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Article

Effects of Konjac Flour and Lactiplantibacillus plantarum on Fermentation Quality, Aerobic Stability, and Microbial Community of High-Moisture Forage Rape Silages

by
Encun Du
1,
Na Zhao
1,
Wanzheng Guo
1,
Qiwen Fan
1,
Jintao Wei
1,* and
Zhiyu Xu
2,*
1
Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
2
Key Laboratory of Technology and Model for Cyclic Utilization from Agricultural Resources, Rural Energy and Environment Agency, Ministry of Agriculture and Rural Affairs, Beijing 100125, China
*
Authors to whom correspondence should be addressed.
Fermentation 2022, 8(8), 348; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation8080348
Submission received: 30 June 2022 / Revised: 19 July 2022 / Accepted: 20 July 2022 / Published: 23 July 2022
(This article belongs to the Section Industrial Fermentation)
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Abstract

:
To obtain high-quality silage and better understand the mechanism underlying silage fermentation, a study was conducted to investigate the effects of konjac flour (KF), Lactiplantibacillus plantarum (LP) and their combination on fermentation quality, aerobic stability, and microbial community of high-moisture forage rape after 60 days of ensiling. Results showed that the KF and LP treatments increased the lactic acid content, decreased the pH value, and inhibited the production of butyric acid in ensiled forage rape (p < 0.05). The additives also altered the bacterial community of forage rape silages, showing reduced Shannon and Simpson indexes (p < 0.05), while the abundance of desirable Lactobacillus was increased, and the abundance of undesirable bacteria, such as enterobacteria and clostridia, was decreased (p < 0.05). In addition, their combination significantly improved the aerobic stability (96 h vs. 28 h, p < 0.05) and exhibited notable influence on the bacterial community, with the highest abundance of Lactobacillus. These results indicated that KF and LP improved the silage quality of high-moisture forage rape, and their combination displayed a beneficial synergistic effect.

1. Introduction

Forage rape (Brassica napus), also known as rapeseed, oilseed rape, or canola, is the second largest oil-producing crop grown worldwide [1]. In addition to oil production, the utilization of forage rape in biofuel or livestock production will help to combat energy crises and feed shortages. Forage rape has a high nutritional value and very high biomass yield (>40 tons fresh matter/hm2), according to Wang et al. [2], which could be used to support the growth of ruminants [3,4]. In China, there is a long-term shortage of high-quality forage, especially in southern China, which challenges the development of ruminant breeding [5]. The high-humidity climatic conditions and acidic soil conditions in southern China are extremely detrimental to the growth of alfalfa, but it is conducive to the growth of forage rape [2]. Therefore, the efficient utilization of forage rape in ruminant production is one of the important solutions to alleviate the shortage of high-quality forage [6].
Ensiling is a traditional technology for preserving green forage, which can extend storage time, improve palatability, and supply year-round availability of moist forage. During ensiling, epiphytic lactic acid bacteria (LAB) ferment water-soluble carbohydrates (WSC) to lactic acid, resulting in a drop in pH and an inhibition of spoilage microorganisms in the ensiled material [7,8]. However, forage rape is difficult to ensile due to its high moisture content (>800 g/kg fresh matter) and high buffer capacity [9]. The pH value of high-moisture silage tends to drop slowly, and it often bears a high risk of clostridial fermentation, resulting in high production of undesirable butyric acid and poor fermentation quality [10,11].
Given the moist and rainy weather during forage rape harvest, it is not practical to reduce moisture content by field wilting. The use of absorbent during ensiling has been suggested as an alternative to wilting and has been reported to improve the quality of silage [12]. According to previous reports, sugar beet pulp, dried barley, and distillers’ dried grains were promising absorbents as they were found to prevent excessive effluent loss and decreased the pH value of ensiled crops with low DM content [12,13]. Konjac flour (KF) is processed from the root of konjac tuber (Amorphophallus konjac), which mainly contains konjac glucomannan (KGM) and displays a very high water-holding capacity [14]. Hence, adding KF could be a possible way to achieve high-quality forage rape silage by inhibiting the water activity. Currently, KF or KGM is generally used as a food additive or dietary supplement because of its considerable potential for enhancing human and animal health [15]. However, little is known about the effectiveness of KF as a silage additive.
Apart from absorbent, inoculating silage with LAB has been proposed as an efficient way to overcome the difficulty in natural ensiling [8,16]. Lactiplantibacillus plantarum (LP) is a homofermentative LAB, which has been widely used in the ensiling of corn, alfalfa, etc., to enhance silage quality by converting WSC to lactic acid and lowering pH [17,18].
Accordingly, it is speculated that adding KF or LP would improve the fermentation quality of high-moisture forage rape, and their combination might show a potential synergistic effect. Nevertheless, there is no information available on the effect of KF or LP on the ensiling of forage rape. Therefore, the present study was conducted to investigate the effect of KF and LP on fermentation quality, chemical composition, aerobic stability, and microbial community of high-moisture forage rape silage.

2. Materials and Methods

2.1. Materials and Silage Preparation

The forage rape (variety Huayouza 62) was harvested from the experimental field of Hubei Academy of Agricultural Sciences (Wuhan, China, 114°01′ E to 114°35′ E and 29°58′ N to 30°32′ N) on 12 March 2021, when the plant was in the flowering stage. Prior to ensiling, the harvested materials were chopped to 2–3 cm in length with a crop chopper.
Four different treatments were conducted as follows: control (no additive, CK), KF (20 g/kg based on fresh matter), LP (5 × 106 cfu/g based on fresh matter), and the combination of KF and LP (KFLP). The KF used in the current study contained 610 g/kg of KGM and 80 g/kg of crude protein (CP). The additives were dissolved in sterile distilled water and then mixed thoroughly with the forage. An equal amount of sterile distilled water was added to the control group. Briefly, 600 g of fresh forage rape was blended with additives, and the mixture was packed into plastic silo bags and sealed by a vacuum sealer. A total of 12 bags (3 replicates each with 4 treatments) were prepared and stored at ambient temperature (15–30 °C). After 60 days of ensiling, aerobic stability, fermentation characteristics, chemical composition, and microbial community were analyzed.

2.2. Assessment of Aerobic Stability

To assess the aerobic stability of the silos after 60 days of ensiling, the temperature was measured by inserting a digital thermometer (TP-101, Honeywell Co., Charlotte, NC, USA) into the geometric center of the silage mass. According to the methods of He et al. [19], approximately 400 g of silages were placed loosely into a clean polystyrene box, and two layers of cheesecloths were placed over each container to decrease moisture volatilization and potential contamination. The buckets were stored in a polystyrene box to inhibit fast heat diffusion. Aerobic stability is defined as the time which elapses before the silage shows clear evidence of heating, that is when the temperature of the silage exceeds the ambient temperature by 2 °C [20].

2.3. Analysis of Chemical Composition and Fermentation Profile

The sample of fresh forage rape prior to ensiling was dried at 65 °C in a forced-air oven for 48 h to determine the DM content. Then the dried sample was ground into particles with a diameter of 1.0 mm for chemical analysis. Standard procedures of the Association of Official Analytical Chemists [21] were used to examine the contents of crude protein (CP), ether extract (EE), and crude ash. The contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed according to the method of Van Soest et al. [22]. The anthrone method [23] was used to analyze the content of WSC. After 60 days of ensiling, 20 g of silage samples were collected from each silo bag, and the contents of CP, NDF, ADF, and crude ash were analyzed.
To determine the fermentation profile, 10 g of silage sample was mixed well with 90 mL sterile water and then filtered through four layers of cheesecloth. The filtrate was used to measure the pH value and the organic acids. The pH value of the filtrate was promptly analyzed using a pH meter. A high-performance liquid chromatography (HPLC, LC-20A, Shimadzu, Japan) with a 210 nm UV detector and a Shodex RSpak KC-811S-DVB gel C column (8.0 mm × 30 cm; Shimadzu, Tokyo, Japan) quantified the levels of organic acids (including lactic acid, acetic acid, propionic acid, isobutyric acid, and butyric acid). 3 mmol/L of HClO4 solution was used as the eluent at the temperature of 50 °C, and the flow rate was 1.0 mL/min.

2.4. Microbial Diversity Analysis

Total DNA was extracted using the E.Z.N.A. Bacterial DNA Kit (Omega Biotek, Norcross, GA, USA) according to the manufacturer’s protocol. Then, the concentration of DNA was determined using spectrophotometry and its quality was evaluated using 2% agarose gel electrophoresis. The microbial 16 s rRNA gene was amplified using the hypervariable V4 region PCR primers (515F: 5′-GTGCCAGCMGCCGCGG-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′) [24] with a unique error-correcting barcode for each sample.
After purification and quantification, the PCR products were sequenced using the Illumina platform (Genewiz Co. Ltd., Suzhou, China). The sequenced reads were processed according to the methods of Wang et al. [25] with a few modifications. Briefly, the QIIME quality-control process was used to obtain the high-quality clean tags (Version 1.9.1) and the UCHIME algorithm was used to detect and remove chimera sequences. Then the effective sequences were aligned into operational taxonomic units (OTUs) analysis using the software VSEARCH (version 1.9.6) based on 97% sequence similarity. Alpha diversity was analyzed by the metrics of Chao1, Ace, Shannon, and Simpson. The representative OTU sequences were then compared with the Silva 132 database using the Ribosomal Database Program Classifier for taxonomic classification (at 80% confidence threshold) at the kingdom, phylum, class, order, family, and genus levels.

2.5. Statistical Analysis

Results are given as mean values and pooled standard errors. One-way ANOVA and Tukey’s multiple comparisons were used to analyze the data. Data were considered significant when p < 0.05. All statistical procedures were performed using SPSS version 22.0 (IBM Inc., Chicago, IL, USA). The LEfSe (linear discriminant analysis effect size) method was utilized to assess the differences in microbial community and explore the specific bacteria in each group (LDA score > 4).

3. Results

3.1. Chemical Composition of Fresh Forage Rape before Ensiling

The average chemical composition of fresh forage rape was analyzed prior to ensiling. Results showed that the fresh forage rape contains 144.0 g/kg of DM, and the content of CP, NDF, ADF, ether extract (EE), and crude ash was 144.5, 490.2, 325.7, 47.3, and 95.1 g/kg based on DM, respectively.

3.2. Effects of KF and LP on Silage Quality

The pH value and organic acid contents of forage rape silages were illustrated in Table 1. Compared with group CK, the three treated groups showed dramatically decreased pH value and increased lactic acid content (p < 0.05). In the present study, the content of butyric acid in group CK was 1.87 g/kg DM, which was decreased to a very low level in all treated silages, even undetectable (p < 0.05). In addition, less isobutyric acid was found in group LP and KFLP (p < 0.05). No difference was found in the content of acetic acid among the four groups (p > 0.05).
The chemical properties of silages were shown in Table 2. The additives did not influence the content of crude ash or ADF (p > 0.05). However, lower CP content was found in group KF and KFLP compared with group CK and LP (p < 0.05). In addition, a higher NDF content was observed in group KF in contrast with group LP (p < 0.05).

3.3. Effects of KF and LP on Aerobic Stability

The aerobic stability of forage rape silages is shown in Figure 1 and Table 3. After an aerobic exposure period of 28.0 h, silages in group CK deteriorated. During the continuous monitoring time of 120 h, CK reached its maximum temperature of 27.5 °C after an aerobic exposure period of 100 h. Compared with CK, group KF and LP did not affect aerobic stability (p > 0.05), while the maximum temperature caused by secondary fermentation was even higher in group KF (p < 0.05), although the time to reach maximum temperature was longer (p < 0.05). On the contrary, group KFLP showed significantly improved aerobic stability (p < 0.05). Meanwhile, silages treated with KFLP took a longer time to reach their maximum temperature (p < 0.05).

3.4. Effects of KF and LP on Microbial Community

In the current study, a coverage value of 1.00 was observed in all the silage samples, indicating that all bacteria were identified. The alpha diversity of bacteria in forage rape silages is shown in Table 4. Though Ace and Chao1 indexes were not influenced by the silage additives (p > 0.05), both Shannon and Simpson indexes decreased significantly in the three additive-treated groups (p < 0.05). In addition, group LP and KFLP displayed reduced Shannon and Simpson indexes as compared with group KF (p < 0.05).
Based on the analysis of the bacterial community at the phylum level (Figure 2A), Firmicutes and Proteobacteria were dominant in group CK. The silage bacterial community was altered by the additives. The relative abundance of Firmicutes was increased remarkably, while the relative abundance of Proteobacteria was decreased to a very low level in the additive-treated groups compared with group CK.
According to the analysis of the bacterial community at the genus level (Figure 2B), Lactobacillus and Serratia were predominant in group CK, accounting for 28.23% and 23.55%, respectively. The subdominant bacteria, were f_Enterobacteriaceae_Unclassified, Clostridium_sensu_stricto_12 and Hafnia-Obesumbacterium in group CK, accounting for 11.74%, 10.56%, and 6.67%, respectively. The additives promoted the relative abundance of Lactobacillus, especially for group KFLP (98.76% vs. 28.23%). Meanwhile, the relative abundances of Serratia, f_Enterobacteriaceae_Unclassified, Clostridium_sensu_stricto_12, and Hafnia-Obesumbacterium were decreased obviously.
The LEfSe method was utilized to assess the differences in microbial communities and explore the specific bacteria in each group (LDA score > 4). Figure 3 shows that in group CK, Enterobacteriaceae was the most abundant family, while Serratia and Clostridium_sensu_stricto_12 were the most abundant genera. Instead, Lactobacillus was found to be the most abundant genus in group KFLP.

4. Discussion

4.1. Chemical Composition of Fresh Forage Rape before Ensiling

The moisture content of fresh forage rape was 856 g/kg DM. It is a challenge to obtain high-quality silage when moisture content exceeds 700 g/kg DM, as this dilutes the LAB count and counteracts the pH drop [26,27]. Moreover, high-moisture silage often bears a high risk of effluent loss and clostridial fermentation, resulting in high DM loss, extensive proteolysis, and high butyric acid production, which will reduce feed palatability [28]. Thus, measures should be taken to improve the fermentation quality of forage rape. Absorbents are suggested to be used in silages with high-moisture content to inhibit water activity and prevent excessive effluent losses [12]. Besides, it is documented that inoculating silage with LAB reduces detrimental microorganisms and prevents spoilage during ensiling [8,16,17]. Therefore, it might be helpful to achieve high silage quality of forage rape by adding KF and LAB.

4.2. Effects of KF and LP on Silage Quality

pH is an important parameter to evaluate the extent of silage fermentation [29]. A lower pH reduces undesirable fermentation and ensures better aerobic stability. In the present study, the pH value of forage rape silage without additives was 4.84, much higher than the benchmark pH of 4.20 for well-fermented silage [10]. This is a common problem with the natural ensiling of high-moisture silage. According to the current study, the pH value of the additive-treated groups was close to or below 4.20, indicating good preservation of high-moisture forage rape. The decreased silage pH contributes to the inhibition of undesirable microbes, thus helping to preserve the forage mass [30]. This is consistent with the reduced abundance of enterobacteria and clostridia according to the 16 s rRNA gene sequencing. Lactic acid is the dominant fermentation product in silage, which is another important evaluation index of fermentation quality. In this study, the additive-treated groups promoted the production of lactic acid, which was the main reason for pH reduction.
LAB inoculants, including LP, are widely used to produce high-quality silage [8,16]. Similar to the present study the LP inoculation was found to decrease the pH value and accelerate the fermentation of lactic acid in high-moisture alfalfa and Napier grass silage, according to Yang et al. [18] and Jaipolsaen et al. [31]. However, little is known about the effectiveness of KF as a silage additive. KF is a water-soluble dietary fiber that displays very high water-holding capacity, good swelling, thickening, and gelation-property [14,32]. Consequently, KF is increasingly used as a stabilizer and emulsifier for processed foods, beverages, and cosmetic products [14,32]. Based on the present study, KF helps improve the fermentation quality of high-moisture forage rape, possibly by inhibiting water activity. Furthermore, the contents of isobutyric acid and butyric acid were decreased in additive-treated groups. Isobutyric acid is an oxidation product of amino acids [33]. Butyric acid is undesirable in silage as its generation is an energy-waste metabolism [10]. When butyric acid exceeds 5 g/kg DM, it indicates substantial clostridial activity and will impair livestock feed intake [10]. In this study, the content of butyric acid in the treated silages was decreased to a very low level, which might be due to the inhibition of clostridial fermentation caused by KF and LP.
The addition of KF, either alone or in combination with LP, decreased the CP content in forage rape silages. This could be explained by the lower CP content of KF compared with forage rape (80 g/kg vs. 144.5 g/kg). In addition, the alteration of microbial activities might result in a difference in nutrient preservation. According to the current study, the NDF content in LP inoculated silages was lower than that in KF treated silages. Similarly, Zhao et al. reported that during the 60-d ensiling process, both NDF and ADF contents were decreased in high-moisture alfalfa treated with a commercial LAB [8]. In addition, in the silage of wilted perennial ryegrass, an inoculant containing LP, L. buchneri and L. casei also reduced the NDF content after ensiling for 60 days and 150 days [34]. It may be due to the fact that LP promotes microbial degradation, which converts plant cell walls into complex sugars during ensiling [35].

4.3. Effects of KF and LP on Aerobic Stability

Aerobic deterioration of silages during the feed-out phase is a significant problem for farm profitability and feed quality worldwide [28,35]. Generally, aerobic deterioration of silage is sponsored by yeasts, which would metabolize lactic acid and WSC into CO2 and water in aerobic conditions [35]. The present study showed that the combination of KF and LP effectively extended the aerobic stability of forage rape silages. On the contrary, silages treated with KF displayed faster temperature rising and higher maximum temperature. According to He et al. [19], mixing Moringa oleifera leaves decreased the aerobic stability and accelerated the temperature rising after aerobic exposure in rice straw silages. The authors speculated that this might be related to the higher lactic acid and WSC contents as well as the lower antifungal components like propionic acid and acetic acid [19]. During aerobic exposure, lactic acid could serve as a carbon source for lactate-assimilating yeasts; thereby, the higher lactic acid would promote yeast proliferation and heat production [16]. Therefore, it could be speculated that the rather high temperature in KF-treated silages could be related to the lactic acid accumulation and the microbial changes that occurred after 48 h of aerobic exposure, such as the proliferation of aerobic bacteria, yeasts, and molds, which requires more research in future.

4.4. Effects of KF and LP on Microbial Community

The application of high-throughput sequencing provided a detailed picture of the microbial community. The Ace and Chao1 indexes were used to characterize the richness of the bacterial community, and the Shannon and Simpson indexes were used to represent the community diversity. In the present study, the decreased Shannon and Simpson indexes caused by KF, LP, and their combination indicated decreased bacterial diversity. It could be explained by the decreased pH, the inhibited growth of undesirable bacteria and the predominance of Lactobacillus caused by the treatments. Similarly, the Shannon index of high-moisture alfalfa silage treated with a commercial LAB inoculant also decreased after 30 days of ensiling [8]. And the combination of L. acidophilus and LP was found to decrease the Chao1, Ace, and Shannon indexes in the whole-plant corn silages [36]. Little is known about the effectiveness of KF on the ensiling process, including microbial community. Despite that, Guan et al. demonstrated that bacterial diversity sharply decreases after successful fermentation [37]. Therefore, KF, LP, and their combination improved the fermentation of high-moisture forage rape silages, evident by the reduction in microbial diversity.
During ensiling, LAB plays a key role in silage fermentation. Lactobacillus is one of the most important LAB, which could ferment WSC to lactic acid, reduce pH and prevent silage spoilage caused by undesirable bacteria [8,38]. Hence, Lactobacillus is often the dominant genus of high-quality silage. In the untreated forage rape silages, Lactobacillus accounted for only 28.23%, while family Enterobacteriaceae, as well as genera Serratia and Clostridium_sensu_stricto_12, were the most abundant bacteria based on the LEfSe analysis. The presence of enterobacteria is undesirable as they may compete with LAB for available sugars [39]. Furthermore, members of Serratia may cause important infections in humans, animals, and insects [40]. Clostridia are also undesirable in silages as many of them ferment carbohydrates and proteins, causing problems such as reduction in feeding value and the production of biogenic amines [39]. Besides, clostridia in silages impair milk quality, and their occurrence and transmission through the dairy chain always cause the death of animals and humans [41]. Ensiling methods that cause a rapid and sufficient drop in silage pH will help to prevent the development of enterobacteria and clostridia [10]. In the present study, the rather high pH value (4.84) of naturally ensiled high-moisture forage rape is not sufficient to inhibit enterobacteria and clostridia.
Lactobacillus became the predominant bacterium in all the treated silages, especially for silages treated with the combination of KF and LP, where Lactobacillus was the most abundant genera based on the LEfSe analysis. The remarkably increased abundance of Lactobacillus caused by KF and LP was in accordance with the decreased pH value and elevated lactic acid content in forage rape silages. In addition, almost no clostridia were found in the treated silages, which was in line with the nearly eliminated butyric acid. It could be speculated that KF and LP might enhance the fermentation quality of high-moisture forage rape by promoting the proliferation of Lactobacillus and inhibiting the proliferation of undesirable bacteria, such as clostridia. Some studies reported that LAB inoculation might reduce the pH value of silage, but the degree of reduction may be insufficient to prevent undesirable bacteria [8,42]. Under the current experimental condition, the inhibition effect of LP on undesirable bacteria is obvious, which was in line with previous studies conducted with high-moisture alfalfa and Napier grass [18,31]. So far, no information is available regarding the effect of KF on silage quality or microbial community. According to the current study, the addition of KF inhibited the proliferation of undesirable bacteria, improved the relative content of Lactobacillus and enhanced the fermentation quality of high-moisture forage rape after 60 days of ensiling. These results indicate that KF can be used as a new type of silage additive to improve the fermentation quality of high-moisture silage. In addition, a better effect can be expected from the combination of KF and LP as their combination displayed a beneficial synergy on aerobic stability and the bacterial community of silages.

5. Conclusions

This study revealed that the addition of KF or LP helps improve the silage quality of high-moisture forage rape, indicated by increased lactic acid content, decreased pH value as well as nearly eliminated butyric acid. The KF and LP treatments also altered the bacterial community of forage rape silages, showing reduced bacterial diversity, while the abundance of desirable Lactobacillus was increased, and the abundance of undesirable bacteria such as enterobacteria and clostridia was decreased. Besides, their combination displayed significant improvement in aerobic stability and exhibited notable influence on the bacterial community. These results indicated that the addition of KF and LP could be a feasible way to improve the silage quality of high-moisture forage rape, and their combination had a beneficial synergistic effect.

Author Contributions

Conceptualization, E.D. and J.W.; methodology, E.D. and N.Z.; investigation, E.D. and W.G.; data curation, E.D. and Q.F.; writing—original draft preparation, E.D.; writing-review and editing, J.W. and Z.X. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the China Agriculture Research System of MOF and MARA (grant number: CARS-12), the project of the Hubei Innovation Center of Agricultural Science and Technology (grant number: 2022-620-004-001), the open project of Hubei Key Laboratory of Animal Embryo and Molecular Breeding (grant number: KLAEMB-2020-02), and Innovation Team Project of Efficient Planting, Silage and Feeding Technology of Winter and Spring Feed of Hubei Provincial Industrial Technology System.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://catalog.data.gov/dataset/sequence-read-archive-sra (accessed on 2 May 2022), PRJNA833919.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Fermentation 08 00348 g001 550
Figure 1. The temperature dynamics of forage rape silages fermented with KF and LP during the aerobic exposure. CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum.
Figure 1. The temperature dynamics of forage rape silages fermented with KF and LP during the aerobic exposure. CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum.
Fermentation 08 00348 g001
Fermentation 08 00348 g002 550
Figure 2. The bacterial community and abundances at the phylum (A) and genus (B) levels in forage rape silages. CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum.
Figure 2. The bacterial community and abundances at the phylum (A) and genus (B) levels in forage rape silages. CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum.
Fermentation 08 00348 g002
Fermentation 08 00348 g003 550
Figure 3. Comparison of bacteria variations using LEfSe analysis. CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum.
Figure 3. Comparison of bacteria variations using LEfSe analysis. CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum.
Fermentation 08 00348 g003
Table
Table 1. The pH and organic acid contents of forage rape silages were fermented with KF and LP for 60 days.
Table 1. The pH and organic acid contents of forage rape silages were fermented with KF and LP for 60 days.
ItempHLactic Acid
(g/kg DM)		Acetic Acid
(g/kg DM)		Isobutyric Acid
(g/kg DM)		Butyric Acid
(g/kg DM)
CK4.84 a8.06 b1.760.08 a1.87 a
KF4.17 b15.83 a1.660.04 abND
LP4.23 b16.13 a1.690.03 bc0.03 b
KFLP4.22 b18.29 a1.63NDND
SEM0.081.210.030.010.24
p-value<0.001<0.0010.3290.001<0.001
DM: dry matter; CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum; SEM: pooled standard error; ND: not detected; Means with different superscripts in the same column differ significantly (p < 0.05).
Table
Table 2. The chemical composition of forage rape silages fermented with KF and LP for 60 days (g/kg DM).
Table 2. The chemical composition of forage rape silages fermented with KF and LP for 60 days (g/kg DM).
ItemCrude AshCPNDFADF
CK96.55146.29 a501.15 ab341.64
KF102.13130.16 b506.49 a345.92
LP99.17150.03 a494.74 b347.06
KFLP95.57129.04 b498.53 ab345.39
SEM1.042.961.631.80
p-value0.084<0.0010.0410.791
DM: dry matter; CP: crude protein; NDF: neutral detergent fiber; ADF: acid detergent fiber; CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum; SEM: pooled standard error; Means with different superscripts in the same column differ significantly (p < 0.05).
Table
Table 3. The aerobic stability of forage rape silages fermented with KF and LP.
Table 3. The aerobic stability of forage rape silages fermented with KF and LP.
ItemAerobic Stability (h)Maximum Temperature (°C)Time to Reach Maximum Temperature (h)
CK28.00 b27.50 bc100.00 c
KF43.33 b30.67 a106.67 ab
LP46.00 b28.40 b104.00 bc
KFLP96.00 a26.40 c111.33 a
SEM8.520.51.37
p-value0.002<0.0010.002
CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum; SEM: pooled standard error; Means with different superscripts in the same column differ significantly (p < 0.05).
Table
Table 4. Alpha diversity of bacteria in forage rape silages fermented with KF and LP for 60 days.
Table 4. Alpha diversity of bacteria in forage rape silages fermented with KF and LP for 60 days.
ItemAceChao1ShannonSimpson
CK67.6466.213.42 a0.86 a
KF62.3260.171.26 b0.35 b
LP45.2143.710.41 c0.09 c
KFLP58.4052.590.27 c0.06 c
SEM3.623.50.390.1
p-value0.140.097<0.001<0.001
CK: the control; KF: konjac flour; LP: Lactiplantibacillus plantarum; KFLP: konjac flour + Lactiplantibacillus plantarum; SEM: pooled standard error; Means with different superscripts in the same column differ significantly (p < 0.05).
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Du, E.; Zhao, N.; Guo, W.; Fan, Q.; Wei, J.; Xu, Z. Effects of Konjac Flour and Lactiplantibacillus plantarum on Fermentation Quality, Aerobic Stability, and Microbial Community of High-Moisture Forage Rape Silages. Fermentation 2022, 8, 348. https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation8080348

AMA Style

Du E, Zhao N, Guo W, Fan Q, Wei J, Xu Z. Effects of Konjac Flour and Lactiplantibacillus plantarum on Fermentation Quality, Aerobic Stability, and Microbial Community of High-Moisture Forage Rape Silages. Fermentation. 2022; 8(8):348. https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation8080348

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Du, Encun, Na Zhao, Wanzheng Guo, Qiwen Fan, Jintao Wei, and Zhiyu Xu. 2022. "Effects of Konjac Flour and Lactiplantibacillus plantarum on Fermentation Quality, Aerobic Stability, and Microbial Community of High-Moisture Forage Rape Silages" Fermentation 8, no. 8: 348. https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation8080348

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