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Article

The Effects of Microorganisms and Cellulases on the Quality and Microbial Diversity of Caragana korshinskii Silage

by
Jingjing Song
1,
Xiu Zhang
1,
Xuefei Wen
2,
Xingguo Tian
1,
Guoping Yang
1,3,* and
Qianru Liu
1
1
Ningxia Key Laboratory for the Development and Application of Microbial Resources in Extreme Environments, College of Biological Science and Engineering, North Minzu University, Yinchuan 750021, China
2
Ningxia Academy of Agricultural and Forestry Sciences, Yinchuan 750002, China
3
Key Laboratory of Agricultural Microbial Applied Technology, Ministry of Agriculture and Rural Affairs, Yinchuan 750021, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(3), 115; https://doi.org/10.3390/fermentation11030115
Submission received: 22 January 2025 / Revised: 14 February 2025 / Accepted: 18 February 2025 / Published: 28 February 2025
(This article belongs to the Section Industrial Fermentation)
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Abstract

:
This study aimed to explore the impacts of Lactiplantibacillus plantarum (L), Saccharomyces cerevisiae (Y), tannase-producing bacteria (D), cellulase (M), and their combined treatment (L + Y + D + M) on the sensory quality, chemical composition, silage quality, and microbial community of Caragana korshinskii. In light of the scarcity of research on the use of tannase-producing bacteria, Lactiplantibacillus plantarum, Saccharomyces cerevisiae, and cellulase as composite silage additives in the utilization of desert shrubs as feed resources, this study centers on this area and endeavors to offer a novel theoretical foundation for relevant fields. Different microorganisms and enzymes were individually added to 500 g of Caragana korshinskii, and anaerobic fermentation was carried out in an incubator at 40 °C for 14 days. The results show that compared to the control group (CK) without any additives, the sensory evaluation of all treatments improved, with the L + Y + D + M treatment being the best (p < 0.05). All treatments reduced the contents of acid detergent fiber, neutral detergent fiber, and tannin (p < 0.05), while increasing the content of crude protein and relative feeding value (p < 0.05), with the L + Y + D + M treatment showing the most significant effect (p < 0.05). Lactic acid levels significantly increased (p < 0.05) and the pH significantly decreased (p < 0.05) in all treatments, with the L + Y + D + M treatment outperforming the other treatments (p < 0.05). The L + Y + D + M treatment increased the abundance of Lactiplantibacillus plantarum and Weissella (p < 0.05), significantly reduced harmful microbial abundance and diversity (p < 0.05), and improved the microbial community structure in silage. In summary, the addition of Lactiplantibacillus plantarum (L), Saccharomyces cerevisiae (Y), tannase-producing bacteria (D), cellulase (M), and their combined treatment (L + Y + D + M) can promote the silage-related characteristics of Caragana korshinskii, with the L + Y + D + M treatment performing better compared to the other treatments. The research shows that the compound bacterial and enzymatic preparation is more effective than the single-component. The components exert synergistic effects and can effectively enhance the quality aspects of silaged Caragana korshinskii. This research provides theoretical underpinnings for the utilization of Caragana korshinskii as feedstuff. This application has the potential to alleviate feed scarcity, reduce reliance on traditional feed, enhance the stability and diversity of the feed supply system, and thereby drive the development of the animal husbandry industry.

1. Introduction

The rapid expansion of animal husbandry has led to a significant shortage of forage resources, with traditional forage and cereal straw proving inadequate to meet the escalating demand for animal feed. Consequently, the development of novel feed resources has become imperative to address the growing requirements of the livestock industry [1]. Caragana korshinskii, a leguminous shrub predominantly cultivated in water-scarce and moderately water-scarce regions of northwest China, covers an area of 8 million mu in Ningxia. Characterized by a well-developed root system and robust stress resistance, this species not only exhibits exceptional capabilities in windbreak, sand fixation, and soil–water conservation but also contains abundant protein and essential amino acids, rendering it highly valuable as a feed resource [2]. However, the high lignin and cellulose content in Caragana korshinskii, coupled with the presence of tannins and certain volatile compounds that form indigestible complexes with minerals, significantly reduces its digestibility. Furthermore, tannins, as polyphenolic polymers, can bind with proteins in Caragana korshinskii, imparting a bitter and astringent taste that diminishes palatability and restricts its application in the feed industry [3].
Fresh feed is commonly preserved by silage, which is a method of anaerobic fermentation dependent on microorganisms. Compared with hay, the loss of nutrients due to adverse weather conditions can be improved by silage. In addition, silage production is not limited by weather conditions and can be successfully carried out in hot or cold areas [4]. The process of silage making, which is complex, mainly determines the silage quality by influencing the structure of the microbial community. During the silage process, moisture content and temperature have a major impact on fermentation quality [5]. Due to the varying mechanisms of action of different additives in silage, the effects produced by applying different additives to feed also differ. Studies have shown that Lactiplantibacillus plantarum, for instance, plays a pivotal role in silage by increasing the population of LAB, thereby facilitating lactic acid fermentation [6]. Saccharomyces cerevisiae, a core component in fermentation processes, significantly contributes to elevating the protein content of fermented feed and improving its palatability [7]. Tannase-producing bacteria are instrumental in hydrolyzing tannins into glucose and gallic acid, mitigating the anti-nutritional effects of tannins and enhancing the overall quality of fermented feed [8]. Through fermentation, the microorganisms’ survival is improved, and their probiotic properties are maintained [9]. Cellulase, widely utilized as a feed additive, effectively catalyzes the degradation of cellulose within cell walls into reducing sugars, thereby enhancing the nutritional value of fermentation substrates [10]. In addition, aerobic stability serves as an indicator for assessing the aerobic deterioration of silaged Caragana korshinskii. Poor aerobic stability can result in the loss of DM and diminish the nutritional value of the silage [11].
Currently, probiotic and enzyme preparations have been demonstrated to exhibit varying degrees of efficacy in enhancing the quality of cereal straw as silage additives. However, there remains a paucity of research on the application of tannase-producing bacteria in conjunction with Lactiplantibacillus plantarum, Saccharomyces cerevisiae, and cellulase as composite silage additives, particularly in the context of utilizing desert shrubs as feed resources. We hypothesized that the fermentation quality and microbial community of silaged Caragana korshinskii could be effectively improved by incorporating mixed microorganisms and cellulases. Consequently, this study aims to investigate the silage performance of a combined bacterial and enzymatic preparation on Caragana korshinskii by systematically comparing the effects of bacterial preparations, enzyme preparations, and their combined application. The findings are expected to furnish a theoretical basis for the future exploration and refinement of bacterial and enzymatic fermentation formulations.

2. Materials and Methods

2.1. Material Preparation

The silage raw materials were collected from Yanchi County, Ningxia Hui Autonomous Region, and were crushed to 2–3 cm at the experimental base of North University for Nationalities. All the strains used in the experiment were preserved by the Key Laboratory of Microbial Resources Development and Utilization in Special Habitats of Ningxia. The cellulase was purchased from Beijing Solabio Technology Co., Ltd., Beijing, China. The viable cell count was 1 × 108 CFU/mL, and the cellulase activity was ≥10,000 U/g.
The experiment was designed with six distinct treatment groups, each administered a specific microbial–enzyme preparation, and a control group (CK) that did not receive any microbial and enzyme treatment. Each treatment, including the control, was replicated three times to ensure statistical reliability and experimental rigor. For every 500 g of caragana, different microbial preparations (Lactiplantibacillus plantarum L, Saccharomyces cerevisiae Y, tannase-producing strain D), enzyme preparations (cellulase M), and microbial–enzyme complex preparations (L + Y + D + M) were added. The cellulase dosage was 1 g/kg (FW), and the silage moisture content was maintained at 60%. The samples were sealed in polyethylene bags, vacuumed, and underwent anaerobic fermentation at 40 °C for 14 days.

2.2. Sensory Quality Assessment and Chemical Composition Analysis of Silage

In order to evaluate the sensory quality of silage feed, scoring was conducted according to the scoring criteria of the German Agricultural Association (DLG), and the final rating is as follows: excellent (20–16 points), good (15–10 points), fair (9–5 points), and poor (4–0 points). The scoring was conducted by ten individuals based on three main indicators: color (0–2 points), odor (2–14 points), and structure (0–4 points) [12].
In an effort to determine the DM content, each stored sample weighing 50 g was dried at 65 °C to the point of constant weight. It was then ground through a 0.20 mm mesh sieve for chemical composition analysis. The CP, EE, ash, and mineral content were determined using the methods of the Official Methods of Analysis of AOAC INTERNATIONAL [13]. The determination of NDF and ADF content was carried out using the method described by Van Soest et al. [14]. The method proposed by Mcdonald et al. was used to determine the content of WSC [15]. The method proposed by Gülçin Demirel Bayik et al. was used to determine the content of TN [16]. An amount of 20 g of the fermented sample was taken out and added to 180 g of ultrapure water. After shaking, it was stored in a refrigerator at 4 °C. The extract obtained by filtering through gauze and qualitative filter paper was centrifuged at 4 °C for 15 min, and the supernatant was measured using a pH meter. High-performance liquid chromatography (HPLC) was used to detect VFA, including LA, AA, PA, and BA [17].
The remaining feed after taking the required amount for the previous chemical analysis was loosened and kept uncovered during air exposure. During the aerobic stability test, the center temperature of the feed was measured with a mercury thermometer at 0, 12, 24, 48, 72, 96, 120, and 144 h [18].
Indicators such as Total Digestible Nutrients (TDNs), Dry Matter Digestibility (DDM), Dry Matter Intake (DMI), Roughage Relative Value (RFV), and Roughage Relative Quality (RFQ) were calculated with reference to methods described by Albayrak, S. [19].

2.3. Sequencing Analysis of Microbial Diversity

Microbial community total genomic DNA was extracted according to the instructions of the E.Z.N.A.® soil DNA kit (Omega Bio-tek, Norcross, GA, USA). The DNA concentration and purity were measured using a NanoDrop2000 (Thermo Scientific, Waltham, MA, USA). The PCR products were recovered using 2% agarose gel electrophoresis, and the recovery products were purified using a DNA gel recovery purification kit (PCR Clean-Up Kit, Yuhua, China). To analyze inter-group differences in Alpha diversity, version 1.46.0 of the mothur software was utilized. Principal coordinate analysis (PCA) utilizing the Bray–Curtis distance algorithm was employed to assess the similarity of microbial community structures across samples. Using the PERMANOVA non-parametric test, the significance of the differences in the structure of the microbial community among sample groups was examined [20].

2.4. Data Processing and Analysis

After preliminary processing of the experimental data using Excel software, SPSS 22.0 was employed for significance analysis and Duncan’s multiple comparison test. The results were expressed in the form of “mean ± standard deviation”, and a p-value less than 0.05 was considered to signify significant differences.

3. Results

3.1. Chemical Composition of Fresh Materials

The chemical components of raw materials can be seen in Table 1. The raw material of Caragana korshinskii contains 88.78% DM, 11.45% CP, 0.83% EE, 7.44% Ash, 55.71% ADF, 63.63% NDF, 70.13% WSC, 0.69% TN, 0.61% Ca, and 0.12% P.

3.2. Sensory Evaluation of Caragana korshinskii Silage Feed

As can be seen from Table 2, the microbial–enzyme compound preparation has a significant impact on the sensory quality of Caragana korshinskii silage. In comparison to the control group CK, the sensory quality of the L, Y, D, M, and L + Y + D + M treatment groups was substantially improved (p < 0.05). Among them, the comprehensive score of L + Y + D + M increased significantly by 10.52%. Comprehensively analyzed, the sensory evaluation grade of the L + Y + D + M treatment is good, while the sensory evaluation grades of the L, Y, D, and M treatment groups are all fair. The control group CK has the lowest comprehensive score and the sensory evaluation grade is medium. This indicates that the addition of microbial preparations, enzyme preparations, and microbial–enzyme compound preparations can significantly improve the sensory quality of Caragana korshinskii straw silage, and the addition of microbial–enzyme compound preparations has a better effect.

3.3. The Nutritional Value of Caragana korshinskii Silage Feed

As illustrated in Table 3, the addition of a bacterial–enzyme compound preparation significantly impacted the nutritional components of Caragana korshinskii. Compared to the CK, the DM and EE contents in all treatment groups increased. Notably, the DM and EE in the L + Y + D + M treatment group increased by 9.39% and 2.16%, respectively, which was statistically significant (p < 0.05). Compared to the CK, the CP content in the L, Y, and L + Y + D + M treatment groups all substantially increased (p < 0.05), with the L + Y + D + M treatment group showing a notable 2.87% increase in CP (p < 0.05). In comparison to the CK, the ash, ADF, NDF, WCS, and TN contents in all treatment groups significantly decreased (p < 0.05). Specifically, in the L + Y + D + M treatment group, the ash, ADF, NDF, WCS, and TN contents decreased by 0.67%, 9.18%, 3.91%, 36.44%, and 0.23%, respectively. Additionally, compared to the CK, the Ca content in the L, Y, and L + Y + D + M treatment groups all significantly increased (p < 0.05), with the L + Y + D + M treatment group showing a notable 0.39% increase in Ca content. This indicates that the addition of different bacterial and enzyme preparations can improve the nutritional components of silaged Caragana korshinskii to varying degrees, and the combined addition of bacterial and enzyme preparations yields better results.
Figure 1 is a visualization model diagram of the contents of various nutrients under different treatments presented by PLS−DA. From the distribution of sample points in the figure, it can be seen that the PLS−DA model can effectively distinguish different treatment groups. In particular, the L + Y + D + M group is distinctly separated from other groups, indicating that the impact of the combined treatment on the contents of nutrients is different from that of single treatments, and it has uniqueness in the composition or content changes in nutrients. The overlapping of the L, Y, D, and M groups suggests that these four single-treatment groups are relatively similar in the content characteristics of certain nutrients, and there may be some commonalities in the influencing mechanisms or outcomes on nutrients. The CK group is far away from the other treatment groups, indicating that there are essential differences in the contents of nutrients between the control group and the treatment groups, and the treatment factors significantly affect the states of nutrients.

3.4. The Relative Feeding Value of Caragana korshinskii Silage

As illustrated in Table 4, the microbial–enzyme complex preparation significantly influenced the relative feeding value of Caragana korshinskii. The TND, DDM, DMI, RFV, and RFQ of the L, Y, D, M, and L + Y + D + M treatment groups were substantially increased (p < 0.05). Specifically, the TND, DDM, DMI, RFV, and RFQ of the L + Y + D + M treatment group were significantly increased by 6.9%, 7.5%, 0.12%, 15.57%, and 15.39%, respectively, compared to the control group CK (p < 0.05). This indicates that the addition of Lactiplantibacillus plantarum, Saccharomyces cerevisiae, tannase-producing bacteria, and cellulase can significantly improve the fiber digestibility of Caragana korshinskii, thereby enhancing the feeding value of silaged Caragana korshinskii. Moreover, the combined addition of microbial and enzyme preparations yields better results.

3.5. VFA Content of Caragana korshinskii Silage

As illustrated in Table 5, the microbial–enzyme complex preparation significantly affects the fermentation quality of Caragana korshinskii. Compared to the control group CK, the pH values in the L, Y, D, M, and L + Y + D + M treatment groups were decreased dramatically (p < 0.05), with the L + Y + D + M group experiencing a notable decrease of 1.66% (p < 0.05). In comparison to the control group CK, the L, Y, and L + Y + D + M treatment groups exhibited significantly increased LA and AA contents (p < 0.05), with the L + Y + D + M group showing notable increases of 3.24% and 0.16%, respectively (p < 0.05). There were no significant differences in BA levels among the L, Y, D, M, and L + Y + D + M treatment groups compared to the control group CK (p > 0.05), and a small amount of PA was detected in all groups, including the CK group. No BA was detected in the CK, L, Y, D, M, and L + Y + D + M treatment groups. This indicates that adding the microbial–enzyme complex preparation during the fermentation process of Caragana korshinskii can increase the LA content, lower the pH, inhibit the growth of BA and PA, and ultimately promote the fermentation quality of Caragana korshinskii.

3.6. Aerobic Stability of Caragana korshinskii Silage

As illustrated in Figure 2a, the microbial–enzyme complex formulation can enhance the aerobic stability of silaged Caragana korshinskii. After aerobic exposure of silage, the pH values of the CK, L, Y, D, M, and L + Y + D + M treatment groups all showed an upward trend. However, after 24 h, 48 h, 72 h, 69 h, 120 h, and 144 h of aerobic exposure, the pH values of the L, Y, D, M, and L + Y + D + M treatment groups were notably lower compared to the CK’s (p < 0.05). Compared to the CK, after 24 h, 48 h, 72 h, 69 h, 120 h, and 144 h of aerobic exposure, the pH values of the L + Y + D + M treatment group decreased by 1.77%, 1.85%, 1.78%, 1.69%, 2%, and 1.97%, respectively. This demonstrates the supplementation of microbial agents and enzyme preparations, and microbial–enzyme complex formulations can significantly inhibit the increase in the pH value after aerobic exposure of Caragana korshinskii fermented feed, thereby improving aerobic stability, but the effect of adding microbial–enzyme complex formulations is superior. As shown in Figure 2b, compared to the CK, the aerobic stability time of the L, Y, D, M, and L + Y + D + M treatment groups was substantially increased (p < 0.05), with the L + Y + D + M treatment group increasing by 40.1%. The feed’s aerobic stability is stronger when the pH is lower and the time it takes for the center temperature to exceed 2 °C above the ambient temperature after exposure to air is longer. Conversely, it is postulated that the aerobic stability of the feed commences to decline when the central temperature of the feed surpasses the ambient temperature by 2 °C. This indicates that the addition of microbial preparations, enzyme preparations, and microbial–enzyme complex formulations can significantly increase the stability time of Caragana korshinskii fermented feed after aerobic exposure, thereby improving aerobic stability, but the effect of adding microbial–enzyme complex formulations is superior.

3.7. Microbial Species Diversity in the Silage of Caragana korshinskii

3.7.1. The Impact of Alpha Diversity and Beta Diversity on the Microbial Community of Caragana korshinskii Silage

To analyze the α diversity of microbial communities in feed, the Shannon index and Simpson index were calculated, and an analysis of the differences between these indices was conducted. The results are presented in Figure 3a,b. When observing these figures, it becomes clear that the Shannon index of the L + Y + D + M group is significantly higher than that of the CK, L, Y, D, and M groups (p < 0.05). Meanwhile, the Simpson index of the L + Y + D + M group is considerably lower than that of the CK, L, Y, D, and M groups (p < 0.05). The above results indicate that the α diversity of microbial communities in the L + Y + D + M group feed is higher than that of the other groups.
At the OTU level, PCoA was performed in accordance with the Bray–Curtis distance. The results are shown in Figure 4. The clustering between Group D and Group Y is not obvious, while the clustering between Group L + Y + D + M and the other groups (CK, L, Y, D, M) is obvious, with marked differences (p < 0.05). The above results indicate that the β diversity of the microbial community in the feed of Group L + Y + D + M is higher than that of the other groups.

3.7.2. The Influence of Microbial Community Composition on Caragana korshinskii Silage

As illustrated in Figure 5a, at the phylum level, the Firmicutes phylum was the most prevalent in untreated Caragana korshinskii, followed by the Proteobacteria, Actinobacteria, and Bacteroidetes. After ensilaging fermentation, the Firmicutes and Actinobacteria were the most dominant bacteria in each treatment group, accounting for 66.56% and 29.14% of the L + Y + D + M group, respectively. As illustrated in Figure 5b, at the genus level, the dominant genera in the L + Y + D + M group are Weissella and Lactobacillus, accounting for 42.44% and 39.21% of the total abundance, respectively. Compared to the CK group, they increased by 39.11% and 34.53%, respectively. In addition, a correlation analysis was conducted between the top ten genera in each treatment group during fermentation and the nutrients of ensiled Caragana korshinskii. The results shown in Figure 5c indicate that DM, CP, EE, and Ca in the feed are positively correlated with Weissella and Lactobacillus, while NDF, ADF, ash, WSC, and TN are negatively correlated with Weissella and Lactobacillus.

4. Discussion

4.1. Effects of Microorganisms and Enzymes on Sensory Quality of Caragana korshinskii Silage

Different types of fermentation agents have varying impacts on the sensory quality of Caragana korshinskii silage. Generally, the better the sensory quality of silage, the less the loss of dry matter and nutrients. In this research, referring to the grading method for the sensory evaluation of silage by the German Agricultural Society, the results showed that the L + Y + D + M treatment group scored the highest, indicating a good grade. The scores of the other treatment groups were all above the level of the control group, indicating a fair grade. The L + Y + D + M treatment group maintained the yellow-green color of Caragana korshinskii, with uniform texture and a wine-like aroma. This may be because the Lactiplantibacillus plantarum in the microbial–enzyme complex agent can promote the production of LA, reduce the pH, and inhibit the growth of pathogenic bacteria such as mold. The apple aroma and wine aroma produced by Saccharomyces cerevisiae can effectively enhance the flavor of the feed. Yao et al. [21] have shown that adding microbial–enzyme complex agents can effectively improve the sensory quality of corn straw, which is consistent with the results of this experiment. However, the aforementioned research indicates that most previous studies have focused on traditional forages such as corn straw. In contrast, the application of desert shrubs, such as Caragana korshinskii, in the forage field represents a relatively novel exploration. Caragana korshinskii is characterized by high contents of lignin, cellulose, and tannin. Its chemical composition is distinct, setting it apart from traditional feeds.

4.2. Effects of Microorganisms and Enzymes on Nutrients of Caragana korshinskii Silage

The content of nutrients is a crucial factor influencing the quality of silage. Research indicates that lactic acid produced after the addition of lactic acid bacteria can facilitate the formation of an acidic environment in feed, inhibit the growth of harmful bacteria, and reduce the loss of dry matter in Caragana korshinskii [22]. In this experiment, the addition of a bacterial–enzyme complex preparation significantly increased the content of DM. This suggests that the bacterial–enzyme complex preparation can enhance the recovery rate of feed dry matter and reduce the loss of nutrients. As an important energy source during silage fermentation, WSC can be rapidly utilized by Lactiplantibacillus plantarum, which may be the primary reason for the decrease in WSC content in silaged Caragana korshinskii in this study. Huang, Y [23] and others found that adding a bacterial–enzyme complex preparation to highland barley straw substantially increased the CP content and decreased the ADF and NDF content, which is consistent with the results of our study. In another study, researchers fermented potato waste using 15 types of additives (such as lactic acid bacteria and enzymes), and found that compared to untreated silage, the experimental group had reduced fiber components and improved nutrient retention [24]. Compared to the CK, the nutritional components of the treatment group L + Y + D + M with the addition of the bacterial–enzyme complex preparation were significantly better than those of the groups with the addition of bacterial or enzyme preparations alone, indicating that the addition of a bacterial–enzyme complex preparation can effectively improve the nutritional quality of Caragana korshinskii. While analogous trends of crude protein increment and fiber content decrement have been observed in prior investigations on other feeds, the extent of change exhibited by Caragana korshinskii is notably pronounced. Caragana korshinskii has a high initial content of anti-nutritional factors. This characteristic renders the microbial and enzyme complex more crucial in reducing the levels of TN, ADF, and NDF. When compared with the research on common silage, the proportional changes in these components in Caragana korshinskii were more substantial. This phenomenon can potentially be attributed to the specific interactions between the added microorganisms and enzymes and the distinctive plant cell-wall structure and chemical components inherent in Caragana korshinskii.

4.3. Effects of Microorganisms and Enzymes on Feeding Value of Caragana korshinskii Silage

In silage fermentation, apart from paying attention to the nutrient content of feed, the feeding quality is also particularly important [25]. Indicators such as TND, DDM, DMI, RFV, and RFQ are crucial for assessing the quality of feed. TND represents the nutrients obtainable from feed for maintaining growth and metabolism. A higher TND value indicates a higher CP content and lower fiber content in the feed, indicating better feed quality. DDM is key for evaluating feed quality and animal nutrient utilization efficiency. DMI is a crucial indicator for measuring animal appetite, feed palatability, satiety, satisfaction of nutritional needs, and health status. RFV is a comprehensive reflection of NDF and ADF. A higher value indicates that the feed has higher digestibility and a higher expected feed intake. RFQ comprehensively evaluates various quality characteristics of forage, including nutritional components, digestibility, palatability, anti-nutritional factors, and their impact on animal production performance and health, accurately quantifying the relative quality of forage [26]. In this experiment, the TND, DDM, DMI, RFV, and RFQ of the L + Y + D + M treatment group were significantly higher than those of the CK, indicating that the Caragana korshinskii supplemented with a microbial–enzyme complex formulation has higher nutritional value, better palatability, easier digestion, better feed quality, and higher feeding value compared to the control group. However, the existing literature on feed quality assessment predominantly concentrates on traditional feeds. In contrast, this study extends the application of these assessment indicators to the novel perspective of evaluating Caragana korshinskii. The high-fiber characteristics of Caragana korshinskii pose greater challenges to the improvement in these feeding quality indicators. Nevertheless, the microbial–enzyme complex can still remarkably enhance these indicators, suggesting the existence of a unique action mechanism.

4.4. Effects of Microorganisms and Enzymes on Fermentation Quality of Caragana korshinskii Silage

The pH and VFA are important indicators for evaluating the quality of feed silage. The lower the pH, the fewer harmful microorganisms in the feed, and the higher the feed quality. During the silage process, microorganisms can produce organic acids by consuming WSC, etc. [27]. Studies have shown that excessive PA and BA content can seriously affect animal feed intake and reduce feed quality. An increase in LA content can effectively lower the feed pH, thereby inhibiting the growth of aerobic spoilage microorganisms such as mold and the production of volatile fatty acids such as PA and BA. AA can not only enhance aerobic stability by lowering the pH but also increase the palatability of feed [28]. Findings from Wang et al.’s research [29] revealed that adding Lactiplantibacillus plantarum and cellulase can significantly increase the LA content in whole-plant silage corn and peanut vines and reduce the pH. Another study showed that, in comparison to fresh oats, the reduction in WSC content at the beginning of fermentation is vital for quickly acidifying the silage environment, preventing harmful microorganisms, and ensuring preservation. After seven days of anaerobic fermentation, the pH values of all treatments decreased to varying degrees, while the LA and AA contents also increased, which is similar to the results of this study [23]. This finding is consistent with the results of this study, which demonstrate that the production of LA reduces the pH value and inhibits the growth of harmful microorganisms. However, the initial microbial community and substrate composition in the silage of Caragana korshinskii diverge from those in common feeds. Tannins present in Caragana korshinskii are likely to exert an influence on the growth and metabolism of microorganisms during the fermentation process. Certain tannins may impede the growth of specific bacteria. However, the tannase-producing bacteria incorporated in this study are capable of counterbalancing this effect. This distinctive interaction is typically overlooked in the research on silages that do not have high tannin content.

4.5. Effects of Microorganisms and Enzymes on Aerobic Stability of Caragana korshinskii Silage

Aerobic stability is one of the important indicators for evaluating feed quality. Under aerobic exposure conditions, soluble sugars and proteins in silage are utilized as nutrient sources by rapidly proliferating aerobic microorganisms. A large amount of heat is generated by this metabolic process, causing the pH value and temperature of the silage to rise rapidly. Ultimately, this leads to the deterioration of the silage and the growth of mold [30]. The aerobic stability of silage is usually evaluated by measuring the time required for the internal temperature of the silage to rise by more than 2 °C above room temperature. The time required for the center temperature of the silage to exceed the ambient temperature by 2 °C after being exposed to air through unsealing can reflect whether the silage has good storage capacity [31]. Liang YC et al. found that inoculating lactic acid bacteria into whole-plant corn significantly improves its aerobic stability compared to other treatment groups [32]. Bai, R et al. [33] found that lactic acid bacteria additives can improve the aerobic stability of fermented feed, which is consistent with the results of this study. This indicates that adding a microbial–enzyme complex can effectively improve the aerobic stability of Caragana korshinskii silage and enhance silage quality. Nevertheless, the high fiber content in Caragana korshinskii silage may offer more complex substrates for aerobic microorganisms during the process of silage deterioration. Additionally, the interaction pattern among tannins, oxygen, and microbial metabolites may also diverge from that in common feeds. These disparities have not been comprehensively investigated in prior studies. Thus, more in-depth research is required to elucidate the specific mechanisms by which microbial and enzyme complexes enhance the aerobic stability within the silage environment of Caragana korshinskii.

4.6. Effects of Microorganisms and Enzymes on Microbial Diversity of Caragana korshinskii Silage

Silage preparation is a fermentation process involving the interaction of multiple microorganisms. The composition of the bacterial community exerts a crucial influence on the quality of silage. Studies show that once the sample coverage goes beyond 97%, it implies that the sample collection is adequate. In the current experiment, the sample coverage of all the six groups surpassed 99%. This indicates that the samples collected are representative, and the vast majority of the microorganisms in the samples have been detected. As a result, it can be inferred that the experimental results precisely mirror the microbial composition of Caragana korshinskii silage [34].
During the silage making process of Caragana korshinskii, the microbial community structures of different treatment groups varied. In this research, the α diversity and β diversity of microorganisms in the L + Y + D + M group fed with silaged Caragana korshinskii were higher than those in other groups. Principal component analysis clearly demonstrated the differences among bacterial communities, indicating that different treatment groups altered the bacterial communities and that the L + Y + D + M group showed significant differences from the other groups. The Shannon index of the L + Y + D + M group was the lowest and lower than that of the control CK, while the Simpson index was the highest and higher than that of the control CK, demonstrating a decrease in the diversity and richness of bacteria. Firmicutes and Actinobacteria were the dominant microorganisms in silaged Caragana korshinskii, which is consistent with Liu’s research results on silage oats with low water-soluble carbohydrates [35]. In order to conduct a more in-depth exploration of the bacterial community during the silage fermentation process, this experiment carried out an analysis of the alterations in the bacterial composition at the genus level. In this experiment, Lactobacillus and Weissella were the dominant genera in silage feed, playing an important role in the decrease in the pH during the later stage of silage fermentation, which has a positive impact on improving silage quality. Generally speaking, Lactobacillus is the most important bacterium in anaerobic fermentation, and Lactiplantibacillus plantarum promotes rapid fermentation to produce lactic acid, preventing further decomposition of sugars and proteins. Studies have shown that a decrease in lactic acid content and an increase in pH in feed can lead to the proliferation of some acid-sensitive microorganisms in the feed, resulting in an increase in microbial diversity [36]. You et al. believed that adding lactic acid bacteria can improve the fermentation quality of feed [37]. Weissella is a heterogeneous fermentation genus that may be beneficial in food preservation and food fermentation, as it can maintain the nutritional quality of feed under anaerobic fermentation [38], in accordance with the research outcomes of Meng et al. [39]. However, the alteration in the microbial community structure in this study is more pronounced, which might be attributable to the synergy among the components within the compound preparation. The unique microbial community structure in Caragana korshinskii silage, with Lactobacillus and Weissella as the dominant genera, is influenced by its specific chemical composition. The interactions between these dominant genera and the degradation processes of tannin, lignin, and cellulose in Caragana korshinskii may differ from those in other feeds. In the context of Caragana korshinskii, which contains high levels of anti-nutritional factors, Weissella may play a more crucial role in maintaining the stability of the anaerobic fermentation environment. This represents a novel discovery when compared with previous research on common feeds.
The results obtained from this study manifested that Lactobacillus and Weissella have a significant correlation with improving the CP and EE content of Caragana korshinskii silage, while reducing ADF, NDF, and TN. This indicates that Lactobacillus and Weissella have a positive impact on the silage quality of Caragana korshinskii. Chen et al. [40] found that the synergistic effect of probiotics and enzymes can effectively improve feed quality. Su et al. discovered that adding Lactiplantibacillus plantarum, Bacillus subtilis, and lignocellulose can significantly enhance the silage quality of fresh waxy corn stalks, which aligns with the findings of this study [41].
In conclusion, while the results of this study share certain similarities with those of traditional silage research, the distinctive characteristics of Caragana korshinskii as a feedstuff give rise to differences in the mechanism and degree of influence exerted by the starter culture. Future research ought to concentrate on these unique aspects to gain a deeper understanding of the silage process of Caragana korshinskii and optimize the utilization of microbial–enzyme complex preparations.

5. Conclusions

This study systematically explored the effects of Lactiplantibacillus plantarum, Saccharomyces cerevisiae, tannase-producing bacteria, cellulase, and their combination treatments on the quality and microbial diversity of Caragana korshinskii silage. The results show that all treatment groups can boost the sensory-related quality of Caragana korshinskii silage, reduce the contents of ADF, NDF, and TN, increase the contents of crude protein and relative feed value, increase the LA content, and reduce the pH value. Among them, the effect of the compound treatment group (L + Y + D + M) is the most significant. In terms of the microbial community, the compound treatment group increases the abundance of Lactiplantibacillus plantarum and Weissella, significantly reduces the microbial abundance and diversity, and optimizes the makeup of the microbial community in the silage. In conclusion, this study showed that the compound microbial–enzyme preparation had a significant effect in improving the quality of Caragana korshinskii silage, which broke through the limitations of traditional silage research. Future research should focus on the unique mechanisms involved in Caragana korshinskii silage and further optimize the application of microbial–enzyme complex preparations, so as to promote the efficient utilization of Caragana korshinskii in the feed field and the sustainable development of animal husbandry.

Author Contributions

Conceptualization, J.S. and G.Y.; methodology, J.S.; validation, J.S., X.W., X.T. and Q.L.; formal analysis, J.S., X.Z. and G.Y.; investigation, J.S., X.Z. and Q.L.; resources, X.Z. and G.Y.; data curation, J.S. and X.Z.; writing—original draft preparation, J.S.; writing—review and editing, X.Z. and G.Y.; visualization, J.S.; funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Science and Technology Leading Talents Project of Ningxia Hui Autonomous Region (2022GKLRLX06), Ningxia Academy of Agricultural and Forestry Sciences Science and Technology Innovation Guidance Project (NKYG-24-07), and Major Science and Technology Achievement Transformation Project of the Autonomous Region (2023CJE09047).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Visualization of the PLS−DA model for the effects of different treatments on nutrients.
Figure 1. Visualization of the PLS−DA model for the effects of different treatments on nutrients.
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Figure 2. (a) Shows the effect of microbial and enzyme complex preparations on the pH value of Caragana korshinskii strips after aerobic exposure; (b) shows the effect of microbial and enzyme complex preparations on the aerobic stability time of Caragana korshinskii. The meaning of “a–d” is that significant differences in nutrient content among different treatments are represented by different letters. The meaning of there being no significant differences among different treatments is represented by the same letters.
Figure 2. (a) Shows the effect of microbial and enzyme complex preparations on the pH value of Caragana korshinskii strips after aerobic exposure; (b) shows the effect of microbial and enzyme complex preparations on the aerobic stability time of Caragana korshinskii. The meaning of “a–d” is that significant differences in nutrient content among different treatments are represented by different letters. The meaning of there being no significant differences among different treatments is represented by the same letters.
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Figure 3. (a) Shows the effect of microbial and enzyme complex preparations on the Shannon index of Caragana korshinskii; (b) shows the effect of microbial and enzyme complex preparations on the Simpson index of Caragana korshinskii. The meaning of “a–e” is that significant differences in nutrient content among different treatments are represented by different letters. The meaning of there being no significant differences among different treatments is represented by the same letters.
Figure 3. (a) Shows the effect of microbial and enzyme complex preparations on the Shannon index of Caragana korshinskii; (b) shows the effect of microbial and enzyme complex preparations on the Simpson index of Caragana korshinskii. The meaning of “a–e” is that significant differences in nutrient content among different treatments are represented by different letters. The meaning of there being no significant differences among different treatments is represented by the same letters.
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Figure 4. Samples were analyzed by principal coordinate analysis (PCoA) using the Bray−Curtis distance metric.
Figure 4. Samples were analyzed by principal coordinate analysis (PCoA) using the Bray−Curtis distance metric.
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Fermentation 11 00115 g005aFermentation 11 00115 g005b
Figure 5. The phylum (a) and genus (b) composition of the bacterial community in ensiled Caragana korshinskii. Heatmap analysis (c) of the correlation between nutrients and bacterial genus levels in Caragana korshinskii silage. In Figure 5c, the symbol “*” indicates that p ≤ 0.05, “**” indicates that p ≤ 0.01, and “***” indicates that p ≤ 0.001.
Figure 5. The phylum (a) and genus (b) composition of the bacterial community in ensiled Caragana korshinskii. Heatmap analysis (c) of the correlation between nutrients and bacterial genus levels in Caragana korshinskii silage. In Figure 5c, the symbol “*” indicates that p ≤ 0.05, “**” indicates that p ≤ 0.01, and “***” indicates that p ≤ 0.001.
Fermentation 11 00115 g005aFermentation 11 00115 g005b
Table 1. Characteristics of Caragana korshinskii.
Table 1. Characteristics of Caragana korshinskii.
ItemContent %
DM88.78 ± 4.57
CP11.45 ± 0.17
EE0.83 ± 0.06
Ash7.44 ± 0.17
ADF55.71 ± 0.02
NDF63.63 ± 0.13
WSC70.13 ± 0.87
TN0.69 + 0.06
Ca0.61 ± 0.08
P0.12 ± 0.01
DM: dry matter; CP: crude protein; EE: ether extract; Ash: coarse ash content; ADF: acid detergent fiber; NDF: neutral detergent fiber; WSC: water-soluble carbohydrates; TN: tannin; Ca: calcium; P: phosphorus.
Table 2. Sensory evaluation of preparation of Caragana korshinskii silage feed with microorganisms and enzyme preparations.
Table 2. Sensory evaluation of preparation of Caragana korshinskii silage feed with microorganisms and enzyme preparations.
ItemTreatment Method
CKLYDML + Y + D + M
Color0.741.381.361.261.291.85
Smell5.7610.659.178.768.4313.87
Structure2.553.133.053.012.873.85
Overall rating9.0515.1613.5813.0312.5919.57
Gradesecondaryfairfairfairfairgood
CK: did not receive any microbial and enzyme treatment; L: Lactiplantibacillus plantarum was added; Y: Saccharomyces cerevisiae was added; D: tannase-producing bacteria added; M: cellulase was added; L + Y + D + M: microorganisms and cellulase were mixed and added. The scores of each group in the table are the average values of the scores given by ten individuals.
Table 3. Preparation of nutrients for Caragana korshinskii silage feed by microorganisms and enzyme preparations.
Table 3. Preparation of nutrients for Caragana korshinskii silage feed by microorganisms and enzyme preparations.
ItemTreatment Method
CKLYDML + Y + D + M
DM%88.78 ± 4.57 a96.18 ± 0.57 a95.44 ± 0.20 a95.07 ± 0.05 a95.08 ± 0.03 a98.17 ± 0.06 b
CP%11.45 ± 0.17 d12.35 ± 0.16 b12.17 ± 0.36 bc11.47 ± 0.25 d11.72 ± 0.28 cd14.32 ± 0.12 a
EE%0.83 ± 0.06 b0.93 ± 0.12 b0.87 ± 0.31 b0.88 ± 0.14 b0.85 ± 0.09 b0.11 ± 1.26 a
Ash%7.44 ± 0.17 a7.12 ± 0.02 b7.19 ± 0.02 b7.17 ± 0.07 b7.18 ± 0.03 b6.77 ± 0.10 c
ADF%55.71 ± 0.02 a51.44 ± 0.17 bc52.80 ± 0.14 b52.21 ± 0.62 b50.46 ± 0.20 c46.53 ± 1.48 d
NDF%63.63 ± 0.13 a62.1 ± 0.40 b62.07 ± 0.21 b62.09 ± 0.08 b60.97 ± 0.58 c59.66 ± 0.09 d
WSC%70.13 ± 0.87 a50.33 ± 0.13 c51.41 ± 0.29 bc52.04 ± 0.52 b52.18 ± 0.12 b33.69 ± 0.72 d
TN%0.69 + 0.06 a0.57 + 0.03 b0.57 + 0.01 b0.52 + 0.05 c0.56 + 0.02 b0.46 + 0.45 d
Ca%0.61 ± 0.08 d0.81 ± 0.03 b0.72 ± 0.02 bc0.68 ± 0.03 cd0.73 ± 0.03 bc1.00 ± 0.02 a
P%0.12 ± 0.010.12 ± 0.010.11 ± 0.010.11 ± 0.020.12 ± 0.030.14 ± 0.02
DM: dry matter; CP: crude protein; EE: ether extract; Ash: coarse ash content; ADF: acid detergent fiber; NDF: neutral detergent fiber; WSC: water-soluble carbohydrates; TN: tannin; Ca: calcium; P: phosphorus. The meaning of “a–d” is that significant differences in nutrient content among different treatments are represented by different letters. The meaning of there being no significant differences among different treatments is represented by the absence letters.
Table 4. Preparation of relative feeding value for Caragana korshinskii silage feed by microorganisms and enzyme preparations.
Table 4. Preparation of relative feeding value for Caragana korshinskii silage feed by microorganisms and enzyme preparations.
ItemTreatment Method
CKLYDML + Y + D + M
TDN%40.43 ± 0.47 a43.64 ± 0.28 b42.62 ± 0.18 c43.07 ± 0.79 b44.38 ± 0.19 b47.33 ± 0.38 d
DDM%45.50 ± 0.76 a48.83 ± 0.28 bc47.77 ± 0.33 c48.23 ± 0.87 bc49.59 ± 0.23 b52.65 ± 0.41 d
DMI%1.89 ± 0.89 a1.93 ± 0.37 b1.93 ± 0.45 b1.93 ± 0.47 b1.97 ± 0.55 a2.01 ± 0.21 c
RFV%66.52 ± 0.53 a73.15 ± 0.32 bc71.60 ± 0.69 c72.26 ± 0.31 bc75.67 ± 0.48 b82.09 ± 0.22 d
RFQ%62.00 ± 0.61 a68.57 ± 0.17 b67.00 ± 0.65 b67.67 ± 0.37 b71.02 ± 0.38 b77.39 ± 0.65 c
TDN: Total Digestible Nutrients; DDM: Digestible Dry Matter; DMI: Dry Matter Intake; RFV: Relative Feed Value; RFQ: Relative Forage Quality. The meaning of “a–d” is that significant differences in nutrient content among different treatments are represented by different letters. The meaning of there being no significant differences among different treatments is represented by the same letters.
Table 5. Preparation of VFA for Caragana korshinskii silage feed by microorganisms and enzyme preparations.
Table 5. Preparation of VFA for Caragana korshinskii silage feed by microorganisms and enzyme preparations.
ItemTreatment Method
CKLYDML + Y + D + M
LA%2.54 ± 0.14 d4.67 ± 0.27 b3.59 ± 0.09 c3.16 ± 0.08 cd3.18 ± 0.01 cd5.78 ± 0.83 a
AA%0.17 + 0.03 b0.20 + 0.04 b0.20 + 0.01 ab0.20 + 0.02 ab0.18 + 0.01 b0.23 + 0.04 a
PA%0.05 ± 0.010.04 ± 0.030.05 ± 0.040.05 ± 0.010.05 ± 0.020.04 ± 0.03
BA%NDNDNDNDNDND
pH5.85 ± 0.09 a4.33 ± 0.2 bc4.45 ± 0.15 bc4.44 ± 0.16 bc4.63 ± 0.08 b4.19 ± 0.10 c
LA: lactic acid; AA: acetic acid; PA: propionic acid; BA: butyric acid. The meaning of “a–d” is that significant differences in nutrient content among different treatments are represented by different letters. The meaning of there being no significant differences among different treatments is represented by the absence of letters. ND indicates that the substance has not been detected.
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Song, J.; Zhang, X.; Wen, X.; Tian, X.; Yang, G.; Liu, Q. The Effects of Microorganisms and Cellulases on the Quality and Microbial Diversity of Caragana korshinskii Silage. Fermentation 2025, 11, 115. https://doi.org/10.3390/fermentation11030115

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Song J, Zhang X, Wen X, Tian X, Yang G, Liu Q. The Effects of Microorganisms and Cellulases on the Quality and Microbial Diversity of Caragana korshinskii Silage. Fermentation. 2025; 11(3):115. https://doi.org/10.3390/fermentation11030115

Chicago/Turabian Style

Song, Jingjing, Xiu Zhang, Xuefei Wen, Xingguo Tian, Guoping Yang, and Qianru Liu. 2025. "The Effects of Microorganisms and Cellulases on the Quality and Microbial Diversity of Caragana korshinskii Silage" Fermentation 11, no. 3: 115. https://doi.org/10.3390/fermentation11030115

APA Style

Song, J., Zhang, X., Wen, X., Tian, X., Yang, G., & Liu, Q. (2025). The Effects of Microorganisms and Cellulases on the Quality and Microbial Diversity of Caragana korshinskii Silage. Fermentation, 11(3), 115. https://doi.org/10.3390/fermentation11030115

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