Next Article in Journal
The Effect of Hydrogen Peroxide on Biogas and Methane Produced from Batch Mesophilic Anaerobic Digestion of Spent Coffee Grounds
Previous Article in Journal
Integrated System of Microalgae Photobioreactor and Wine Fermenter: Growth Kinetics for Sustainable CO2 Biocapture
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Fermentation
Volume 11
Issue 2
10.3390/fermentation11020059
fermentation-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effects of a Mountain Honeysuckle (Lonicerae flos) Extract on Fermentation Characteristics, Antioxidant Capacity and Microbial Community of Alfalfa Mixed Silage

by
Yating Wang
,
Lin Mu
,
Xin Cao
,
Qinglan Wang
and
Zhifei Zhang
*
Grassland Discipline, Department of Agriculture, Hunan Agricultural University, Changsha 410128, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(2), 59; https://doi.org/10.3390/fermentation11020059
Submission received: 13 December 2024 / Revised: 16 January 2025 / Accepted: 24 January 2025 / Published: 28 January 2025
(This article belongs to the Special Issue The Use of Lactobacillus in Forage Storage and Processing, 2nd Edition)
Browse Figures
Versions Notes

Abstract

:
The aim of this study was to examine the impact of a mountain honeysuckle (Lonicerae flos) extract on the fermentation characteristics, antioxidant capacity and microbial community of silage composed of a mixture of alfalfa, soybean meal and distiller’s dried grains with solubles (DDGS). Compared to the Control group, the application of the Lonicerae flos extract (0.05 to 0.25%) expressly improved the fermentation quality of the mixed alfalfa, as indicated by a reduced pH and increased concentrations of crude protein (CP). Notably, butyric acid (BA) was not detected in any treatment group. Additionally, an appropriate concentration of the extract enhanced the antioxidant capacity and active components of the silage. The abundance of L. acetotolerans exhibited an increasing trend corresponding to the rise in honeysuckle extract concentration. In conclusion, this Lonicerae flos extract has potential to improve anaerobic fermentation quality by promoting the growth of beneficial Lactobacillus spp. and inhibiting that of undesirable microbes. This study provides new insights into novel applications of herbal medicine extracts.

1. Introduction

Chinese herbal medicines contain many active compounds commonly regarded as safe phytogenic substances [1], many with antimicrobial effects [2]. One potential strategy to take advantage of these properties is to incorporate natural plant-derived compounds into the diets of ruminants [3]. The application of products of metabolism sourced from medicinal plants in the care and management of animal husbandry has increasingly gained attention in both studies and practical applications within the food production industry [4,5]. This trend underscores a growing interest in exploring the benefits of natural compounds sourced from plants in enhancing animal health and productivity. Moreover, traditional Chinese herbal medicines are being utilized not only to address various diseases affecting livestock but also in the formulation of fermented feeds. These herbal medicines contribute to improving the overall quality and flavor of feeds, while simultaneously elevating the levels of beneficial metabolites that can promote positive outcomes in livestock [6]. Therefore, herbal extracts can be used to enhance silage. Currently, the majority of research focuses on the adjustment of mixed silage through herbal extracts themselves or their residues, with relatively limited studies on the application of these extracts.
Lonicerae flos consists of the initial flowers or dried buds of L. macranthoides Hand.-Mazz., L. hypoglauca Miq., L. confusa DC., L. fulvoto-mentosa Hsu et S.C.Cheng. It has the functions of clearing away heat, detoxifying and dispelling wind. It is used for carbuncles, swelling and sores, laryngeal arthralgia, erysipelas, heat and blood dysentery, wind–heat colds, warm disease and fever. The main components of Lonicerae flos extract include chlorogenic acid, flavonoids, phenols and other substances that are closely related to its pharmacological effects [7]. Its medicinal value generally includes antibacterial, antioxidants and antivirals [8].
A mixed silage of 80% alfalfa + 10% soybean meal + 10% distiller’s dried grains with solubles is a good-quality feed and protein source. During ensiling, alfalfa is susceptible to deterioration by pathogenic microorganisms (e.g., Enterobacter, Clostridium and Salmonella), which greatly reduces the feed quality [9,10]. However, appropriate additives can promote fermentation and reduce losses.
We proposed the hypothesis that an extract of Lonicerae flos, when used as an ensiling additive, could enhance the quality of anaerobic fermentation by suppressing the growth of detrimental microorganisms. The primary objective of our research was to investigate how a Lonicerae flos extract affected various aspects of alfalfa mixed silage, including fermentation quality, nutritional value, aerobic stability, antioxidant properties and the diversity of microbial populations. Through this study, we aimed to have a comprehensive understanding of the impacts of this natural additive on the overall silage quality and its potential benefits in improving the preservation and nutritional characteristics of ensiled forage.

2. Materials and Methods

2.1. Preparation of Fresh Materials and Silage

Alfalfa was cultivated in Changde West Lake management zone (Hunan, China) and harvested at the budding stage in the third cutting, using a hand sickle and leaving a 5 cm stubble; then, a grinder was used to crush the samples into lengths of 1~2 cm. Soybean meal was purchased from COFCO Grain and Oil Industry Co., Ltd. (Jingzhou, China). The DDGS was purchased from Shijiazhuang Liyuan Bioprotein Co., Ltd. (Shijiazhuang, China). The Lonicerae flos extract was purchased from the Haiaosi Baicao essence shop (Shanxi, China), and its characteristics are listed in Table 1. The commercial lactic acid bacteria (Taiwan Yaxin Co., Ltd., Taiwan, China) used had a live bacterial count of 1.0 × 1011 cfu·g−1 and label instructions recommending the addition of 2 g·t−1.
Alfalfa, soybean meal and DDGS were mixed at a ratio of 8:1:1, with the addition of varying amounts of Lonicerae flos extract, as follows: 0 (Control), 0.05% (ST1), 0.10% (ST2), 0.15% (ST3), 0.20% (ST4), and 0.25% (ST5). All samples (500 g fresh materials) were placed in polyvinyl vacuum silage packages (20 × 30 cm2; Huaguan Printing Co., Ltd., Zhejiang, China), vacuumed, sealed by a vacuum sealer (Dafeng Machinery Co., Ltd., Zhejiang, China) and stored at room temperature (25~32 °C). After 30 days of ensiling, silage quality, aerobic stability, antioxidant capacity and bacterial communities were determined.

2.2. Analysis of Silage Quality

The pH of the silage extract was determined by a pH meter. Ammonia nitrogen (NH3-N) was determined by the phenol hypochlorous acid method [11]. The analysis of organic acids was performed using high-performance liquid chromatography [12]. Silage samples weighing 100 g were subjected to drying at 65 °C for 48 h to evaluate their dry matter (DM) content. Subsequently, DM were crushed and passed through a 40-mesh sieve. The water-soluble carbohydrate (WSC) content was obtained through a colorimetric analysis [13]. The determination of crude protein content (CP) was carried out using the Kjeldahl method [14]. The analysis of both neutral detergent fiber (NDF) and acid detergent fiber (ADF) was conducted according to the methodology outlined by Van Soest et al. [15,16].

2.3. Analysis of Mixed Silage Aerobic Stability

After a fermentation period of 30 days, the assessment of aerobic stability involved placing 1 kg of a sample into a 2 L plastic bucket. A high-precision temperature sensor (Smowo MDL-1048A, Shanghai Tianhe Automation Instrument Co., Ltd., Shanghai, China) was installed in the bucket to continuously monitor the temperature every 2 h. The assessment of aerobic stability depended on how long the temperature remained 2 °C above room temperature [17]. The impact of aerobic exposure on the pH level was evaluated on days 0, 2, 4, 6 and 8, with three samples being tested per treatment.

2.4. Determination of Chlorogenic Acid, Total Phenols, Total Flavonoids and Antioxidant Ability

2.4.1. Extraction Solution Preparation

The extraction solution was obtained by ethanol extraction, as described by Aloo et al. [18], with 1g of sample (processed through a 40-mesh sieve) put into 20 mL of 70% ethanol, mixed well and then incubated in the dark for 12 h at 4 °C with constant oscillation. Subsequently, the extract was obtained via centrifugation at 4000 rpm for 10 min and preserved at −20 °C pending determination of total flavonoids, total phenols and antioxidant ability. Under the same conditions, the process was replicated twice, with all samples analyzed three times each.

2.4.2. Determination of Chlorogenic Acid

The chlorogenic acid standard (Beijing Solaibao Technology Co., Ltd., Beijing, China) was prepared into a 100 μg/mL mother liquor with 70% ethanol. The mother liquor was diluted into 0, 3.297, 6.522, 9.677, 12.766, 15.789, 18.75, 21.649 μg/mL chlorogenic acid standard storage solution. A sample weighing 0.5 g was added to 30 mL of 70% ethanol. This mixture was thoroughly stirred for 5 min, followed by a 25-min ultrasound extraction (ultrasonic frequency of 40 KHz, power set at 100 W). After letting the mixture stand for 30 min, it was vortex-mixed again for an additional 5 min. The resulting extract was then separated via centrifugation at 6000 rpm and subsequently placed at -20 °C for future analysis [19].
We adjusted the pH of the standard or extract to 3 with 12 mol/L HCl, added activated carbon (solid/liquid ratio of 1.4%) and decolorized the solution in a 60 °C water bath for one hour. Finally, the absorbance measurement was carried out at 329 nm using a Varioskan reader (Varioskan, Biotek, Epoch 2, Winosky, VT, USA) [20].

2.4.3. Determination of Total Flavonoids

The rutin standard (Shanghai Yuanye Technology Co., Ltd., Shanghai, China) was prepared into 0.5 mg/mL mother liquor with 70% ethanol and diluted into 0, 0.0313, 0.0625, 0.125, 0.1875, 0.25 mg/mL rutin standard storage solution.
We took 2 mL of standard or sample extract and 5 mL of 1 mol/L CH3COOK solution (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), mixed them well, added 5 mL of 0.1 mol/L AlCl3 solution (wt/Vol.) and stored the obtained solution at 20 °C for 20 min; then, the absorbance was measured at 410 nm (Varioskan) [21].

2.4.4. Determination of Total Phenols

The gallic acid standard (Shanghai Yuanye Technology Co., Ltd., Shanghai, China) was prepared into 0.5 mg/mL mother liquor with 70% ethanol, and diluted into 0, 0.0151, 0.0294, 0.0429, 0.0758, 0.0882 mg/mL standard storage liquor.
A 1 mL sample extraction or standard was combined with 0.5 mL of Folin–Ciocalteu’s phenol reagent and allowed to stand for 5 min. Subsequently, it was mixed thoroughly with 75 g/L Na2CO3 (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), and then the mixture was stored at room temperature in darkness for 2 hours. After this period, the absorbance measurement was carried out at 760 nm (Varioskan) [21].

2.4.5. Analysis of DPPH Radical Scavenging Activity

The Trolox standard (Shanghai Yuanye Technology Co., Ltd., Shanghai, China) was prepared into 0.5 mg/mL mother liquor with absolute ethanol and then diluted into 0, 19.23, 37.04, 53.57, 68.97, 83.33, 109.38 μg/mL Trolox standard storage solution.
The extraction solution or Trolox standard solution was mixed with 0.1 mM DPPH in methanol. Incubation in the dark of this solution was essential to ensure that the components of the mixture interacted effectively without the interference of light. Following this waiting period, the optical density (OD) at 517 nm of this solution was measured according to [22].

2.4.6. Analysis of ABTS Radical Scavenging Activity

Trolox mother liquor (Nanjing Biyuntian Biotechnology Co., Ltd., Nanjing, China) with a concentration of 10 mM was prepared using 0, 0.146, 0.283, 0.536, and 0.763 mM Trolox standard storage solution.
A fresh preparation of ABTS solution was produced and then diluted by a factor of 10. To the reaction system, 0.01 mL of the standard or extract was added, and then the mixture was incubated in the dark for about 5 min. After that, the OD at 734 nm of this solution was measured following the method described in [22].

2.4.7. Analysis of the Reducing Power (FRAP)

We used 27.8 mg of ferrous sulfate heptahydrate (Nanjing Biyuntian Biotechnology Co., Ltd., Nanjing, China) to prepare 100 mM ferrous sulfate heptahydrate mother liquor, which was diluted to 0.1757, 0.3509, 0.6994, 1.0457, 1.3896 mM standard storage solution.
The was analysis was conducted using the approach of He et al. [22]. The OD at 593 nm was determined. Ultimately, the results were expressed in terms of Trolox equivalents, quantified as mg TE/g DM.

2.5. Microbial Diversity Analysis

Fresh and fermented samples (50 mL) underwent centrifugation at 8500 rpm for 5 min. Following this process, the supernatant was carefully removed, and the remaining residue was preserved at a temperature of −80 °C to maintain its integrity for future analysis. The extraction of DNA was performed following the manufacturer’s protocol using a DNA isolation kit (DP812, Tiangen, Beijing, China) on frozen–thawed fermented silage samples. Microbial diversity sequencing was performed by Biomark Technology (Beijing, China). The analytical procedures utilized the PacBio Sequel platform [23]. Bacterial community diversity and correlation analyses were performed according to the method of Mu Lin et al. [24], and Venn diagrams aided in the comparison of bacterial communities. Furthermore, the evaluation of microbial functions was facilitated by using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt v2.4.2) software.

2.6. Statistical Analyses

The data analyses for this study were conducted using the BMK Cloud platform. To evaluate the differences between the groups, one-way analysis of variance (ANOVA) was utilized. This statistical test was carried out using IBM SPSS Statistics version 27.

3. Results and Discussion

3.1. Characteristics of the Fresh Material Before Ensiling

The composition of the silage raw material is described below (Table 1). The crude protein (CP) level in alfalfa (199.27 g/kg DM) exceeded the values of previous studies [13], yet fell short when compared to the results of Yang et al. [25]. Factors such as climate, fertilization and timing of harvest have an impact on forage quality [26]. The water-soluble carbohydrate (WSC) concentration serves as an important metric for evaluating the fermentation quality; in this research, the amount present in the fresh mixture (88.50 g/kg DM) surpassed the expected range (60–70 g/kg DM) [27], which increased its vulnerability to spoilage and heightened the requirement for additives.

3.2. Fermentation Characteristics

During fermentation, important indicators for assessing the fermentation quality include pH, NH3-N and organic acids (primarily, lactic and acetic acids) [28]. The pH of silage treated with the Lonicerae flos extract was lower than that of the Control (p < 0.05), with greater reductions measured with increasing extract concentration (Table 2). The ST4 group had the lowest pH and NH3-N content after 30 days of ensiling. The pH of legume silage is usually between 4.3 and 5.0 [29]. In these studies, the pH of all silages was above the acceptable value for well-fermented silage (pH 4.2) [30], which led to a negative impact on aerobic stability and long-term storage. This may have been due to the lower organic acid concentrations of the mixed silage. However, after 30 days of ensiling, the content of lactic acid (LA) and acetic acid (AA) was increased, with no significant difference among the extract treatments (p < 0.05), ranging, across all treatments, from 169.10 to 178.44 and from 22.90 to 24.58 g/kg DM for LA and AA, respectively. This contributed to the lower pH (p < 0.05) of the mixed silage supplemented with the Lonicerae flos extract. Furthermore, AA, an antifungal that can improve the aerobic stability of silage, was likely the reason why molds and yeasts were not detected.
The butyric acid (BA) content decreased significantly after 30 days of ensiling, and BA was undetectable in ST2 to ST5, probably due to their lower pH suppressing the growth of Clostridia spp., the usual source of BA. The fermentation of Clostridium is not recommended because it can lead to nutrient losses and potential health problems [30]. Furthermore, the reproduction of Clostridium reduced the livestock silage intake, especially when the BA content was higher than 5 g/kg DM [31].
NH3-N is typically a key value used to assess fermentation quality and is commonly linked to the breakdown of protein [32,33]. A threshold of NH3-N under 100 g/kg DM is regarded as indicative of properly preserved silage. Despite the increase in additional extract leading to a rise (p < 0.05) in the NH3-N levels (Table 2), all experimental treatments exhibited acceptable concentrations. A comparable pattern of elevated NH3-N content was noted when alfalfa was ensiled at a moisture level of 72% [34].
In this research, the incorporation of the Lonicerae flos extract created a microenvironment distinguished by a reduced pH and increased levels of bioactive compounds, enabling the swift growth of beneficial microorganisms like lactic acid bacteria (LAB) in anaerobic conditions.

3.3. Chemical Composition

The content of DM decreased after adding the Lonicerae flos extract (Table 2), which indicated that this addition improved the content of water in the silage.
Effects on the WSC, NDF, HC and ash contents were observed due to the treatments and the length of ensiling (p < 0.05), while the interaction between treatments and ensiling time also influenced CP and ADF (p < 0.05). Additionally, ADL was influenced (p < 0.05) by both treatments and their interaction with the ensiling time. Only the length of ensiling impacted DM (p < 0.05). The DM, WSC, NDF, ADF, and HC contents decreased (p < 0.05) after 30 days of ensiling with the Lonicerae flos extract. Given that both ST3 and ST4 exhibited a greater DM content than the Control after 30 days of ensiling, it is possible that the extract successfully reduced DM loss. An elevated DM level in raw forage may inhibit the development of microbes that cause spoilage by supplying adequate substrates for the production of organic acids, promoting a rapid reduction in pH [35,36] that limits the growth and proliferation of spoilage microorganisms.
The CP and ash contents increased (p < 0.05) after 30 days of ensiling, with the CP content in the ST3 and ST4 treatment groups being higher (p < 0.05) than that in the Control. This is thought to be due to the fact that the Lonicerae flos extract contains phenols, and these phenols have an extraordinary ability to combine with proteins because they feature a polyhydroxyl structure. It is highly likely that this feature will assist in warding off the hydrolysis of proteins during ensilage [37]. Furthermore, the NDF and ADF contents in the Control were significantly higher than in ST1 and ST2, whereas the ADL content in the Control was expressly higher than in all treatment groups. The low fiber content can be attributed to microbial acid hydrolysis and potential cellulase enzyme production during fermentation [38].

3.4. Aerobic Stability of Alfalfa Mixed Silage

After 30 days, both the Control and extract-treated groups had aerobic stability lasting over 360 h (Table 3). Although the pH increased slightly on the second day, it thereafter remained relatively stable (range, 4.45 to 4.64 in the first 8 days of aerobic exposure; Table 3). Therefore, the Lonicerae flos extract did not diminish the aerobic stability of silage and, to some extent, inhibited the growth of aerobic spoilage microorganisms.

3.5. Chlorogenic Acid, Total Flavonoids, Total Phenols, and Antioxidant Ability of Alfalfa Mixed Silage

As oxidative stress is associated with various health issues in ruminants and can adversely impact animal productivity, enhancing the antioxidant potential of silage is essential [39,40]. It has been highlighted that plant extracts play a significant role in promoting antioxidant capacities in livestock and the utilization of the derived products [41,42]. Typically, the reducing power of FRAP, the activity of ABTS in scavenging radicals and the capacity to scavenge DPPH radicals are associated with enzymes that carry out processes like dismutating superoxide radicals (O2−), decomposing hydrogen peroxide (H2O2) and transforming hydroperoxides into non-toxic substances (H2O/alcohol and O2) [43]. Based on the concentration of chlorogenic acid, total phenols, FRAP reducing activity and ABTS radical scavenging ability were remarkably influenced (p < 0.05) by treatment, ensiling time and their interaction, whereas total flavonoids were only influenced (p < 0.05) by silage time (Table 4). The DPPH radical scavenging activity was dramatically impacted (p < 0.05) by ensiling time and its interaction with treatment. All indices increased (p < 0.05) after ensiling for 30 days. Though there were no statistically significant deviations (p > 0.05) in DPPH free radical scavenging ability among the treatment groups, for the ST2 group, it was higher than for the other treatment groups. The reducing power measured by FRAP in the Control group exceeded that of the groups receiving the extract treatment (p < 0.05). Additionally, the ABTS radical scavenging activity in the ST3 was notably better than that in the other treatments, reaching a peak of 76.54 mg/g.
The chlorogenic acid concentration increased with the extract content. There was no difference (p > 0.05) among the treatment groups in the concentrations of total flavonoids and total phenols, but the total flavonoid concentration in ST3 and ST5 exceeded that in the Control. Moreover, the antioxidant indexes (ABTS, FRAP and DPPH) were correlated with chlorogenic acid content, total flavonoids and total phenols (Table 5). Among them, ABTS was significantly and positively correlated with the chlorogenic acid content, implying that the higher the concentration of chlorogenic acid, the stronger the radical scavenging activity of ABTS. Flavonoids have high antioxidant activity, due to their polyhydroxy structure [44,45]. Based on in vitro assays, the antioxidant activity of fermented dandelion flavonoids is higher than that of non-fermented dandelion flavonoids, which is due to the ability of fermented flavonoids to scavenge and reduce DPPH free radicals [40]. These results support the hypothesis that the Lonicerae flos extract acts as a chemical antioxidant and improves the antioxidant capacity of silage.

3.6. Microbial Community of Alfalfa Mixed Silage

Table 6 displays the alpha diversity of bacteria found in the alfalfa mixed silage. The sequencing data, according to Good’s coverage values, sufficiently represented the majority of bacterial communities. The values of Chao1, Simpson index, and Shannon index for the fresh materials were significantly greater (p < 0.05) than those determined after a period of 30 days, indicating that the multiplicity and richness of bacteria decreased during ensiling, especially in all ST samples. Based on Venn analysis (Figure 1), 39 bacterial OTUs were shared by the honeysuckle extract addition treatment groups and the Control. Furthermore, after 30 days of ensiling, ST1, ST2, ST3, ST4 and ST5 had 3, 4, 3, 1 and 5 specific OTUs, respectively, whereas no specific OTUs were detected in the Control. According to Polley et al. [46], the multiplicity of a microbial community decreases when there is a high abundance of the dominant bacteria. This phenomenon could be attributed to antibacterial actions that modify the competition among bacteria, thus facilitating the growth of beneficial bacterial species in the current study.
The bacterial relative abundances (at the phylum, genus and species levels) after 30 days can be seen in Figure 2, Figure 3 and Figure 4. In all treatment groups, the most abundant phyla were Firmicutes and Proteobacteria (Figure 2), aligning with earlier studies [47,48,49].
The primary genera associated with LA fermentation are Lactobacillus, Pedicoccus, Weissella and Leuconostoc [50]. Lactobacillus is crucial in silage and has beneficial properties, comprising the main strains that promote lactic acid fermentation during silage [51]. In our study, before ensiling, the dominant microorganism of the alfalfa mixture was Weissella. However, after 30 days of silage, the number of Weissella cells decreased, and the dominant microorganism was Lactobacillus among the inoculated silages (Figure 3), consistent with Weissella being an early colonizer that is replaced by acid-resistant LAB as fermentation progresses and pH decreased [52,53].
Weissella are heterofermentative LAB that cannot grow at pH < 4.5 [53]. The abundance of Lactobacillus acetotolerans, Lactobacillus coleohominis and Lactobacillus formosensis increased, whereas that of Weissella bombi and Weissella cibaria decreased after 30 days. In particular, the abundance of L. acetotolerans showed an upward trend as the content of Lonicerae flos extract increased (Figure 4). Similarly, according to Li et al. [54], residues of mint and purple perilla could play a role in enhance the enrichment of L. acetotolerans during the ensiling process.
Unlike the general beneficial microbial behavior in silage, L. acetotolerans showed higher relatively abundance in the ST4 silage based on linear discriminant analysis effect size (LEfSe) (Figure 5), which might indicate that the Lonicerae flos extract had a positive effect, promoting the accumulation of L. acetotolerans during ensiling. Perhaps this was due to high temperatures and active ingredients in the extract, although the specific reasons need to be further explored. As predicted by FAPROTAX and the BUGBASE function (Figure 6), the addition of Lonicerae flos extract can inhibit the growth of aerobic bacteria and promote the growth of facultative anaerobes (such as lactic acid bacteria), thereby further improving the fermentation quality. Therefore, the incorporation of the Lonicerae flos extract appeared to be an effective strategy for promoting the beneficial effects of ensiling, affecting both the silage environment and microbial dynamics. Perhaps, other Chinese traditional herbal drugs for relieving heat and eliminating toxic material have similar effects.

3.7. Correlation Between Bacterial Composition and Fermentation Quality and Antioxidant Capacity of Alfalfa Mixed Silage

The heatmap, which is based on Spearman’s correlation at the species level (Figure 7), illustrates the connections between the microbial community and fermentation characteristics (such as pH, AN, LA, AA, DM, WSC, NDF, ADL, HC and Ash). The fermentation of silage is a complex biological process, in which Lactobacillus plays a crucial role. This genus positively impacts lactic acid synthesis, while also influencing the pH and NH3-N levels [25,38]. Consistent with previous studies, alterations in the community of lactic acid bacteria could significantly impact of the quality of silage fermentation. A negative correlation (p < 0.05) was observed between L. acetotolerans and the pH level, whereas a positive correlation (p < 0.05) was noted between pH and the presence of Pediococcus pentosaceus, Weissella cibaria, Lactiplantibacillus plantarum and L. timonensis, suggesting that the observed decrease in pH was largely due to Lactobacillus activity. During the initial phases of ensiling, Lactococci initiate the lactic acid fermentation process [54]; however, their survival rate declines in acidic environments. Therefore, lactic acid-producing rods, such as Lactobacillus, are of primary importance for the decline in pH in the later phases of ensiling. The were positive correlations (p < 0.05) between LA content and Lactobacillus formosensis, L. namurensis and L. spicheri and positive correlations (p < 0.05) between AA content and Lactobacillus paralimentarius, Lactobacillus formosensis, L. namurensis and L. spicheri. In addition, there existed an effective negative correlation (p < 0.05) between CP and L. namurensis, L. paralimentarius, L. spicheri, L. secaliphilus, L. timonensis and Pediococcus pentosaceus and a significant positive correlation (p < 0.05) between CP and L. acetotolerans and L. pontis. In addition, there was a negative correlation between DM and L. timonensis, and a negative correlation between WSC and L. secaliphilus, implying that microbes, especially Lactobacillus, significantly affected the nutritional quality of fermentation.
FRAP was positively correlated with L. spicheri, Lactobacillus formosensis and Lactobacillus timonensis, and a negative correlation was found between FRAP and Lactobacillus acetotolerans. Furthermore, there was a positive correlation between total phenols and L. spicheri, L. namaensts, L. parallmetartus, Weissella cibaria and Pediococcus pentosaceus and a negative correlation between total phenols and L. pontis and L. acetotolerans. Finally, the total flavonoid content was positively correlated with Weissella cibaria (Figure 8). Clearly, different lactic acid bacteria affected the active components and the antioxidant capacity of silage. To gain a deeper insight into this phenomenon, it is crucial to examine the mechanisms through which these bacteria communities influence active components, as well as to investigate whether they generate metabolites that impact antioxidant defense systems in silage.
The current study provides insights into how a Lonicerae flos extract may affect alfalfa mixed silage quality and how fermentation microbes, active ingredients, and antioxidant capacity interacted. In the group treated with the Lonicerae flos extract, the content of LA, CP and crude fiber was ≥ 173.83 g·kg−1, 26.39% DM, and 15.92% DM, respectively. After 30 days of ensiling, aerobic stability was good (>360 h). The antioxidant content increased by adding the Lonicerae flos extract; although the Control group’s FRAP, Fe ion reduction/antioxidant capacity, was considerably higher than that of the treatments (p < 0.05), there were no effects on DPPH, ABTS, total flavonoids, or total phenols. Furthermore, the Lonicerae flos extract increased the abundance of L. acetotolerans, with a correlation between L. acetotolerans content and silage quality. Specifically, a higher number of L. acetotolerans as associated with increased protein content and decreased pH.
The findings of this research diverge from those attained in the silage study where Lonicerae flos was added directly [55]. To investigate the underlying reasons, we compared the active ingredients in Lonicerae flos and in the Lonicerae flos extract. The chlorogenic acid, total flavonoid, and total phenolic contents in Lonicerae flos were significantly higher than in the Lonicerae flos extract (Table 7). This suggests that, in addition to chlorogenic acid, other active compounds have a crucial role in silage. Furthermore, the content and interactions of these active ingredients may also influence silage quality.

4. Conclusions

Incorporating 0.25% Lonicerae flos extract into 80% alfalfa + 10% soybean meal + 10% DDGS mixed silage produced a very good quality silage with high protein content and antioxidant capacity. Active substances present in Lonicerae flos and its extracts, including chlorogenic acid, total flavonoids, and total phenols, are crucial for regulating the fermentation environment, improving the chemical composition of silage, and enhancing its antioxidant capacity. These findings provide novel insights into using Chinese herbal medicine additives in silage production and lay the ground for producing safe and high-quality silage. In addition, metagenomics could be used to identify the functional bacteria promoted by the Lonicerae flos extract and similar medicinal herbs that could be used to promote silage production.

Author Contributions

Conceptualization, Y.W. and Z.Z.; methodology, Y.W. and L.M.; software, Y.W. and X.C.; validation, Y.W. and Q.W.; formal analysis, Y.W.; investigation, Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

National Key Research and Development Program of China (2024YFD1301204), Special Project for the Construction of an Innovative Province in Hunan (Grant No. 2024ZYQ085) and Interdisciplinary research of Hunan Agricultural University Youth Guidance Project (2024XKJC01).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Hou, Y.; Jiang, J.-G. Origin and Concept of Medicine Food Homology and Its Application in Modern Functional Foods. Food Funct. 2013, 4, 1727. [Google Scholar] [CrossRef] [PubMed]
  2. Reddy, P.R.K.; Elghandour, M.M.M.Y.; Salem, A.Z.M.; Yasaswini, D.; Reddy, P.P.R.; Reddy, A.N.; Hyder, I. Plant Secondary Metabolites as Feed Additives in Calves for Antimicrobial Stewardship. Anim. Feed. Sci. Technol. 2020, 264, 114469. [Google Scholar] [CrossRef]
  3. Chen, L.; Bao, X.; Guo, G.; Huo, W.; Xu, Q.; Wang, C.; Liu, Q. Treatment of Alfalfa Silage with Tannin Acid at Different Levels Modulates Ensiling Characteristics, Methane Mitigation, Ruminal Fermentation Patterns and Microbiota. Anim. Feed. Sci. Technol. 2021, 278, 114997. [Google Scholar] [CrossRef]
  4. Hristov, A.N.; Oh, J.; Firkins, J.L.; Dijkstra, J.; Kebreab, E.; Waghorn, G.; Makkar, H.P.S.; Adesogan, A.T.; Yang, W.; Lee, C.; et al. SPECIAL TOPICS—Mitigation of Methane and Nitrous Oxide Emissions from Animal Operations: I. A Review of Enteric Methane Mitigation Options. J. Anim. Sci. 2013, 91, 5045–5069. [Google Scholar] [CrossRef] [PubMed]
  5. Mertenat, D.; Cero, M.D.; Vogl, C.R.; Ivemeyer, S.; Meier, B.; Maeschli, A.; Hamburger, M.; Walkenhorst, M. Ethnoveterinary Knowledge of Farmers in Bilingual Regions of Switzerland—Is There Potential to Extend Veterinary Options to Reduce Antimicrobial Use? J. Ethnopharmacol. 2020, 246, 112184. [Google Scholar] [CrossRef]
  6. Lin, Z.; Ye, L.; Li, Z.; Huang, X.; Lu, Z.; Yang, Y.; Xing, H.; Bai, J.; Ying, Z. Chinese Herb Feed Additives Improved the Growth Performance, Meat Quality, and Nutrient Digestibility Parameters of Pigs. Animal Model. Exp. Med. 2020, 3, 47–54. [Google Scholar] [CrossRef] [PubMed]
  7. Li, Y.; Li, W.; Fu, C.; Song, Y.; Fu, Q. Lonicerae Japonicae Flos and Lonicerae Flos: A Systematic Review of Ethnopharmacology, Phytochemistry and Pharmacology. Phytochem. Rev. 2020, 19, 1–61. [Google Scholar] [CrossRef] [PubMed]
  8. Park, E.; Kum, S.; Wang, C.; Park, S.Y.; Kim, B.S.; Schuller-Levis, G. Anti-Inflammatory Activity of Herbal Medicines: Inhibition of Nitric Oxide Production and Tumor Necrosis Factor-α Secretion in an Activated Macrophage-like Cell Line. Am. J. Chin. Med. 2005, 33, 415–424. [Google Scholar] [CrossRef] [PubMed]
  9. Zielinska, K.; Fabiszewska, A.; Stefanska, I. Different Aspects of Lactobacillus Inoculants on the Improvement of Quality and Safety of Alfalfa Silage. Chil. J. Agric. Res. 2015, 75, 298–306. [Google Scholar] [CrossRef]
  10. Ni, K.; Wang, X.; Lu, Y.; Guo, L.; Li, X.; Yang, F. Exploring the Silage Quality of Alfalfa Ensiled with the Residues of Astragalus and Hawthorn. Bioresour. Technol. 2020, 297, 122249. [Google Scholar] [CrossRef]
  11. Broderick, G.A.; Kang, J.H. Automated Simultaneous Determination of Ammonia and Total Amino Acids in Ruminal Fluid and In Vitro Media. J. Dairy. Sci. 1980, 63, 64–75. [Google Scholar] [CrossRef] [PubMed]
  12. Wang, Y.; Wang, C.; Zhou, W.; Yang, F.; Chen, X.; Zhang, Q. Effects of Wilting and Lactobacillus Plantarum Addition on the Fermentation Quality and Microbial Community of Moringa Oleifera Leaf Silage. Front. Microbiol. 2018, 9. [Google Scholar] [CrossRef] [PubMed]
  13. Wang, C.; He, L.; Xing, Y.; Zhou, W.; Yang, F.; Chen, X.; Zhang, Q. Fermentation Quality and Microbial Community of Alfalfa and Stylo Silage Mixed with Moringa Oleifera Leaves. Bioresour. Technol. 2019, 284, 240–247. [Google Scholar] [CrossRef]
  14. Association of Official Analytical Chemists. Official Methods of Analysis, 15th ed.; Chemical and Functional Properties of Food Saccharides; AOAC: Washington, DC, USA, 1990; Volume 1. [Google Scholar]
  15. Van Soest, P.J.; Robertson, J.B.; Lewis, B.A. Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. J. Dairy. Sci. 1991, 74, 3583–3597. [Google Scholar] [CrossRef]
  16. Licitra, G.; Hernandez, T.M.; Van Soest, P.J. Standardization of Procedures for Nitrogen Fractionation of Ruminant Feeds. Anim. Feed. Sci. Technol. 1996, 57, 347–358. [Google Scholar] [CrossRef]
  17. Guan, H.; Ran, Q.; Li, H.; Zhang, X. Succession of Microbial Communities of Corn Silage Inoculated with Heterofermentative Lactic Acid Bacteria from Ensiling to Aerobic Exposure. Fermentation 2021, 7, 258. [Google Scholar] [CrossRef]
  18. Aloo, S.-O.; Ofosu, F.-K.; Oh, D.-H. Effect of Germination on Alfalfa and Buckwheat: Phytochemical Profiling by UHPLC-ESI-QTOF-MS/MS, Bioactive Compounds, and In-Vitro Studies of Their Diabetes and Obesity-Related Functions. Antioxidants 2021, 10, 1613. [Google Scholar] [CrossRef]
  19. Mangiapelo, L.; Blasi, F.; Ianni, F.; Barola, C.; Galarini, R.; Abualzulof, G.W.; Sardella, R.; Volpi, C.; Cossignani, L. Optimization of Ultrasound-Assisted Extraction of Chlorogenic Acid from Potato Sprout Waste and Enhancement of the In Vitro Total Antioxidant Capacity. Antioxidants 2023, 12, 348. [Google Scholar] [CrossRef] [PubMed]
  20. Zeng, G.; Ran, Y.; Huang, X.; Li, Y.; Zhang, M.; Ding, H.; Ma, Y.; Ma, H.; Jin, L.; Sun, D. Optimization of Ultrasonic-Assisted Extraction of Chlorogenic Acid from Tobacco Waste. Int. J. Environ. Res. Public. Health 2022, 19, 1555. [Google Scholar] [CrossRef] [PubMed]
  21. Sytar, O.; Bośko, P.; Živčák, M.; Brestic, M.; Smetanska, I. Bioactive Phytochemicals and Antioxidant Properties of the Grains and Sprouts of Colored Wheat Genotypes. Molecules 2018, 23, 2282. [Google Scholar] [CrossRef]
  22. He, L.; Zhou, W.; Wang, C.; Yang, F.; Chen, X.; Zhang, Q. Effect of Cellulase and Lactobacillus Casei on Ensiling Characteristics, Chemical Composition, Antioxidant Activity, and Digestibility of Mulberry Leaf Silage. J. Dairy. Sci. 2019, 102, 9919–9931. [Google Scholar] [CrossRef]
  23. Mu, L.; Wang, Q.; Cao, X.; Li, H.; Zhang, Z. The Potential of Pre-Fermented Juice or Lactobacillus Inoculants to Improve the Fermentation Quality of Mixed Silage of Agro-Residue and Lucerne. Front. Microbiol. 2022, 13. [Google Scholar] [CrossRef] [PubMed]
  24. Mu, L.; Wang, Q.; Cao, X.; Zhang, Z. Effects of Fatty Acid Salts on Fermentation Characteristics, Bacterial Diversity and Aerobic Stability of Mixed Silage Prepared with Alfalfa, Rice Straw and Wheat Bran. J. Sci. Food Agric. 2022, 102, 1475–1487. [Google Scholar] [CrossRef] [PubMed]
  25. Yang, L.; Yuan, X.; Li, J.; Dong, Z.; Shao, T. Dynamics of Microbial Community and Fermentation Quality during Ensiling of Sterile and Nonsterile Alfalfa with or without Lactobacillus Plantarum Inoculant. Bioresour. Technol. 2019, 275, 280–287. [Google Scholar] [CrossRef]
  26. Zhang, Q.; Yu, Z.; Yang, H.; Na, R.S. The Effects of Stage of Growth and Additives with or without Cellulase on Fermentation and in Vitro Degradation Characteristics of Leymus Chinensis Silage. Grass Forage Sci. 2016, 71, 595–606. [Google Scholar] [CrossRef]
  27. Smith, L.H. Theoretical Carbohydrates Requirement for Alfalfa Silage Production. Agron. J. 1962, 54, 291–293. [Google Scholar] [CrossRef]
  28. Hashemzadeh-Cigari, F.; Khorvash, M.; Ghorbani, G.R.; Ghasemi, E.; Taghizadeh, A.; Kargar, S.; Yang, W.Z. Interactive Effects of Molasses by Homofermentative and Heterofermentative Inoculants on Fermentation Quality, Nitrogen Fractionation, Nutritive Value and Aerobic Stability of Wilted Alfalfa (Medicago sativa L.) Silage. J. Anim. Physiol. Anim. Nutr. 2014, 98, 290–299. [Google Scholar] [CrossRef] [PubMed]
  29. Kung, L.; Shaver, R.D.; Grant, R.J.; Schmidt, R.J. Silage Review: Interpretation of Chemical, Microbial, and Organoleptic Components of Silages. J. Dairy. Sci. 2018, 101, 4020–4033. [Google Scholar] [CrossRef] [PubMed]
  30. McDonald, P.; Henderson, N.; Heron, S. The Biochemistry of Silage, 2nd ed.; Chalcombe Publications: Devon, UK, 1991. [Google Scholar]
  31. Muck, R.E. Silage Microbiology and Its Control through Additives. Revista Brasileira de Zootecnia 2010, 39, 183–191. [Google Scholar] [CrossRef]
  32. Pahlow, G.; Muck, R.E.; Driehuis, F.; Elferink, S.J.W.H.O.; Spoelstra, S.F. Microbiology of Ensiling. In Silage Science and Technology; American Society of Agronomy: Madison, WI, USA, 2015; pp. 31–93. [Google Scholar]
  33. Wang, S.; Li, J.; Zhao, J.; Dong, Z.; Dong, D.; Shao, T. Dynamics of the Bacterial Communities and Predicted Functional Profiles in Wilted Alfalfa Silage. J. Appl. Microbiol. 2022, 132, 2613–2624. [Google Scholar] [CrossRef] [PubMed]
  34. Wang, B.; Yu, Z. Effects of Moisture Content and Additives on the Ensiling Quality and Vitamins Changes of Alfalfa Silage with or without Rain Damage. Anim. Sci. J. 2020, 91. [Google Scholar] [CrossRef]
  35. Nishino, N.; Li, Y.; Wang, C.; Parvin, S. Effects of Wilting and Molasses Addition on Fermentation and Bacterial Community in Guinea Grass Silage. Lett. Appl. Microbiol. 2012, 54, 175–181. [Google Scholar] [CrossRef] [PubMed]
  36. Agarussi, M.C.N.; Pereira, O.G.; da Silva, V.P.; Leandro, E.S.; Ribeiro, K.G.; Santos, S.A. Fermentative Profile and Lactic Acid Bacterial Dynamics in Non-Wilted and Wilted Alfalfa Silage in Tropical Conditions. Mol. Biol. Rep. 2019, 46, 451–460. [Google Scholar] [CrossRef] [PubMed]
  37. Huang, Y.; Qiu, C.; Wang, Y.; Zhang, W.; He, L. Effect of Tea Polyphenols on the Fermentation Quality, Protein Preservation, Antioxidant Capacity and Bacterial Community of Stylo Silage. Front. Microbiol. 2022, 13. [Google Scholar] [CrossRef]
  38. Chen, L.; Qu, H.; Bai, S.; Yan, L.; You, M.; Gou, W.; Li, P.; Gao, F. Effect of Wet Sea Buckthorn Pomace Utilized as an Additive on Silage Fermentation Profile and Bacterial Community Composition of Alfalfa. Bioresour. Technol. 2020, 314, 123773. [Google Scholar] [CrossRef]
  39. Miller, J.K.; Brzezinska-Slebodzinska, E.; Madsen, F.C. Oxidative Stress, Antioxidants, and Animal Function. J. Dairy. Sci. 1993, 76, 2812–2823. [Google Scholar] [CrossRef]
  40. Liu, Q.H.; Shao, T.; Bai, Y.F. The Effect of Fibrolytic Enzyme, Lactobacillus Plantarum and Two Food Antioxidants on the Fermentation Quality, Alpha-Tocopherol and Beta-Carotene of High Moisture Napier Grass Silage Ensiled at Different Temperatures. Anim. Feed. Sci. Technol. 2016, 221, 1–11. [Google Scholar] [CrossRef]
  41. Tian, X.Z.; Paengkoum, P.; Paengkoum, S.; Chumpawadee, S.; Ban, C.; Thongpea, S. Short Communication: Purple Corn (Zea maysL.) Stover Silage with Abundant Anthocyanins Transferring Anthocyanin Composition to the Milk and Increasing Antioxidant Status of Lactating Dairy Goats. J. Dairy. Sci. 2019, 102, 413–418. [Google Scholar] [CrossRef] [PubMed]
  42. Khosravi, M.; Rouzbehan, Y.; Rezaei, M.; Rezaei, J. Total Replacement of Corn Silage with Sorghum Silage Improves Milk Fatty Acid Profile and Antioxidant Capacity of Holstein Dairy Cows. J. Dairy. Sci. 2018, 101, 10953–10961. [Google Scholar] [CrossRef]
  43. Ighodaro, O.M.; Akinloye, O.A. First Line Defence Antioxidants-Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPX): Their Fundamental Role in the Entire Antioxidant Defence Grid. Alex. J. Med. 2018, 54, 287–293. [Google Scholar] [CrossRef]
  44. Treml, J.; Šmejkal, K. Flavonoids as Potent Scavengers of Hydroxyl Radicals. Compr. Rev. Food Sci. Food Saf. 2016, 15, 720–738. [Google Scholar] [CrossRef] [PubMed]
  45. Bao, T.; Xu, Y.; Gowd, V.; Zhao, J.; Xie, J.; Liang, W.; Chen, W. Systematic Study on Phytochemicals and Antioxidant Activity of Some New and Common Mulberry Cultivars in China. J. Funct. Foods 2016, 25, 537–547. [Google Scholar] [CrossRef]
  46. Wayne Polley, H.; Wilsey, B.J.; Derner, J.D. Dominant Species Constrain Effects of Species Diversity on Temporal Variability in Biomass Production of Tallgrass Prairie. Oikos 2007, 116, 2044–2052. [Google Scholar] [CrossRef]
  47. Xu, Z.; He, H.; Zhang, S.; Kong, J. Effects of Inoculants Lactobacillus Brevis and Lactobacillus Parafarraginis on the Fermentation Characteristics and Microbial Communities of Corn Stover Silage. Sci. Rep. 2017, 7, 13614. [Google Scholar] [CrossRef] [PubMed]
  48. Dong, Z.; Li, J.; Chen, L.; Wang, S.; Shao, T. Effects of Freeze-Thaw Event on Microbial Community Dynamics during Red Clover Ensiling. Front. Microbiol. 2019, 10. [Google Scholar] [CrossRef]
  49. Zi, X.; Li, M.; Chen, Y.; Lv, R.; Zhou, H.; Tang, J. Effects of Citric Acid and Lactobacillus Plantarum on Silage Quality and Bacterial Diversity of King Grass Silage. Front. Microbiol. 2021, 12. [Google Scholar] [CrossRef]
  50. Ni, K.; Zhao, J.; Zhu, B.; Su, R.; Pan, Y.; Ma, J.; Zhou, G.; Tao, Y.; Liu, X.; Zhong, J. Assessing the Fermentation Quality and Microbial Community of the Mixed Silage of Forage Soybean with Crop Corn or Sorghum. Bioresour. Technol. 2018, 265, 563–567. [Google Scholar] [CrossRef] [PubMed]
  51. Ni, K.; Wang, F.; Zhu, B.; Yang, J.; Zhou, G.; Pan, Y.; Tao, Y.; Zhong, J. Effects of Lactic Acid Bacteria and Molasses Additives on the Microbial Community and Fermentation Quality of Soybean Silage. Bioresour. Technol. 2017, 238, 706–715. [Google Scholar] [CrossRef]
  52. Graf, K.; Ulrich, A.; Idler, C.; Klocke, M. Bacterial Community Dynamics during Ensiling of Perennial Ryegrass at Two Compaction Levels Monitored by Terminal Restriction Fragment Length Polymorphism. J. Appl. Microbiol. 2016, 120, 1479–1491. [Google Scholar] [CrossRef] [PubMed]
  53. Cai, Y.; Benno, Y.; Ogawa, M.; Ohmomo, S.; Kumai, S.; Nakase, T. Influence of Lactobacillus Spp. from an Inoculant and of Weissella and Leuconostoc Spp. from Forage Crops on Silage Fermentation Fermentation. Appl. Environ. Microbiol. 1998, 64, 2982–2987. [Google Scholar] [CrossRef]
  54. Li, X.; Chen, F.; Xu, J.; Guo, L.; Xiong, Y.; Lin, Y.; Ni, K.; Yang, F. Exploring the Addition of Herbal Residues on Fermentation Quality, Bacterial Communities, and Ruminal Greenhouse Gas Emissions of Paper Mulberry Silage. Front. Microbiol. 2022, 12. [Google Scholar] [CrossRef] [PubMed]
  55. Mu, L.; Cao, X.; Wang, Y.; Wang, Q.; Zhang, Z. Inclusion of Lonicerae Flos Improved Anaerobic Fermentation and Antioxidant Activity of Mixed Silage (Agro-Residue and Alfalfa). Anim. Feed. Sci. Technol. 2024, 315, 116007. [Google Scholar] [CrossRef]
Fermentation 11 00059 g001
Figure 1. Venn analysis of each group after 30 days of silage.
Figure 1. Venn analysis of each group after 30 days of silage.
Fermentation 11 00059 g001
Fermentation 11 00059 g002
Figure 2. Relative abundance of bacterial communities after 30 days of mixed silage (phylum level).
Figure 2. Relative abundance of bacterial communities after 30 days of mixed silage (phylum level).
Fermentation 11 00059 g002
Fermentation 11 00059 g003
Figure 3. Relative abundance of bacterial communities after 30 days of mixed silage (genus level).
Figure 3. Relative abundance of bacterial communities after 30 days of mixed silage (genus level).
Fermentation 11 00059 g003
Fermentation 11 00059 g004
Figure 4. Relative abundance of bacterial communities after 30 days of mixed silage (species level).
Figure 4. Relative abundance of bacterial communities after 30 days of mixed silage (species level).
Fermentation 11 00059 g004
Fermentation 11 00059 g005
Figure 5. Analysis of microbial variations during the ensiling process was conducted utilizing the latent Dirichlet allocation effect size (LEfSe) method, which involved the Kruskal–Wallis test (p < 0.05) alongside a linear discriminant analysis (LDA) score greater than 3.0.
Figure 5. Analysis of microbial variations during the ensiling process was conducted utilizing the latent Dirichlet allocation effect size (LEfSe) method, which involved the Kruskal–Wallis test (p < 0.05) alongside a linear discriminant analysis (LDA) score greater than 3.0.
Fermentation 11 00059 g005
Fermentation 11 00059 g006
Figure 6. Prediction of bacterial community function after 30 days of silage. (a) FAPROTAX-based function analysis; (b) BUGBASE-based phenotypic difference analysis.
Figure 6. Prediction of bacterial community function after 30 days of silage. (a) FAPROTAX-based function analysis; (b) BUGBASE-based phenotypic difference analysis.
Fermentation 11 00059 g006
Fermentation 11 00059 g007
Figure 7. Heatmap showing the relationship between key bacterial species (15 most abundant species) and silage parameters. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 7. Heatmap showing the relationship between key bacterial species (15 most abundant species) and silage parameters. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fermentation 11 00059 g007
Fermentation 11 00059 g008
Figure 8. Heatmap depicting the key bacterial species (the 15 most prevalent species) associated with antioxidant activities and three active ingredients. CA, chlorogenic acid; TF, total flavonoids; TP, total phenols. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 8. Heatmap depicting the key bacterial species (the 15 most prevalent species) associated with antioxidant activities and three active ingredients. CA, chlorogenic acid; TF, total flavonoids; TP, total phenols. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fermentation 11 00059 g008
Table 1. Chemical composition of fresh silage components and Lonicerae flos extract.
Table 1. Chemical composition of fresh silage components and Lonicerae flos extract.
ItemMixtureAlfalfaSoybean MealDDGSLonicerae flos ExtractSEM
DM (g/kg FM)380.78255.29906.27910.051000.0010.88
NDF (g/kg DM)296.15329.89229.89415.98ND2.87
ADF (g/kg DM)166.36213.3590.14171.42ND1.82
ADL (g/kg DM)17.8528.35ND14.00ND0.33
Ash (g/kg DM)88.6694.8963.4166.3429.400.70
WSC (g/kg DM)88.5092.23187.4628.19588.7066.50
CP (g/kg DM)275.09199.27454.94398.8619.875.19
NH3-N (g/kg TN)201.515.052.26724.82ND12.02
Mixture, alfalfa, soybean meal, DDGS on a fresh matter basis in a 8:1:1 mixture; DDGS, distiller’s dried grains with solubles; FM, fresh matter; DM, dry matter; NDF, neutral detergent fiber; ADF, acid detergent fiber; ADL, acid detergent lignin; WSC, water-soluble sugar; CP, crude protein; NH3-N, ammonia nitrogen; TN, total nitrogen; ND, not detected.
Table 2. Effects of Lonicerae flos extract on fermentation indicators of mixed alfalfa silage ensiled for 0 and 30 days.
Table 2. Effects of Lonicerae flos extract on fermentation indicators of mixed alfalfa silage ensiled for 0 and 30 days.
Item (g/kg DM)Day(d)TreatmentSEMp Value
ControlST1ST2ST3ST4ST5TDT × D
NH3-N03.38 Bb3.57 Bab3.55 Bab3.72 Ba3.76 Ba3.67 Ba0.520.007<0.0010.236
304.46 Aa4.43 Aa4.57 Aa4.76 Aa4.49 Aa4.75 Aa
pH05.45 Aabc5.47 Aa5.47 Aab5.45 Abc5.43 Ac5.44 Ac0.50<0.001<0.001<0.001
304.51 Ba4.47 Bb4.47 Bb4.47 Bb4.45 Bc4.47 Bb
LA013.54 Bd15.13 Bb14.32 Bc15.13 Bb16.27 Ba14.94 Bb81.380.314<0.0010.171
30176.19 Aa169.10 Aa177.53 Aa178.44 Aa174.34 Aa175.66 Aa
AA01.04 Ba1.22 Ba0.90 Ba1.17 Ba1.38 Ba0.99 Ba11.640.454<0.0010.150
3024.37 Aab22.90 Ab24.58 Aa23.85 Aab24.11 Aab24.44 Aa
PA01.14 Aa0.86 Ac0.90 Abc0.72 Ad0.97 Abc0.99 Ab0.15<0.001<0.0010.082
300.88 B0.73 A0.77 B0.69 ANDND
BA00.97 A0.99 ANDNDNDND0.050.647--
30NDNDNDNDNDND
DM0380.78 Aa380.83 Aa388.03 Aa382.04 Aa382.54 Aa382.73 Aa8.510.558<0.0010.161
30368.33 Aabc366.11 Bbc365.68 Bc369.63 Bab370.90 Ba366.29 Bbc
WSC088.50 Aab89.20 Aab89.32 Aab81.53 Ac84.58 Abc90.02 Aa35.590.013<0.0010.004
3017.40 Ba16.18 Ba16.90 Ba17.40 Ba17.79 Ba17.67 Ba
CP0275.09 Ba275.61 Ba273.06 Bab272.71 Bab271.29 Bb273.39 Bab9.230.310<0.0010.001
30289.09 Ab289.92 Aab288.56 Ab293.65 Aa293.69 Aa291.84 Aab
NDF0296.15 Ab299.44 Ab299.17 Ab312.61 Aa290.63 Ab291.39 Ab10.380.007<0.0010.008
30289.77 Aa277.58 Bb278.50 Bb290.25 Ba288.69 Aa287.84 Aa
ADF0169.65 Bab181.74 Aa176.18 Aab171.91 Bab166.61 Bb164.21 Bb7.330.1730.0030.002
30182.66 Aa174.73 Ab171.24 Ab176.57 Ab183.26 Aa175.80 Ab
ADL017.85 Bab21.46 Aab16.09 Bb16.68 Ab22.32 Aa17.75 Aab4.220.0020.0630.015
3028.54 Aa21.07 Ab18.75 Ab15.96 Ab21.19 Ab18.06 Ab
HC0126.50 Ab117.71 Ab122.99 Ab140.70 Aa124.02 Ab127.19 Ab12.200.004<0.0010.589
30107.11 Ba102.85 Aa107.25 Aa113.68 Ba105.43 Ba112.04 Ba
Ash088.66 Bbc89.17 Ba88.41 Bc88.58 Bbc88.78 Bb86.02 Bd3.85<0.001<0.001<0.001
3094.80 Ad95.80 Ab96.84 Aa96.56 Aa95.31 Ac94.23 Ae
Treated samples contained varying amounts of Lonicerae flos extract, as follows: 0 (Control), 0.05% (ST1), 0.10% (ST2), 0.15% (ST3), 0.20% (ST4), and 0.25% (ST5). HC, hemicellulose. a–e Within a row, different letters indicate a remarkable difference (p < 0.05). A,B Within a column, different letters indicate remarkable differences between day 0 and day 30 (p < 0.05).
Table 3. Changes in pH and aerobic stability of silage during aerobic exposure.
Table 3. Changes in pH and aerobic stability of silage during aerobic exposure.
TreatmentpHAerobic Stability (h)
0d2d4d6d8d
Control4.51 A4.63 C4.63 B4.62 A4.61 A>360
ST14.47 B4.64 B4.64 AB4.58 B4.60 AB
ST24.47 B4.65 B4.65 A4.58 B4.61 A
ST34.47 B4.67 A4.64 AB4.58 B4.60 AB
ST44.45 C4.62 C4.61 C4.55 C4.58 B
ST54.47 B4.61 C4.63 B4.56 C4.61 A
SEM0.020.020.010.010.01
p value<0.001<0.001<0.001<0.0010.033
A–C Within a column, different letters indicate remarkable differences between day 0 and day 30 (p < 0.05).
Table 4. Dynamic changes in alfalfa mixed silage properties after 0 and 30 days.
Table 4. Dynamic changes in alfalfa mixed silage properties after 0 and 30 days.
Item Day(d)TreatmentSEMp Value
ControlST1ST2ST3ST4ST5TDT × D
DPPH (mg TE/g DM)01.92 Bb1.98 Bb1.92 Bb2.08 Bab2.09 Bab2.21 Ba0.370.431<0.0010.001
302.79 Aab2.71 Ab2.84 Aa2.75 Aab2.66 Ab2.67 Ab
FRAP (mg TE/g DM)08.91 Bb9.75 Aa8.34 Bb8.86 Ab9.64 Aa8.63 Bb1.5<0.001<0.001<0.001
3013.83 Aa10.70 Ab10.48 Ab9.70 Ab10.01 Ab10.86 Ab
ABTS (mg TE/g DM)064.35 Ba63.07 Bab62.39 Bab64.42 Ba61.69 Bb61.78 Bb5.58<0.001<0.001<0.001
3072.41 Abc70.75 Acd74.38 Aab76.54 Aa69.00 Ab74.96 Ab
Chlorogenic acid (mg/g DM)03.29 Ac3.45 Aabc3.38 Abc3.61 Aa3.53 Aab3.49 Aab0.460.005<0.0010.045
302.52 Bab2.62 Bab2.65 Ba2.68 Ba2.45 Bb2.61 Bab
Total flavonoids (mg RE/g DM)011.05 Aa10.48 Bb10.37 Bb10.38 Bb10.64 Bab10.60 Bab0.590.143<0.0010.09
3011.59 Aab11.55 Aab11.50 Aab11.74 Aab11.32 Ab11.82 Aa
Total phenols (mg GAE/g DM)04.73 Bab4.52 Bc4.62 Bbc4.86 Ba4.74 Bab4.85 Ba0.520.028<0.0010.008
305.79 Aa5.68 Aa5.85 Aa5.68 Aa5.26 Ab5.68 Aa
Different letters indicate remarkable differences between day 0 and day 30 (p < 0.05).
Table 5. Pearson correlation study of the antioxidant activity of mixed alfalfa silage and three active ingredients.
Table 5. Pearson correlation study of the antioxidant activity of mixed alfalfa silage and three active ingredients.
ItemDPPHFRAPABTSChlorogenic AcidTotal FlavonoidsTotal Phenols
DPPH1
FRAP0.1861
ABTS0.369−0.0741
Chlorogenic acid0.066−0.2330.486 *1
Total flavonoids−0.0380.1300.3550.0951
Total phenols0.4630.3030.3660.2730.4471
* p < 0.05.
Table 6. Alpha diversity of microbes of mixed raw materials and alfalfa mixed silage after 30 days of ensiling.
Table 6. Alpha diversity of microbes of mixed raw materials and alfalfa mixed silage after 30 days of ensiling.
Sample IDMControlST1ST2ST3ST4ST5SEMP
Sequences2333829338264552428727054265302564123338-
ACE87.73 a72.30 ab66.51 ab70.58 ab86.31 a54.79 b57.36 b16.170.085
Chao187.48 a66.72 b62.71 b60.84 bc67.00 b49.08 c59.75 bc12.16<0.001
Simpson0.84 a0.82 a0.65 bc0.76 ab0.61 c0.59 c0.65 bc0.11<0.001
Shannon4.08 a3.14 b2.36 cd2.91 bc2.29 d2.17 d2.47 cd0.66<0.001
Good’s Coverage1.001.001.001.001.001.001.00--
Different letters indicate remarkable differences between day 0 and day 30 (p < 0.05). M, mixed raw materials. Sequences and Good’s coverage were not analyzed for significant differences.
Table 7. Comparison of active ingredients in mountain honeysuckle (Lonicerae flos) and its extract (± SD, n = 3).
Table 7. Comparison of active ingredients in mountain honeysuckle (Lonicerae flos) and its extract (± SD, n = 3).
Component (mg/g DM)Lonicerae flosLonicerae flos Extract
Chlorogenic acid40.04 ± 0.19 a19.14 ± 0.10 b
Total flavonoids27.32 ± 0.25 a8.05 ± 0.09 b
Total phenols33.8 ± 1.21 a12.71 ± 0.34 b
a,b Differences (p < 0.05) between the native compound and its extract for all components.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wang, Y.; Mu, L.; Cao, X.; Wang, Q.; Zhang, Z. Effects of a Mountain Honeysuckle (Lonicerae flos) Extract on Fermentation Characteristics, Antioxidant Capacity and Microbial Community of Alfalfa Mixed Silage. Fermentation 2025, 11, 59. https://doi.org/10.3390/fermentation11020059

AMA Style

Wang Y, Mu L, Cao X, Wang Q, Zhang Z. Effects of a Mountain Honeysuckle (Lonicerae flos) Extract on Fermentation Characteristics, Antioxidant Capacity and Microbial Community of Alfalfa Mixed Silage. Fermentation. 2025; 11(2):59. https://doi.org/10.3390/fermentation11020059

Chicago/Turabian Style

Wang, Yating, Lin Mu, Xin Cao, Qinglan Wang, and Zhifei Zhang. 2025. "Effects of a Mountain Honeysuckle (Lonicerae flos) Extract on Fermentation Characteristics, Antioxidant Capacity and Microbial Community of Alfalfa Mixed Silage" Fermentation 11, no. 2: 59. https://doi.org/10.3390/fermentation11020059

APA Style

Wang, Y., Mu, L., Cao, X., Wang, Q., & Zhang, Z. (2025). Effects of a Mountain Honeysuckle (Lonicerae flos) Extract on Fermentation Characteristics, Antioxidant Capacity and Microbial Community of Alfalfa Mixed Silage. Fermentation, 11(2), 59. https://doi.org/10.3390/fermentation11020059

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop