignificantly decreased the concentration of tartaric,malic, and citric acids, with succinic and ascorbic acidscompletely oxidized. Moreover, the microorganismsconsumed succinic acid and converted it into fumaricand formic acids, disrupting the pomace aroma. Tartaricacid decomposed under the action of Debaryomycesyeast and various lactic acid bacteria (Lactobacillusbrevis, Lactobacillus hilgardii, Lactobacillus plantarum,and heterofermentative cocci), producing diacetyl,acetic, propionic, and lactic acids [29].The above process consumed a large amount ofglycerin. The growth in lactic acid bacteria increased theconcentration of lactic acid and ethyl lactate ester. Citricacid decomposed under the action of enzymes of lacticacid bacteria and molds, producing acetoin and acetone.The growth in acetic acid bacteria and moldssignificantly changed concentrations of volatilecomponents, namely:– ethanol decomposed under the action of enzymesystems of acetic acid bacteria into acetic acid and itsderivatives in the stored pomace extracts, making theirfurther use in wine distillation impossible;– glycerol, which is used by the pomace microflorain the biochemical processes to synthesize newcomponents, decreased 4.4–6.0-fold;– acetaldehyde, acetic acid, and ethyl acetate increased7.3–7.7, 4.2–4.8, and 4.5–5.2 times respectively, allhaving a smell of acetic acid and thus giving the extractsan unbalanced tangy taste;– propionic acid and its ethyl ester were identified in thestored pomace extracts, unlike the fresh extracts;– higher alcohols, especially isoamylol and butanol,significantly increased, making the pomace unsuitablefor distilling grape alcohol due to their pronounced fuseltones.Acetic acid bacteria can oxidize mono- andpolyhydric alcohols (as well as ethyl alcohol),carbohydrates and other substances in the extracts.Monohydric alcohols are oxidized to the correspondingacids (e.g., propanol to propionic acid, butyl alcohols tobutyric acid), increasing their concentrations (Table 2).Non-volatile (extractive) components, namelypolysaccharides, phenolic compounds, and anthocyaninsdecreased 1.7–1.9, 3.7–4.0, and 4.0–4.5 times, dietary fiber and extracts of phenolic compounds fromthe pomace stored under those conditions, lowering itsefficiency 4.5–6.8 times.Thus, our experimental data showed a need todevelop a pomace storage technology that would makepomace suitable for further use in production.Microbiological pomace parameters versus prestoragetreatment methods. Various methods can beused to prepare pomace for storage. They include dryingat various temperatures, treatment with ultraviolet andinfrared rays, electromagnetic waves, regulating thegaseous environment, using chemical preservatives,alcoholization, and others [30–32].Alcoholization is obviously the best preserver ofbioactive components in grapes, but it requires largeamounts of min 25% ethanol.Our microbiological assays involved all types ofthe pomace samples treated by different methods:drying at 60–65°C, infrared drying at 60–65°C, addingsulfur dioxide and sodium metabisulfite (Table 3). Wefound that all the methods decreased contaminationduring storage. Drying at 60–65°C was most effectivein reducing the activity of micromycetes, especiallyin red pomace. Infrared drying had the same effect,but to a lesser extent. It may be necessary to work outoptimal processing modes, in particular, with highertemperatures.Sulfur dioxide and its derivatives decreased thegrowth in micromycetes 75–100 times during onemonth. Bacterial contamination also decreased, butto a lesser extent. Noteworthily, both drying methodswere more efficient than sulfur dioxide and sodiummetabisulfite. Most samples, including alcoholized andsulfitized ones, showed an increase in acetic and lacticacid bacteria at the end of the treatments. This indicatedthat these modes of sulfitation and drying did not ensurecomplete inhibition of the pomace microflora.CONCLUSIONOur experimental data led us to the followingconclusions. The pomace samples were contaminatedwith various microorganisms, whose growth spoiled thepomace. Significant changes in its chemical compositionduring long-term storage can make it unsuitable forfurther use in food production. Available treatmentmethods decreased microorganism contamination, butdid not ensure long-term preservation of the pomace.Sulfur dioxide or sodium metabisulfite can be used forshort-term storage (up to a month). However, thermaltreatment is required for longer storage to inhibitmicroorganism growth.CONTRIBUTIONThe authors were equally involved in writing themanuscript and are equally responsible for plagiarism.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterest.