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Article

The Effect of Dietary Fermented Grape Pomace Supplementation on In Vitro Total Gas and Methane Production, Digestibility, and Rumen Fermentation

by
Kanber Kara
1,* and
Mehmet Akif Öztaş
2
1
Department of Animal Nutrition and Nutritional Diseases, Faculty of Veterinary Medicine, Erciyes University, 38280 Kayseri, Turkey
2
Department of Animal Nutrition and Nutritional Diseases, Health Sciences Institute, Erciyes University, 38280 Kayseri, Turkey
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(8), 741; https://doi.org/10.3390/fermentation9080741
Submission received: 14 June 2023 / Revised: 13 July 2023 / Accepted: 14 July 2023 / Published: 7 August 2023
(This article belongs to the Section Industrial Fermentation)
Review Reports Versions Notes

Abstract

:
The aim of this study comprises the effect of fermented grape pomace (FGP) in experimental total mixed rations (TMR) at different rates (0, 7.5%, 15%, and 22.5%) on the in vitro cumulative gas production (6th, 12th, 18th and 24th hours), methane production, ruminal fermentation values, pH and ammonia-nitrogen and straight and branched short-chain fatty acids (SCFA and BCFA) concentration. The method of in vitro total gas production was carried out in glass syringes. Ruminal in vitro methane production linearly decreased by adding up to 22.5% FGP in experimental TMR (p < 0.05). The molarities of acetic, propionic, butyric, and valeric acids in the in vitro fermentation fluid linearly decreased with the addition of FGP to TMR (p < 0.05). FGP up to 22.5% in experimental TMRs decreased the molarity of iso-valeric acid and iso-butyric acid from BSCFAs (p < 0.05). As a result, it was concluded that the use of FGP, containing a low level of total condensed tannins (TCTs), up to 22.5% in the experimental TMR based on dry matter (DM) did not adversely affect the in vitro ruminal fermentation value and had an anti-methanogenic effect. In addition, some SCFA (acetic, propionic, butyric, and valeric acids) molarities and iso-acid BSCFA (iso-butyric and iso-valeric acid) did not change up to 15% rate of FGP in the ration. Still, these values decreased by using a 22% rate of FGP. The dose-dependent effect of FGP on ruminal iso-acids has been associated with the ability of TCTs to inhibit ruminal protein degradation partially.

1. Introduction

Grape pomace represents 45–62% of organic waste from grapes’ pressing and fermentation stages. Approximately 30 kg of FGP is waste for every 100 L of white wine produced from grapes (Vitis vinifera) used during wine production. This pulp contains grape skins, fruit pulp, grape stem, and grape seeds [1,2,3]. Grape pomace can be sweet, fermented, and alcoholic, depending on grape processing technology [4]. The pomace that emerges as a by-product of wine or vinegar production is FGP. If grape juice is to be obtained from grapes, the by-product obtained as a waste product is sweet and called sweet grape pulp [4]. More than 50% of the grape stems are polysaccharides (15–40% cellulose and 12–20% hemicellulose), and the most abundant hemicellulose is xylans [2,3,5]. In the use of FGP or sweet grape pomace, a waste product, as a functional feedstuff in animal nutrition, especially the product’s fiber content (total dietary fiber: TDF) and antioxidant components are concentrated [6]. Grape pomace consists mainly of TDF (43–75% of dry pomace), crude protein (CP) (8.4–13.9% in DM), soluble sugars, and functional compounds (e.g., polyphenols-TCTs) and oil (6–9.1% in DM)) [6,7,8,9]. The grape pomace ranged from approximately 45 to 80% TDF, 0.4 to 18% soluble dietary fiber (SDF), and 30 to 75% insoluble dietary fiber (IDF) [6]. The TDF substance in the grape pomace consists of cellulose, hemicellulose, lignin, and uronic acids [10]. Studies have been carried out on the use of grape pomace in poultry nutrition [11,12,13,14]. However, some studies have been carried out in recent years about the effectiveness of adding grape pomace to the ruminant ration. Some studies have been conducted to investigate the effects of FGP on performance and animal product quality in ruminants [15,16]. The microbiota of FGP, a wine production by-product, can be considered a microbial community. Nearly all of the samples stored for one month in an open area may contain the yeasts of Saccharomyces cerevisiae, higher concentrations of filmy fungi of the genera Candida, Pichia, Hansenula, Hanseniaspora/Kloeckera and Torulaspora, as well as conidia of Mucor, Aspergillus niger and Penicillium [4]. In addition, acetic acid (mainly Acetobacter aceti) and lactic acid (Lactobacillus plantarum, Pediococcus, Leuconostoc) bacteria were identified as common bacteria in fermented grape pomace [4]. Kafantaris et al. [17]. observed that the intake of FGP in lambs inhibited pathogenic populations such as E. coli by accompanying the development of facultative probiotic bacteria in the fecal microbiota. This study hypothesizes that FGP containing fermentative yeast and bacteria, high unsaturated fatty acids, and moderate CP and CT contents may affect the fermentation of feeds and fermentation end products (such as methane production and short-chain fatty acid) in the rumen. FGP’s effects as a co-substrate in vitro fermentation were evaluated using different rates in dairy cattle total mix ration (TMR). This study aimed to use the FGP, which is an industrial (wine production) by-product, as an alternative feedstuff to experimental TMR at different rates (0, 7.5%, 15%, and 22.5%), in vitro cumulative total gas and methane productions and the concentrations of ammonia-nitrogen and short-chain fatty acids (straight and branched chain) of ruminal fermentation fluid.

2. Material and Methods

2.1. Fermented Grape Pomace

The FGP to be used in this study was obtained from two private wine-producing factories in Nevşehir (38.631078, 34.719787) and Tokat (40.313971, 36.551784) provinces (Türkiye). The grapes used in the enterprises were Kalecik karasi, Öküzgözü, and Boğazkere red grape varieties. The 10 kg fresh wet FGP for each factory taken were combined. The wet FGP was taken while fresh without losing liquid and brought to the laboratory in a lidded container.

2.2. Experimental TMRs for In Vitro Treatment

Experimental TMRs were set for dairy cattle in vitro and were prepared at 1 kg as DM for each TMR group. The daily energy and nutrient requirements of the Simmental breed experimental TMR, which was 630 kg live weight at the 12th week of lactation and 54 months of age, produced 23 L/day (with 3.8% fat and 3.5% protein) were adjusted according to NRC [18]. Experimental TMRs containing maize silage (29–33% DM), wheat straw, alfalfa hay (17–19% CP, 40–44% NDF), barley flake, cracked corn, sunflower meal (with 28% CP) cottonseed meal (with 28% CP) were in dried form. The experimental TMRs with 0, 7.5%, 15 and 22.5% of dried FGP were prepared (Table 1).

2.3. Chemical Analysis of Fermented Grape Pomace and TMRs

The DM content of wet FGP was determined in an oven at 60 °C for 48 h (Nüve, Ankara, Türkiye). Experimental TMR samples to be used in the dry form in the composition of experimentally prepared dairy cattle, on the other hand, were dried and ground in a mill with a maximum sieve diameter of 1.0 mm (IKA Mf 10 Basic Microfine grinder, IKA Werke GmbH & Co. KG, Staufen im Breisgau, Germany). The CP, diethyl ether extract (EE), and ash levels of FGP and TMRs were determined according to the Association of Official Analytical Chemists [19]. The neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin contents of the samples were analyzed according to the method of Van Soest et al. [20]. The NDF analyses were performed with 0.5 g sodium sulfite and 200 µL heat-stable alpha-amylase (Megazyme, Wiclow, Ireland) (aNDF). These fiber contents were expressed on an ash-free basis (aNDFom, ADFom, and lignin). All nutrient analyses mentioned above were performed in three replications. The TCT and bound condensed tannin (BCT) contents of the samples were determined by Makkar et al. [21] in UV-VIS spectrophotometer (SI Analytics–Xylem Analytics Germany Sales GmbH & Co. KG, Mainz, Germany) according to the butanol-HCl + FeSO4 method [22]. Chemical analyses were performed in triplicate.
The fatty acids in FGP were methylated [23]. Fatty acid methyl esters (FAMEs) were detected in gas chromatography with flame ionization detector (GC-FID) (TRACE™, GC-FID, Thermo Scientific, Whaltham, MA, USA), which had the fatty acid methyl esters (FAME’s), used for the separation of fatty acid (cyanopropylphenyl-based phase) (length: 60 m, inside diameter: 0.25 mm, film: 0.25 µm and maximum temperature 250–260 °C) (Thermo Scientific TRACETM, TR-FAME GC Columns, catalog number: 260M153P, Whaltham, MA, USA). The FAMEs into n-hexane were taken in amber vials (1.5 mL screw neck ND-9, silicon white/PTFE cap), and then they loaded GC-FID using an auto-sampler (Thermo Scientific TRACETM, Whaltham, MA, USA). Peak identification of each FAME was used in a standard mixture solution of a commercial FAME in dichloromethane (Chem-Lab, catalog number: CL.40.13093,0001, Zedelgem, Belgium) to define retention times and areas [24].

2.4. Detection of In Vitro Total Gas and Methane Production

Rumen fluid was taken from two dairy cattle (Simmental), which consumed approximately 65% concentrated feed mixture (cottonseed meal, soybean meal, wheat bran, DDGS, barley grain, corn bran, and corn flake) + 35% forage (wheat straw, alfalfa hay, and wet sugar beet pulp) on DM basis, by rumen tube two hours after morning feeding. Donor cattle had a live weight of about 600 kg at the 12th week of lactation and 72 months of age, producing 23 L milk per day. The rumen fluid was put into a glass bottle with a screw cap (Isolab Laborgeräte GmbH, Eschau, Germany) at approximately 39 ± 1 °C. It was brought to the laboratory in a lidded thermos carrier. Rumen liquid was used in the in vitro gas production after filtering through four layers of cheesecloth under CO2 gas. The 200 ± 10 mg dried sample and chemical mixture (buffer + macro mineral + micro mineral + reduction + resazurin solutions) (20 mL) and filtered rumen fluid inoculum (10 mL) in 100 mL glass syringes (Model Fortuna, Haeberle Labortechnik, Fortuna Spezialmaschinen GmbH Eisenbahnstraße, Weil der Stadt, Germany) were incubated [25]. In this study, in vitro gas production was performed with 12 repetitions for each TMRs and FGP. Twelve fermentation syringes were used as blank (without feed sample, containing only buffer, solutions, and rumen fluid) and used in the calculation as a correction value for in vitro gas production. In this in vitro study, the amount of total gas produced (mL) in each syringe at the 6, 12, 18, and 24 h of incubation was determined by reading the syringes. After reading the in vitro total gas produced at 24 h of incubation, approximately 50 mL was used to determine the methane amount. The total gas in the in vitro fermentation syringes was taken into a feeding syringe (50 mL volume) with a three-way tap connection. All the gas was injected with the slow flow from these anaerobic syringes into a computer-connected infrared-type methane measuring device (Sensor, Europe GmbH, Erkrath, Germany). The in vitro methane volume in the in vitro total gas was determined on the computer (Vestel, Manisa, Türkiye) [26].

2.5. Detection of Metabolic Energy, Net Energy Lactation, and Organic Matter Digestion

The total gas levels produced in the 24th hour of the TMRs containing FGP at different rates used in this study were determined by Menke et al. [25]. using the following equations developed by ME, NEL, and OMD values were calculated [27].
ME (MJ/kg DM) = 2.20 + 0.136 × GP + 0.057 × CP
NEL (MJ/kg DM) = 0.115 × GP + 0.0054 × CP + 0.014 × EE − 0.0054 × Ash − 0.36
OMD (% DM) = 14.88 + 0.889 × GP + 0.45 × CP + 0.0651 × Ash
GP = Total gas production as mL for 200 mg DM at 24th h of incubation, CP = Crude protein as g for kg DM, Ash = Ash as g for kg DM, EE = Diethyl ether extract as g for kg DM.

2.6. Determination of In Vitro Fermentation Fluid Variables

The 15 mL of in vitro fermentation fluid of all syringes at the 24th of incubation were taken to determine the pH value and the concentration of SCFA, BSCFA, and ammonia-nitrogen. The pH levels were determined with a digital pH meter (Mettler Toledo, S220 pH/ion meter, Columbus, OH, USA). The ammonia-nitrogen concentration was determined using a commercial kit (Megazyme, Ammonia Assay Kit–Catalogue no: Product code: K-AMIAR, Wicklow, Ireland) in a UV-VIS spectrophotometer (SI Analytics–Xylem Analytics Germany Sales GmbH & Co. KG, Mainz, Germany).
The SCFA composition [acetic acid—AA (C2:0), propionic acid—PA (C3:0), butyric acid—BA (C4:0), valeric acid—VA (C5:0), hexanoic acid—HEXA (C6:0), heptanoic acid—HEPA (C7:0) and BSCFA [iso-butyric acid—IBA (C4:0i), iso-valeric acid—IVA (C5:0i) (iso-valeric is the sum of 3-methylbutyric and 2-methylbutyric acids) and iso-caproic acid—ICA (C6:0i)] of fermentation fluid were determined in a GC-FID (TRACETM1300, Thermo Fisher Scientific, Orlando, FL, USA). Helium was used as a carrier gas, and nitrogen and hydrogen were used for combustion. A polyethylene glycol column (length: 60 m, i.d: 0.25 mm, film thickness: 0.25 µm; TG-WAXMS, Thermo Scientific, Orlando, FL, USA) was used for this analysis in the device, and the analysis procedure was performed according to Ersahince and Kara [28]. Total short-chain fatty acids (T-SCFA = SCFA + BSCFA) composition (as mmol/L of fermentation fluid and as % in T-SCFA), acetic acid/propionic acid (A/P), and (acetic acid + butyric acid)/propionic acid (A + B)/P) values were calculated.

2.7. Statistical Analysis

In the present study, the in vitro ruminal fermentation variables of TMRs contained at different ratios (0, 7.5, 15, and 22.5%) of FGP were determined by one-way analysis of variance in the SPSS 17.0 package program. When statistical significance was determined, the significance level was determined with the Tukey multiple comparison test. The significance level was taken as p < 0.05. Polynomial Contrast analysis (linear, quadratic, and cubic effects) revealed the dose-effect relationship of increasing use of FGP (0, 7.5, 15, and 22.5%) in experimental TMRs. The data’s mean, standard error, and mean standard deviation are also given in the tables.
This study performed in vitro gas production and fermentation values (methane, ME, NEL, OMD, SCFA, and ammonia-N) in 12 replicates for each experimental ration. Chemical analyses of feed raw materials and FGP samples were performed in three replicates. Twelve fermentation syringes were used as blank (without feed sample, containing only buffer, solutions, and rumen fluid) to calculate the correction value for in vitro gas production.

3. Results

3.1. Chemical Composition of Fermented Grape Pomace

The FGP contained an average of 36.88% DM, 94.6% OM, 10.92% CP, 7.01% EE, 56.5% aNDFom, 51.49% ADFom, 35.76% lignin, 5.6% ash, 19.98% NFC, 15.75% cellulose, 4.99% hemicellulose and 3.66 g/DM TCT in DM (Table 2). Among the total fatty acids of FGP used in this study presented, 60.54% linoleic acid (ω-6), 17.01% oleic acid (ω-9), 12.39% palmitic acid, 5.80% stearic and 2.22% α-linolenic acid (ω-3) in total fatty acids. The FGP included 80.33% unsaturated fatty acids, 17.24% monounsaturated fatty acids (MUFA), 63.09% polyunsaturated fatty acids (PUFA), and 99.37% long chain fatty acids (LCFA) fatty acids in the total fatty acids. The total fatty acids of the FGP consisted of 2.34% ω-3, 60.75% ω-6, and 17.07% ω-9 fatty acids (Table 3).

3.2. Chemical Composition of TMRs

The starch contents of TMRs (0, 7.5, 15, and 22.5%) with FGP were approximately 21% and 17%. The CP contents in TMRs in the present study were in the range of 11.22% and 12.55%. While the EE content was 2.27% in the TMR without FGP, it increased to 3.88% with 22.5% FGP. The aNDFom value of the prepared TMRs was between about 41.4% and 45.7%, and the hemicellulose contents were in a range of about 11–16%. The ADFom content of the TMRs increased from about 30% to 32% due to FGP utilization. The lignin contents of the TMRs with and without FGP were approximately 14–15%, and their cellulose contents were around 16–17%. The NFC contents of the TMRs in the present study ranged from 32 to 36%. The non-fiber carbohydrate (NFC) content differed between the TMRs and was found to be higher than the control TMR (approximately 11%) compared to the treatment TMRs (about 15–16%). While the TCT content of the TMRs was 1.16 g/DM in the control TMR, it increased with the addition of FGP to 2.32 g/DM (Table 4).

3.3. In Vitro Ruminal Fermentation Values

In vitro cumulative gas production values were similar at the 6th, 12th, 18th, and 24th hours of the TMRs with and without FGP (p > 0.05). In vitro cumulative gas production values did not change with increasing FGP addition (Table 5). In vitro methane production (mL and %) and other ruminal fermentation values of adding FGP to experimental TMRs are given in Table 6. The in vitro methane production decreased linearly with the addition of FGP up to 22.5% to experimental TMRs (p < 0.05). The addition of FGP up to 22.5% in the experimental TMRs did not change (for linear, quadratic, and cubic contrasts) the calculated in vitro ME, NEL, and OMD values, as well as NH3-N concentration and pH value of the in vitro fermentation fluid. In the present study, in vitro methane production values of experimental TMRs were approximately 19–20% in total gas, ME value is approximately 8.3–8.5 MJ/kg DM, NEL value is approximately 4.7–4.9 MJ/kg DM, OMD values approximately 61–63%, and the NH3-N concentration was found to be approximately 82–87 mg/L (Table 6).

3.4. In Vitro Fermentation Fluid Variables

The molarity of T-SCFA in the in vitro fermentation fluid was approximately 124 mmol/L for the control TMR, and it was approximately 128, 106, and 88 mmol/L for the TMRs with 7.5%, 15%, and 22.5% FGP, respectively. The T-SCFA decreased with the addition of FGP (p < 0.05) (Linear p value = 0.027, Quadratic p value = 0.001, and Cubic p value = 0.027). However, the T-SCFA molarity in the control experimental TMR fermentation fluid was similar to experimental TMR with 7.5% FGP. The T-SCFA molarity in fermentation fluid of the TMRs with 15% and 22.5% FGP was lower than that of the control TMR (p < 0.05). The molarities of AA, PA, BA, and VA in the in vitro fermentation fluid linearly decreased with the addition of FGP to TMR (p < 0.05), but the molarities of these acids did not change in quadratic and cubic contrasts (p > 0.05). With the use of FGP up to 22.5% in experimental TMRs, the molarity of IVA and IBA from BSCFAs in the in vitro fermentation fluid decreased (p < 0.05) (linear contrast p values = 0.004 and 0.012, respectively). The FGP supplementation to experimental TMRs did not change the molarity of ICA (p > 0.05). The HEPA and HEXA molarities in the in vitro fermentation fluid were not affected (contrast p values >0.05) by the FGP ratio in the experimental TMRs (p < 0.05) (Table 7).

4. Discussion

4.1. The Chemical Composition of Fermented Grape Pomace

The nutrient composition of the FGP was generally compatible with previous studies [29,30,31]. The FGP used in the present study had a CP content of approximately 11%. This value was similar to some studies [13] or higher [30,32] or lower [33]. It was determined that FGP was a good source of structural carbohydrates (56.5% aNDFom, 51.49% ADFom, 35.76% lignin, 15.75% cellulose, and 4.99% hemicellulose). These findings were supported by previous study results [6,29,30,31]. The proportion of grape seed, which constitutes most of the grape pomace, was the factor affecting the nutritional value of the pomace. The most crucial nutrient substances in grape seeds are carbohydrates and fats. The EE content of FGP in the present study was 7.01%, similar to previous studies [13,32]. The ash content, which expresses the total inorganic substances, is about 5% in the FGP used; in some studies, it was higher [13,32] or lower [33]. A meta-analysis determined that the ash content of grape pomace varied between 2% and 23% depending on the type, the process applied, and whether it was fermented or sweet pulp [6]. Beres et al. [30] reported that red grape pomace flour contained 6.4% ash, 13.87% CP, 2.12% EE and 59.13% TDF. Another study reported that grape pomace flour had 2.75% EE, 6.58% CP, 2.75% ash, and 71.41% carbohydrate content [33]. Although the FGP in the study contained approximately 36% lignin, it was similar to previous studies [8,9]. The high lignin content in the FGP used in the present study could be related to the seed rate and seed size of the grape species and the amount of grape stem. The NFC content of FGP in the present study was approximately 20%, most of which was composed of sugar and pectin. Researchers reported that grape pomace contained 8.04–15% glucan, 4.1–7.05% xylan, and pectineus polysaccharide (less than 3%) [8,9]. The fatty acids found at the highest level in the total fatty acids of FGP used in the present study were linoleic acid > oleic acid > pamitic acid > stearic acid > α-linolenic acid, which was consistent with previous studies [34,35]. The FGP contained 60.54% linoleic acid (ω-6), 17.01% oleic acid (ω-9), 12.39% palmitic acid, 5.80% stearic, and 2.22% α-linolenic acid (ω-3) in the total fatty acids. In the present study, FGP obtained from Kalecik karasi, Öküzgözü, and Boğazkere (red grape) varieties of the grape contained 7.01% EE. Researchers reported that the EE content of grape seeds contained range from 6% (red grape varieties such as Cinsaut and Gamay) to 20% (white sweet grape seeds such as Isabella and Muscat of Hamburg) per 100 g dry weight [36,37,38]. It has been stated that this grape seed oil’s principal components were triacylglycerol, which consists of mainly omega-6 linoleic acid [39]. Tangolar et al. [40] found that linoleic acid was the dominant fatty acid among the fatty acids identified in the seeds of seven grape varieties (Alphonse Lavallée, Muscat of Hamburg, Alicante Bouschet, Razaki, Narince, Öküzgözü, and Horoz karasi), followed by oleic acid (C18:1) and palmitic acid (C16:0) in all varieties. Vlaicu et al. [32] reported that total fatty acids in grape pomace comprised approximately 30% unsaturated fatty acids, 66% PUFA, and 65% w-6 fatty acids. The FGP used in the present study contained 80.33% unsaturated fatty acids, 17.24% MUFA, 63.09% PUFA, and 99.37% LCFA fatty acids in total fatty acids. The nutrient composition of the FGP used in the present study was relatively similar to those of previous studies despite substantial differences in soil structure, environmental conditions, type and variety of grapes, and the processes applied according to the purpose of grape processing (sweet, fermented, and alcoholic grape pomace). In addition, the microbial community in the chemical structure of wet grape pomace may change depending on the waiting times and conditions in the environment.

4.2. In Vitro Ruminal Fermentation Values

In vitro gas production technique is a practical method used to measure the ruminal degradation of feed in ruminants [25]. In this study, it was understood that the in vitro gas production and calculated digestion parameters of different TMRs were similar, possibly due to the similarity of easily fermented carbohydrates (starch, NFC), aNDFom, and hemicellulose in the content of the substrates. The in vitro total gas production values of experimental TMR without FGP at the 6th, 12th, 18th, and 24th hours of incubation did not change with supplementation of FGP up to 22.5% to TMRs. Therefore, ME (approximately 8.3–8.5 MJ/kg DM), NEL (4.7–4.9 MJ/kg DM), OMD (approximately 61–63%) values, and NH3-N (82–87 mg/L) concentration in the in vitro fermentation fluid of experimental TMRs were similar to those of dairy cattle TMRs used up to 22.5% FGP.
Previous researchers stated that the most abundantly identified microbiota taxa in rumen fluid were members of the genera Prevotella, Ruminococcus, and Butyrivibrio, as well as unclassified members of the orders Clostridiales and Bacteroidales and of the families Ruminococcaceae and Lachnospiraceae [41]. Prevotella is a very versatile microbe capable of processing a wide range of proteins and polysaccharides, and one of its fermentation products is propionate [42]. In addition, Kara et al. [43] reported that the bacterial genus with the highest relative abundance in the rumen environment is Rikenellaceae_RC9_gut_group (about 15.5–18.5%) and Prevotella (about 4–10%). Rabee et al. [44] stated that the dominant bacterial genera in the rumen are Rikenellaceae_RC9_gut_group, Ruminococcus, Saccharofermentans, Butyrivibrio, Succiniclasticum, Selenomonas, and Streptococcus. In the present study, the condensed tannins and fatty acids in FGP could be affected by the in vitro fermentation value due to effects on the fibrolytic, pectinolytic, cellulolytic, and proteolytic bacteria [45,46].
Methane is produced naturally by the fermentation of feedstuffs materials in the rumen. Ruminal methane production is carried out by Methanobrevibacter and Methanosphaera genera from the order Methanobacteriales from Euryarchaeota [43]. These archaea use H2/CO2 or H2/methanol produced by fermentative bacteria as a by-product of carbohydrate metabolism [47]. Methanobrevibacter is active in the hydrogenotrophic pathway that catalyzes the conversion of CO2 to methane in the rumen [48]. The TGP could effectively reduce ruminal methane emission due to the phenolic compounds in their content. It was determined that the FGP used in this study contained 3.66 g/DM TCT. When the condensed tannins in feedstuff are above 2–3%, they can prevent the breakdown of ruminal protein and enable proteinaceous compounds to be broken down and digested in the abomasum and duodenum [18]. Depending on the condensed tannin content, ruminal methane production can be reduced [26]. In the present study, in vitro methane production decreased from 8.3 mL to 7.1 mL for 0.2 g DM of TMR, with a decrease of approximately 15% with 22.5% FGP in TMR. It is thought to be caused by the suppressive effect on the rumen’s methanogenic archaea and ciliated protozoa [43,47,49] due to the TCT and BCT in the grape pomace. Moate et al. [50] showed that feeding a diet (total 18.3 kg/d DM) containing 5.0 kg of the dried-pelleted or ensiled FGP DM/d (about 27.3% FGP in TMR) of dairy cattle decreased methane emissions and methane yield by approximately 20%, altered ruminal bacterial and archaeal communities, but did not change ruminal fungal and protozoan communities. Other researchers found that the grape pomace powder at 2% of DM intake of dairy steers decreased ruminal AA concentration, cellulolytic bacteria count, the number of protozoa, and methane production [51]. O’Brien et al. [52] stated that adding oleic acid to grass silage reduced in vitro ruminal methane production. Jayanegara et al. [53] reported that condensed and hydrolyzable tannins reduced methane production. The BCT is also found in plants with fibrous compounds [54]. In the present study, the anti-methanogenic activity of FGP suggests that BCT, which is linked to fibrous compounds, affects the fermentation of FGP in the wine production process and plant cell wall compounds positively affect ruminal fermentation and that condensed tannin compounds may be more effective on the microbial ecosystem in the rumen environment [45,46]. Poulsen et al. [55] demonstrated that ruminal fermentation of pectin resulted in significantly lower methane production rates during the first 10 h of fermentation compared to the other carbohydrate sources (wheat and corn starch and inulin). In addition, Liu et al. [56] reported that hemicellulose was hydrolyzed more quickly than cellulose. These researchers determined that the higher concentrations of acetic, n-butyric, and n-valeric acids hydrolyzed from the hemicellulose resulted in a lower pH and more severe inhibition of methane production than that of cellulose. In the present study, the anti-methanogenic activity of FGP utilization in TMR may also be related to an increase in starch-free NFC (mainly pectin) and a decrease in hemicellulose.
The indicator of the fermentation level of the feedstuffs or ration in the rumen is the total gas amount produced, as well as the level of the end-products in the fermentation liquid. The molarities of T-SCFA, AA, PA, BA, VA, and BSCFAs (IBA and ICA) in the in vitro fermentation fluid of experimental TMRs with and without FGP were consistent with reference values [43]. In this study, the T-SFCA and AA, PA, BA, and VA from SCFA in the in vitro fermentation fluid linearly decreased with the FGP supplementation doses, especially at high doses (22.5%). This could be related to the TCT content, and the high level of fatty acids (linoleic acid, palmitic acid, and oleic acid) in content [57,58]. Moate et al. [50] demonstrated that the dried-pelleted FGP (about 27.3% in TMR as DM) in experimental TMRs decreased ruminal AA molarity, but ensiled FGP did not change ruminal AA molarity. The reducing effect on acids, which is the end product of ruminal fermentation, showed that the tannins or fatty acids of FGP could have a suppressive effect on bacteria that decompose the carbohydrates (cell wall components and starch components) in the rumen [52,59].
The BSCFA concentration of experimental TMRs with and without FGP differed, although the ICA concentration did not change. A previous study stated that the decrease in the molarity of BSCFAs in the rumen fluid was associated with a decrease in the relative ratio of Provetella bacteria in the rumen fluid [43]. The decrease in BSCFA concentration in fermentation fluid in the present study was consistent with the decreased abundance of Prevotella, which are considered to be major fermenters of peptides and amino acids in the rumen by previous researchers [42,60]. The reduction in the molarity of IBA and IVA in the present study could indicate reducing protein degradation in the rumen. The TCT and BCT in FGP could reduce the molarity of ruminal iso-acids by partially reducing ruminal protein breakdown.
In conclusion, the FGP had a good nutrient composition (except for lignin), functional fatty acid profile, and superior in vitro ruminal fermentation value for ruminants. The FGP had a ruminal anti-methanogenic effect on dairy cattle. Using FGP up to 22.5% in experimental TMR (by reducing corn silage and grains) based on DM did not adversely affect in vitro cumulative gas production, ME, NEL, OMD, and ammonia-nitrogen. Using up to 15% FGP in TMR did not change the concentrations of ruminal SCFA (AA, PA, BA, and VA) and BSCFA (IBA and IVA). In addition, 15% and 22.5% FGP used in TMR decreased ruminal T-SCFA molarities, which are the energetic products of the ruminal fermentation for the animal. The FGP could be recommended to use up to 15% of the experimental TMRs. However, in vivo feeding trials also need to investigate these results.

Author Contributions

Conceptualization: K.K.; Methodology: K.K.; Formal analysis and investigation: K.K. and M.A.Ö.; Writing—original draft preparation: K.K. and M.A.Ö.; Writing—review and editing: K.K.; Resources: K.K. and M.A.Ö.; Supervision: K.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by The Research Fund of Erciyes University (Kayseri, Türkiye), Project ID TYL-2021-10900. This study was produced from Mehmet Akif Öztaş’s master’s thesis (thesis number: 786268) named “The effect of fermented grape pomace using at different ratios in dairy cattle ration on the in vitro total gas and methane production, digestibility and rumen fluid variables”.

Institutional Review Board Statement

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to, and the appropriate ethical review committee approval has been received. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes and feed legislation.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data will be provided to any interested part upon its requesting.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Table 1. Fermented grape pomace addition to experimental total mixed ration used in in vitro gas production technique.
Table 1. Fermented grape pomace addition to experimental total mixed ration used in in vitro gas production technique.
TMRs Containing FGP, kg/day DM
Feedstuffs0%7.5%15%22.5%
FGP0.001.683.375.05
Corn silage6.204.523.002.80
Wheat straw3.593.593.332.11
Alfalfa hay3.683.683.683.50
Barley flake1.381.381.752.12
Wheat grain1.381.381.381.38
Corn flake1.381.381.381.38
Sunflower meal, 28% CP2.762.762.482.21
Cottonseed meal, 28% CP2.302.302.302.12
Total (kg)22.6722.6722.6722.67
CP: Crude protein, DM: Dry matter, FGP: fermented grape pomace.
Table 2. Chemical composition and in vitro fermentation values of FGP.
Table 2. Chemical composition and in vitro fermentation values of FGP.
Chemical Composition%, in DM
DM, % (as fed)36.88
OM 94.60
CP10.92
EE7.01
aNDFom56.50
ADFom51.49
Lignin35.76
Ash5.60
NFC19.98
Cellulose15.75
Hemicellulose4.99
TCT0.37
BCT0.33
In vitro ruminal fermentation values
In vitro GP44.25
In vitro Methane, %17.96
In vitro Methane, mL8.48
ME10.61
NEL5.51
OMD57.59
CP: crude protein, EE: Diethyl ether extract, aNDFom: neutral detergent-insoluble fibrous compounds detected by alpha amylase and without ash, ADFom: acid-free detergent-insoluble fibrous compounds without ash, Lignin: acid detergent lignin = aNDFom − ADFom, NFC: Non-fibrous carbohydrate, Cellulose: ADFom − Lignin, TCT: Total condensed tannin, BCT: Bound condensed tannin. In vitro GP: Total gas produced as mL by 0.2 g feed DM in 24 h incubation, In vitro methane %: % methane in total gas produced by 0.2 g feed DM in 24 h incubation, In vitro methane mL: Methane volume produced by 0.2 g feed DM in 24 h incubation, ME: in vitro metabolic energy as Mcal for kg DM, NEL: in vitro net energy lactation as Mcal for kg DM, OMD: in vitro organic matter digestion as % in DM.
Table 3. The fatty acid content of fermented grape pomace.
Table 3. The fatty acid content of fermented grape pomace.
Fatty AcidsAs % in Total Fatty Acid
Myristic acid (C14:0)0.21
Myristoleic acid (C14:1)0.02
Pentadecanoic acid (C15:0)0.06
Palmitic acid (C16:0)12.39
Palmitoleic acid (C16:1) 0.10
Heptadecanoic acid (C17:0) 0.60
cis-10-Heptadecenoic acid (C17:1) 0.06
Stearic acid (C18:0) 5.80
Oleic acid (C18:1 w9 cis) 17.01
Linoleic acid (C18:2 w6 cis) 60.54
Arachidic acid (C20:0) 0.23
cis-11-Eicosenoic acid (C20:1) 0.01
α-Linolenic acid (C18:3 w3) 2.22
Heneicosanoic acid (C21:0) 0.02
cis-11,14,17-Eicosadienoic acid (C20:2) 0.05
cis-8,11,14-Eicosatrienoic acid (C20:3 w6) 0.03
Erucic acid (C22:1 w9) 0.05
cis-11,14,17-Eicosatrienoic acid (C20:3 w3) 0.00
Arachidonic acid (C20:4 w6) 0.04
Trichosanoic acid (C23:0) 0.00
cis-13,16-Docosadienoic acid (C22:2) 0.10
Lignoceric acid (C24:0) 0.20
cis-5,8,11,14,17-Eicosapentaenoic acid (C20:5 w3)0.04
Nervonic acid (C24:1) 0.01
cis-4,7,10,13,16,19-Docosahexaenoic acid (C22:6 w3) 0.08
Saturated fatty acid, %19.66
Unsaturated fatty acid, %80.33
MUFA17.24
PUFA63.09
ω-32.34
ω-660.75
ω-917.07
ω-3/ω-60.04
MCFA0.09
LCFA99.37
VLCFA0.45
MUFA: Monounsaturated fatty acid, PUFA: Polyunsaturated fatty acid, MCFA: Medium chain fatty acid (6–12 C), LCFA: Long-chain fatty acid (14–20 C), VLCFA: Very long-chain fatty acid (>20 C).
Table 4. Chemical compositions of TMRs with added fermented grape pomace.
Table 4. Chemical compositions of TMRs with added fermented grape pomace.
%, DM
Chemical Compositions, % in DM0% FGP7.5% FGP15% FGP22.5% FGP
Starch20.8321.1919.3717.26
CP11.2211.2911.5012.55
EE2.272.813.123.88
aNDFom45.6941.3541.3543.78
ADFom29.9430.2630.7432.30
Lignin13.7414.4613.6015.25
Ash8.958.368.087.48
NFC31.8536.1735.9532.29
Starch-free NFC11.0214.9816.5715.03
Cellulose16.2015.8017.1417.05
Hemicellulose15.7511.0910.6011.47
TCT0.120.130.190.23
BCT0.110.120.150.20
CP: crude protein, EE: Diethyl ether extract, aNDFom: neutral detergent-insoluble fibrous compounds detected by alpha amylase and without ash, ADFom: acid-free detergent-insoluble fibrous compounds without ash, Lignin: acid detergent lignin = aNDFom − ADFom, NFC: Non-fibrous carbohydrate, Starch-free NFC = NFC − Starch, Cellulose: ADFom − Lignin, TCT: Total condensed tannin, BCT: Bound condensed tannin.
Table 5. In vitro cumulative total gas production of dairy cattle TMR with fermented grape pomace.
Table 5. In vitro cumulative total gas production of dairy cattle TMR with fermented grape pomace.
In Vitro Cumulative Total Gas Production, mL/0.2 g DM
6th h12th h18th h24th h
TMR with 0% FGP 21.1431.4135.7741.42
TMR with 7.5% FGP 20.8430.4936.1740.16
TMR with 15% FGP 22.4831.6736.0940.56
TMR with 22.5% FGP 19.4028.3132.4740.09
SD 2.394.225.373.51
SEM 0.480.861.090.50
p-value (combined) 0.1700.5320.6080.792
p-valueL0.3990.3140.3330.448
Q0.1500.4960.3830.707
C0.1240.4070.7650.583
L: Linear, Q: Quadratic, C: Cubic, effects of polynomial contrast.
Table 6. In vitro ruminal methane and other fermentation values of the addition of fermented grape pomace to dairy cattle TMR.
Table 6. In vitro ruminal methane and other fermentation values of the addition of fermented grape pomace to dairy cattle TMR.
In Vitro Methane %In Vitro Methane mLMENELOMDNH3-NpH
TMR with 0% FGP19.87 a8.27 a8.524.8962.7781.996.62
TMR with 7.5% FGP19.70 a7.72 ab8.334.7361.0284.626.64
TMR with 15% FGP18.72 ab7.33 ab8.374.8461.0486.956.65
TMR with 22.5% FGP18.25 b7.07 b8.344.9360.5082.586.63
SD1.020.350.4730.393.145.460.05
SEM0.300.190.060.050.451.090.01
p-value (combined)0.0350.0250.7430.6440.3160.3840.951
p-valueL0.0270.0210.4320.6660.0990.6740.728
Q0.7840.6930.5470.2860.5050.1240.666
C0.8870.9980.6220.5710.5630.5260.938
L: Linear, Q: Quadratic, C: Cubic, in vitro methane %: as % methane in the total gas produced by 0.2 g feed DM in 24 h of incubation, In vitro methane mL: methane level in total gas produced by 0.2 g feed DM in 24 h incubation, NH3-N: as mg/L ammonia nitrogen of in vitro fermentation fluid, ME: in vitro metabolic energy as Mcal in kg DM, NEL: in vitro net energy lactation as Mcal in kg DM, OMD: in vitro organic matter digestion as % in DM. a,b: The difference between the means shown with different lettering in the same column is significant according to the Tukey multiple comparison test.
Table 7. The molarities of short-chain fatty acid in the in vitro ruminal fermentation of dairy cattle TMR with fermented grape pomace.
Table 7. The molarities of short-chain fatty acid in the in vitro ruminal fermentation of dairy cattle TMR with fermented grape pomace.
mmol/L, In Vitro Ruminal Fermentation
SCFABSCFAT-SCFAA/P(A + B)/P
AAPABAVAHEXAHEPAIBAIVAICA
TMR with 0% FGP89.79 a16.04 a12.65 ab1.44 ab0.390.231.23 a2.23 a0.26124.01 a5.776.56
TMR with 7.5% FGP91.31 a17.13 a13.47 a1.52 a0.410.351.23 a2.39 a0.39127.86 a5.426.21
TMR with 15% FGP75.74 ab14.20 ab11.16 ab1.26 ab0.350.341.04 ab2.05 ab0.26106.13 b5.386.16
TMR with 22.5% FGP62.34 c12.60 b9.43 b1.18 b0.360.340.87 b1.88 b0.3988.21 c5.085.83
SD12.502.662.480.200.090.100.260.330.1517.190.870.87
SEM2.500.530.490.040.010.020.050.060.043.440.170.17
p-value (combined)<0.0010.0070.0140.0070.7680.4280.0280.0330.586<0.0010.5480.548
p-valueL<0.0010.0020.0050.0020.7490.2450.0040.0120.5500.0270.1710.934
Q<0.0010.1340.1400.2360.4600.2920.3620.1750.9860.0010.9800.976
C0.0150.1840.3380.1060.4600.6280.6290.2190.2330.0270.7160.716
L: Linear, Q: Quadratic, C: Cubic. ICA: Iso-caproic acid, IBA: Iso-butyric acid, IVA: Iso-valeric acid, HEPA: Heptanoic acid, HEXA: Hexanoic acid, VA: Valeric acid, BA: Butyric acid, PA: Propionic acid, AA: Acetic acid, SCFA: Short chain fatty acids = AA + PA + BA + VA + HEXA + HEPA, BSCFA: Branched short-chain fatty acid = ICA + IBA + IVA, T-SCFA: Total short-chain fatty acid = SCFA + BSCFA, A/P = acetic acid/propionic acid, (A + B)/P = (acetic acid + butyric acid)/propionic acid. a–c: The difference between the means shown with different lettering in the same column is significant according to the Tukey multiple comparison test.
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Kara, K.; Öztaş, M.A. The Effect of Dietary Fermented Grape Pomace Supplementation on In Vitro Total Gas and Methane Production, Digestibility, and Rumen Fermentation. Fermentation 2023, 9, 741. https://doi.org/10.3390/fermentation9080741

AMA Style

Kara K, Öztaş MA. The Effect of Dietary Fermented Grape Pomace Supplementation on In Vitro Total Gas and Methane Production, Digestibility, and Rumen Fermentation. Fermentation. 2023; 9(8):741. https://doi.org/10.3390/fermentation9080741

Chicago/Turabian Style

Kara, Kanber, and Mehmet Akif Öztaş. 2023. "The Effect of Dietary Fermented Grape Pomace Supplementation on In Vitro Total Gas and Methane Production, Digestibility, and Rumen Fermentation" Fermentation 9, no. 8: 741. https://doi.org/10.3390/fermentation9080741

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