This study aimed to evaluate the microbiota, phytochemical, antioxidant profile and DNA fingerprinting of Cabernet Sauvignon grapes from Slovakia and R. North Macedonia. There were used two samples of grape berries (one sample from Slovakia and one from Macedonia). Each sample was analyzed in triplicate. The bacteria were cultivated on Plate count agar (PCA), microscopic filamentous fungi were cultivated on Malt extract agar (MEA). MALDI-TOF MS Biotyper mass spectrometry was used for the identification of microorganisms (bacteria and yeasts) and microscopic filamentous fungi with manuals. DPPH method was used to determine of antioxidant activity of grape berries. Phytochemical and antioxidant profiles were evaluated in grape berries samples. Total genomic DNA was extracted from mature grapes by GeneJET Plant Genomic DNA Purification Kit. The number of bacteria was higher in the sample of Macedonian grape (4.13 log CFU.g-1) in comparison to the grape from Slovakia as well as the number of yeasts was also higher in the Macedonian sample (2.57 log CFU.g-1). Antioxidant activity of Slovak grape berries was 0.55 mg TEAC.g-1 and of Macedonian grape, berries was 0.51 mg TEAC.g-1. Total polyphenol content was higher in grape from Slovakia (0.81 mg GAE.g-1) than in grape from Macedonia (0.77 mg GAE.g-1), while total flavonoid content was 0.57 and 0.17 mg QE.g-1 in Slovak grape and Macedonian grape, respectively. Total phenolic acid content was higher in the sample from Macedonia (0.40 mg CAE.g-1) compared to the grape from Slovakia (0.24 mg CAE.g-1). Total anthocyanin content was also higher in Macedonian grape (0.46 mg.g-1) compared to the Slovak sample (0.05 mg.g-1). The total polymorphism for all of the used primers of 87.5% was obtained for the Macedonian sample of Cabernet Sauvignon and 89.4% for the Slovak sample.
Grapes have been used for winemaking since the ancient Greek and Roman civilizations (
Biological activities and health-promoting benefits are mostly possessed by polyphenols, which are considered to be the most important phytochemicals of grape. The phenolic compounds mainly include anthocyanins, flavanols, flavonols, stilbenes (resveratrol) and phenolic acids (
From the vineyard to the winery, microorganisms play key roles in wine production and quality. The grapevine (
Microbes could originate from the vineyard soil (
Microbes that are grapevine-associated and are transferred to the must-have a profound influence on wine composition, flavor and quality (
In the study of
Inter Primer Binding Site (iPBS) polymorphism is generated on the biological background of plant pararetroviruses, which primer binding site (PBS) is complementary to the 3′ end of the primer tRNA. In plant retrotransposons, the PBS is either complementary to the 3′ end or an internal region of the primer tRNA. The method of whole genome iPBS amplification is based on the virtually universal presence of a PBS in LTR retrotransposons (
Grape berries contain various microorganisms. Bacteria, yeasts and molds could be identified with MALDI TOF mass spectrometry.
There are many biologically active compounds in grape berries – flavonoids, polyphenols, phenolic acid and anthocyanins.
Two types of grapes were studied in this work: one from Slovakia and one from Macedonia.
The fresh grape berries were used for the preparation of ethanolic extract; 1 g of each sample was extracted with 20 mL of 80% ethanol for 2 h and centrifuged at 4000 g (Rotofix 32 A, Hettich, Germany) for 10 min. The supernatant was used for the measurement of antioxidant activity (DPPH) and the detection of total polyphenol, total flavonoid, and phenolic acid content.
All chemicals were of analytical grade and purchased from Reachem (Slovakia) and Sigma Aldrich (USA).
Radical scavenging activity of samples was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to the procedures described by
The total polyphenol content of extracts was measured by the method of
Total flavonoids were determined using the modified
Total phenolic acid content was determined using a method of
Anthocyanin content was measured according to the method of
A [mg.g-1] = (A∗Mw∗1000)/(
where: A is the absorbance difference = (A520 − A700) pH 1.0 − (A520 − A700), pH 4.5; MW is the molecular weight of (Cy-3-glc) = 449.2 g.mol-1; ε is the extinction coefficient of (Cy-3-glc) = 1700 cm.mol-1; L the absorption; path length : 1 cm.
Five grams of berries from each grape samples were diluted in 45 mL of sterile physiological saline (0.85%). Berries were stirred on a horizontal shaker for 30 minutes. After that, the dilutions of 10-2 and 10-3 were prepared for cultivation with the spread plate method. A 0.1 mL of each dilution (10-2, 10-3) was placed on the surface of a solid cultivation medium. Bacteria were cultivated on Plate count agar (PCA) (Oxoid, UK), yeasts on Malt extract agar base (MEA) (Oxoid, UK) supplemented with bromocresol green (0.020 g.L-1) (Centralchem®, Slovakia). Bacteria were cultivated at 37 °C for 24 – 48 h in aerobic condition, yeasts at 25 °C for five days in aerobic conditions. Growing colonies with macroscopic morphological differences were recultivated on TSA (Tryptic Soy agar, Oxoid®). Inoculated plates were cultivated at 30 °C for 48 h (TSA). After cultivation, the proteins were extracted from fresh bacterial colonies.
One colony of each bacterial and yeast isolate was transferred into an Eppendorf vial and mixed in 300 μL of sterile water. After the addition of ethanol (900 μL), the suspension was mixed and centrifuged (13 000 g, 2 min). After removal of the supernatant, the pellets were dried at room temperature at least for 5 min. The bacterial and yeast pellets were resuspended in 20 – 50 μL of formic acid (70%) and the same amount of acetonitrile. After centrifugation (2 min at 13,000 g), a 1 μL of supernatant was spotted onto a sample position of a polished steel MALDI target plate and dried at room temperature. A 1 μL of MALDI matrix (solution of α-cyano-4- hydroxycinnamic acid (HCCA) in 50% acetonitrile/2.5% trifluoro-acetic acid) was added to the spot and dried.
The MALDI target plate was introduced into the MALDI-TOF mass spectrometer (Bruker, Germany) for automated measurement and data interpretation. MALDITOF profile mass spectra were imported into the MALDI Biotyper 3.0 software and processed automatically after measurement. The logarithm of the score (log[score]) was displayed as the matching result. The MALDI Biotyper output was a log(score) between 0 and 3.0, which was calculated from a comparison of the peak list from an unknown isolate with the reference MSP in the database. A log(score) ≥1.7 indicated identification at the genus level, log(score) ≥2.0 was set as the threshold for a match at the species level. Isolates with ≥2.0 were accepted as a correct identification.
Total genomic DNA was extracted from mature grapes by GeneJET Plant Genomic DNA Purification Kit (Thermo Fisher) following the instructions of the manufacturer. The iPBS primers 1845, 1846 and 1886 were used for the fingerprints amplification (
All experiments were carried out in triplicate and standard deviations for replication as well as T-tests were calculated using MS Excel.
According to many authors, the antioxidant activity of grape berries and wines results mainly from their phenolics, whereas the phenolic content and composition depend on the grape variety, vineyard location, cultivation system, climate, soil types, vine cultivation practices, harvesting time, production process and aging (
DPPH·method is one of the most popular methods for detecting the antioxidant activity of wine (
The antioxidant activity of Slovak grape berries was 0.55 mg TEAC.g-1 and antioxidant activity of Macedonian grape were 0.51 mg TEAC.g-1.
The value of total polyphenols was 0.81 mg GAE.g-1 in grape from Slovakia and 0.77 mg GAE.g-1 in grape berries from Macedonia. Total flavonoids were 0.57 mg and 0.17 QE.g-1 in Slovak and Macedonian grape berries, respectively. Phenolic compounds, which are abundant in grape berries and wines, play one of the most important roles in the quality of grapes and wines. They strongly contribute to the color, mouthfeel and palatability of red wines (
Anthocyanins are natural pigments, responsible for a wide range of colors in grapes and red wines. The anthocyanins in red grapes vary greatly with the species, maturity, production area, seasonal conditions, and yield of the fruit (
Antioxidant activity, total polyphenol, flavonoid, phenolic acid and anthocyanin content of analyzed grape.
Samples | DPPH mg TEAC.g-1 | TPC mg GAE.g-1 | TFC mg QE.g-1 | TPAC mg CAE.g-1 | TAC mg.g-1 |
---|---|---|---|---|---|
|
|||||
0.51 ±0.15 | 0.77 ±0.09 | 0.17 ±0.02 | 0.40 ±0.01 | 0.46 ±0.03a |
Note:
The surface of grape berries represents a comprehensive natural reservoir of bacterial microbiota originating from the surrounding environment (
Microorganisms counts isolated from wine grapes in log CFU.g-1.
Sample | Bacteria | Yeasts |
---|---|---|
|
||
3.57 ±0.29 | 2.34 ±0.27 | |
4.13 ±0.08 | 2.57 ±0.18 |
Grapes have a complex microbial ecology including filamentous fungi, yeasts, and bacteria with different physiological characteristics and effects upon wine production. Some species are only found in grapes, such as parasitic fungi and environmental bacteria, while others can survive and grow in wines, constituting the wine microbial consortium. This consortium covers yeast species, lactic acid bacteria, and acetic acid bacteria (
Bacterial populations are usually several orders of magnitude lower than those of yeasts in sound grapes. Lactic acid bacteria have counts lower than 102 CFU.g-1 (
Table
Microorganisms isolated from wine grape berries.
Grapevine bacteria play a key role not only in plant health but also in crop quality and yields, which can influence the winemaking process (
The yeast populations of grapes generally comprise between 102 and 104 cells.g-1 (
The microbiota of grapes also includes fungi that may dominate under favorable weather conditions accompanied by inefficient phytochemical utilization. Fungal obligate parasites can penetrate through the intact grape skin by their own biochemical and mechanical activities and are responsible for high economic losses. The main species are the oomycete
The variability in polymorphism length was inspected among the Macedonian and Slovak Cabernet Sauvignon grapes using an iPBS markers 1845, 1846 and 1886. The total number of obtained iPBS fragments was 57 which were distributed to 21 levels. The average number of fragments per primer was 9.5. Their size ranged from 378 bp up to the 882 bp. The level of the shortest fragments was present in both of the analyzed varieties for all of the used primers (Figure
Amplification profiles of analysed samples of Cabernet Sauvignon.
The total polymorphism for all of the used primers of 87.5% was obtained for the Macedonian sample of Cabernet Sauvignon and 89.4% for the Slovak sample. The most similar iPBS profiles of Slovak and Macedonian samples of Cabernet Sauvignon grapes were obtained for the primer 1846 (Figure
Analysis of length of obtained 1846 fragments for samples from Moldavia(A) and Slovakia (B) evaluated by software GelAnalyzer.
The analysis of the relationships of obtained iPBS amplicon profiles was performed by the clustering method using the UPGMA analysis (Figure
Dendrogram of obtained iPBS profiles of analysed samples of Cabernet-Sauvignon.
The PBS primed PCR generated markers are reported to be very effective for extensive intraspecific polymorphism detecting, including in the study of clonal variability. Genotyping by iPBS markers was used for finding differences between varieties and their clones as well as one of the tools for grapevine collection management (
In our study, the Slovak grape berries sample contained a higher concentration of polyphenols and flavonoids, but a lower concentration of phenolic acids and anthocyanins in comparison to the Macedonian grape. The number of yeasts and bacteria was higher in grape berries from Macedonia. Weather and cultivation conditions can affect the content of biologically active components as well as microorganisms in grape berries.
The study was supported by the European Community project No 26220220180: Building Research Centre „AgroBioTech".