fermentation
Article
Effect of Mixing Peanut Vine on Fermentation Quality,
Nitrogen Fraction and Microbial Community of High-Moisture
Alfalfa Silage
Yu Sun 1,† , Chunhui Wu 1,† , Xiaowei Zu 2 , Xiaolin Wang 1 , Xiaomeng Yu 1 , Huan Chen 1 , Ling Xu 1 ,
Mingya Wang 1, * and Qiufeng Li 1, *
Citation: Sun, Y.; Wu, C.; Zu, X.;
Wang, X.; Yu, X.; Chen, H.; Xu, L.;
Wang, M.; Li, Q. Effect of Mixing
Peanut Vine on Fermentation Quality,
Nitrogen Fraction and Microbial
Community of High-Moisture Alfalfa
Silage. Fermentation 2023, 9, 713.
https://doi.org/10.3390/
fermentation9080713
Academic Editor: Qing Zhang
Received: 14 June 2023
Revised: 5 July 2023
Accepted: 10 July 2023
Published: 27 July 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Fermentation 2023, 9, 713. https://doi.org/10.3390/fermentation9080713
1 College of Animal Science and Technology, Hebei Agricultural University, Baoding 071000, China;
SunYu82475@163.com (Y.S.)
2 Hebei Provincial Animal Husbandry Station, Shijiazhuang 050000, China
* Correspondence: hebeigrass@163.com (M.W.); lqf582@126.com (Q.L.)
† These authors contributed equally to this work.
Abstract: Fresh alfalfa is difficult to ensile successfully because of its high moisture content and
greater susceptibility to spoilage by Clostridia, Bacilli or Enterobacter. In this study, we evaluated the
effects of mixing high-moisture alfalfa with peanut vine in different proportions on the bacterial
communities and fermentation characteristics of silage. The high-moisture alfalfa and peanut vine
were mixed at ratios of 10:0 (CK), 8:2 (TI), 7:3 (T2), 6:4 (T3) and 5:5 (T4), respectively. For each
treatment, silos (25 × 35 cm) were anaerobically fermented in darkness at room temperature and
analyzed after 45 days. The results showed that the CK silage was weakly fermented, as indicated by
a low lactic acid concentration, a high pH value, and high levels of propionic acid (PA), butyric acid
(BA) and ammonia nitrogen (NH3 -N). As the proportion of peanut vine in the mixture increased, the
pH level decreased, and levels of BA, propionic acid, NH3 -N, crude protein(CP), nonprotein nitrogen
and soluble protein also declined (p < 0.05), while the lactic acid concentration increased and levels of
neutral detergent fiber (NDF) and water-soluble carbohydrates (WSC) also rose (p < 0.05). A protein
component analysis of silage mixtures using the Cornell Net Carbohydrate and Protein System
(CNCPS) showed that the content of the nonprotein nitrogen component (PA) decreased when the
proportion of peanut vine increased, whereas the content of rapidly degraded protein (PB1) increased.
Mixing with peanut vine also influenced the distribution of the bacterial community. Compared with
the CK silage, the relative abundances of Enterococcus, Garciella and Anaerosporobacter in T2, T3 and
T4 were significantly lower, while the relative abundance of Lactobacillus was significantly higher.
In the T2, T3 and T4 groups, Garciella and Anaerosporobacter were not detected. In summary, in this
study, we ensiled high-moisture alfalfa, which was weakly fermented. We found that mixing with
peanut vine improved fermentation quality and optimized the structure of the bacterial community.
Therefore, to improve the fermentation quality and nutritional value of silage, high-moisture alfalfa
should be ensiled with at least 30% peanut vine.
Keywords: alfalfa; peanut vine; fermentation quality; nitrogen fraction; microbial community
1. Introduction
Alfalfa (Medicago sativa L.) is planted widely across the world due to its high levels
of protein, vitamins and many essential minerals [1,2]. However, harvesting alfalfa as hay
involves a high risk of damage from adverse weather and possible leaf loss by shatter-
ing. Ensiling has been adopted worldwide as a method for preserving green forage and
supplying moist feedstock all year [3]. However, alfalfa is difficult to ensile successfully
due to its high buffering capacity, high natural moisture content, and low concentration of
water-soluble carbohydrates (WSC) [4]. High moisture content makes alfalfa silage more
susceptible to being spoiled by Clostridia, Bacilli or Enterobacter, resulting in high levels of
https://www.mdpi.com/journal/fermentation
Fermentation 2023, 9, 713 2 of 12

butyric acid accumulation and proteolysis, thus reducing animal DM intake and increasing
contamination in milk [5]. Therefore, natural wilting and mixing with other forage crops
have become the conventional methods used to improve the ensilability of alfalfa. However,
it is very difficult to completely avoid unexpected rainfall events during wilting in actual
production processes. The authors of [6] reported that the quality of alfalfa silage was
reduced and the loss of nutrients increased as a result of rain damage. To counter the risks
associated with rainfall and improve the success rate of the ensiling process, alfalfa can be
mixed with forage crops with high dry matter content in a certain proportion to increase
the content of the silage fermentation substrate and ultimately obtain high-quality silage,
especially in rainy seasons.
Peanut vine, as a by-product of peanut cultivation, is usually abandoned or burned,
resulting in significant environmental pollution and a waste of resources [7]. In recent years,
peanut vine has been gradually applied to the production of ruminants due to its high
feeding value, characterized by high levels of crude protein (CP), digestible DM, neutral
detergent fiber (NDF), and acid detergent fiber (ADF) [8–10]. Alfalfa silage mixed with
peanut vine could increase the fermentation substrate and avoid wilting in the field, result-
ing in more successful silage production and lower levels of crushing loss and respiratory
consumption. Moreover, alfalfa and peanut vines exhibit a positive associative effect to
improve the utilization of feed [10]. To date, research on mixed silage using high-moisture
alfalfa has focused on whole-plant corn [11,12], jujube powder [13], red clover [14], Moringa
oleifera leaves [15] and Neolamarckia cadamba leaves [16]. However, there has been little
information so far regarding the effect of high-moisture alfalfa mixed with peanut vine on
the microbial community, fermentation quality and nitrogen fraction.
Ensiling is an anaerobic, microbial-based fermentation process [17], in which microbial
communities and biochemical reactions are positively associated with the quality of silage.
Further research on the microbial communities of co-ensiled alfalfa and peanut vine would
broaden our knowledge of the silage process.
In this study, therefore, we aimed to evaluate the effects of mixing high-moisture alfalfa
with peanut vine in different proportions on the bacterial communities and fermentation
characteristics of silage. It was hypothesized that combining alfalfa with peanut vine would
increase the lactic acid concentration of the silage and decrease levels of pH and NH3 -N.
We also sought to confirm that the relative abundance of the major lactic acid bacteria
involved in silage fermentation would be increased with a higher proportion of peanut
vine in the mixture.

2. Materials and Methods

2.1. Raw Materials and Silage Preparation
Alfalfa (cultivar “WL358”) was cultivated at the experimental field of Hebei Agri-
cultural University (Baoding City, Hebei Province, China). Second-regrowth alfalfa was
harvested during the early flowering period on 29 June 2022. This process was carried
out by hand using a sickle, leaving 8 cm of stubble, which was subsequently chopped to
approximately 2 cm with a forage cutter. Chopped peanut vine (about 2 cm) was purchased
from a village in Tang County, Baoding City, Hebei Province, China. Alfalfa (without
wilting) was immediately mixed with peanut vine at ratios of 10:0 (CK), 8:2 (T1), 7:3 (T2),
6:4 (T3) and 5:5 (T4), respectively. The alfalfa moisture content was 76.63%, while the
peanut vine moisture content was 7.14%. Table 1 shows the chemical composition of the
materials used. Next, 400 g amounts of forage mass were packed into plastic silo bags
(25 × 35 cm) and then vacuumed and sealed using an automatic vacuum sealer (Aoba
brand P-290; Guangdong Dongguan Qingye Company, Dongguan, China). A total of
20 bags (5 treatments × 4 repeats) were filled and then kept at room temperature (25–32 ◦ C).
After 45 days of ensiling, the silage bags were opened to determine fermentation quality,
chemical composition, and bacterial communities.
Fermentation 2023, 9, 713 3 of 12

Table 1. Chemical composition and microbial population of peanut vine and fresh alfalfa prior to
ensiling ( ± SD, n = 3, dry-matter basis).

Item Alfalfa Peanut Vine

DM (g/kg FW) 233.67 ± 1.86 928.57 ± 0.75
CP (g/kg DM) 205.84 ± 0.48 72.36 ± 0.77
NDF (g/kg DM) 434.25 ± 8.76 575.20 ± 7.69
ADF (g/kg DM) 342.72 ± 5.54 466.70 ± 2.73
WSC (g/kg DM) 35.90 ± 0.22 39.28 ± 1.09
Ash (g/kg DM) 145.39 ± 2.07 86.52 ± 0.87
TP (g/kg CP) 685.81 ± 2.08 852.58 ± 1.15
NPN/(g/kg CP) 314.19 ± 2.08 147.42 ± 1.15
SP (g/kg CP) 402.10 ± 1.62 240.55 ± 0.92
NDIP (g/kg CP) 197.21 ± 0.44 273.03 ± 1.17
ADIP (g/kg CP) 191.05 ± 0.15 231.88 ± 2.61
PA (g/kg CP) 314.19 ± 2.08 147.42 ± 1.15
PB1 (g/kg CP) 87.92 ± 0.64 93.13 ± 1.95
PB2 (g/kg CP) 400.69 ± 1.93 486.42 ± 1.30
PB3 (g/kg CP) 6.16 ± 0.31 41.15 ± 1.72
PC (g/kg CP) 191.05 ± 0.15 231.88 ± 2.61
Lactic acid bacteria (log10 cfu/g FW) 5.25 ± 0.01 3.76 ± 0.09
Aerobic bacteria (log10 cfu/g FW) 6.00 ± 0.05 4.94 ± 0.05
Yeast (log10 cfu/g FW) 4.43 ± 0.07 4.06 ± 0.09

2.2. Analysis
The bags were opened on a clean bench; one subsample (20 g) was taken from each
silo and immediately blended with 180 mL of sterile distilled water, then shock and serially
diluted. Quantities of lactic acid bacteria, aerobic bacteria and yeasts were incubated and
counted using De Man, Rogosa and Sharpe (MRS) agar; nutrient agar; and potato dextrose
agar, respectively [18].
Another 20 g subsample was taken from each silo and mixed with 180 mL of distilled
water, stored at 4 ◦ C for 24 h, and then filtered. The filtrate was for organic acid, pH
and ammonia nitrogen (NH3 -N) analysis. The pH of this filtered sample was measured
immediately using a UB-7 (Denver Instruments Co., Ltd. Denver, CO, USA) precision
acidity meter. Content levels of organic acids (lactic acid, acetic acid, propionic acid and
butyric acid) were determined by high-performance gas chromatography (Agilent 7890A
gas chromatograph, purchased from Agilent, CA, USA), and the NH3 -N was determined
colorimetrically, all as previously described by Song [19].
The remaining silage samples were dried in an air-drying oven for 48 h at 65 ◦ C to DM,
followed by crushing with a pulverizer to 10 mesh and 40 mesh. Concentrations of NDF
and ADF were measured using heat-stable amylase and sodium sulfite by an ANKOM
A2000i fully automatic fiber meter according to the method of Van Soest et al. [20]. WSC
was determined by the anthrone sulfate method of Murphy [21]. CP was analyzed using
a Kjeldahl nitrogen analyzer according to the methods described by the Association of
Official Analytical Chemists [22] and converted to total nitrogen (TN) by a coefficient of 6.25.
Soluble protein (SP), NDF insoluble protein (NDIP) and acid detergent insoluble protein
(ADIP) were determined as described by Licitra et al. [23]. Protein fractions of PA, PB1,
moderately degraded proteins (PB2), lowly degraded proteins (PB3) and insoluble proteins
(PC) were calculated according to the Cornell Net Carbohydrate and Protein System [24].
Silage bacterial community was analyzed by the next-generation sequencing technique,
according to Wang et al. [16]. The 16S rDNA V3-V4 (a) regions were amplified using primers
338F (ACTCCTACGGGAGGCAGCA) and 806R (GGACTACHVGGGTWTCTAAT).

2.3. Data Statistics and Analysis

The experiment was conducted using a completely randomized design with a 5 × 4
(5 treatments and 4 duplicates) factorial arrangement. Each silo was used as an experimental
Fermentation 2023, 9, 713 4 of 12

unit. All data were analyzed using one-way analysis of variance (ANOVA, General Linear
Models) by SPSS (version 23), with the proportion of peanut vine in the mixture as a fixed
effect and duplicates as a random effect. The means were compared for significance using
Duncan’s method at p < 0.05. The results were expressed as the average mean plus or minus
the standard error. The data from high-throughput sequencing were analyzed using the
OmicShare tool, a free online platform for data analysis (http://www.omicshare.com/tools
(accessed on 20 December 2022)).

3. Results
3.1. Characteristics of Fresh Alfalfa and Peanut Vine before Ensiling
The chemical composition of the silage material in this experiment is shown in Table 1.
Briefly, DM and WSC concentrations in fresh forages were as follows: for alfalfa, 233.67 g/kg
FW and 35.90 g/kg DM; for peanut vine, 928.57 g/kg FW and 39.28 g/kg DM. The CP
concentrations in fresh alfalfa and peanut vine were 205.84 g/kg DM and 72.36 g/kg DM,
respectively. The NDF and ADF concentrations in peanut vine were significantly higher
than those in alfalfa, at 32.46% and 36.18%, respectively.

3.2. Fermentation Quality and Chemical Composition of Silage

The fermentation and chemical parameters of silage are provided in Tables 2 and 3.
The pH values, content levels of NH3 -N organic acids and microbial populations of alfalfa
silage with peanut vine are presented in Table 2. The pH values, PA concentration and
BA concentration of alfalfa silage all significantly decreased after mixing with peanut vine
(p < 0.05), whereas the LA and AA content of alfalfa silage significantly increased with
more peanut vine in the mixture (p < 0.05). The amounts of lactic acid bacteria and yeast in
the CK and T4 groups were significantly lower than those in T1, T2 and T3 (p < 0.05), and
the quantities of aerobic bacteria in the CK and T3 groups were significantly lower than
those in T1, T2 and T4 (p < 0.05). As shown in Table 3, levels of DM, WSC, NDF and ADF
gradually increased, but concentrations of CP and ash both progressively decreased in all
silages as the proportion of peanut vine increased. Among these, the CP content in the T2
and T3 groups was significantly higher than that in the T4 group but lower than that in the
CK and T1 groups (p < 0.05). Among the groups T1–T4, NDF content was lowest in the T2
group, and ADF content was lowest in the T1 group. Levels of WSC in the T2, T3 and T4
groups were significantly lower than in the CK and T1 groups (p < 0.05). Compared with
the CK group, the ash content in the experimental group was significantly lower (p < 0.05).

3.3. Nitrogen Fractions and CNCPS Composition of Silage

The nitrogen fractions of silages with different mixing ratios of alfalfa and peanut
vine exhibited dramatic changes during fermentation (Tables 4 and 5). In the experimental
group, TP increased significantly with an increase in the proportion of peanut vine in the
silage mixture (p < 0.05), while NPN, PA and SP decreased significantly (p < 0.05). The
NDIP content in the T1, T2, T3 and T4 groups was significantly higher than in the CK group
(p < 0.05), and the T4 group had the highest CP content, at 209.86 g/kg CP. The PB2 levels
of the CK and T2 groups were significantly higher than those of the T1 group but lower
than that of the T3 and T4 groups (p < 0.05).

3.4. Analysis of Microbial Diversity Results

3.4.1. Microbial Composition at Phyla Level
At the phyla classification level (Figure 1), there were differences in the microbial
community composition of mixed-silage samples with different proportions of alfalfa
and peanut vine. The microorganisms in alfalfa raw materials are mainly Proteobacteria
and Firmicutes, while the microorganisms in peanut vine are mainly Proteobacteria and
Actinobacteriota. In the CK, T3 and T4 groups, the relative abundances of Firmicutes reached
97.09%, 52.29% and 65.90%, respectively. Therefore, in the CK and T4 groups, Firmicutes
were the absolute dominant flora, while the dominant flora in the T3 group were Firmicutes
Fermentation 2023, 9, 713 5 of 12

and Proteobacteria. The relative abundances of Proteobacteria in the T1 and T2 groups were
53.74% and 61.41%, respectively, making these the absolute dominant bacteria. The relative
indexing of Actinobacteria in the CK, T2, T3 and T4 groups was below 1.00% but reached
1.87% in the T1 group. In addition to the two raw materials, Bacteroidetes were also detected
in the T1 group, while Planctomycetes were only detected in peanut stalks.
Table 2. Effect of different mixing ratios of alfalfa and peanut vine on silage fermentation quality
(±SD, n = 4, dry-matter basis).

Item CK T1 T2 T3 T4
pH 7.36 ± 0.03 a 5.56 ± 0.05 b 5.26 ± 0.01 c 5.06 ± 0.03 d 4.86 ± 0.01 e
AA (g/kg DM) 20.11 ± 0.41 c 18.47 ± 0.17 d 23.25 ± 0.22 b 24.78 ± 0.75 a 25.77 ± 1.45 a
PA (g/kg DM) 10.50 ± 0.49 a 2.30 ± 0.03 b 0.75 ± 0.01 c 0.27 ± 0.09 d 0d
BA (g/kg DM) 17.69 ± 0.30 a 3.37 ± 0.10 b 0.40 ± 0 c 0c 0c
LA (g/kg DM) 10.36 ± 0.30 d 12.86 ± 0.36 c 26.74 ± 0.33 b 29.72 ± 0.82 a 31.05 ± 0.64 a
NH3 -N (g/kg TN) 65.02 ± 2.20 a 36.38 ± 1.02 b 23.96 ± 0.95 c 22.75 ± 1.03 c 21.27 ± 1.53 c
Lactic acid bacteria (log10 cfu/g FW) 4.86 ± 0.04 d 5.57 ± 0.02 a 5.38 ± 0.03 b 5.35 ± 0.01 b 4.97 ± 0.03 c
Aerobic bacteria (log10 cfu/g FW) 4.87 ± 0.01 d 5.12 ± 0.06 a 4.75 ± 0.01 b 4.07 ± 0.08 d 4.42 ± 0.05 c
Yeast (log10 cfu/g FW) 4.67 ± 0.05 d 5.56 ± 0.02 a 5.31 ± 0.02 b 5.34 ± 0.01 b 5.00 ± 0.03 c
Note: a–e Means in the same row followed by different superscript letters are significant differences (p < 0.05).

Table 3. Effect of different mixing ratios of alfalfa and peanut vine on the nutritional quality of silage
(±SD, n = 4, dry-matter basis).

Item CK T1 T2 T3 T4
205.16 ± 3.63 e d 424.14 ± 2.68 c b 594.76 ± 9.12 a
DM (g/kg FW) 349.59 ± 2.16 516.37 ± 7.36
CP (g/kg DM) 171.65 ± 0.44 a 151.23 ± 2.15 b 136.22 ± 1.24 c 133.32 ± 2.30 c 124.68 ± 2.29 d
NDF (g/kg DM) 492.84 ± 2.92 d 549.88 ± 6.82 bc 541.52 ± 5.13 c 562.23 ± 7.86 b 582.88 ± 3.70 a
ADF (g/kg DM) 379.55 ± 15.30 c 435.44 ± 12.71 b 441.85 ± 7.47 ab 458.88 ± 10.03 ab 476.15 ± 5.64 a
WSC (g/kg DM) 12.19 ± 0.50 d 13.27 ± 0.46 d 15.74 ± 0.60 c 20.83 ± 0.65 b 29.80 ± 1.11 a
Ash (g/kg DM) 137.86 ± 1.47 a 120.61 ± 0.99 b 115.61 ± 0.93 c 117.46 ± 2.56 bc 109.93 ± 0.54 d
Note: a–e Means in the same row followed by different superscript letters are significant differences (p < 0.05).

Table 4. Nitrogen fractions of the different mixture ratios of alfalfa and peanut vine ( ± SD, n = 4,
dry-matter basis).

Item CK T1 T2 T3 T4
TP (g/kg CP) 396.39 ± 2.39 d 373.92 ± 5.60 e 428.55 ± 5.17 c 492.08 ± 2.74 b 524.33 ± 7.56 a
NPN (g/kg CP) 603.61 ± 2.39 b 626.08 ± 2.42 a 571.45 ± 5.17 c 507.92 ± 2.73 d 475.67 ± 7.56 e
SP (g/kg CP) 603.58 ± 3.33 b 643.00 ± 2.28 a 596.11 ± 5.80 b 542.46 ± 5.23 c 519.79 ± 7.37 d
NDIP (g/kg CP) 142.49 ± 3.50 c 170.51 ± 4.93 b 171.26 ± 1.27 b 179.34 ± 4.36 b 209.86 ± 8.76 a
ADIP (g/kg CP) 144.85 ± 4.42 a 127.35 ± 1.56 b 147.49 ± 1.52 a 152.55 ± 6.06 a 156.99 ± 6.26 a
Note: a–e Means in the same row followed by different superscript letters are significant differences (p < 0.05).
TP—true protein; NPN—non-protein nitrogen; SP—soluble protein; NDIP—neutral detergent insoluble protein;
ADIP—acidic detergent insoluble protein.

Table 5. Analysis of the components of CNCPS after ensiling with different mixture ratios of alfalfa
and peanut vine ( ± SD, n = 4, dry-matter basis).

Item CK T1 T2 T3 T4
b 626.08 ± 2.42 a 571.45 ± 5.17 c d 475.67 ± 7.56 e
PA (g/kg CP) 603.61 ± 2.39 507.92 ± 2.73
PB1(g/kg CP) 0.58 ± 0 e 15.18 ± 0.39 d 29.62 ± 1.48 c 35.63 ± 0.78 b 40.07 ± 0.45 a
PB2 (g/kg CP) 250.46 ± 5.24 b 190.64 ± 8.48 c 229.58 ± 8.17 b 277.12 ± 7.12 a 280.01 ± 9.42 a
PB3 (g/kg CP) 0.50 ± 0.04 d 37.39 ± 1.16 b 24.72 ± 0.45 c 26.75 ± 1.39 c 47.26 ± 6.64 a
PC (g/kg CP) 144.85 ± 4.42 a 127.35 ± 1.65 b 147.49 ± 1.52 a 152.55 ± 6.06 a 156.99 ± 6.26 a
Note: a–e Means in the same row followed by different superscript letters are significant differences (p < 0.05).
PA—the proportion of NPN to CP; PB1—the proportion of rapidly degrading protein to CP; PB2—the proportion
of moderately degrading protein to CP; PB3—the proportion of slow-degrading protein to CP; PC—the proportion
of bound protein to CP.

Fermentation 2023, 9, 713
were Firmicutes and Proteobacteria. The relative abundances of Proteobacteria in the T1 and
T2 groups were 53.74% and 61.41%, respectively, making these the absolute dominant
bacteria. The relative indexing of Actinobacteria in the CK, T2, T3 and T4 groups was below
1.00% but reached 1.87% in the T1 group. In addition to the two raw materials,
Bacteroidetes were also detected in the T1 group, while Planctomycetes were only detected
6 of 12
in peanut stalks.

Microbial composition of alfalfa mixed silage at the

Figure 1. Microbial the backdoor
backdoor level in
in different
different proportions
of alfalfa and peanut vine.

3.4.2. Microbial Composition

3.4.2. Microbial Composition at at the
the Taxonomic
Taxonomic Level
Level of
of the
the Genus
Genus
rmentation 2022, 8, x FOR PEER REVIEW There
There were differences in the species composition at the taxonomic
were differences in the species composition at the taxonomic level
level of
of the genus
the genus 7
in different treatment groups (Figure 2). Prior to ensiling, alfalfa raw materials
in different treatment groups (Figure 2). Prior to ensiling, alfalfa raw materials mainly mainly
contained Staphylococcus,
contained Staphylococcus, accounting
accounting forfor 15.61%, and Pantoea,
15.61%, and Pantoea, accounting
accounting for 15.62%, while
for 15.62%, while
peanut vine mainly contained Methylovirgula, accounting for 11.52%, and
peanut vine mainly contained Methylovirgula, accounting for 11.52%, and Massilia, Massilia, account-
ing for 9.68%.
Enterococcus,
accounting forAfter
Garciellasilage,
9.68%. and in
After the CK ingroup,
Anaerosporobacter;
silage, the CKthe group,
main genera
in thethe of
T1,main
T2,bacteria
T3 and
genera were
ofT4 Enterococcus,
groups,
bacteria Enterococ
were
Garciella and Anaerosporobacter; in the T1, T2, T3 and T4 groups, Enterococcus, Lactobacillus
Lactobacillus and were
and Enterobacter Enterobacter wereLactobacillus,
the main genera; the main therefore,
genera;had Lactobacillus, therefore, had
an absolute advantage
absolute advantage
in the T4 group. in the T4 group.

Figure 2. Microbial
Figure 2. Microbialcomposition
composition ofof alfalfa,
alfalfa, peanut
peanut strawstraw andmixed
and silage silageinmixed
differentinproportions.
different proporti

3.5. Correlation between Fermentation Characteristics and Microbial Communities in

3.5. Mixed-Alfalfa-and-Peanut-Straw
Correlation between FermentationSilage Characteristics and Microbial Communities in Mixed
Alfalfa-and-Peanut-Straw
Correlations betweenSilage
silage fermentation quality indicators and microbial communi-
ties were analyzed between
Correlations and presented in the form
silage of heat maps quality
fermentation (Figure 3). indicators
The results showed
and micro
that Lactobacillus and Enterobacter were significantly negatively correlated with pH, NH3 -N,
communities were analyzed and presented in the form of heat maps (Figure 3). The res
PA and BA content (p < 0.05); Lactobacillus exhibited a significant positive correlation with
showed that (p
LA content Lactobacillus
< 0.05). and Enterobacter were significantly negatively correlated w
pH, NH3-N, PA and BA content (p < 0.05); Lactobacillus exhibited a significant pos
correlation with LA content (p < 0.05).
 Alfalfa-and-Peanut-Straw Silage
Correlations between silage fermentation quality indicators and microbial
communities were analyzed and presented in the form of heat maps (Figure 3). The results
showed that Lactobacillus and Enterobacter were significantly negatively correlated with
Fermentation 2023, 9, 713 pH, NH3-N, PA and BA content (p < 0.05); Lactobacillus exhibited a significant positive
7 of 12
correlation with LA content (p < 0.05).

Heat
Figure3.3.Heat
Figure map
map of of
thethe correlation
correlation between
between silage
silage fermentation-quality
fermentation-quality indicators
indicators and and micro-
microbial
bial communities.
communities.
4. Discussion
4. Discussion
4.1. Characteristics of Fresh Alfalfa and Peanut Vine before Ensiling
4.1. Characteristics of Fresh Alfalfa and Peanut Vine before Ensiling
There are great differences in the chemical composition of alfalfa and peanut straw raw
There are
materials. great
In this differences
study, the DMin the chemical
content composition
of the two materials of
wasalfalfa
233.70and peanut
and 928.57straw
g/kg,
raw
respectively. Therefore, mixing the two raw materials in different proportionsand
materials. In this study, the DM content of the two materials was 233.70 can 928.57
reduce
the moisture content—and thus improve the quality—of the resulting silage. The CP
content of peanut vine (72.00 g/kg DM) is higher than that of sorghum straw, wheat
straw [25], corn stover [26] and oat hay [27]. At the same time, peanut vine is relatively low
in NDF and ADF content, and its nutritional value is relatively high, which is conducive to
improving the quality of silage after mixing.

4.2. Fermentation Quality and Chemical Composition of Silage

High-moisture alfalfa silage is more vulnerable to damage from clostridial spores,
leading to the accumulation of butyric acid in periods of cloudy weather during wilting [28].
To avoid this, combining high-moisture alfalfa with another crop at ensiling is an alternative
way to enhance its fermentability. In the present study, mixing high-moisture alfalfa with
peanut vine in silage resulted in lower levels of pH, NH3 -N, propionic acid and butyric acid
as the proportion of peanut vine increased, indicating that mixing with peanut vine helps
improve fermentation quality. NH3 -N, pH, VFA and lactic acid are important indicators in
the evaluation of silage fermentation quality [29]. A lower pH indicates a better quality of
silage feed. In this experiment, pH decreased significantly with increased proportions of
peanut vine, and the pH values of the T2, T3 and T4 groups were significantly lower than
those of the CK and T1 groups. This is because the DM content in the silage increased as the
proportions of peanut vine increased, thereby providing sufficient fermentation substrate
for lactic acid bacteria and accelerating the production of lactic acid, thus reducing pH,
in a result that was similar to that reported by Zhang Jinxia [30]. NH3 -N reflects the
degradation of protein in silage, which is usually caused by the combined action of plant
protease activity and microbial activity. In addition, an increase in NH3 -N content increases
the production of ammonia during silage fermentation, which accelerates the growth of
decay substances, lowers the quality of silage, and affects the palatability of feed [31]. In
this experiment, the NH3 -N content of the T2, T3 and T4 groups with mixed ratios of alfalfa
Fermentation 2023, 9, 713 8 of 12

and peanut vine was significantly lower than that of the CK and T1 groups, indicating that
mixed silage of high-moisture alfalfa and peanut vine could reduce protein degradation
and that this may be due to the inhibition of protease and microbial activities resulting
from a low level of pH. Fairbairn showed that silage with a low pH could effectively
inhibit protease activity and thus reduce protein degradation and NH3 -N content; these
findings are consistent with the results of the present study [32]. Fermentation is mainly
carried out under anaerobic conditions. The anaerobic environment is also conducive
to the production of organic acids. Silage fermentation involves mainly lactic acid and
acetic acid, but the process is also accompanied by the growth of decaying acids such as
propionic acid and butyric acid. The higher the content of lactic acid and acetic acid in
the fermentation process, the better the silage quality. Lactic acid in silage is produced by
lactic acid bacteria using WSC as a substrate. A high content of lactic acid can inhibit the
growth of harmful microorganisms and improve fermentation quality [33]. In the present
study, as the proportion of peanut vine increased, the content of lactic acid also increased
significantly, while levels of propionic acid and butyric acid decreased significantly. These
results may be related to the activities of both beneficial and harmful microorganisms in
silage materials. High DM content and high WSC content can increase the fermentation
substrate. Moreover, when water content is low, the cell-juice osmotic pressure of silaged
raw materials increases, which can inhibit the growth activity of Clostridium and reduce
butyric acid production. The normal life activities of other microorganisms can also be
easily restricted [34]. Researchers have previously reported that, as the water content of
raw materials decreases, the number of microorganisms decreases significantly and the
fermentation quality of alfalfa silage increases, and these findings are consistent with the
results of the present study [35,36].

4.3. Effects of Different Proportions of Alfalfa and Peanut Vine on the Nutritional Quality of Silage
Previous studies have shown that the DM content of silage is the key to obtaining
high-quality fermented feed. Both high and low levels of DM content, as well as low
density and poor stability of fermented feed, can result in Clostridium fermentation [37,38].
In this experiment, the DM content of alfalfa raw materials was less than 30%; such a level
is not conducive to good fermentation quality and results in poor silage. However, the
DM content of peanut vine was as high as 90%, and its CP content was 7.2%. After mixing,
therefore, the differing DM levels of the two materials complemented each other, and the
CP content of the silage could be maintained at a high level. Studies by Zhao Mengdi [39]
have shown that Clostridium fermentation can be effectively avoided when the content of
DM in silage is higher than 30%. In this experiment, although the DM content of the T1
group was less than 30%, the butyric acid content of the T1 group was significantly higher
than that of the T2, T3 and T4 groups. This may have been because alfalfa and peanut
vine were legumes; the WSC content decreased as a result, and silage was difficult. CP
is the most important index for evaluating nutritional value, being positively correlated
with the nutritional value of forage grass. The higher the content of CP, the higher the
nutritional value of forage grass [40]. In this experiment, compared with the control group,
the CP content of each treatment group was lower by 2.04%, 3.54%, 3.84% and 4.70%,
respectively. Because the CP content of peanut vine was lower than that of alfalfa, the CP
content decreased as the proportion of peanut vine increased. NDF and ADF are effective
indicators of the digestibility of silage [41]. In feed, NDF is mainly composed of plant cell
walls, while ADF consists mainly of lignin and cellulose [42]. There is a negative correlation
between ADF levels in feed and the digestibility of the latter by ruminants. The lower
the level of ADF, the higher the nutritional value of the feed. Among the raw materials,
the content levels of NDF and ADF in peanut vine were, respectively, 14.09% and 12.40%
higher than in alfalfa, so that NDF and ADF content increased significantly with increases
in the proportion of peanut vine, indicating that the nutritional quality decreased as the
proportion of peanut vine increased. This finding is related to the nutritional characteristics
of alfalfa and peanut vine.
Fermentation 2023, 9, 713 9 of 12

4.4. Effects of Different Proportions of Alfalfa and Peanut Vine on Nitrogen Composition and
CNCPS of Silage
The purpose of silage is to prolong the storage life of forage and reduce the loss
of nutrition. Protein degradation is one of the most important biochemical reactions in
silage [43]. Protein is converted to NPN (such as small peptides, free amino acids and
ammonia nitrogen) under the action of microorganisms and proteases [44]. The conversion
of true protein to NPN (PA component) affects nitrogen utilization in ruminants, increasing
losses of urinary and fecal nitrogen and leading to environmental pollution [45]. Therefore,
it is very important to reduce the loss of protein in the silage process, especially when
it involves legume forage. In this study, we found that when the proportion of peanut
vine increased, the content of TP increased significantly, while NPN and SP decreased
significantly. SP was composed of PA and PB1 components. With increases in the peanut
seedling mixture ratio, PA decreased significantly, while PB1 increased significantly. Among
these, PA is the main source of protein in roughage, and its nutritional value is consistent
with that of real protein. PB1 is a kind of true protein that can degrade rapidly so that
it is lost rapidly in the rumen and cannot be used further [46]. Therefore, as the peanut
seedling content increased, the protein quality decreased. NDIP consists of PB3 and PC.
NDIP and ADIP belong to a group of proteins that bind to plant cell walls. Compared
with NDIP, ADIP is less easily digested and used by ruminants [23]. Therefore, the PC
part is mainly the unavailable binding protein part, which cannot be degraded by rumen
bacteria and cannot be digested by the hindgut. In this experiment, the content levels of
PC and PB3 both increased as the proportion of peanut vine increased, indicating that the
higher the proportion of peanut vine, the lower their nutritional value. This may have
been due to the different proportions of the two raw materials in the mixed silage, which
resulted in different levels of protein composition in the feed. Sun Honghong found that
the nutritional value of peanut vine was lower than that of alfalfa and that its content levels
of NDF, ADF, NDIP and ADIP were relatively high [47]. This finding may explain why
protein quality decreased as the proportion of peanut vine increased.

4.5. Effects of Different Proportions of Alfalfa and Peanut Vine on the Microbial Composition
of Silage
Microorganisms play an important role in the silage process. Therefore, in order to
make high-quality silage, it is necessary to clearly understand the dynamic changes in
bacterial communities during the fermentation process. In recent years, 16S rRNA high-
throughput sequencing technology has been widely used in silage. Changes in community
structures are always quantified by a series of nonparametric ecological indexes [48]. The
composition of microflora is closely related to the quality of silage. In this experiment,
the dominant flora at the phylum level was found to be Streptomyces and Proteus, a result
similar to that reported by Wang [49]. Streptomyces can secrete a variety of cellulases, lipases
and proteases and can survive in anaerobic and low-pH environments [50]. At the same
time, the diversity and composition of alfalfa silage were evaluated, and it was found that
the dominant genera were Lactobacillus, Enterococcus and Enterobacter. However, Wang [11]
pointed out that Lactobacillus, Weissiella and Enterobacter are the dominant bacteria in alfalfa
silage. Differences in microbial communities may be related to geographical location, forage
variety, growth stage, DM content and other factors.
The bacterial community reflects the fermentation characteristics of silage. The authors
of [44] found that the more Lactobacillus in silage, the higher the fermentation quality. In
this study, the silage quality of the CK group was the poorest, the community structure
of this group was complex, and the abundance of lactic acid bacteria was very low. The
emergence of Enterobacter in silage is not desirable because they compete with lactic acid
bacteria in the process of silage as a consequence of their facultative anaerobic nature. In
addition, Enterobacter species can degrade protein and reduce NO3 to form NH3 -N, thus
increasing the buffering capacity of silage and resulting in a slow decline in pH [0], which
is not conducive to inhibiting the growth of harmful bacteria. Lactobacillus plays a key role
Fermentation 2023, 9, 713 10 of 12

in increasing lactic acid concentration and reducing pH value and can inhibit the activity of
harmful microorganisms, such as Enterobacter [51]. Lactic acid bacteria, such as Weissiella or
Enterococcus, are considered early colonizers [52] because they outperform other bacteria in
acid tolerance. The authors of [53] found that, with increasing duration of the silage period,
levels of Lactobacillus decreased due to the decrease in pH. In the present study, it was found
that the addition of peanut vine to alfalfa significantly changed the bacterial community in
silage; the relative abundance of Lactobacillus was increased and that of Enterobacter was
decreased. Similarly, Wang [54] found that the addition of corn increased the abundance of
lactic acid bacteria in silage when alfalfa and corn were mixed.

4.6. Analysis of the Correlation between Fermentation Quality and Microbial Communities of
Mixed Silage with Different Ratios of Alfalfa and Peanut Seedlings
Low pH is beneficial to the growth of Lactobacillus. At the initial stage of fermenta-
tion, the growth of aerobic bacteria and the respiration of plant cells lead to high oxygen
consumption. Lactobacillus can then grow rapidly in a completely anaerobic environment.
Muck [55] reported that various enterobacteria can use nitrate as an electron acceptor
instead of oxygen to reduce nitrate to nitrite or nitrogen oxide. These bacteria are the main
source of gas (a mixture of various NOx gases) in the silage process, and Enterobacter also
competes with lactic acid bacteria for fermentation substrates such as WSC. Their main
product is AA, not LA. Therefore, their fermentation effect is not as good as that of lactic
acid bacteria. Garciella is a kind of thermophilic anaerobe that belongs to Clostridium. Its
existence in silage environments increases the difficulty of silage production [18]. In this
experiment, the content level of Garciella in the CK and T1 groups was relatively high, and
the abundance of anaerobic bacillus in the CK group reached 31.37%, so the silage effect of
these two groups was not ideal. After adding peanut vine, the abundance of Enterococci and
Lactobacillus in the T2, T3 and T4 groups increased, LA content increased, pH decreased,
and the silage effect was better.

5. Conclusions
This study revealed that mixed ensiling of high-moisture alfalfa and peanut vine is
useful for improving fermentation quality and nutrition. When the mixed proportion
of peanut seedlings was between 30% and 50%, high-quality silage was produced, as
evidenced by a relatively high DM, an abundance of Lactobacillus, PB1 and TP, and low
levels of pH, N-NH3 and PA.

Author Contributions: Y.S.: conceptualization, methodology, software, validation, formal analy-

sis, data curation, and writing—original draft preparation. M.W.: conceptualization, methodology,
software, investigation, resources, data curation, writing—review and editing, visualization, supervi-
sion, project administration, and funding acquisition. Q.L.: investigation, resources, data curation,
visualization, supervision, project administration, and funding acquisition. C.W.: methodology,
investigation, writing—review and editing, visualization, and supervision. X.Z.: investigation, vi-
sualization, supervision, and project administration. X.W.: validation, software, and investigation.
X.Y.: validation, software, and investigation. H.C.: validation, software, and investigation. L.X.:
validation, software, and investigation. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was financially supported by the S&T Program of Hebei (20326608D) and the
Hebei Natural Science Foundation (C2020204065).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Fermentation 2023, 9, 713 11 of 12

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