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Article

Effects of Wilting and Exogenous Lactic Acid Bacteria on the Fermentation Quality and Microbial Community of Plantago lanceolata Silage

by
Yanhua Tang
1,2,
Qing Dou
2,3,
Bin Luo
2,
Lili Zhao
1,
Puchang Wang
4,
Xuedong Yang
2,* and
Yi Xi
1,*
1
College of Animal Science, Guizhou University, Guiyang 550025, China
2
Guizhou Extension Station of Grassland Technology, Guiyang 550025, China
3
Corn Research Institute, Sichuan Agricultural University, Chengdu 625014, China
4
School of Life Science, Guizhou Normal University, Guiyang 550025, China
*
Authors to whom correspondence should be addressed.
Fermentation 2024, 10(11), 536; https://doi.org/10.3390/fermentation10110536
Submission received: 26 August 2024 / Revised: 8 October 2024 / Accepted: 15 October 2024 / Published: 22 October 2024
(This article belongs to the Special Issue Functional Properties of Microorganisms in Fermented Foods)
Browse Figures
Review Reports Versions Notes

Abstract

:
This study aimed to evaluate the effects of wilting and exogenous lactic acid bacteria treatments on the chemical composition, fermentation quality, and microbial community composition of Plantago lanceolata silage (PS). This experiment was carried out in the Guizhou Extension Station of Grassland Technology (25°38′48″ N, 106°13′6″ E). The PS samples were divided into four treatment groups, namely control PS (C-PS), wilting-treated PS (W-PS), Lactobacillus brucei-treated PS (LB-PS), and wilting + L. brucei-treated PS (WLB-PS) groups, and analyzed after 60 d of treatment. The W-PS and WLB-PS groups showed significantly lower ether extract, ash, and Neutral detergent fiber contents but significantly higher water-soluble carbohydrate content compared to the C-PS and LB-PS groups (p < 0.05). Additionally, the W-PS group had significantly lower propionic acid content but significantly higher butyric acid content compared to the other groups (p < 0.05). Meanwhile, the WLB-PS group had the highest lactic acid content, the lowest pH, and no butyric acid content (p < 0.05). Additionally, the WLB-PS group showed a high proliferation of beneficial bacterial species (Lactobacillus buchneri and Lactobacillus plantarum) and decreased proliferation of undesirable bacterial species (Clostridium lutlcellarli and Clostridium tyrobutyricum). In conclusion, the combination treatment with wilting and L. brucei increased beneficial microorganisms and inhibited undesirable microorganisms during ensiling, thereby improving the fermentation quality of PS. Therefore, the combination treatment with wilting and L. brucei may be an effective Plantago lanceolata silage modulation technique.

1. Introduction

In recent years, more and more countries have banned the use of antibiotic feed additives, progressively exploring herbal medicines as viable alternatives in animal husbandry [1,2]. Plantago lanceolata has the effect of treating malaria and diarrhea in livestock, with high mineral content (phosphorus, calcium, magnesium, sulfur, iron, etc.), good adaptability, and the stronger competition for nutrients than other common forages [3]. P. lanceolata grows rapidly and has high yield in spring, summer, and autumn. It can be planted alone or as part of a seed mixture for transforming and establishing permanent pasture [4]. For grazing animals, feeding with feed containing the P. lanceolata can significantly increase the fattening rate of sheep and milk yield of cattle. Adding P. lanceolata to the diet of two-month-old, weaned lambs for 12 weeks can increase the average dry matter intake of lambs and positively affect meat quality [5]. In the grazing system, the milk production of dairy cows fed with forage from the grazing P. lanceolata replacing perennial ryegrass-white clover pasture increased from 17.1 kg/cow/day to 19.7 kg/cow/day, while the daily weight gain also significantly increased (0.91 g/cow/day to 1.08 g/cow/day) [6]. In addition, P. lanceolata has high palatability. Studies have shown that P. lanceolata has low fiber content, high vitamins, good digestibility, and enhances the palatability of livestock due to its rich taste of sorbitol [7]. Its excellent seasonal production, feed quality, and palatability to livestock make it a common forage plant for livestock such as sheep and beef cattle [8]. Due to the problem of its slow growth in winter and excess production in other seasons, preservation as silage is a way to rationally utilize the resources of P. lanceolata.
Silage fermentation is an efficient storage measure. Silage is not affected by climate and season, reduces pests and diseases, and maintains quality and nutritional value for a long time [9]. The moisture content of raw materials is a key factor in silage fermentation quality. For instance, excessive moisture content can lead to the loss of some nutrients during the compaction process, which can further affect the silage quality [10]. Additionally, excessive moisture content can increase the propagation of harmful bacteria such as Clostridium, while low moisture content can inhibit or delay the fermentation process [11]. Wilting treatment can limit proteolysis and fermentation of silage, leading to an increase in water-soluble carbohydrate (WSC) content and a decrease in fermented acid content, thereby improving the silage quality [12,13]. The addition of lactic acid bacteria (LAB) to silage is another effective measure to improve silage quality [14,15,16]. One study determined the feasibility of P. lanceolata as silage without additives through ideal crude nutrient content, fermentation parameters, and excellent sensory evaluation [17]. At the same time, it was found that adding 3% honey could increase the dry matter content of P. lanceolata silage (PS) [17]. Another study proved that the silage quality in the early stage of P. lanceolata was higher than that in the late stage, and the silage storage period could reach 120 days [18]. In addition, after silage, the total gas production and acetic acid concentration in vitro were significantly increased, and the ruminal fibrolytic bacteria community was significantly improved [19]. The moisture content of P. lanceolata is high. Controlling the moisture content and adding LAB may be important methods to obtain high-quality silage of P. lanceolata, but the effects of wilting and adding LAB on the quality of P. lanceolata silage are still unclear.
In this study, the fermentation quality and microbial community of silage were evaluated to explore the feasibility of wilting treatment and adding LAB to improve the quality of PS. In order to provide a technical reference for the development and utilization of P. lanceolata resources in the development of ecological animal husbandry, the modulation methods for improving the quality of PS were screened.

2. Materials and Methods

2.1. Test Materials

P. lanceolata cultivar Tonic was bred by the PGG Wrightson Seeds Corporate (Lincoln, New Zealand) and introduced by the Guizhou Extension Station of Grassland Technology to be planted on the farm in Shangsi Town, Dushan County, Guizhou Province. The chemical composition of P. lanceolata cv Tonic is shown in Table 1.
The silage additive Lactobacillus brucei (1 × 1010 cfu/g) was obtained from Taiwan Asian Chip Biotechnology Co., Ltd. (Taipei, China).

2.2. Experimental Design

The experimental treatments were conducted in September 2023 using P. lanceolata (Tonic, mean leaf height: 38.5 cm, ear height: 56.8 cm) regrown to the final flowering stage after mowing. The P. lanceolata samples were cut into 2 cm sections and divided into four groups based on the treatments: control PS (C-PS), wilting-treated PS (W-PS), L. brucei-treated PS (LB-PS), and wilting + L. brucei-treated PS (WLB-PS). For the wilting treatment (water content: 62.07%), fresh P. lanceolata samples were spread on a cement floor and dried under natural conditions for 2 h (Tightly pinch the sample, with a small amount of juice dripping down), while for the LB treatment, fresh P. lanceolata samples were treated with 0.025 g/kg of L. brucei. Lastly, for the wilting + L. brucei treatment, fresh P. lanceolata samples were subjected to both wilting and LAB treatments, as described above. L. brucei was diluted with deionized water to a concentration of 0.0025 g/mL. The LB-PS group and WLB-PS group were sprayed with 10 mL of additives each. During the spraying process, the grass samples were thoroughly mixed to ensure even distribution of the additives. The same volume of deionized water was added to the C-PS and W-PS groups.

2.3. Silage Preparation

After the treatments, the P. lanceolata samples were mixed and added to 35 × 50 cm polyethylene silage bags, which were then vacuum sealed. Each bag weighed approximately 1 kg and was stored at ambient temperature (20–30 °C). The nutritional quality and fermentation quality of the PS samples were determined after 60 d [19]. The experiments were conducted in triplicate.

2.4. Analysis of the PS Samples

2.4.1. Determination of the Chemical Composition of the PS Samples

The dry matter (DM) contents of fresh samples and silages were determined in a 65 °C oven for 48 h. The dried samples were ground and passed through a 0.42 mm sieve for chemical analysis. The crude ash (Ash) and crude protein (CP) contents were determined using the crucible and Kjeldahl methods, respectively. The ether extract (EE) content was measured according to the AOAC method [20]. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined using a method described by Van Soest method [21]. The water-soluble carbohydrate (WSC) content was determined by the anthrone sulfate colorimetric method [22].

2.4.2. Determination of the Fermentation Characteristics of the PS Samples

After 60 d of storage, the silage bags were opened and mixed evenly. Thereafter, 30 g of PS was added to a 250-mL conical flask and mixed with 100 mL of deionized water. The sample was incubated at 4 °C for 24 h and then filtered using filter paper. The filtrate was then added to a sealable plastic bottle for the determination of pH and organic acid contents. The pH of the PS samples was determined using a pH meter (Thunder Magnetic). The content of organic acids, including lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA), was measured using high-performance liquid chromatography (HPLC, KC-811 column, Shodex; Shimadzu Co., Ltd., Tokyo, Japan; oven temperature 50 °C; mobile phase 3 mmol/L pechlorate solution; flow rate 1.0 mL/min; flame photometric detector 210 nm; sample size 5.0 μL).

2.4.3. Determination of the Bacterial Community of the PS Samples

The total genomic DNA was extracted from the PS samples using the cetyltrimethylammonium bromide/sodium dodecyl sulfate method. The concentration and purity of the DNA were determined using 1% agarose gel electrophoresis. Thereafter, the DNA samples were diluted to 1 ng/μL with sterile water for polymerase chain reaction (PCR). The V4 region of the 16S rRNA gene was amplified by using specific primers with barcodes. PCR was performed using the Phusion® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, Ipswich, MA, USA). Thereafter, equal volumes of 1× loading buffer (containing SYBR green) and PCR products were mixed and loaded on a 2% agarose gel for electrophoresis. The PCR products were mixed in equidensity ratios. The separated PCR products were then eluted and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Sequencing libraries were generated using the NEB Next®UltraTMDNA Library Prep Kit for Illumina (Omaha, NE, USA), following the manufacturer’s instructions. Subsequently, the index codes were added and the library quality was assessed on the Qubit 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 2100 system (Santa Clara, CA, USA). Lastly, the library was sequenced on an Illumina MiSeq platform, and 250/300 bp paired-end reads were generated. The original sequences were processed by the Novogene Bio-Technology Co., Ltd. (Beijing, China) to annotate classification information and evaluate the phylogenetic relationship with the Silva SSU rRNA database. The microbial community structure and non-metric multidimensional scaling (NMDS) were analyzed by the NovoMagic platform (NovoGene Biotechnology Co., Ltd., Beijing, China).

2.5. Statistical Analysis

Statistical analysis of the chemical composition and fermentation parameters was conducted using analysis of variance with the SPSS v25.0 software (SPSS, Inc., Chicago, IL, USA). Based on the Duncan test, the results are expressed as the “mean ± SE”. A p-value < 0.05 was considered statistically significant. In addition, Spearman correlation was analyzed among bacterial community compositions, function prediction, and silage parameters.

3. Results and Discussion

3.1. Chemical Composition and Fermentation Characteristics of the PS Samples

The chemical compositions of the PS samples are shown in Table 2. The LB-PS group did not show significant variations in the chemical composition, except for the characteristics (Crude fat, %DM and Crude ash) compared to the C-PS group. The W-PS groups had decreased CP content, while the WLB-PS group had increased CP content, compared to the C-PS group, although the difference was not significant. Additionally, the W-PS and WLB-PS groups had significantly lower EE, Ash, and NDF contents compared to the C-PS and LB-PS groups, while the content of WSC was significantly higher than that in C-PS and LB-PS groups (p < 0.05). It has been reported that the Ash content of the PS with 3% honey was almost the same as that of the WLB-PS group in this experiment (15.61%) [17]. Moreover, the high EE and Ash content of the C-PS group and the LB-PS group have the same value in the P. lanceolata cultivated on the Loess Plateau (22.47%, 3.81%) [23]. The significant difference in EE, Ash, NDF, and WSC between wilted and non-wilted samples may be due to the water content in silage. High moisture content is conducive to the reproduction of harmful bacteria, causing the loss of DM, consuming a large amount of WSC for fermentation, and diluting any silage additives, causing poor silage fermentation [24]. Due to the loss of DM, the relative ratio of EE and Ash in the control group and the LB-PS group increased, while the wilting treatment greatly preserved the nutrients of the P. lanceolata silage by controlling the water content. There was no significant difference in WSC content between W-PS group and WLB-PS group, which was similar to the results of other researchers on alfalfa [25]. The significant difference in WSC between wilted and non-wilted samples may be that wilting treatment kills some microorganisms and reduces cell activity by reducing water, resulting in inhibition of plant and microbial respiration and thus reducing WSC consumption.
The W-PS, LB-PS, and WLB-PS groups showed decreased pH and increased LA contents compared to the C-PS group, with the W-PS and WLB-PS groups showing significant differences compared to the C-PS group (p < 0.05). The LB-PS group had significantly higher AA content, while the W-PS group had significantly lower PA content compared to the other groups (p < 0.05). Additionally, the W-PS group had the highest BA content (p < 0.05). Previous studies have shown that the presence of high levels of lactic acid and propionic acid can effectively reduce pH and inhibit the growth of unwanted bacteria [26]. High levels of acetic acid produced by heterotypic lactic acid bacteria (such as L. buchneri) can improve the stability of silage under anaerobic conditions [27]. Although the LB-PS group had better fermentation characteristics, BA was still produced. The pH value of WLB-PS group was the lowest, with high levels of LA, AA, PA contents, and no BA content. These results indicated that the combined wilting and L. brucei treatment reduced the pH, increased LA, PA, and AA contents, and decreased BA content of the PS, thereby improving the silage quality. Similar results were reported in the study of wilting of Lolium multiflorum silage and treatment with microbial additives [28]. In addition, the organic acid content of P. lanceolata silage in this study was higher, but pH could not reflect such acidity. It is speculated that the mineral content in the ash was higher, and these buffer substances could resist changes in pH [29].

3.2. Bacterial Community of the PS Samples

Principal coordinate analysis (PCoA) was conducted to investigate the effects of wilting and/or L. brucei treatments on the bacterial community of the PS samples. The PCoA plot revealed clear differences between the bacterial communities of different PS groups, with components 1 and 2 explaining 46.80% and 26.36% of the total variance, respectively. The PS samples were divided into two groups along the first principal coordinate: with and without wilting treatment (Figure 1a). These results indicated that wilting treatment significantly altered the bacterial community composition of the PS samples. The samples were analyzed by NMDS and compared based on the weighted UniFrac distances (Figure 1b). The results revealed that there were significant differences in the microbial communities of all the PS samples (p < 0.05). The WLB-PS group showed a significantly lower Shannon index compared to other PS samples (Figure 1c), which may be attributed to the inhibitory effects of low pH levels on bacterial growth [30]. This finding is consistent with the results of inoculation of Lactobacillus plantarum in wilting alfalfa [31].
At the phylum level, Firmicutes were significantly abundant in all the PS samples, accounting for >60% of their bacterial communities (Figure 1d). Moreover, the relative abundance of Firmicutes increased to 78.15% in the WLB-PS sample. Firmicutes are usually found in high abundance in samples with good fermentation performance, as the LAB in Firmicutes inhibit the nutrient consumption of fermented samples by other microorganisms [32]. Meanwhile, compared with the C-PS, the relative abundance of Proteobacteria increased from 32.73% to 38.28% in the W-PS group and decreased from 32.73% to 15.46% in the WLB-PS group. These results indicated that wilting treatment promoted the proliferation of Proteobacteria, while L. brucei treatment inhibited its proliferation. Additionally, the relative abundances of Bacteroidetes and Cyanophyta increased in the W-PS, LB-PS, and WLB-PS groups compared to the C-PS group.
At the genus level, unidentified_Clostridiales, Enterobacter, Caproiciproducens, and SporoLactobacillus were relatively abundant in the C-PS group, accounting for 27.63%, 15.10%, 22.51%, and 4.41% of its bacterial community, respectively (Figure 1e). The relative abundance of Lactobacillus increased from 0.80% to 15.34% and 0.80% to 18.49% in the W-PS and LB-PS groups, respectively. Additionally, the relative abundance of unidentified_Clostridiales decreased from 27.63% to 21.55% and 27.63% to 17.81% in the W-PS and LB-PS groups, respectively. These results indicated that both wilting and L. brucei treatments inhibited Clostridiales and that wilting treatment promoted the proliferation of Lactobacillus. The relative abundances of Novosphingobium, Sphingobacterium, and Weissella increased significantly, while the relative abundances of Caproiciproducens and SporoLactobacillus decreased significantly in the W-PS group, indicating that wilting inhibited the proliferation of Caproiciproducens and SporoLactobacillus.
The WLB-PS group showed a lower Shannon index, indicating reduced bacterial community diversity (Figure 1c,e). Lactobacillus was the dominant bacterial genera in the WLB-PS group (75.01%). The relative abundances of Novosphingobium and Sphingomonas increased, while the relative abundance of Enterobacter decreased significantly in the WLB-PS group. Lactobacillus is abundant in good-quality silage, as it promotes LA production and lowers pH [33]. Novosphingobium and Sphingomonas have gained increasing research attention owing to their positive effects [34]. Enterobacter is widely considered to be an undesirable microorganism in silage, as it is believed to deaminate and decarboxylate certain amino acids, resulting in the production of ammonia and biogenic amines, which reduce the nutritional value and palatability of silage [35]. In addition, Sphingobacterium and Weissella, which were relatively abundant in the W-PS group, were absent in the LB-PS group, indicating that Lactobacillus competed with Sphingobacterium and Weissella for nutrients, thereby inhibiting their proliferation.
Further analysis of the abundance of specific microorganisms in the PS samples was conducted (Figure 1f). Lactobacillus plantarum can inhibit yeast and mold damage in silage and reduce its nutrient loss caused by aerobic degradation. Therefore, the fermentation quality of silage increases with an increase in L. plantarum abundance [36]. The relative abundance of L. plantarum increased from 0.43% in C-PS to 5.06% in W-PS, 9.23% in LB-PS, and 5.78% in WLB-PS. Additionally, the relative abundance of Lactobacillus buchneri was the highest in the LB-PS (12.77%) and WLB-PS (68.79%) groups. SporoLactobacillus inulinus and Lactococcus lactis are used as beneficial bacteria in the food fermentation process. Stenotrophomonas rhizophila can adsorb heavy metals and emit volatile organic compounds to directly inhibit the growth of plant pathogenic fungi and bacteria [37]. The abundances of S. inulinus, L. lactis, and S. rhizophila were relatively high in Plantago silage. Clostridium can multiply and produce high concentrations of BA, resulting in odorous and low-quality silage [38]. Clostridium lutlcellarli and Clostridium tyrobutyricum accounted for 3.03% and 1.62% of the bacterial community of the C-PS group, respectively. The relative abundance of C. lutlcellarli increased by 0.66% in the LB-PS group and decreased by 3.02% in the W-PS and WLB-PS groups. Meanwhile, the relative abundance of C. tyrobutyricum increased by 6.12% and 19.45% in the LB-PS and W-PS groups, respectively, but decreased by 1.57% in the WLB-PS group. These results indicated that the combined wilting + L. brucei treatment enriched the beneficial bacterial population and decreased the undesirable bacterial population, thereby improving the fermentation quality of the PS. After wilting treatment, the moisture content of silage raw materials decreased, which provided a powerful environment for the fermentation of LAB. When the moisture content is moderate, LAB can grow rapidly and produce lactic acid, thereby reducing the pH value of silage and inhibiting the growth of bad microorganisms [39]. It has been reported that wilting treatment and inoculation of LAB can improve the quality of alfalfa silage to a certain extent [40].

3.3. Interspecific Correlation Analysis of the Dominant Bacteria and Correlation Analysis of Fermentation Characteristics

Interspecific correlation analysis revealed that L. buchneri was negatively correlated with all the dominant bacterial strains, except L. plantarum (R = 0.41), and that C. tyrobutyricum was positively correlated with all the dominant bacterial strains, except L. buchneri (R = −0.61). Notably, there was a significant negative correlation between L. buchneri and C. tyrobutyricum (p < 0.05; Figure 2a). A Spearman correlation heat map of the potential strains and fermentation parameters showed that C. tyrobutyricum was positively correlated with BA (R = 0.79, p < 0.05) and that L. buchneri was positively correlated with LA (R = 0.76) and negatively correlated with BA (R= −0.76, p < 0.05). Additionally, LA was also positively correlated with L. plantarum (R = 0.34) and Flavobacterium akiainvivens (R = 0.049) and negatively correlated with the other bacterial strains, although the correlation was not significant. Furthermore, the results showed that C. tyrobutyricum increased BA content, while L. buchneri inhibited C. tyrobutyricum activity, increased LA content, and decreased BA content. Clostridium can be classified as protein- or sugar-fermenting Clostridium, based on the substrate. The protein-fermenting Clostridium primarily metabolizes proteins and a few types of WSC, while the sugar-fermenting Clostridium metabolizes various forms of WSC [41]. In this study, S. inulinus (R = −0.73) and C. luticellarii (R = −0.79) were negatively correlated with WSC and positively correlated with pH (p < 0.05). C. luticellarii may be a sugar-fermenting Clostridium, and S. inulinus may have a competitive advantage in sugar fermentation. These species may compete with LAB for the available WSC and hinder the LA fermentation process, resulting in a slow decrease in the pH value and a loss of nutrients in the silage. This was confirmed by Muck’s research, who reported that LAB produce lactic acid by fermenting sugars, C. tyrobutyricum ferments WSC and some sugars to produce butyric acid, while Bacillus ferments organic acids into water and carbon dioxide [42]. Therefore, C. tyrobutyricum, S. inulinus, and C. luticellarii compete with LAB for fermentation substrates. The low quality of PS may be attributed to the decrease in LA content and pH. These findings are also consistent with previous studies [43].

3.4. Functional Prediction of the Dominant Bacteria in the PS Sample

The functional profiles of silage microbial communities are shown in Figure 3a. A study found that chemoheterotrophy and fermentation are the prevalent functions of the bacterial communities in silage [44]. Consistently, functional prediction revealed that chemoheterotrophy (19.91%) and fermentation (16.27%) were the prevalent functions of the bacterial communities in all the PS samples. The functions of the LB-PS and W-PS groups increased, while that of the WLB-PS group decreased, compared to the C-PS group. The dynamic changes in the functions of the PS groups were consistent with the dynamic changes in their bacterial communities. The W-PS and LB-PS groups showed decreased “Aerobic_chemoheterotrophy” and increased “Fermentation” function compared to the C-PS group, which may be attributed to the decreased proliferation of unwanted aerobic microbes and increased proliferation of anaerobic microbes in the W-PS and LB-PS samples [45]. Spearman correlation analysis (Figure 3b) showed that L. buchneri was positively correlated with “Nitrogen_respiration”, “Nitrite_respiration”, and “Nitrite_ammonification” and negatively correlated with “Nitrate_reduction” and “Animal_parasites_or_symbionts” (p < 0.05). Additionally, correlation analysis revealed that L. plantarum was positively correlated with “nitrate_respiration”. These functions may be associated with nitrate and nitrite degradation by L. brucei and L. plantarum, which can improve the quality of Plantago silage. The nitrite in the silage process mainly comes from the conversion of nitrogen-containing substances such as nitrate, which is facilitated by the participation of microorganisms. The degradation of nitrite during silage is mainly related to the growth of LAB [46]. During silage, lactic acid bacteria can produce organic acids, nitrite reductase, lactobacillin, and other products by metabolism, which play a direct or indirect role in the degradation of nitrite [47]. It was found that LAB such as L. plantarum, L. brevis and Lactococcus lactissubsp. lactis have good acid-producing ability, which can rapidly reduce the pH value in the fermentation process to degrade nitrite and inhibit the reproduction of other harmful microorganisms [48].
The metabolic pathways related to silage microbial communities were analyzed by Tax4Funt. The functional prediction of all the silage samples at the ‘first pathway’ level was primarily related to metabolism (Figure 4a). Compared with the C-PS group, the LB-PS and W-PS groups showed higher relative abundance of metabolism (45.26%). “Membrane_transport”, “Carbohydrate_metabolism”, and “Amino_acid_metabolism” were highly abundant at the second level (Figure 4b). Total carbohydrate metabolism is composed of glycolysis and gluconeogenesis, and amino acids are essential for protein synthesis and primary metabolism [49]. However, the enrichment of “Carbohydrate_Metabolism” and “Amino_acid_metabolism” was not significantly different between the PS samples.
At the third level, the functional spectrum heat map showed a clear division of metabolic pathways between the wilted and un-wilted PS samples (Figure 4c). The C-PS group was highly enriched in “Alanine,_aspartate,_and_glutamate_metabolism”, “Starch_and_sucrose_metabolism”, and “Amino_acid_related_enzymes”. The functional aggregation in the LB-PS group showed slight variations compared to the C-PS group; however, the LB-PS group was primarily enriched in amino acid metabolism-related pathways. The W-PS group was enriched in “ABC_transporters” and “Quorum_sensing”. ABC transporters are involved in the uptake of essential substances from the external environment, including sugars, amino acids, peptides, proteins, metabolites, and antibiotics [50]. Quorum sensing involves the use of small signal molecules to respond to fluctuations in the population density to communicate and coordinate various cell behaviors and colony-wide functions, such as biofilm formation, antibiotic resistance, and bioluminescence [51]. The WLB-PS group was enriched in “RNA_degradation”, “Messenger_RNA_biogenesis”, and “Glycine, serine, and threonine metabolism”. The amino acids glycine, serine, and threonine play key roles in various biological processes and are associated through various metabolic pathways, including protein synthesis, nitrogen metabolism, and energy metabolism. A study has shown that the energy metabolism (mainly amino acid metabolism) of LAB is crucial for promoting the production of lactic acid during silage [52]. Based on this, some scholars believe that the energy metabolism of high-quality silage should be up-regulated [53].

4. Conclusions

This study showed that wilting treatment and addition of L. brucei would significantly affect the fermentation quality of P. lanceolata. The wilting treatment reduced the loss of DM and WSC, reduced the pH value, and greatly preserved the nutrients. The addition of L. brevis produced more lactic acid and acetic acid, reduced butyric acid, and promoted the fermentation process. The combined treatment of wilting and L. brucei has both advantages of the two, promoting the proliferation of beneficial bacteria (L. plantarum and L. brevis) and reducing the survival of bad microorganisms (C. tyrobutyricum). Altogether, the results indicate that combination treatment with wilting and L. brucei may be an effective silage modulation technique. However, due to the small sample size, there results are only used as evidence for preliminary research. Future studies with expanded sample sizes and repeated experiments will provide a more solid scientific basis.

Author Contributions

X.Y., Y.X. and Y.T. designed the experiments and revised the manuscript. Y.T., B.L. and Q.D. performed the experiments. Y.T. and X.Y. wrote the manuscript. Y.T. and P.W. performed the data analysis. X.Y., Y.X., L.Z. and P.W. contributed to the grammar and language revision of the paper and contributed to this experiment. All authors have read and agreed to the published version of the manuscript.

Funding

This project was supported by the Guizhou Province Key Research and Development Program of China (Qiankehezhicheng [2021]Yiban143), Guizhou high-level innovative talents project (Qiankehepingtairencai-GCC [2022]022-1), the project of forest and grass seed cultivation in Guizhou Province (2024-ZM-02), the Program forestry and grassland reform and development fund in China (Gui [2024]TG 23).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.

Acknowledgments

We thank the personal Biotechnology Co., Ltd. (Shanghai, China) for providing technical support.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Analysis of the bacterial community structure of the Plantago silage (PS) samples treated with wilting and/or exogenous lactic acid bacteria (LAB). (a) Principal coordinate analysis (PCoA) of bacterial communities in the control PS (C-PS), wilting-treated PS (W-PS), Lactobacillus brucei-treated PS (LB-PS), and wilting + L. brucei-treated PS (WLB-PS) groups. The blue and red dashed circles indicate the unwilted and wilted groups, respectively. (b) Non-metric multidimensional scaling (NMDS) diagram of the C-PS, W-PS, LB-PS, and WLB-PS groups (stress = 0.053). Each point in the diagram represents a sample. The distance between points indicates the degree of difference, and samples from the same group are represented using the same color. NMDS can accurately reflect the degree of difference between the samples when stress is <0.2. (c) Shannon index of the C-PS, W-PS, LB-PS, and WLB-PS groups. (d–f) Relative abundance of phylum (d), genus (e), and species (f) in the C-PS, W-PS, LB-PS, and WLB-PS groups.
Figure 1. Analysis of the bacterial community structure of the Plantago silage (PS) samples treated with wilting and/or exogenous lactic acid bacteria (LAB). (a) Principal coordinate analysis (PCoA) of bacterial communities in the control PS (C-PS), wilting-treated PS (W-PS), Lactobacillus brucei-treated PS (LB-PS), and wilting + L. brucei-treated PS (WLB-PS) groups. The blue and red dashed circles indicate the unwilted and wilted groups, respectively. (b) Non-metric multidimensional scaling (NMDS) diagram of the C-PS, W-PS, LB-PS, and WLB-PS groups (stress = 0.053). Each point in the diagram represents a sample. The distance between points indicates the degree of difference, and samples from the same group are represented using the same color. NMDS can accurately reflect the degree of difference between the samples when stress is <0.2. (c) Shannon index of the C-PS, W-PS, LB-PS, and WLB-PS groups. (d–f) Relative abundance of phylum (d), genus (e), and species (f) in the C-PS, W-PS, LB-PS, and WLB-PS groups.
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Figure 2. Correlation analysis between bacterial species and fermentation parameters of the PS samples treated with wilting and/or exogenous LAB. (a) Spearman correlation analysis was used to establish the correlation between dominant strains. (b) A correlation heat map of different bacterial species and fermentation parameters of the C-PS, W-PS, LB-PS, and WLB-PS groups. R values are presented in different colors. The legend on the upper left side is a range of colors for different R values. * p < 0.05.
Figure 2. Correlation analysis between bacterial species and fermentation parameters of the PS samples treated with wilting and/or exogenous LAB. (a) Spearman correlation analysis was used to establish the correlation between dominant strains. (b) A correlation heat map of different bacterial species and fermentation parameters of the C-PS, W-PS, LB-PS, and WLB-PS groups. R values are presented in different colors. The legend on the upper left side is a range of colors for different R values. * p < 0.05.
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Figure 3. Functional prediction of the dominant bacteria in the PS samples treated with wilting and/or exogenous LAB. (a) Relative abundance of the bacterial functional spectrum in the C-PS, W-PS, LB-PS, and WLB-PS groups, as predicted by FAPROTAX analysis. (b) Correlation heat maps of the dominant bacteria and bacterial functions based on Spearman correlation analysis.
Figure 3. Functional prediction of the dominant bacteria in the PS samples treated with wilting and/or exogenous LAB. (a) Relative abundance of the bacterial functional spectrum in the C-PS, W-PS, LB-PS, and WLB-PS groups, as predicted by FAPROTAX analysis. (b) Correlation heat maps of the dominant bacteria and bacterial functions based on Spearman correlation analysis.
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Figure 4. Dynamic prediction of microbial functional spectrum in the PS samples treated with wilting and/or exogenous LAB by Tax4Funt analysis. Primary (a) and secondary (b) metabolic pathway dynamics predicted from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. (c) Heat maps of the tertiary metabolic pathways predicted from the KEGG database.
Figure 4. Dynamic prediction of microbial functional spectrum in the PS samples treated with wilting and/or exogenous LAB by Tax4Funt analysis. Primary (a) and secondary (b) metabolic pathway dynamics predicted from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. (c) Heat maps of the tertiary metabolic pathways predicted from the KEGG database.
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Table 1. Chemical composition of Plantago lanceolata cultivar Tonic.
Table 1. Chemical composition of Plantago lanceolata cultivar Tonic.
ItemsData
Dry matter, % Fresh weight24.89 ± 1.24
Crude ash, %DM12.15 ± 0.43
Crude protein, %DM16.19 ± 0.19
Water Soluble Carbohydrate, %DM12.23 ± 2.61
Crude fat, %DM1.96 ± 0.02
Neutral detergent fiber, %DM52.80 ± 0.49
Acid detergent fiber, %DM32.19 ± 0.77
Table 2. Chemical composition and fermentation characteristics of the P. lanceolata silage (PS) samples.
Table 2. Chemical composition and fermentation characteristics of the P. lanceolata silage (PS) samples.
ItemsC-PSLB-PSW-PSWLB-PS
Chemical composition
Dry matter, % Fresh weight22.95 ± 0.43 c23.31 ± 1.18 c34.29 ± 0.77 a28.07 ± 1.11 b
Crude protein, %DM16.52 ± 0.33 ab17.29 ± 0.33 a16.02 ± 0.25 b17.06 ± 0.93 ab
Crude fat, %DM3.55 ± 0.03 a2.99 ± 0.15 b2.35 ± 0.35 c2.53 ± 0.04 c
Crude ash, %DM18.54 ± 0.73 b19.93 ± 0.06 a16.73 ± 0.71 c15.49 ± 0.79 d
Neutral detergent fiber, %DM60.88 ± 2.04 a59.40 ± 0.70 a55.93 ± 1.81 b56.17 ± 1.33 b
Acid detergent fiber, %DM32.39 ± 1.47 a32.59 ± 1.66 a31.83 ± 0.50 a31.29 ± 1.28 a
Water Soluble Carbohydrate, %DM2.47 ± 0.83 b2.24 ± 1.00 b10.21 ± 4.10 a10.68 ± 3.38 a
Fermentative characteristics
pH5.05 ± 0.02 a4.99 ± 0.02 a4.56 ± 0.11 b4.27 ± 0.02 c
Lactic acid, %DM6.47 ± 0.18 c7.23 ± 0.82 bc7.42 ± 0.30 b8.49 ± 0.33 a
Acetic acid, %DM5.31 ± 0.75 b6.42 ± 0.39 a4.46 ± 0.08 b5.20 ± 0.39 b
Propionic acid, %DM1.87 ± 0.11 a1.70 ± 0.21 a1.38 ± 0.07 b1.69 ± 0.08 a
Butyric acid, %DM0.03 b0.02 ± 0.01 b0.05 ± 0.01 aND
PS, control group; LB-PS, L. brucei-treated group; W-PS, wilting-treated group; and WLB-PS, wilting + L. brucei-treated group. Different letters in the same row indicate significant differences (p < 0.05).
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Tang, Y.; Dou, Q.; Luo, B.; Zhao, L.; Wang, P.; Yang, X.; Xi, Y. Effects of Wilting and Exogenous Lactic Acid Bacteria on the Fermentation Quality and Microbial Community of Plantago lanceolata Silage. Fermentation 2024, 10, 536. https://doi.org/10.3390/fermentation10110536

AMA Style

Tang Y, Dou Q, Luo B, Zhao L, Wang P, Yang X, Xi Y. Effects of Wilting and Exogenous Lactic Acid Bacteria on the Fermentation Quality and Microbial Community of Plantago lanceolata Silage. Fermentation. 2024; 10(11):536. https://doi.org/10.3390/fermentation10110536

Chicago/Turabian Style

Tang, Yanhua, Qing Dou, Bin Luo, Lili Zhao, Puchang Wang, Xuedong Yang, and Yi Xi. 2024. "Effects of Wilting and Exogenous Lactic Acid Bacteria on the Fermentation Quality and Microbial Community of Plantago lanceolata Silage" Fermentation 10, no. 11: 536. https://doi.org/10.3390/fermentation10110536

APA Style

Tang, Y., Dou, Q., Luo, B., Zhao, L., Wang, P., Yang, X., & Xi, Y. (2024). Effects of Wilting and Exogenous Lactic Acid Bacteria on the Fermentation Quality and Microbial Community of Plantago lanceolata Silage. Fermentation, 10(11), 536. https://doi.org/10.3390/fermentation10110536

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