Expression of Speciﬁc Class I Chitinase mRNA Levels in Di ﬀ erent Grape Varieties and Their Antimicrobial Activity

: The aim of this study was to compare the natural variability of the expression of class I chitinase gene in grapes of Vitis vinifera L. varieties as well as investigate their antimicrobial activity. Further, the e ﬀ ect of antimicrobial activity in chitinase expression was analyzed. Matured berries of thirteen grape varieties were used in the study—Alibernet, Dornfelder, Cabernet Sauvignon, Blaufränkisch, Sauvignon Blanc, Welschriesling, Weisser Riesling, Irsai Oliver, Pinot Blanc, P á lava, Müller-Thurgau, Gr˝uner Veltliner, and Feteasca Regala, grown in the Small Carpathians wine region of Slovakia, Vrbov é . Chitinase I gene expression was found to be similar in most of the analyzed varieties with the except of two varieties, where the expression of chitinase I was signiﬁcantly higher—Pinot Blanc and Savignon Blanc. By comparing the varieties between the lowest and highest expression fold change, they were statistically di ﬀ erent in the level of its chitinase I gene expression. Grape pomace extracts of analyzed Vitis vinifera L. were e ﬀ ective against Gram-positive, Gram-negative bacteria and yeasts, but a weak correlation tendency was found between chitinase I gene expression and antimicrobial activity, only against Staphylococcus aureus subsp. aureus , Candida albicans , and Candida glabrata .


Introduction
Vitis vinifera L., belongs to the oldest cultivated plants and is well adapted to a wide range of climates. The best agroclimatic conditions for its growth are those of the temperate Mediterranean regions up to the continental areas in central Europe. Western Europe is the world's biggest producer of grapes and they are consumed either directly or as wine and the [1]. The health beneficial effects of grapes and wine consumption are well-known. Vitis vinifera L. is valuable/rich source of polyphenols, flavonoids, anthocyanins, phenolic acids, stilbenes, vitamins (A and C), minerals (phosphorus, calcium), and carbohydrates [2,3]. Recently, it has been observed that moderate consumption of grapes or

Chitinase Expression Analysis
A two-step qPCR protocol was used for comparative determination of chitinase mRNA levels where actin (GenBank accession AY847627) was used as the housekeeping gene. Amplification was performed by 5x Hot FirePol EvaGreen (Solis BioDyne, Tartu, Estonia) in Stratagene Mx3005P (Agilent, Santa Clara, CA, USA) under the following cycling conditions: 95 • C for 2 min followed by 40 cycles of 95 • C-10 s, 60 • C-40 s, and final analysis of amplicons dissociation curves. Chitinase primers were designed on the base of class I chitinase [8] with checking the specificity in silico as well as for unique fragment amplification.

Pomace Extract Preparation
The pomace extracts were prepared from a portion of the pomace samples (100 g) immediately freeze-dried after receiving. The samples were extracted with 96% ethanol at 1:10 ratio (m/V) under overnight shaking. The extracts were filtered through Whatman No. 2 filter paper to remove unwanted residues. After evaporating off the organic solvent, the filtrates were dissolved in dimethyl sulfoxide (DMSO) at 40 mg/mL −1 as the stock solution and stored at −20 • C for further investigation [27].

Microorganisms
In our study, nine strains of microorganisms were tested, including three Gram-positive bacteria

Disc Diffusion Method
The agar disc diffusion method was used for the determination of antimicrobial activity of the pomace extracts. Briefly, a suspension of the tested microorganism (0.1 mL of 10 5 cells/mL) was spread onto Mueller Hinton Agar (MHA, Oxoid, Basingstoke, United Kingdom) and Sabouraud dextrose agar (Oxoid, Basingstoke, United Kingdom) at 25 • C. Filter paper discs (6 mm in diameter) were impregnated with 15 µL of the pomace extract and placed on the inoculated plates. Ciprofloxacin (10 µg/disc) and Fluconazole (25 µg/disc) were used as a positive control to determine the sensitivity of the microorganisms under study. The plates were kept at 4 • C for 2 h and after incubated aerobically at 37 • C for 24 h and 25 • C for 48 h for bacteria and yeast, respectively. The diameters of the inhibition zones were measured in millimetres. All the tests were performed in triplicate [28].

Determination of Minimum Inhibitory Concentration
The minimum inhibitory concentration (MIC) is the lowest concentration of the sample that will inhibit the visible growth of microorganisms. Pomace grape extracts were dissolved in DMSO (conc. 20 mg/mL). MICs were determined by the microbroth dilution method according to the Clinical and Laboratory Standards Institute recommendation [28] in Mueller Hinton broth (Oxoid) for bacteria and Sabouraud dextrose broth (Oxoid) for yeasts. Briefly, the DMSO extracts solutions were prepared as serial two-fold dilutions to obtain a final concentration ranging from 3.9 to 2000 µg/mL. The range of resveratrol concentrations tested was 2-512 µg/mL, before the addition of the cells. Each well was then inoculated with microbial suspension at the final density of 0.5 McFarland. After 24 h incubation at 37 • C for bacteria and 25 • C for yeasts, the inhibition of microbial growth was evaluated by measuring the well absorbance at 570 nm in an absorbance microplate reader Biotek EL808 with shaker (Biotek Instruments, Winooski, VT, USA). The 96 microwell plates were measured before and after the experiment. Wells without resveratrol and pomace extract were used as positive controls of growth. Pure DMSO was used as negative control. This experiment was done in eight-replicates for a higher accuracy of the MICs of used pomace grape extracts. The results were expressed in µg/mL [28].

Data Analysis
A qPCR analysis with the biological triplicates was used in the study and the relative expression values were calculated by the delta delta Ct method when the expression of specific chitinase was determined as the number of amplification cycles obtained in the reaching of the threshold during the exponential phase of the PCR. Blaufränkish grape variety was used as a comparator in the analysis. Two factor ANOVA with replication and the correlation of antimicrobial activity for individual microorganisms and expression differences of chitinase I gene were performed. All the calculations were prepared and performed in Microsoft Excel for Windows. A parametric two-tailored T-test was performed for obtaining class I chitinase Ct values [29] using the online platform T-Test Calculator at the significance level 0.05 [30].

Results and Discussion
The expression profiles of class I chitinase in matured grapes of thirteen different varieties (Alibernet, Dornfelder, Cabernet Sauvignon, Blaufränkisch, Sauvignon Blanc, Welschriesling, Weisser Riesling, Irsai Oliver, Pinot Blanc, Pálava, Müller-Thurgau, Grűner Veltliner, Feteasca Regala) were analyzed by delta delta Ct method, firstly. Actin was used for the purposes of correction of sample-to-sample differences as an internal control. Both of the amplicons, actin, as well as chitinase I were amplified in a linear mode with reaching the plateau phase. Dissociation curves of both amplicon types returned single melting peak with a Tm of 87 • C for actin and 86 • C for grape chitinase I (Figure 1), indicating the specific and unique product.
Agronomy 2020, 10, x; doi: www.mdpi.com/journal/agronomy of resveratrol concentrations tested was 2-512 µg/mL, before the addition of the cells. Each well was then inoculated with microbial suspension at the final density of 0.5 McFarland. After 24 h incubation at 37 °C for bacteria and 25 °C for yeasts, the inhibition of microbial growth was evaluated by measuring the well absorbance at 570 nm in an absorbance microplate reader Biotek EL808 with shaker (Biotek Instruments, Winooski, VT, USA). The 96 microwell plates were measured before and after the experiment. Wells without resveratrol and pomace extract were used as positive controls of growth. Pure DMSO was used as negative control. This experiment was done in eight-replicates for a higher accuracy of the MICs of used pomace grape extracts. The results were expressed in µg/mL [28].

Data Analysis
A qPCR analysis with the biological triplicates was used in the study and the relative expression values were calculated by the delta delta Ct method when the expression of specific chitinase was determined as the number of amplification cycles obtained in the reaching of the threshold during the exponential phase of the PCR. Blaufränkish grape variety was used as a comparator in the analysis. Two factor ANOVA with replication and the correlation of antimicrobial activity for individual microorganisms and expression differences of chitinase I gene were performed. All the calculations were prepared and performed in Microsoft Excel for Windows. A parametric twotailored T-test was performed for obtaining class I chitinase Ct values [29] using the online platform T-Test Calculator at the significance level 0.05 [30].

Results and Discussion
The expression profiles of class I chitinase in matured grapes of thirteen different varieties (Alibernet, Dornfelder, Cabernet Sauvignon, Blaufränkisch, Sauvignon Blanc, Welschriesling, Weisser Riesling, Irsai Oliver, Pinot Blanc, Pálava, Müller-Thurgau, Grűner Veltliner, Feteasca Regala) were analyzed by delta delta Ct method, firstly. Actin was used for the purposes of correction of sample-to-sample differences as an internal control. Both of the amplicons, actin, as well as chitinase I were amplified in a linear mode with reaching the plateau phase. Dissociation curves of both amplicon types returned single melting peak with a Tm of 87 °C for actin and 86 °C for grape chitinase I (Figure 1), indicating the specific and unique product. in Blaufränkish: Alibernet and Cabernet Savignon. Expression change between 1% up to the 10% of overexpression was obtained for the varieties Irsai Oliver, Dornfelder, Pálava, and Welshriesling. In two of the analyzed grape varieties, the expression of chitinase I was significantly higher than in others, in Pinot Blanc matured grapes, it was 51.7% and in Savignon Blanc, it was 41.72%. In four of the analyzed grape varieties, the underexpression of chitinase I was found: Feteasca Regala (−5.32%), Weisser Riesling (−1.93%), Grüner Veltliner (−1.84%), and Müller Thurgau (−0.19%). When comparing the varieties with the lowest and highest expression fold change, they were statistically different in chitinase I mRNA expression at the level of confidence 0.05 with the Tuckey HSD p-value of 0.02.
Agronomy 2020, 10, x 5 of 12 Agronomy 2020, 10, x; doi: www.mdpi.com/journal/agronomy Chitinase I expression (Figure 2) was found to be similar with a very small changes up to the 1 % of overexpression for two of analyzed grape varieties compared to the expression of chitinase I in Blaufränkish: Alibernet and Cabernet Savignon. Expression change between 1% up to the 10% of overexpression was obtained for the varieties Irsai Oliver, Dornfelder, Pálava, and Welshriesling. In two of the analyzed grape varieties, the expression of chitinase I was significantly higher than in others, in Pinot Blanc matured grapes, it was 51.7 % and in Savignon Blanc, it was 41.72 %. In four of the analyzed grape varieties, the underexpression of chitinase I was found: Feteasca Regala (−5.32 %), Weisser Riesling (−1.93 %), Grüner Veltliner (−1.84 %), and Müller Thurgau (−0.19 %). When comparing the varieties with the lowest and highest expression fold change, they were statistically different in chitinase I mRNA expression at the level of confidence 0.05 with the Tuckey HSD p-value of 0.02. To date, very limited information exists in literature for grape specific class I chitinase expression and those that exist are in contrary. It was found to be not expressed or to obtain only a faint band after longer exposure in berries of Shiraz, Riesling, Semillon, Muscat Gordo Blanco, Sultana, Pinot Noir, Chardonnay, and Cabernet Savignon by northern blots of total RNA [16]. Another study reported chitinase I as strongly expressed in the roots and stem-internodes, lower expressed in berries, and absent of expression in the leaves [14]. The most recent results of grape class I chitinase in the Cardinal variety under the CO2 pre-treatment show its variable expression in the skin tissues of non-treated grape berries and the accumulation of Vcchit1b transcript during low temperature storage was paralleled by the change in total decay [8].
Using the same matured grapes, antimicrobial activity was investigated further. The diameters of the inhibition zones (in mm) corresponding to the tested pomace extracts are shown in Table 1. All assays were carried out in triplicate. Results are expressed as means ± SD. When regarding individual varieties, different antimicrobial activity was obtained for individual tested microorganisms among themselves with significant differences mainly among bacteria and yeasts data obtained. To date, very limited information exists in literature for grape specific class I chitinase expression and those that exist are in contrary. It was found to be not expressed or to obtain only a faint band after longer exposure in berries of Shiraz, Riesling, Semillon, Muscat Gordo Blanco, Sultana, Pinot Noir, Chardonnay, and Cabernet Savignon by northern blots of total RNA [16]. Another study reported chitinase I as strongly expressed in the roots and stem-internodes, lower expressed in berries, and absent of expression in the leaves [14]. The most recent results of grape class I chitinase in the Cardinal variety under the CO 2 pre-treatment show its variable expression in the skin tissues of non-treated grape berries and the accumulation of Vcchit1b transcript during low temperature storage was paralleled by the change in total decay [8].
Using the same matured grapes, antimicrobial activity was investigated further. The diameters of the inhibition zones (in mm) corresponding to the tested pomace extracts are shown in Table 1. All assays were carried out in triplicate. Results are expressed as means ± SD. When regarding individual varieties, different antimicrobial activity was obtained for individual tested microorganisms among themselves with significant differences mainly among bacteria and yeasts data obtained. Both factors, microorganism specie as well as grape variety, were returned in two factor ANOVA as a significant effect in antimicrobial activity (Table 2) at the level 0.05. The most inhibitory activity of GPE was found against Listeria monocytogenes, which corresponds to the results where a strong inhibitory effect against Listeria monocytogenes has been found by grape juice and grape extracts derived from Vitis vinifera variety "Ribier" [31]. The minimum inhibitory concentration that was count as is the lowest concentration of the sample that will inhibit the visible growth of microorganisms are summarized in Table 3.         Only a few chitinases are reported in the literature as proteins with antimicrobial effect [32]. Chitinases were found in many of the organisms, including seed plants such as Carica papaya [33], Arachis hypogaea [34], and Hevea brasiliensis [35]. In the case of Arachis hypogaea, antimicrobial activity against Staphylococcus aureus was described, similar to our results, [36] and chitinases of Carica papaya exhibited antibacterial activity towards E. coli [33]. Class I basic chitinase, which is reported in literature to be generally more antifungal in nature compared to other chitinase classes, is present in vacuoles [35]. Validation of the occurrence of chitinase activity in berry skin extracts was performed and confirmed previously [37]. The present study has demonstrated that GPEs were effective against Gram-positive, Gram-negative bacteria and yeasts. Red wine has been shown to prevent damage to the gastric mucosa induced by Helicobacter pylori, possibly through inhibition of the vacA gene [38]. Another study [39] demonstrated that grape seed extracts have antimicrobial potential, Gram-positive bacteria being more sensitive than Gram-negative bacteria. Our study has also demonstrated that Gram-positive strains were more susceptible to GPE compared with Gram-negative bacteria and yeasts. Recently, it has been reported [40] an antibacterial effect of grape pomace extracts mainly against S. aureus and E. coli: the activity was directly related to the polar phenolic content. Grape seed extracts obtained from wine and table cultivars of Vitis vinifera L. were found to be active against Candida albicans and their activity was related to the presence of polymeric flavan-3-ols [41].

Conclusions
In this work, a first study of natural variability of expression of grape class I chitinase mRNA levels were analyzed among thirteen different Vitis vinifera L varieties as well as antimicrobial activity of their matured berries. Our results indicate, that the expression of the class I chitinase gene is stable among the grape varieties. However, in two of the analyzed varieties, it was expressed higher. Further, it has been demonstrated that grape pomace extracts were effective against Gram-positive, Gram-negative bacteria and yeasts in the case of analyzed varieties and for antimicrobial activity against Staphylococcus aureus susbp. aureus, Candida albicans, and Candida glabrata, weak correlation tendency was found. Further analysis is needed to ascertain the tendencies as well as define it more specifically.
Author Contributions: J.Ž. and M.K. were responsible for the design of the study; J.S., J.Ž. and M.K. conducted the study and collected the samples. L.Z., J.B., V.F., J.S. and S.K. performed the laboratory analysis. J.Ž., L.Z. and M.K. were responsible for writing and editing the manuscript. All authors have read and agreed to the published version of the manuscript.