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Article

Fermentation Properties and Bacterial Community Composition of Mixed Silage of Mulberry Leaves and Smooth Bromegrass with and without Lactobacillus plantarum Inoculation

by
Weihan Yang
1,†,
Fengyuan Yang
2,†,
Changsong Feng
3,
Shanshan Zhao
1,
Xueying Zhang
1 and
Yanping Wang
1,*
1
Henan Key Laboratory of Ion Beam Bio-Engineering, School of Agricultural Science, Zhengzhou University, Zhengzhou 450000, China
2
Basic Medical Research Center, Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450052, China
3
Institute of Animal Husbandry and Veterinary Science, Henan Academy of Agricultural Sciences, Zhengzhou 450000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2023, 9(3), 279; https://doi.org/10.3390/fermentation9030279
Submission received: 16 February 2023 / Revised: 9 March 2023 / Accepted: 10 March 2023 / Published: 13 March 2023
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
To evaluate the fermentation properties and bacterial community composition of mulberry leaves when ensiled with smooth bromegrass, and the effects of Lactobacillus plantarum inoculation on the mixed silage of mulberry leaves and smooth bromegrass, mulberry leaves were mixed with smooth bromegrass at ratios of 100:0, 90:10, 80:20, 70:30 and 60:40, and ensiled for 60 d with and without L. plantarum inoculant. The results showed that the sole fermentation of mulberry leaves failed to achieve optimum fermentation quality. Silage with a mulberry leaf ratio of 80% performed better fermentation quality compared with other non-inoculated groups, indicated by lower pH value, adequate lactic acid accumulation, and enriched proportion of Lactobacillus in the bacterial community. L. plantarum inoculation dramatically improved fermentation quality of mulberry leaf silage compared with the non-inoculated control. However, the fermentation quality of the inoculated silage decreased along with the reduction in the ratio of mulberry leaves. In conclusion, L. plantarum inoculation has the capability to improve the silage quality of mulberry leaves. Combined ensiling with smooth bromegrass could also aid in improving silage quality of mulberry leaves, with the optimum ratio of mulberry leaves being 80%.

1. Introduction

Mulberry (Morus alba) is a perennial, deep-rooted woody plant of the Moraceae family, and has been cultivated in China for more than 5000 years [1]. Its leaves are rich in protein (15–35% dry matter (DM)), minerals and vitamins, with a biomass yield of approximately 25–30 tons of fresh matter/ha/year [2,3]. Mulberry leaves have been served as an optimal protein supplement in the diets of terrestrial farm animals [4,5,6]. Moreover, mulberry leaf polysaccharides have been reported to be promoters of the ecology of gut microbiota, and effectively reduced diarrhea rate in early weanling pigs [6]. Mulberry leaves, thus, might also be a potential “green” additive to improve the growth and disease resistance of livestock.
Similar to Broussonetia papyrifera, another member of the Moraceae family, seasonal production of mulberry might hinder its continuous and extensive application in feed rations [7]. Ensiling might be an effective technology for the preservation of mulberry leaves [8], especially during the rainy season. However, as in the case of other high-protein forage, such as alfalfa and soybean, the sole fermentation of mulberry leaves could be detrimental to the aim of achieving optimum fermentation quality [9,10,11]. The incorporation of a second substrate with a high WSC content has been reported to be an effective strategy to improve fermentation qualities of these high-protein forage [7,12,13]. Smooth bromegrass (Bromus inermis Leyss) is widely cultivated in temperate regions of the world [14]. It has a crude protein (CP) content of 11−16% DM, a WSC content of 6−12% DM, and a neutral detergent fiber (NDF) content of 40−50% DM [15]. Previous research has shown that the fermentation quality of high-protein materials like alfalfa improves when co-ensiled with smooth bromegrass [15]. Enhanced WSC content of smooth bromegrass might be an efficient strategy for improving fermentation quality when co-ensiled with mulberry leaves. LAB inoculants can effectively improve silage quality [16]. Lactobacillus plantarum is a widely applied bacterial inoculant in forage ensiling studies [17], while knowledge of its effects on mulberry leaf silage and mixed silage of mulberry leaves and smooth bromegrass remains poor.
The objective of this study Is to evaluate the fermentation properties and bacterial community composition of mulberry leaves when ensiled with smooth bromegrass at different ratios, with or without L. plantarum inoculation, and to determine the optimum ratio of the mixture for good fermentation quality.

2. Materials and Methods

2.1. Raw Materials and Silage Preparation

Mulberry leaves were harvested on 11 June 2020 from an experimental field belonging to Henan Academy of Agricultural Sciences in Lankao, Henan, China (34.82° N, 114.82° E) with an experimental area of 0.3 ha. Smooth bromegrass was harvested on 11 June 2020 from an experimental field belonging to Henan Academy of Agricultural Sciences in Zhengzhou, Henan, China (34.76° N, 113.65° E) with an experimental area of 0.2 ha. The raw materials (approximately 15 kg of mulberry leaves and 5 kg of smooth bromegrass) were immediately taken to the laboratory and chopped to an approximate length of 2 cm using a handy cutter. Chopped smooth bromegrass was manually mixed with mulberry leaves at ratios of 0% (ML100), 10% (ML90), 20% (ML80), 30% (ML70), and 40% (ML60) on a fresh matter (FM) basis. Six kilograms of each homogenous mixture was equally divided into 6 parts. Approximately 1 kg for each of three replicates of the homogenous mixture was treated with the following: (1) distilled water control (CK); (2) 1 × 106 colony forming units (cfu)/g of L. plantarum A345 (LP). L. plantarum A345 is an alfalfa epiphytic strain isolated from Shanxi, Beijing, China, and is stored in the Henan key laboratory of ion beam bioengineering. Treated samples were then separately vacuum packed in vacuum-sealed polyethylene plastic bags (dimensions: 200 mm × 300 mm, Dongda, Zhengzhou, China). Silages were stored at ambient temperature (24−28 °C) in the dark room, and ensiled for 60 d.

2.2. Analyses of Fermentation Products, Chemical Composition and Microbial Population

A sample of 10 g was taken from each bag of silage after manually and homogeneously mixed within the bag and mixed with 90 mL sterilized water by vigorous shaking at 180 r/min for 2 h at 4 °C, and then filtered through a 0.45 μm membrane. The pH of samples was determined via a pH meter (Mettler Toledo CO., Ltd., Greifensee, Switzerland). The organic acid contents (LA, acetic acid (AA), propionic acid (PA) and butyric acid (BA)) were determined using high-performance liquid chromatography (Waters Alliance e2695, Waters, MA, USA) following the method described by Zhao et al. (2020) [18]. The ammoniacal nitrogen (NH3-N) concentration was determined using Berthelot colorimetry [19].
A sample of approximately 150 g was taken from each bag for the determination of oven DM content at 65 °C for 48 h in a drying oven (FX101-0, Shanghai Shuli Yiqi YIbiao Co., Ltd., Shanghai, China). The dried samples were then pulverized to pass through a 1 mm sieve using a laboratory knife mill (FW100, Taisite Instrument Co., Ltd., Tianjin, China) for chemical analysis. CP concentration was calculated according to the Kjeldahl method [20] using a Kjeldahl nitrogen analyzer (K1160, Hanon, Shanghai, China). The NDF and acid detergent fiber (ADF) concentration was calculated according to Van Soest et al. (1991) [21] using an Ankom 200 Fiber Analyzer System (Ankom Technology Corp., Fairport, NY, USA). The WSC concentration was calculated using anthrone colorimetry [22].
A sample of 10 g was taken from each bag of the raw materials and mixed with 90 mL sterilized saline solution (0.85% NaCl) and serially diluted from 10−1 to 10−5 in sterilized saline solution. Microbial population was determined by plate counting method as described by Zhao et al. (2020) [18]. The number of LAB, coliform bacteria and molds was determined on de Man Rogosa Sharpe agar, Violet Red Bile agar and potato dextrose agar, respectively (Beijing Dingguo Changsheng Bio-tech CO., Ltd., Beijing, China).

2.3. Analyses of Bacterial Community

Samples were collected after being ensiled for 60 d and stored at −20 °C before DNA extraction. Pre-treatments of the samples for DNA extraction were performed as previously described by Yang et al. (2021) [23]. Total DNAs of the samples were extracted using a Bacterial DNA Kit D3350-02 (Omega Biotek, Norcross, GA, USA) according to the manufacturer’s instructions. DNA quality was evaluated by 1% agarose gel electrophoresis and a NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA with concentration greater than 20 ng/μL, total amount greater than 500 ng, and 260/280 nm ratio of 1.8−2.0 were recognized as good-quality DNAs.
The V3−V4 regions of 16S rDNA were amplified with primers 341F (5′-CCTACGGGNGGCWGCAG) and 805R (5′-GACTACHVGGGTATCTAATCC). An absolute quantification 16S-seq method using spike-in sequences was applied for bacterial community analyses [24,25]. The procedures of Yang et al. (2021) [23] were followed.
The spike-in sequences were filtered out, and reads were counted. High-quality sequences were output into amplicon sequence variants using QIIME 2 (https://qiime2.org/ (accessed on 15 October 2021)) [26]. The copy numbers were rectified based on ribosomal RNA operons (rrn) DataBase according to the procedure described by Stoddard et al. (2015) and Wu et al. (2017). The taxonomy assignment of representative sequences was performed with the Ribosome Database Project [27]. The sequence data have been submitted to the NCBI database under accession number PRJNA930053.

2.4. Statistical Analyses

Effects of ratio of mulberry leaves (R), L. plantarum inoculation (I), and their interactions (R*I) on fermentation properties and alpha diversity indices were analyzed using general liner model in IBM SPSS Statistics for Windows, version 21.0. (IBM Corporation, Armonk, NY, USA). Alpha diversity indices including observed species, Chao 1 and Shannon indices were calculated using mothur (version 1.9.0; https://mothur.org/wiki/mothur_v.1.9.0/ (accessed on 15 October 2021)) [28]. Principal coordinate analyses (PCoA) based on weighted unifrac distance matrix and linear discriminant analysis effect size (LEfSe) was performed using python (version 2.7.14; https://www.python.org/ (accessed on 15 October 2021)). Correlations of fermentation properties with bacterial community were analyzed using two-tailed Spearman correlations in IBM SPSS Statistics for Windows, version 21.0. Significant differences between means were identified by Duncan’s multiple range tests in which a probability (p) value of <0.05 was designated as significant.

3. Results and Discussion

3.1. Chemical Composition and Microbial Population of Raw Materials

Chemical composition and microbial population of the fresh mulberry leaves and smooth bromegrass are listed in Table 1. The nutritional components of mulberry leaves are highly dependent on their varieties, cultivation areas, and conservation strategies [29,30,31]. The CP and WSC contents of raw mulberry leaves were comparable with the CP (13.61−24.97% DM) and WSC (3.99−17.44% DM) contents of mulberry leaves reported by Zheng et al. (2017) [32]. The NDF and ADF contents of raw mulberry leaves were comparable with those reported by He et al. (2020) [33]. The WSC content and population of epiphytic LAB in raw mulberry leaves were below the optimum level (>5% DM [34], 2018; >105 colony-forming unit/g FM [17]), and were considered insufficient for adequate fermentation during ensiling. A lack in the count of epiphytic LAB in mulberry leaves was also reported by Wang et al. (2019) [9] and He et al. (2020) [33].
Smooth bromegrass possessed higher content of WSC, NDF and ADF, and lower content of DM and CP compared with mulberry leaves (p < 0.05). Smooth bromegrass also exhibited higher counts of LAB and Coliform bacteria compared with mulberry leaves (p < 0.05). The WSC content and population of epiphytic LAB in raw smooth bromegrass were above the optimum level. These results indicated that the addition of smooth bromegrass might aid in improving the fermentation quality of mulberry leaf silage. However, the DM content was lower, and the undesirable coliform bacteria population was higher in smooth bromegrass compared with fresh mulberry leaves. The DM content of fresh smooth bromegrass was low compared with previous studies (31.7% and 33.9%, respectively) [15,35]. This might be due to the rainy weather conditions during the harvest season in our study. A low DM content of the raw material was also reported in previous studies in stylo (22.5%), bur clover (16.15%), and annual ryegrass (18.88%) [8,13]. Gao et al. (2021) [36] reported that gene expression was mainly altered in the early stage during the succession of bacterial communities, and significant changes in gene expression could occur at the very beginning. The proportion of smooth bromegrass, would therefore greatly affect the fermentation process in the mixed silage. It is necessary to determine the optimum proportion of smooth bromegrass in the inoculated and non-inoculated mixed silage.

3.2. Chemical Composition and Fermentation Properties of Silage

The chemical compositions of the inoculated and non-inoculated mixed silages at different ratios are listed in Table 2. The DM content is important in silage fermentation, due to its effects on the growth and reproduction of microbes during ensiling [37]. The DM content varied with different ratios of mulberry leaves, in both the inoculated and non-inoculated silages (p < 0.05). Non-inoculated silage, with mulberry leaf ratios of 80% and 70%, presented higher DM content compared than the other non-inoculated treatments (p < 0.001). As for the inoculated silage, the DM content decreased with increasing proportion of smooth bromegrass in silage (p = 0.001). The CP content decreased, while the NDF and ADF content increased, following the increase in the proportion of smooth bromegrass in both the inoculated and non-inoculated silages (p < 0.05). This is in line with the results of Li et al. (2018) [13], who reported decreased CP content and increased NDF and ADF content with increasing proportion of annul ryegrass in bur clover and annul ryegrass mixed silage. No significant DM loss was observed in the silages (p >0.05).
The fermentation properties of the inoculated and non-inoculated mixed silage at different ratios are shown in Figure 1. The ratio of mulberry leaves (R), inoculation (I), and their interactions (R*I) had significant effects on pH, LA, AA, and NH3-N concentration in silage (p < 0.05). Among the non-inoculated silages, the ML80 silage exhibited a lower pH value than the other groups (p = 0.04), and a more abundant LA accumulation compared with the ML100, ML70 and ML60 silages (p < 0.001). This indicates that more abundant LA fermentation was observed in the ML80 silage in the non-inoculated group. NH3-N is recognized as an indicator of decarboxylation [38]. Meanwhile, little effect of the mulberry leaf ratio on NH3-N concentration was observed in the non-inoculated silage (p = 0.29). The response of L. planturum inoculant to silage fermentation varied in silages with different mulberry leaves’ proportions. As for the inoculated silage, the optimum fermentation quality was observed in the ML100 silage, with a pH value of 4.18, an LA concentration of 200.26 g/kg DM, and an NH3-N concentration of 9.82 g/kg total nitrogen (TN). The enhancement of LA fermentation in the sole ensiling of mulberry leaves by L. planturum inoculation was in line with the results of Wang et al. (2018) [9] and Yang et al. (2019) [10] in Moringa oleifera leaves and in alfalfa silage, respectively. Unlike the effects of LAB inoculants on the fermentation quality of mixed silage reported in previous studies [13,39,40], the pH level (p < 0.001) and NH3-N concentration increased (p < 0.001), following the increase in proportion of smooth bromegrass in the inoculated silage. LA accumulation also decreased in the inoculated silage with the addition of smooth bromegrass compared with the ML100 silage (p < 0.001). Variation in the response of the inoculant to silage fermentation was also discovered in our previous study between wilted and non-wilted alfalfa silage [41]. This might indicate poor adaption of the inoculant to the environmental conditions of the silage, and a failure to dominate the bacterial community [18]. Meanwhile, substantial evidence would be needed to explain the poor adaption of the inoculant. The increasing content of NH3-N in the inoculated group reflected the enhanced growth and activity of undesirable microorganisms, along with the increased proportion of smooth bromegrass in the silage. PA and BA were not detected in the silage samples. This is a welcome finding, as PA and BA accumulation are generally the result of Clostridia fermentation in silage, which is undesirable [42].

3.3. Bacterial Community Analyses of Silage

In total, 5,656,019 reads were acquired for the bacterial community analyses of 30 samples, with spike-in reads accounting for 32.62% ± 6.39%. These valid sequences were output into 691 amplicon sequence variants. The number of gene copies per ng of DNA was calculated from the standard curves with fitting coefficients (R2) > 0.99.
The richness of the bacterial community represented by observed species and Chao 1 indices, as well as diversity, represented by Shannon index, are shown in Figure 2. In the non-inoculated silage, silage with mulberry leaf ratios of 100% and 80% exhibited lower numbers of observed species (p = 0.01) and Chao 1 (p = 0.01) indices compared with the ML90 and ML60 silages. This indicates that the presence of larger numbers of microbial species was observed in the ML90 and ML60 silages compared to the ML100 and ML80 groups. The Shannon index of the non-inoculated silage with mulberry leaf ratios of 80% and 70% was lower than in the other non-inoculated mixed silages (p < 0.001). Although the non-inoculated ML100 and ML80 silages had similar richness indices of bacterial communities, the variation in Shannon indices indicated different proportions of bacterial species in the two groups. As for the inoculated silage, the observed species (p < 0.001), Chao 1 (p < 0.001) and Shannon (p = 0.01) indices increased, following the increase in the proportion of smooth bromegrass in silage. This might partly explain the decreased fermentation quality along with the reduction in the proportion of mulberry leaves in silage. The growth and activities of undesirable microorganisms resulted in the high diversity of bacterial community. further leading to the silage having poor fermentation quality [43].
PCoA based on weighted unifrac distance matrix clearly reflected the variation within the bacterial community (Figure 3). Divisions in the plots representing the non-inoculated silages indicated that the proportion of smooth bromegrass affected the distribution of the bacterial community in non-inoculated silage. The clear separations between the non-inoculated and inoculated groups in the ML100, ML90 and ML60 silage indicated that L. planturum inoculation altered the bacterial community compositions in these silages. The distribution of the bacterial community in the inoculated silage tended to be more stable, regardless of the proportion of smooth bromegrass compared to the non-inoculated group. This indicated that the inoculant exerted a potential effect, stabilizing the bacterial community in silage.
Among the non-inoculated silages, Lactobacillus predominated among the bacterial community in silage, with a proportion greater than 85%, with the mulberry leaf ratio being 80% and 70% (Figure 4). The highest proportion of Lactobacillus was observed in the ML80 silage (93.44%). The dominance of Lactobacillus in the bacterial community has also been reported in previous studies in well-preserved silages [7,44,45]. Some Enterobacteriaceae species transform nitrogen into alkaline products in silage, such as biogenic amines and other NH4+-containing compounds, and therefore, their presence is undesirable in silage [46]. A high abundance of Enterobacter and an unassigned genus belonging to Enterobacteriaceae were observed in the non-inoculated silage with mulberry leaf ratios of 100% (2.54 × 108 and 4.54 × 108 copies/ng DNA, respectively) and 90% (1.73 × 108 and 3.94 × 108 copies/ng DNA, respectively). LEfSe also exhibited significantly higher abundance of Enterobacteriaceae in ML100CK silage than in other non-inoculated silages (Figure 5A). The major bacteria involved in LA fermentation of alfalfa silage belong to the genera Lactobacillus, Pediococcus, Weissella, and Leuconostoc [34,47]. Signifcant abundance of Weissella and Lactococcus was observed in ML90CK silage compared with other non-inoculated silages (Figure 5A). These genera were thought to be active at the early stage of ensiling, before being outcompeted following prolonged ensiling time due to their poor acid tolerance [43]. Meanwhile, the pH level of ML90CK silage allowed the presence of the two genera in this study (5.0−6.5) [48,49].
As for the inoculated silage, Lactobacillus predominated among the bacterial community in the silage, with a proportion greater than 85% in all groups. However, the proportion of Lactobacillus decreased, while its abundance increased, following the increase in the proportion of smooth bromegrass in the silage (p < 0.05) (Figure 4B). The proportion of an undesirable unassigned genus belonging to Enterobacteriaceae increased in the inoculated silage when the proportion of smooth bromegrass in the silage increased (p < 0.05). LEfSe also exhibited significance with respect to the abundance of Enterobacteriaceae in the ML60LP silage compared with other inoculated silages (Figure 5B). The apparent abundance of Clostridium sensu stricto was observed in the inoculated silage, with mulberry leaf ratios of 70% and 60% (Figure 4B). The growth of these undesirable genera may partly explain the increasing accumulation of NH3-N in silage. In general, the total microbial population decreased with prolonged ensiling time owing to the declining pH and the lack of WSCs, finally achieving a balanced level [50]. In this study, the total population of the bacterial community increased along with the increase in pH value of the inoculated silage, indicating that LA fermentation was insufficient to inhibit the growth of microorganisms in the silages with lower proportions of mulberry leaves after 60 d ensiling.
Notably, while co-ensilement with smooth bromegrass and L. planturum inoculation both improved fermentation quality of mulberry leaf silage, the two strategies failed to exert a stacking effect in this study. Li et al. (2018) [13] reported improvement in fermentation quality as a result of L. planturum inoculation in bur clover and annul ryegrass mixed silage. In this study, however, the proportion of Lactobacillus and LA accumulation decreased (p < 0.05) in the inoculated ML80 silage compared to the non-inoculated group. One potential explanation is that the inoculant had poor adaption to the environmental conditions with the incorporation of smooth bromegrass [18]. It failed to exhibit a synergistic effect with the epiphytic LAB of smooth bromegrass, which led to poorer acidification in silage compared with the ML100 group, finally resulting in the growth of undesirable microbes such as Enterobacter and Clostridium. The screening of adapted LAB inoculants is necessary for further improving the fermentation quality of the mixed silage.

3.4. Spearman Correlations of Bacterial Community with Fermentation Properties in Silage

Spearman correlations of fermentation properties with alpha diversity indices of bacterial community are listed in Table 3. Observed species and Chao 1 indices exhibited positive correlations with pH, AA and NH3-N concentration, and negative correlations with LA concentration in silage (p < 0.05). Shannon index was positively correlated with pH and NH3-N concentration, and negatively correlated with LA concentration in silage (p < 0.05). The close correlations of richness and diversity of bacterial community with fermentation properties in silage indicated that bacterial community played a key role in silage fermentation. A negative correlation of fermentation quality with the diversity of the bacterial community was illustrated. This is based on general recognition that LA bacteria outcompeted other microorganisms and mainly produced LA for pH decline to optimize the ensiling process. The AA content had a strong correlation with the richness of the bacterial community, while a correlation was hardly detected of the AA content with the diversity of the bacterial community. A probable explanation for this is that AA was mainly produced by some specific species in silage, so the concentrations of these products were mainly dependent on the population and activity of its producers rather than the composition of microbes [10].
Spearman correlations further illustrated the interactions of bacterial genera with fermentation properties in silage (Table 3). The pH and NH3-N concentrations were positively correlated with the top 10 most abundant genera in the bacterial community, including the LA producing genera, Lactobacillus, Enterococcus, Weissella, Pediococcus and Lactococcus (p < 0.05). LA concentration was negatively correlated with Lactobacillus, Enterobacter, Enterococcus, Weissella and Lactococcus (p < 0.05), while AA concentration was positively correlated with Citrobacter (p < 0.05). This was due to the low total abundance of the bacterial community in the well-preserved silages in this study. Several previous studies have reported a positive correlation of Lactobacillus proportion with the fermentation quality of silage [10,45,51]. This perhaps indicates that high proportion, rather than abundance, of Lactobacillus in the bacterial community is essential for the optimum fermentation of silage. Negative correlations of fermentation quality with the abundance of undesirable and pathogenic microorganisms, including Enterobacter, Clostridium sensu stricto, Klebsiella and Citrobacter, were also illustrated [46,52,53].

4. Conclusions

Mulberry leaves ensiled with L. planturum inoculation achieved optimum fermentation quality after 60 d ensiling. L. planturum inoculation could thus be a potential strategy for ensiling of mulberry leaves. Combined ensiling with smooth bromegrass could also aid in improving silage quality of mulberry leaves, with the optimum ratio of mulberry leaves being 80%. However, the two strategies failed to exhibit a synergistic effect.

Author Contributions

W.Y.: Conceptualization, Writing—Original Draft. F.Y.: Formal analysis, Writing—Original Draft, Visualization. C.F.: Funding Acquisition, Resources. S.Z.: Methodology, Formal Analysis, Validation, Investigation. X.Z.: Resources, Methodology. Y.W.: Writing—Review and Editing, Supervision, Project Administration, Funding Acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China, grant number: 31772672; Zhengzhou Municipal Science and Technology Bureau, grant number: 2021KJHM0008; Henan Academy of Agricultural Sciences, grant number 2023XK07; and Science and Technology Department of Henan Province, grant number: 222102110478.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The pH value (A), LA (B), AA (C), and NH3-N (D) concentration in the non-inoculated and inoculated mixed silage with different mulberry leaf proportions. CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. Means with different capital letters represent significant difference among different mixed ratios in the non-inoculated silage at p < 0.05. Means with different small letters represent significant difference among different mixed ratios in the inoculated silage at p < 0.05. R, ratio of mulberry leaves and smooth bromegrass; I, inoculation. SEM, standard error of means The text continues here (Figure 2 and Table 2).
Figure 1. The pH value (A), LA (B), AA (C), and NH3-N (D) concentration in the non-inoculated and inoculated mixed silage with different mulberry leaf proportions. CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. Means with different capital letters represent significant difference among different mixed ratios in the non-inoculated silage at p < 0.05. Means with different small letters represent significant difference among different mixed ratios in the inoculated silage at p < 0.05. R, ratio of mulberry leaves and smooth bromegrass; I, inoculation. SEM, standard error of means The text continues here (Figure 2 and Table 2).
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Figure 2. Boxplots of observed species (A), Chao1 (B) and Shannon (C) indices of bacterial communities in mixed silage. CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. The numbers after “ML” indicate the proportion of mulberry leaves in the silage. Means with different capital letters represent significant difference among different mixed ratios in non-inoculated silage at p < 0.05. Means with different small letters represent significant difference among different mixed ratios in the inoculated silage at p < 0.05 (n = 3, bars indicate standard error of means).
Figure 2. Boxplots of observed species (A), Chao1 (B) and Shannon (C) indices of bacterial communities in mixed silage. CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. The numbers after “ML” indicate the proportion of mulberry leaves in the silage. Means with different capital letters represent significant difference among different mixed ratios in non-inoculated silage at p < 0.05. Means with different small letters represent significant difference among different mixed ratios in the inoculated silage at p < 0.05 (n = 3, bars indicate standard error of means).
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Figure 3. Cluster analysis of bacterial communities in mixed silage as assessed by a Principal Coordinate Analysis. CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. The numbers after “ML” indicate proportion of mulberry leaves in silage.
Figure 3. Cluster analysis of bacterial communities in mixed silage as assessed by a Principal Coordinate Analysis. CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. The numbers after “ML” indicate proportion of mulberry leaves in silage.
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Figure 4. Bar plots of bacterial community structures in non-inoculated (A) and inoculated (B) silage. CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. The numbers after “ML” indicate proportion of mulberry leaves in silage.
Figure 4. Bar plots of bacterial community structures in non-inoculated (A) and inoculated (B) silage. CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. The numbers after “ML” indicate proportion of mulberry leaves in silage.
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Figure 5. Comparison of microbial variations using LEfSe analysis in non-inoculated (A) and inoculated silage (B). CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. The numbers after “ML” indicate the proportion of mulberry leaves in silage.
Figure 5. Comparison of microbial variations using LEfSe analysis in non-inoculated (A) and inoculated silage (B). CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. The numbers after “ML” indicate the proportion of mulberry leaves in silage.
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Table
Table 1. Chemical compositions and microbial populations in mulberry leaves and smooth bromegrass before ensiling.
Table 1. Chemical compositions and microbial populations in mulberry leaves and smooth bromegrass before ensiling.
Mulberry LeavesSmooth Bromegrass
Dry matter (%)26.6714.79
Crude protein (% DM)25.5116.47
Neutral detergent fiber (% DM)19.2949.18
Acid detergent fiber (% DM)13.8225.83
Water-soluble carbohydrate (% DM)3.907.45
Lactic acid bacteria (lg cfu/g FM)3.855.69
Coliform bacteria (lg cfu/g FM)5.156.79
Mold (lg cfu/g FM)3.853.65
DM, dry matter; FM, fresh matter; cfu, colony-forming units.
Table
Table 2. Chemical compositions in mixed silage after 60 d ensiling.
Table 2. Chemical compositions in mixed silage after 60 d ensiling.
InoculationItemRatio of Mulberry Leavesp-ValueSEM
ML100ML90ML80ML70ML60
CKDM (% FM)29.66 ab27.62 b30.48 a30.08 a23.52 cp < 0.0010.73
CP (% DM)26.69 a26.98 a25.05 b23.41 c23.20 cp < 0.0010.30
NDF (% DM)23.11 d25.18 c29.18 b28.62 b31.02 ap < 0.0010.39
ADF (% DM)20.53 c22.73 b21.53 bc24.70 a24.60 ap < 0.0010.39
LPDM (% FM)31.53 a30.02 a30.28 a26.78 b25.21 b0.0010.82
CP (% DM)27.34 a25.26 b24.48 b24.30 b23.12 cp < 0.0010.31
NDF (% DM)23.27 c26.12 b28.34 a27.78 ab27.58 ab0.0010.58
ADF (% DM)19.97 c19.97 c24.53 b23.47 b25.90 ap < 0.0010.40
CK, silage without inoculation; LP, silage inoculated with L. plantarum A345. DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber. FM, fresh matter. SEM, standard error of means. Values with different letters in a row indicate significant differences among treatments (p < 0.05).
Table
Table 3. Spearman’s correlation analyses of fermentation properties with alpha diversity indices and abundance of the top 10 most abundant genera.
Table 3. Spearman’s correlation analyses of fermentation properties with alpha diversity indices and abundance of the top 10 most abundant genera.
pHLAAANH3-N
rprprprp
Correlations of fermentation properties with alpha diversity indices
Observed0.590.001−0.500.0050.400.0280.550.002
Chao 10.590.001−0.500.0050.400.0280.540.002
Shannon0.66<0.001−0.420.0190.220.2370.580.001
Correlations of fermentation properties with the top 10 most abundant genera
Lactobacillus0.460.011−0.370.0450.010.9660.64<0.001
Unassigned0.75<0.001−0.350.0560.110.5730.78<0.001
Enterobacter0.71<0.001−0.380.0360.080.6780.73<0.001
Enterococcus0.74<0.001−0.390.0350.250.1850.70<0.001
Weissella0.69<0.001−0.430.0170.300.1080.65<0.001
Pediococcus0.62<0.001−0.290.1240.150.4320.520.003
Lactococcus0.71<0.001−0.410.0260.230.2330.67<0.001
Clostridium sensu stricto0.530.003−0.150.445−0.080.6600.430.017
Klebsiella0.70<0.001−0.280.1310.100.5850.73<0.001
Citrobacter0.530.003−0.360.0540.410.0260.570.001
r, correlation coefficient.
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Yang, W.; Yang, F.; Feng, C.; Zhao, S.; Zhang, X.; Wang, Y. Fermentation Properties and Bacterial Community Composition of Mixed Silage of Mulberry Leaves and Smooth Bromegrass with and without Lactobacillus plantarum Inoculation. Fermentation 2023, 9, 279. https://doi.org/10.3390/fermentation9030279

AMA Style

Yang W, Yang F, Feng C, Zhao S, Zhang X, Wang Y. Fermentation Properties and Bacterial Community Composition of Mixed Silage of Mulberry Leaves and Smooth Bromegrass with and without Lactobacillus plantarum Inoculation. Fermentation. 2023; 9(3):279. https://doi.org/10.3390/fermentation9030279

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Yang, Weihan, Fengyuan Yang, Changsong Feng, Shanshan Zhao, Xueying Zhang, and Yanping Wang. 2023. "Fermentation Properties and Bacterial Community Composition of Mixed Silage of Mulberry Leaves and Smooth Bromegrass with and without Lactobacillus plantarum Inoculation" Fermentation 9, no. 3: 279. https://doi.org/10.3390/fermentation9030279

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