Introduction. Grape pomace is the most important by-product of winemaking that can be used as an additional raw material. There is a need for an optimal storage technology so that pomace can be further processed to obtain new types of products. We aimed to study the effect of grape pomace treatment on its microflora. Study objects and methods. We identified and quantified microflora on the fresh and one-month-stored pomace samples from white and red grape varieties. The samples were exposed to conventional drying at 60–65°C, infrared drying at 60–65°C, as well as sulfitation with sulfur dioxide and sodium metabisulfite. Results and discussion. The pomace microflora can be considered a microbial community. Almost all the samples stored for one month in an open area contained Saccharomyces cerevisiae yeasts, higher concentrations of filmy yeasts of the Candida, Pichia, Hansenula, Hanseniaspora/Kloeckera, and Torulaspora genera, as well as conidia of Mucor, Aspergillus niger, and Penicillium molds. Prevalent bacteria included acetic acid (mainly Acetobacter aceti) and lactic acid (Lactobacillus plantarum, Pediococcus, Leuconostoc) bacteria. These microorganisms significantly changed concentrations of volatile and non-volatile components, decreasing total polysaccharides, phenolic compounds, and anthocyanins 1.7–1.9, 3.7–4.0, and 4.0–4.5 times, respectively. The contents of micromycetes and bacteria in the one-month-stored samples were significantly higher than in the fresh pomace. Predrying and sulfitation decreased bacterial contamination, but to a lesser extent compared to micromycetes. Conclusion. Long-term storage spoiled pomace, leading to significant changes in its chemical composition. Sulfitation reduced microorganism growth during storage, but did not provide long-term preservation (over a month), while pre-drying at 60–65°C promoted longer storage.
Winemaking by-products, grape pomace, yeast, bacteria, microflora, storage conditions
INTRODUCTION
The accumulation of wine production waste has an
adverse effect on the environmental situation in grapegrowing
regions. Grape pomace is a key winemaking
by-product that can be used as an additional raw
material [1].
Grape pomace is rich in biologically valuable
components, including polyphenols, pectin substances,
and microelements [2, 3]. About 10–15% of this byproduct
is used as a biofertilizer to improve the soil
structure [4]. Grape pomace can also be a source of
dietary fiber, natural food colors, grape alcohol, tartaric
acid, as well as extracts and concentrates [5–8]. Grape
seeds are used to extract grape oil, which is widely used
in cosmetology [9, 10]. Therefore, there is an urgent need
for 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 grapes
after the juice has separated. Such pomace contains
microorganisms and major components of grape berries,
including sugars. Sweet pomace is usually derived from
white grapes during the production of table wines and wine base for sparkling wines and champagne, as well
as from red grapes processed like white grapes (without
maceration or fermentation).
Fermented pomace results from pressing the
fermented grapes during red table wine production. It
contains ethanol (a product of natural fermentation of
grape sugars), organic acids, phenolic, nitrogenous and
pectin substances, aromatic components of wine base, as
well as wine yeast and malolactic fermentation bacteria
used for fermentation and acidity reduction.
Alcoholized pomace is produced through pressing
fermented and alcoholized grapes in the production of
liqueur wines, especially Kagor (fortified dessert wine)
and Muscat wines. The last 15–20 years have seen a
significant decrease in these wines due to a need to use
grape alcohols in their production. Alcoholized pomace
contains ethanol, sugars, and other components of
grapes and wine base, including yeast. According to the
Russian Ministry of Agriculture, alcoholized pomace
accounts for 2.0–3.4% of total pomace, depending on the
volume of liqueur wine production.
Various types of pomace differ in their components
and microflora. Pomace can rapidly deteriorate during
storage due to a combination of nutrients (sugars,
nitrogenous compounds, organic acids, vitamins,
etc.) and air exposed natural grape microflora (sweet
pomace), 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 processed
immediately after its separation. However, sometimes
it 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 must
be stored under appropriate conditions, depending
on the amount, type, and the physiological state of its
microflora.
Grape pomace is usually stored on special
sites, covered with tarpaulin or other material, if
any. 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 others
microorganisms [11–14]. In this regard, the assessment
of its microbiological state is an important part of
pomace disposal, which depends on grape processing
technology and storage conditions.
Our aim was to study the influence of storage
conditions on the microflora of white and red grape
pomace treated with different methods.
STUDY OBJECTS AND METHODS
Sampling and preparation for microbiological
research. We studied fresh and one-month stored
pomace from Vitis vinifera grapes produced in
Krasnodar Krai (Russia), namely: sweet (Chardonnay,
Riesling, Sauvignon Blanc, Traminer Rose, and Pinot
Noir), fermented (Cabernet Sauvignon, Merlot, and
Saperavi), and alcoholized (Traminer Rose, Cabernet
Sauvignon, and Saperavi). The pomace came from
the production of white and red table and liqueur
wines. Some grape processing technologies used
pectoproteolytic enzyme preparations – Trenolin
Blanc and Trenolin Rouge (Erbsloeh Geisenheim AG,
Germany) – in optimal manufacturer-recommended
amounts. The storage temperature varied from 14°C
(at night) to 26°C (at daytime). An average sample was
obtained by mixing equal amounts of samples taken
from the surface of the pomace mass. The samples
were taken from a depth of 0.5 and 1.0 m, placed in
glass flasks, filled with distilled water, and incubated at
22–25°C for two days.
Isolation of microorganisms. The samples were
inoculated and passed on yeast-peptone agar containing
10 g yeast extract, 20 g peptone, 20 g agar-agar, and
20 g glucose per 1 L of water (Research Center for
Pharmacotherapy, Russia). The elective test was
performed on Lysine Medium Base (Himedia, India).
Those isolates that were incapable of growing on the
elective medium were considered as belonging to the
genus Saccharomyces.
We also used solid nutrient media, such as grape
juice agar (2%) alcoholized with ethanol (14% alcohol) –
to identify saccharomycetes, and OFS-agar (Erbsloeh
Geisenheim AG, Germany) – to quantify yeast, mold
fungi, as well as lactic and acetic acid bacteria.
Chloramphenicol (50 mg/L) was introduced into
the media to improve yeast growth and suppress
bacterial growth. Yeast colonies were cultivated at
24 ± 2°C for 6–7 days. Some of them were inoculated on
selective solid nutrient media. During the cultivation, we
monitored the presence of other genera yeast, including
Saccharomyces, Pichia, Hansenula, and Hanseniaspora.
The colonies were microscoped to identify
saccharomycetes and other microorganisms based on
their cultural and morphological characteristics [13, 15].
Generic identification of the isolates was based on their
morphological and biochemical characteristics.
Physical and chemical parameters. The
pomace was extracted with hot water (65–70°C) at a
hydromodule of 1:5. The extracts were analyzed to
determine:
– organic acids: by capillary electrophoresis State
Standard 52841-2007. Wine production. Determination
of 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 it
producing. Method of ethyl alcohol determination;
– phenolic substances: by the Folin-Ciocalteu colorimetric
method [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 study
the effect of storage conditions on microbiological
parameters, the pomace samples were treated using the
following methods:
– drying at 60–65°C to constant weight in a laboratory
drying oven with forced air convection (AB UMEGAGROUP,
Lithuania);
– drying at 60–65°C to constant weight in a drying oven
with infrared radiation (Radiozavod, Russia);
– exposing to sulfur dioxide (sulfitation) introduced as a
concentrated solution (at least 1g SO2/kg pomace); and
– treating with sodium metabisulfite introduced in tablet
form into the lower part of pomace (when decomposed,
it produces sulfur dioxide that evenly spreads throughout
the pomace).
RESULTS AND DISCUSSION
Microbiological studies of fresh and stored grape
pomace. We compared the microbiological indicators
for fresh and one-month stored pomace samples from
various grape varieties obtained by different methods
(Table 1). As we can see, fresh sweet pomace had a
significantly smaller amount of micromycetes (including
yeast fungi) and bacteria than fermented pomace. This
was because the fermented samples contained wine
yeast, which is used for alcoholic fermentation, and
lactic acid-reducing bacteria, which are often introduced
at the final stage of fermentation. The smallest amount
of microorganisms was found in the alcoholized pomace,
which is associated with the inhibitory effect of ethyl
alcohol.
We found that the pomace microflora included
microorganisms of various classes, species, and genera.
Their metabolic interactions involved the transfer
of metabolites between partners, a producer and a
metabolizer. For example, yeast converted residual
sugars to ethyl alcohol that was consumed by acetic acid
bacteria to produce acetaldehyde and acetic acid. Lactic
acid bacteria and yeast have a symbiotic relationship.
Yeast stimulates growth in lactic acid bacteria, fortifies
foods with vitamins, as well as ferments lactose and
other sugars to produce antibiotic substances acting
against pathogenic microorganisms.
With changes in environmental conditions,
some microorganisms can suspend the processes of
reproduction and fermentation of other species. Some
lactic acid bacteria, mainly rod-shaped (a threat to
wine production), can act antagonistically and destroy
yeast cells, for example, in nitrogen-depleted media
(pH < 6) [17]. Yeast and acetic acid bacteria stimulate
growth in lactic acid bacteria. Thus, some biochemical
processes that occur during storage can significantly
change the chemical composition of grape pomace
and make it unsuitable for production. In particular,
pomace microorganisms destroy organic acids and
polysaccharides, basic components of dietary fiber.
Moreover, they consume vitamins and vitamin-like
substances, leading to a significant decrease in bioactive
components, so important for the production of extracts
and concentrates.
The Chardonnay samples can be used to show a
correlation between the method of pressing and the
number of microorganisms (Table 1). Different pressing
equipment produces pomace that varies in moisture. The
Busher Vaslin press (France) provided a higher degree
of pressing and, therefore, a higher mechanical effect
on grapes (fresh, fermented or alcoholized) compared
to other presses, resulting in less active microorganisms
and fewer colonies.
The use of enzyme preparations to produce sweet
and fermented pomace led to a decomposition of
many high-molecular grape components (proteins,
polysaccharides, complex compounds) into lowmolecular
substances easily assimilated by the
microflora. The fermentation increased the concentration
of sugars and nitrogenous substances, stimulating the
growth of micromycetes and bacteria cells on nutrient
media.
Storing the pomace samples in tarpaulin-covered
cement pits with air access activated microorganism
cells, leading to their significant increase, especially
bacteria, in all the experiments.
Figure 1 shows colonies of microorganisms in the
pomace samples stored for one month in an open area.
They were isolated by inoculation on solid nutrient
media. The average pomace sample contained yeast of the Saccharomyces cerevisiae genus, characteristic of
wine production. Its colonies varied in shape (round,
with or without septa, radial or feathery, some with a
well-defined inner ring), appearance (shiny or matte, dry
or wet, smooth or wrinkled, with smooth or deformed
edges), surface relief, and thickness. Such a variety
was due to their belonging to different species [12, 14,
18–20].
Growing on the pomace surface, Candida
mycoderma consumes extractives and releases volatile
compounds that give the pomace a pungent taste and
unpleasant odor, making it unsuitable for further
processing [12, 14]. Moreover, its enzyme systems can
break down high-molecular compounds (including
pectin substances), significantly reducing the value of
the pomace as a secondary raw material.
Almost all the samples contained filmy yeasts of the
Candida, Pichia, Hansenula, Hanseniaspora/Kloeckera,
аnd Torulaspora genera, with their greatest amount in
fresh pomace of white grape varieties and the smallest
amount in alcoholized pomace. Noteworthily, yeasts of
the Brettanomyces and Schizosaccharomyces genera,
which are always present on grape berries, were low in
our samples, under 0.7–1.0% [21]. Yeasts of the Pichia
and Hansenula genera were under 1.2%, depending
on the technology of pomace production. The growth
of these microorganisms in our pomace samples
significantly changed their aroma, giving them the tones
of fermentation, ethyl acetate, and sour milk.
Debaryomyces yeasts, which we identified in the
average pomace sample, have a poor ability to absorb
sugars, metabolize tartaric, lactic, and citric acids into
esters, synthesize extracellular enzymes, and decompose
toxins [22, 23]. They make the pomace unsuitable for
further processing.
Molds were clearly visible on the pomace surface
(3.5–6.4%), namely Mucor, Aspergillus niger, and
Penicillium. They are highly undesirable since they can
produce mycotoxins and compounds with unpleasant
odors and tastes [24, 25].
Prevailing bacteria included acetic acid bacteria
(mainly Acetobacter aceti) and lactic acid bacteria
(including Lactobacillus plantarum, Pediococcus, and
Leuconostoc) amounting to 6–9%, with their greatest
increase in sweet pomace during storage.
The greatest growth in microorganisms was in
the sweet pomace samples during storage: yeast cells
converted sugars to ethanol, which was then used by
acetic acid bacteria to synthesize acetic acid. Lactic acid
bacteria were especially frequent in fermented pomace.
We found that microorganism growth was much greater
in white grape pomace compared to red grape pomace,
which is rich in phenolic compounds with antiseptic and
antibacterial action [26–28].
Microflora also increased in alcoholized pomace,
despite 7–10% ethyl alcohol, although not as much as
in the other types of samples. With acetic and lactic
acid fermentation, alcoholized pomace (e.g. Cabernet-
Sauvignon) still retained grape-wine tones in its aroma.
Thus, we found that red grape pomace did better
during storage than white pomace due to the presence of
polyphenols with antiseptic effects. Alcoholized pomace
showed the smallest growth in micromycetes.
Physicochemical parameters of fresh and onemonth
stored pomace extracts. Changes in the
physicochemical parameters of the Traminer Rose
pomace extracts (sweet and fermented) are presented
in Table 2.
The chemical composition of the extracts (Table 2)
showed that microorganism growth in the stored pomace
significantly decreased the concentration of tartaric,
malic, and citric acids, with succinic and ascorbic acids
completely oxidized. Moreover, the microorganisms
consumed succinic acid and converted it into fumaric
and formic acids, disrupting the pomace aroma. Tartaric
acid decomposed under the action of Debaryomyces
yeast and various lactic acid bacteria (Lactobacillus
brevis, Lactobacillus hilgardii, Lactobacillus plantarum,
and heterofermentative cocci), producing diacetyl,
acetic, propionic, and lactic acids [29].
The above process consumed a large amount of
glycerin. The growth in lactic acid bacteria increased the
concentration of lactic acid and ethyl lactate ester. Citric
acid decomposed under the action of enzymes of lactic
acid bacteria and molds, producing acetoin and acetone.
The growth in acetic acid bacteria and molds
significantly changed concentrations of volatile
components, namely:
– ethanol decomposed under the action of enzyme
systems of acetic acid bacteria into acetic acid and its
derivatives in the stored pomace extracts, making their
further use in wine distillation impossible;
– glycerol, which is used by the pomace microflora
in the biochemical processes to synthesize new
components, decreased 4.4–6.0-fold;
– acetaldehyde, acetic acid, and ethyl acetate increased
7.3–7.7, 4.2–4.8, and 4.5–5.2 times respectively, all
having a smell of acetic acid and thus giving the extracts
an unbalanced tangy taste;
– propionic acid and its ethyl ester were identified in the
stored pomace extracts, unlike the fresh extracts;
– higher alcohols, especially isoamylol and butanol,
significantly increased, making the pomace unsuitable
for distilling grape alcohol due to their pronounced fusel
tones.
Acetic acid bacteria can oxidize mono- and
polyhydric alcohols (as well as ethyl alcohol),
carbohydrates and other substances in the extracts.
Monohydric alcohols are oxidized to the corresponding
acids (e.g., propanol to propionic acid, butyl alcohols to
butyric acid), increasing their concentrations (Table 2).
Non-volatile (extractive) components, namely
polysaccharides, phenolic compounds, and anthocyanins
decreased 1.7–1.9, 3.7–4.0, and 4.0–4.5 times, dietary fiber and extracts of phenolic compounds from
the pomace stored under those conditions, lowering its
efficiency 4.5–6.8 times.
Thus, our experimental data showed a need to
develop a pomace storage technology that would make
pomace suitable for further use in production.
Microbiological pomace parameters versus prestorage
treatment methods. Various methods can be
used to prepare pomace for storage. They include drying
at various temperatures, treatment with ultraviolet and
infrared rays, electromagnetic waves, regulating the
gaseous environment, using chemical preservatives,
alcoholization, and others [30–32].
Alcoholization is obviously the best preserver of
bioactive components in grapes, but it requires large
amounts of min 25% ethanol.
Our microbiological assays involved all types of
the pomace samples treated by different methods:
drying at 60–65°C, infrared drying at 60–65°C, adding
sulfur dioxide and sodium metabisulfite (Table 3). We
found that all the methods decreased contamination
during storage. Drying at 60–65°C was most effective
in reducing the activity of micromycetes, especially
in red pomace. Infrared drying had the same effect,
but to a lesser extent. It may be necessary to work out
optimal processing modes, in particular, with higher
temperatures.
Sulfur dioxide and its derivatives decreased the
growth in micromycetes 75–100 times during one
month. Bacterial contamination also decreased, but
to a lesser extent. Noteworthily, both drying methods
were more efficient than sulfur dioxide and sodium
metabisulfite. Most samples, including alcoholized and
sulfitized ones, showed an increase in acetic and lactic
acid bacteria at the end of the treatments. This indicated
that these modes of sulfitation and drying did not ensure
complete inhibition of the pomace microflora.
CONCLUSION
Our experimental data led us to the following
conclusions. The pomace samples were contaminated
with various microorganisms, whose growth spoiled the
pomace. Significant changes in its chemical composition
during long-term storage can make it unsuitable for
further use in food production. Available treatment
methods decreased microorganism contamination, but
did not ensure long-term preservation of the pomace.
Sulfur dioxide or sodium metabisulfite can be used for
short-term storage (up to a month). However, thermal
treatment is required for longer storage to inhibit
microorganism growth.
CONTRIBUTION
The authors were equally involved in writing the
manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interest.
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