Ayuso, S.1 Fernández-Pérez, R.2 Moreta, C.1 Tenorio, Carmen and 1Ruiz-Larrea, Fernanda.
1Universidad de La Rioja, ICVV (Instituto de Ciencias de la Vid y del Vino: CSIC, Universidad de La Rioja, Gobierno de La Rioja), Av. Madre de Dios 53, 26006 Logroño (Spain).
2Unidad de Análisis Instrumental, ICVV, Finca La Grajera, 26071 Logroño (Spain).
*Corresponding author: rocio.fernandez@unirioja.es
Article extracted from work presented at Enoforum Web Scientists (March 13, 2023)
Introduction
In the context of circular economy, wineries generate large amounts of grape pomaces whose efficient exploitation is currently an important challenge, and they may become suitable candidates for developing viable and up-graded food and feed products. Grape pomace is estimated to represent around 13 % of the total grape weight used in the winemaking process, and it means that 612,000 tonnes were generated in Spain in 2021 (OIV, 2022). Grape pomace can be used to obtain ethanol and grape seed oil; nevertheless, phenolic compounds still remain in the waste product. These grape phenolic compounds are secondary metabolites synthesized by the plant cells to act as natural defences against ultraviolet radiation or aggression by pathogens. The antioxidant properties of these phenolic compounds and their benefits for human health have been demonstrated (Ferrer-Gallego and Silva, 2022). However, currently no systematic study has been reported on their activity against the growth of antibiotic-resistant commensal bacteria that are found in the intestine of humans and animals. These resistant bacterial strains are carriers of antibiotic resistance genes, which can be disseminated to other commensal and pathogenic bacteria.
Escherichia coli is an important Gram-negative species of saprophytic bacteria of the intestinal microbiota of humans and animals; nevertheless, they may act as opportunistic pathogens under certain circumstances and may develop problematic antibiotic resistances that hinder treatments against bacterial infections. E. coli is as well an indicator microorganism of faecal contamination, antibiotic resistance and dissemination through the food chain (Sanz et al., 2021). Among the numerous properties reported for phenolic compounds, antibacterial activity is included (Álvarez-Martínez et al., 2020). Few studies have been reported on the effect on the growth of antibiotic resistant bacteria, among them phenolic extracts from fresh grape skins and seeds have been shown to possess effect on their growth (Fernández-Pérez et al. 2018, 2020).
The objective of this study was to analyse the chemical composition of six grape phenolic extracts, obtained from grape pomaces generated as winery by-products, and to evaluate their antibacterial activity against susceptible and multidrug resistant E. coli strains with the phenotype of extended spectrum beta-lactamases.
Material and methods
1. Bacterial strains
All strains included in this study (Table 1) were of intestinal origin and were isolated from healthy animals. They belonged to E. coli species and two of them were multidrug resistant and ESBLs (extended-spectrum beta-lactamases) producers.
2. Grape phenolic extracts
A total of six extracts shown in Table 2 were studied. T and M extracts were obtained at laboratory scale from pomaces of red grape vinifications of li L. cv. Tempranillo (2019 vintage in D.O.Ca. Rioja, Spain) and Merlot (2013 vintage in D.O. Ribera del Duero, Spain) respectively. Vinifications were conducted following the traditional method, i.e. fermentations took place in presence of the crushed red grapes, seeds and stems. Pomaces were frozen and stored at -20 ºC until the extraction. These monovarietal T and M extracts were obtained by maceration with the pectolytic product Lallzyme EXV (Lallemand Inc.), homogenization in the Ultraturrax (IKA Werke GmbH & Co. KG. Germany) high shear dispersing machine, and conventional ethanolic extraction with sonication (14 kHz, 20 min). The obtained monovarietal extracts were dried in a laboratory centrifugal evaporator with cold trap Jouan RCT-60. Four commercial natural food and feed supplements of red and white grape phenolic extracts (generously provided by Alvinesa Natural Ingredients, Daimiel, Ciudad Real, Spain) were included in this study as well. These dry phenolic extracts from Alvinesa Natural Ingredients are entirely constituted by phenolic compounds (premium selected blending of monomers, dimers, oligomers, and polymers) from grapes. These extracts are currently approved by the EFSA either for human (55705, 55709 and 55710) or animal (55707) consumption.
3. Chemical analysis of the extracts
Chemical analyses of the six extracts were performed by the Service of Instrumental Analysis of the Institute of Science of Vine and Wine (ICVV, Logroño, Spain). Phenolic profiles were obtained using an UHPLC (ultra-high performance liquid chromatography) chromatograph (Shimadzu Nexera) equipped with a triple quadrupole/ion trap mass spectrometer ABSciex 3200QTRAP®, and an atmospheric pressure ionization source (ESI). A Waters Acquity UPLC BEH C18 (100 mm x 2.1 mm, 1.7 mm) column was used. Data processing was performed with the software package Analyst® 1.6.2 and MultiQuant® 3.0.2 (AB Sciex, USA). Chemical profiles of the phenolic extracts included 32 phenolic compounds, which were grouped into eight families: flavonols, anthocyans, hydroxybenzoic acids, hydroxycinnamic acids, flavan-3 ols, procyanidins, stilbenes and lignans.
4. Antibacterial activity
The potential antibacterial activity of the phenolic extracts of our study was tested by the microtiter dilution assay method (Rojo-Bezares et al. 2007) against the susceptible and multidrug resistant E. coli strains shown in Table 1, and minimal inhibitory concentration (MIC) values were determined. Briefly, double serial dilutions of the phenolic extracts in water were prepared in the concentration range from 6.3 to 0.2 mg/ml for T and M extracts, and from 8 to 0.13 mg/ml for the commercial extracts, in concordance with their solubility in water. They were tested in bacterial cultures in BHI broth at 30 ºC in the Tecan Spark® 10M multimode microplate reader for 24 h. Optical density at 600 nm was measured to determine bacterial growth. Experiments were performed in triplicate, and positive and negative controls were included in all the assays. Bactericide activity was determined by subculturing the samples for 48 h onto BHI-agar plates without the tested extract, and minimal bactericide concentration was defined as the minimal concentration of the antimicrobial agent that had killed more than 99.9% of the initial inoculum after incubation for 48 onto BHI-agar plates.
5. Statistical analysis
Statistical analysis of the results obtained in triplicate was performed to determine mean values, standard deviations, and significant differences. Principal component analysis (PCA), analysis of variance (ANOVA) and post-hoc analysis using Tukey’s test at a significance level of p=0.05 were used. IBM-SPSS Statistics 22.0 for Windows (IBM-SPSS Inc., Chicago, IL, USA) was used for data processing.
Results and Discussion
1. Phenolic composition of the extracts
Chemical analysis by UHPLC of the six studied extracts identified 32 phenolic compounds that were grouped into the following phenolic families: hydroxycinnamic acids, hydroxybenzoic acids, flavonols, flavan 3-ols, procyanidins, stilbenes, lignans and anthocyans (Table 3).
2. Antibacterial activity of the phenolic extracts
All the phenolic extracts of our study showed antibacterial activity against the intestinal E. coli strains of animal origin that were tested: two multidrug resistant E. coli strains (C6840 and C7577) and two antibiotic susceptible E. coli strains (C7067 and C7023). Figures 2 and 3 show the resulting growth curves of the antibacterial assays of each of the phenolic extracts against the resistant strain C6840. These are representative growth curves of the assays that were obtained against the four E. coli strains of our study. A direct correlation between the concentration of the phenolic extract in the culture broth and the decrease of bacterial growth was evidenced by the results in all the assays.
It should be noted that the observed effect was bacteriostatic in all cases, and no bactericidal activity was detected in the studied range of extract concentrations, as bacterial growth was recovered after removing the phenolic extract from the culture broth and subculturing the sample for 48 h. Gram-negative bacteria possess an additional outer membrane containing lipopolysaccharides that covers the bacterial cell wall (Silhavy et al., 2010), therefore they can show higher resistance to antimicrobial agents that should reach the cell wall and the membrane to exert their bactericidal activity. This could be the case of the E. coli strains of our study, which were inhibited in their growth in presence of the phenolic extracts, but recovered after removing the active polyphenols from the culture broth.
Table 4 shows the MIC values obtained for each phenolic extract against our collection of E. coli strains. Results were slightly different depending on the assayed phenolic extract. Thus, extract 55710 showed higher antibacterial activity than the other extracts, followed by extract 55707, and both extracts were especially active against the resistant E. coli strains. These antibacterial activities correlated with the results of phenolic composition in that extract 55707 contained the highest total amount of phenolic compounds, it comprised the highest contents of flavan 3-ols and procyanidins, and both phenolic families were discriminating variables in the PCA. Extract 55710 followed in total phenolic content and showed the highest contents of anthocyans and stilbenes, both discriminating variables that followed flavan 3-ols and procyanidins in relevance in the PCA.
These results are in agreement with previous studies of our group that reported inhibitory activity of Tempranillo skin extracts from fresh grapes (MIC 6.3 mg/mL) against E. coli strains (Fernández-Pérez et al., 2018). Our results are also in accordance with studies on the activity of polyphenols against E. coli strains, that reported MIC values in the range of 6-50 mg/mL for flavan-3-ols and slightly higher values, in the range of 10-100 mg/mL, for anthocyans from a variety of vegetable sources (Ma et al., 2019).
In summary, the overall results of this work show that the studied phenolic extracts exert an inhibitory effect on the bacterial growth of intestinal E. coli strains, both sensible and resistant to antibiotics, and that it is not a bactericide effect. In addition, it is shown that grape pomaces are rich sources of bioactive phenolic extracts, and they can become important feedstocks for additives and other upgraded products of interest in animal farming.
Acknowledgments
Financed with the project ADER2019-I-IDD-00048 of the Government of the Autonomous Region of La Rioja and FEDER of the E.U. We also acknowledge Alvinesa Natural Ingredients for generously providing their grape pomace extracts, Lallemand Inc. for providing their pectolytic product, and the Technology Transfer Office (OTRI) of the University of La Rioja for their administrative support.
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