Evaluation of the Antimicrobial Capacity of a White Grape Marc Extract Through Gastrointestinal Digestion

Abstract

Polyphenols are extensively studied for their antimicrobial and prebiotic properties, but concerns about their stability persist. In order to elucidate the antimicrobial stability of such molecules in the gastrointestinal environment and their potential effect as antimicrobials and microbiota modulators, a white grape marc extract from the variety Albariño has been exposed to simulated digestions. In vitro digestions were performed following the INFOGEST protocol and samples were taken after each digestive phase and submitted to bacterial resazurin viability assays. The results reveal that the extract presents a potential antimicrobial effect against foodborne pathogens, such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella enterica, which is enhanced during the intestinal phase. Modulation of the bacterial growth at concentrations below 2% (v/v) of the extract against pathogenic bacteria was observed. Although gastrointestinal digestion reduces the extract’s polyphenolic content, with procyanidin and quercetin-3-glucoside identified as the most unstable compounds, cell viability assays confirmed that its antimicrobial efficacy is maintained. In conclusion, the Albariño marc extract demonstrates a promising microbial modulation capacity, which persists during the digestive process despite variations in the polyphenolic composition.

1. Introduction

Polyphenols are chemical compounds produced by plants as secondary metabolites to be used as self-defenders [1]. Because of self-protection activities against ultraviolet radiation or pathogenic microorganisms among other factors, polyphenols have become a focal point of interest in nutrition and new drug design [2]. Polyphenols have also proved their antimicrobial activity via different mechanisms of action, such as the inhibition of the cell wall synthesis, the disruption of the cell membrane permeability with the subsequent leakage of cytoplasmic content, DNA damage, or the inactivation of key resistant enzymes and surface proteins [3]. Preclinical and clinical studies strongly suggest that the long term consumption of polyphenol-rich diets offers protection against the development of various chronic diseases, due to the crucial role these compounds play in metabolism regulation and cell proliferation [4]. Flavanols, such as catechins and their derivates, are the most abundant polyphenols in human diets, mainly consumed in fruits and beverages [5], with the average total polyphenol intake being approximately 1 g per day [6].

Moreover, one of the biggest challenges related to the consumption of polyphenols is their low bioaccessibility and bioavailability [7]. Polyphenol bioaccessibility depends on the food matrix, basically conformed by dietary fibre [8], which has a fundamental role in the metabolism of the gut microbiota and the beneficial metabolites it produces [9]. On the other hand, low polyphenol bioavalability is not only related to molecular size and chemical composition, but also to interactions with other food components, the effect of the gastrointestinal environment, and degradation by the microbiota [10]. Bioavailability studies on humans have detected very low plasma concentrations of polyphenols after ingestion, rounding to 1 μM after the ingestion of 10–100 mg of an individual phenolic compound [5].

Among dietary sources of polyphenols, grapes and their derivatives are some of the most widely studied [11]. Various studies have proved their bioactivities, which include anticancer [12], anti-inflammatory, antioxidant [13], antimicrobial, and neurodegenerative and antiaging protection [14,15]. Although fruits and wine are extensively consumed, non-commonly consumed parts of the grape, such as the seeds and skins, contain the majority of polyphenols [16], mainly catechins, proanthocyanidins in all kinds of grapes [9], and tannins and stilbenes in red ones [17]. Because of the large number of polyphenols retained in the usually wasted grape parts, rich polyphenolic extracts produced using grape byproducts are becoming very popular. Previous research conducted on the antimicrobial and antioxidant activity of polyphenol-rich grape extracts suggests promising applications as antibiotic coadjutants, foodborne pathogen controllers, and potential sources of nutraceuticals [18].

A previous study assessed the antimicrobial activity of an Albariño grape marc extract produced using a hydro-organic solvent. This extract, which was reported to contain a high catechin concentration, showed good potential as a bacterial controller in nosocomial environments due to its effectiveness against multidrug-resistant bacteria [16].

Even though plenty of in vitro investigations to assess the bioactivities of grape extracts are being conducted, very few of them consider the behaviour and stability of the polyphenolic compounds under digestive conditions. As mentioned above, the polyphenols found in abundance in our diets may not necessarily possess the optimal bioavailability [5,7,8]. Therefore, it is important not only to know the quantity of polyphenols in a particular food or extract, but also to understand their potential bioavailability and behaviour once consumed. Although it is well known that gastrointestinal digestion influences and transforms polyphenol stability and bioaccessibility [19], no previous research has analyzed the antimicrobial behaviour of a grape marc extract during gastrointestinal digestion.

The present study analyzes the behaviour of an Albariño grape marc extract during in vitro digestion to assess its bioaccesibility and antimicrobial activity under gastrointestinal conditions. Samples of the gastrointestinal content harbouring the extract taken at different digestive stages were tested for their antimicrobial activity against some relevant foodborne pathogens, i.e., Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Listeria monocytogenes.

The findings of this study highlighted the potential of white grape marc as a valuable source of bioactive and antimicrobial compounds, exhibiting good bioaccessibility and stability throughout in vitro gastrointestinal digestion.

2. Materials and Methods

2.1. Materials

MS grade methanol, MS water, and formic acid were purchased from Merck (Steinheim, Germany). Individual polyphenol standard stock solutions (1000–10,000 μg mL⁻¹) were prepared in methanol. Working solutions were prepared via dilution in 50:50 (v/v) methanol/water. The stock and working solutions were stored in a freezer at −20 °C and protected from light. All solvents and reagents were of analytical grade. The target polyphenols and their identification CAS numbers are summarized in Table S1.

In vitro digestions were designed following the INFOGEST protocol. CaCl₂, HCl, Pepsin (0.8 FIP U/mg), and Pancreatin (36,000 FIP U/g) were purchased from ITW reagents (Barcelona, Spain). Bile salts were mimicked using bile extract porcine from Sigma-Aldrich (St. Louis, MO, USA). Bacterial culture broth and Tryptone Soy Agar (TSA) were purchased from Condalab (Madrid, Spain). Cation-Adjusted Müller–Hinton II broth (CAMHB) was from Becton Dickinson (BBL, Sparks, NV, USA). AlamarBlue from ThermoFisher Scientific (Waltham, MA, USA), was employed as an enzymatic substrate for the resazurin cell viability tests.

2.2. Albariño Marc Extract

The grape marc extract was produced by i-Grape Laboratory S.L. using Albariño grape marc from the Mar de Frades winery (Pontevedra, Spain). The extract was obtained through the Medium Ambient Scale Temperature (MSAT) system, under the patented procedure by Lores et al. [20].

2.3. INFOGEST Static In Vitro Simulations

In vitro gastrointestinal digestion was performed following the INFOGEST^® 2.0 protocol under strict sterile conditions [21].

The oral phase was skipped because of the liquid nature of the extract. The extract (25 mL) was mixed with simulated gastric fluids (1:1 v/v), the pH was adjusted to 3.0 with 1 N HCl and 12.5 μL of 0.3 M CaCl₂, and 2000 U mL⁻¹ of pepsin was added. Gastric phase digestion was carried out for 2 h under constant shaking (100 rpm) at 37 °C using an Innova 4340 incubator (Rome, Italy). The intestinal phase started with the resulting volume (20 mL), which was mixed with an equal volume of simulated intestine fluid: 40 μL of 0.3 M CaCl₂, 5 mL of a pancreatin solution (800 U mL⁻¹), and bile salts at a concentration of 10 mM in the final mixture. The pH was adjusted to 7.0 and the assay was incubated for 2 h in the same conditions previously described for the gastric phase. Aliquots of 5 mL were recovered after each phase, filtered through 0.22 μm membranes, kept in ice to inactivate the enzymes, and subsequently exposed to analytical and antimicrobial tests.

Samples of both gastric and intestinal fluids were used as blanks for chromatographic and antimicrobial analysis.

2.4. Target Polyphenols’ LC-MS/MS Characterization

The quantification of the polyphenols in the extracts and in the digestates was performed via LC-MS/MS using a Thermo Scientific (San Jose, CA, USA) instrument based on a TSQ Quantum Ultra^TM triple quadrupole mass spectrometer equipped with a heated electrospray ionization (HESI) source, and an Accela Open autosampler with a 20 µL loop. The optimal instrumental conditions were previously optimized by Celeiro et al. [22] to obtain the best chromatographic separation of the target polyphenols. The chromatographic separation was performed employing a Kinetex C18 column (100 mm 2.1 mm 100 Å) obtained from Phenomenex (Torrance, CA, USA). The mobile phase was composed of water (A) and methanol (B), both with 0.1% formic acid. The chromatographic gradient was set at 5% B, reaching 90% B in 11 min and kept constant for 3 min. The initial conditions were achieved in 6 min. The injection volume was 10 µL, with a flow rate of 0.2 mL min⁻¹. The column temperature was set at 50 °C. Compound identification and detection were performed via selected reaction monitoring (SRM) working simultaneously in both negative and positive modes, monitoring two or three MS/MS transitions for each compound. Other HESI source parameters were the spray voltage: 3000 V, vaporizer temperature: 350 °C, sheath gas pressure: 35 au (arbitrary units), ion sweep and auxiliar gas pressure: 0 and 10 au, respectively, and the capillary temperature: 320 °C. The system was operated by Xcalibur 2.2. and Trace Finder 3.1. software. External calibration was used for the quantification of polyphenols. Linearity was evaluated at a wide range of concentrations from 0.01 to 10 μg mL⁻¹ (8 levels and 3 replicates per level), employing standard solutions prepared in water/methanol (50:50 v/v). The obtained coefficients of determination (R²) were, in all cases, higher than 0.9900. Three replicates of each sample were processed for analysis.

2.5. Bacterial Strains and Culture

Bacterial strains of Escherichia coli ATCC 25922, Salmonella enterica subsp. Enterica CECT 554, Staphylococcus aureus ATCC 25923, and Listeria monocytogenes CECT 4032 were purchased from the Spanish Type Culture Collection. They were cultured on TSA plates and incubated for 24 h at 37 °C.

2.6. Antimicrobial Activity

In order to determine the antibacterial activity of the extract, a viable cell assessment via fluorometric reading was employed. EUCAST inhibitory assay considerations were followed with slight modifications to avoid pH and bile salt resazurin interferences [23,24]. A bacterial suspension was prepared by diluting a 0.5 McFarland standard (~10⁸ CFU/mL) in CAMHB to a final concentration of 10⁶ CFU/mL. Subsequently, 100 μL of this bacterial suspension was mixed with 40 μL of extract or digestate at different concentrations (0%, 0.625%, 1.25%, 2.5%, 5%, and 10%, v/v), and 60 μL of PBS (1 M), resulting in a final well volume of 200 μL. The final number of viable bacterial cells per well was 5 × 10⁵ CFU, as recommended by the EUCAST guidelines. Concentrations of the extract and digestates were calculated as a percentage relative to this final volume. The microplate was incubated for 21 h at 37 °C. A blank of the extract was employed by incubating 100 μL of CAMH broth instead of the bacterial inoculum. After the incubation, 100 μL of fresh culture broth, 60 μL of phosphate-buffered saline (PBS, 1 M), 20 μL of AlamarBlue, and 20 μL of each well from the overnight incubated plate were mixed in a new 96-well microplate. The plates were incubated at 37 °C until blue to pink colorimetric change was observed, normally from 30 min to 2 h. Fluorometric reading was performed to determine the viability of the cells, as previously described by multiple authors [25,26,27,28,29,30]. Fluorescence assays were conducted with measurements taken at an excitation wavelength of 544 nm and an emission wavelength of 590 nm, utilizing the FLUOstar microplate reader (BMG Labtech, Ortenberg, Germany). The same procedure was followed for the evaluation of the gastrointestinal content and control samples. Each sample was tested in triplicate in order to obtain statistical significance data to calculate the IC50 (minimum concentration required to inhibit 50% of the bacterial population) and MBC values (minimum bactericidal concentration) [25,26,27,28,29,30].

2.7. Statistical Analysis and Visualization

The concentration of target polyphenols in the samples was expressed as the mean concentration (mg/L ± SD) measured in each sample as illustrated in Figure 1. Statistical differences between the target polyphenol concentrations in each digestive phase were analyzed via a two-way ANOVA Tukey’s test,

Table 1. Target polyphenol variance observed during Albariño grape marc extract gastrointestinal digestion. Data are expressed in mg/L as average and standard deviation of three measured samples. Ce; crude extract. GDe; gastric digestion of the extract. IDe; intestinal digestion of the extract. Different letters in the same row indicate that the mean values are significantly different between them (p < 0.05).

Polyphenol	Ce	GDe	IDe
Gallic acid	31 ± 1.8 a	19 ± 1.4 b	13 ± 0.25 b,c
2-4-6-trihydrobenzoic acid	18 ± 4 a	10 ± 1.5 b	7 ± 0.91 b,c
Caftaric acid	4 ± 0.7 a	2 ± 0.2 a	1 ± 0.06 a
Procyanidine B1 + B2 + C1	302 ± 8.1 a	241 ± 18.5 b	104 ± 2.52 c
Catechin	50 ± 1 a	37 ± 1.2 b	21 ± 0.48 c
Epicatechin	30 ± 0.2 a	27 ± 0.8 a	17 ± 0.38 b
Epicatechingallate	14 ± 0.2 a	12 ± 0.9 a	4 ± 0.22 b
Quercetin-3-glucuronide	54 ± 1.3 a	23 ± 1.2 b	11 ± 0.24 c
Quercetin-3-rutinoside	2 ± 0.1 a	2 ± 0.4 a	1 ± 0.37 a
Quercetin-3-glucoside	167 ± 4.6 a	71 ± 4.1 b	24 ± 0.45 c

. The IC50 values (minimum concentration required to inhibit 50% of the bacterial population) and MBC values (minimum bactericidal concentration) were compared to the untreated control and expressed as means ± SD of bacterial growth (%). The IC50 values were calculated using Graphpad Prism 9.0. Statistical differences in the antimicrobial activity of digestive treatments were analyzed using a one-way ANOVA test (Figure 2c–f). The bacterial growth trends across crude extract (Ce), gastric digestion of the extract (GDe), and intestinal digestion of the extract (IDe) treatments were analyzed via two-way ANOVA followed by Tukey’s test (Figure 2g–j). The Pearson correlation matrix (Figure 3) was produced using the concentration of target polyphenols detected in each phase (mg/L) and their corresponding IC50 values. The MBC values were not included in the correlation analysis, as the data were presented in intervals. The IC50 interval data (e.g., Ce E. coli IC50) were reported using the full interval values. Statistical significance was considered at p < 0.05 for all analyses and graphically represented as ns, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; and **** p ≤ 0.0001. All statistical tests were performed using GraphPad Prism version 9.0.

The illustrative schemes in Figure 2 and Figure 3 were created in BioRender (https://www.biorender.com/). Licence agreement FK286W1S36.

3. Results and Discussion

Dietary polyphenols are a diverse group of compounds found in fruits, vegetables, and beverages [31]. They are widely studied due to their broad range of biological activities, largely attributed to their inherent antioxidant properties [32]. Due to their association with promoting overall health, there has been a heightened demand for polyphenolic nutraceutical compounds [33]. Consequently, there has been growing research into identifying new polyphenol sources, developing innovative extraction methods [18], and improving compound stability [34]. These improvements have enhanced the value of polyphenol-rich extracts derived from agro-industrial byproducts [35,36,37]. Despite well-documented bioactivities, the application of polyphenols is often limited by their low bioavailability [10]. To address this challenge, in vitro digestion models are favoured due to their speed, cost-effectiveness, and reproducibility, often showing good correlation with in vivo results [38]. Therefore, the effectiveness of food fortification and the use of polyphenols as antimicrobial agents should be evaluated using in vitro bioaccessibility and bioavailability simulations to determine the optimal formulations and concentrations.

This study aimed to assess the antimicrobial activity of an Albariño grape marc extract across different stages of in vitro gastrointestinal digestion, focusing on the relationship between polyphenol bioaccessibility and antimicrobial efficacy.

Figure 1a and Table 1 summarize the quantification of key polyphenols in the extract. The undigested extract exhibited a rich polyphenolic profile, particularly high in procyanidins (302 mg/L) and quercetin-3-glucoside (167 mg/L), followed by quercetin-3-glucuronide (54 mg/L) and catechin (50 mg/L). Epicatechin gallate and phenolic acids such as gallic acid (31 mg/L) were also present in lower concentrations. These results are consistent with previous evaluations of white grape marc extracts, which generally contain more flavan-3-ols (e.g., procyanidins) than flavonols [16]. Previous work by our group found that the bioaccessibility of polyphenols from white grape marc increased during gastric digestion, but declined during intestinal digestion [39], possibly due to interactions with bile salts, which prevents polyphenol detection. Although white grape marc extracts have previously been evaluated for their antimicrobial properties [16,40] and bioaccessibility, there are no studies, to our knowledge, that specifically assess the persistence of antimicrobial activity after digestion. To better understand the present study, it is important to take into consideration that the polyphenol quantification of the samples is affected by two factors acting in the opposite way: firstly, the dilution of the samples after gastric (×2) and intestinal (×4) digestion would dilute the concentrations of the molecules; secondly, as has been previously reported [39], the amount of some polyphenols can increase during gastric digestion, due to their liberation from insoluble complexes present in the extract when it is exposed to acidic conditions. Since the goal of this research was to assess the antimicrobial activity at each digestion stage, no dilution corrections were applied and only the soluble fractions, representing the potentially bioavailable polyphenols, were analyzed for their composition and antimicrobial activity (Figure 2b).

Table 1 and Figure 1b show the variation in target polyphenols during digestion. Despite a reduction in the phytochemical content throughout digestion, the amount of many phenolics, particularly catechins, remained relatively stable after gastric exposure. These findings align with those of Sánchez-Velázquez et al., who noted increased polyphenol and flavonoid contents after the gastric digestion of blackberry extracts, likely due to the pH and enzymatic effects [19]. However, intestinal digestion consistently reduced polyphenol bioaccessibility, a trend supported by multiple in vitro studies [20,41,42,43,44].

Regarding the antimicrobial activity, the extract exhibited strong bactericidal effects. As shown in Figure 2a, its effects were more pronounced against Gram-positive bacteria (L. monocytogenes and S. aureus), with the complete eradication of S. aureus at concentrations below 1% (v/v), and approximately 5% for L. monocytogenes. On the other hand, Gram-negative strains (E. coli and S. enterica) were less susceptible, requiring concentrations exceeding 10% (v/v). This aligns with other studies that also reported the greater sensitivity of Gram-positive bacteria to white grape pomace extracts [45]. This differential sensitivity may be linked to the protective lipopolysaccharide layer in Gram-negative bacteria [40]. However, previous work by Rama et al. [17] demonstrated that catechin-rich white grape extracts effectively inhibited E. coli and S. enterica at concentrations below 1%, suggesting polyphenol–bacterium specificity. According to a recent review on the polyphenols’ antimicrobial potential, this antimicrobial activity is influenced not only by bacterial cell wall structures, but also by the polyphenols’ chemical composition and structure, including hydroxyl radicals, molecular size, and degree of polymerization, which affect their ability to disrupt bacterial membranes and inhibit key enzymes [3]. Furthermore, polyphenols may generate oxidative stress within bacterial cells, interfere with quorum sensing, and disrupt biofilm formation [3,40]. These multifaceted modes of action explain why certain polyphenol-rich extracts show selective efficacy depending on the bacterial species and even strain.

During this research, the bactericidal activity was mostly maintained after in vitro gastrointestinal digestion, especially at certain concentrations; however, slight differences were observed among phases and strains. The antimicrobial activity of the samples taken directly after the digestive phases are collected in Figure 2c–f. Control tests using gastric (GF) and intestinal fluids (IF) revealed increased bacterial growth, particularly for Gram-positive bacteria and S. enterica. Concretely, resistance to gastric fluids was observed for L. monocytogenes (Figure 2c) and S. enterica (Figure 2f) during fluidic control assessment. This phenomenon was previously reported by Akritidou et al., who observed that these foodborne pathogens are very resistant to the gastrointestinal environment, proving that the effects of gastric acidity and bile acids alone could not prevent bacterial survival [46]. Gastric fluids are traditionally considered bactericidal at pH < 3.0 [47], although bacterial overgrowth can occur if the pH exceeds 4.0 [48]. Although digestive fluids alone can inhibit the growth of most pathogenic bacteria, for the particularly resistant strains we tested, the presence of the polyphenolic extract both in the stomach and the intestine presents marked antimicrobial effects in vitro. In order to compare the antimicrobial stability of the digested extract with the crude sample, conventional inhibitory values, the IC50 and MBC values, were calculated. The inhibitory values are collected in

Table 2. Antimicrobial activity observed after foodborne pathogens’ exposure to the Albariño grape marc extract before and after gastrointestinal digestions. The first column collects the date for the crude extract (Ce), and subsequent columns refer to the assays performed after the extract’s exposure to each digestive process. GDe: gastric digestion of the extract; IDe: intestinal digestion of the extract. IC50 refers to the minimum concentration needed to eradicate 50% of the pathogen population. MBC refers to the minimal bactericidal concentration. Data are expressed in % of extract volume. Assays were performed in triplicate.

Strains		Ce	GDe	IDe
E. coli	IC50	>10%	4.58%	≤0.625%
	MBC	>10%	>10%	≥10%
S. aureus	IC50	<0.625%	1.22%	0.72%
	MBC	≤0.625%	<2.5%	<2.5%
S. enterica	IC50	4.98%	4.84%	1.67%
	MBC	>10%	>10%	≥10%
L. monocytogenes	IC50	3.81%	7.19%	2.04%
	MBC	≤5%	≤10%	≥10%

. Additionally, Figure 2g–j display the bacterial growth trends under different concentrations of crude and digested samples, the measurements needed for inhibitory value determination. Notably, S. aureus was the most sensitive bacteria, showing strong inhibition and bactericidal effects across all conditions. In contrast, E. coli displayed resistance to the crude extract (IC50 and MBC > 10%), but its susceptibility increased following intestinal digestion (IC50 ≤ 0.625%). Similarly, the inhibitory effect against S. enterica improved post-intestinal digestion, although the bactericidal activity remained invariable (MBC ≥ 10%). L. monocytogenes showed moderate sensitivity, with some fluctuations throughout the digestive phases.

In general, the inhibitory activity of the extract increased post digestion (Table 2), especially against Gram-negative strains, where the IC50 values decreased to a third. However, no differences were observed in the MBC values. Nevertheless, in the case of Gram-positive foodborne bacteria, the inhibitory activity after gastric exposure to L. monocytogenes decreased notably (Figure 2g). And the same phenomenon was observed for S. aureus (Figure 2h). These findings suggest that digestion can alter the bioactivity of the compounds present in the extract, enhancing or reducing their antimicrobial potential depending on the bacterial target. Interestingly, a quantitative reduction in polyphenols did not correlate directly with a decrease in the antimicrobial activity, especially against Gram-negative bacteria. Figure 3 shows Pearson’s correlation matrix of polyphenols’ variation and the antimicrobial IC50 values in crude and digested extracts. A negative correlation, e.g., higher polyphenol concentration–lower IC50 values, is represented in lighter colours, meanwhile a positive correlation is represented in dark greyish green. As has been observed, the extract’s antimicrobial activity against S. aureus is potentially linked to quercetin-3-glucoside (Figure 3a). However, the IC50 value was maintained after digestion despite quercetin-3-glucoside reduction. This suggests a synergistic effect among polyphenols, where the combined presence of multiple compounds maintains the antimicrobial activity even when individual concentrations decline (Figure 3c). Such synergistic interactions have been reported for grape-derived polyphenols, enhancing antimicrobial efficacy through cooperative effects that may influence bacterial growth and metabolism [11,31]. E. coli’s sensitivity to the extract increased after digestion. This may be linked on the one hand to the presence of gallic acid, procyanidins, epicatechingallate, and quercetin-3-glucuronide in IDe, but on the other hand to a higher sensitivity to the gastrointestinal conditions with respect to the rest of the tested strains, which is reflected in Figure 2e. These same polyphenols have been linked to L. monocytogenes IC50 maintenance after IDe digestion (Figure 3c) despite the overgrowth observed because of gastric fluid stimulation (Figure 2c,g). The IC50 values for S. enterica improved after digestion, with this sensitivity being linked to the presence of procyanidins, epicatechingallate, and quercetin-3-glucuside (Figure 3c).

This rational analysis, in conjunction with the data presented in Table 1 and Table 2, suggests that while digestion reduced the overall polyphenol content, especially of procyanidins and quercetin glycosides, no clear decrease in antimicrobial efficacy was observed, especially against Gram-negative bacteria. This implies that lower polyphenol levels might still be enough for bactericidal effects, with procyanidins, epicatechingallate, and quercetin glucosides likely contributing most significantly. The persistence of the antimicrobial activity despite compositional changes supports the hypothesis that polyphenol efficacy is strongly influenced by environmental factors like digestion and solubility, and suggests that these compounds may act synergistically with digestive fluids to offer a safe environment for the host. This has strong evolutionary support, since the human body, whose digestive system we are simulating in this study, has evolved in the presence of a polyphenol-rich diet.

Although no previous evaluation of the variations in the antimicrobial activity of a polyphenol-rich grape marc extract after gastrointestinal digestion had been performed before this research, the effect of digestion on antimicrobial stability was previously assessed for two other natural extracts. Khochapong et al. [49] and González-Montiel et al. [50] described in their studies the impact of in vitro gastrointestinal digestion on coffee pulp and propolis extracts, respectively. Both teams observed a reduction in both the phytochemical and antimicrobial activity, and a maintenance of the prebiotic potential. However, both authors employed the agar diffusion technique for the antimicrobial determination, which is a semi-quantitative method not appropriate for determining minimum inhibitory concentrations when using complex substances [51], because the molecules present in such extracts may present different solubilities, affecting their capability for diffusion and producing false-negative results [52].

Considering the limitations of this in vitro research, the complexity of natural extracts, and the active response of gut microbiota to prebiotics and pathogenic bacteria, future studies should include both bioaccessible and non-soluble extract fractions to better reflect in vivo conditions. Additionally, the role of polyphenol-derived metabolites in modulating microbial metabolic pathways and biofilm development inhibition effects should be further investigated.

4. Conclusions

This study highlights the antimicrobial potential of a white grape marc extract and its stability throughout in vitro gastrointestinal digestion. Despite a progressive reduction in polyphenol content, especially for procyanidins and quercetin glycosides, following gastric and intestinal phases, the extract retained its bactericidal activity. Notably, the antimicrobial efficacy increased against Gram-negative pathogens after intestinal digestion, suggesting that digestive conditions may enhance the bioactivity of specific compounds. In contrast, slight reductions in antimicrobial performance were observed against Gram-positive bacteria following gastric exposure, particularly for L. monocytogenes. Pearson correlation analysis suggests that certain key polyphenols are involved in maintaining antimicrobial activity. These results demonstrate that high polyphenol concentrations are not necessary to maintain antimicrobial activity, reinforcing the importance of compound solubility and structural transformations during digestion. Overall, the Albariño grape marc extract used in this study appears to be a promising functional ingredient with potential applications in food preservation and nutraceutical development, especially considering its retained and, in some cases, enhanced antimicrobial properties post digestion.

Because only potentially bioavailable fractions of the digestates were evaluated, future experiments should be performed assessing the effect of the gastrointestinal process on non-bioavailable polyphenols, as well as studies of its potential prebiotic and microbiota modulation effect.