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Article

Effects of Fresh Corn Stover to Corn Flour Ratio on Fermentation Quality and Bacterial Community of Mixed Silage

by
Jintong Li
†,
Ke Wu
†,
Jiaxuan Wu
,
Chuang Yang
,
Baoli Sun
,
Ming Deng
,
Dewu Liu
,
Yaokun Li
,
Guangbin Liu
and
Yongqing Guo
*
College of Animal Science, South China Agricultural University, Guangzhou 510642, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2024, 10(12), 654; https://doi.org/10.3390/fermentation10120654
Submission received: 13 November 2024 / Revised: 12 December 2024 / Accepted: 12 December 2024 / Published: 17 December 2024
(This article belongs to the Section Industrial Fermentation)
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Abstract

:
Due to the high price of whole-plant corn silage in southern China, many dairy farms are attempting to prepare whole-plant corn silage using corn stover and corn flour, but the mixing ratio has not yet been determined. Therefore, we mixed fresh corn stover and corn flour at the proportions of 100:0 (F0 group) to 80:20 (F20 group), using five groups with three replicates each. The optimal mixing ratio was determined by assessing the nutritional composition, fermentation quality, and bacterial community of silage after 45 days. The results showed that dry matter and water-soluble carbohydrates in silage increased linearly with the increasing ratio of corn flour (p < 0.01) while crude protein, true protein, neutral detergent fiber, acidic detergent fiber, and crude ash content decreased linearly (p < 0.01). The F0 group had the highest pH, and the mixing ratio quadratically affected ammonia nitrogen concentration (p < 0.05). Additionally, at the phylum level, the relative abundances of Proteobacteria and Cyanobacteria in the F15 group were significantly higher than in other groups (p < 0.05). At the genus level, Lactobacillus increased with corn flour inclusion compared to the F0 group. In conclusion, the silage quality is the best when the mixing ratio of fresh corn stover and corn flour is 85:15.

1. Introduction

With the rapid development of China’s cattle and goat industry, the traditional extensive farming model is gradually transitioning to intensive and large-scale farming models. Therefore, the shortage of roughage is becoming increasingly prominent in the southern region. Roughage is an important source of ruminant animal feed, and typically accounts for 40% to 100% of diet and plays an irreplaceable role in maintaining rumen health and providing nutrition [1,2]. High-quality herbage such as alfalfa, oats, and whole-plant corn silage currently cannot fully meet the production needs of cattle and goats, resulting in higher feed production costs. Therefore, finding new types of roughage resources is urgent.
Fresh corn mainly includes sweet, glutinous, and sweet glutinous corn. Sweet corn is widely planted and has a high yield in Guangdong Province. The corn can be planted throughout the year, resulting in abundant corn stover. Fresh corn stover is rich in nutrients and has practical value [3,4]. However, most of the fresh corn stover has not been properly utilized, and instead it gradually rots in the fields, resulting in waste. In addition, the quality of whole-plant corn silage varies greatly in southern regions, and the price is relatively high. Therefore, exploring the utilization of fresh corn stover as feed is necessary, and can not only contribute to the development of feed and animal husbandry, reducing resource waste, but also attract a large number of farmers to produce silage feed and participate in cattle and goat production, promoting the socio-economic development of rural areas.
Fresh corn stover silage can effectively improve its utilization rate and provide green and juicy feed for livestock throughout the four seasons. Silage is a microbial-driven method of preserving fresh feed under anaerobic conditions. It can help extend the feed storage time through fermentation mainly with lactic acid bacteria. If fresh forage is ensiled promptly, the loss of nutrients such as protein and vitamins is usually only 10% to 15% [5]. Adding multiple additives before ensiling can obtain high-quality and easily digestible feed by increasing nutrient contents and promoting fermentation. The commonly used silage additives mainly include fermentation stimulants, fermentation inhibitors, aerobic deterioration inhibitors, as well as nutrients and absorbents, each with its functional characteristics [6]. Ensiling fresh corn stover is difficult due to its high moisture content. Excessive moisture content is beneficial for the fermentation of spoilage microorganisms such as Clostridium and Bacillus, easily leading to mold and rot. In addition, the high temperature and humidity in the southern region makes it difficult to dry the corn stover [7,8]. Corn flour is an important nutrient substrate with high levels of water-soluble carbohydrates and dry matter. Mixing corn flour with high-moisture corn stover can balance the moisture content. Moreover, the lactic acid bacteria in the silage can use nutrients from corn flour as fermentation substrates [8,9]. Therefore, mixing corn flour with fresh corn stover has the potential to improve the fermentation quality of silage.
The present study was conducted to investigate the optimal mixing ratio of fresh corn stover and corn flour. The fermentation quality, nutritional composition, and bacterial community of silage will be analyzed. We expect to provide a scientific basis and practical reference for improving the fermentation quality of mixed silage of fresh corn stover and corn flour.

2. Materials and Methods

2.1. Raw Materials and Silage Preparation

The fresh corn stover used were harvested during the milk stage with a stubble height of 20–30 cm from Boluo County, Huizhou City, Guangdong Province, and the variety was sweet corn. They were cut into 1–2 cm using a stover cutter. The finely crushed corn flour (passing through a 1.5 mm sieve) was purchased from the farmer’s market.

2.2. Experimental Design and Sampling

This experiment adopted a single-factor experimental design, with 5 treatment groups and 3 replicates within each group. The control group was the fresh corn stover without corn flour (F0), and the test groups contained different proportions of corn flour of 5% (F5), 10% (F10), 15% (F15), and 20% (F20), respectively. Fresh corn stover and corn flour were mixed in different proportions and put into polyethylene bags. Each bag was about 1 kg. Then, a vacuum packaging machine (Deli 14886, Ningbo, China) was used to vacuum, and the silage bag was sealed. All the fermentation bags were stored in the dark at room temperature (25–28 °C).
After the silage was fermented for 45 days, the samples were opened for sampling, and the on-site evaluation was carried out first, and scored according to the German Agricultural Association silage quality sensory scoring standard [10]. Each sample was accurately weighed (~5 g) from each package into a 50 mL centrifuge tube. Then 45 mL of deionized water was added, and the tubes were kept in a refrigerator at 4 °C for 24 h. The samples were filtered through four layers of gauze to separate the solid and liquid. The resulting liquid was used for the determination of fermentation indicators. Another 5 g of sample was pretreated using the same method and stored in a −80 °C freezer for subsequent 16S rDNA sequencing. The remaining silage from each bag was dried in a ventilation drying oven (105 °C) for ten minutes to inactivate enzymes [11]. Then, the temperature was adjusted to 65 °C for 48 h until a constant weight was reached. After cooling, the sample was taken out and weighed. All of the dried samples were ground and sieved through a 40-mesh sieve for later analysis.

2.3. Fermentation Parameters

The pH value of the silage extract was measured using a pB-10 pH meter (Sartorius, Guangzhou, China). Ammonia nitrogen (NH3-N) was determined using the phenol sodium hypochlorite colorimetric method [12]. Lactic acid (LA) was measured using the hydroxyphenyl colorimetric method [13]. Acetic acid (AA), propionic acid (PA), and butyric acid (BA) were determined using a high-performance gas chromatograph (Agilent 7890B, Santa Clara, CA, USA) [14], and the settings for the instrumental parameters were the same as reported in Xian et al. [15].

2.4. Nutritional Composition

Dry matter (DM) (method 934.01) and crude ash (Ash) (method 942.05) were determined according to AOAC [16]. The pre-treatment of true protein (TP) was performed using the trichloroacetic acid method [17], and the content of crude protein (CP) and TP were measured using an automatic Kjeldahl nitrogen analyzer (Hanon K9840, Jinan, China). The water-soluble carbohydrates (WSC) were determined by the sulfuric acid phenol colorimetric method [18]. Finally, according to Van Soest et al. [19], the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were measured using a semi-automatic fiber analyzer (ALVA F1600, Jinan, China).

2.5. Bacterial Community Analysis

Total DNA was extracted using the DNeasy Power Water Kit from Mo Bio/QIAGEN Company (Venlo, The Netherlands) and then quantified using a Nanodrop to detect its quality (Madison, WI, USA). PCR amplification was performed on the V3-V4 hypervariable region of 16S using primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHGGGTATCTAAT-3′). The amplified product was purified and recovered using Vazyme VAHTSTM DNA Clean Beads and then subjected to fluorescence quantification. The sequencing library was constructed using Illumina’s TruSeq Nano DNA LT Library Prep Kit at Personalbio Technology (Shanghai, China). The library quality was assessed on Vsearch. The bar-coded amplicons were sequenced on an Illumina NovaSeq system and 250 bp paired-end reads were generated. Raw sequence data were demultiplexed using the demux plugin, followed by primer cutting using the Cutadapt plugin. The sequence was then subjected to quality filtering, denoising, merging, and chimerism removal using the DADA2 plugin. Multiple indicators were estimated using diversity plugins and the samples were rarefied. Sequence data analysis was performed using QIIME2 (v2021.2) and R package (v3.2.0). Based on OTU sequence and abundance data, the ASV-level alpha diversity index was calculated using the ASV table in QIIME2 and visualized as a box plot. Beta diversity analysis was performed using multiple indicators such as Jaccard, and a visual analysis of classification composition and abundance was performed using MEGAN (v 6.19.4) [20].

2.6. Data Statistical Analysis

The experimental data were analyzed using the PROC GLM procedure of SAS 9.4 (SAS Institute, Inc., Cary, NC, USA) including treatment as fixed effects in the model. A difference would be detected between two groups when the treatment effect was significant. Preplanned contrasts were used to evaluate linear and quadratic treatment effects. Differences were declared significant at p < 0.05 and trends at p < 0.10. Results were reported as least squares means. Spearman correlation was conducted to evaluate the nutritional composition, fermentation parameters, bacterial community, and correlation analysis between different groups of silage.

3. Results

3.1. The Nutritional Composition of Each Group of Silage Materials and Corn Flour

The nutritional compositions of each group of test materials and corn flour are shown in Table 1. The contents of DM, CP, WSC, NDF, ADF, and ash in the F0 group were 19.24%, 11.14%, 11.31%, 56.95%, 34.22%, and 9.86%, respectively. According to Table 1, fresh corn stover has high NDF and ADF content and low WSC. The DM content of corn flour was 92.64%, which was much higher than that of fresh corn stover. Therefore, the DM content of the experimental group increased with the increase in the proportion of corn flour. The DM content of F5, F10, F15, and F20 was 21.82%, 25.95%, 29.70%, and 33.90%, respectively.

3.2. Sensory Evaluation of Silage

After 45 days of adding corn flour silage in different proportions to fresh corn stover for fermentation, the sensory score results were obtained by observing the color, texture, and smell of each treatment. Table 2 shows that the score grade of the five treatments is Excellent. In terms of smell, all treatments have an aromatic sour taste, but the aromatic sour taste of the F0 group and F20 group is slightly worse than that of other groups. In terms of color, all treatments are yellow green. In terms of texture, all processed stem and leaf structures are well preserved without signs of deterioration or mold.

3.3. Fermentation Parameters

The results showed that the mixing ratio of fresh corn stover and corn flour had a significant impact on the pH, LA, and AA content of silage (p < 0.01) (Table 3). With the increasing amount of corn flour, the pH decreased firstly and then increased in a quadratic curve (p < 0.01). The treatment had a quadratic effect on the NH3-N content (p < 0.05). The F15 group has the highest value of lactic acid but is not statistically different from F5 and F10 groups. The mixing ratio had a linear effect on the content of acetic acid (p < 0.001). In addition, PA and BA were not detected in all groups.

3.4. Nutritional Composition

With the increasing amount of corn flour, the contents of DM and WSC decreased linearly (p < 0.001) and the contents of CP, TP, NDF, ADF, and ash decreased linearly with increasing corn flour (p < 0.001) (Table 4). The treatment also had quadratic effects on contents of DM, NDF, ADF, and ash (p < 0.001).

3.5. Bacterial Community and Correlation Analysis

Adding different proportions of corn flour silage to fresh corn stover had a greater impact on bacterial α diversity. The Goods index representing sequencing coverage is all greater than 0.99, indicating that all sequencing results are authentic and reliable. The Chao1 index of the F15 group was significantly higher than all of the other groups (p < 0.05) (Table 5). The Shannon and Simpson indices of the F0 and F15 groups were significantly higher than those of the other groups except the F0 group (p < 0.05). There was no significant difference in the Shannon index among the F5 group, F10 group, and F20 group. The Simpson index of the F5 group was significantly higher than that of the F10 and F20 groups (p < 0.05).
Principal coordinate (PCoA) analysis revealed that the first principal component and the second principal component explained 36.6 and 27.7% of the variation in bacterial diversity, respectively (Figure 1). There was obvious separation between each group, indicating that the bacterial community differed with different proportions of corn flour.
The highest relative abundance of the bacteria in all groups was Firmicutes (Figure 2A, Table 6). The relative abundance of Firmicutes in the F15 group was significantly lower than that in other groups (p < 0.05). The relative abundance of Proteobacteria in the F15 group was significantly higher than all other groups (p < 0.05), reaching 16.13%. The relative abundance of Cyanobacteria in the F15 and F20 groups tended to be higher than in the rest of the groups (p = 0.051).
The dominant bacterial genera were Lactobacillus, Limosilactobacillus, or Lacticaseibacillus. Adding corn flour to fresh corn stover can increase the relative abundance of Lactobacillus, and the relative abundance of Lactobacillus in the F20 group is the highest (88.44%; Figure 2B, Table 7). Compared with the F0 group, the relative abundance of Limosilactobacillus in the other groups was significantly lower (p < 0.05). The relative abundance of Lacticaseibacillus, Klebsiella, Lactiplantibacillus, Weissella, and Bacillus in the F15 group was significantly higher than in the other groups (p < 0.05). The relative abundance of Secundilactobacilli in the F0 group was significantly higher than in the other groups (p < 0.05).
LEfSe analysis showed that Secundilactobacillus was enriched in the F0 group. Lactobacillales, Lactobacillaceae, Bacilli, and Firmicutes were enriched in the F10 group, and Proteobacteria, Gammaproteobacteria, Lacticaseibacillus, and Weissella were enriched in the F15 group (Figure 3). Similarly, the cladogram also presents the key bacterial clades in a taxonomic tree specified by the hierarchical feature in each respective treatment group (Figure 3B).
Pearson correlation analysis was conducted to study the fermentation quality of silage with the top ten bacterial genera (Figure 4). It was found that pH is positively correlated with Secundilactobacillus (p < 0.01), and CP, TP, NDF, ADF, and ash are all positively correlated with Limosilactobacillus (p < 0.05). The content of NH3-N is positively correlated with Bacillus, and pH, AA, CP, TP, NDF, ADF, and ash are negatively correlated with Lactobacillus. However, these were not statistically significant (p > 0.05).

4. Discussion

4.1. The Chemical Composition of Each Group of Silage Materials and Corn Flour

The quality of silage feed is largely influenced by the characteristics of the raw materials before ensiling, such as moisture content, water-soluble carbohydrate (WSC) content, and bacterial count. The moisture content of raw materials affects the growth rate of microorganisms during fermentation. Generally speaking, a DM content of 30–35% is ideal for obtaining high-quality silage feed, whereas the dry matter content of fresh corn stover is lower than this value [21]. Thus, adding additives to high moisture corn stover could effectively prevent the extensive growth of spoilage bacteria, and promote the preparation of high-quality fresh corn silage. The high content of NDF and ADF might affect the production performance of ruminants. Ensiling can effectively reduce the content of NDF and ADF and improve the palatability and nutritional value of fresh corn stover [15]. The WSC content of fresh corn was higher than the ideal content of 60 to 70 g/kg DM. Silage feed can be led to excessive fermentation if Lactobacillus with strong acid production and acid resistance are added [22].

4.2. Fermentation Parameters

The pH value of silage feed is one of the important indicators for evaluating its fermentation. The pH value of high-quality silage feed is normally below 4.2 [23]. Mcdonald et al. [24] found that well-preserved silage often had a pH of 3.7 to 4.2 and a high LA concentration. In this experiment, the F0 group had a pH value of 4.40, indicating suboptimal fermentation. Zhang et al. [25] found that a WSC content higher than 5% was required to ensure good fermentation efficiency of silage feed. The WSC content of the F0 group did not meet this standard. By adding corn flour to fresh corn stover, the pH values of silage could reach 3.76~3.88. This indicated that the silage was well preserved, which was consistent with the research results of Yi et al. [26].
The generation of NH3-N during ensiling is closely related to the activity of plant proteases and the protein degradation process induced by microorganisms. NH3-N is usually a product formed by the deamination of amino acids [27,28]. During fermentation, an acidic environment can inhibit the growth of microorganisms and reduce enzyme activity, thereby preventing protein breakdown and lowering NH3-N content [29]. Niu et al. [30] found that the pH value and NH3-N content of smooth brome silage with 9% corn flour decreased significantly after 56 days of fermentation compared with the control group. Zhang et al. [9] found that, compared with the control group, adding 10% corn flour to banana pseudo stem silage could significantly reduce the pH value and NH3-N content. Obviously, adding the proper amount of corn flour was conducive to reducing the pH value and inhibiting protein hydrolysis through the action of enzymes and microorganisms. The NH3-N content of fresh corn stover silage with an added 5~15% corn flour was lower than that of the control group, and the NH3-N content of corn flour with an added 20% corn flour was higher than that of the control group. It may be that the increase in corn flour content makes the protein easier for microorganisms to decompose and utilize, which leads to the increase in NH3-N content, while the addition of 5~15% corn flour inhibits the decomposition of protein by microorganisms due to the decrease in pH, which results in the difference in NH3-N content.
Organic acids, such as LA and AA, are products of microbial metabolism during ensiling. LA is mainly produced by lactic acid bacteria by fermenting glucose, which dominates the pH in the early stage of ensiling and is an ideal fermentation product in silage feed [24,31]. In this study, with the increase in the proportion of corn flour, the available fermentation substrate of lactic acid bacteria gradually increased. The LA content in the F15 group was significantly higher than that in the F0 and F20 groups, which positively correlated with the silage pH. Heterofermentative lactic acid bacteria can produce AA by fermentation, which has an inhibitory effect on yeast and other bacteria in silage feed [32]. Our study found that AA content decreased with increasing corn flour, indicating that heterotypic lactic acid bacteria attached to the plant surface did not dominate. Wu et al. [33] found that adding 9% corn flour to the whole Broussonetia papyrifera silage significantly increased the content of LA compared with the control group. Jiang et al. [8] found that adding 10% corn flour to high moisture alfalfa silage significantly increased LA content and significantly reduced AA content compared with the control group, which is similar to our study. Therefore, corn flour can promote the fermentation of lactic acid bacteria, inhibit the proliferation of spoilage bacteria, and effectively improve the fermentation parameters of silage.

4.3. Nutritional Composition

One of the important purposes of making silage is to preserve the feed [34]. In this study, compared with the control group, the DM content gradually increased with increasing proportions of corn flour addition. The DM content in silage is the key factor affecting the fermentation quality, feed intake, and animal production [35]. The ideal dry matter content for silage should be at 30–35%, the DM contents in the F0, F5, and F10 groups were lower than 30%, mainly due to the low DM content of fresh corn stover and the low amount of corn flour added. The WSC content gradually increased with the increase in corn flour addition because corn flour contains a certain amount of WSC, which can maintain a high level of WSC during fermentation. The lack of WSC in the silage material can be compensated by the addition of maize flour [36], which was consistent with our results. The ash content of corn flour is low, thus the increasing addition of corn flour decreased the ash content of mixed silage in the current study. Wu et al. [33] found that adding 9% corn flour to the whole Broussonetia papyrifera silage can significantly reduce the content of ash, which is mainly related to the characteristics of corn flour. Zhang et al. [37] found that the CP content of nettle and corn flour silage at the ratio of 5:1 was significantly reduced, due to the characteristics of corn flour. In this experiment, the CP contents of silage decreased with the increase in corn flour due to the low CP in corn flour. Moreover, the addition of corn flour can reduce the NDF and ADF in silage, which is mainly because the lower fiber content of corn and the rapid decline of acidity in the silage process promotes the anaerobic bacteria activity to decompose the plant cell wall [38].

4.4. Bacterial Community and Correlation Analysis

The α diversity can be used to evaluate the richness, diversity, and evenness of species [39]. In this study, the Goods coverage index in all samples was greater than 99%, indicating that the sequencing depth is sufficient for a reliable analysis of microbial communities [40]. In this experiment, the Chao1 index of the F15 group was the highest, indicating a high species richness. The addition of corn flour promotes the growth of bacteria, thus leading to changes in bacterial α diversity. The PCoA analysis showed that the addition of different proportions of corn flour had an impact on the composition of the bacterial community in silage. As shown in the PCoA diagram, different treatments improve separation significantly, especially the F15 group and the F0 group, which is consistent with the research results of Zeng et al. [41].
Adding corn flour can change not only the richness and diversity of the bacterial community, but also the composition and structure of the bacterial community. In the current study, Firmicutes and Proteobacteria were the main bacterial phyla involved in silage fermentation, consistent with the research results of Zhang et al. [42]. Gavande et al. [43] found that Firmicutes play an important role in the degradation of cellulose and hemicellulose, while Proteobacteria can promote the degradation of cellulose and lignin. In this experiment, the fiber content decreased significantly with the increase in the proportion of corn flour added, which indicates that the content change of fiber components has a relationship with the Firmicutes and Proteobacteria bacteria. Proteobacteria may have the ability to adapt to the acidic conditions, and thus proliferate during the ensiling process. Studies have shown that Bacteroidetes and Cyanobacteria are also important members in silage [44]. In this experiment, Bacteroidetes and Cyanobacteria accounted for a relatively small proportion of the total bacterial community. In addition, Cyanobacteria are usually replaced by Lactobacillus and Enterobacter during the fermentation process [45].
The genus level results showed that, except for the F0 group, the dominant bacterial genus in all other groups was Lactobacillus, Limosilactobacillus, and Lacticaseibacillus. Lactobacillus can ferment carbohydrates to produce LA, creating an environment that is unfavorable for the growth of spoilage microorganisms [46]. With the increase in the corn flour proportion from 5% to 15%, the relative abundance of Lactobacillus declined. The F20 group had the highest content of Lactobacillus, but the pH value in the F20 group was not the lowest. This may be due to other types of microorganisms present in the F20 group competing for resources with Lactobacilli or producing different metabolites, thereby affecting the pH value. The dominant bacterial genus in the F0 group is Limosilactobacillus, which is a heterofermentative genus of lactic acid bacteria created in 2020 by splitting from Lactobacillus. Its metabolites include AA, ethanol, and CO2 in addition to lactic acid; this may explain why the F0 group has a higher content of AA [47]. Compared with the F0 group, the relative abundance of Limosilactobacillus decreased significantly, which may be because the increase in the relative abundance of Lactobacillus inhibited the activity of Limosilactobacillus [48]. Lacticaseibacillus in the F15 group was also significantly higher than in the other groups. It belongs to facultative heterotrophic fermentation lactic acid bacteria and can produce acid rapidly and strongly. It can effectively reduce the pH of silage feed and inhibit the activity of harmful bacteria. These characteristics help improve the quality of silage fermentation and shorten the fermentation process. Therefore, an increase in the relative abundance of dominant bacterial genera is beneficial for the quality of silage, while there is a mutually inhibitory relationship between multiple dominant bacterial genera, indicating that high diversity may lead to a decrease in silage quality [49]. In this experiment, adding 15% corn flour can increase the relative abundance of Lacticaseibacillus, Lactiplantibacillus, Weissella, and Bacillus in silage, in comparison with the control group.
Microorganisms influence the quality of silage by altering its metabolites. In this experiment, the pH, AA, CP, TP, NDF, ADF, and ash of silage were negatively correlated with the genus Lactobacillus. Studies have shown that Lactobacillus mainly affects the production of LA [50], which is consistent with our result. The significantly positive correlation between pH and Lactobacillus may be because as the pH value decreases during the ensiling process, Secundilactobacillus are replaced by Lactobacillus due to their poor acid resistance. The content of NH3-N is positively correlated with Bacillus, because Bacillus competes with lactic acid bacteria for soluble carbohydrates in the early stage of ensiling and is one of the main microbial competitors. Its existence directly affects the rapid accumulation of lactic acid during the ensiling process, leading to protein degradation [51]. The CP, TP, NDF, ADF and ash are positively correlated with Limosilactobacillus; this suggests that Limosilactobacillus may influence the nutritional composition during the ensiling process.

5. Conclusions

The study demonstrated that varying the proportion of corn flour to fresh corn stover influenced both the fermentation parameters and nutritional composition of mixed silage. Additionally, the bacterial community was affected by the inclusion of corn stover. Among the mixing ratios examined, the F15 group (fresh corn stover/corn flour = 85:15) exhibited the highest fermentation quality. Therefore, this ratio is recommended for preparing mixed silage from fresh corn stover and corn flour.

Author Contributions

Conceptualization, K.W.; methodology, K.W.; software, J.L.; validation, K.W.; formal analysis, J.W.; investigation, C.Y.; resources, K.W. and M.D.; data curation, K.W.; writing—original draft preparation, K.W. and J.L.; writing—review and editing, J.L. and J.W.; visualization, Y.L. and G.L.; supervision, D.L. and Y.G.; project administration, Y.G. and B.S.; funding acquisition, Y.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Modern Agricultural Industrial Technology System of Guangdong Province (2022KJ127) and Dairy Herd Improvement and Construction of Efficient Service System program (h20230549).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 10 00654 g001
Figure 1. Principal coordinate analysis (PCoA) of bacterial communities for fresh corn stover and corn flour mixed silage.
Figure 1. Principal coordinate analysis (PCoA) of bacterial communities for fresh corn stover and corn flour mixed silage.
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Fermentation 10 00654 g002aFermentation 10 00654 g002b
Figure 2. Accumulation map of bacterial communities at the phylum (A) and genus (B) levels for mixed silage.
Figure 2. Accumulation map of bacterial communities at the phylum (A) and genus (B) levels for mixed silage.
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Fermentation 10 00654 g003aFermentation 10 00654 g003b
Figure 3. LEfSe analysis of bacterial biomarkers with different mixing ratios. (A) LDA score assessments of the size of differentiation among five groups with a threshold of two. (B) Cladogram of LEfSe analysis of bacterial abundance.
Figure 3. LEfSe analysis of bacterial biomarkers with different mixing ratios. (A) LDA score assessments of the size of differentiation among five groups with a threshold of two. (B) Cladogram of LEfSe analysis of bacterial abundance.
Fermentation 10 00654 g003aFermentation 10 00654 g003b
Fermentation 10 00654 g004
Figure 4. Correlation analysis between quality of silage and the top ten bacterial genera. (Positive correlation is shown in red, and negative correlation is shown in blue; “*” denotes p < 0.05, and “**” denotes p < 0.01; and the color depth is proportional to the correlation value). Abbreviations: pH (Pondus Hydrogenii), NH3-N (Ammonia nitrogen), LA (Lactic acid), AA (Acetic acid), DM (Dry matter), CP (Crude protein), TP (True protein), WSC (Water-soluble carbohydrate), NDF (Neutral detergent fiber), ADF (Acid detergent fiber), and Ash (Crude ash).
Figure 4. Correlation analysis between quality of silage and the top ten bacterial genera. (Positive correlation is shown in red, and negative correlation is shown in blue; “*” denotes p < 0.05, and “**” denotes p < 0.01; and the color depth is proportional to the correlation value). Abbreviations: pH (Pondus Hydrogenii), NH3-N (Ammonia nitrogen), LA (Lactic acid), AA (Acetic acid), DM (Dry matter), CP (Crude protein), TP (True protein), WSC (Water-soluble carbohydrate), NDF (Neutral detergent fiber), ADF (Acid detergent fiber), and Ash (Crude ash).
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Table 1. Nutritional composition of each group of silage materials and corn flour.
Table 1. Nutritional composition of each group of silage materials and corn flour.
ItemsTreatmentCorn Flour
F0F5F10F15F20
DM (%)19.2421.8225.9529.7033.9092.64
CP (% DM)11.148.7610.298.789.549.90
WSC (% DM)11.3113.1914.4214.6115.0815.85
NDF (% DM)56.9547.2238.5331.5124.712.70
ADF (% DM)34.2227.6023.7717.7912.760.94
Ash (% DM)9.866.685.534.674.005.51
Table 2. The sensory evaluation of fresh corn stover and corn flour mixed silage.
Table 2. The sensory evaluation of fresh corn stover and corn flour mixed silage.
ItemsTreatment
F0F5F10F15F20
Odor1112121211
Structural44444
Color22222
Scores1718181817
GradeExcellentExcellentExcellentExcellentExcellent
A total score of 16~20 was rated as excellent, 10~15 as good, 5~9 as medium, and 0~4 as corrupt.
Table 3. The fermentation parameters of fresh corn stover and corn flour mixed silage.
Table 3. The fermentation parameters of fresh corn stover and corn flour mixed silage.
ItemsTreatmentSEMp-Value
F0F5F10F15F20TreatmentLinearQuad
pH4.40 a3.86 bc3.83 bc3.76 c3.88 b0.06<0.001<0.001<0.001
Ammonia nitrogen
(g/kg TN)		0.0550.0470.0330.0530.0690.000.0720.8410.046
Lactic acid
(g/kg DM)		10.48 b21.38 ab20.13 ab30.37 a15.82 b0.220.0170.0020.943
Acetic acid
(g/kg DM)		29.76 a27.23 b25.68 bc24.79 c22.01 d0.73<0.001<0.0010.076
a–d Mean values within a row with different superscripts differ significantly (Tukey’s test; p < 0.05).
Table 4. The nutrient composition of fresh corn stover and corn flour mixed silage.
Table 4. The nutrient composition of fresh corn stover and corn flour mixed silage.
ItemsTreatmentSEMp-Value
F0F5F10F15F20TreatmentLinearQuad
DM (%)16.39 e20.44 d25.57 c30.83 b36.24 a1.91<0.001<0.001<0.001
CP (%DM)11.55 a10.73 b10.51 c9.76 d9.66 d0.23<0.001<0.0010.002
TP (%DM)6.68 a5.97 b5.84 b4.97 c4.28 d0.28<0.001<0.0010.878
WSC (%DM)1.70 d4.33 cd7.49 bc8.97 b12.60 a1.08<0.001<0.0010.133
NDF (%DM)61.87 a43.94 b30.90 c21.66 d20.10 d4.19<0.001<0.001<0.001
ADF (%DM)39.14 a27.14 b19.16 c11.92 d12.93 d2.73<0.001<0.001<0.001
Ash (%DM)8.99 a6.77 b5.51 c4.64 d4.29 e0.32<0.001<0.001<0.001
a–e Mean values within a row with different superscripts differ significantly (Tukey’s test; p < 0.05). Abbreviations: DM (Dry matter), CP (Crude protein), TP (True protein), WSC (Water-soluble carbohydrate), NDF (Neutral detergent fiber), ADF (Acid detergent fiber), Ash (Crude ash).
Table 5. The α-diversity of bacterial community of fresh corn stover and corn flour mixed silage.
Table 5. The α-diversity of bacterial community of fresh corn stover and corn flour mixed silage.
ItemsTreatmentSEMp-Value
F0F5F10F15F20TreatmentLinearQuad
Chao1214.01 c184.68 d133.69 d570.18 a337.46 b52.42<0.0010.106 0.994
Goods0.9998 a0.9998 a0.9999 a0.9994 b0.9996 ab0.000.6800.707 1.000
Shannon4.17 a2.60 b1.77 b4.25 a1.82 b0.370.0020.004 0.702
Simpson0.88 a0.71 b0.56 c0.91 a0.44 d0.06<0.0010.014 0.350
a–d Mean values within a row with different superscripts differ significantly (Tukey’s test; p < 0.05). SEM, standard error of the mean.
Table 6. The top ten phyla in the relative abundance among bacterial communities of mixed silage.
Table 6. The top ten phyla in the relative abundance among bacterial communities of mixed silage.
ItemsTreatmentSEMp-Value
F0F5F10F15F20AllLinearQuad
Firmicutes99.15 a98.90 a99.51 a82.98 b96.26 a1.850.0010.9000.865
Proteobacteria0.51 b0.97 b0.39 b16.13 a2.90 b1.760.0010.9650.823
Actinobacteriota0.150.070.050.240.240.040.2270.3600.724
Bacteroidota0.080.020.010.260.250.040.1450.5780.796
Cyanobacteria0.04 b0.02 b0.02 b0.27 a0.15 ab0.040.0510.8170.858
Verrucomicrobiota0.020.01<0.010.020.050.010.3290.4660.671
Bdellovibrionota<0.01 b<0.01 b<0.01 b0.01 b0.04 a0.010.1261.0001.000
Patescibacteria<0.01<0.01<0.010.020.010.000.2021.0001.000
Acidobacteriota0.01 b<0.01 b<0.01 b0.02 a0.01 b0.000.0370.5880.754
Myxococcota0.01<0.01<0.010.010.010.000.2740.1450.383
a,b Mean values within a row with different superscripts differ significantly (Tukey’s test; p < 0.05). SEM, standard error of the mean.
Table 7. The top ten genera in the relative abundance among bacterial communities of mixed silage.
Table 7. The top ten genera in the relative abundance among bacterial communities of mixed silage.
ItemsTreatmentSEMp-Value
F0F5F10F15F20AllLinearQuad
Lactobacillus21.30 d70.38 b60.42 c54.26 c88.44 a7.390.0010.0710.052
Limosilactobacillus72.97 a22.84 c37.92 b4.72 d3.27 d8.60<0.0010.0420.005
Lacticaseibacillus2.34 b1.28 bc0.58 c11.63 a2.02 bc1.37<0.0010.3020.943
Klebsiella0.20 b0.54 b0.19 b12.28 a0.25 b1.60<0.0010.9810.905
Lactiplantibacillus0.08 b0.12 b0.10 b9.40 a0.31 b1.25<0.0011.0000.976
Weissella0.06 b0.25 b0.04 b3.05 a0.18 b0.410.0120.9500.899
Companilactobacillus0.480.400.031.030.020.180.4220.2150.761
Levilactobacillus1.40 a0.43 b0.03 b1.33 a0.05 b0.200.0010.4810.825
Secundilactobacillus0.49 a0.03 b0.04 b0.01 b0.03 b0.060.0040.1020.322
Bacillus0.09 c0.10 c0.05 c1.85 a0.72 b0.23<0.0011.0000.849
a–d Mean values within a row with different superscripts differ significantly (Tukey’s test; p < 0.05). SEM, standard error of the mean.
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Li, J.; Wu, K.; Wu, J.; Yang, C.; Sun, B.; Deng, M.; Liu, D.; Li, Y.; Liu, G.; Guo, Y. Effects of Fresh Corn Stover to Corn Flour Ratio on Fermentation Quality and Bacterial Community of Mixed Silage. Fermentation 2024, 10, 654. https://doi.org/10.3390/fermentation10120654

AMA Style

Li J, Wu K, Wu J, Yang C, Sun B, Deng M, Liu D, Li Y, Liu G, Guo Y. Effects of Fresh Corn Stover to Corn Flour Ratio on Fermentation Quality and Bacterial Community of Mixed Silage. Fermentation. 2024; 10(12):654. https://doi.org/10.3390/fermentation10120654

Chicago/Turabian Style

Li, Jintong, Ke Wu, Jiaxuan Wu, Chuang Yang, Baoli Sun, Ming Deng, Dewu Liu, Yaokun Li, Guangbin Liu, and Yongqing Guo. 2024. "Effects of Fresh Corn Stover to Corn Flour Ratio on Fermentation Quality and Bacterial Community of Mixed Silage" Fermentation 10, no. 12: 654. https://doi.org/10.3390/fermentation10120654

APA Style

Li, J., Wu, K., Wu, J., Yang, C., Sun, B., Deng, M., Liu, D., Li, Y., Liu, G., & Guo, Y. (2024). Effects of Fresh Corn Stover to Corn Flour Ratio on Fermentation Quality and Bacterial Community of Mixed Silage. Fermentation, 10(12), 654. https://doi.org/10.3390/fermentation10120654

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