The Effects of Dried Apple Pomace on Fermentation Quality and Proteolysis of Alfalfa Silages
Institute of Ensiling and Processing of Grass, College of Agro-Grassland Science, Nanjing Agricultural University, Nanjing 210095, China
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Author to whom correspondence should be addressed.
Agronomy 2025, 15(2), 438; https://doi.org/10.3390/agronomy15020438
Submission received: 4 January 2025
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Revised: 7 February 2025
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Accepted: 7 February 2025
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Published: 11 February 2025
(This article belongs to the Section Grassland and Pasture Science)
Abstract
:This work aimed to evaluate the effects of dried apple pomace (DAP) on the fermentation characteristics and proteolysis of alfalfa silages. The alfalfa was ensiled with (1) no additives (control), (2) 5% DAP, (3) 10% DAP and (4) 15% DAP based on fresh weight (FW) for 1, 3, 7, 14, 30 and 60 days, respectively. With the increasing proportion of DAP, lactic acid bacteria (LAB) count, lactic acid (LA) and dry matter (DM) content linearly (p < 0.05) increased, while the pH, the content of acetic acid (AA), propionic acid (PA), butyric acid (BA) and ammonia nitrogen (NH3-N) linearly (p < 0.05) decreased during ensiling. The 10% and 15% DAP silages had significantly (p < 0.05) lower aerobic bacteria (AB), yeast and enterobacteria counts than the control during ensiling. The contents of nonprotein nitrogen (NPN), peptide nitrogen (peptide-N) and free amino acid nitrogen (FAA-N) and activities of carboxypeptidase, aminopeptidase and acid proteinase linearly (p < 0.05) decreased as DAP proportion increased during ensiling. On day 60, the addition of DAP significantly (p < 0.05) decreased the contents of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, spermine and total biogenic amines compared with the control. As the DAP ratio increased, the contents of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine, aspartic acid, serine, glutamic, total amino acids, crude protein (CP) and water-soluble carbohydrates (WSCs) linearly (p < 0.05) increased, while the contents of glycine, alanine, cysteine, and proline linearly (p < 0.05) decreased on day 60. Overall, the addition of 15% DAP was optimal as indicated by better fermentation quality and less proteolysis than other treatments.
1. Introduction
During ensiling, extensive proteolysis of alfalfa would cause low availability of true protein. According to [1], proteolysis mainly resulted in true protein being degraded to nonprotein nitrogen (NPN), which included peptide nitrogen (peptide-N) and free amino acid nitrogen (FAA-N), biogenic amines and ammonia nitrogen (NH3-N). The ruminal microbes could use ammonia as nitrogen sources; however, excess ammonia was absorbed through the rumen wall and largely excreted as urea in urine into environment [2]. Meanwhile, the production of biogenic amines might be responsible for depressed intake and ketonemia of ruminants [3]. Therefore, it is essential to suppress proteolysis of alfalfa during ensiling. Li et al. (2018) has reported that protein breakdown could be reduced by the formation of protein–quinone complexes [4]. O-quinones were the oxidative product of polyphenols catalyzed by polyphenol oxidase (PPO), and proteins were cross-linked with o-quinones, thus leading to the restraint of protein breakdown [5]. Nguyen et al. (2020) suggested that enzyme browning in fruit waste like apple pomace easily occurred via the enzymic oxidation of polyphenols [6].
Apple pomace is the typical by-product of the apple processing industry, and its global yield exceeds 3600 kilotons per year [7]. It is rich in water-soluble carbohydrates (WSCs), phenolic compounds, protein and vitamins, and could meet the requirement of ruminant production [8]. Furthermore, the high sugar content of apple pomace makes it a great substrate for fermentation. However, the high moisture content makes apple pomace prone to microbial contamination, thus resulting in a waste of resources and pathogen infection of animals. Meanwhile, fresh apple pomace faces the challenge of long-distance transportation, which limits its widespread utilization. To sustain quality and storage stability, the moisture content of apple pomace was reduced to less than 10%. Dried apple pomace (DAP) has a low pH and high metabolizable energy and is used as quality feedstuff for ruminants [9]. In recent years, ensiling has also become a way to utilize by-products in livestock production. Considering its fermentability and polyphenolic compounds, we hypothesized that the addition of DAP might promote fermentation quality and suppress protein degradation of alfalfa silages.
To better understand the effects of DAP addition on fermentation quality, nitrogen distribution, biogenic amines and amino acid content of alfalfa silages, we conducted an experiment using alfalfa silages with different additions of DAP (5%, 10% and 15%). It was hypothesized that addition of DAP to alfalfa silages within a typical range would improve fermentation quality and result in less nonprotein nitrogen and biogenic amine production during ensiling. Additionally, it was hypothesized that the response to less proteolysis would be higher for alfalfa supplemented with a high content of DAP compared to alfalfa silages without any DAP addition.
2. Materials and Methods
2.1. Silage Preparation
The DAP was obtained from Jiangsu Chishan Lake Agricultural Technology Co., Ltd. (Zhen Jiang, China). Due to its low moisture, DAP was stored at ambient temperature and transported a long distance. Alfalfa (Medicago sativa L.) was cultivated in the experimental field and harvested at the full bloom stage in second cutting; it was then wilted for 1 h to obtain a dry matter (DM) content of 30% DM. The wilted alfalfa was chopped into pieces of about 2 cm with a fodder chopper and was randomly divided into 100 subsamples (about 150 g for each subsample). Four subsamples were chosen to analyze their chemical compositions immediately. The remaining 96 subsamples were subjected to one of the following treatments: (1) without DAP (control), (2) with 5% DAP (5% DAP), (3) with 10% DAP (10% DAP) and (4) with 15% DAP (15% DAP) on a FW basis. In detail, the control (150 g alfalfa + 0 g DAP), 5% DAP (150 g alfalfa + 7.5 g DAP), 10% DAP (150 g alfalfa + 15 g DAP) and 15% DAP (150 g alfalfa + 22.5 g DAP) were prepared, respectively. All treatments were packed into polyethylene plastic bags (30 × 40 cm) and sealed with a vacuum sealer (DZD-400; Aomitai Instrument Co., Ltd., Nanjing, China). After sealing, a total of 96 vacuum bags (4 treatments × 6 ensiling days × 4 replicates) were stored at ambient temperature (33 ± 2 °C), and four bags of each treatment were opened after 1, 3, 7, 14, 30 and 60 days, respectively.
2.2. Chemical and Microbial Composition Analysis
A subsample of fresh and ensiled alfalfa was oven-dried at 65 °C for 48 h to determine the DM content and then ground to pass a 1 mm screen for further chemical analysis. The content of WSCs was determined by the modified phenol–sulfuric acid method [10]. Thermostable α-amylase and sodium sulfite were prepared to measure the content of acid detergent fiber (ADF) and neutral detergent fiber (NDF) as described by Van Soest et al. (1991) [11]. Hemicellulose (HC) was calculated as the difference between NDF and ADF. Total phenolic content (TPC) was evaluated according to Michalczyk et al. (2019) [12]. A calibration curve of gallic acid (0–400 mg/L) was constructed, which served as a phenolic standard (R2 = 0.998), and the TPC was expressed as mg of gallic acid equivalent (GAE) in 1 g of DM. The activity of PPO was determined from the slope of the linear part of the curves (absorbance vs. time) and expressed as U/mL min. One unit of enzyme activity was measured by a 0.001-unit increase in absorbance per min and mL of the enzyme [13].
Sixty grams of silage sample was blended with 180 mL of distilled water and macerated at 4 °C for 24 h; subsequently, the extract was filtered through four layers of medical gauze and Whatman filter paper (11 μm pore size, Xinhua Co., Hangzhou, China). The pH was determined by a glass electrode pH meter (HANNA pH 211, Hanna Instruments Italia Srl, Villafranca Padovana, Italy). The buffering capacity (BC) of the raw materials was determined as described by Playne and McDonald (1966) [14]. Organic acid measurement was performed in a high-performance liquid chromatography system (1260 HPLC, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a refractive index detector (column: CarbomixVR H-NP5, Sepax Technologies, Inc., Newark, DE, USA; eluent: 2.5 mM H2SO4, 0.5 mL/min; temperature: 55 °C).
Approximately 10 g of fresh alfalfa or silages was serially diluted 10-fold with sterilized saline solution (0.85% sodium chloride). The lactic acid bacteria (LAB) were enumerated on deMan, Rogosa and Sharp agar medium after incubation in an anaerobic incubator at 37 °C for 48 h. Aerobic bacteria (AB) were enumerated on nutrient agar medium (Qingdao Haibo Biotechnology Co., Ltd., Qingdao, China) after incubation at 37 °C for 24 h. Yeasts were enumerated on potato dextrose agar after incubation at 30 °C for 72 h. Enterobacteria were enumerated on the Violet Red Bile Glucose Agar medium after 48 h of incubation at 37 °C. Microbial compositions can be found in the Supplementary Materials.
2.3. Nitrogen Distribution Analysis
Total nitrogen (TN) was determined by a Kjeldahl nitrogen apparatus (Kjeltec 8200; FOSS, Karlskoga, Sweden) and multiplied by 6.25 to calculate the content of crude protein (CP). Sixty grams of silage sample of each treatment was blended with 180 mL of distilled water and macerated at 4 °C for 24 h. The extract was filtered through medical gauze and filter paper. To assess the content of nitrogen distribution, 3 mL of 10% (w/v) trichloroacetic acid (TCA) was blended with the filtrate (12 mL) at a ratio of 1:4 at 4 °C for 24 h to precipitate true protein. The solution was centrifugated for 10 min at 9000× g and 4 °C, and the supernatant was used to determine the content of FAA-N and NH3-N. The content of FAA-N and NH3-N was measured following the method described by Broderick and Kang (1980) [15]. The NPN content was determined as per Licitra et al. (1996) using 10 mL of deproteinizing solution [16]. Peptide-N was determined by the increase in FAA-N in the TCA supernatant after digestion with 6 M HCl for 22 h at 110 °C under an N2 atmosphere [17].
2.4. Protease Activity Analysis
About 20 g of fresh materials or silage samples was homogenized in 40 mL of prechilled 0.1 M sodium phosphate buffer (pH 6.0) containing 5 mM sodium thiosulfate. The homogenate was filtered through four layers of gauze and centrifuged at 10,000× g for 10 min at 4 °C. An aliquot (5 mL) of each sample was stored at −80 °C for the measurement of enzymatic activities. The activities of carboxypeptidase, aminopeptidase and acid proteinase were determined according to the method of McKERSIE (1981) by using a microplate reader (Multiskan Sky, Thermo Scientific, Waltham, MA, USA) [18]. The substrates of carboxypeptidase, aminopeptidase and acid proteinase were Hippuryl-L-phenyl-alanine, L-leucine-p-nitroanilide and azocasein, respectively.
2.5. Biogenic Amine Content Analysis
Biogenic amines were detected according to the description of Jia et al. (2021) [19]. Lyophilized powder (1 g) of fresh alfalfa or alfalfa silages was added to 15 mL of 5% (w/v) TCA, and then the solution was shocked and incubated at 37 °C for 60 min. The extract was centrifuged at 8000× g for 10 min, and the supernatant was filtered through a layer of filter paper into a volumetric flask. The total volume was adjusted to 10 mL with 5% TCA. Afterwards, 1 mL of the solution was transferred into a 5 mL volumetric flask. Meanwhile, 200 μL of 2 M NaOH, 300 μL of saturated NaHCO3 and 1 mL of 10 mg/mL dansyl chloride were added into the volumetric flask, and the sample was incubated at 45 °C for 45 min in the dark. To remove residual dansyl chloride, the reactant was mixed with 100 μL of 25% aqueous ammonia and incubated at ambient temperature for 30 min. At the end of incubation, the volume of the reactant was brought to 5 mL with acetonitrile, and then the solution was centrifuged for 5 min at 10,000× g and 4 °C. The supernatant was subjected to a high-performance liquid chromatography system (1260 HPLC, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a diode array detector (C18 column: ZORBAX SB-Aq, 5 μm, 4.6 × 250 mm, Agilent Technologies Inc., USA; eluent A: 0.1 M ammonium acetate; eluent B: acetonitrile; 0.8 mL/min; wavelength: 254 nm; temperature: 30 °C).
2.6. Amino Acid Content Analysis
The oven-dried powder of fresh alfalfa or silages was soaked with 6 M HCl at 110 °C. After incubation for 22 h, all solutions were transferred into a 50 mL volumetric flask, and the total volume was increased to 50 mL with ultrapure water. One milliliter of the extract was placed in a glass bottle, and the sample was dried under an N2 atmosphere. The remnant was redissolved in 2 mL of 0.02 M HCl and filtered through a 0.22 μm syringe filter to analysis amino acids using an LA8080 high-speed amino acid analyzer (Hitachi Corp., Tokyo, Japan). Amino acids were expressed on a DM base.
2.7. Statistical Analyses
Statistical analyses were performed using Statistical Analysis System (SAS Inst. Inc., Cary, NC, USA). Data on chemical compositions of raw materials, content of biogenic amines and amine acid of fresh alfalfa on day 60 of ensiling were analyzed by a one-way analysis of variance (ANOVA). Data on fermentation quality, nitrogen distribution and protease activities of alfalfa silages during ensiling were analyzed by a two-way ANOVA. Statistical differences between means were determined by using Tukey’s multiple comparisons and were reported as significant at p < 0.05.
3. Results
3.1. Enzymatic Activity, Nitrogen Distribution and Chemical and Microbial Compositions of Fresh Alfalfa and Dried Apple Pomace
As shown in Table 1, DAP had significantly higher DM content and TPC (p < 0.05), and WSCs in DAP were almost quintuple those in alfalfa (p < 0.05). Lower pH, BC, lactic acid bacteria count, aerobic bacteria count, yeast count, enterobacteria count, carboxypeptidase activity, aminopeptidase activity, acid proteinase activity and HC, CP, TN, FAA-N and NH3-N contents were found in DAP (p < 0.05), while similar NDF, ADF, NPN and peptide-N contents were found between DAP and alfalfa (p > 0.05). The PPO activity was only detected in DAP.
The biogenic amine and amine acid contents of fresh alfalfa are presented in Table 2. Eight biogenic amines including tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine were analyzed, and the contents were 4.18, 30.28, 3.98, 15.08, 37.74, 11.84, 20.95 and 10.63 mg/kg DM, respectively. The content of total biogenic amines was 134.66 mg/kg DM. Seventeen amino acids including threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine, aspartic acid, serine, glutamic, glycine, alanine, cysteine, tyrosine and proline were analyzed, and the contents of them were 9.20, 10.15, 0.51, 8.15, 15.50, 10.71, 12.55, 4.73, 9.19, 24.29, 9.00, 19.37, 9.60, 11.38, 1.29, 6.06 and 8.60 g/kg DM, respectively. The total amine acid content in fresh alfalfa was 170.27 g/kg DM.
3.2. Dry Matter and Fermentation Quality of Alfalfa Silages During Ensiling
Treatments, ensiling days and their interaction had significant (p < 0.05) impacts on pH, lactic acid, acetic acid and propionic acid content, while DM and butyric acid content was only significantly (p < 0.05) affected by treatments and ensiling days (Table 3). The lactic acid, acetic acid, propionic acid and butyric acid contents significantly (p < 0.05) increased, and pH significantly (p < 0.05) decreased in all silages. Compared with the control, the DM and lactic acid content linearly (p < 0.05) increased with the increase in DAP ratio, while the pH, acetic acid, propionic acid and butyric acid content linearly (p < 0.05) decreased during ensiling.
3.3. Nitrogen Distribution of Alfalfa Silages During Ensiling
The dynamics of NPN, peptide-N, FAA-N and NH3-N are shown in Table 4, and they were significantly (p < 0.05) affected by treatments, ensiling days and their interaction, except peptide-N, which was not affected by the interaction (p > 0.05). The NPN, FAA-N and NH3-N content significantly (p < 0.05) increased in all silages throughout the whole period of ensiling. The most rapid increase in NPN in all silages was observed in the first 3 days of ensiling, while the lowest increase was found in 15% DAP silages. With the increasing proportion of DAP, NPN content significantly (p < 0.05) decreased compared with the control during ensiling. Peptide-N content in all silages increased first and thereafter decreased during the ensiling progress (p < 0.05). A significant (p < 0.05) decrease in peptide-N content was observed in DAP-treated silages compared with the control on the first day, while no significant (p > 0.05) difference was observed in all silages during 3–7 days of ensiling. Thereafter, the 10% and 15% DAP silages had significantly (p < 0.05) lower peptide-N content compared with the control during 14–60 days. During the first 7 days of ensiling, 15% DAP silage had significantly (p < 0.05) higher FAA-N content compared with the control, while no significant difference (p > 0.05) was observed among the control, 5% DAP and 10% DAP silages. During 14–60 days of ensiling, the application of DAP linearly (p < 0.05) decreased FAA-N content. Compared with the control, the NH3-N content linearly (p < 0.05) decreased with the increase in DAP ratio during ensiling.
3.4. Protease Activities of Alfalfa Silages During Ensiling
Treatments, ensiling days and their interaction had significant (p < 0.05) effects on the activities of carboxypeptidase, aminopeptidase and acid proteinase (p > 0.05) (Table 5). Their protease activities significantly (p < 0.05) decreased over the course of ensiling. During the first 7 days of ensiling, the 10% and 15% DAP silages had significantly (p < 0.05) lower carboxypeptidase activity compared with the control, whereas no significant (p > 0.05) difference was found between the control and 5% DAP silages. Treating with DAP linearly (p < 0.05) decreased carboxypeptidase activity during 14–60 days of ensiling. With the increase in DAP proportions, aminopeptidase activity linearly (p < 0.05) decreased relative to the control during ensiling. Silages treated with 10% and 15% DAP had significantly (p < 0.05) lower acid proteinase activity than the control during the first 14 days, and subsequently, DAP-treated silages significantly (p < 0.05) decreased acid proteinase activity during 30–60 days of ensiling.
3.5. The Biogenic Amine Content of Alfalfa Silage on Day 60 of Ensiling
Addition of DAP had significant (p < 0.05) effects on eight biogenic amines and total biogenic amines (Table 6). Putrescine, cadaverine, tyramine and phenylethylamine were the dominant biogenic amines produced in alfalfa silage. On day 60 of ensiling, all silages had low spermine and spermidine content, while low tryptamine and histamine were only observed in DAP-treated silages. Treating with DAP significantly (p < 0.05) decreased the content of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, spermidine, spermine and total biogenic amines compared with the control. The 10% and 15% DAP silages had significantly (p < 0.05) lower tyramine content, while no significant (p > 0.05) difference was observed between the control and 5% DAP silage.
3.6. The Amino Acid Content of Alfalfa Silages on Day 60 of Ensiling
As the DAP proportion increased, the content of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine, aspartic acid, serine, glutamic and total amino acids linearly (p < 0.05) increased, while the content of glycine, alanine, cysteine and proline linearly (p < 0.05) decreased (Table 7). Meanwhile, there were no significant (p > 0.05) differences in valine, isoleucine, leucine, lysine, arginine, glutamic, glycine, alanine and proline content between the control and 5% DAP silages. Only 15% DAP silage had significantly (p < 0.05) higher total amino acid content compared with the control. Treating with DAP had numerically (p > 0.05) higher tyrosine than the control. No significant (p > 0.05) difference in methionine content was observed among all silages.
3.7. Chemical Composition of Alfalfa Silages on Day 60 of Ensiling
The WSCs and CP contents were significantly (p < 0.05) affected by the addition of DAP, whereas no obvious (p > 0.05) difference was found in NDF, ADF and HC content (Table 8). Though a linear increase in WSCs and CP content was found in DAP-treated silages (p < 0.05), no significant (p > 0.05) difference was observed between the control and 5% DAP silages. There were no significant (p > 0.05) differences in NDF, ADF and HC content among all silages.
3.8. Correlation Relationship Between Fermentation Characteristics and Nitrogen Distribution
Based on Pearson correlation analysis, NPN was positively corrected with acetic acid (p < 0.001), propionic acid (p < 0.001), butyric acid (p < 0.001), carboxypeptidase (p < 0.001), pH (p < 0.001), acid proteinase (p < 0.01) and aminopeptidase (p < 0.05), while it was negatively corrected with DM (p < 0.001) (Figure 1). Peptide-N had significant positive correlations with acetic acid (p < 0.001), propionic acid (p < 0.001), butyric acid (p < 0.01), carboxypeptidase (p < 0.001), pH (p < 0.001) and acid proteinase (p < 0.05), while it was negatively corrected with DM (p < 0.001). FAA-N was positively correlated with acetic acid (p < 0.001), propionic acid (p < 0.001), butyric acid (p < 0.001), carboxypeptidase (p < 0.001), pH (p < 0.001) and acid proteinase (p < 0.01), while it was negatively corrected with DM (p < 0.001). NH3-N showed significant positive correlations with acetic acid (p < 0.001), propionic acid (p < 0.001), butyric acid (p < 0.01), carboxypeptidase (p < 0.001), pH (p < 0.001), acid proteinase (p < 0.01) and aminopeptidase (p < 0.05), while it was negatively corrected with DM (p < 0.001).
4. Discussion
4.1. Analysis of Raw Materials
It is often difficult to obtain quality silage from legume forages like alfalfa due to their high BC and low WSC and DM contents [1]. In this work, the WSC content of alfalfa was lower than 5% DM, which was not beneficial for quality fermentation. However, the population of epiphytic LAB in fresh alfalfa was more than 5.0 log10 cfu/g FW, which was conducive to efficient lactic acid fermentation. Furthermore, high WSC content in DAP could provide adequate substrates for LAB fermentation and contribute to a rapid decrease in pH at the initial period of ensiling. Biogenic amines were a series of endogenous metabolites in forages, which derived from decarboxylation of free amino acids [20,21]. Eight biogenic amines were found in fresh alfalfa of the experiment, and their contents were higher than those in earlier cuttings of alfalfa, which was reported by Mlejnkova et al. (2016) [22]. This may be the case because the longer growth time promoted the accumulation of biogenic amines. The contents of CP and essential amino acids were lower compared to the earlier study of Guo et al. (2008), in which alfalfa was harvested at the early bloom stage [23]. We speculated that harvesting at the early bloom stage might result in higher CP and essential amino acid content, which should be aspired to in silage making.
4.2. Analysis of Fermentation Characteristics During Ensiling
The pH was a critical parameter to assess fermentation quality, and a low pH was favorable for the suppression of undesirable microbes [24]. Compared with the control, the DAP-treated silages had lower pH and relatively better fermentation quality. The pH in all silages significantly decreased during ensiling, which might be due to the formation of lactic acid. The increased lactic acid content in response to DAP addition might result from more transformation of substrates into lactic acid by LAB fermentation. The increase in DM content in DAP-treated silages compared with the control was notable. The probable reason was that DAP provided more fermentation substrates for LAB. Furthermore, the increasing DM could inhibit the activities of proteases and undesirable bacteria, thus reducing the degree of proteolysis [17,25]. As expected, DM had negative correlations with NPN, peptide-N, FAA-N and NH3-N. The accumulation of lactic acid and acetic acid had the capacity to inhibit the growth of undesirable microorganisms [26]. In the experiment, the content of lactic acid and acetic acid in all silages increased during ensiling, while the counts of aerobic bacteria, yeast and enterobacteria synchronously decreased. The addition of DAP linearly decreased acetic acid content, which possibly resulted from the inhibition of some enterobacteria, which could principally produce acetic acid [27]. Compared with the control, lower NH3-N content in DAP-treated silages indicated that the addition of DAP contributed to inhibiting proteolysis. Dai et al. (2022) had reported that DAP had abundant malic acid, which was a favorable factor for the quick formation of an acidification environment [28].
4.3. Analysis of Nitrogen Distribution During Ensiling
According to McDonald et al. (1991), protein degradation mainly underwent two steps, i.e., true protein was primarily degraded into peptides and free amino acids by plant proteases and subsequently degraded into amines and ammonia by microbial enzymes during ensiling [1]. The NPN, which mainly included peptide-N, FAA-N and NH3-N, served as a direct indicator to reflect the degree of proteolysis during ensiling. This has been reported by Khejornsart et al. (2025), who suggested that rumen microbial protein synthesis was more efficient by supplying silage with protein-N than NPN [29]. Therefore, the accumulation of NPN resulted in the inefficient utilization of nitrogen in ruminants [30]. It has been claimed that PPO could catalyze the reaction of o-phenols to o-quinones, which could further generate quinone–protein complexes to resist proteolysis during ensiling [5]. Polyphenols were oxidized by PPO to quinone compounds, which were associated reversibly or irreversibly with amino acids and proteins. The formation of protein–quinones could resist the hydrolysis of proteolytic enzymes on amino acids or proteins.
The application of DAP decreased NPN content compared with the control, which indicated that the addition of DAP enhanced protein preservation of alfalfa silages. One possible reason was that DAP had the protein binding ability endowed by o-quinones, which could preserve true protein against the hydrolysis of proteases, thus leading to less degradation of true protein to NPN. The presence of malic acid and citric acid in apple pomace contributed to the foundation of acidification environment in DAP-treated silages. Lower pH value and its faster decline led to restraining the activities of proteases, such as carboxypeptidase, aminopeptidase and acid proteinase. Their activities in 5% DAP, 10% DAP and 15% DAP silages were significantly lower than that in the control. The decline in protease activities resulted in the decreased hydrolysis of true protein. Correspondingly, 5% DAP, 10% DAP and 15% DAP silages had significantly lower NPN content than the control.
In the experiment, DAP had high PPO activity and TPC. Furthermore, the deamination activity of microorganisms like proteolytic clostridia might be inhibited by lower pH in DAP-treated silages, as indicated by lower NH3-N content and butyric acid content. It is likely that the low pH might decrease the activities of plant proteases or the rate of deamination or decarboxylation [4,31].
4.4. Protease Activity During Ensiling
Tao et al. (2011) has reported that the main proteases in alfalfa were carboxypeptidase, aminopeptidase and acid proteinase, and their optimal pH was 5.4–5.5 [32]. Our results were consistent with Yuan et al. (2017), who reported that the decline in aminopeptidase activity was antecedent to carboxypeptidase and acid proteinase [33]. The activity of aminopeptidase had a drastic decline in DAP-treated silages during the first 14 days of ensiling, since the pH was far from the optimum. However, the control had a significant decrease in aminopeptidase activity during the first 3 days, and then kept stable during 7–14 days of ensiling. The control had stable activities of carboxypeptidase and acid proteinase during the first 14 days of ensiling and was accompanied by the optimal pH with the same ensilage time. Significant decreases in carboxypeptidase and acid proteinase activities were observed in 10% and 15% DAP silages during the first 14 days, which mainly result from a lower pH than 5.5. Compared with the control, the activities of carboxypeptidase, aminopeptidase and acid proteinase linearly decreased with the increasing ratio of DAP during ensiling. The lower activities of carboxypeptidase, aminopeptidase and acid proteinase might have contributed to the inhibition of protease activities by low pH or o-quinones binding to proteases [4]. The activities of protease still did not cease at the end of ensiling. This seems to imply that decreased protein degradation in DAP-treated silages might be the mutual result of the combination of o-quinones and proteinases and the deactivation of protease activities under sub-optimal conditions.
4.5. Analysis of Biogenic Amines on Day 60 of Ensiling
The formation of biogenic amines was related to proteolysis, which was mainly influenced by plant enzymes and microbial enzymes [34]. In fact, biogenic amines were largely end-products of microbial activities rather than plant proteases [1]. In the experiment, the control had greater tyramine and cadaverine content (>1000 mg kg/DM), and the content of total biogenic amines was approximately 5000 mg kg/DM. The result was consistent with Nishino et al. (2007), who reported a large amount of tyramine (1063 mg kg/DM) and cadaverine (1233 mg kg/DM) in festulolium silages [35]. Phenylethylamine and putrescine were also a primary biogenic amine formed in alfalfa silages, and the contents of phenylethylamine and putrescine in the control were 917.42 and 882.94 mg kg/DM, respectively. The large formation of phenylethylamine, putrescine, tyramine and cadaverine might be related to the abundant degradation of phenylalanine, arginine, tyrosine and lysine, respectively [19]. In the control, more than 50% of arginine, tyrosine, lysine and phenylalanine (84.33%, 74.59%, 58.00% and 51.07%, respectively) was degraded during ensiling. The application of DAP significantly decreased the content of eight and total biogenic amines on day 60 of ensiling. According to Fijałkowska et al. (2015), amino acids were degraded into biogenic amines by decarboxylation, and enterobacteria played an important role in decarboxylation [31]. In the experiment, lower pH in DAP-treated silages inhibited the growth of enterobacteria, thereby inactivating the decarboxylation activities.
4.6. Analysis of Amino Acids on Day 60 of Ensiling
A large loss of amino acids was observed in the control on day 60 of ensiling. This was consistent with the consequence of Pobednov and Kosolapov (2018) regarding the extensive degradation of amino acids in alfalfa silages [36]. The content of total amine acids in DAP-treated silages was higher than that in the control, which indicated that DAP was more effective at restraining proteolysis during ensiling. Meanwhile, significant increases in threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine, aspartic acid, serine and glutamic content in DAP-treated silages might be due to the lower pH. Since Guo et al. (2008) demonstrated that the decrease in pH could decrease the degradation of amino acids in forage silages [23]. In addition, apple pomace was rich in essential amino acids, aspartic acid and glutamic, which also related to the higher amino acid content in DAP silages than the control [37,38]. Furthermore, the decrease in proline and the increase in glutamic suggested that the transformation progress of glutamic had been hindered in DAP-treated silages [39].
4.7. Analysis of Chemical Composition on Day 60 of Ensiling
The DAP-treated silages linearly increased WSC content compared with the control, which might relate to the high WSC content of DAP. More DAP was supplied to alfalfa silages. The significant increase in CP content in DAP-treated silages might result from the linear increase in DAP addition. Meanwhile, the significant decrease in protein breakdown in DAP-treated silages could retain more protein, which was also attributed to the significant increase in CP content in DAP-treated silages.
5. Conclusions
The results demonstrated that proteolysis and proteinase activity weakened as the DAP ratio increased, accompanied by positive effects on fermentation characteristics in alfalfa silages. The content of NPN, peptide-N, FAA-N and NH3-N, and the activities of carboxypeptidase, aminopeptidase and acid proteinase decreased as the DAP ratio increased. The DAP-treated silages had significantly lower biogenic amine content and higher essential amino acid, aspartic acid and serine content than the control on day 60 of ensiling. Among conditions tested, 15% was the optimal addition for alfalfa silages as indicated by the better fermentation quality and less proteolysis than other silages during ensiling.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15020438/s1. Table S1. Effects of dried apple pomace on the microbial composition of alfalfa silages during ensiling.
Author Contributions
Data curation, T.D., J.L. and G.Z.; funding acquisition, Z.D. and X.Y.; investigation, T.D., Z.D. and G.Z.; methodology, T.D., Z.D. and X.Y.; validation, Z.D., J.L. and G.Z.; writing—original draft, T.D.; writing—review and editing, Z.D. and X.Y. All authors have read and agreed to the published version of the manuscript.
Funding
This research was supported by the Demonstration of Ecological Grass Husbandry Technology in Tibet High-cold Region (XZ202301YD0012C) and National Key Research and Development Program of China (2024YFD13003033).
Data Availability Statement
All data and materials are available upon request to the corresponding author.
Conflicts of Interest
The authors have declared no conflicts of interest.
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Figure 1.
Heatmap of Pearson correlation analysis of fermentation characteristics and nitrogen distribution during ensiling. DM, dry matter; LAB, lactic acid bacteria; AB, aerobic bacteria; FAA-N, free amino acid nitrogen; NH3-N, ammonia nitrogen; NPN, nonprotein nitrogen; peptide-N, peptide nitrogen. Control, without DAP; 5% DAP, with 5% dried apple pomace of FW; 10% DAP, with 10% dried apple pomace of FW; 15% DAP, with 15% dried apple pomace of FW. All data were the means of three biological replicates. Red color represents a positive correlation, while blue color represents a negative correlation. Asterisks (***, ** and *) indicate significant differences between fermentation characteristics and nitrogen distributions with p < 0.001, p < 0.01 and p < 0.05, respectively.
Figure 1.
Heatmap of Pearson correlation analysis of fermentation characteristics and nitrogen distribution during ensiling. DM, dry matter; LAB, lactic acid bacteria; AB, aerobic bacteria; FAA-N, free amino acid nitrogen; NH3-N, ammonia nitrogen; NPN, nonprotein nitrogen; peptide-N, peptide nitrogen. Control, without DAP; 5% DAP, with 5% dried apple pomace of FW; 10% DAP, with 10% dried apple pomace of FW; 15% DAP, with 15% dried apple pomace of FW. All data were the means of three biological replicates. Red color represents a positive correlation, while blue color represents a negative correlation. Asterisks (***, ** and *) indicate significant differences between fermentation characteristics and nitrogen distributions with p < 0.001, p < 0.01 and p < 0.05, respectively.
Table 1.
Enzymatic activity, nitrogen distribution and chemical and microbial compositions of fresh alfalfa and dried apple pomace.
Table 1.
Enzymatic activity, nitrogen distribution and chemical and microbial compositions of fresh alfalfa and dried apple pomace.
| Items | Alfalfa | Dried Apple Pomace | p Value |
|---|---|---|---|
| DM (g/kg FW) | 307.18 | 951.76 | <0.001 |
| pH | 6.05 | 3.76 | <0.001 |
| BC (mEq/kg DM) | 358.69 | 72.29 | <0.001 |
| NDF (g/kg DM) | 487.15 | 458.11 | NS |
| ADF (g/kg DM) | 314.12 | 324.69 | NS |
| HC (g/kg DM) | 173.03 | 133.42 | 0.013 |
| WSCs (g/kg DM) | 32.69 | 157.06 | <0.001 |
| CP (g/kg DM) | 193.13 | 53.44 | <0.001 |
| TPC (mg GAE/g DM) | 3.75 | 6.13 | 0.038 |
| LAB (log10 cfu/g FW) | 5.86 | ND | - |
| AB (log10 cfu/g FW) | 7.29 | 1.20 | 0.007 |
| Yeast (log10 cfu/g FW) | 6.82 | 0.87 | 0.007 |
| Enterobacteria (log10 cfu/g FW) | 7.14 | ND | - |
| TN (g/kg DM) | 30.90 | 8.55 | <0.001 |
| NPN (g/kg TN) | 34.29 | 36.85 | NS |
| Peptide-N (g/kg TN) | 8.48 | 21.84 | NS |
| FAA-N (g/kg TN) | 22.99 | 13.71 | 0.004 |
| NH3-N (g/kg TN) | 2.82 | 1.31 | 0.036 |
| Carboxypeptidase activity (μmol/g of free amino acids released/h·g DM) | 40.52 | 2.19 | <0.001 |
| Aminopeptidase activity (units/h·g DM) | 24.75 | 0.65 | <0.001 |
| Acid proteinase activity (units/h·g DM) | 21.25 | 0.68 | <0.001 |
| PPO (U/mL·min) | ND | 400.00 | - |
Note: DM, dry matter; FW, fresh weight; BC, buffering capacity; mEq, milligram equivalent; NDF, neutral detergent fiber; ADF, acid detergent fiber; HC, hemicellulose; WSCs, water-soluble carbohydrates; CP, crude protein; TPC, total phenolic content; GAE, gallic acid equivalents; LAB, lactic acid bacteria; AB, aerobic bacteria; cfu, colony-forming unit; TN, total nitrogen; NPN, nonprotein nitrogen; peptide-N, peptide nitrogen; FAA-N, free amino acid nitrogen; NH3-N, ammonia nitrogen; PPO, polyphenol oxidase. ND, not detected; NS, non-significant; U/mL·min, a 0.001-unit change in absorbance per minute per mL of the enzyme.
Table 2.
The content of biogenic amines and amino acids of fresh alfalfa.
| Items | Alfalfa |
|---|---|
| Tryptamine (mg/kg DM) | 4.18 |
| Phenylethylamine (mg/kg DM) | 30.28 |
| Putrescine (mg/kg DM) | 3.98 |
| Cadaverine (mg/kg DM) | 15.08 |
| Histamine (mg/kg DM) | 37.74 |
| Tyramine (mg/kg DM) | 11.84 |
| Spermidine (mg/kg DM) | 20.95 |
| Spermine (mg/kg DM) | 10.63 |
| Total biogenic amines (mg/kg DM) | 134.66 |
| Threonine (g/kg DM) | 9.20 |
| Valine (g/kg DM) | 10.15 |
| Methionine (g/kg DM) | 0.51 |
| Isoleucine (g/kg DM) | 8.15 |
| Leucine (g/kg DM) | 15.50 |
| Phenylalanine (g/kg DM) | 10.71 |
| Lysine (g/kg DM) | 12.55 |
| Histidine (g/kg DM) | 4.73 |
| Arginine (g/kg DM) | 9.19 |
| Aspartic acid (g/kg DM) | 24.29 |
| Serine (g/kg DM) | 9.00 |
| Glutamic (g/kg DM) | 19.37 |
| Glycine (g/kg DM) | 9.60 |
| Alanine (g/kg DM) | 11.38 |
| Cysteine (g/kg DM) | 1.29 |
| Tyrosine (g/kg DM) | 6.06 |
| Proline (g/kg DM) | 8.60 |
| Total amino acids (g/kg DM) | 170.27 |
Note: DM, dry matter.
Table 3.
Effects of dried apple pomace on the dry matter and fermentation characteristics of alfalfa silages during ensiling.
Table 3.
Effects of dried apple pomace on the dry matter and fermentation characteristics of alfalfa silages during ensiling.
| Items | Ensiling Days | Treatments | SEM | p Value | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | 5% DAP | 10% DAP | 15% DAP | T | D | T × D | A-L | A-Q | |||
| DM (g/kg FW) | 1 | 299.21 c | 349.65 Ab | 378.43 Aab | 415.35 Aa | 4.632 | <0.001 | <0.001 | NS | <0.001 | 0.013 |
| 3 | 296.06 d | 341.16 ABc | 369.13 ABb | 403.77 ABa | |||||||
| 7 | 295.33 d | 340.18 ABc | 367.65 ABb | 395.01 ABa | |||||||
| 14 | 294.79 d | 336.87 ABc | 366.55 ABb | 391.53 ABa | |||||||
| 30 | 291.02 d | 328.75 Bc | 362.91 ABb | 389.55 ABa | |||||||
| 60 | 289.83 d | 327.93 Bc | 353.40 Bb | 379.82 Ba | |||||||
| pH | 1 | 6.11 Aa | 5.66 Ab | 5.57 Ab | 5.20 Ac | 0.066 | <0.001 | <0.001 | 0.034 | <0.001 | NS |
| 3 | 5.75 Ba | 5.51 Ab | 5.35 ABbc | 5.06 ABc | |||||||
| 7 | 5.64 BCa | 5.32 ABb | 5.16 Bb | 4.96 ABb | |||||||
| 14 | 5.48 Ca | 4.95 BCb | 4.79 Cb | 4.56 BCb | |||||||
| 30 | 5.21 Da | 4.78 Cb | 4.49 CDc | 4.31 Cc | |||||||
| 60 | 5.18 Da | 4.20 Db | 4.14 Db | 4.07 Cb | |||||||
| LA (g/kg DM) | 1 | 1.40 Eb | 1.46 Db | 1.80 Fab | 3.67 Da | 4.626 | <0.001 | <0.001 | 0.001 | 0.045 | NS |
| 3 | 24.67 Dc | 29.33 Cb | 31.08 Eab | 33.05 Ca | |||||||
| 7 | 45.52 Cb | 53.71 Ba | 54.49 Da | 58.12 BCa | |||||||
| 14 | 56.37 BCb | 63.87 Ba | 71.00 Ca | 95.20 ABa | |||||||
| 30 | 67.10 Bb | 90.01 Aa | 100.72 Ba | 116.86 Aa | |||||||
| 60 | 88.65 Ab | 100.44 Aa | 112.64 Aa | 133.31 Aa | |||||||
| AA (g/kg DM) | 1 | 4.53 Ca | 2.72 Eb | 1.55 Ec | 0.72 Cd | 0.674 | <0.001 | <0.001 | <0.001 | <0.001 | NS |
| 3 | 12.49 Ba | 6.36 Db | 4.95 Db | 2.68 BCc | |||||||
| 7 | 15.61 Ba | 8.94 Cb | 7.81 Cb | 5.04 ABc | |||||||
| 14 | 15.78 Ba | 10.84 Bb | 8.28 BCb | 6.53 Ab | |||||||
| 30 | 19.61 Aa | 10.92 Bb | 9.76 Bb | 6.48 Ac | |||||||
| 60 | 23.38 Aa | 15.18 Ab | 12.07 Ac | 6.70 Ad | |||||||
| PA (g/kg DM) | 1 | ND | ND | ND | ND | 0.371 | <0.001 | <0.001 | <0.001 | <0.001 | 0.019 |
| 3 | 4.28 | 1.32 | 1.61 | ND | |||||||
| 7 | 7.81 | 1.89 | 1.91 | ND | |||||||
| 14 | 8.19 | 2.03 | 2.08 | ND | |||||||
| 30 | 8.24 a | 2.43 b | 2.23 b | 0.83 c | |||||||
| 60 | 9.60 a | 4.48 b | 3.73 bc | 0.84 c | |||||||
| BA (g/kg DM) | 1 | 0.19 B | ND | ND | ND | 0.093 | <0.001 | <0.001 | NS | <0.001 | NS |
| 3 | 1.36 AB | 0.80 | 0.08 | 0.05 | |||||||
| 7 | 1.79 Aa | 1.59 ab | 1.21 b | 0.61 c | |||||||
| 14 | 1.80 Aa | 1.62 ab | 1.20 b | 0.59 c | |||||||
| 30 | 1.84 Aa | 1.72 ab | 1.58 ab | 0.90 b | |||||||
| 60 | 2.02 Aa | 1.83 a | 1.75 a | 1.03 b | |||||||
Note: DM, dry matter; FW, fresh weight; LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid. ND, not detected. Control, without DAP; 5% DAP, with 5% DAP of FW; 10% DAP, with 10% DAP of FW; 15% DAP, with 15% DAP of FW. Different capital letters indicate significant differences among different ensiling days under the same treatment (p < 0.05). Different lowercase letters indicate significant differences among different treatments on the same ensiling days (p < 0.05). The same letter indicates no significant difference (p > 0.05). NS, non-significant; SEM, standard error of the mean. T, treatments; D, ensiling days; T × D, interaction between treatments and ensiling days; A-L, linear effect of DAP proportions; A-Q, quadratic effect of DAP proportions.
Table 4.
Effects of dried apple pomace on the nitrogen distribution of alfalfa silages during ensiling (g/kg TN).
Table 4.
Effects of dried apple pomace on the nitrogen distribution of alfalfa silages during ensiling (g/kg TN).
| Items | Ensiling Days | Treatments | SEM | p Value | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | 5% DAP | 10% DAP | 15% DAP | T | D | T × D | A-L | A-Q | |||
| NPN | 1 | 245.28 Ea | 214.07 Db | 209.13 Db | 192.58 Cb | 12.412 | <0.001 | <0.001 | <0.001 | <0.001 | NS |
| 3 | 379.46 Da | 346.80 Cb | 321.47 Cbc | 275.25 Bc | |||||||
| 7 | 442.42 Ca | 400.12 BCb | 378.26 BCbc | 334.36 Ac | |||||||
| 14 | 506.96 Ba | 424.45 Bb | 396.54 ABb | 338.96 Ac | |||||||
| 30 | 564.74 ABa | 443.70 ABb | 407.75 ABbc | 354.58 Ac | |||||||
| 60 | 593.61 Aa | 499.08 Ab | 442.99 Abc | 376.25 Ac | |||||||
| Peptide-N | 1 | 190.06 Da | 168.11 Cb | 166.89 Cb | 161.10 Cb | 5.991 | <0.001 | <0.001 | NS | <0.001 | NS |
| 3 | 266.24 C | 261.35 B | 248.32 B | 220.36 B | |||||||
| 7 | 292.46 BC | 287.07 A | 278.76 A | 254.89 A | |||||||
| 14 | 322.13 ABa | 298.08 Aab | 280.56 Ab | 243.59 ABb | |||||||
| 30 | 348.52 Aa | 296.15 Aab | 275.02 Ab | 249.55 ABb | |||||||
| 60 | 275.24 BCa | 255.63 Bab | 236.98 Bb | 211.38 Bb | |||||||
| FAA-N | 1 | 42.42 Ea | 40.04 Ea | 36.93 Ea | 27.48 Eb | 5.126 | <0.001 | <0.001 | <0.001 | 0.025 | NS |
| 3 | 60.59 DEa | 58.80 DEab | 54.92 Dab | 44.24 Db | |||||||
| 7 | 75.73 CDa | 69.93 CDab | 64.22 CDab | 57.92 Cb | |||||||
| 14 | 97.47 BCa | 80.21 BCb | 77.13 BCb | 63.49 BCc | |||||||
| 30 | 111.47 Ba | 91.48 Bb | 87.94 Bb | 71.22 Bc | |||||||
| 60 | 200.91 Aa | 171.36 Ab | 155.68 Abc | 129.48 Ac | |||||||
| NH3-N | 1 | 12.68 Ea | 5.94 Eb | 5.70 Eb | 4.00 Db | 3.559 | <0.001 | <0.001 | <0.001 | <0.001 | 0.034 |
| 3 | 52.62 Da | 26.65 Db | 18.23 Dc | 10.65 Cd | |||||||
| 7 | 74.22 Ca | 43.12 Cb | 35.28 Cb | 21.54 Bc | |||||||
| 14 | 87.35 Ba | 46.16 BCb | 38.85 BCbc | 31.88 Ac | |||||||
| 30 | 104.75 Aa | 56.07 Bb | 44.79 ABc | 33.82 Ac | |||||||
| 60 | 117.46 Aa | 72.09 Ab | 50.33 Ac | 35.39 Ad | |||||||
Note: NPN, nonprotein nitrogen; peptide-N, peptide nitrogen; FAA-N, free amino acid nitrogen; NH3-N, ammonia nitrogen; TN, total nitrogen. Control, without DAP; 5% DAP, with 5% DAP of FW; 10% DAP, with 10% DAP of FW; 15% DAP, with 15% DAP of FW. Different capital letters indicate significant differences among different ensiling days under the same treatment (p < 0.05). Different lowercase letters indicate significant differences among different treatments on the same ensiling days (p < 0.05). The same letter indicates no significant difference (p > 0.05). NS, non-significant; SEM, standard error of the mean. T, treatments; D, ensiling days; T × D, interaction between treatments and ensiling days; A-L, linear effect of DAP proportions; A-Q, quadratic effect of DAP proportions.
Table 5.
Effects of dried apple pomace on the activities of carboxypeptidase, aminopeptidase and acid proteinase of alfalfa silages during ensiling.
Table 5.
Effects of dried apple pomace on the activities of carboxypeptidase, aminopeptidase and acid proteinase of alfalfa silages during ensiling.
| Items | Ensiling Days | Treatments | SEM | p Value | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | 5% DAP | 10% DAP | 15% DAP | T | D | T × D | A-L | A-Q | |||
| Carboxypeptidase activity (μmol/g of free amino acids released/h·g DM) | 1 | 33.05 Aa | 32.40 Aa | 20.75 Ab | 17.15 Ab | 1.254 | <0.001 | <0.001 | <0.001 | <0.001 | NS |
| 3 | 32.90 Aa | 30.33 Ab | 19.57 Ab | 14.74 Bb | |||||||
| 7 | 31.63 ABa | 29.59 Aa | 12.99 Bb | 11.02 Cb | |||||||
| 14 | 24.30 Ba | 19.89 Bb | 10.12 Bc | 8.68 Dc | |||||||
| 30 | 13.61 Ca | 8.67 Cb | 3.96 Cc | 1.84 Ec | |||||||
| 60 | 10.48 Ca | 6.70 Cb | 2.97 Cbc | 1.15 Ec | |||||||
| Aminopeptidase activity (Units/h·g DM) | 1 | 22.82 Aa | 19.89 Ab | 18.18 Ac | 16.42 Ad | 0.748 | <0.001 | <0.001 | <0.001 | 0.034 | NS |
| 3 | 16.38 Ba | 14.09 Bb | 12.77 Bbc | 11.53 Bc | |||||||
| 7 | 12.68 Ca | 11.09 Cb | 10.78 Bb | 10.09 Bb | |||||||
| 14 | 11.22 Ca | 4.86 Db | 4.55 Cb | 2.53 Cb | |||||||
| 30 | 4.93 Da | 3.88 Db | 2.77 Cbc | 2.39 Cc | |||||||
| 60 | 3.67 Da | 2.95 Db | 2.26 Cbc | 1.94 Cc | |||||||
| Acid proteinase activity (Units/h·g DM) | 1 | 17.73 Aa | 16.10 Aab | 13.72 Abc | 11.71 Ac | 0.580 | <0.001 | <0.001 | <0.001 | 0.001 | NS |
| 3 | 16.06 Aa | 15.44 Aa | 12.53 Ab | 11.24 Ab | |||||||
| 7 | 15.33 Aa | 14.82 Aa | 8.27 Bb | 6.85 Bc | |||||||
| 14 | 8.04 Ba | 7.44 Bab | 6.16 BCb | 5.54 Bb | |||||||
| 30 | 6.98 Ba | 5.39 Cb | 4.05 CDbc | 2.86 Cc | |||||||
| 60 | 5.95 Ba | 4.25 Cb | 2.95 Dbc | 2.24 Cc | |||||||
Note: DM, dry matter. Control, without DAP; 5% DAP, with 5% DAP of FW; 10% DAP, with 10% DAP of FW; 15% DAP, with 15% DAP of FW. Different capital letters indicate significant differences among different ensiling days under the same treatment (p < 0.05). Different lowercase letters indicate significant differences among different treatments on the same ensiling days (p < 0.05). The same letter indicates no significant difference (p > 0.05). NS, non-significant; SEM, standard error of the mean. T, treatments; D, ensiling days; T × D, interaction between treatments and ensiling days; A-L, linear effect of DAP proportions; A-Q, quadratic effect of DAP proportions.
Table 6.
Effects of dried apple pomace on the biogenic amine content of alfalfa silages on day 60 of ensiling (mg/kg DM).
Table 6.
Effects of dried apple pomace on the biogenic amine content of alfalfa silages on day 60 of ensiling (mg/kg DM).
| Items | Treatments | SEM | p Value | |||||
|---|---|---|---|---|---|---|---|---|
| Control | 5% DAP | 10% DAP | 15% DAP | T | A-L | A-Q | ||
| Tryptamine | 124.25 a | 34.58 b | 28.73 b | 5.56 b | 14.191 | 0.001 | <0.001 | 0.007 |
| Phenylethylamine | 917.42 a | 384.40 b | 276.41 b | 117.08 b | 94.599 | <0.001 | <0.001 | 0.019 |
| Putrescine | 822.94 a | 391.42 b | 264.78 bc | 109.18 c | 81.636 | <0.001 | <0.001 | 0.007 |
| Cadaverine | 1601.88 a | 736.05 b | 490.45 bc | 247.75 c | 156.886 | <0.001 | <0.001 | 0.002 |
| Histamine | 366.82 a | 120.75 b | 36.68 b | 24.61 b | 46.681 | 0.004 | 0.001 | 0.048 |
| Tyramine | 1128.91 a | 882.07 ab | 743.08 b | 653.32 b | 60.022 | 0.003 | <0.001 | NS |
| Spermidine | 25.88 a | 20.96 b | 13.56 c | 10.00 d | 1.895 | <0.001 | <0.001 | NS |
| Spermine | 22.66 a | 18.27 b | 15.05 c | 10.34 d | 1.373 | <0.001 | <0.001 | NS |
| Total biogenic amines | 5010.76 a | 2588.51 b | 1868.74 c | 1177.85 c | 440.901 | <0.001 | <0.001 | 0.001 |
Note: DM, dry matter. Control, without DAP; 5% DAP, with 5% DAP of FW; 10% DAP, with 10% DAP of FW; 15% DAP, with 15% DAP of FW. Different lowercase letters indicate significant differences among different treatments (p < 0.05). NS, non-significant; SEM, standard error of the mean. T, treatments; A-L, linear effect of DAP proportions; A-Q, quadratic effect of DAP proportions.
Table 7.
Effects of dried apple pomace on the amino acid content of alfalfa silages on day 60 of ensiling (g/kg DM).
Table 7.
Effects of dried apple pomace on the amino acid content of alfalfa silages on day 60 of ensiling (g/kg DM).
| Items | Treatments | SEM | p Value | |||||
|---|---|---|---|---|---|---|---|---|
| Control | 5% DAP | 10% DAP | 15% DAP | T | A-L | A-Q | ||
| Threonine | 2.65 b | 5.20 a | 5.57 a | 5.85 a | 0.422 | 0.002 | 0.001 | 0.026 |
| Valine | 8.22 c | 8.63 bc | 9.99 ab | 11.47 a | 0.418 | 0.001 | <0.001 | NS |
| Methionine | 0.61 | 0.69 | 0.34 | 0.57 | 0.083 | NS | NS | NS |
| Isoleucine | 6.70 c | 7.04 c | 8.40 b | 9.27 a | 0.356 | 0.006 | 0.001 | NS |
| Leucine | 11.31 c | 12.03 bc | 14.18 ab | 15.51 a | 0.573 | 0.005 | 0.001 | NS |
| Phenylalanine | 5.24 b | 6.64 a | 6.75 a | 6.98 a | 0.233 | 0.005 | 0.002 | NS |
| Lysine | 5.27 b | 7.75 ab | 7.94 a | 8.03 a | 0.419 | 0.022 | 0.009 | NS |
| Histidine | 2.29 b | 3.11 a | 3.18 a | 3.62 a | 0.159 | 0.002 | <0.001 | NS |
| Arginine | 1.44 c | 2.03 bc | 2.41 ab | 2.81 a | 0.168 | 0.002 | <0.001 | NS |
| Aspartic acid | 8.88 b | 16.29 a | 16.60 a | 17.41 a | 1.098 | <0.001 | <0.001 | 0.004 |
| Serine | 2.66 c | 4.03 b | 4.81 ab | 5.23 a | 0.313 | <0.001 | <0.001 | NS |
| Glutamic | 10.31 c | 10.62 bc | 11.45 b | 12.61 a | 0.285 | <0.001 | <0.001 | NS |
| Glycine | 8.80 a | 8.19 ab | 7.05 b | 6.60 b | 0.305 | 0.008 | 0.001 | NS |
| Alanine | 12.84 a | 12.12 ab | 10.09 bc | 9.06 c | 0.508 | 0.003 | <0.001 | NS |
| Cysteine | 11.71 a | 6.30 b | 3.44 bc | 1.53 c | 1.218 | <0.001 | <0.001 | NS |
| Tyrosine | 1.54 | 2.16 | 2.37 | 2.44 | 0.140 | NS | 0.016 | NS |
| Proline | 8.64 a | 8.02 ab | 6.92 bc | 6.41 c | 0.302 | 0.006 | 0.001 | NS |
| Total amino acids | 109.12 b | 120.85 ab | 121.50 ab | 125.40 a | 2.256 | 0.028 | 0.007 | NS |
Note: DM, dry matter. Control, without DAP; 5% DAP, with 5% DAP of FW; 10% DAP, with 10% DAP of FW; 15% DAP, with 15% DAP of FW. Different lowercase letters indicate significant differences among different treatments (p < 0.05). NS, non-significant; SEM, standard error of the mean. T, treatments; A-L, linear effect of DAP proportions; A-Q, quadratic effect of DAP proportions.
Table 8.
Effects of dried apple pomace on chemical compositions of alfalfa silages on day 60 of ensiling (g/kg DM).
Table 8.
Effects of dried apple pomace on chemical compositions of alfalfa silages on day 60 of ensiling (g/kg DM).
| Items | Treatments | SEM | p Value | |||||
|---|---|---|---|---|---|---|---|---|
| Control | 5% DAP | 10% DAP | 15% DAP | T | A-L | A-Q | ||
| NDF | 467.75 | 464.45 | 462.14 | 438.90 | 4.708 | NS | 0.030 | NS |
| ADF | 333.61 | 3331.86 | 331.79 | 329.29 | 4.596 | NS | NS | NS |
| HC | 134.14 | 132.59 | 130.35 | 109.61 | 5.018 | NS | NS | NS |
| WSCs | 12.45 c | 13.81 bc | 18.47 ab | 23.72 a | 1.428 | 0.001 | <0.001 | NS |
| CP | 163.14 c | 171.01 bc | 178.49 ab | 184.09 a | 2.609 | 0.002 | <0.001 | NS |
Note: DM, dry matter; NDF, neutral detergent fiber; ADF, acid detergent fiber; HC, hemicellulose; WSCs, water-soluble carbohydrates; CP, crude protein. Control, without DAP; 5% DAP, with 5% DAP of FW; 10% DAP, with 10% DAP of FW; 15% DAP, with 15% DAP of FW. Different lowercase letters indicate significant differences among different treatments (p < 0.05). NS, non-significant; SEM, standard error of the mean. T, treatments; A-L, linear effect of DAP proportions; A-Q, quadratic effect of DAP proportions.
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MDPI and ACS Style
Dai, T.; Long, J.; Zhang, G.; Yuan, X.; Dong, Z. The Effects of Dried Apple Pomace on Fermentation Quality and Proteolysis of Alfalfa Silages. Agronomy 2025, 15, 438. https://doi.org/10.3390/agronomy15020438
AMA Style
Dai T, Long J, Zhang G, Yuan X, Dong Z. The Effects of Dried Apple Pomace on Fermentation Quality and Proteolysis of Alfalfa Silages. Agronomy. 2025; 15(2):438. https://doi.org/10.3390/agronomy15020438
Chicago/Turabian StyleDai, Tongtong, Jiangyu Long, Guanjun Zhang, Xianjun Yuan, and Zhihao Dong. 2025. "The Effects of Dried Apple Pomace on Fermentation Quality and Proteolysis of Alfalfa Silages" Agronomy 15, no. 2: 438. https://doi.org/10.3390/agronomy15020438
APA StyleDai, T., Long, J., Zhang, G., Yuan, X., & Dong, Z. (2025). The Effects of Dried Apple Pomace on Fermentation Quality and Proteolysis of Alfalfa Silages. Agronomy, 15(2), 438. https://doi.org/10.3390/agronomy15020438
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