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Article

Bale Ensiling Preserves Nutritional Composition and Phenolic Compounds of Red Grape Pomace

by
Gema Romero
1,*,
Lidia Nieddu
2,
Aymane Mouhssine
1,
Paulina Nowicka
3,
Joel Bueso-Ródenas
4,
Nemesio Fernández
5 and
José Ramón Díaz
1
1
Institute of Agri-Food and Agro-Environmental Research (CIAGRO-UMH), Miguel Hernández University of Elche, Ctra. De Beniel, km 3.2, 03312 Orihuela, Spain
2
Department of Agricultural Science, University of Sassari, Viale Italia 39, 07100 Sassari, Italy
3
Department of Fruit, Vegetable and Plant Nutraceutical Technology, Faculty of Biotechnology and Food Sciences, Wroclaw University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
4
Department of Animal Production and Public Health, Faculty of Veterinary Medicine and Experimental Sciences, Catholic University of Valencia San Vicente Mártir, 46001 Valencia, Spain
5
Institute of Animal Science and Technology, Polytechnic University of Valencia, Camino de Vera s/n, 46022 Valencia, Spain
*
Author to whom correspondence should be addressed.
AgriEngineering 2025, 7(6), 172; https://doi.org/10.3390/agriengineering7060172
Submission received: 16 April 2025 / Revised: 20 May 2025 / Accepted: 26 May 2025 / Published: 3 June 2025
(This article belongs to the Section Pre and Post-Harvest Engineering in Agriculture)
Versions Notes

Abstract

:
Reusing agro-industrial by-products is a successful strategy that aligns with the 2030 Sustainable Development Goals. Red grape pomace poses a significant environmental challenge, particularly for wine-producing nations. Due to its high moisture content and seasonal availability, ensiling emerges as a potential method to prolong the nutritional value of red grape pomace, supporting the need for research into its application in animal nutrition. This study analyzed the bale ensiling process for red grape pomace by assessing its potential integration into ruminant diets and comparing its storage stability to untreated preservation methods. Baled silage units (approximately 300 kg each) were employed for this purpose. Analytical evaluations were conducted at 0, 7, 14, 35, 60, and 180 days of storage to monitor microbial and fermentation activity, nutritional composition, and bioactive attributes. Bale silage preserved the nutritional and microbial quality of red grape pomace for ruminant feed over a storage period of 180 days. The results demonstrated that bale silage successfully maintained the macro-composition, bioactive compounds, and antioxidant properties while reducing the fatty acid profile’s susceptibility to oxidation. By contrast, untreated storage led to significant spoilage. We concluded that bale ensiling is a suitable and effective technique that preserves red grapes for ruminant feed over a long period.

1. Introduction

The wine industry generates a significant amount of waste, known as pomace. About 25 percent of the total mass of grapes used in wine production is turned into organic waste, with a global estimate of about 9 million tons each year [1,2]. Managing and preserving pomace represent some of the most significant challenges for the wine industry, not only because of the large amount of waste produced but also because of the potential value these by-products could have if properly treated. Pomace is composed mainly of skins, seeds, and stems and is rich in bioactive compounds such as phenols, antioxidants, and aromatic compounds, which, if properly valorized, could find application in numerous industries.
However, inadequate management can lead to serious environmental consequences, including pollution in soil and water resources, with negative impacts on the surrounding ecosystem. Therefore, it is crucial to adopt strategies that reduce the amount of waste resulting from winemaking and promote the reuse of these by-products through valorization processes. Currently, a small portion of pomace is used for oil production and polyphenol extraction or processed into animal feed [1,3]. These practices help reduce environmental impacts and offer new economic opportunities for the wine industry, fostering sustainability and innovation in the sector. However, effectively maintaining these by-products is essential to preserving their quality and preventing their degradation due to microbiological and chemical processes during their long-term storage.
A promising technique for pomace preservation is ensiling, a process that promotes anaerobic environments, limiting oxidation and the growth of undesirable microorganisms [1,4]. Ensiling is a biomass preservation process based on lactic fermentation under anaerobic conditions. Epiphytic lactic acid bacteria (LAB) ferment water-soluble carbohydrates in the biomass, converting them into lactic acid and, to a lesser extent, acetic acid and other organic acids. This process rapidly decreases pH, which inhibits and/or eliminates the microorganisms responsible for spoilage, thus preventing biomass decomposition. The success of ensiling depends on various factors, including storage techniques, compaction, sealing, and the use of inocula to stimulate lactic fermentation, thus ensuring a quality product and long-term storage [1,5].
Grape pomace is particularly suitable for ensiling due to its already low pH and high polyphenol and lipid contents, helping prevent protein degradation [1,6]. The effects of polyphenols on animal diets have been studied recently, showing benefits such as improving the oxidative stability of meat, modulating the gut microbiota, and reducing the need for synthetic antioxidants such as vitamin E [1,7]. During ensiling, monitoring polyphenol content is crucial to ensuring the quality of the process, as this influences nutritional and microbiological quality.
The present study aims to characterize the effects of two storage methods on red grape pomace for a long period (180 d): ensiling using commercial plastic bales (300 kg each) and storage without any treatment (buckets). Changes in chemical, microbiological, and physical parameters; antioxidant capacity; phenolic compounds; and fatty profile were studied to provide an in-depth understanding of the ensiling process and to evaluate its effectiveness in optimizing the long-term storage and enhancement of red grape pomace.

2. Materials and Methods

2.1. Experimental Design

This study examined two storage methods for red grape pomace obtained at POD “Vinos de Alicante” (Alicante, Spain) during the 2022 harvest season: silage in commercial plastic bales (300 kg) and open buckets (0.5 m3). The latter was used to simulate bulk storage without additional treatment. Open buckets were placed under a covered barn with limited ventilation. Both treatments were indirectly exposed to the region’s environmental conditions, characterized by a Mediterranean climate, simulating uncontrolled storage conditions. During the storage period, mean environmental temperatures fluctuated between 10 °C and 23 °C, with moderate rainfall and a relative humidity of 58–65% RH, as is usual for the location.
To monitor changes over time, samples were collected at six intervals: day 0 (the day of silo preparation) and days 7, 14, 35, 60, and 180 of conservation. On day 0, three samples were taken from three sections of the entire batch of material intended for storage. For subsequent sampling days, two bales were selected, and a 1 kg sample was extracted using a manual auger from three zones of each bale: the middle, the upper section, and 20 cm from the base. These three sub-samples from each bale were then combined into a composite sample. Two buckets were also sampled on each occasion, with collection from three levels—top, middle, and bottom—using a method similar to that used for the bales. Globally, 10 bales and buckets were used (5 sampling days × 2 sampling units) as the 0 sampling day was the raw material. Samples were transported to the laboratory, where microbiological assays and pH measurements were performed immediately. The remaining samples were stored at −80 °C for subsequent analyses, including ruminant nutritional composition, phenolic compounds, antioxidant capacity using ABTS and DPPH methods, and the fatty profile.
Commercial microsilos in a bale format (approximately 300 kg each) were manufactured from red grape pomace in a pilot plant at the facilities of the “Granja Caprina”, part of the Polytechnic School of Orihuela (EPSO) at Miguel Hernández University of Elche (UMH) of Spain. The manufacturing process followed the method described in the patent by Díaz et al. [8]. Briefly, silos were formed using an Agronic MR 820 rotary baler (AGRONIC OY, Haapavesi, Finland) with a capacity of 0.64 m3 and a weight of 300 kg per bale. To ensure proper compaction and firmness, each bale was wrapped with five layers of netting. Additionally, 13 layers of plastic film (Karatzis, Heraklion, Greece) were applied to maintain an airtight seal and prevent oxygen infiltration. The red grape pomace comprised a heterogeneous mixture of skins, pulp residues, and seeds. To enhance compaction and promote anaerobic conditions within the silos, 10% chopped cereal straw was added. This raw material was chopped and homogenized using a FASTER MIX 1 V mixer wagon (Compar, Sant Pere de Torelló, Spain).

2.2. Microbiological Determinations

Samples were transported to the laboratory in aseptic plastic bags. Subsequently, 25 g of each sample was placed in aseptic plastic bags with a lateral strainer and homogenized with 225 mL of peptone water using a stomacher (BagMixer® 400, Interscience, Saint Nom la Bretêche, France). Microbiological cultures were prepared to enumerate enterobacteria, aerobic mesophilic bacteria, lactic acid bacteria, yeast, molds, and clostridia spores. For enterobacteria and aerobic mesophilic bacteria analyses, samples were diluted with peptone water and directly incubated on EB and LAC 3M™ Petrifilm plates (3M Microbiology, St. Paul, MN, USA) at 37 °C for 24 h. For lactic acid bacteria counts, samples were diluted in MRS broth (Liofilchem, Roseto degli Abruzzi, Italy) and incubated on LAC 3M™ Petrifilm plates at 37 °C for 48 h in an anaerobic jar. Molds and yeasts were cultured on YM Petrifilm plates and incubated for 72 and 120 h, respectively, after dilution in peptone water. A spore count of the Clostridium genus was performed using the most probable number technique (MPN) and Bryant–Burkey broth (BBB, Merck, Darmstadt, Germany), following the methodology indicated by Arias et al. [9]. All microbiological analyses were conducted at the Animal Science Laboratory, part of the CIAGRO-UMH.

2.3. Chemical Analysis

2.3.1. Quantification of Fermentation Products (Sugars, Ethanol, and Organic Acids)

The key metabolites involved in fermentative dynamics were quantified using frozen samples. We analyzed sugars naturally present in the fruit (sucrose, fructose, and glucose), which act as substrates in silage fermentation; short-chain organic acids (volatile fatty acids, VFAs), including tartaric, acetic, propionic, and butyric acids; lactic acid; and ethanol. Quantification was performed following the method described by Feng-Xia et al. [10] using high-performance liquid chromatography (HPLC; Agilent 1200, Santa Clara, CA, USA). The analysis employed a 30 cm × 7.8 mm DI C610H column (Supelcogel, Sigma-Aldrich Co, Darmstadt, Germany) with 0.1% orthophosphoric acid as the mobile phase. The results are reported as g/kg of dry matter (DM).

2.3.2. Assessment of Physicochemical Properties

Physicochemical properties were evaluated on the same day as sampling. The pH was measured using a pH meter (GLP 21, Crison, L’Hospitalet de Llobregat, Spain), and dry matter (DM) content was determined as g/kg according to AOAC Method 948.12 (1990) [11]. Flieg scores were calculated for each sample using the equation proposed by [12]:
Flieg score = 220 + (2 × DM (%) − 15) − 40 × pH
Samples were categorized based on Flieg scores as follows: <20 points indicate very low-quality silage; 21–40 points indicate low-quality silage; 41–60 points indicate medium-quality silage; 61–80 points indicate high-quality silage; and >81 points indicate very high-quality silage. Further analyses were conducted on samples dehydrated at 60 °C and ground to a 1 mm particle size. The following parameters were analyzed following AOAC protocols: ash (g/kg DM, 934.01), crude protein (CP, g/kg DM, 988.05), ether extract (EE, g/kg DM, 920.39), crude fiber (CF, g/kg DM, 978.10), and total sugars (g/kg DM, 974.06). The levels of neutral detergent fiber (NDF, g/kg DM), acid detergent fiber (ADF, g/kg DM), and acid detergent lignin (ADL, g/kg DM) were determined according to the method by Van Soest et al. (1991) [13]. Non-protein nitrogen (NPN, g/kg DM) was quantified using the Cornell method for feed nitrogen fractionation described by Licitra et al. [14]. Starch content was determined using the polarimetric method by Ewers (ISO 10520:1997) [15].

2.3.3. Antioxidant Capacity and Polyphenolic Compounds

ABTS: We followed the method described by Leite et al. [16] to conduct the ABTS analysis. To create the ABTS solution, we dissolved 0.1920 g of ABTS in 50 mL of water, resulting in a 7 mM solution of ABTS reagent in 2.45 mM K2S2O8 (sol 1). Next, we initiated a reaction by mixing 1 mL of 2.45 mM K2S2O8 with 10 mL of ABTS solution, which formed SOL I. The reaction mixture was then kept in the dark at room temperature with continuous agitation for 12–16 h. Sol 1 remained stable for up to 2 days and could be stored in Eppendorf tubes in the freezer for 1 month. To prepare the working solution (ws), we combined sol 1 with 80% ethanol and adjusted the absorbance to 0.7 ± 0.005 at 734 nm. The amount of ws needed for each analysis, including a calibration curve and blank samples, was determined based on the absorbance measurement. We prepared 4.875 mL of ws for each analysis. The results were expressed in meq trolox/g.
DPPH: The procedure for DPPH analysis was adapted from Cheng et al. [17], originally developed by Brand-Williams et al., 1995 [18]. A DPPH solution in 80% ethanol (sol 1) with a concentration of 600 μM was prepared. The ws was created by combining sol 1 with 80% ethanol and adjusting the absorbance to 0.98 ± 0.005 at 515 nm. The appropriate amount of ws for each analysis, including a calibration curve and blank samples, was determined by measuring the absorbance. For sample analysis, 10 μL of the extract was mixed with 990 μL of WS in disposable cuvettes. The reaction mixture was then kept in the dark at room temperature for 6 min. The absorbance at 515 nm was measured using a spectrophotometer, and the antioxidant capacity was expressed as a percentage of inhibition or as the mMol equivalent of trolox per gram of dry weight. All samples were examined in triplicate, and their absorbance was verified to fall within the calibration curve range and maintain an absorbance higher than 0.05. If the absorbance was lower, implying a high concentration of antioxidants, the sample was diluted with extractant (70% acetone), and the procedure was repeated. The results were expressed in meq trolox/g.
Polyphenolic compounds: Samples stored at −80 °C were used for the analysis. The extraction procedure for polyphenolics in grape pomace followed the method of Wojdyło, Oszmiański, and Bielicki (2013) [19], with slight modifications. Approximately 0.7 g (±0.1 g) of lyophilized sample was weighed and transferred to tubes. To each tube, 5 mL of 30% methanol containing ascorbic acid (2 g/L) and acetic acid (1 mL/L) was added. The mixture was subjected to ultrasound treatment for 30 min. Subsequently, the samples were refrigerated for 20–24 h. After refrigeration, the samples underwent an additional 30 min of ultrasound treatment. Following the ultrasound treatment, the samples were centrifuged, and the supernatants were collected. The supernatants were then filtered and prepared for further analysis. Polyphenol identification and quantification were conducted using an Acquity UPLC system with photodiode array (PDA) and fluorescence (FL) detectors (Synergy™ H1 microplate reader, BioTek, Winooski, VT, USA). Separation was performed on an Acquity UPLC BEH C18 column using a gradient elution system comprising formic acid in water and acetonitrile as the mobile phases. Individual phenolic compounds detected, namely flavan-3-ols, phenolic acids, flavonols, and anthocyanins, were analyzed at specific wavelengths of 280 nm, 320 nm, 360 nm, and 520 nm, respectively. In addition, polymeric procyanidin content was analyzed following the phloroglucinol method [20]. Total polyphenol content was calculated based on peak areas in the chromatograms. The results were processed using the Empower 3.0 software and expressed in milligrams per 100 g of DM. These analyses were performed at the Wroclaw University of Environmental and Life Sciences (Poland).

2.3.4. Lipid Profile

The lipid profile was determined on days 0 and 180 by separating isomers of polyunsaturated fatty acids through the direct methylation of the lyophilized sample without prior fat extraction, per Kramer et al. [21]. The fatty acid composition was determined using a gas chromatograph (GC-17A, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FID) and a CP Sil 88 capillary column (100 m × 0.25 mm internal diameter × 0.20 mm film thickness; Agilent, Santa Clara, CA, USA). A mix of methylated fatty acid esters (catalog number 18912-1AMP, Sigma-Aldrich, Saint Louis, MO, USA) was employed as a reference standard. Nutritional and health indices for assessing fatty acids were calculated according to Chen and Liu [22].

2.4. Statistical Analysis

All the determined variables were analyzed following a general linear model (Proc.GLM, SAS V 9.4, 2022) according to the following equation:
Y = μ + Bi + Dk + (Bi × Dk) + e
where Y is the dependent variable, μ is the intercept, Bi is the fixed effect of the conservation day (i = 0, 7, 14, 30, 60, and 180 days), Dk is the fixed effect of the treatment (k = buckets or silos), Bi × Dk is the interaction of both, and e is the residual error.
To interpret the differences between fixed effect levels, least-squares means were calculated, representing model-adjusted estimates. Mean comparisons between fixed effect levels were performed by testing the null hypothesis: H0: LSMean (h) = LSMeann (j), where (h) and (j) represent different fixed effect levels. The significance of these comparisons was evaluated using Pr > t. If p < 0.05, the null hypothesis is rejected, indicating that the differences between levels are statistically significant.

3. Results

3.1. Microbiological Analyses

Trends in microbial counts by treatment and conservation time are shown in Table 1. Generally, all microbial groups, except for molds and clostridia spores, were significantly affected by the interaction between the treatment and conservation time. The population of mesophilic aerobes was higher in buckets than in silos: in buckets, the population increased from day 0 to day 35 (7.12 to 9.71 log10 cfu/g) and then decreased slightly until day 180 (9.22 log10 cfu/g), but the population remained almost equal in silos at all times, from 6.39 to 6.66 log10 cfu/g on days 0 and 180, respectively. Treatment did not affect lactic acid bacteria counts significantly, demonstrating a marked increase from day 0 to 7, with slight fluctuations after the latter in both treatments. The effect of treatment and time was strong on the enterobacterial population: it increased in the buckets until day 35 (8.08 log10 cfu/g) and then slightly decreased until day 180 (6.50 log10 cfu/g), but in the silos, the enterobacterial population increased until day 14 (5.13 log10 cfu/g), decreasing and almost disappearing by day 60 to the end of the experiment (1.27 log10 cfu/g at 180 days). Molds and yeasts demonstrated lower levels in silos than buckets after day 14. The clostridia spore count was significantly affected by treatment (p = 0.001), being lower in buckets than in silos, albeit with slight differences.

3.2. Fermentation Products

Table 2 shows the effect of treatment and conservation time on sugars (sucrose, glucose, and fructose), volatile organic compounds, and other organic acids. Regarding sugars, glucose showed the highest level at day 0, and sucrose showed the lowest. The three sugars were significantly affected by treatment and conservation time. The trend was almost identical between treatments, although the values were lower in silos, where a sharp decrease in these compounds was observed from day 14 to day 35, with a reduction of about four-fold. Regarding acids and ethanol, a different trend was observed between treatments on lactic, acetic, and ethanol, resulting in significant interaction. Lactic acid sharply increased in silos from day 0 (2.10 g/kg DM) to day 7 (10.65 g/kg DM), which was preserved until almost the end of the experiment (13.68 and 6.33 g/kg DM on days 60 and 180, respectively) but not in buckets where levels did not increase. An opposite trend was observed for acetic acid: it increased in buckets from day 0 (4.72 g/kg DM) to day 7 (24.61 g/kg DM) until day 14 (25.56 g/kg DM), and it almost disappeared on day 60 and later, demonstrating no relevant variation in silos. Regarding ethanol, the results were very different between treatments: although similar levels were observed at day 0 for both treatments (34.91 and 35.96 g/kg DM), silos preserved the content until day 60 (27.57 g/kg DM), with a decrease on day 180 (16.14 g/kg DM); contrarily, buckets lost all ethanol from day 7 (0.40 g/kg DM) to the end of the experiment. Propionic and butyric acids showed very low levels in both treatments from day 0 onward.

3.3. Chemical–Physical Parameters

The effects of treatment and conservation time on the chemical and physical parameters are reported in Table 3. The DM content was not significantly affected by treatment or conservation time, showing values ranging from 53.78 to 66.85%. pH was affected (p < 0.001) by all the effects considered: it remained close to 4.00 until day 14 in both treatments; from day 35 onward, it increased markedly in buckets (pH > 7.7) but not in silos. The Flieg score was significantly (p < 0.001) affected by all the effects considered, with a trend similar to the one observed in pH: In both treatments, the score was higher than 154 until day 7, indicating very high quality. In the silos, the score remained almost stable at these quality levels, varying between 160.94 and 143.85 from day 0 to day 180, respectively. However, in the buckets, the Flieg score sharply decreased below 36 from day 35 to day 180 as the pH remained at high levels.
Nutritional composition was preserved during the experiment by both treatments, with slight non-relevant variations between days of conservation. Protein content fluctuated throughout with no significant effect, with slightly higher values in buckets than silos (p < 0.01). NDF content was only significantly (p = 0.0038) affected by conservation time; a slight increase was observed in both treatments.

3.4. Antioxidant Capacity and Polyphenolic Compounds

Regarding polyphenolic compounds (Table 4), the greatest changes were observed in procyanidin polymers: as time went on, a higher decrease was observed in buckets than silos, with the latter treatment preserving 50% of the initial content after 180 days (2264 vs. 1435 mg/100 g DM), whereas buckets only preserved 5% (3170 vs. 172 mg/100 g DM). Similar trends were observed for phenolic acids and flavonols. After 180 days of conservation, flavonols in buckets were 0.11 mg/100 g DM (13% of the initial value), and phenolic acids were not present at all. Conversely, in silos, total phenolic acid content was 58% of the initial content (0.66 mg/100 g DM) and 60% of the initial flavonol content (1.28 mg/100 g). Anthocyanins were only significantly affected by conservation time (p = 0.002), demonstrating an increase in the first few days (in the case of buckets, 7 days, and in the case of silos, 14 days) and a later decrease until day 180, when they degraded in both buckets and silos. Generally, after 180 days of conservation, the total polyphenols were much lower in buckets than in silos (174.97 vs. 1444.29 mg/100 g DM, respectively). An interesting phenomenon was also observed in the buckets after 14 days. The polyphenolic compounds polymerized, which resulted in an increase in total polyphenolic compounds (3192 mg/100 g dm to 3439 mg/100 g dm) due to increased polymeric proanthocyanidins (from 3170 mg/100 g dm to 3412 mg/100 g dm). However, at the turn of the 14th and 35th days, there was a significant degradation in the polyphenols in the buckets (by almost 70%). In the samples stored in the silos, peak polymerization was observed after 35 days, and polyphenolic compound degradation occurred after 60 days of storage. However, in the silos, this process was much less intensive than in the buckets; the decrease in polyphenolic compounds was slightly over 10%.
Table 5 shows the results for antioxidant capacity by treatment and ensiling time: both the DPPH and ABTS tests were significantly affected by treatment, conservation time, and their interaction: silos demonstrated a high increase on days 7 to 60 compared with day 0 in both variables and a later decrease resulting in values at 180 days similar to day 0. In buckets, a decrease was observed starting on day 35, resulting in lower values on day 180 than on day 0 for both DPPH (103.7 vs. 419 mg eq trolox/100 g DM) and ABTS (109.3 vs. 751 mg eq trolox/100 g DM).

3.5. Lipid Profile

The treatment influenced the fatty acid profile, with small differences observed in most variables, except for short-chain fatty acids (SCFAs), which were not significantly affected (Table 6). Overall, the bucket samples exhibited slightly higher unsaturated fatty acid (UFA) and lower saturated fatty acid (SFA) values from the beginning (p < 0.001). The impact of conservation time, depending on the treatment, was more pronounced for the Omega-6–Omega-3 ratio (n6/n3), the linoleic acid–alpha-linolenic acid ratio (LA/ALA), and trans-fatty acids (TFA), all of which were higher in buckets, whereas the oleic acid–stearic acid ratio (OLESTE), hypocholesterolemic–hypercholesterolemic fatty acid ratio (HH), and health-promoting index (HPI) were higher in silos.

4. Discussion

Lactic acid bacteria (LAB) were the dominant microbial populations in the silos from day 7 onward. These bacteria are epiphytic to plant material and represent the primary microflora responsible for spontaneous silage fermentation, underscoring the importance of maintaining a sufficient LAB population for optimal fermentation [23]. The primary role of these microorganisms is converting carbohydrates into organic acids, which effectively reduce pH levels and promote the preservation and stability of silage material [24]. In this study, the LAB population in silos consistently outnumbered other microbial populations throughout the experimental period, mirroring observations in other grape pomace ensiling studies [6,25]. Enterobacteria populations in silos decreased dramatically after 35 days, according to previous studies by Schnürer et al. [26] and Zheng et al. [27], which highlighted the antimicrobial potential of LAB species. These species synthesize specific substances, including bacteriocins, hydrogen peroxide, and organic acids, that inhibit the growth of pathogenic and spoilage bacteria. The reduced mold and yeast populations in the silos likely stemmed from limited substrate availability and elevated concentrations of volatile organic acids, which are antifungal and bactericidal [28]. Their count remained below 6 log10 CFU/g of fresh matter in the silos, a threshold suggested by Kung et al. [29] for ensuring high silage stability once a silo is opened. By contrast, the buckets did not exhibit similar acidity or anaerobiosis conditions, failing to inhibit the proliferation of enterobacteria, molds, and yeasts. These microorganisms still suggest some vulnerability to microbial contamination, which could undesirably affect by-product quality and preservation in the long term [30].
Kung et al. [28] found that the predominant microbial populations, fermentable substrates, and the fermentation types occurring throughout the ensiling process are key factors influencing the synthesis of fermentation products. Lactic acid is the primary acid found in high-quality silages due to its crucial role in pH reduction [31]. Its synthesis primarily depends on the presence of LAB populations and the availability of easily fermentable substrates (monosaccharides and disaccharides) under anaerobic conditions [32]. In the silo treatment, the concentration of fermentable sugars (glucose, fructose, and sucrose) decreased as lactic acid concentrations increased. This pattern of sugar consumption favoring lactic acid accumulation has also been described by several authors in grape pomace ensiling studies [1,6]. The lactic acid concentration observed in the silos aligns with the 65–70% proportion of total acids mentioned by Kung [33] for high-quality silages. However, as the ensiling period progressed, the lactic acid concentration decreased, and the acetic acid levels increased. This phenomenon can be attributed to a shift toward a higher proportion of heterofermentative colonies [24], which synthesize acetic acid as a fermentation metabolite, including the conversion of lactic acid into acetic acid, as indicated by Ni et al. [34]. Carrasquillo [35] described how the ensiling process’s progress toward fermentation mediated by heterofermentative lactic acid bacteria is favorable for maintaining aerobic stability upon opening baled silage. In addition, according to Der Bedrosian et al. [36], some strains of lactic acid bacteria can consume lactic acid under anaerobic conditions when sugar availability is limited, resulting in reduced lactic acid and increased acetic acid. It is important to highlight that a moderate acetic acid concentration can be advantageous for livestock, as it is absorbed into the rumen and transformed into energy or fat that is incorporated into milk or body reserves [37]. The lactic fermentation carried out by LAB colonies during the ensiling process preserved ethanol concentrations in the silos, reflecting a predominance of this energy-efficient pathway over less conservative energy routes [38]. Conversely, aerobic conditions in the buckets rapidly facilitated ethanol oxidation, transforming it into acetic acid [39]. Due to its volatile nature, the acetic acid produced in the buckets eventually disappeared. The tartaric acid naturally present in grape pomace maintained its stability in silos, creating a favorable environment for ensiling and its conservation [40]. However, the bucket environment likely favored its metabolism as a carbon and energy source for the present microorganisms [41]. The absence of propionic and butyric acids after the ensiling process indicates good fermentation quality.
Ensiling is a feed preservation technique that maintains nutrient content while minimizing dry matter (DM) and energy losses [24]. The DM losses observed in silos align with those attributed to the activity of lactic acid bacteria (2–5%), as reported by [24]. Furthermore, Chen et al. [42] note that these losses are converted into CO2 during fermentation mediated by heterofermentative bacteria. The DM content observed in this study is consistent with the findings of Massaro et al. [4] and De Bellis et al. [7] in grape pomace. Silo remained at a pH between 3.8 and 4.2 over six months, a critical and desirable indicator of high-quality silage production [43,44]. By contrast, this was not the case for the bucket treatment, where a loss of acidity was observed. This reduction is due to the generation of alkalizing metabolites during plant organic matter degradation [45]. The Fleig score for both treatments corresponded with the pH results, with silos maintaining excellent quality up to day 180. The scores were significantly higher than those reported for silage from other plant by-products [46,47], reaffirming the suitability of this by-product for the ensiling process. According to Chen et al. [48], the acidity and maintenance of dry matter (DM) observed in the silos during the study are critical factors for the stability of other nutritional components. Over six months, the silos effectively preserved starch, EE, CP, CF, NDF, ADF, ADL, and ash contents, and total sugars were consumed as substrates in the lactic acid fermentation process associated with ensiling [49]. Zhang et al. [45] highlight that during plant material decomposition, significant nitrogen and dissolved organic carbon losses occur through leaching alongside the volatilization of compounds such as ammonia. These losses contribute to the observed increase in the proportion of fibrous and proteinaceous content in bucket treatments. Non-structural carbohydrates were identified as the most readily and efficiently degraded macronutrients, serving as the most accessible energy source during the decomposition process in bucket conditions [50].
Regarding the phenolic compounds and antioxidant capacity (ABTS and DPPH), the increase in polyphenolic compounds in the first phase of fermentation with the participation of LAB was directly related to the ongoing process. Several factors interact, including bacteria directly, and with them, their enzymes, which break down cell walls (cellulases and pectinases), phenolic glycosides (β-glucosidases), or strong bonds between polyphenolic compounds and fiber. In addition, reduced pH denatures cell structures [51,52]. All of these components consequently promote the extraction of polyphenolic compounds from deeper layers of plant tissue, which would be inaccessible under normal conditions. The increased concentration of polyphenolic compounds, especially procyanidin polymers, was visible faster in buckets than silos, as the fermentation was more acute at the beginning and lower pH was achieved faster, while silos progressed slower. In subsequent days, the more favorable conditions for fermentation in the silos (anaerobiosis and additives) provided good conditions for lower pH and the subsequent extraction and stabilization of polyphenolic compounds [52], especially anthocyanins, flavonols, and phenolic acids. By contrast, increasing this parameter, as observed in buckets, degraded polyphenols and disconnected ester bonds, resulting in lower biological activity for these compounds [53,54]. The potential of the bale silage treatment observed for the intensive and effective extraction of polyphenolic compounds and stabilization during storage is extremely important, as polyphenolic compounds have significant health-promoting potential: they have anti-inflammatory, antibacterial, and anti-clotting properties and improve blood vessel function, among other things [55,56]. Additionally, the best-known property of polyphenolic compounds is their antioxidant potential, which protects cells against oxidative stress, i.e., damage caused by free radicals, which was also demonstrated by the higher ABTS and DPPH results in the silos on the latter days of our research.
The fatty acid profile of this by-product aligns with most of the related literature [6,57,58]. This matrix is particularly rich in unsaturated fatty acids (UFAs), with a significant predominance of polyunsaturated fatty acids (PUFAs), compounds known for their health benefits when they are consumed [59,60]. The silo treatments had a positive impact over conservation time on the health-promoting indices, such as the n6/n3 ratio, the LA/ALA ratio, and TFA levels. The lower concentration of Omega-3 fatty acids (n3) can be attributed to the high susceptibility of these compounds to oxidation relative to other fatty acids [61]. The high susceptibility of n3 to oxidation was mitigated in the silos by the anaerobic environment [62] and the pH reduction caused by lactic fermentation [31]. Natural antioxidant compounds can also prevent lipid oxidation [63]. Given the impact of silage treatment on antioxidant potential over time, this effect was to be expected. Furthermore, the positive effect of encapsulation on n3 oxidation [63] is provided by the matrix formed within the bale silage. The increase in TFAs shown in the bucket treatment during the study period is due to the isomerization of cis–trans-double-bond configurations, which can be catalyzed by free radicals under oxidative conditions, per [64], such as those present in the bucket conditions. The same author [64] notes that a vegetal matrix deficient in natural antioxidants, which happened in the buckets over time, is also more prone to forming TFAs. In summary, while buckets were slightly richer in UFA, the silo treatment might be preferable in terms of lipid stability and quality, with lower susceptibility to oxidation over time.

5. Conclusions

Bale silage can preserve red grape pomace macro-composition for ruminant nutrition and microbial quality for almost 180 days, whereas storage without any conservation treatment causes spoilage. In addition, other properties, such as antioxidant capacity and bioactive compounds like polymeric procyanthocyanidins, are better preserved with bale silage, providing adequate stability and lower susceptibility to oxidation in the fatty profile. Future studies on ensiled by-products in animal diets could explore these effects on production and animal health status.

Author Contributions

Conceptualization, G.R., N.F. and J.R.D.; Data curation, A.M. and J.B.-R.; Formal analysis, G.R. and J.B.-R.; Funding acquisition, G.R., N.F. and J.R.D.; Investigation, A.M.; Methodology, G.R. and P.N.; Project administration, J.R.D.; Supervision, G.R. and J.R.D.; Writing—original draft, L.N.; Writing—review and editing, G.R. and P.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science and Innovation of Spain and FEDER/EU, grant number PID2021-122962OB-C31, MCIN/AEI/10.13039/501100011033/FEDER, EU.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the findings of this study are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
ABTS2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)
ADFAcid detergent fiber
ADLAcid detergent lignin
ALAAlpha-linolenic acid
AOACAssociation Of Analytical Communities
CPCrude protein
DMDry matter
DPPH1,1-diphenyl-2-picrylhydrazyl
ECEuropean Commission
EEEther extract
FAMEFatty Acid Methyl Ester
FMFresh matter
HLPCHigh-Liquid-Pressure Chromatography
LALinoleic acid
LABLactic acid bacteria
LCFALong-chain fatty acid
MUFAMonounsaturated fatty acid
NDFNeutral detergent fiber
OBCFAsOdd- and branched-chain fatty acids
PODProtected Origin Denomination
PUFAPolyunsaturated fatty acid
SCFAShort-chain fatty acid
SFASaturated fatty acid
TFATrans-fatty acid
TIThrombogenicity index
UFAUnsaturated fatty acid
VFAVolatile fatty acid

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Table 1. Effect of treatment and conservation time on the counts of different microbial groups investigated.
Table 1. Effect of treatment and conservation time on the counts of different microbial groups investigated.
Microbial Group (log10 cfu/g)TreatmentDays of ConservationSEEffect of Model 1
07143560180 TreatmentDayTreat × Day
AerobesBucket7.127.128.639.719.479.220.18*********
Silo6.395.526.496.425.916.66
LacticBucket6.067.247.768.047.938.130.220.8897****
Silo5.848.457.578.357.247.81
EnterobacteriaBucket4.115.107.258.087.926.500.56*********
Silo3.453.435.134.280.001.27
MoldsBucket6.005.346.868.228.147.401.03****0.097
Silo5.194.542.184.671.744.09
YeastsBucket7.676.287.487.718.545.900.60***0.146*
Silo5.754.303.035.143.055.08
Clostridia 2Bucket3.311.952.792.562.712.870.24***0.2270.223
Silo3.263.263.333.333.333.27
1 The effect of the model is significant for * p < 0.05; ** p < 0.01; and *** p < 0.001. 2 log10 spores/g.
Table 2. Effect of treatment and conservation time on sugars, acids, and volatile organic compounds.
Table 2. Effect of treatment and conservation time on sugars, acids, and volatile organic compounds.
g/kg DMTreatDays of Conservation Effect of Model 1
07143560180SETreatDayTreat × Day
GlucoseBucket19.5316.3018.295.483.080.270.60****0.07
Silo20.0216.2314.993.841.940.28
FructoseBucket6.734.713.761.440.910.480.42*******
Silo7.841.551.541.150.650.51
SucroseBucket0.470.500.390.400.290.070.05*****0.0731
Silo0.340.250.200.280.230.13
Lactic acidBucket2.052.051.480.320.180.090.78*********
Silo2.1010.6513.6611.9613.586.33
Acetic acidBucket4.7224.6125.560.830.480.152.950.05****
Silo4.564.534.818.624.657.20
EthanolBucket34.910.400.290.070.070.061.65*********
Silo35.9624.8027.5923.7327.5716.14
Tartaric acidBucket17.7015.9615.615.396.250.630.57*********
Silo16.3114.9613.7110.1611.4912.03
Butyric acidBucket0.000.000.000.000.000.230.070.550.450.46
Silo0.040.030.000.000.000.02
Propionic acidBucket0.380.180.190.000.000.740.080.11******
Silo0.660.140.000.100.000.10
1 The effect of the model is significant for * p < 0.05; ** p < 0.01; and *** p < 0.001.
Table 3. Effect of treatment and conservation time on chemical–physical parameters.
Table 3. Effect of treatment and conservation time on chemical–physical parameters.
Item 1TreatDays of Conservation Effect of Model 2
07143560180SETreatDayTreat × Day
DM, %Bucket55.1659.6756.5166.8564.6654.473.450.16020.30560.362
Silo55.8258.9858.6855.6856.4853.78
pHBucket4.033.803.977.577.907.620.16 *** *** ***
Silo3.893.913.913.744.104.22
Flieg Score Bucket154.3179.5159.435.818.49.037.63 *** *** ***
Silo160.9166.7166.1166.9154.1143.8
StarchBucket3.352.752.452.952.701.300.400.0550.3190.146
Silo2.953.052.953.402.803.30
SugarsBucket0.500.400.500.300.300.300.120.194 ** *
Silo1.200.400.300.500.300.20
EEBucket9.4010.8510.109.459.359.600.60 ** 0.1610.348
Silo7.459.009.058.509.907.70
CPBucket11.1511.0511.4512.8512.2014.250.77 ** 0.0660.484
Silo9.7510.0010.2011.0012.0510.90
NPNBucket0.650.851.450.501.350.800.230.0910.1970.153
Silo1.301.251.201.401.250.70
CFBucket34.6536.8038.9539.4540.1036.452.41 ** 0.2710.639
Silo31.3033.7032.1029.9038.1532.30
NDFBucket46.7048.8051.0562.1555.7570.154.190.115 * 0.110
Silo48.9548.6048.6055.4059.1549.20
ADFBucket40.1042.4544.0555.4046.6562.854.070.2930.243 *
Silo46.8545.5045.4546.2552.3539.60
ADLBucket27.7730.8932.6040.0532.3351.294.35*0.363*
Silo29.2129.6630.7431.3137.6622.87
AshBucket6.104.854.956.556.156.750.470.7220.1370.365
Silo6.105.505.705.405.956.10
1 DM: dry matter; Starch (% DM); EE: ether extract, % DM; CP: crude protein,% DM; NPN: non-protein nitrogen,% DM; CF: crude fiber,% DM; NDF: neutral detergent fiber,% DM,); ADF: acid detergent fiber, % DM,); ADL: acid detergent lignin, % DM. 2 The effect of the model is significant for * p < 0.05; ** p < 0.01; and *** p < 0.001.
Table 4. Effect of treatment and conservation time on polyphenolic compounds.
Table 4. Effect of treatment and conservation time on polyphenolic compounds.
Item (mg/100 g DM)TreatmentDays of Conservation Effect of Model (p < 0.05) 1
07143560180SETreatmentDayTreat × Day
Polymeric proanthocyanidinsBucket31702358341211389421722560.05 * 0.05
Silo226423952894311527181435
Total anthocyaninsBucket0.730.880.460.000.000.000.160.56 ** 0.28
Silo0.340.500.650.190.060.00
Total flavan-3-olsBucket20.6630.6524.8111.163.352.867.520.140.240.50
Silo7.498.9613.977.276.707.35
Total flavonolsBucket0.832.771.730.970.260.110.650.090.130.20
Silo2.151.033.261.711.421.28
Total phenolic acidsBucket0.320.320.160.000.000.000.12 * 0.38 **
Silo1.140.630.940.610.600.66
Total polyphenolsBucket31922392343911509451742257** ** 17.36
Silo227524062912312427261444
1 The effect of the model is significant for * p < 0.05; ** p < 0.01.
Table 5. Effect of treatment and conservation time on antioxidant capacity.
Table 5. Effect of treatment and conservation time on antioxidant capacity.
mg eq Trolox/
100 g DM		TreatmentDay of Conservation Effect of Model 1
07143560180SETreatmentDayTreat × Day
DPPHBucket471.9429.5418.4208.9173.1103.734.37*********
Silo335.8588.7714.6432.0541.4325.5
ABTSBucket751.0837.2792.6332.6350.7109.382.77*******
Silo646.01290.31164.9806.7993.0607.1
1 The effect of the model is significant for * p < 0.05; ** p < 0.01; and *** p < 0.001.
Table 6. Effect of treatment and conservation time on fatty acids (percentage of methylated fatty acid area in the fatty acid profile).
Table 6. Effect of treatment and conservation time on fatty acids (percentage of methylated fatty acid area in the fatty acid profile).
Item 1TreatmentDay of Conservation Effect of Model 2
0180SETreatmentDayTreat × Day
SFABucket16.7714.800.6054 ***0.6172*
Silo17.7219.07
MUFABucket17.2316.340.1989 ****0.1732
Silo18.3518.04
PUFABucket66.0168.850.6176 ***0.1698**
Silo63.9362.89
UFABucket83.2385.190.6055 ***0.6209*
Silo82.2880.93
SCFABucket0.450.090.13670.14960.9926*
Silo0.300.66
MCFABucket1.268.690.4871 ***0.1492**
Silo11.8712.94
LCFABucket88.3091.210.5862 ***0.2271**
Silo87.8386.41
n3Bucket1.300.300.1651 ***0.173***
Silo1.301.82
n6Bucket64.5768.400.7675 ***0.1849**
Silo62.4160.73
n6/n3Bucket62.41229.037.0833*********
Silo49.9135.13
IABucket0.140.100.0075 ***0.1944**
Silo0.150.16
TIBucket0.340.320.3383 ***0.7990.0945
Silo0.360.38
HHBucket7.8110.220.3241*******
Silo7.336.64
HPIBucket5.705.430.1838 *******
Silo6.925.03
LA/ALABucket55.72265.396.4981 *********
Silo51.2837.80
TFABucket0.010.520.0900 ***
Silo0.050.01
OBCFABucket0.280.290.0388 *0.74890.9785
Silo0.370.38
OLESTEBucket3.382.650.0421 *********
Silo3.393.35
1 SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; UFA, unsaturated fatty acid; SCFA, short-chain fatty acid; MCFA, medium-chain fatty acid; LCFA, long-chain fatty acid; n3, Omega-3 fatty acids; n6, Omega-6 fatty acids; n6n3, Omega-6–Omega-3 ratio; IA, index of atherosclerosis; TI, thrombogenicity index; HH, hypocholesterolemic–hypercholesterolemic fatty acid ratio; HPI, health-promoting index; LA/ALA, linoleic acid–alpha-linolenic acid ratio; TFA, trans-fatty acids; OBCFA, odd- and branched-chain fatty acid; OLESTE, oleic acid–stearic acid ratio. 2 * p < 0.05; ** p < 0.01; *** p < 0.001.
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MDPI and ACS Style

Romero, G.; Nieddu, L.; Mouhssine, A.; Nowicka, P.; Bueso-Ródenas, J.; Fernández, N.; Díaz, J.R. Bale Ensiling Preserves Nutritional Composition and Phenolic Compounds of Red Grape Pomace. AgriEngineering 2025, 7, 172. https://doi.org/10.3390/agriengineering7060172

AMA Style

Romero G, Nieddu L, Mouhssine A, Nowicka P, Bueso-Ródenas J, Fernández N, Díaz JR. Bale Ensiling Preserves Nutritional Composition and Phenolic Compounds of Red Grape Pomace. AgriEngineering. 2025; 7(6):172. https://doi.org/10.3390/agriengineering7060172

Chicago/Turabian Style

Romero, Gema, Lidia Nieddu, Aymane Mouhssine, Paulina Nowicka, Joel Bueso-Ródenas, Nemesio Fernández, and José Ramón Díaz. 2025. "Bale Ensiling Preserves Nutritional Composition and Phenolic Compounds of Red Grape Pomace" AgriEngineering 7, no. 6: 172. https://doi.org/10.3390/agriengineering7060172

APA Style

Romero, G., Nieddu, L., Mouhssine, A., Nowicka, P., Bueso-Ródenas, J., Fernández, N., & Díaz, J. R. (2025). Bale Ensiling Preserves Nutritional Composition and Phenolic Compounds of Red Grape Pomace. AgriEngineering, 7(6), 172. https://doi.org/10.3390/agriengineering7060172

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