Evaluation of the Quality and Nutritional Value of Modified Corn Wet Distillers’ Grains Plus Solubles (mcWDGS) Preserved in Aerobic and Anaerobic Conditions

Abstract

To enhance the effectiveness of sustainable preservation of modified corn wet distillers’ grains plus solubles (mcWDGS), various additives were tested under aerobic and anaerobic conditions. In Experiment I, the mcWDGS was stored under aerobic conditions for 5 days at 25 °C. Treatments included different organic acids applied at 0.3% or 0.6% of fresh matter (FM). In Experiment II, the mcWDGS was ensiled anaerobically for 8 weeks at 25 °C using organic acids, a commercial acid mixture, or a microbial inoculant at 0.2% FM. In aerobic conditions, the best preservability was achieved with propionic and formic acids at 0.6% FM, as indicated by the lowest temperature, pH, and microbial counts on days 3 and 5 (p ≤ 0.01). Under anaerobic storage, the highest lactic acid concentrations were recorded in the control, citric acid, and commercial acid mixture variants (p ≤ 0.01). Acetic acid levels were highest in the control (p ≤ 0.01). The highest NH3-N content was found in the formic acid variant and the lowest in the inoculant variant (p ≤ 0.01). Aerobic stability after ensiling was greatest in the control and propionic acid groups (p ≤ 0.01). Nutritional analysis showed that the citric acid group had the highest dry matter content (p ≤ 0.01), while the control group contained the most crude protein (p ≤ 0.01) and saturated fatty acids (p ≤ 0.05). The propionic acid and commercial acid mixture variants had the highest unsaturated fatty acids (p ≤ 0.05). Antioxidant capacity was also greatest in the control (p ≤ 0.01). In conclusion, mcWDGS can be effectively preserved aerobically with 0.6% FM of propionic or formic acid, and anaerobically via ensiling, even without additives. These findings support its potential as a stable and nutritious feed ingredient.

1. Introduction

Many by-products from the agricultural, food, or biofuel industries are excellent sources of nutrients in animal nutrition [1]. In some cases, certain by-products, such as post-extractive soybean meal, potato protein, or distillers’ grains, can be more valuable and expensive to feed producers or farmers than the primary produce from which they originated. Even though some industrial by-products possess beneficial characteristics such as higher protein and/or rumen undegradable protein content, they frequently face underutilization by farmers due to a variety of reasons. The most common reason is high moisture content, which often creates a need for costly drying processes. Drying can be effective, as it improves storability and enables use in feed rations. However, as energy costs are gradually rising, so are the costs of feed drying, which subsequently leads to increased prices [2,3]. Therefore, a more economical and sustainable solution is required to facilitate and promote the use of wet by-products in animal feeding, thereby avoiding high drying costs. One of these by-products, which could be fed in wet form, is wet distillers’ grains plus solubles (WDGS). WDGS, alongside wet distillers’ grains (WDG), dried distillers’ grains (DDG), dried distillers’ grains plus solubles (DDGS), condensed corn distillers solubles (CCDS), and non-normative fraction of corn (NNCF), is created as a by-product during bioethanol production [4,5,6]. WDGS is a mixture of WDG-CCDS at an unstandardized ratio that usually oscillates between 80:20 and 90:10 per fresh matter (FM) [7]. DDG and DDGS are dehydrated WDG and WDGS, respectively. DDGS and WDGS can be utilized as organic fertilizers, soil amendments, or feedstuff [8]. CCDS can also be considered a resource for biodiesel production or as feed [9], while NNCF is a corn fraction that is unused in bioethanol production and therefore either discarded or, if possible, used as livestock feed [10,11]. To date, the inclusion of non-normative corn fraction (NNCF) in WDG-CCDS mixtures has not been explored. However, this approach could offer a sustainable solution for modifying WDGS, potentially benefiting both producers and consumers by serving as an alternative replacement to corn grain, hay, or straw. WDGS and DDGS are the most frequently used bioethanol by-products in livestock nutrition [12,13]. However, the use of DDGS in animal nutrition is more prevalent, mainly due to the many storability issues surrounding WDGS, as spoilage can occur after only 2 days at 32 °C [14]. As a result, among the many challenges for WDGS utilization as feed are a low dry matter (DM) content of 35–45%, high water activity, and microbial instability [15]. Moreover, the distillers’ grains are characterized by a high energetic value. As such, the concentration of nutrients such as starch is around 9% DM [16], crude protein 25–30% DM, and crude fat 7–12% DM [17]. Low dry matter content and high energetic value lead to an increased growth of detrimental and harmful microbiota, particularly yeasts and molds. Among the most common mold species found in corn grain and WDGS are Alternaria sp., Fusarium sp., and Penicillium sp. [18]. Due to rapid spoilage and decreased storability, WDGS requires immediate and effective preservation methods. Most bioethanol-producing plants utilize drying as the main preservation method for WDGS. However, during this process, temperatures can reach up to 300 °C, requiring significant energetic and financial input, thus making it an impractical solution for some complexes and farmers [19]. Even though DDGS is a more popular option in livestock feeding, WDGS can still be utilized in cattle feeding [20] and might even be successfully implemented into pigs’ diets under a wet lot feeding system [21]. Suffice it to say utilizing WDGS at the expense of corn silage may prove beneficial from both environmental and economic points of view [20].

Nevertheless, the main obstacle to widespread WDGS utilization is its rapid perishability [22]. Consequently, WDGS requires adequate preservation immediately after its production at the bioethanol plant. From the perspective of WDGS producers, it is essential to find a short-term, inexpensive, and sustainable method during aerobic preservation. The producers’ main interests lie with selling WDGS, so it must not spoil under any circumstances during storage or transportation (oxygen-rich environment). However, from the farmers’ perspective, WDGS is a type of roughage that requires long-term preservation through ensiling (oxygen-deficient environment), with an emphasis on its use at a later date. Therefore, WDGS storage requires a two-tier preservation strategy, accounting for the short-term requirements to be employed by producers and the long-term need to be utilized by the farmers.

To prevent WDGS spoilage and effectively decelerate the growth of harmful microorganisms during aerobic and anaerobic preservation conditions, pH must be maintained below 4 [23] or between 4 and 4.5 [24]. For this purpose, technological feed additives such as acidifiers or inoculants may be added to WDGS to extend its shelf-life and utility, similarly to how they are currently used for other wet by-products [25,26]. Propionic and formic acids are the most effective acidifiers in feed preservation [27]. Moreover, propionic, formic, acetic, lactic, and citric acids have a range of inhibitory effects against fungi and bacterial activity [28,29].

The preservation method of WDGS can affect its chemical composition and nutritional value. WDGS stored under aerobic conditions exhibited higher dry matter, organic matter, and crude fat losses than in anaerobic conditions [30]. However, WDGS storage and preservation conditions do not affect the content of crude protein, ruminal undegradable protein, or NDF levels, even if feed spoilage and secondary fermentation have already occurred under aerobic conditions [22,30].

This study aimed to analyze the nutritional value and quality of WDGS preserved under aerobic and anaerobic conditions. It can be assumed that these preservation methods are essential in decelerating WDGS spoilage from both producer and consumer perspectives. Moreover, we hypothesize that it should be possible to prolong mcWDGS storability by applying acidifiers or other technological additives for up to 5 days under aerobic conditions and 8 weeks under anaerobic conditions. However, the addition of NNCF could potentially affect storability due to changes in chemical composition, e.g., increasing dry matter and starch contents or decreasing crude protein concentration. Therefore, we conducted physical and chemical analyses to determine the feasibility of aerobic and anaerobic preservation of WDGS and to assess whether any changes in the nutritional value or quality of the feed occurred due to the storage conditions.

2. Materials and Methods

2.1. Experimental Design and Research Procedures

Two experiments were performed in November 2021 to determine the preservation methods of modified corn WDGS (mcWDGS) under aerobic and anaerobic conditions. The experimental mixture—mcWDGS—was prepared at the Bioagra SA bioethanol plant (Bioagra SA, Nysa, Poland). It consisted of WDG, NNCF, and CCDS blended in a ratio of 75:15:10 on an FM basis, which corresponds to 61:32:7 on a DM basis. A self-made experimental mixer equipped with a liquid dispenser, created and provided by the Institute of Wood Sciences and Furniture of Warsaw University of Life Sciences in Warsaw, was used for this process. The mixer, with a volume of 30 L, operated at 25 rpm. While the industrial standard for WDGS typically involves mixing WDG and CCDS in an 80:20 or 90:10 FM ratio [4], this study incorporated an additional 15% of NNCF (FM basis) to further enrich the WDGS. NNCF is a corn by-product that originates from unused grain in bioethanol production due to kernel damage. The ingredients used in this study were confirmed to be safe for feed processing and livestock consumption, in accordance with EU Regulation (EC) No 1881/2006 of 19 December 2006 [31]. The addition of NNCF can potentially enhance WDGS by increasing its dry matter and nutrient content, specifically starch and energy. The average chemical compositions of components and the final product (mcWDGS) are presented in

Table 1. The chemical composition of mcWDGS and its constituents, i.e., WDG, NNCF and CCDS (g/kg DM).

Item	Bioethanol By-Products
	WDG	NNCF	CCDS	mcWDGS
Dry Matter (FM)	412.5	906.6	792.7	435.0
Crude Protein	238.5	68.9	242.4	247.2
Ether Extract	79.8	34.6	132.0	69.8
Crude Fiber	204.2	36.4	18.0	158.0
Starch	56.1	743.0	16.2	275.5
Crude Ash	29.6	19.0	24.7	29.1

WDG—wet distillers’ grain; NNCF—non-normative fraction of corn; CCDS—condensed corn distillers’ solubles; mcWDGS—mixture of WDG:NNCF:CCDS at 61:32:7 per DM; FM—in fresh matter.

.

Experiment I: mcWDGS underwent aerobic preservation. The material was placed in open, non-sealed plastic mini-silos of 15 L capacity in a storage chamber and maintained for 5 days at 25 °C, which is in line with Alvarez et al. (2019) [22] and Lehman and Rosentrater (2012) [14]. Each WDGS variant received a distinct additive and was prepared in triplicate, utilizing separate mini-silos for each replicate. A total of five different additives were used, each at two concentrations. All additives were incorporated during the mixing phase of the mcWDGS ingredients. Each additive was prepared by diluting it 50:50 (w/w) with distilled water. The final inclusion rates for these additives were 0.3% or 0.6% on a fresh matter (FM) basis, consistent with our preliminary findings and values recommended in the literature [25,32,33]. These additives included propionic (PA0.3%; PA0.6%), formic (FA0.3%; FA0.6%), citric (CA0.3%; CA0.6%), and lactic (LA0.3%; LA0.6%) acids, or a commercial acidifier mixture (CAM0.3%; CAM0.6%) (consisting of glycerin, propylene glycol propionic, lactic, orthophosphoric, formic, acetic, citric, and muriatic acid). The control variant (C1) was also prepared in triplicate, with mcWDGS preserved without any acidifier addition (Figure 1). Measurements in Experiment I were primarily performed on day 3 of preservation. This timeframe was selected based on practical considerations, such as typical transport to farms and storage duration, and aligns with the findings of Lehman and Rosentrater (2012) [14]. During the sampling procedure (at 0, 3, and 5 days of storage), mcWDGS was transferred from the mini-silos. Samples were either frozen at −20 °C for oxidative capacity analysis or dehydrated at 105 °C for chemical composition analysis. Analyses conducted on fresh material examined temperature, pH, total bacteria count (TBC), and total fungi count (TFC). For day 0, analyses such as TBC, TFC, chemical composition, and oxidative capacity were performed on a combined mean sample, assuming that these parameters would be uniform across all variants immediately after mixing due to the homogenous nature of mcWDGS. Subsequently, all variants were analyzed on the 3rd day. On day 5, however, analyses were performed exclusively on the mcWDGS variants that had not spoiled. The TBC, TFC, chemical composition, and oxidative capacity were analyzed on days 0, 3, and 5 of Experiment I; however, for day 0, only a mean value representing all the variants was given due to the practical impossibility of simultaneously collecting and preparing individual samples for each mcWDGS variant and replicate immediately upon additive application. Also, these chemical-physical analyses should not have been affected at day 0 in the same manner as pH was. Still, future studies should try to improve and incorporate collection time at day 0. Moreover, some samples appear to be missing by the 5th day of Experiment I; this was implemented on purpose, as these variants had already spoiled and would have been considered unsafe for livestock consumption. Analyzing feedstuff that could not be used in livestock nutrition would serve little to no purpose. The analyses performed during Experiment I were specifically designed to best illustrate and focus primarily on the effectiveness of conservation under aerobic conditions.

Experiment II: mcWDGS underwent ensiling via anaerobic preservation. The material was packed into an airtight vacuum-sealed polyethylene silobag (25 µm thickness) (vidaXL, Venlo, The Netherlands) at a packing density of approximately 800–875 g/L. These silobags were then sealed within 15 L plastic mini-silos and stored in a chamber at 25 °C. The ensiling process spanned 8 weeks, in line with Alvarez et al. (2019) [22]. Similar to Experiment I, each WDGS variant received a distinct additive and was independently ensiled in triplicate, utilizing separate mini-silos for each replicate. A total of five additives were employed, all incorporated during the mixing phase of the mcWDGS, in which ingredients were combined using a custom-made mixer equipped with a liquid dispenser. Each additive was prepared by diluting it 50:50 (w/w) with distilled water. The final inclusion rate for each additive was 0.2% of fresh matter (FM), which aligned with the values recommended in the literature [26] and the manufacturer’s recommendations for the inoculant and commercial acidifier mixture. The additives used were as follows: propionic (PA0.2%), formic (FA0.2%), and citric acids (CA0.2%), a commercial acidifier mixture (CAM0.2%) (consisting of glycerin, propylene glycol, propionic, lactic, orthophosphoric, formic, acetic, citric, and muriatic acid), or heterolactic bacterial inoculant (I0.2%) (consisting of 6.0 × 1010/1 g of homolactic bacteria species such as Lactobacillus plantarum, L. brevis, L. buchneri, Pediococcus acidilactici, and Enterococcus faecium) (Canagri, Dąbrówka, Poland). Similar to the first experiment, a control variant (C2) was also prepared in triplicate, where WDGS was preserved without additives (Figure 1). The sampling procedure took place during the opening of the silages, so that the WDGS could be transferred from the mini-silos to be frozen at −20 °C for short-chain fatty acid composition, lactic acid, fatty acid composition, ethanol content, ammonia nitrogen content, and oxidative capacity, or dehydrated at 105 °C for analyses of chemical and non-starch polysaccharide composition. The aerobic stability and pH were measured on fresh material. In contrast to Experiment I, the analyses performed during Experiment II were specifically structured to more comprehensively determine the quality and nutritional value of mcWDGS, with a main focus being on its suitability as a livestock feedstuff.

2.2. Chemical and Physical Analyses

2.2.1. Temperature Measurements and Aerobic Stability

Temperature and later aerobic stability were measured using an LB-755A temperature sensor (LAB-EL Elektronika Laboratoryjna Sp. J., Warsaw, Poland), while all the data were collected, transferred, and analyzed by LOGGER software version 2.1.32 (LAB-EL Elektronika Laboratoryjna Sp. J., Warsaw, Poland).

For the first experiment on the aerobic preservation of mcWDGS, temperature was measured on days 0, 3, and 5 of the experiment. However, aerobic stability was analyzed for the second anaerobic preservation experiment (ensiling). mcWDGS was unsealed and its content was transferred to different mini-silos and disturbed, allowing for free air exchange and deep penetration within the mass. The aerobic stability was measured according to the time required to increase the temperature by at least 1 °C.

2.2.2. pH Level

The pH level was measured using a CP-411 pH sensor (Elmetron, Zabrze, Poland) with the IJ44A combination pH electrode (Elmetron, Zabrze, Poland). For the first experiment on the aerobic preservation of WDGS, pH was measured on days 0, 3, and 5 of the experiment. However, for the second anaerobic preservation experiment, pH was measured after unsealing the silage.

2.2.3. Microbial Analyses

To determine the total bacteria count (TBC) and total fungi count (TFC), 10 g of each aerobically preserved mcWDGS variant (the first experiment) was suspended in 90 mL of 0.9% NaCl (w/v). Afterwards, tenfold dilutions were prepared from suspensions in the diluting medium. Following the spread plate method, 100 µL of each WDGS variant suspension was pipetted and spread evenly over the surface of the plate with growing media such as Plate Count Agar LAB-AGAR™ (Biomaxima, Lublin, Poland) for TBC and DRBC LAB-AGAR™ (Biomaxima, Lublin, Poland) for TFC. Then, the plates were placed flat in an incubator. Incubation was performed under aerobic conditions for 24 h at 35 °C. After incubation, the numbers of bacterial and fungal colonies were calculated to express the final mean value as log CFU/g.

2.2.4. Short-Chain Fatty Acid Composition and Lactic Acid, Ethanol, and Ammonia Nitrogen Content

To determine short-chain fatty acid, lactic acid, and ethanol content concentrations, 5 g of the ensilaged mcWDGS (the second experiment) was homogenized, and 5 g was mixed with 60 mL of distilled water. Subsequently, 2 mL of Carrez I 3.6% (w/v), 2 mL of Carrez II 7.2% (w/v), and 4 mL of NaOH 0.4% (w/v) solution were added to each vial. Finally, vials were filled with water to the 100 mL mark, vortexed, and filtered using Whatman 1 filter paper.

The concentrations of L-lactic, D-lactic, acetic, and butyric acids and ethanol were determined using Megazyme assay kits such as K-DLATE 08/18 for both lactic acids and, respectively, K-ACET 04/18, K-HDBA 04/18, and K-ETOH 08/18 (Megazyme, Wicklow, Ireland). The measurements and subsequent calculations were performed according to the manufacturer’s protocols. Afterwards, the absorbance value of the samples was read using a spectrophotometer (INFINITE M NANO; TECAN™, Männedorf, Switzerland) at A = 340 nm; 340 nm; 340 nm; 492 nm; and 340 nm wavelengths, respectively.

To determine the ammonia nitrogen (NH3-N) content, 20 g of the ensilaged mcWDGS was homogenized and extracted with 50 mL of 0.1 M H₂SO₄. The samples were then vortexed for 1 h and left overnight for the solution to settle. From each sample, 10 mL of the supernatant was collected and analyzed via the direct distillation method using a Kjeltec System 1026 Distilling Unit (Foss Tecator, Hilleroed, Denmark).

2.2.5. Chemical Composition

The chemical composition of the feeds was determined according to AOAC (2012) [34]: dry matter content via drying at 105 °C to constant weight, crude ash via incineration at 550 °C for 6 h, crude protein (N × 6.25) by using the micro-Kjeldahl technique (Kjeltec System 1026 Distilling Unit, Foss Tecator, Hilleroed, Denmark), and crude fat after extraction with petroleum ether via the Soxhlet method. Crude fiber, NDF, and ADF in feed were determined according to Van Soest et al. (1991) [35] with ANKOM200 Fiber Analyzer A200l (ANKOM Technology, Macedon, NY, USA). The total water-soluble carbohydrate content was determined using the Luff-Schoorl method, a titrimetric procedure based on calculating the equivalent of the sum of reducing sugars and sucrose according to the PN-R-64784:1994 norm. The starch content was subsequently calculated by multiplying the glucose equivalent by a constant factor [36].

2.2.6. Non-Starch Polysaccharide Composition

The quantitative and qualitative determination of soluble and insoluble non-starch polysaccharide content in ensilaged mcWDGS (the second experiment) was performed with a Clarus 600 GC (PerkinElmer, Waltham, MA, USA) equipped with an autosampler, split injector, and flame ionization detector (FID). After enzymatic starch hydrolysis, samples were centrifuged and separated into a soluble fraction (ethanol precipitates from the supernatant) and an insoluble fraction (the remaining pellet). Afterwards, each fraction underwent acid hydrolysis with 1 mol/ L sulfuric acid (100 °C, 2 h). The resulting monomeric sugars—arabinose, xylose, mannose, galactose, and glucose—were derivatized into volatile alditol acetate derivatives according to Englyst et al. (1982) [37] and Englyst and Cummings (1984) [38]. The separation was conducted with an Rtx-225 fused silica column, 0.53 mm × 30 m (Restek, Lisses, France), at a mobile phase flow rate of 2 mL/min. The mobile phase consisted of helium. Injection and detection temperatures were set at 275 °C. The chromatograms were processed by identifying the polysaccharides based on standards and areas of chromatographic peaks. Moreover, the retention times and the peak area ratio for the dominating electrode to that of neighboring electrodes were considered.

2.2.7. Fatty Acid Composition

The fatty acid composition in extracted ensilaged mcWDGS fat samples (the first experiment) was analyzed using the gas chromatography flame ionization detection method (GC/FID) according to PN-EN ISO 12966-1:2015 [39] + AC:2015-06, PN-EN ISO 12966-2:2017-05 pkt. 5.2 [40], PN-EN ISO 12966-4:2015-07 [41]. The analysis was performed using a Bruker 456 SCION-GC (Bruker Corporation, Billerica, MA, USA) with Restek Rt-2560 (Restek, Lisses, France) GC Capillary Column, 100 m, 0.25 mm ID, 0.20 µm. The analysis preceded the preparation of fatty acid methyl esters by alkaline transesterification. The following groups of fatty acids were determined: saturated fatty acids (SFAs)—C16:0, C18:0, C20:0, C21:0, C22:0, C24:0; unsaturated fatty acids (UFAs)—MUFAs, PUFAs; monounsaturated fatty acids (MUFAs)—C16:1, C18:1n9c, C20:1n9; polyunsaturated fatty acids (PUFAs)—PUFA ω-3, PUFA ω-6; PUFA ω-3—C18:1n7c, C18:3n3; PUFA ω-6—C18:2n6c.

2.2.8. Oxidative Capacity

The oxidative capacity of WDGS was determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay (Glentham Life Sciences, Corsham, UK). First, an extraction was performed by adding 30 mL of 100% pure methanol to 3 g of the mcWDGS samples. Subsequently, mcWDGS samples were vortexed for 30 min, left to settle for 1 h, and treated with ultrasounds with ultrasonic cleaner proclean 10.0DSP (Ulsonix Cleaning Instruments, Berlin, Germany) for 30 min at 37 kHz. Then, the samples were centrifuged with MPW-352R (MPW MED. INSTRUMENTS, Warsaw, Poland) for 10 min at 9000 rpm. The supernatant was then collected and frozen at −20 °C until analyzed. The DPPH solution (Glentham Life Sciences, Corsham, UK) was prepared at a concentration of 39.4 mg/L by diluting DPPH in 100% pure methanol. Afterwards, 280 µL of DPPH solution and 20 µL of samples were pipetted into each of the wells of the 96-well plate. After that, samples were read using a spectrophotometer (Infinite M Nano, Tecan™, Männedorf, Switzerland) at A = 517 nm.

2.3. Statistical Analysis

All statistical analyses were conducted using Statistica version 13.3 (Statsoft Polska, Cracow, Poland). Data were assessed for normality via the Shapiro-Wilk test and for homogeneity of variances using Levene’s test. For data meeting the assumptions for parametric testing, a one-way analysis of variance (ANOVA) was applied. Conversely, if these assumptions were not met, a non-parametric Kruskal-Wallis test was employed. Results are presented as mean values and standard error (SE), along with corresponding p-values. Statistical significance was set at p < 0.05, with p < 0.01 indicating high significance; p > 0.05 was considered non-significant. Post hoc comparisons between groups were performed using Tukey’s HSD test following ANOVA, or the Dunn-Bonferroni test after the Kruskal-Wallis test. Specifically, one-way ANOVA with subsequent Tukey’s HSD test was used for almost all of the analyses of ensiled WDGS, and included chemical composition, nutritional value, non-starch polysaccharide composition, short-chain fatty acid composition, antioxidant potential, pH, and aerobic stability. However, due to unmet ANOVA assumptions, the Kruskal-Wallis test followed by the Dunn-Bonferroni test was applied for the fatty acid composition of ensiled mcWDGS and for all analyses of aerobically stored mcWDGS, such as chemical composition, yeast/mold colony count, antioxidant potential, pH, and temperature.

3. Results

3.1. The First Experiment—Aerobic Storage of mcWDGS

3.1.1. Temperature Measurements

The data concerning the temperature measurements of mcWDGS preserved aerobically are presented in

Table 2. Temperature of mcWDGS preserved under aerobic conditions (°C).

Treatment Group	Experimental Groups											SEM	p-Value
	C1	PA0.3%	PA0.6%	FA0.3%	FA0.6%	CA0.3%	CA0.6%	LA0.3%	LA0.6%	CAM0.3%	CAM0.6%
day 0	24.0	23.7	23.1	23.8	23.6	23.4	23.8	23.3	23.6	23.7	24.0	0.061	0.052
3rd day	47.8 DE	31.7 BC	22.7 A	22.4 A	22.7 A	47.9 DE	49.7 E	46.1 D	50.9 E	34.2 C	29.3 B	1.153	<0.001
5th day	39.8 CD	26.1 A	24.1 A	44.0 D	24.5 A	37.8 BC	42.5 D	36.1 BC	39.8 CD	48.6 E	33.8 B	0.832	<0.001

C1—control group; PA0.3%—propionic acid at 0.3% DM; PA0.6%—propionic acid at 0.6% DM; FA0.3%—formic acid at 0.3% DM; FA0.6%—formic acid at 0.6% DM; CA0.3%—citric acid at 0.3% DM; CA0.3%—citric acid at 0.6% DM; LA0.3%—lactic acid at 0.3% DM; LA0.6%—lactic acid at 0.6% DM; CAM0.3%—commercial acidifier mixture at 0.3% DM; CAM0.6%—commercial acidifier mixture at 0.6% DM; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters ABCDE differ at p ≤ 0.01.

. The temperature was affected by different additives during aerobic preservation, but significantly only on the 3rd and 5th days. The highest temperature on the 3rd day of measurement was observed for the CA0.6% and LA0.6% variants, but the lowest for PA0.6%, FA0.3%, and FA0.6% (p ≤ 0.01). For the 5th day, the highest temperature was found for the CAM0.3% variant, while the lowest values were found for the PA0.3%, PA0.6%, and FA0.6% variants (p ≤ 0.01).

3.1.2. pH Measurements

The data in

Table 3. pH level of mcWDGS preserved under aerobic conditions.

Treatment Group	Experimental Groups											SEM	p-Value
	C1	PA0.3%	PA0.6%	FA0.3%	FA0.6%	CA0.3%	CA0.6%	LA0.3%	LA0.6%	CAM0.3%	CAM0.6%
day 0	4.69 F	4.29 DE	4.26 DE	3.75 B	3.34 A	4.20 CDE	4.10 CD	4.33 E	4.13 CD	4.26 DE	4.00 C	0.036	<0.001
3rd day	7.2 EF	5.01 C	4.50 B	4.20 AB	3.79 A	6.77 DE	6.89 DE	7.54 F	6.98 E	6.47 D	4.50 B	0.138	<0.001
5th day	4.74 AB	4.74 AB	4.75 AB	4.79 B	4.7 A	4.71 A	4.70 A	4.72 A	4.68 A	4.74 AB	4.71 A	0.005	<0.001

C1—control group; PA0.3%—propionic acid at 0.3% DM; PA0.6%—propionic acid at 0.6% DM; FA0.3%—formic acid at 0.3% DM; FA0.6%—formic acid at 0.6% DM; CA0.3%—citric acid at 0.3% DM; CA0.3%—citric acid at 0.6% DM; LA0.3%—lactic acid at 0.3% DM; LA0.6%—lactic acid at 0.6% DM; CAM0.3%—commercial acidifier mixture at 0.3% DM; CAM0.6%—commercial acidifier mixture at 0.6% DM; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters ABCDEF differ at p ≤ 0.01.

present changes in pH levels within the different variants of mcWDGS. The pH levels of WDGS were affected by the use of different additives during aerobic preservation. Therefore, for day 0, the highest pH value was observed for the C1 variant and the lowest for the FA0.6% variant (p ≤ 0.01). However, for the 3rd day of measurement, the highest pH level was found for the LA0.3% variant, while the lowest was for FA0.6% (p ≤ 0.01). The 5th day of WDGS storage exhibited a statistically higher pH value for the FA0.3% variant but lower values for the FA0.6%, CA0.3%, CA0.6%, LA0.6%, and CAM0.6% variants (p ≤ 0.01).

3.1.3. Microbial Analyses

The data on the microbial analyses of mcWDGS are expressed in

Table 4. Total bacteria count of mcWDGS preserved under aerobic conditions (CFU/g mcWDGS).

Treatment Group	Experimental Groups											SEM	p-Value
	C1	PA0.3%	PA0.6%	FA0.3%	FA0.6%	CA0.3%	CA0.6%	LA0.3%	LA0.6%	CAM0.3%	CAM0.6%
day 0	4.8 × 102											N/A	N/A
3rd day	2 × 104 C	5 × 103 AB	3 × 102 A	2.9 × 102 A	4 × 101 A	1.3 × 104 ABC	1.7 × 104 BC	2 × 104 C	8.2 × 103 ABC	3.4 × 103 AB	6.7 × 102 A	1538	<0.001
5th day	N/A	1.3 × 104 b	5.4 × 103 ab	1.3 × 103 ab	2 × 101 a	N/A	N/A	N/A	N/A	4.8 × 103 ab	4.2 × 103 ab	1340	0.042

C1—control group; PA0.3%—propionic acid at 0.3% DM; PA0.6%—propionic acid at 0.6% DM; FA0.3%—formic acid at 0.3% DM; FA0.6%—formic acid at 0.6% DM; CA0.3%—citric acid at 0.3% DM; CA0.3%—citric acid at 0.6% DM; LA0.3%—lactic acid at 0.3% DM; LA0.6%—lactic acid at 0.6% DM; CAM0.3%—commercial acidifier mixture at 0.3% DM; CAM0.6%—commercial acidifier mixture at 0.6% DM; N/A—not applicable; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters ab differ at p ≤ 0.05 while values with letters ABC differ at p ≤ 0.01.

and

Table 5. Total fungi count of mcWDGS preserved under aerobic conditions (CFU/g mcWDGS).

Treatment Group	Experimental Groups											SEM	p-Value
	C1	PA0.3%	PA0.6%	FA0.3%	FA0.6%	CA0.3%	CA0.6%	LA0.3%	LA0.6%	CAM0.3%	CAM0.6%
day 0	1 × 102											N/A	N/A
3rd day	7.7 × 103 BC	2.5 × 103 AB	8.5 × 102 A	3.3 × 101 A	0 A	4.8 × 103 AB	1.4 × 104 C	3.2 × 103 AB	4.7 × 103 AB	5.1 × 103 AB	2.4 × 102 A	740	0.002
5th day	N/A	5.7 × 103 ab	3.6 × 102 a	1.9 × 102 a	2.8 × 102 a	N/A	N/A	N/A	N/A	7.4 × 103 b	6.8 × 103 b	881	0.023

C1—control group; PA0.3%—propionic acid at 0.3% DM; PA0.6%—propionic acid at 0.6% DM; FA0.3%—formic acid at 0.3% DM; FA0.6%—formic acid at 0.6% DM; CA0.3%—citric acid at 0.3% DM; CA0.3%—citric acid at 0.6% DM; LA0.3%—lactic acid at 0.3% DM; LA0.6%—lactic acid at 0.6% DM; CAM0.3%—commercial acidifier mixture at 0.3% DM; CAM0.6%—commercial acidifier mixture at 0.6% DM; N/A—not applicable; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters ab differ at p ≤ 0.05 while values with letters ABC differ at p ≤ 0.01.

. The TBC (Table 4) and TFC (Table 5) were affected by the additive used in the aerobic preservation of mcWDGS.

The data for TBC on day 0 are expressed as an average value of 4.8 × 10² CFU/g mcWDGS. However, for the 3rd day, the TBC values were the highest for the C1 and LA0.3% variants. On the other hand, variants PA0.6%, FA0.3%, FA0.6%, and CAM0.6% displayed the lowest TBC values (p ≤ 0.01). During the 5th day, the highest TBC value was exhibited by the PA0.3% variant while FA0.6% was the lowest (p ≤ 0.05) (Table 4).

The data for TFC on day 0 are expressed as an average value of 1 × 10² CFU/g mcWDGS. For the 3rd day of the measurement, the highest TFC was exhibited in the CA0.6% variant, while the lowest was found for PA0.6%, FA0.3%, FA0.6%, and CAM0.6% variants (p ≤ 0.01). The highest TFC value on the 5th day was found for CAM0.3% and CAM0.6% variants, but the lowest for PA0.6%, FA0.3%, and FA0.6% variants (p ≤ 0.05) (Table 5).

3.1.4. Chemical Composition

The chemical composition of mcWDGS, detailed in

Table 6. Chemical composition of mcWDGS (g/kg DM).

Treatment Group	Item	Experimental Groups											SEM	p-Value
		C1	PA0.3%	PA0.6%	FA0.3%	FA0.6%	CA0.3%	CA0.6%	LA0.3%	LA0.6%	CAM0.3%	CAM0.6%
3rd day	Dry Matter (FM)	514.6	448.2	460.3	443.7	453.7	438.0	381.0	414.8	435.3	453.6	443.0	7.260	0.051
	Crude Protein	281.7 C	246.7 AB	224 A	235.2 A	238.2 A	272.7 BC	297.7 C	282.3 C	274.7 BC	238.7 A	233.8 A	4.515	0.002
	Ether Extract	50.2	66.9	65.2	67.7	69.1	65.5	64.5	55.2	62.1	58.1	65.4	1.520	0.273
	Crude Fiber	123.7	125.0	124.1	123.8	123.1	106.1	113.7	104.4	104.6	98.9	116.7	3.184	0.372
	Crude Ash	34.4 C	28.7 AB	27.4 A	27.6 AB	26.7 A	31 BC	29.9 AB	33.6 C	33.9 C	30.0 ABC	29.4 AB	0.486	0.002
5th day	Dry Matter (FM)	N/A	460.3	465.3	446	465.4	N/A	N/A	N/A	N/A	490	466.3	5.218	0.620
	Crude Protein	N/A	245.6 ab	222.5 a	238.4 a	224.8 a	N/A	N/A	N/A	N/A	269.7 b	238.7 a	4.183	0.013
	Ether Extract	N/A	64.4	64.6	58.2	57.5	N/A	N/A	N/A	N/A	45	65.6	2.209	0.172
	Crude Fiber	N/A	95.0 ab	99.4 b	92.5 ab	107.1 b	N/A	N/A	N/A	N/A	80.7 a	106.7 b	2.444	0.014
	Crude Ash	N/A	28.8 B	26.5 A	31.6 C	26.5 A	N/A	N/A	N/A	N/A	32.3 C	30 B	0.555	0.008

C1—control group; PA0.3%—propionic acid at 0.3% DM; PA0.6%—propionic acid at 0.6% DM; FA0.3%—formic acid at 0.3% DM; FA0.6%—formic acid at 0.6% DM; CA0.3%—citric acid at 0.3% DM; CA0.3%—citric acid at 0.6% DM; LA0.3%—lactic acid at 0.3% DM; LA0.6%—lactic acid at 0.6% DM; CAM0.3%—commercial acidifier mixture at 0.3% DM; CAM0.6%—commercial acidifier mixture at 0.6% DM; FM—in fresh matter; N/A—not applicable; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters ab differ at p ≤ 0.05, while values with letters ABC differ at p ≤ 0.01.

, was affected by the additives used during WDGS aerobic preservation as measured on the 3rd and 5th days. At the start of the study, the composite samples of mcWDGS were collected for chemical composition analysis. As such, on day 0, the composite sample of WDGS exhibited the following average values: dry matter 43.5%, crude protein 24.7%, crude fat 6.98%, crude fiber 15.8%, and crude ash 2.9%.

The utilization of different additives to WDGS stored under aerobic conditions affected the crude protein and crude ash content on the 3rd day (Table 6). The highest value of crude protein was found for the C1, CA0.6%, and LA0.3% variants, while the lowest was found for PA0.6%, FA0.3%, FA0.6%, CAM0.3%, and CAM0.6% variants (p ≤ 0.01). Moreover, the C1, LA0.3%, and LA0.6% variants exhibited the highest crude ash content, while PA0.6% and FA0.6% variants exhibited the lowest (p ≤ 0.01).

The utilization of different additives to WDGS stored under aerobic conditions affected the crude protein, crude fiber, and crude ash content at the 5th day (Table 6). WDGS exhibited a statistically higher crude protein value for the CAM0.3% variant and lower values for PA0.6%, FA0.3%, FA0.6%, and CAM0.6% variants (p ≤ 0.05). Crude fiber was significantly higher for CAM0.3% but lower for PA0.6%, FA0.6%, and CAM0.6% (p ≤ 0.05). The highest content of crude ash was determined for FA0.3% and CAM0.3% variants, and the lowest values were found for FA0.6% and PA0.6% variants (p ≤ 0.01).

3.1.5. Oxidative Capacity

The data on the inhibition of DPPH radicals in the presence of mcWDGS was significantly influenced by the variant used (p ≤ 0.01), as presented in

Table 7. Percent inhibition of DPPH radical in the presence of different additives to mcWDGS stored under aerobic conditions (% inhibition).

Treatment Group	Experimental Groups											SEM	p-Value
	C1	PA0.3%	PA0.6%	FA0.3%	FA0.6%	CA0.3%	CA0.6%	LA0.3%	LA0.6%	CAM0.3%	CAM0.6%
day 0	31.09											N/A	N/A
3rd day	82.14 CDE	70.99 B	65.03 A	83.61 DE	86.9 E	80.18 CD	79.64 CD	77.47 C	79.98 CD	78.84 CD	77.75 C	0.760	<0.001
5th day	N/A	51.62 AB	50.9 AB	55.98 B	70.76 C	N/A	N/A	N/A	N/A	64.92 C	46.49 A	1.584	<0.001

C1—control group; PA0.3%—propionic acid at 0.3% DM; PA0.6%—propionic acid at 0.6% DM; FA0.3%—formic acid at 0.3% DM; FA0.6%—formic acid at 0.6% DM; CA0.3%—citric acid at 0.3% DM; CA0.3%—citric acid at 0.6% DM; LA0.3%—lactic acid at 0.3% DM; LA0.6%—lactic acid at 0.6% DM; CAM0.3%—commercial acidifier mixture at 0.3% DM; CAM0.6%—commercial acidifier mixture at 0.6% DM; N/A—not applicable; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters ABCDE differ at p ≤ 0.01.

. At the beginning of the experiment on day 0, the average antioxidant properties of WDGS were similar, at slightly above 30%. By the 3rd day of WDGS storage, the inhibition of DPPH was shown to be at the highest for the FA0.6% variant, while the lowest for PA0.6% (p ≤ 0.01). However, for the 5th day, FA0.6% and CAM0.3% variants demonstrated the strongest antioxidant properties, while CAM0.6% showed the weakest (p ≤ 0.01).

3.2. The Second Experiment—Anaerobic Storage (Ensiling) of mcWDGS

3.2.1. Aerobic Stability

Figure 2 presents the aerobic stability of ensiled mcWDGS, which is measured according to the time required for the silage to increase its ambient temperature by 1 °C after its initial exposure to oxygen. Using different additives to WDGS stored under anaerobic conditions affected the aerobic stability. The C2 variant exhibited the highest aerobic stability, while FA0.2% and CA0.2% or I0.2% showed the lowest stability (p ≤ 0.01).

3.2.2. pH Measurement

The data in Figure 3 present pH levels of the different variants of ensiled modified corn WDGS. No effect or statistical differences were detected for the pH levels of ensiled mcWDGS (p ≥ 0.05).

3.2.3. Short-Chain Fatty Acid Composition and Lactic Acid and Ethanol Content

The contents of short-chain fatty acids, lactic acid, and ethanol in ensiled mcWDGS varied significantly between variants, and are presented in

Table 8. Contents of short-chain fatty acids, lactic acid, ethanol, and ammonia nitrogen of ensiled mcWDGS (g/100 g WDGS) and lactic acid-to-acetic acid ratio.

Treatment Group	Experimental Groups						SEM	p-Value
	C2	PA0.2%	FA0.2%	CA0.2%	I0.2%	CAM0.2%
L-lactic acid	4.65 BC	4.19 AB	4.02 A	5.81 D	5.18 C	5.18 D	0.122	<0.001
D-lactic acid	6.28 C	5.61 BC	3.56 A	6.17 C	4.83 B	6.17 C	0.175	<0.001
total lactic acid	10.92 bcd	9.80 b	7.57 a	11.99 d	10.01 c	11.95 d	0.257	0.013
acetic acid	4.77 B	4.14 AB	4.19 AB	4.13 A	4.12 A	4.55 AB	0.067	0.003
butyric acid	ND	ND	ND	ND	ND	ND	N/A	N/A
ethanol	0.27 A	0.06 A	0.07 A	0.09 A	0.06 A	0.59 B	0.039	<0.001
LA/AA	2.3 AB	2.4 B	1.8 A	2.9 C	2.4 BC	2.6 BC	0.074	<0.001
NH3-N	0.037 BC	0.035 ABC	0.031 A	0.034 AB	0.04 C	0.037 BC	0.001	<0.001

C2—control group; PA—propionic acid at 0.2% DM; FA—formic acid at 0.2% DM; CA—citric acid at 0.2% DM; I—inoculant at 0.2% DM; CAM—commercial acidifier mixture at 0.2% DM; LA/AA—lactic acid-to-acetic acid ratio; NH₃-N—ammonia nitrogen; ND—not detected; N/A—not applicable; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters abcd differ at p ≤ 0.05, while values with letters ABCD differ at p ≤ 0.01.

. mcWDGS exhibited the highest concentration of L-lactic acid for the CA0.2% and CAM0.2% variants, while having the lowest for the FA0.2% variant (p ≤ 0.01). D-lactic acid was determined to be highest for the C2, CA0.2%, and CAM0.2% variants, but the lowest for the FA0.2% variant (p ≤ 0.01). Similarly, L-lactic acid and total lactic acid concentrations were the highest for CA0.2% and CAM0.2% variants, yet the lowest for FA0.2% (p ≤ 0.05). The concentration of acetic acid was significantly higher for the C2 variant but lower for CA0.2% and I0.2% variants (p ≤ 0.01). Butyric acid was not detected in any of the ensiled mcWDGS variants. However, ethanol was detected and found to be significantly higher for the CAM0.2% variant, but lower for the C2, PA0.2%, FA0.2%, CA0.2%, and I0.2% variants (p ≤ 0.01). Moreover, a significantly higher lactic acid-to-acetic acid ratio was found for the CA0.2% variant, whereas the FA0.2% variant showed the lowest value (p ≤ 0.01). The ammonia nitrogen concentration was found to be the highest for I0.2% and lowest for FA0.2% variants (p ≤ 0.01).

3.2.4. Chemical Composition

The data on the chemical composition, NDF, ADF, total reducing sugars, and starch contents of the ensiled mcWDGS are showcased in

Table 9. Chemical composition and NDF and ADF contents of ensiled mcWDGS.

Treatment Group	Experimental Groups						SEM	p-Value
	C2	PA0.2%	FA0.2%	CA0.2%	I0.2%	CAM0.2%
Dry Matter (FM)	413.1 AB	416.5 AB	411.5 A	425.0 B	405.2 A	415.3 AB	8.098	0.004
Crude Protein	252.0 C	246.3 BC	234.0 AB	235.3 AB	233.3 A	244.3 ABC	1.933	<0.001
Ether Extract	83.4	87.4	82.4	84.4	84.5	86.3	0.762	0.485
Crude Fiber	118.0	117.0	145.0	122.5	120.3	115.3	3.752	0.192
Crude Ash	27.9 A	28.1 A	30.5 AB	29.0 A	32.8 B	29.8 AB	0.477	0.007
NDF	337.2 A	350.8 AB	378.3 B	362.1 AB	339.0 A	353.9 AB	3.745	0.007
ADF	184.4	192.7	180.3	192.5	184.5	188.0	1.446	0.068
WSC	6.7 a	7.6 a	24.4 b	6.6 a	22.6 b	7.3 a	2.004	0.040
Starch	252.0	253.4	212.1	229.1	262.7	216.8	6.172	0.106

C2—control group; PA—propionic acid at 0.2% DM; FA—formic acid at 0.2% DM; CA—citric acid at 0.2% DM; I—inoculant at 0.2% DM; CAM—commercial acidifier mixture at 0.2% DM; FM—in fresh matter; NDF—neutral detergent fiber; ADF—acid detergent fiber; WSC—water soluble carbohydrates; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters ab differ at p ≤ 0.05, while values with letters ABC differ at p ≤ 0.01.

. The utilization of different additives to WDGS stored under anaerobic conditions affected the dry matter, crude protein, crude ash, and NDF contents (p ≤ 0.01). WDGS exhibited statistically higher dry matter content for FA0.2% and I0.2% variants but lower for CA0.2% (p ≤ 0.01). Moreover, the highest concentration of crude protein was determined for the C2 variant, while the lowest was for the I0.2% variant (p ≤ 0.01). Crude ash content was statistically higher for the C2, PA0.2%, and CA0.2% variants but lower for I0.2% (p ≤ 0.01). Moreover, NDF content was statistically higher for the FA0.2% variant and lower for the C2 and I0.2% variants (p ≤ 0.01). Total water-soluble carbohydrate content was significantly higher for the FA0.2% and I0.2% variants compared to all other variants (p ≤ 0.05). The starch concentration was unaffected by the used additives (p ≥ 0.05).

3.2.5. Non-Starch Polysaccharide Composition

The data in

Table 10. The non-starch polysaccharide composition of ensiled mcWDGS (g/100 g DM).

Treatment Group	Experimental Groups						SEM	p-Value
	C2	PA0.2%	FA0.2%	CA0.2%	I0.2%	CAM0.2%
S-NSP
arabinose	0.44	0.32	0.34	0.36	0.31	0.39	0.017	0.186
xylose	0.32 A	0.49 B	0.47 B	0.51 B	0.31 A	0.37 A	0.020	<0.001
mannose	0.98	0.99	0.99	0.88	0.89	0.85	0.024	0.334
galactose	0.17	0.15	0.16	0.16	0.14	0.16	0.003	0.343
glucose	0.51 b	0.38 a	0.39 a	0.39 a	0.40 ab	0.45 ab	0.014	0.014
I-NSP
arabinose	4.85	4.98	4.73	4.68	4.73	4.74	0.042	0.364
xylose	7.31	7.95	7.56	7.41	7.35	7.39	0.082	0.202
mannose	0.82	0.79	0.72	0.72	0.74	0.81	0.017	0.374
galactose	1.54	1.56	1.48	1.49	1.51	1.50	0.020	0.884
glucose	9.10	9.53	8.66	8.72	8.59	9.08	0.139	0.386

C2—control group; PA—propionic acid at 0.2% DM; FA—formic acid at 0.2% DM; CA—citric acid at 0.2% DM; I—inoculant at 0.2% DM; CAM—commercial acidifier mixture at 0.2% DM; S-NSP—soluble non-starch polysaccharide; I-NSP—insoluble non-starch polysaccharide; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters ab differ at p ≤ 0.05, while values with letters AB differ at p ≤ 0.01.

present the composition of non-starch polysaccharides (NSPs) of ensiled mcWDGS. The soluble non-starch polysaccharide (S-NSP) contents were largely not influenced by the additive used in the anaerobic preservation of WDGS, except for xylose and glucose contents. However, the contents of insoluble non-starch polysaccharides (I-NSPs) were unaffected by the additives used. Within the S-NSP, soluble xylose exhibited statistically higher contents for PA0.2%, FA0.2%, and CA0.2% variants, while lower for C2, I0.2%, and CAM0.2% variants (p ≤ 0.01). Similarly, soluble glucose was significantly higher for the C2 variant but lower for PA0.2%, FA0.2%, and CA0.2% variants (p ≤ 0.05).

3.2.6. Fatty Acid Profile

Table 11. Effects of additives on the fatty acid profile of ensiled mcWDGS fat (g/100 g of fatty acids).

Treatment Group	Experimental Groups						SEM	p-Value
	C2	PA0.2%	FA0.2%	CA0.2%	I0.2%	CAM0.2%
SFA	12.95 abc	12.74 a	13.08 bc	12.86 abc	13.14 c	12.80 ab	0.041	0.043
C16:0	10.39 ab	10.25 a	10.54 b	10.37 ab	10.58 b	10.30 a	0.033	0.035
C18:0	1.76	1.70	1.74	1.70	1.76	1.71	0.009	0.091
C20:0	0.40 ab	0.39 a	0.41 b	0.40 ab	0.40 ab	0.39 a	0.001	0.033
C21:0	0.13	0.12	0.12	0.13	0.13	0.14	0.002	0.073
C22:0	0.12	0.12	0.12	0.12	0.12	0.12	0.001	0.416
C24:0	0.15	0.15	0.15	0.15	0.16	0.15	0.001	0.149
UFA	82.25 ab	82.48 b	82.16 ab	82.35 ab	82.07 a	82.41 b	0.041	0.045
MUFA	26.56	26.58	26.50	26.6	26.45	26.69	0.034	0.262
C16:1	0.13	0.12	0.12	0.12	0.13	0.13	0.002	0.556
C18:1n9c	25.57	25.61	25.51	25.42	25.41	25.64	0.026	0.051
C20:1n9	0.25	0.25	0.24	0.25	0.25	0.26	0.003	0.384
PUFA	55.68	55.9	55.66	55.75	55.63	55.72	0.035	0.312
ω-3	1.60 CD	1.51 AB	1.49 A	1.57 BC	1.59 CD	1.65 D	0.013	0.009
C18:1n7c	0.61	0.61	0.61	0.62	0.66	0.6	0.005	0.077
C18:3n3	1.49	1.51	1.49	1.57	1.59	1.65	0.022	0.051
ω-6	54.2	54.38	54.17	54.18	54.03	54.08	0.042	0.162
C18:2n6c	54.2	54.38	54.17	54.18	54.03	54.08	0.042	0.162

C2—control group; PA—propionic acid at 0.2% DM; FA—formic acid at 0.2% DM; CA—citric acid at 0.2% DM; I—inoculant at 0.2% DM; CAM—commercial acidifier mixture at 0.2% DM; SFAs—saturated fatty acids; UFAs—unsaturated fatty acids; PUFAs—polyunsaturated fatty acids; ω-3—omega 3 fatty acids; ω-6—omega 6 fatty acids; C16:0—palmitic acid; C18:0—stearic acid; C20:0—arachidic acid; C21:0—heneicosylic acid; C22:0—behenic acid; C24:0—lignoceric acid; C16:1—palmitoleic acid; C18:1n9c—oleic acid; C20:1n9—gondoic acid; C18:1n7c—vaccenic acid; C18:3n3—α-linolenic acid; C18:2n6c—linoleic acid; SEM—standard error of the mean. Numerical values in the same row marked in pairs with letters abc differ at p ≤ 0.05, while values with letters ABCD differ at p ≤ 0.01.

presents the results of ensiled mcWDGS fatty acid composition. The fatty acid profile of WDGS was influenced by adding the preservatives before the ensiling process. The fatty acid groups whose contents changed were SFAs, UFAs, and ω-3, while of all the individual fatty acids, only palmitic acid (C16:0) and arachidic acid (C20:0) were affected. Ensiled WDGS exhibited the highest content of SFAs for the I0.2% variant and the lowest for the PA0.2% variant (p ≤ 0.05). C16:0 and C20:0 were the highest for the FA variant, while the lowest for PA0.2% and CAM0.2% variants (p ≤ 0.05). The highest content of UFAs was found for PA0.2% and CAM0.2% variants, yet the lowest was exhibited by the I0.2% variant (p ≤ 0.05). ω-3 was the highest for the CAM0.2% variant and the lowest for the FA0.2% variant (p ≤ 0.01).

3.2.7. Oxidative Capacity

The ability to inhibit the oxidation of DPPH in the presence of ensiled mcWDGS was significantly influenced by the variant used (p ≤ 0.01), as shown in Figure 4. The WDGS C2 variant exhibited the highest inhibition of the DPPH radical, while the lowest value was found for the FA0.2% variant.

4. Discussion

The current study aimed to investigate the efficacy of preservation strategies for modified corn wet distillers’ grains plus solubles (mcWDGS), a by-product derived in its entirety from the bioethanol industry. This research contributes to the broader understanding of optimizing distillers’ grain management, especially given that domestic EU production was estimated at approximately 3.77 million tonnes in 2023 [42]. This aligns with a strategy of maximizing by-product utilization in livestock nutrition and promoting preservation methods that are more economical and environmentally sustainable. Wet distillers’ grains plus solubles (WDGS) is a mixture of WDG and CCDS, but its mixing is unstandardized, meaning that each bioethanol-producing plant uses its own mixing ratio [7]. In this study, WDGS was further modified through the incorporation of non-normative corn fiber (NNCF). NNCF, often underutilized or even discarded, was introduced to create mcWDGS with a distinct WDG:NNCF:CCDS ratio of 75:15:10 per FM (61:32:7 DM). While the inclusion of CCDS is known to reduce crude protein, crude fat, and crude fiber in WDGS [7], our findings suggest that NNCF incorporation enhanced the overall nutritional value of WDGS. Specifically, an increase in dry matter and carbohydrates was achieved, although this was accompanied by a decrease in crude protein. This alteration in the nutritional profile highlights NNCF’s potential to beneficially influence the composition of distillers’ grains, further cementing its utility as a valuable feed ingredient and not a waste product in a similar fashion as legume/cereal husks, cereal brans, hay, or straw [43,44,45].

4.1. Aerobic Preservation of mcWDGS

Experiment I investigated the efficacy of mcWDGS aerobic preservation using various organic acids such as formic, propionic, lactic, and citric acids, and a commercial acidifier mixture was also used. These technological additives were added to the mcWDGS at either 0.3% or 0.6% FM based on our preliminary findings and values recommended in the literature [25,32,33]. The organic acid addition affected the quality and preservation of mcWDGS. The temperature dynamics were used as one of the indicators of spoilage, as increased temperature causes accelerated microbial activity due to exothermic processes [22]. mcWDGS exhibited temperatures exceeding 40 °C by the 3rd day for the control (C1), citric acid (CA0.3%, CA0.6%), and lactic acid (LA0.3%, LA0.6%) variants, indicating rapid deterioration and spoilage. In contrast, propionic acid (PA0.6%) and formic acid (FA0.3%, FA0.6%) variants consistently maintained temperatures below 25 °C throughout the 5-day aerobic preservation period. This phenomenon indicates that the microbial activity was minimal for propionic and formic acid variants as the temperature resembled initial ambient conditions. Moreover, this demonstrates the superiority of propionic and formic acids as additives in mcWDGS preservation. Contrary to the temperature measurements, pH levels varied immediately after the application of technological additives. Formic acid variants consistently exhibited the lowest pH values across all measurements throughout the five-day experiment. This phenomenon is consistent with formic acid having a lower negative logarithm of the acid dissociation constant (pKα), of approximately 3.75, which compared to other organic acids, such as lactic acid (3.86), citric acid (4.80), and propionic acid (4.87), is considerably lower [46,47], further reinforcing formic acid as having the highest acidifying potential. While pH generally increased for all variants by the 3rd day, the highest increases in pH (above 6) indicated microbial spoilage and corresponded well with variants exhibiting higher temperature. Conversely, PA0.6%, FA0.3%, FA0.6%, and CAM0.6% maintained pH levels below 4.5. As such, by the 5th day, pH values finally stabilized for all mcWDGS variants, remaining slightly above 4.5. Moreover, the PA0.6% and FA0.6% variants maintained the most stable pH level throughout the entirety of the experiment. This study confirms that mcWDGS variants with a pH exceeding 4.5 on the 3rd day exhibited not only poorer quality but also reduced shelf-life and overall worse preservation in general. Similar to the findings of other authors, the WDGS exhibiting pH levels below 4.5 was shown to prevent WDGS deterioration and spoilage [22,24]. Microbial analyses on the 3rd day also reinforce these findings, demonstrating that the lowest bacterial and fungal counts were found for PA0.6%, FA0.3%, and FA0.6% variants. These variants exhibited values well below 10³ CFU/g mcWDGS, resembling the initial, untreated material. The inhibitory and antimicrobial properties of formic and propionic acids persisted until the 5th day of the experiment, confirming the strong preservation capacity [28,48]. In addition, the observed interconnectedness of temperature, pH, and microbial analyses can serve as an indicator and predictor of the WDGS spoilage rate, especially during aerobic preservation [17,22,23,24]. Changes in the chemical composition of mcWDGS variants under aerobic conditions were also evident, particularly in crude protein, crude fiber, and crude ash content. The highest crude protein content on the 3rd day was observed in the rapidly spoiling variants. This aligns with Reed et al. (2007) [49], who suggest that microbial respiration during spoilage consumes non-structural carbohydrates, leading to a relative increase in protein concentration. Similar increases in crude fiber and crude ash in spoiled variants likely stem from the same process. Furthermore, the oxidative capacity was affected by organic acidifiers. The FA0.6% variant demonstrated the highest inhibition of DPPH on both the 3rd and 5th days, correlating with its consistently low temperatures. It is thought that the antioxidant potential of formic acid is a thermodynamically favorable reaction, meaning that the higher the antioxidant properties, the lower the temperature [50].

4.2. Anaerobic Preservation of mcWDGS

Experiment II investigated the efficacy of mcWDGS preserved under anaerobic conditions using various technological additives such as organic acids (formic, propionic, and citric acids), a commercial acidifier mixture, or a heterofermentative inoculant. These additives were used in the mcWDGS at 0.2% FM, which aligned with the values recommended in the literature [26] and the manufacturer’s recommendations for the inoculant and commercial acidifier mixture. The technological additives affected the quality and preservation of mcWDGS. Aerobic stability, a crucial indicator for silage perishability and quality, displayed values spanning from 21 ± 3 h to 61 ± 12 h. Interestingly, the highest stability was observed for the control variant, followed by the propionic acid variant. Conversely, the inoculant, formic acid, and citric acid treatments demonstrated the lowest stability. These findings align with Zhang et al. (2019) [51], who reported that the addition of exclusively one type of technological additive, e.g., propionic acid or inoculant, does not increase the aerobic stability of the silage. However, these results contradict those found in some other studies [32,33]. Despite these differences in aerobic stability, the pH levels across all ensiled mcWDGS variants remained consistently low (slightly below 4), which is consistent with previous reports for ensiled WDGS mixtures [23] and indicative of adequate overall preservation. The short-chain fatty acid, lactic acid, and ethanol concentrations were also influenced by the additives. Lactic acid values were comparable to those reported for WDGS [22] and higher than in typical sorghum and corn silages [52,53]. However, the acetic acid concentration in our ensiled mcWDGS was considerably higher than in WDGS, sorghum, and corn silages [52,53,54]. The superior aerobic stability of the control variant is likely attributable to its elevated acetic acid concentration, an organic acid known for its fungistatic and bacteriostatic properties [55] which heavily inhibits secondary fermentation. The increased acetic acid and ethanol in the control also suggest a more prominent heterofermentative process [56,57]. In contrast, the high stability observed in the propionic acid variant was a direct result of the additive itself. These findings align well with Zhang et al. (2019) [51] and Chen et al. (2016) [33], who reported that the addition of propionic acid does not affect or might even slightly decrease the concentration of lactic and acetic acids. This phenomenon is best explained by the heterofermentative-inhibiting properties of propionic acid [51]. Our findings corroborate that a high lactic acid concentration alone is insufficient to prevent secondary fermentation due to yeast metabolizing this acid. Notably, butyric acid was not detected in any variant, indicating successful ensiling. The ammonia nitrogen (NH₃-N) content across all mcWDGS variants was lower than values previously reported in the literature [22,52,58,59]. The NH₃-N content was observed to be the lowest for the formic acid variant. This result can likely be attributed to formic acid having a strong bacteriostatic effect on detrimental microorganisms, in particular the population of Clostridium sp., which is responsible for the production of ammonia nitrogen in silages [60]. Similarly, the lowest lactic acid-to-acetic acid ratio (LA/AA) was also found for the formic acid variant. The LA/AA ratio, which was below 2 for the formic acid variant, indicates a stronger effect of this additive on the growth and development of heterofermentative bacteria at the expense of homofermentators [61]. Regarding the nutritional profile under anaerobic conditions, technological additives influenced dry matter, crude protein, crude ash, and NDF content. Generally, the dry matter content was higher while the crude protein content was lower for mcWDGS compared to some previously reported values for WDGS, which likely stems from the addition of NNCF [22,62,63]. The use of NNCF can also increase the starch content in mcWDGS [16,64]. Compared to the effect of the additives used, the crude protein content showed an interesting relationship, with the highest concentrations being observed in the control variant. This contradicts with studies suggesting lower protein losses when organic acids are used [65]. NDF and ADF in the present research were also notably higher than in some reports [62], with the lowest NDF content found in the control and inoculant variants. These differences in NDF likely resulted from hemicellulose breakdown during fermentation, as heterofermentative lactic acid bacteria can metabolize xylose to produce lactic and acetic acid [66,67], affecting NDF while leaving ADF relatively unchanged. Non-starch polysaccharide analysis further supported this finding, as only soluble xylose and glucose among the soluble non-starch polysaccharides differed significantly. Interestingly, in this study, insoluble glucose content was found to be notably higher compared to the values reported by Pedersen et al. (2014) [68], which indicates a direct impact of NNCF addition on the feedstuff’s carbohydrate profile. Moreover, the highest water-soluble carbohydrate (WSC) content was observed for the formic acid and inoculant variants. While the increased WSC content is likely due to the deceleration of homofermentation by the formic acid [61], the same explanation cannot be applied for the inoculant variant. The higher WSC content in the inoculant likely stems from a combination of factors, including the presence of enzymes and the microbiota composition. Enzymes such as xylanase and cellulase present in the inoculant aid in the breakdown of NDF and non-starch polysaccharides, thereby increasing WSC content [69]. Furthermore, the inoculant contained Lactobacillus buchneri, a microorganism known for lactic acid conversion into acetic acid, which further reduced its reliance on soluble carbohydrates [70]. Fatty acid composition was also influenced, with C18:1 (oleic acid) and linoleic acid being predominant. The inoculant variant displayed the highest saturated fatty acid (SFA) content and lowest unsaturated fatty acid (UFA) content, while the propionic acid and commercial acid mixture variants had the highest UFA. This inverse relationship in the inoculant variant might be explained by the microbial conversion of UFA to SFA through biohydrogenation [71,72]. Finally, the use of technological additives also influenced the oxidative capacity. The control variant exhibited the highest antioxidant properties, while the formic acid variant displayed the lowest. This may be due to formic acid’s high reduction potential and thermodynamic favorability [50,73,74].

5. Conclusions

Our study confirms that mcWDGS can be effectively preserved under both aerobic and anaerobic conditions. Aerobic preservation offers a short-term solution, which is essential to ensuring that the feed remains suitable for sale and transportation. On the other hand, anaerobic preservation provides a long-term mcWDGS storage method that cattle and pig farmers can successfully implement. Both methods offer distinct solutions for the prevention of mcWDGS spoilage, thereby enabling the sale and subsequent utilization of this by-product as animal feed. During aerobic storage, the untreated mcWDGS deteriorated rapidly after only 3 days at 25 °C. However, the addition of formic and propionic acids at 0.6% FM significantly decelerated this deterioration, thus prolonging the mcWDGS shelf-life to 5 days. The mcWDGS treated with these two organic acid additives effectively maintained stable temperatures, pH, and microbial growth. Additionally, formic and propionic acids had no major effect on the nutritional value of mcWDGS. Contrary to aerobic storage, all mcWDGS variants preserved under anaerobic conditions underwent successful ensiling. Although the inclusion of propionic acid at 0.2% FM proved superior among the technological additives, the control variant exhibited the most effective preservation. Furthermore, all ensiled variants maintained comparable or slightly different compositional values, indicating that this feedstuff’s nutritional quality was generally unaffected. In summary, the perishability of mcWDGS can be extended under both aerobic and anaerobic conditions without negative changes in quality or nutritional value. However, aerobic preservation of mcWDGS requires the use of an additive, with formic and propionic acids proving the most effective in this study. Our investigation revealed that mcWDGS can be effectively preserved under anaerobic conditions, even in the absence of preserving additives. From the perspective of livestock farmers, anaerobic preservation (ensiling) can be an advantageous preservation method. This approach enables the long-term storage of WDGS, a product otherwise prone to rapid spoilage. This facilitates sustainable year-round feeding and can act as a substitute for some concentrates and roughage in the diet as a good source of energy and protein. Future studies should expand upon the combined effects of temperature and relative humidity on WDGS preservation. Additionally, further focus should be placed on optimizing the formulation ratio of either WDGS or mcWDGS.