Open AccessArticle

Exploring the Fermentation Products, Microbiology Communities, and Metabolites of Big-Bale Alfalfa Silage Prepared with/without Molasses and Lactobacillus rhamnosus

Baiyila Wu

^1,†,

Tong Ren

^1,†,

Changqing Li

²,

Songyan Wu

³,

Xue Cao

¹,

Hua Mei

¹,

Tiemei Wu

¹,

Mei Yong

¹,

Manlin Wei

^1,*

and

Chao Wang

^2,*

College of Animal Science and Technology, Inner Mongolia Minzu University, Tongliao 028000, China

Inner Mongolia Academy of Agriculture and Animal Husbandry Science, Hohhot 010031, China

Naiman Banner Animal Disease Prevention and Control Center, Tongliao 028000, China

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Agriculture 2024, 14(9), 1560; https://doi.org/10.3390/agriculture14091560

Submission received: 8 August 2024 / Revised: 3 September 2024 / Accepted: 6 September 2024 / Published: 9 September 2024

(This article belongs to the Section Farm Animal Production)

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Abstract

The influence of molasses (M) and Lactobacillus rhamnosus (LR) on fermentation products, microbial communities, and metabolites in big-bale alfalfa silage was investigated. Alfalfa (Medicago sativa L.) was harvested at the third growth stage during the flowering stage in the experimental field of Linhui Grass Company from Tongliao City, Inner Mongolia. An alfalfa sample without additives was used as a control (C). M (20 g/kg) and LR (10⁶ cfu/g) were added either alone or in combination. Alfalfa was fermented for 7, 14, and 56 d. Lactic acid content in the M, LR, and MLR groups increased, whereas the pH value and butyric acid, 2,3-butanediol, and ethanol contents decreased compared to those of C group after 7, 14, and 56 d of fermentation. A two-way analysis of variance (ANOVA) was performed to estimate the results. The LR group exhibited increased Lactobacillus abundance, whereas the M and MLR groups showed increased Weissella abundance compared to the C group. The relative contents of amino acids (tyrosine, isoleucine, threonine, arginine, valine, and citrulline) in the M and MLR groups were higher than those in the C group. During fermentation, the M, LR, and MLR groups showed decreased phenylalanine, isoleucine, and ferulic acid contents. Amino acids such as isoleucine and L-aspartic acid were positively correlated with Lactobacillus but negatively correlated with Weissella. In conclusion, combining high-throughput sequencing and liquid chromatography–mass spectrometry during anaerobic alfalfa fermentation can reveal new microbial community compositions and metabolite profiles, supporting the application of M, LR, and MLR as feed fermentation agents.

Keywords:

alfalfa silage; high-throughput sequencing; microbial communities; metabolites; fermentation product

1. Introduction

Feed is fundamental to animal husbandry. Given China’s large population, limited cultivated land, and insufficient food supply, developing the feed industry is essential for advancing animal husbandry in China [1]. Alfalfa, a high-quality forage crop, is widely grown and fed in China because it has strong growth adaptability and high protein content [2,3]. The main methods of utilizing alfalfa in China are to make hay or silage [4]. Alfalfa planting areas in China are mainly distributed in semi-arid and arid areas of the north [5]. Rain and mildew often affect hay production because of the high heat and humidity. In addition, the nutritional loss caused by mechanical extrusion during hay baling can be as high as 20%, directly affecting farmers.

Ensiling preserves nutrients and prolongs forage preservation time [6], making it an efficient method of forage processing, utilization, and storage. Producing high-quality silage from alfalfa is challenging without wilting or adding additives due to its high buffering capacity and low water-soluble carbohydrate (WSC) content [7]. Molasses is a nutritional additive that provides a fermentation substrate for silage. Adding molasses accelerates the fermentation process. Molasses improves the fermentation quality and in vitro dry matter digestibility of soybean, king grass, and corn silage [8,9,10].

Microorganisms naturally present on fresh forage initiate silage fermentation [11]. Naturally occurring lactic acid bacteria (LAB) are the main probiotics in silage fermentation, quickly producing lactic acid to lower pH, thereby inhibiting harmful microbes and reducing nutrient loss [12]. Good fermentation quality and long-term preservation of forage can be ensured when the number of LAB reaches at least 10⁵ cfu/g in fresh material [13]. However, when LAB levels in fresh material are below 10⁵ cfu/g, adding exogenous lactic acid bacteria is necessary to obtain high-quality silage. We isolated several LAB strains with good fermentation quality from alfalfa, Caragana korshinskii, and Leymus chinensis silage. We found that Lactobacillus plantarum and Lactobacillus buchneri in Leymus chinensis silage promoted fermentation while inhibiting yeast and mold growth [14]. The metabolites produced by LAB during feed fermentation regulate the unique flavor [15]. Metabolites, including flavonoids, lipids, amino acids, and carbohydrates, are inextricably linked with microorganisms during anaerobic fermentation of alfalfa [16,17]. However, information regarding the characteristics of Lactobacillus rhamnosus isolated from big-bale alfalfa silage and its effects on the metabolite and microbial community structure of big-bale alfalfa silage is lacking. Hence, we isolated an L. rhamnosus strain from big-bale alfalfa silage using phenotyping and 16S ribosomal RNA (rRNA) gene sequencing. It is necessary to study the changes in metabolites and microbes during fermentation when L. rhamnosus is added to big-bale alfalfa silage. A comprehensive understanding of the microorganisms involved in silage metabolism and their correlation can provide rich information for the feed industry, including silage quality assessment, nutritional value, fermentation effects, and safety.

Although some studies have examined the fermentation quality and bacterial communities in laboratory-scale alfalfa silage [18,19], few have evaluated the metabolites and microbial community structure of big-bale alfalfa silage during fermentation. Herein, the effects of molasses (M), L. rhamnosus (LR), and molasses + L. rhamnosus (MLR) were examined on the metabolites and microbial community structure of big-bale alfalfa silage during fermentation, using high-throughput sequencing technology combined with liquid chromatography–mass spectrometry (LC-MS).

2. Materials and Methods

2.1. Silage Preparation

On 25 July 2023, we collected the third growth stage of alfalfa (Medicago sativa L.) in the flowering stage in the experimental field of Linhui Grass Company (120°25′—121°50′ E, 43°18′—44°9′ N) from Tongliao City, Inner Mongolia. The sample was later prepared in segments with the length of 20–30 mm with a cutting machine for obtaining the needed dry matter content at about 327 g/kg. Molasses was a byproduct of the sugarcane industry. The density of molasses was 45%, while the sugarcane content of molasses was 48%. L. rhamnosus was isolated from big-bale alfalfa silage (Inner Mongolia, China). L. rhamnosus cultures were prepared through 72 h of anaerobic incubation within the MRS broth under 30 °C and diluted using sterile physiological saline. Thereafter, 10⁶ cfu/g LR and 20 g/kg M were introduced separately or together. An additive-free alfalfa sample served as the control. Samples with and without additives were tightly wrapped in ten layers of plastic film to ensure anaerobic fermentation of the big bales. The bales (1.1 m diameter × 1.1 m length and average weight 700 kg) were stored outside in two layers. Every treatment was carried out thrice. Additionally, alfalfa fermentation was lasted for 1, 2, and 8 weeks.

2.2. Fermentation Characteristics, Microbial Counts, and Chemical Composition Analyses

To be specific, 30 g fresh sample was added into 270 mL sterile water within a blender for 1 min of homogenization, and the homogenate was later passed through the 0.22 μm membrane for preparing sample water extracts to measure pH, alcohol level, and short-chain fatty acid contents. The extract pH was determined with the glass electrode pH meter (ARH-10; Xima, Suzhou, China), whereas high-performance liquid chromatography (HPLC) was conducted to measure fermentation product [6].

We conducted diverse dilutions on the clean bench for quantifying diverse microbes. Mold and yeast were quantified using the potato dextrose agar (02-032, Aoboxing Ltd., Beijing, China; pH 3.4), whereas enterobacteria were counted with violet red bile agar (11C02, Luqiao Ltd., Jinan, China). We counted lactic acid bacteria (LAB) using de Man, Rogosa, and Sharpe agar (02-293, Aoboxing Ltd., China). The number of colonies was measured through calculating live microorganism number within samples.

Dry matter (DM) contents within big-bale alfalfa silage were analyzed after 48 h of drying under 65 °C, followed by pulverizing within the 1 mm sieve shredder (SUS304, Juchuang Ltd., Jinan, China). Crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), and water-soluble carbohydrates (WSC) were determined according to descriptions by Van Soest et al. [20] and Wu and Nishino [21].

2.3. Microbial Sequencing Analysis

Next, 20 g big-bale alfalfa silage was introduced into the 80 mL sterile phosphate-buffered saline (pH 7.4) with 2 h oscillation at 120 rpm using an electronic oscillator, and the resultant sample was passed through the two-layer gauze. Later, the resulting filtrate was centrifuged at 4 °C and 13,000× g for a 10 min period. Pellets were maintained on dry ice when supernatants were discarded. Biomarker Technologies (Beijing, China) was responsible for conducting PCR amplification, extracting DNA, as well as conducting metagenomic sequencing. Additionally, data were processed, and Illumina MiSeq sequencing was carried out. UPARSE version 7.1 was employed to cluster operational taxonomic units (OTUs) at the similarity threshold of 97% [22]. When chimeric sequences were identified and eliminated, we adopted Ribosomal Database Project classifier (version 2.2) for classifying and analyzing feature OTU sequences (at a confidence level of 0.7) by using 16S rRNA databases (with Silva v138) [23]. We then imported raw sequence data into NCBI Sequence Read Archive (accession number, PRJNA1112470).

2.4. Metabolite Analysis by LC-MS

Samples were collected after 7, 14, and 56 d fermentation and immediately frozen in a minus 80 °C freezer. As previously described by Zou et al. [15], 50 mg of sample was placed in a 1.5 mL EP tube, and 20 μL of L-2-chlorophenylalanine (0.3 mg/mL) and 600 μL of methanol/water (v/v = 4:1) were added and vortexed for 30 s. The samples were placed in a frozen tissue grinder (CTM-192, Shandong Laiyin Optoelectronic Technology Co., Ltd., Weifang, Shandong, China) along with 100 mg glass beads and were ground at 60 Hz and −10 °C for 2 min. They were then subjected to lower-temperature ultrasound for 10 min and then centrifuged at 13,000 rpm and 4 °C for 10 min. Next, the 200 μL supernatant was removed and put into the LC-MS injection vial. Quality control samples were prepared using a 10 μL aliquot of all samples, and the remaining samples were examined by LC-MS.

The LC-MS system for metabolite analysis consisted of a liquid chromatograph (ACQUITY UPLC I-Class PLUS) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and a mass spectrometer (Xevo G2-XS QTof). The chromatographic column was the ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm i.d., 1.8 μm) (Waters Inc., Milford, MA, USA) purchased from the Waters (Milford, MA, USA). The liquid chromatograph and mass spectrometer settings followed previously established conditions [24,25].

2.5. Statistical Analyses

John’s Macintosh Project software (version 16; SAS Institute, Tokyo, Japan) was applied to analyze how addition and fermentation time influenced fermentation characteristics, microbial counts, and chemical composition of big-bale alfalfa silage. Moreover, Results were estimated by two-way analysis of variance (ANOVA), and one-way ANOVA was conducted to predict the influence of addition, whereas Tukey’s test was carried out for multiple comparisons. p < 0.05 represented statistical significance. Microbial diversity, metabolite analysis, and correlations between microbial communities and metabolite were examined with a public web-based platform (https://www.biocloud.net/) (accessed on 16 December 2015) of Biomarker.

3. Result and Discussion

3.1. Chemical and Microbial Composition of Pre-Ensiling Alfalfa

The chemical and microbial compositions of the pre-ensiled alfalfa are shown in Table 1. The DM content in alfalfa was 327 g/kg of fresh material, which was relatively high compared with the fresh material of alfalfa [26]. CP content was observed to be 164.42 g/kg of DM, which was higher than the 153 g/kg of DM found by Wu and Nishino [21]. Different varieties may lead to differences of protein content in alfalfa silage. WSC content in fresh material is an important substrate to silage fermentation, and WSC content of DM of more than 6% can ensure the desired fermentation. In this study, the WSC level of pre-ensiled alfalfa was 45.78 g/kg DM, indicating that it was difficult to ensure fermentation quality in alfalfa silage without additives. LAB, yeast, and enterobacteria counts in fresh alfalfa were 4.16, 5.17, and 5.28 log cfu/g of FM, respectively. When the number of LAB attached to the surface of fresh materials is less than 10⁵ cfu/g, silage often fails to be stored [27]. Therefore, the addition of molasses and LAB to alfalfa silage not only improves the nutritional value but also enhances the fermentation quality during anaerobic fermentation.

3.2. Roles of Molasses and Lactobacillus rhamnosus in Fermentation Products and Microbial Counts of Big-Bale Alfalfa Silage

After 7 days of fermentation, the lactic acid content in the control silage was 8.28 g/kg DM, whereas the acetic acid content was 4.87 g/kg DM (Table 2). With prolonged ensiling time, the concentrations of lactic acid and acetic acid increased in the control silage. The 1,2-propanediol and ethanol levels in control silage were 1.43 and 1.65 g/kg of DM, respectively. It was reported that 1,2-propanediol and ethanol can be produced from sugar by heterofermentative LAB species including Lactobacillus hilgardii, Lactobacillus buchneri, and Lactobacillus brevis [28]. The LAB count and lactic acid contents in the LR group were increased, while the pH value and butyric acid, 2,3-butanediol, and ethanol contents were decreased compared with that of the C group. The lactic acid and acetic acid contents in the M and MLR groups increased, while the pH value and butyric acid, 2,3-butanediol, and ethanol contents decreased after 7, 14, and 56 d of fermentation. Hashemzadeh-Cigari et al. [29] discovered that the addition of molasses to alfalfa silage improved the acetic acid content and inhibited mold and yeast growth after 90 days of storage. The enterobacterial and yeast counts in group C were higher than those in the other groups during anaerobic fermentation.

3.3. Roles of Molasses and Lactobacillus rhamnosus in Bacterial Diversity and Bacterial Community Structure of Big-Bale Alfalfa Silage

A Venn plot displays the number of common and unique genera in the different silage samples (Figure 1A). All the samples included 65 common and 528 exclusive genera, which were classified as follows: fresh material, 37; 7-day fermentation, 108; 14-day fermentation, 103; and 56-day fermentation, 280. Figure 1B illustrates the spatial distribution and principal component analysis of the microbial community in the alfalfa silage treated with molasses and L. rhamnosus. The microbial community in the raw materials was separated from that in the alfalfa silage. In our previous study, Leymus chinensis silage treated with or without Lactiplantibacillus plantarum or Lentilactobacillus buchneri exhibited similar results [14]. LR7 and MLR14 groups crossed and deviated from other groups, indicating that the microbial flora structure of alfalfa silage was similar between the two groups, even though different additives were added to the alfalfa silage.

Figure 2A shows the bacterial communities at the phylum level before and after alfalfa silage fermentation. Before alfalfa silage fermentation, the abundance of Firmicutes was the highest, followed by Proteobacteria, Cyanobacteria, and Bacteroidota. Alfalfa silage produced in farm bunker silos has shown different results because different environmental factors and alfalfa varieties affect the microorganisms attached to the surface of alfalfa [2]. The main phyla are Firmicutes and Proteobacteria during the fermentation process, accounting for more than 88% of the bacterial composition. Firmicutes and Proteobacteria could be found in other silages, such as whole-crop corn, Italian ryegrass, Moringa oleifera leaf, and Leymus chinensis silage [14,30,31].

At the genus level, high-abundance microorganisms such as Weissella, Lactobacillus, Enterococcus, Enterobacter, Lactococcus, and Pediococcus were detected during alfalfa fermentation (Figure 2B). Weissella, Pediococcus, Lactobacillus, and Enterococcus were the dominant genera in the M, LR, and MLR groups, which led to the special sensory characteristics and strong flavor of alfalfa silage. Consistent with the findings of Zou et al. [15], Pantoea was the main genus before alfalfa fermentation, whereas Lactobacillus and Weissella were the main genera after 7, 14, and 56 d, respectively (Figure 2C,D). After 7 d of fermentation, the poor fermentation quality of the control sample may be related to Enterobacter, which accounted for 6.1% of the total microorganisms. Enterobacter is a bacterium belonging to the Enterobacteriaceae family and is found in animal feed and the environment. After 7, 14, and 56 d of ensiling, the LR group exhibited an increased Lactobacillus abundance compared than that in the C group. The increase in Lactobacillus may have increased the lactic acid content in the LR group. This result is consistent with previous findings on fermented vegetables [32]. Moreover, Lactobacillus converts fermentation substrates into lactic and acetic acids, and then, this acidic environment can inhibit the growth of Enterobacter and Pseudomonas, thereby allowing LAB to have a benefit in competition. After 7, 14, and 56 d of ensiling, the M and MLR groups exhibited increased Weissella abundance compared to the C group. Weissella, a heterofermentative LAB, can ferment WSC into acetic acid during fermentation. This could explain why the acetic acid content increased in the M and MLR groups.

3.4. Roles of Molasses and Lactobacillus rhamnosus in Metabolites of Big-Bale Alfalfa Silage

During silage fermentation, microorganisms decompose nutrients such as sugars and proteins in raw materials, producing various compounds, such as amino acids, lactic acid, acetic acid, alcohols, and esters. Each of these compounds has a unique odor and taste; when mixed together, they form a unique silage flavor. In this study, non-targeted LC-MS and one-way statistical methods were used to reveal the diversity of metabolites in big-bale alfalfa silage treated with molasses and Lactobacillus rhamnosus. A total of 980 metabolites were identified using positive and negative ionization modes, including 148 flavonoids, 150 lipids, 354 amino acids, 149 carbohydrates, 65 alkaloids and derivatives, 100 nucleosides and analogs, and 14 lignans and neolignans. A total of 962 (308 upregulated and 265 downregulated, Figure 3A), 974 (301 upregulated and 277 downregulated, Figure 3B), and 951 (308 upregulated and 265 downregulated, Figure 3C) metabolites were detected in the LR, M, and MLR groups, respectively, compared to the control silage after 56 days of fermentation. This indicates that the addition of molasses and Lactobacillus rhamnosus improved the fermentation quality and nutritional value of alfalfa silage.

Heatmap analysis revealed the relative contents of some differential metabolites in the different treatment groups (Figure 4). Amino acids play a crucial role in silage, as they not only affect the growth and metabolism of microorganisms but also determine the quality and flavor of silage [33]. The relative contents of amino acids (tyrosine, isoleucine, threonine, arginine, valine, and citrulline) in the M and MLR groups increased compared with that of the C group, which is different from the report of Zou et al. [15]. According to the previous study, microbes can produce acetic acid through the degradation of tyrosine, isoleucine, and valine [34], which might explain the high content of acetic acid in the M and MLR groups. Proteins in anaerobic fermentation are decomposed by microorganisms into free amino acids and peptides; however, excessive acid and microbial activity in anaerobic environments also leads to an increase in other substances and amino acids. Umami amino acids included alanine, phenylalanine, glutamic acid, and aspartic acid, which are the main free amino acids in silage [16]. The rich free amino acids in silage not only provide unique flavor for silage but also provide various nutrients for ruminants, with high nutritional value. Among the aroma substances in many fermented food, essential amino acids such as valine and phenylalanine play a crucial role in the human body and are important precursors for synthesizing other proteins and bioactive substances [35]. The M, LR, and MLR groups decreased the content of phenylalanine, isoleucine, and ferulic acid during fermentation process. Phenylalanine and tyrosine are closely related to bitterness; [36] thus, the addition of molasses and Lactobacillus rhamnosus improved the taste of alfalfa silage. Ferulic acid has anti-aging, antioxidant, antibacterial, anti-cancer, and cardiovascular disease prevention effects and maintains bone health [37]. Ferulic acid has been detected in corn stalk silage [38]. The main microorganism responsible for synthesizing ferulic acid is yeast [39]. Under suitable conditions, other microorganisms can also synthesize ferulic acid [39]. The yeast counts in alfalfa fermentation were much lower, which may be related to the dominance of lactic acid bacteria in anaerobic fermentation. Therefore, reducing the yeast that produces ferulic acid leads to a decrease in ferulic acid content. Li et al. [38] found that Lactobacillus plantarum can be used in the industrial production of ferulic acid. In this study, M and MLR groups decreased the abundance of Lactobacillus, resulting in decreased ferulic acid production, which may explain the reduced amount of ferulic acid detected during long-term fermentation. In conclusion, intake of beneficial amino acids from silage may improve ruminant health. Moreover, their good flavor is one of the reasons why ruminants choose fermented feeds.

Water-soluble carbohydrates such as glucose and fructose were lower in the M and MLR groups than in the C group. This might be because the more abundant Weissella in these groups converted fermentable carbohydrates into lactic acid, acetic acid, and ethanol. Similar results were reported in Broussonetia papyrifera leaves treated with Lactobacillus plantarum by He et al. [33]. Malonic acid is a fermentation product of harmful microorganisms such as Enterobacter, Clostridium, Pseudomonus, and Pantoea [40] and was more frequently detected in the C group than in the M, LR, and MLR groups. This may be because the addition of molasses and Lactobacillus rhamnosus inhibits the activity of these microorganisms. Succinic acid enhances the unique flavor of alfalfa silage and has many benefits for ruminant health. Succinic acid was detected in alfalfa silage, and the succinic acid content was increased by the addition of molasses and Lactobacillus rhamnosus. These results suggest that relevant animal health products can be obtained from alfalfa silage.

3.5. The Relationship between the Microbial Community and Metabolites

The Mantel test was used to study the possible relationship between metabolites and microbial communities in alfalfa silage. After 56 d of ensiling, the metabolites in the alfalfa silage were associated with microbes, particularly Lactobacillus, Weissella, Enterococcus, and Enterobacter (Figure 5). According to the correlation results, amino acids such as isoleucine and L-aspartic acid were positively correlated (p < 0.05) with Lactobacillus but negatively correlated with Weissella (p < 0.05). In this study, the addition of Lactobacillus rhamnosus decreased the pH index, and Lactobacillus dominated the fermentation process, which may have played an important role in increasing the amino acid content so that the alfalfa silage had a higher nutritional value and flavor. Sucrose, syringic acid, ferulic acid, and itaconic acid were positively correlated (p < 0.05) with Lactobacillus but negatively correlated with Weissella (p < 0.05). Stevioside exhibited a negative correlation (p < 0.05) with Enterococcus and Enterobacter. In conclusion, Weissella and Lactobacillus had the greatest impact on the metabolic products of alfalfa silage, which is in accordance with previous study [41]. This research provides new information on the relationship between microorganisms and metabolites in alfalfa silage; however, biochemical and molecular biological methods were used to reveal the mechanisms behind this relationship.

4. Conclusions

This study investigated the fermentation products, microbial community structure, metabolites, and their correlations during the fermentation of alfalfa silage with or without M, LR, and MLR. The lactic acid content in the M, LR, and MLR groups increased, while the pH value and butyric acid, 2,3-butanediol, and ethanol contents decreased compared to those in the C group after 7, 14, and 56 days of fermentation. The LR group exhibited increased Lactobacillus abundance, whereas the M and MLR groups exhibited increased Weissella abundance compared to the C group. The relative contents of amino acids (tyrosine, isoleucine, threonine, arginine, valine, and citrulline) in the M and MLR groups were higher than those in the C group. The M, LR, and MLR groups showed decreased phenylalanine, isoleucine, and ferulic acid content during the fermentation process. Amino acids such as isoleucine and L-aspartic acid were positively correlated (p < 0.05) with Lactobacillus but negatively correlated with Weissella (p < 0.05). In summary, the results reveal the influence of M, LR, and MLR on fermentation products, microbial communities, and metabolites. Moreover, this result provides a theoretical basis for improving the fermentation quality of big-bale alfalfa silage through microbial control.

Author Contributions

Conceptualization, B.W. and T.R.; methodology, S.W.; formal analysis, X.C.; investigation, H.M.; data curation, T.W.; writing—original draft preparation, B.W.; writing—review and editing, C.W. and M.W.; visualization, M.Y.; supervision, C.L.; project administration, B.W.; funding acquisition, B.W., C.W. and M.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the China Agriculture Research System (grant number CARS-38); the Natural Science Foundation of Inner Mongolia (grant number 2024QN03072, 2023QN03028, 2022MS03072).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. (A) Venn diagram of taxonomic units (OTUs) before and after big-bale alfalfa fermentation. (B) Spatial distribution—principal component analysis of operational taxonomic unit before and after big-bale alfalfa fermentation. FM, fresh material; C, control group; M, molasses treatment group; LR, L. rhamnosus treatment group; MLR, molasses and L. rhamnosus treatment group; Numbers after C, M, LR, and MLR denote fermentation times. The numbers represent OTUs.

Figure 2. Microbial community at the phylum level (A), genus level (B), and error bar plot (C,D) before and after big-bale alfalfa fermentation. FM, fresh material; C, control group; M, molasses treatment group; LR, L. rhamnosus treatment group; MLR, molasses and L. rhamnosus treatment group; Numbers after C, M, LR, and MLR denote fermentation times.The dot of different colors in the Figure 2C represent the Lactobacillus abundance. The dot of different colors in the Figure 2D represent the Weissella abundance.

Figure 3. Metabolites in big-bale alfalfa silage. (A) Volcano plot analysis of C vs. LR. (B) Volcano plot analysis of C vs. M. (C) Volcano plot analysis of C vs. MLR. C, control group; M, molasses treatment group; LR, L. rhamnosus treatment group; MLR, molasses and L. rhamnosus treatment group.

Figure 4. Heatmap of differentially accumulated metabolites in big-bale alfalfa silage. C, control group; M, molasses treatment group; LR, L. rhamnosus treatment group; MLR, molasses and L. rhamnosus treatment group; Numbers after C, M, LR, and MLR denote three repetitions.

Figure 5. Mantel test analyses of microbial communities and metabolites in big-bale alfalfa silage.

Table 1. Chemical composition and microbial composition of pre-ensiling alfalfa.

	Alfalfa
Dry matter (g/kg)	327 ± 2.52
pH	5.62 ± 0.27
Crude protein (g/kg DM)	164.42 ± 1.15
Water-soluble carbohydrate (g/kg DM)	45.78 ± 0.76
Acid detergent fiber (g/kg DM)	287.62 ± 6.32
Neutral detergent fiber (g/kg DM)	356.34 ± 8.92
Lactic acid bacteria (log cfu/g)	4.16 ± 0.82
Enterobacteria (log cfu/g)	5.28 ± 0.67
Yeasts (log cfu/g)	5.17 ± 0.53

Data are mean of duplicate analyses.

Table 2. Fermentation products and microbial counts of big-bale alfalfa silage treated with/without molasses and Lactobacillus rhamnosus.

	7 Days					14 Days					56 Days					2-Way ANOVA
	C	M	LR	MLR	SE	C	M	LR	MLR	SE	C	M	LR	MLR	SE	I	S	I × S
Dry matter (g/kg)	328	332	326	335	1.53	332	329	336	322	1.28	329	332	335	338	1.37	NS	NS	NS
pH	5.37A	4.46C	5.03B	4.12C	0.08	5.13a	4.28c	4.87b	4.08c	0.15	4.75x	4.13z	4.37y	3.95z	0.23	**	**	*
Lactic acid (g/kg DM)	8.28C	12.56B	15.76A	16.86A	0.87	13.27c	17.48b	19.49a	20.78a	1.26	21.76z	24.87y	28.93x	31.78x	2.86	**	**	**
Acetic acid (g/kg DM)	4.87C	6.68A	5.23B	7.27A	0.56	6.98b	8.94a	7.23b	9.14a	0.85	8.91y	13.65x	9.32y	14.98x	0.96	**	**	NS
Butyric acid (g/kg DM)	2.23A	0.43C	0.76B	0.58C	0.33	3.21a	0.86c	1.35b	0.67c	0.37	4.12x	1.45yz	1.68y	1.23z	0.48	**	NS	**
1,2-Propanediol (g/kg DM)	1.43	1.34	1.58	1.51	0.27	1.89	1.79	1.87	1.72	0.23	2.13	2.08	2.25	2.28	0.32	NS	NS	NS
2,3-Butanediod (g/kg DM)	1.12A	0.44B	0.59B	0.43B	0.31	1.96a	0.83c	1.16b	0.78c	0.32	2.34x	0.95z	1.21y	0.78z	0.34	**	**	**
Ethanol (g/kg DM)	1.65A	0.87B	0.95B	0.78B	0.22	2.13a	1.32b	1.46b	1.27b	0.41	2.76x	1.14yz	1.36y	0.97z	0.37	**	**	**
Lactic acid bacteria (log cfu/g)	6.45C	7.35B	7.89A	7.76A	0.79	6.28b	7.65a	7.89a	7.83a	0.93	6.38x	7.32y	7.48y	7.82y	0.56	**	**	**
Enterobacteria (log cfu/g)	6.87A	4.38C	4.67B	4.27C	0.83	5.38a	4.28b	4.35b	4.18b	0.76	5.53x	4.29y	4.15y	4.38y	0.87	**	NS	**
Yeasts (log cfu/g)	6.49A	4.76C	5.13B	4.38C	0.76	5.75a	4.36b	4.27b	4.07b	0.84	5.82x	4.34y	4.22y	4.36y	0.73	**	**	**

Means from triplicate samples; values for identical fermentation times with varying following letters (A–C, a–c, and x–z) differ significantly (p < 0.05). I and S represent the effects of additions and fermentation times, and I × S refers to the interaction of additions and fermentation times. C, control group; M, molasses treatment group; LR, L. rhamnosus treatment group; MLR, molasses and L. rhamnosus treatment group. **, p < 0.01; *, p < 0.05; NS, p ≥ 0.05.

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Wu, B.; Ren, T.; Li, C.; Wu, S.; Cao, X.; Mei, H.; Wu, T.; Yong, M.; Wei, M.; Wang, C. Exploring the Fermentation Products, Microbiology Communities, and Metabolites of Big-Bale Alfalfa Silage Prepared with/without Molasses and Lactobacillus rhamnosus. Agriculture 2024, 14, 1560. https://doi.org/10.3390/agriculture14091560

AMA Style

Wu B, Ren T, Li C, Wu S, Cao X, Mei H, Wu T, Yong M, Wei M, Wang C. Exploring the Fermentation Products, Microbiology Communities, and Metabolites of Big-Bale Alfalfa Silage Prepared with/without Molasses and Lactobacillus rhamnosus. Agriculture. 2024; 14(9):1560. https://doi.org/10.3390/agriculture14091560

Chicago/Turabian Style

Wu, Baiyila, Tong Ren, Changqing Li, Songyan Wu, Xue Cao, Hua Mei, Tiemei Wu, Mei Yong, Manlin Wei, and Chao Wang. 2024. "Exploring the Fermentation Products, Microbiology Communities, and Metabolites of Big-Bale Alfalfa Silage Prepared with/without Molasses and Lactobacillus rhamnosus" Agriculture 14, no. 9: 1560. https://doi.org/10.3390/agriculture14091560

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