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Article

Lycium barbarum Residue Enhances Fermentation Quality and Antioxidant Activity of Alfalfa Silage

by
Yuanzhen Cheng
,
Tao Shao
,
Haobo Chen
,
Jie Zhao
,
Junfeng Li
and
Zhihao Dong
*
Institute of Ensiling and Processing of Grass, College of Agro-Grassland Science, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(12), 2839; https://doi.org/10.3390/agronomy15122839
Submission received: 10 November 2025 / Revised: 3 December 2025 / Accepted: 9 December 2025 / Published: 10 December 2025
(This article belongs to the Special Issue Innovative Solutions for Producing High-Quality Silage)
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Abstract

This study explored the potential application of Lycium barbarum residue (LBR) in alfalfa silage, particularly focusing on its synergistic effects when combined with silage additives. Two controlled experiments were conducted. In Experiment 1, four treatment groups were established with different LBR addition levels (0, 70, 140, 210 g/kg fresh weight, FW). Experiment 2 used the optimal LBR level identified (210 g/kg FW), and further investigated the effects of additive combinations. The treatments in this experiment included: (1) 210 g/kg FW LBR (CK), (2) a combination of 210 g/kg FW LBR with lactic acid bacteria (ALL), (3) a combination of 210 g/kg FW LBR with molasses (ALM), and (4) a combination of 210 g/kg FW LBR with both lactic acid bacteria and molasses (ALLM). The silage was ensiled for 7, 15, 30, and 90 days. The results demonstrated that the incorporation of LBR significantly enhanced silage fermentation quality. The 210 g/kg treatment exhibited the most favorable outcomes, characterized by the lowest pH, reduced ammonia nitrogen content, and the highest concentration of lactic acid. Additionally, 210 g/kg treatment showed increased levels of total phenolics and flavonoids, as well as enhanced antioxidant activities as measured by DPPH (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity), and FRAP (ferric-reducing antioxidant power) assays. These improvements in bioactive compounds were positively correlated with lactic acid content and negatively associated with pH. Furthermore, in Experiment 2, the combined application of LAB and molasses along with LBR further optimized the silage quality, resulting in the lowest pH and ammonia nitrogen content, alongside a marked improvement in antioxidant capacity during the later ensiling stages. Overall, the study concludes that the inclusion of 210 g/kg LBR in combination with lactic acid bacteria and molasses effectively enhances both the fermentation process and the functional value of alfalfa silage, providing a scientific foundation for the utilization of agricultural byproducts.

1. Introduction

Oxidative stress, a key pathological process resulting from an imbalance between the production and scavenging of free radicals within animal bodies, has become a major contributing factor that negatively impacts both the productivity and general health of animals [1]. Studies have shown that incorporating dietary antioxidants into animal feed can effectively reduce oxidative damage and enhance the functionality of the body’s own antioxidant enzyme systems [2]. With the growing worldwide emphasis on eliminating the use of antibiotics in livestock farming due to stricter regulatory measures, there is increasing interest in the use of natural, plant-based bioactive compounds as feed additives. These additives are being recognized for their strong safety profiles and wide range of beneficial properties, making them a promising area of focus in the field of animal nutrition research.
Lycium barbarum L., a well-known plant with both medicinal and edible applications, is extensively cultivated across multiple provinces in China. A byproduct of its industrial processing, Lycium barbarum residue (LBR), primarily comprises the peels and seeds remaining after juice extraction. This residue is abundant in underexploited nutrients and residual bioactive compounds, which can promote the utilization of nutrients, rumen digestion, and the growth and immunity of animals [3]. At the same time, it was found that LBR as a feed additive improved the feed intake and rumen fermentation characteristics of brown sheep [4]. Currently, large amounts of LBR are discarded as waste, leading not only to substantial resource losses but also contributing to notable environmental contamination. Identifying effective strategies for the valorization of LBR has become an urgent priority. Scientific investigations have revealed that LBR is rich in various bioactive constituents, including polysaccharides, polyphenols, and flavonoids, all of which hold promise for enhancing the antioxidant activity of animal feed [5,6]. Furthermore, studies have shown that Lycium barbarum polysaccharides-key components of LBR-can stimulate the proliferation of lactic acid bacteria (LAB), a characteristic that may confer advantages during the silage process, particularly in promoting lactic acid fermentation [7].
Alfalfa (Medicago sativa L.), valued for its exceptionally high crude protein content, plays an irreplaceable role in the nutritional support of ruminant animals. Silage technology, which leverages the metabolic activities of lactic acid bacteria to generate substantial quantities of organic acids, rapidly lowers the pH within the silage environment, and the low pH resulting from lactic acid stabilizes silage fermentation by suppressing microbes that are intolerant of acidic conditions [8]. This anaerobic, low-pH condition effectively preserves the nutritional integrity of the forage, establishing silage as a cornerstone method for feed preservation in contemporary animal husbandry [9]. Nevertheless, the ensiling of alfalfa presents several intrinsic challenges: its naturally high buffering capacity, coupled with low levels of water-soluble carbohydrates and limited populations of epiphytic lactic acid bacteria [10], often results in unregulated fermentation dynamics and undesirable nutrient degradation, thereby hindering the large-scale application of alfalfa silage. The dominance of facultative heterofermentative lactic acid bacteria during the early, active phase of fermentation serves to inhibit enterobacteria, clostridia, and other microorganisms, ultimately leading to reduced proteolysis and fermentation dry matter losses [11]; as such, the inclusion of lactic acid bacteria as silage additives has become essential. Moreover, research indicates that LAB-mediated fermentation can facilitate the biotransformation and dynamic redistribution of bioactive compounds in Lycium barbarum [12], implying a potential synergistic interaction between LAB and LBR during ensiling. Notably, LAB and their metabolic byproducts not only enhance feed palatability via biochemical conversion but also exhibit intrinsic antioxidant properties [13]. Nevertheless, the synergistic effect of combining LBR with LAB on these parameters has not been fully elucidated.
Based on the above background, we hypothesized that LBR would not only improve alfalfa silage quality on its own but also act synergistically with LAB and molasses to enhance both fermentation quality and antioxidant activity. The aim of this study is to systematically investigate the effect of LBR as a natural additive on fermentation quality and antioxidant activity of alfalfa silage. Further exploration was conducted on the effects of LAB, molasses, and their combination on LBR–alfalfa silage at the optimal LBR level, ultimately constructing a quality improvement technology system based on LBR and LAB composite reinforcement. This study provides a scientific basis and technical approach for food waste utilization, eco-friendly feed development, and high-value use of unconventional feed materials.

2. Materials and Methods

2.1. Description of Materials

Alfalfa (Medicago sativa L. cv. ‘Maverick’) was cultivated in the experimental field of Yangzhou University, Yangzhou, China, located at 32.39° N latitude, 119.42° E longitude, and an altitude of 8 m. The alfalfa plants (third cutting) were harvested manually at the early flowering stage and wilted in the field for 6 h to ensure optimal biomass quality. The LBR utilized in this study was sourced from a processing facility in the Ningxia Hui Autonomous Region, China. This residue is the solid byproduct generated during the industrial extraction of Lycium barbarum juice. The LAB inoculant used in this study is the CM4 community, a defined consortium of laboratory isolated strains including P. acidilactici B2, L. plantarum B19, L. pentosus A2, W. cibaria F1, L. rhamnosus M41, P. pentosaceus K3, and E. faecalis L1. The inoculant was prepared by mixing the individual strains at a 1:1 ratio based on OD600 values. It exhibits consistent and reliable fermentation performance, meeting the stringent standards required for scientific research [14]. Molasses was procured from a certified commercial supplier in Guangxi Zhuang Autonomous Region, China, to ensure product quality and traceability; the main component is sucrose.

2.2. Experimental Design

Experiment 1: The experimental design followed a completely randomized design. Four treatments were established, each differing in the inclusion level of LBR. The specific treatments were as follows: (1) Control group (CK) with 0 g/kg LBR; (2) Treatment AL1 with 70 g/kg LBR; (3) Treatment AL2 with 140 g/kg LBR; and (4) Treatment AL3 with 210 g/kg LBR.
Experiment 2: The experimental design followed a completely randomized design with four treatment groups. Based on the optimal LBR inclusion level in Experiment 1. Firstly, prepared the base mixture by adding LBR at 210 g/kg FW to alfalfa and thoroughly mixing. The treatments comprised: (1) 210 g/kg FW LBR (CK), a combination of 210 g/kg FW LBR with 1 × 106 cfu/g FW of LAB (ALL), a combination of 210 g/kg FW LBR with 30 g/kg FW of molasses (ALM), and a combination of 210 g/kg FW LBR with both 1 × 106 cfu/g FW of LAB and 30 g/kg FW molasses (ALLM). Silage samples from each treatment were subjected to fermentation periods of 7, 15, 30, and 90 days. At the end of each ensiling period, three silos from each treatment group were opened for subsequent analyses.

2.3. Silage Preparation

After harvest, the alfalfa was manually chopped into 2–3 cm segments using a laboratory forage chopper. The prescribed amounts of LBR were then added and thoroughly mixed with the alfalfa. The mixture was packed into pre-sterilized (UV-treated) polyethylene silage bags (40 cm × 20 cm), which were vacuum-sealed using an Omite DZD-400 sealer (Omite Technology Co., Ltd., Nanjing, China) to ensure anaerobic conditions. Sealed bags were stored at ambient temperature (18 °C to 22 °C) to simulate practical silage fermentation.

2.4. Sampling and Chemical Analyses

For each treatment, silage samples were collected and transferred to ethanol-sterilized plastic containers. After thorough homogenization, 20 g of each sample was weighed and extracted with 60 mL of distilled water at 4 °C for 24 h. The extracts were filtered through four layers of medical-grade sterile gauze and qualitative filter paper. The resulting filtrate was used to determine pH, ammonia nitrogen (NH3-N), organic acids (lactic acid, acetic acid, propionic acid, butyric acid), and ethanol content. The pH was measured using an HI 2221 pH meter (HANNA Instruments, Smithfield, RI, USA). The ammonia nitrogen content was determined by the phenol-hypochlorite colorimetric (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) method and expressed as g/kg total nitrogen (TN) [15]. Organic acids and ethanol were quantified using a 1260 Infinity high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA) equipped with a refractive index detector [16]. The buffering capacity (BC) was measured using a standard acid-base titration method [17].
Approximately 60 g of silage was weighed into envelopes and dried to constant weight in a 65 °C oven to determine dry matter (DM) content. The dried samples were ground to a 1 mm screen with a laboratory knife mill (FW100; Taisite Instrument Co., Tianjin, China). This powder was used to analyze water-soluble carbohydrates (WSC), neutral detergent fiber (NDF), acid detergent fiber (ADF) and total nitrogen (TN). The WSC content was determined using the sulfuric acid–anthrone colorimetric method [18]. The neutral detergent fiber and acid detergent fiber content of fresh samples were measured following Van Soest et al. [19] with an Ankom 200 fiber analyzer (Ankom Technology, Macedon, NY, USA). Total nitrogen was measured by using an automatic N analyzer (Kjeltec-TM2300; FOSS, Hillerød, Denmark) according to the Kjeldahl method, and crude protein (CP) content was calculated as total N × 6.25.
Fresh samples were homogenized with sterile saline solution (1:9 ratio) and shaken at 37 °C and 120 rpm for 2 min. Following this, 1 mL of the homogenate was serially diluted for microbial enumeration. LAB was counted on MRS agar after 3 days of anaerobic incubation at 37 °C. Under aerobic conditions, aerobic bacteria and fungi (yeasts and molds) were enumerated on NA and PDA medium after 2 days of incubation at 30 °C, respectively, while enterobacteria were counted on VRBGA after 1 day at 30 °C. All media used were purchased from Shanghai Shengsi Co., Ltd. (Shanghai, China). All microbial counts were logarithmically transformed and expressed as Log10 colony-forming units (CFU) per gram of fresh matter (FM) [20].
The sample (1 g) was weighed and placed in 20 mL of 70% (v/v) ethanol, followed by vortexing to ensure thorough mixing. The mixture was then subjected to extraction in a constant-temperature shaker (300 rpm, 50 °C, 2 h). After extraction, samples were centrifuged at 4000× g for 10 min at room temperature, and the supernatant was carefully collected [21]. The pooled supernatants were stored at −20 °C for subsequent analysis of total phenolics (TP) (Folin–Ciocalteu method) [22], the result is described as gallic acid equivalent (GAE/g DM); total flavonoids (TF) (AlCl3 colorimetric method) [22], the result is described as rutin equivalent (RE/g DM); and antioxidant activities [23]: (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity (ABTS), and ferric reducing antioxidant power (FRAP), the result is described as Trolox equivalent (mg TE/g DM).All experimental materials used in this procedure were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

2.5. Statistical Analyses

All experimental data were analyzed using one-way analysis of variance (ANOVA) with the General Linear Model (GLM) procedure in IBM SPSS Statistics 19.0. Post hoc multiple comparisons of means were conducted using the Waller-Duncan test to determine statistically significant differences, with statistical significance set at p < 0.05.

3. Results

3.1. Characteristics of Fresh Materials

The chemical and microbial compositions of the raw materials are presented in Table 1. Compared to alfalfa, LBR exhibited a significantly lower pH and BC. In terms of nutrients, LBR possessed higher DM and WSC contents, albeit with lower crude protein (CP) levels. Epiphytic microbial analysis revealed higher populations of LAB, yeasts, and molds in LBR. Notably, LBR was substantially richer in bioactive compounds, characterized by significantly higher levels of TP, TF, and corresponding superior antioxidant activities (DPPH, ABTS, and FRAP).

3.2. Effects of LBR Inclusion on the Fermentation Characteristics and Chemical Compositions of Alfalfa Silage

The fermentation characteristics and chemical compositions of silage after 90 days are shown in Figure 1 and Figure 2. LBR inclusion and ensiling duration significantly affected pH, LA, ethanol, NH3-N, and WSC (p < 0.05), with significant interactions observed for PA, ethanol, NH3-N, and WSC (p < 0.05). Increasing the LBR proportion resulted in a linear decrease (p < 0.001) in pH, PA, BA, ethanol, and NH3-N, alongside a linear increase (p < 0.001) in DM and LA contents. Prolonging ensiling duration reduced pH and WSC levels, while LA, AA, and NH3-N accumulated gradually. Overall, AL3 consistently exhibited the most desirable fermentation profile.

3.3. Effects of LBR Inclusion on the Antioxidant Activities of Alfalfa Silage

Both LBR inclusion proportion and ensiling duration significantly affected TP, TF, DPPH, ABTS, and FRAP (p < 0.001). Significant interactions between these two factors were observed for TP, TF, ABTS, and FRAP (p < 0.001). Specifically, TF content increased linearly (p < 0.001) with rising LBR inclusion levels. By day 90, TP and TF contents in all LBR-treated silages were notably higher than in the control, peaking in the AL3 group. As shown in Figure 3, LBR inclusion significantly enhanced antioxidant activity. Throughout the ensiling period, DPPH, ABTS, and FRAP activities exhibited a linear increase (p < 0.05) corresponding to increased LBR proportions. The AL3 group consistently displayed the highest values, with the exception of FRAP activity at day 90. Pearson correlation analysis indicated that TP, TF, and antioxidant activities (DPPH and FRAP) were negatively correlated with pH (p < 0.001) but positively correlated with LA (p < 0.001). Furthermore, a strong positive correlation was observed between antioxidant activity and the contents of total phenolics and flavonoids.

3.4. Effects of Additives on the Fermentation Quality of LBR–Alfalfa Silage

Based on the fermentation characteristics and antioxidant profiles observed in Experiment 1, the 210 g/kg FW LBR dosage (AL3) was selected for the subsequent experiment.
Additive, ensiling duration, and their interaction significantly influenced LA, AA, ethanol, NH3-N, and pH (p < 0.05). Distinct pH dynamics were observed: CK and ALM groups exhibited a gradual pH decline, whereas ALL and ALLM groups underwent rapid acidification, reaching their lowest values (3.72 and 3.61, respectively) by day 7. Overall, ALL and ALLM silages maintained superior fermentation quality, characterized by significantly lower pH, NH3-N, and AA levels, alongside higher LA content compared to CK and ALM groups (p < 0.05) (Figure 4). Both DM and WSC contents were significantly affected by additive and ensiling duration (p < 0.001), with a significant interaction observed for WSC (p < 0.05) (Figure 5). While DM and WSC levels decreased significantly (p < 0.05) across all treatments during ensiling, the ALM group consistently preserved higher WSC content than all other silages throughout the fermentation process (p < 0.05).

3.5. Effects of Additives on the Antioxidant Activities of LBR–Alfalfa Silage

Ensiling significantly increased TP, TF, and antioxidant activity compared to fresh material. Additives, ensiling duration, and their interaction significantly affected these parameters (p < 0.05). The LAB-inoculated silages (ALL, ALLM) had higher TP levels than non-inoculated groups (CK, ALM) throughout ensiling process (p < 0.05). After 30 days of ensiling, TF content in all treated silages exceeded that of the control (p < 0.05). Also, the treated groups exhibited consistently higher DPPH and FRAP activities during ensiling. Molasses supplementation significantly enhanced antioxidant activity during the later stages of ensiling. Pearson correlation analysis indicated that TP, TF and antioxidant activities of DPPH and FRAP were negatively correlated with pH (p < 0.01) while positively correlated with LA (p < 0.001). Furthermore, strong positive correlations were observed between TP/TF contents and antioxidant activities of DPPH and FRAP (p < 0.01).

4. Discussion

4.1. Effects of LBR Inclusion on the Fermentation Characteristics and Chemical Compositions of Alfalfa Silage

This study demonstrates that the inclusion of LBR substantially enhances alfalfa silage quality by leveraging its intrinsic nutritional attributes. Alfalfa ensiling is inherently challenging due to its high buffering capacity and low indigenous WSC content, factors that often lead to suboptimal lactic acid fermentation and accelerated proteolysis [24,25]. The addition of LBR effectively mitigated these limitations; its abundant WSC provided essential fermentable substrates that promoted LAB proliferation and facilitated a dose-dependent acceleration in acidification (Figure 1). This rapid pH decline, in turn, effectively suppressed spoilage microorganisms and proteolysis, as evidenced by the linear reductions in NH3-N, PA, and BA (p < 0.001). These findings align with previous studies reporting that agricultural by-products rich in WSC and LAB improve alfalfa silage fermentation [26,27].
Among the treatments, AL3 group (210 g/kg FW LBR inclusion) demonstrated the most favorable fermentation profile, characterized by the lowest pH (4.15) and NH3-N (38.30 g/kg TN), and highest LA (62.51 g/kg DM). Well-preserved silage is characterized by low proteolysis, which is reflected in an ammonia nitrogen concentration of less than 100 g/kg TN [28]. Thus, higher LBR inclusion effectively suppresses spoilage microorganisms (e.g., clostridia, yeasts), thereby reducing protein degradation and the production of PA, BA, and ethanol [27]. Moreover, it is plausible that the bioactive compounds present in LBR, which are known to inhibit protease activity and undesirable microbes, likely also contributed to these beneficial effects [29,30,31,32].

4.2. Effects of LBR Inclusion on the Antioxidant Activities of Alfalfa Silage

The LBR-mediated enhancement of fermentation quality coincided with a significant increase in antioxidant activity, attributed to the reducing and radical-scavenging properties of its phenolic and flavonoid constituents [33]. Fresh LBR exhibited substantially higher TP and TF levels than alfalfa (Table 1), resulting in superior antioxidant performance across DPPH, ABTS, and FRAP assays. Notably, these bioactive components were preserved and further released during ensiling (Figure 4) [34]. Although FRAP activity overall exhibited a positive linear response to LBR inclusion, the significant interaction with ensiling duration may have resulted in the AL3 treatment did not present the highest FRAP value at the 90-day time point, as visualized in Figure 3. Pearson correlation analysis confirmed a significant positive relationship between TP/TF content and antioxidant activity, identifying LBR-derived phenolics as the primary contributors. This corroborates previous findings that plant phenolics retain biological activity in anaerobic systems [35]. Furthermore, antioxidant activity in LBR-treated silage increased progressively during fermentation. While polyphenols are typically bound to cell walls [36] or proteins [37,38], the acidic ensiling environment may play a role in facilitates their liberation via acid hydrolysis or enzymatic degradation [39,40,41]. This mechanism explains the observed accumulation of free bioactive forms and the consequent boost in antioxidant potential.

4.3. Effects of Additives on the Fermentation Quality of LBR–Alfalfa Silage

Combining LBR with LAB inoculants significantly improved fermentation quality compared to LBR alone, characterized by a rapid pH decline (<3.9), reduced NH3-N (<20 g/kg TN), and elevated LA production (>70 g/kg DM). This synergistic enhancement stems from the immediate dominance of exogenous LAB, which effectively outcompete the indigenous microbiome for LBR-derived water-soluble carbohydrates (WSC). The resulting accelerated acidification suppresses spoilage microorganisms (e.g., clostridia and enterobacteria) and inhibits proteolysis, consistent with previous studies [42,43]. Conversely, while molasses increased WSC levels in ALM and ALLM silages (Figure 5), it conferred no significant advantage over LAB inoculation alone [44]. This indicates that the WSC supplied by LBR (68.22 g/kg DM) is sufficient to meet the theoretical requirement for optimal alfalfa ensiling (60–70 g/kg DM) [45], rendering additional molasses redundant.

4.4. Effects of Additives on the Antioxidant Activity of LBR–Alfalfa Silage

Beyond acidification, LAB confer functional benefits by secreting enzymes such as phenolic acid decarboxylase and β-glucosidase. It is hypothesized that these enzymes facilitate the release and biotransformation of LBR-derived polyphenols into more bioavailable forms, thereby enhancing antioxidant activity [46]. The observed increases in total phenolics and flavonoids align with established fermentation mechanisms involving depolymerization and bioconversion [47,48,49]. Consequently, the boosted antioxidant capacity is attributed to both the accumulation of free bioactive compounds and specific structural modifications, such as hydrolysis and glycosylation [50]. Notably, molasses supplementation (ALM) significantly enhanced late-stage antioxidant activity (Figure 6) without a corresponding increase in TP or TF content. This uncoupling suggests that the enhanced activity is not driven by phenolic accumulation, but rather by alternative mechanisms. These may include structural modifications (e.g., methylation) that boost the radical-scavenging efficiency of existing phenolics [51], or the preservation of non-phenolic antioxidants (e.g., ascorbic acid, carotenoids) protected by exogenous sucrose [52]. In conclusion, combining LBR with LAB not only optimizes fermentation and nutrient preservation but also actively regulates phenolic bioconversion, underscoring the synergistic value of this strategy for high-quality silage production.

5. Conclusions

This study demonstrates that LBR serves as a promising multifunctional additive for alfalfa silage. The inclusion of LBR at 210 g/kg FW, particularly in combination with LAB and molasses, which boosted lactic acid fermentation efficiency, and promoted the biotransformation of bioactive compounds, synergistically enhanced fermentation quality and simultaneously boosting the silage’s antioxidant activity. This strategy provides a viable approach for the valorization of food-processing waste and the production of high-quality, functional silage.

Author Contributions

Conceptualization, Z.D. and T.S.; Methodology, T.S., Y.C., H.C., J.Z. and J.L.; Formal analysis, Y.C.; Investigation, Y.C.; Writing—original draft, Y.C.; Writing—review and editing, Y.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by National Natural Science Foundation of China [Grant No. 32171690] and National Key Research and Development Program of China [Grant No. 2024YFD1300303].

Data Availability Statement

All relevant data are contained within the paper. Contact the corresponding author if further explanation is required.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 15 02839 g001
Figure 1. The dynamics of fermentation characteristics during ensiling. LA (g/kg DM), lactic acid; AA (g/kg DM), acetic acid; PA (g/kg DM), propionic acid; BA (g/kg DM), butyric acid; ethanol (g/kg DM); NH3-N (g/kg TN), ammonia nitrogen; TN, total nitrogen. Values are presented as mean ± standard deviation. The error bar represents the standard deviation. T, effect of LBR inclusion proportion; D, effect of ensiling day; T × D, interaction effect between LBR inclusion proportion and ensiling day; L and Q represent linear and quadratic effects of LBR inclusion proportion.
Figure 1. The dynamics of fermentation characteristics during ensiling. LA (g/kg DM), lactic acid; AA (g/kg DM), acetic acid; PA (g/kg DM), propionic acid; BA (g/kg DM), butyric acid; ethanol (g/kg DM); NH3-N (g/kg TN), ammonia nitrogen; TN, total nitrogen. Values are presented as mean ± standard deviation. The error bar represents the standard deviation. T, effect of LBR inclusion proportion; D, effect of ensiling day; T × D, interaction effect between LBR inclusion proportion and ensiling day; L and Q represent linear and quadratic effects of LBR inclusion proportion.
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Agronomy 15 02839 g002
Figure 2. The dynamics of DM and WSC during ensiling. Means with different superscripts in the same ensiling day (A–C) differ significantly (p < 0.05). T, effect of LBR inclusion proportion; D, effect of ensiling day; T × D, interaction effect between LBR inclusion proportion and ensiling day; L and Q represent linear and quadratic effects of LBR inclusion proportion.
Figure 2. The dynamics of DM and WSC during ensiling. Means with different superscripts in the same ensiling day (A–C) differ significantly (p < 0.05). T, effect of LBR inclusion proportion; D, effect of ensiling day; T × D, interaction effect between LBR inclusion proportion and ensiling day; L and Q represent linear and quadratic effects of LBR inclusion proportion.
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Agronomy 15 02839 g003
Figure 3. The dynamics of total phenolics, total flavonoids, antioxidant activities (DPPH, ABTS, and FRAP) during ensiling and Pearson correlations between total phenolics, total flavonoids, antioxidant activities (DPPH, ABTS, and FRAP), and fermentation parameters during ensiling. Values are presented as mean ± standard deviation. The error bar represents the standard deviation. Significant differences (p < 0.05) between samples processed differently are indicated by capital letters. T, effect of LBR inclusion proportion; D, effect of ensiling day; T × D, interaction effect between LBR inclusion proportion and ensiling day; L and Q represent linear and quadratic effects of LBR inclusion proportion.
Figure 3. The dynamics of total phenolics, total flavonoids, antioxidant activities (DPPH, ABTS, and FRAP) during ensiling and Pearson correlations between total phenolics, total flavonoids, antioxidant activities (DPPH, ABTS, and FRAP), and fermentation parameters during ensiling. Values are presented as mean ± standard deviation. The error bar represents the standard deviation. Significant differences (p < 0.05) between samples processed differently are indicated by capital letters. T, effect of LBR inclusion proportion; D, effect of ensiling day; T × D, interaction effect between LBR inclusion proportion and ensiling day; L and Q represent linear and quadratic effects of LBR inclusion proportion.
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Agronomy 15 02839 g004
Figure 4. The dynamics of fermentation characteristics during ensiling. LA (g/kg DM), lactic acid; AA (g/kg DM), acetic acid; PA (g/kg DM), propionic acid; BA (g/kg DM), butyric acid; ethanol (g/kg DM); NH3-N (g/kg TN), ammonia nitrogen; TN, total nitrogen. Values are presented as mean ± standard deviation. The error bar represents the standard deviation. T, effect of additives; D, effect of ensiling day; T × D, interaction effect between additives and ensiling day.
Figure 4. The dynamics of fermentation characteristics during ensiling. LA (g/kg DM), lactic acid; AA (g/kg DM), acetic acid; PA (g/kg DM), propionic acid; BA (g/kg DM), butyric acid; ethanol (g/kg DM); NH3-N (g/kg TN), ammonia nitrogen; TN, total nitrogen. Values are presented as mean ± standard deviation. The error bar represents the standard deviation. T, effect of additives; D, effect of ensiling day; T × D, interaction effect between additives and ensiling day.
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Agronomy 15 02839 g005
Figure 5. The dynamics of DM and WSC during ensiling. Means with different superscripts in the same ensiling day (A–D) differ significantly (p < 0.05). T, effect of additives; D, effect of ensiling day; T × D, interaction effect between additives and ensiling day.
Figure 5. The dynamics of DM and WSC during ensiling. Means with different superscripts in the same ensiling day (A–D) differ significantly (p < 0.05). T, effect of additives; D, effect of ensiling day; T × D, interaction effect between additives and ensiling day.
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Agronomy 15 02839 g006
Figure 6. The dynamics of total phenolics, total flavonoids, antioxidant activities (DPPH, ABTS, and FRAP) during ensiling and Pearson correlations between total phenolics, total flavonoids, antioxidant activities (DPPH, ABTS, and FRAP), and fermentation parameters during ensiling. Values are presented as mean ± standard deviation. The error bar represents the standard deviation. Significant differences (p < 0.05) between samples processed differently are indicated by capital letters. T, effect of additives; D, effect of ensiling day; T × D, interaction effect between additives and ensiling day.
Figure 6. The dynamics of total phenolics, total flavonoids, antioxidant activities (DPPH, ABTS, and FRAP) during ensiling and Pearson correlations between total phenolics, total flavonoids, antioxidant activities (DPPH, ABTS, and FRAP), and fermentation parameters during ensiling. Values are presented as mean ± standard deviation. The error bar represents the standard deviation. Significant differences (p < 0.05) between samples processed differently are indicated by capital letters. T, effect of additives; D, effect of ensiling day; T × D, interaction effect between additives and ensiling day.
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Table 1. Chemical compositions of raw materials prior to ensiling.
Table 1. Chemical compositions of raw materials prior to ensiling.
ItemsExperiment 1Experiment 2
AlfalfaLBRMixture Raw Matter
pH6.11 ± 0.04 A4.04 ± 0.02 B5.76 ± 0.05
DM (g/kg FW)323.50 ± 13.21 B415.83 ± 2.19 A350.65 ± 12.23
NDF (g/kg DM)339.58 ± 23.65348.89 ± 9.11329.05 ± 2.80
ADF (g/kg DM)244.53 ± 10.95249.60 ± 2.67236.74 ± 8.89
CP (g/kg DM)235.94 ± 4.66 A171.81 ± 2.90 B217.85 ± 2.32
WSC (g/kg DM)49.45 ± 1.28 B114.19 ± 8.04 A68.22 ± 2.05
BC (mEq/kg DM)259.51 ± 17.14 A112.27 ± 6.08 B228.21 ± 4.54
LAB (lg CFU/g FW)5.97 ± 0.12 B6.40 ± 0.07 A5.96 ± 0.05
Aerobic bacteria (log10 CFU/g FW)7.82 ± 0.067.85 ± 0.047.65 ± 0.05
Yeasts and molds (log10 CFU/g FW)6.43 ± 0.08 B7.99 ± 0.02 A6.74 ± 0.06
Enterobacteria (log10 CFU/g FW)6.28 ± 0.086.53 ± 0.086.28 ± 0.05
TP (mg GAE/g DM)10.36 ± 0.02 B12.43 ± 0.45 A10.48 ± 0.06
TF (mg RE/g DM)10.70 ± 0.20 B15.06 ± 0.30 A10.94 ± 0.33
DPPH (mg TE/g DM)5.36 ± 0.28 B10.79 ± 0.51 A6.55 ± 0.50
ABTS (mg TE/g DM)13.98 ± 0.4814.70 ± 0.0813.85 ± 0.23
FRAP (mg TE/g DM)8.77 ± 0.339.18 ± 0.478.52 ± 0.19
Note: DM, dry matter; FW, fresh weight; NDF, neutral detergent fiber; ADF, acid detergent fiber; CP, crude protein; WSC, water-soluble carbohydrate; BC, buffering capacity; LAB, lactic acid bacteria; TP, total phenolics; TF, total flavonoids; DPPH, 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activity; ABTS, 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity; FRAP, ferric reducing antioxidant power. Values are presented as mean ± standard deviation. Significant differences (p < 0.05) between samples differently are indicated by capital letters.
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MDPI and ACS Style

Cheng, Y.; Shao, T.; Chen, H.; Zhao, J.; Li, J.; Dong, Z. Lycium barbarum Residue Enhances Fermentation Quality and Antioxidant Activity of Alfalfa Silage. Agronomy 2025, 15, 2839. https://doi.org/10.3390/agronomy15122839

AMA Style

Cheng Y, Shao T, Chen H, Zhao J, Li J, Dong Z. Lycium barbarum Residue Enhances Fermentation Quality and Antioxidant Activity of Alfalfa Silage. Agronomy. 2025; 15(12):2839. https://doi.org/10.3390/agronomy15122839

Chicago/Turabian Style

Cheng, Yuanzhen, Tao Shao, Haobo Chen, Jie Zhao, Junfeng Li, and Zhihao Dong. 2025. "Lycium barbarum Residue Enhances Fermentation Quality and Antioxidant Activity of Alfalfa Silage" Agronomy 15, no. 12: 2839. https://doi.org/10.3390/agronomy15122839

APA Style

Cheng, Y., Shao, T., Chen, H., Zhao, J., Li, J., & Dong, Z. (2025). Lycium barbarum Residue Enhances Fermentation Quality and Antioxidant Activity of Alfalfa Silage. Agronomy, 15(12), 2839. https://doi.org/10.3390/agronomy15122839

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