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Article

Fibrolytic Enzymes and Lactic Acid Bacteria Improve the Ensiling Characteristics of Ramie and Elephant Grass Mixed Silage

by
Mengwei Li
1,†,
Faiz-ul Hassan
2,†,
Muhammad Uzair Akhtar
2,
Lijuan Peng
1,
Fang Xie
1,
Qian Deng
1,
Huapei Zhong
1,
Kelong Wei
1 and
Chengjian Yang
1,*
1
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Nanning 530001, China
2
Faculty of Animal Production and Technology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur 63100, Pakistan
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2024, 14(10), 1746; https://doi.org/10.3390/agriculture14101746
Submission received: 6 August 2024 / Revised: 26 September 2024 / Accepted: 1 October 2024 / Published: 3 October 2024
(This article belongs to the Special Issue Silage Preparation, Processing and Efficient Utilization)
Versions Notes

Abstract

:
Understanding the effects of bacteria and enzyme addition on mixed crop silage is imperative for dairy producers to make informed decisions. The current study evaluated the chemical changes in silage prepared from different ramie and elephant grass ratios (30:70, 50:50, 70:30, and 100:0) in response to bacteria (0, 100, 200, and 300 mg/kg) and enzyme addition (0, 10, 20, and 30 mg/kg) in a complete randomized design. The results indicated that the proportion of ramie in silage (p < 0.01), level of bacteria (p < 0.05), and level of enzyme added (p = 0.05) affected the CP, fiber, volatile fatty acids, and lactic acid contents and pH of silage. By comprehensive analysis, low lignin content and pH of silage with high lactic acid content was observed with a 30% ramie proportion. High CP and lactic acid contents with low ADF, lignin, and pH values were observed with the addition of bacteria (p < 0.05). High lactic acid with low ADF content was observed with the addition of enzyme in silage (p ≤ 0.05). The optimum quality of silage was observed when the ramie, bacteria, and enzymes were added at the levels of 30%, 200 mg/kg, and 20 mg/kg, respectively, in ramie and elephant grass mixed silage.

1. Introduction

Ruminant nutritionists are in a continuous search for quality forages for sustainable livestock production. The efficient utilization of forages not only enhances animal productivity but also reduces production costs. In this context, the use of mixed crop silage has been observed to improve silage quality and the stability of fermentation conditions [1,2,3], exhibiting a positive impact on the quantity and quality of milk [4].
Ramie (Boehmeria nivea L. Gaud) is a non-conventional fiber crop that is used for livestock feeding due to its important nutritional profile. It is produced in various countries including China, India, Indonesia, Brazil, Japan, Korea, and Vietnam [5]. The crude protein (CP), lysine, and calcium contents of ramie are 22, 1.02, and 4.07%, respectively, on a dry matter (DM) basis, which indicates its potential as a suitable alternative to alfalfa [6]. It was reported that replacing alfalfa hay with ramie up to a certain limit in the diet of black goats did not compromise their growth performance [7]. A study conducted by Chen et al. [8] indicated that the quality of mixed silage was improved when a proportion of 20:80 rice straw and fresh ramie was used. Moreover, mixed silage prepared by the addition of ramie beneficially modulated the ruminal pH and increased DM digestibility in Chinese Holstein cows [4]. Elephant grass or Napier (Pennisetum purpureum Schum.) is a perennial, tall erect grass. This grass is extensively cultivated in different regions and is primarily grown in cut and carry systems in various dairy and feedlot operations. Napier grass holds many advantages such as high DM content (25–40%), excellent yield per cultivated area, water use efficiency, and high resistance to drought, in addition to the presence of reasonable soluble carbohydrates, making it convenient to preserve as silage for successful ruminant production systems [9,10].
Inoculation with bacterial strains and the addition of fibrolytic enzymes has been introduced as another strategy to improve ensiling characteristics, ultimately leading to the improved digestibility of silage [11,12,13]. Various inoculants and enzymes are being used for this purpose to improve silage quality by stimulating the fermentation process and improving preservation. A combination of organic acids, lactic acid bacteria, and fibrolytic enzymes may promote the silage quality of different grasses [14,15]. It has been reported that the lignin content of silage is reduced by a combination of lactic acid bacteria and cellulase [16]. The oxidation of lignin-related compounds is catalyzed by laccase [17], especially when combined with lactic acid bacteria [18]. Bacterial inoculants may ensure a decrease in pH and reduce proteolysis [19], while fibrolytic enzymes may increase carbohydrate availability separately and synergistically with bacterial combinations [20,21].
Most previous studies investigated the effects of bacteria and enzymes on silage prepared from different but single crops [22,23]. The literature is lacking on the response of mixed crop silage to the inclusion of bacterial inoculants and enzymes and their optimum dose levels, especially for ramie- and elephant grass-based silage. We hypothesized that the addition of bacteria and enzymes in mixed crop silage would improve the ensiling characteristics of the silage. The present study aimed to evaluate the effects of lactic acid-producing bacteria and fibrolytic enzymes and find the optimum combinations of ramie, enzyme, and bacteria levels in terms of mixed crop silage quality prepared from ramie and elephant grass.

2. Materials and Methods

2.1. Experimental Materials

Three cultures of bacterial species (viz: Lactobacillus plantarum, Lactobacillus brucelli, and Pediococcus pentosaceus, known as Lactiplantibacillus plantarum, Apilactobacillus, and Lactobacillus pentosaceus according to Zheng et al. [24]) were used (ratio of 1:1:1) as fermentation strains in this study. The total number of viable bacteria was 1.35 × 1010, 1.39 × 1010, and 1.38 × 1010 cfu/g for Lactobacillus plantarum, Lactobacillus brucelli, and Pediococcus pentosaceus, respectively. These strains were sourced in freeze-dried form and purchased from Shandong Zhongke Jiayi Bio-engineering Co. Ltd., Weifang, China, for the execution of the current study. Three fibrolytic enzymes, i.e., cellulase, xylanase, and laccase (ratio of 7:7:1) with enzymatic activities of 50,000, 50,000, and 1000 units/g, respectively, were taken from the Solarbio Science and Technology Co., Beijing, China.

2.2. Experimental Design

The experiment was designed in a 4 × 4 × 4 factorial arrangement in a completely randomized design with five replicates per treatment group. Details of the experimental arrangement are presented in Table 1. Fermentation conditions varied, four ratios of ramie to elephant grass in silage (30:70, 50:50, 70:30, and 100:0), four levels of bacterial addition (0, 100, 200, and 300 mg/kg), and four levels of compound enzyme addition (0, 10, 20, and 30 mg/kg). Chopped elephant grass was thoroughly mixed with ramie according to the treatment proportions before packing it in plastic bags. The composite bacteria (LAB) and fibrolytic enzymes used in the experiment were dissolved in ultrapure water and uniformly sprayed on the silage. The spraying volume for each group was balanced by water simultaneously. An equivalent quantity of ultrapure water was sprayed on the samples in the control group. After uniform mixing, the plastic bags were vacuum-packed and stored at a temperature of around 20–22 °C. The samples were then ensiled for 45 days.

2.3. Sampling and Chemical Analysis

The fresh samples of ramie and elephant grass were obtained by harvesting from 5 cm above ground level and chopped to the length of 1–2 cm at the Guangxi Buffalo Research Institute pasture (Nanning, China). A cutter mill (Puverisette 15, Fritsch GmbH, Idar-Oberstein, Germany) was used for this purpose. The plantation was sown in the month of April and harvesting was performed in September. The average temperature during the growing phase was 28.7 °C and relative humidity was 79.5%. The planting land was hilly terrain with an average rainfall of 1304.2 mm. These samples (500 g each) were stored in silage bags (150 mm × 250 mm) and sealed using a vacuum sealer (DZ500, Gzrifu Co. Ltd., Guangzhou, China) after the respective treatments. Raw material and silage samples were analyzed in duplicate for DM, CP, neutral detergent fiber (NDF), acid detergent fiber (ADF), crude ash, and lignin contents following AOAC [25]. Samples were analyzed for DM at 65 °C in a forced oven (LABO-250, STIK Co. Ltd., Shanghai, China) until a constant weight was achieved [26]. The dried samples were ground to pass a 1 mm screen size (FE220, Beijing Zhongxingweiye Instrument Co. Ltd., Beijing, China) for the analysis of DM (method 934.01) and ash (method 942.05) contents. The CP content was analyzed through the total nitrogen (method 954.01) using an automatic Kjeldahl apparatus (Kjeltec-8400, FOSS, Hillerød, Denmark). The NDF, ADF, and lignin (method 973.18) contents were analyzed using the fiber analyzer (ANKOM Technology Corp., Macedon, NY, USA) without ash correction or treatment with amylase and sodium sulfite, following the method described by Van Soest et al. [27]. To determine the pH value after fermentation, each sample (20 g) was stirred with distilled water (180 mL) in a blender for one minute and filtered with four layers of cheesecloth [28]. The filtrates were used to measure the pH of samples using a pH meter (Hanna Instruments Italia Sril, Padova, Italy). The gross energy of the samples was determined using a bomb calorimeter (PARR-6400, automatic bomb calorimeter, Moline, Illinois, USA), as mentioned previously [29]. Metabolizable energy was calculated using gross energy and NDF content following Sung and Kim [30]. The lactic acid content was estimated by the enzymatic method using commercially available kits (A019-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Briefly, 0.02 mL H2O, 1 mL enzyme working solution, and 0.2 mL chromogen were added to a blank tube, while 0.02 mL standard, 1 mL enzyme working solution, and 0.2 mL chromogen were added to the test tube. After 10 min of incubation in the water bath at 37 °C, 2 mL terminal solution was added to each tube and the absorbance was noted at 530 nm [31]. The following formula was used for the calculations:
Lactic acid content (mmol/L) = (Test tube OD − blank tube OD)/(standard tube OD − blank tube OD) × 3 mmol/L

2.4. Statistical Analysis

The obtained data were subjected to the generalized linear model using SAS 9.4 software using a completely randomized design with a 4 × 4 × 4 factorial arrangement of the experimental treatments. Means were compared using Tukey’s multiple comparison test. Range analysis and the comprehensive balance method were used to analyze the total score of silage fermentation quality. Ki was the factor I (I = 1, 2, 3, 4); the greater the Ki, the better the level was. The range was the difference between the maximum and minimum of Ki. The greater the range, the greater the influence of this factor on silage quality. Data were expressed as means and results were declared significant at p < 0.05.

3. Results

For the nutrient composition of feed samples on an air-dried basis, the DM, CP, NDF, ADF, and ash contents were 21.9, 15.6, 42.4, 39.6, and 12.5% in ramie and 17.2, 6.89, 75.7, 48.5, and 8.89% in elephant grass, respectively.

3.1. Changes in Chemical Composition of Silage under Different Fermentation Conditions

The effects of different fermentation conditions on the chemical composition of silage are presented in Table 2. The proportion of ramie in the silage (p = 0.001) and the level of enzyme addition (p = 0.001) not only had independent effects but also interacted with each other and the bacteria level to affect the DM content. Interactions of R × B, E × B, and R × B × E were observed for the DM content of the silage (p < 0.05). Although silage CP content responded to the proportion of ramie in the silage (p = 0.046) and the level of bacterial inoculation (p < 0.001), with no direct effect of the level of enzyme addition (p = 0.28), interactions were observed for R × B, R × E, E × B, and R × B × E (p < 0.05). Silage energy content changed with the proportion of ramie (p = 0.011), level of bacteria added (p = 0.012), and interactions of R × E and E × B (p < 0.05). Ash content did not change by the enzyme and bacteria levels; however, effects of ramie proportion and E × B were observed (p < 0.05). The NDF content responded to bacteria level and R × B, R × E, and R × B × E interactions (p < 0.05). Silage ADF and lignin contents changed with ramie proportion, bacteria and enzyme levels, and their interactions (p ≤ 0.05).

3.2. Best Combinations of Fermentation Conditions for Silage Nutrient Composition

Ideal combinations of ramie proportion with bacteria and enzyme levels for different silage composition parameters are presented in Table 3. The factor ranking regarding the effect on DM content was ramie > enzyme > bacteria, where a ramie proportion of 100 with a bacteria level of 0 and a 20 mg/kg enzyme level was the best combination. The ranking of factors affecting the CP content was bacteria > ramie > enzyme, where a bacteria level of 200 mg/kg with a ramie proportion of 100 and a 20 mg/kg enzyme level was the ideal combination for CP content. The factor ranking regarding the effect on energy content was ramie > bacteria > enzyme, where a ramie proportion of 70 with 0 mg/kg bacteria and enzyme levels was the ideal combination for energy content. The factor ranking regarding the effect on ash content was ramie > bacteria > enzyme, where a ramie proportion of 0 with a 100 mg/kg bacteria level and a 20 mg/kg enzyme level was the best combination. The ranking of factors affecting NDF content was bacteria > enzyme > ramie, where a bacteria level of 300 mg/kg with a 0 mg/kg enzyme level and a ramie proportion of 50 was the best combination. The factor ranking regarding the effect on ADF content was ramie > enzyme > bacteria, where a ramie proportion of 50 with a 20 mg/kg enzyme level and a 200 mg/kg bacteria level was the ideal combination. The factor ranking regarding the effect on ash content was ramie > bacteria > enzyme, where a ramie proportion of 0 with a 100 mg/kg bacteria level and a 20 mg/kg enzyme level was the best combination. The factor ranking regarding the effect on lignin content was ramie > bacteria > enzyme, where a ramie proportion of 0 with 200 mg/kg bacteria and 30 mg/kg enzyme levels was the ideal combination for lignin content.

3.3. Changes in the pH, Lactic Acid, Ammonia-N, and VFAs of Silage under Different Fermentation Conditions

The effects of different fermentation conditions on the pH, lactic acid, ammonia-N, and VFAs of silage are presented in Table 4. Silage pH responded to ramie proportion, bacteria level, and R × E and B × E interactions (p < 0.05). The proportion of ramie, bacteria level, enzyme level, and their interactions affected the lactic acid and ammonia-N contents of the silage (p < 0.05). Acetic acid and butyric acid changed with ramie proportion, enzyme level, their interactions with each other, and bacteria level (p < 0.05). Propionic acid content was affected by ramie proportion and the B × E interaction (p < 0.05).

3.4. Best Combinations of Fermentation Conditions for Silage Quality Parameters

Ideal combinations of ramie proportion with bacteria and enzyme levels for the pH, lactic acid, ammonia-N, and VFAs of mixed silage are presented in Table 5. The factor ranking regarding the effect on pH and lactic acid was ramie > bacteria > enzyme, where a ramie proportion of 0 with 200 mg/kg bacteria and 30 mg/kg enzyme levels was the ideal combination for silage pH and lactic acid content. The factor ranking regarding the effect on ammonia-N content was bacteria > ramie > enzyme, where a ramie proportion of 100 with a 200 mg/kg bacteria level and a 20 mg/kg enzyme level was the best combination. The factor ranking regarding the effect on acetic acid content was ramie > enzyme > bacteria, where a ramie proportion of 0 with a 30 mg/kg enzyme level and a bacteria level of 0 was the best combination. The factor ranking regarding the effect on propionic acid was ramie > enzyme > bacteria, where a ramie proportion of 100 with 0 mg/kg bacteria and 0 mg/kg enzyme levels was the ideal combination for propionic acid content. The factor ranking regarding the effect on butyric acid content was ramie > enzyme > bacteria, where a ramie proportion of 0 with a 20 mg/kg enzyme level and a 100 mg/kg bacteria level was the ideal combination for butyric acid.

3.5. Comprehensive Score Analysis of Silage Quality

The results of the comprehensive analysis are presented in Table 6. Silage pH, lactic acid, ash, lignin, acetic acid, and butyric acid were primarily affected by the ramie proportion in the silage, and a proportion of 30 was best for these indices. The DM, CP, and propionic acid contents were high with a ramie proportion of 100. A bacterial dose of 200 mg/kg was best for silage CP, ADF, lignin, pH, lactic acid, and ammonia-N. A bacterial dose of 100 mg/kg was ideal for ash and butyric acid contents. An enzyme level of 20 mg/kg was ideal for silage DM, CP, ash, ADF, and butyric acid, while 30 mg/kg enzyme was ideal for lignin, pH, lactic acid, and acetic acid. The best silage combination was achieved with a ramie proportion of 30%, compound bacteria of 200 mg/kg, and compound enzyme of 20 mg/kg.
Table 4. Analysis of pH, lactic acid, ammonia-N, and volatile fatty acid of mixed silage under different fermentation conditions 1.
Table 4. Analysis of pH, lactic acid, ammonia-N, and volatile fatty acid of mixed silage under different fermentation conditions 1.
ItemRamie-to-Elephant-Grass RatioBacteria, mg/kgEnzyme, mg/kgSEMp-Value 2
01002003000102030RBER × BR × EB × ER × B × E
pH30:704.953.973.664.444.954.443.973.660.2170.0010.0170.4490.1920.0240.0040.136
50:504.884.874.544.604.604.884.544.87
70:304.824.484.564.604.484.564.824.60
100:04.984.844.924.864.924.844.864.98
Lactic acid, g/Kg DM30:702.3712.519.02.202.372.2012.519.00.451<0.001<0.001<0.001<0.001<0.001<0.001<0.001
50:503.743.193.292.702.703.743.293.19
70:302.612.582.832.452.582.832.612.45
100:05.023.614.073.694.073.613.695.02
NH3-N, g/Kg total-N30:704.372.333.043.694.373.692.333.040.212<0.001<0.001<0.001<0.001<0.001<0.001<0.001
50:503.463.311.421.451.453.461.423.31
70:303.082.451.142.422.451.143.082.42
100:02.541.960.902.170.901.962.172.54
Acetic acid, g/Kg DM30:7010.94.954.459.4810.99.484.954.451.515<0.0010.0980.0190.0060.017<0.0010.026
50:5012.415.717.818.318.312.417.815.7
70:3016.617.819.518.817.819.516.618.8
100:015.720.821.319.421.320.819.415.7
Propionic acid, g/Kg DM30:702.961.672.322.442.962.441.672.320.372<0.0010.7880.3220.2010.353<0.0010.189
50:504.174.483.694.024.024.173.694.48
70:304.894.564.354.194.564.354.894.19
100:06.667.257.596.987.597.256.986.66
Butyric acid, g/Kg DM30:703.311.775.293.453.313.451.775.290.441<0.0010.1070.026<0.001<0.001<0.001<0.001
50:506.885.715.284.654.656.885.285.71
70:305.365.314.415.245.314.415.365.24
100:04.905.175.324.975.325.174.974.90
1 DM, dry matter; SEM, standard error of the mean. 2 R = proportion of ramie in silage, B = level of bacterial inoculation, E = level of enzyme added.
Table 5. The optimal combination of ramie-to-elephant-grass ratio, enzyme level, and bacteria inoculation level for the pH, lactic acid, ammonia-N, and volatile fatty acid of mixed silage.
Table 5. The optimal combination of ramie-to-elephant-grass ratio, enzyme level, and bacteria inoculation level for the pH, lactic acid, ammonia-N, and volatile fatty acid of mixed silage.
Item 1Fermentation Conditions 2Optimum Combination 3
ABC
pHK1 44.254.914.74Factor ranking A > B > C
A1B3C4
K24.724.524.67
K34.614.424.54
K44.904.614.53
Range0.650.490.18
Lactic acid, g/Kg DMK19.033.442.93Factor ranking A > B > C
A1B3C4
K23.235.563.07
K32.627.315.62
K44.152.717.43
Range6.414.604.50
NH3-N, g/Kg total-NK13.463.362.29Factor ranking B > A > C
B3A4C3
K22.412.542.59
K32.271.632.26
K41.872.452.83
Range1.591.730.57
Acetic acid, g/Kg DMK17.4413.8917.06Factor ranking A > C > B
A1C4B1
K216.0614.4815.25
K318.1515.7514.43
K419.2016.3513.67
Range11.762.463.39
Propionic acid, g/Kg DMK12.354.674.78Factor ranking A > C > B
A4C1B1
K24.094.344.41
K34.504.494.17
K47.124.274.41
Range4.770.400.61
Butyric acid, g/Kg DMK13.465.114.65Factor ranking A > C > B
A1C3B2
K25.634.454.97
K35.085.074.31
K45.094.565.28
Range2.170.660.97
1 DM, dry matter. 2 Fermentation conditions represented by the ratio of ramie to elephant grass in silage (30:70, 50:50, 70:30, and 100:0; Factor A), level of bacteria addition (0, 100, 200, and 300 mg/kg; Factor B), and level of enzyme addition (0, 10, 20, and 30 mg/kg; Factor C). 3 A1, A2, A3, and A4 represent the proportion of ramie in the silage (30, 50, 70, and 100, respectively); B1, B2, B3, and B4 represent the level of added bacteria (0, 100, 200, and 300 mg/kg, respectively); and C1, C2, C3, and C4 represent the level of added enzyme (0, 10, 20, and 30 mg/kg, respectively). 4 Ki represents the average value at that specific level in an increasing order from K1 to K4.
Table 6. Levels of ramie, bacteria, and enzyme added in mixed silage corresponding to the highest values for silage quality parameters, 1 estimated through the comprehensive balance method.
Table 6. Levels of ramie, bacteria, and enzyme added in mixed silage corresponding to the highest values for silage quality parameters, 1 estimated through the comprehensive balance method.
ItemsDMCPMetabolizable EnergyAshNDFADFLigninpHLactic AcidNH3-NAcetic AcidPropionic AcidButyric Acid
Ramie proportion (%)100100703050503030301003010030
Compound bacteria dose (mg/kg)0200010030020020020020020000100
Compound enzyme dose (mg/kg)20200200203030302030020
1 DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber.

4. Discussion

The nutrient profiles of ramie and elephant grass were close to those of previous reports [32]. However, the DM content of ramie was relatively lower than previously reported values, which might be attributable to the maturity level and cutting stage of the plant. Ensiling is a practical approach to preserve forages with high moisture content using lactic acid bacteria, which anaerobically convert carbohydrates into organic acids to inhibit the activity of spoilage organisms by decreasing the pH [22]. It is reported that the water-soluble carbohydrate content of ramie is low, which causes low-quality silage after fermentation [33]. This results from the lower multiplication and dominance of lactic acid bacteria [34]. Therefore, a combination of different forages could provide sufficient fermentation conditions and promote lactic acid bacteria. Several studies have reported that bacterial inoculation and enzyme addition affect carbohydrate content and decrease the fiber content of silage [35,36,37]. Therefore, it might be inferred that enzymes degrade the fiber content to improve the sugar content, promoting lactic acid bacteria, which ultimately decreases the pH, leading to the inhibition of non-lactic acid bacterial activity and plant enzymes for proteolysis [20]. Numerous previous studies have reported that enzymes and/or bacteria could improve fermentation by decreasing the pH and improving the aerobic stability of silage [38,39,40].
The proportion of ramie in the silage and the level of bacterial inoculation affected the DM content of the silage in the present study. Although no independent effect of the enzyme was detected, two-way and three-way interactions indicated that the enzyme levels interacted with both ramie proportion and bacteria levels to affect the DM content. The optimal combination for DM was observed when the ramie proportion was 100%, bacterial quantity was 0 mg/kg, and enzyme addition was 20 mg/kg. However, the DM content of silage was greater with 100% ramie due to the relatively higher initial DM of ramie compared with elephant grass, and no additional beneficial effect of bacterial quantity and enzyme addition was observed on DM content. Previously, improved ruminal degradability of silage or no change in the ruminal degradability of silage with bacteria and enzyme addition was reported. These differences in silage quality were attributed to the roughages used, as bacteria and enzymes affect the degradability of different roughages differently [20,21]. In the present study, the response of silage characteristics was also changed with the proportion of ramie in the mixed silage. Interestingly, different interactions observed in this study indicated that the introduction of enzymes and bacteria at the same time could be more beneficial through their synergistic effects than individual treatments with bacteria and enzymes [20]. For CP content, the optimum bacterial quantity was 200 mg/kg when the proportion of ramie silage was 100% and enzyme addition was 20 mg/kg. During the ensiling, changes in protein fractions may occur due to extensive proteolysis by plant and microbial proteases [8]. Similar to previous reports, the addition of bacteria might have reduced the proteolysis of high-moisture silage through decreased pH [8]. Better protein preservation in different silages is reported with bacterial addition compared with controls in the literature [41]. Another interesting finding of the current study observed through the three-way interaction of R × B × E revealed that the ability of bacteria to preserve the protein content of silage is influenced by silage enzyme addition and forage type. According to the best of our knowledge, this is the first study reporting the effects of forage type × bacteria × enzyme interaction on mixed crop silage quality, and further studies using different ratios of various forage sources are required for further understand the interaction of bacteria and enzymes with forage type and level. For energy, the optimum proportion of ramie silage was 70% when no bacteria and enzymes were added. Moreover, for crude ash, the optimum proportion of ramie silage was 30% when the quantity of bacterial culture and enzyme was 100 and 20 mg/kg, respectively. Crude ash and ADF contents were significantly decreased with enzyme addition, which is in agreement with the previous studies [42,43]. However, it is noteworthy that the role of forage type and bacteria level also needs to be considered for optimum ADF content when adding enzymes to mixed crop silage, as indicated by their interaction in the present study. For ADF, optimum conditions were achieved by 50% ramie silage with 200 mg/kg bacterial quantity and 20 mg/kg enzyme addition, whereas, for NDF, optimum conditions were achieved by 50% ramie silage with 300 mg/kg bacterial quantity, without any enzyme addition. For lignin content, the optimum proportion of ramie silage was 30% when the quantity of bacteria and enzyme was 200 mg/kg and 30 mg/kg, respectively. The enzymatic hydrolysis of plants is primarily controlled by the diffusion movements of enzyme solutions to the specific target spots of plant cells. Differences in moisture content may result in interference with the transportation system and promote the enzymatic hydrolysis of silages with optimum moisture contents, leading to a greater release of soluble carbohydrates during the fermentation process [43]. Furthermore, pH is also a crucial factor that affects enzymatic activity.
The pH of silage is an important indicator to evaluate the quality of ensiling [44]. The addition of bacteria accelerates the decrease in pH and effectively inhibits the activity of undesirable organisms [45]. No interaction of ramie with bacteria but a two-way interaction of ramie with enzyme level and an interaction between bacteria and enzyme level indicated that a lower ramie proportion provided better conditions for enzyme and bacteria to reduce the pH of silage by increasing the production of lactic acid. Consequently, the same optimal combination was observed for the lactic acid and pH of silage. Moreover, propionic acid and butyric acid contents increased with increasing ramie proportion in the silage, which is considered undesirable for silage quality [46] as their production results in extensive energy losses [43]. Butyric acid is generally produced by certain anaerobic bacteria, particularly clostridium [47], and increased butyric acid with a high ramie proportion was indicative of increased clostridium levels in this study. Although no direct effect of bacteria on butyric acid or enzyme on propionic acid was observed, both bacteria and enzyme interacted to affect the production of butyric acid and propionic acid in the present study. The lowest ammonia-N content as an indicator of the lowest protein degradation [26] was observed with a high ramie proportion combined with a 200 mg/kg bacteria level. This indicated that this bacterial combination was strong enough to keep working, even with the highest ramie level in the silage, and was not dependent upon the silage CP content. For pH and lactic acid content, the optimum proportion of ramie in the silage was 30% when the bacterial quantity was 200 mg/kg and the enzyme addition was 30 mg/kg in this study. Bacterial addition ensured a sufficient number of lactic acid-producing bacteria to decrease the pH, undesirable fermentation, and proteolysis [48], thereby improving forage preservation [49].

5. Conclusions

In the current study, the proportion of ramie, bacterial inoculation, and enzyme addition not only affected the silage quality independently but also interacted to alter the silage fermentation characteristics. Silage dry matter and protein contents increased with an increase in the ramie proportion and bacterial inoculation level in the silage. Acid detergent fiber and lignin contents decreased when bacteria and enzymes were added to the silage. Overall, the optimum quality of silage was observed when the ratio of ramie to elephant grass was 30:70 and the amounts of compound bacteria and enzyme were 200 mg/kg and 20 mg/kg, respectively. These findings infer that ensiling characteristics are affected by forage type/ratio, while bacteria and enzymes interact not only with each other but also with forage type to affect the composition of mixed silage prepared from ramie and elephant grass.

Author Contributions

Conceptualization, M.L., F.-u.H. and C.Y.; Data curation: M.L., F.-u.H., L.P. and F.X.; Validation, C.Y. and K.W.; Formal analysis, M.L., F.-u.H., L.P., H.Z. and F.X.; Investigation, M.L., F.-u.H., Q.D. and C.Y.; Methodology, M.L., F.-u.H., H.Z. and M.U.A.; Resources, C.Y., Q.D. and K.W.; Writing—original draft preparation, M.L., F.-u.H., M.U.A. and L.P.; writing—review and editing, F.-u.H., M.U.A. and C.Y.; Visualization, H.Z., Q.D. and K.W.; Supervision, C.Y.; Funding acquisition, M.L., F.-u.H. and C.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Guangxi Science and Technology Major Project under grant number GuiKeAA22068099, the Guangxi Natural Science Foundation Youth Science Fund under grant number 2021GXNSFBA196060, and the Guangxi Milk Buffalo Innovation Team at the National Modern Agricultural Industry Technology System under grant number nycytxgxcxtd-2021-21-03.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Experimental grouping of the treatments.
Table 1. Experimental grouping of the treatments.
Ramie/Elephant Grass RatioBacteria Added (mg/kg)Enzyme Added (mg/kg)Ramie/Elephant Grass RatioBacteria Added (mg/kg)Enzyme Added (mg/kg)
30:700070:3000
30:7001070:30010
30:7002070:30020
30:7003070:30030
30:70100070:301000
30:701001070:3010010
30:701002070:3010020
30:701003070:3010030
30:70200070:302000
30:702001070:3020010
30:702002070:3020020
30:702003070:3020030
30:70300070:303000
30:703001070:3030010
30:703002070:3030020
30:703003070:3030030
50:5000100:000
50:50010100:0010
50:50020100:0020
50:50030100:0030
50:501000100:01000
50:5010010100:010010
50:5010020100:010020
50:5010030100:010030
50:502000100:02000
50:5020010100:020010
50:5020020100:020020
50:5020030100:020030
50:503000100:03000
50:5030010100:030010
50:5030020100:030020
50:5030030100:030030
Table 2. Effects of different fermentation conditions on silage characteristics.
Table 2. Effects of different fermentation conditions on silage characteristics.
Item 1Ramie-to-Elephant-Grass RatioBacteria, mg/kgEnzyme, mg/kgSEMp-Value 2
01002003000102030RBER × BR × EB × ER × B × E
DM, %30:7020.322.521.618.620.318.622.521.61.150.0010.760.0010.0010.0510.0010.017
50:5017.315.819.820.020.017.319.815.8
70:3023.920.518.719.020.518.723.919.0
100:021.023.519.323.919.323.523.921.0
CP, %30:709.5512.612.911.19.5411.112.612.90.440.046<0.0010.280.01<0.0010.0080.008
50:5010.310.311.711.111.110.311.710.3
70:3011.311.711.711.811.711.711.311.8
100:010.713.112.011.712.013.111.710.8
Metabolizable energy, cal/g30:702145213022452152214521522130224528.40.0110.0120.510.120.0080.0090.078
50:5022382322213422052205223821342320
70:3023992321219822002321219823992200
100:022932295220721492207229521492293
Ash, %30:7015.313.014.315.915.315.913.014.30.252<0.0010.150.720.250.09<0.0010.13
50:5015.415.915.215.515.515.415.215.8
70:3016.015.815.616.315.815.616.016.3
100:016.216.817.217.817.216.817.816.2
NDF, %30:7057.254.451.156.357.256.354.451.10.990.780.030.110.020.0080.080.03
50:5054.654.054.855.855.854.654.854.1
70:3052.954.554.755.154.554.752.955.1
100:053.652.154.456.454.452.156.453.6
ADF, %30:7039.134.633.938.139.138.134.633.90.78<0.0010.010.050.0170.005<0.0010.048
50:5039.839.538.339.739.739.838.339.5
70:3039.440.739.439.340.739.439.439.2
100:041.640.040.641.840.039.941.841.7
Lignin, %30:7014.412.211.012.414.412.412.211.00.740.0010.0010.002<0.001<0.001<0.001<0.001
50:5015.915.012.415.815.815.912.415.0
70:3015.915.012.712.415.012.715.912.4
100:013.620.215.012.715.020.212.713.7
1 DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; SEM, standard error of the mean. 2 R = proportion of ramie in silage, B = level of bacterial inoculation, E = level of enzyme added.
Table 3. The optimal combination of ramie-to-elephant-grass ratio, enzyme level, and bacteria inoculation level for various characteristics of mixed silage.
Table 3. The optimal combination of ramie-to-elephant-grass ratio, enzyme level, and bacteria inoculation level for various characteristics of mixed silage.
Item 1Fermentation Conditions 2Optimum Combination 3
ABC
DM, %K1 420.7220.6220.03Factor ranking A > C > B
A4C3B1
K218.2420.5819.51
K320.5219.8422.53
K421.9520.4019.37
Range3.710.783.16
CP, %K111.5410.4511.08Factor ranking B > A > C
B3A4C3
K210.8311.9011.53
K311.5912.0711.81
K411.8811.4211.42
Range1.051.620.73
Metabolizable energy, cal/gK1216822682219Factor ranking A > B > C
A3B1C1
K2222422662220
K3227821962203
K4223521762264
Range78.5170.2530.74
Ash, %K114.6015.7115.94Factor ranking A > B > C
A1B2C3
K215.5015.3715.93
K315.9315.5715.51
K416.9916.3615.65
Range2.390.990.43
NDF, %K154.7854.6155.51Factor ranking B > C > A
B4C1A2
K254.8453.8054.46
K354.3753.7854.65
K454.1555.9353.50
Range0.692.152.01
ADF, %K136.4640.0140.08Factor ranking A > C > B
A2C3B3
K239.3638.7338.73
K339.7238.0738.07
K441.0239.7539.75
Range4.561.942.01
Lignin, %K112.5214.9915.09Factor ranking A > B > C
A1B3C4
K214.8315.6215.33
K314.0212.8313.33
K415.4413.3713.05
Range2.922.792.28
1 DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber. 2 Fermentation conditions represented by the ratio of ramie to elephant grass in silage (30:70, 50:50, 70:30, and 100:0; Factor A), level of bacteria addition (0, 100, 200, and 300 mg/kg; Factor B), and level of enzyme addition (0, 10, 20, and 30 mg/kg; Factor C). 3 A1, A2, A3, and A4 represent the proportion of ramie in the silage (30, 50, 70, and 100, respectively); B1, B2, B3, and B4 represent the level of added bacteria (0, 100, 200, and 300 mg/kg, respectively); and C1, C2, C3, and C4 represent the level of added enzyme (0, 10, 20, and 30 mg/kg, respectively). 4 Ki represents the average value at that specific level in an increasing order from K1 to K4.
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Li, M.; Hassan, F.-u.; Akhtar, M.U.; Peng, L.; Xie, F.; Deng, Q.; Zhong, H.; Wei, K.; Yang, C. Fibrolytic Enzymes and Lactic Acid Bacteria Improve the Ensiling Characteristics of Ramie and Elephant Grass Mixed Silage. Agriculture 2024, 14, 1746. https://doi.org/10.3390/agriculture14101746

AMA Style

Li M, Hassan F-u, Akhtar MU, Peng L, Xie F, Deng Q, Zhong H, Wei K, Yang C. Fibrolytic Enzymes and Lactic Acid Bacteria Improve the Ensiling Characteristics of Ramie and Elephant Grass Mixed Silage. Agriculture. 2024; 14(10):1746. https://doi.org/10.3390/agriculture14101746

Chicago/Turabian Style

Li, Mengwei, Faiz-ul Hassan, Muhammad Uzair Akhtar, Lijuan Peng, Fang Xie, Qian Deng, Huapei Zhong, Kelong Wei, and Chengjian Yang. 2024. "Fibrolytic Enzymes and Lactic Acid Bacteria Improve the Ensiling Characteristics of Ramie and Elephant Grass Mixed Silage" Agriculture 14, no. 10: 1746. https://doi.org/10.3390/agriculture14101746

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