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Article

Revealing the Potential Advantages of Plectasin Through In Vitro Rumen Fermentation Analysis

by
Qinwu Li
,
Baozhen Zhu
,
Fei Li
*,
Zhiyuan Ma
,
Long Guo
and
Xiuxiu Weng
State Key Laboratory of Grassland Agro-Ecosystems, Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, Engineering Research Center of Grassland Industry, Ministry of Education, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730000, China
*
Author to whom correspondence should be addressed.
Fermentation 2024, 10(11), 542; https://doi.org/10.3390/fermentation10110542
Submission received: 23 August 2024 / Revised: 17 October 2024 / Accepted: 21 October 2024 / Published: 24 October 2024
(This article belongs to the Special Issue In Vitro Digestibility and Ruminal Fermentation Profile, 2nd Edition)
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Abstract

:
Plectasin, a novel antimicrobial peptide, has the potential to disrupt bacterial cell walls and alter the rumen fermentation mode, making it a superior alternative to antibiotics. However, there is limited research on the effects of plectasin on rumen microbiota. This study aimed to evaluate the effects of plectasin (0.057 μmol/L) on in vitro rumen fermentation characteristics and select groups of rumen bacterial communities in comparison with monensin (5 μmol/L), one of the most commonly used ionophores in ruminants, and as a control treatment with the basal substrate. Unlike monensin, plectasin was found to increase the molar proportions of butyrate and acetate/propionate ratio (p < 0.001) while decreasing pH and the molar proportions of propionate (p < 0.05). Principal component analysis of bacterial 16S rRNA gene amplicons clearly showed a separation between the bacteria shaped by plectasin and monensin. Comparative analysis also revealed differences in the relative abundance of certain bacteria in different taxa between plectasin and monensin. The divergent effects of plectasin and monensin on bacterial communities are likely responsible for the differences in their ability to alter rumen fermentation. Plectasin may have advantages over monensin in modulating ruminal bacterial communities and increasing the butyrate and the acetate/propionate ratio. Therefore, it may be considered as a potential additive for ruminant feed.

1. Introduction

In the current intensive breeding process of ruminants, the issue of low energy and nitrogen utilization efficiency in animal diets has garnered significant attention [1]. Ruminants currently face two major challenges due to the metabolic processes in the rumen: approximately 2 to 12% of the feed energy is wasted as methane, and more than 60% of dietary nitrogen is excreted as urea through urine [2]. These challenges limit the production performance of ruminants. Previous studies have indicated that adding antibacterial agents in animal feed can improve animal production performance [3]. However, due to their incomplete metabolism in the body and excretion through urine and feces, they not only enhance the spread of antibiotic resistance genes (pre-existing) through the bacterial community but also cause environmental pollution [4]. The European Union banned antibiotics as growth promoters in 2005 [5] as did China in 2020 [6]. The ban on antibiotic use, combined with consumers’ preferences, provoked scholars to look for antibiotic alternatives [7].
Monensin, as an ion carrier antibiotic [8], is beneficial for improving the feed digestibility of ruminants, inhibiting the growth of H2-producing bacteria [9]. It also causes a shift in volatile fatty acid (VFA) profiles, increasing propionate and decreasing amino acid fermentation and ruminal ammonia concentration [10]. However, since the late 1990s, some countries have frequently reported residual monensin in meat and eggs [11]. A recent study has shown that monensin exhibits strong cytotoxicity and has a narrow safety margin. Improper clinical use can result in lamb mortality events [12].
Antimicrobial peptides (AMPs) have gained increasing attention for their broad-spectrum antimicrobial properties against Gram-positive bacteria, Gram-negative bacteria, fungi, and viruses [13]. Among AMPs, plectasin was the first AMP that was extracted from a fungus in 2005 [14]. It has verified advantages, such as strong pH stability, high-thermal stability, and potent antimicrobial activity against Gram-positive bacteria, including Staphylococcus aureus, S. pneumoniae, and Streptococcus suis, even against some antibiotic-resistant strains [15]. The application of plectasin in growth and finishing pigs [16], weaned piglets [17], and broilers [18] has shown certain advantages in promoting nutrient absorption and improving production performance. However, there are currently no reports on the effects of plectasin on digestion in ruminants. The antimicrobial peptides have a distinct bactericidal mechanism compared to traditional antibiotics. They offer advantages such as targeted action and non-toxicity, making them more promising for future development than traditional antibiotics [19]. Therefore, this experiment compares the effects of monensin and plectasin on the rumen fermentation function and microbial community of Hu sheep through in vitro fermentation to determine whether plectasin can replace monensin to achieve the same effect due to their activity against Gram-positive bacteria and to explore the potential of antimicrobial peptides as feed additives.

2. Materials and Methods

2.1. Experimental Design and In Vitro Fermentation Process

The experimental design was a single-factor design. A total of three treatments were designed: CON treatment with the basal substrate (Table 1), MON treatment with the basal substrate + 5 μmol/L monensin (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China), which is equivalent to the widely used in vivo dose in ruminants [10], and PLE treatment with the basal substrate + 0.057 μmol/L plectasin (Guangdong Hainachuan Biotechnology Co., Ltd., Guangzhou, China), following the dose methodology described in the study by Afolabi et al. [20]. Each treatment had five replicates (n = 5).
Three healthy wethers of Hu sheep, with similar body weights and fitted with a ruminal fistula were used as ruminal fluid donors for this in vitro study. The sheep were fed a full mixed pellet feed twice daily at 08:00 and 18:00, with free access to feed and water. All sheep were individually housed in single pens (1.8 × 1.25 × 1.0 m3). It is confirmed that this study followed the recommendations of the Biological Studies Animal Care and Use Committee of Gansu Province, China (2005–2012). The experiment was approved by the animal care committee of Lanzhou University on 2 March 2018 (Protocol number: LZU 201803002), and it was conducted according to their established guidelines. All efforts were made to minimize animal suffering. Before morning feeding, a total of approximately 1 L of rumen fluid was collected from three different locations in the rumen of each Hu sheep and strained through four layers of gauze into a bottle, while maintaining a temperature of 39 °C and CO2 stream protection. The collected rumen fluid was then brought back to the laboratory for future use.
The in vitro batch fermentation was carried out in 250 mL ferment bottles, with each bottle containing 1.2 g of basal substrate. The specific preparation and storage methods of the buffer required for in vitro fermentation were as described by Menke et al. [21]. The rumen fluid and anaerobic buffer medium were mixed in a 1:2 (v/v) ratio under anaerobic conditions. Then, 90 mL of this mixture was stored, and CO2 was used as the headspace gas in each bottle at 39 °C. The samples were incubated and covered with a rubber stopper, while being continuously injected with CO2, for 24 h at 39 °C in a water bath with intermittent shaking.

2.2. Sample Collection and Item Determination

Gas production was measured at 24 h using a gas collecting bag. After gas collection, all fermentation bottles were transferred from the constant temperature water bath to a prepared ice water mixture and cooled for 15 min to terminate the fermentation process. At the end of the 24-h incubation period, the pH value of each in vitro culture was measured using a portable pH meter (Leici PHB-4, Shanghai, China). Then, 5 mL of each culture was preserved by adding 2 mL of 25% HPO3 in a 10 mL centrifuge tube for VFA analysis using gas chromatography (Thermo Scientific, TRACE 1300, Milano, Italy) according to the method described by F. Li et al. [22]. Another 10 mL of each culture was collected for DNA extraction and subsequent microbial analysis. The culture samples and microbial samples were stored at a temperature of −20 °C and −80 °C separately until analysis.

2.3. DNA Extraction

The bacterial DNA extraction method described by Ma et al. [23] follows the RBB + C extraction method. For each bottle, 200 μL of the fermentation fluid sample was vibrated using a CEBO-48 vibrator (Chebo BioTech Co., Shanghai, China) at a frequency of 50 Hz for 3 min. The purified DNA was obtained using spin columns from the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, CA, USA). The concentration and purity of the DNA were measured using a spectrophotometer (ND-2000; NanoDrop Technologies, Wilmington, DE, USA), and the samples were stored at −20 °C until analysis.

2.4. 16S rDNA Gene Sequencing and Bioinformatics Analysis

After extracting the total DNA of the sample, for bacterial analysis, V3 to V4 region amplicons of 16S rRNA genes were achieved by using primers of 341F (forward, 5′-CCTAYGGGRBGCASCAG-3′) and 806R (reverse, 5′-GGACTACNNGGGTATCTAAT-3′). Sequencing adapters were added to the end of the primers to enable PCR amplification. Following amplification, the products were purified and dissolved in Elution Buffer using Agencourt AMPure XP to create a sequencing library. The concentration and fragment range of the library were assessed using the Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). The library that met the detection criteria was then sequenced on the HiSeq 2500 platform (Wuhan Huada Medical Laboratory Co., Ltd., Wuhan, China) based on the size of the inserted fragment. After performing quality control on the original sequencing sequence, including low-quality filtering, length filtering, removed interdigitation sequence, and removed singletons, to obtain high-quality sequences, FLASH (Fast Length Adjustment of Short reads, v1.2.11) was used to concatenate clean reads into tags. OTUs were clustered at 97% sequence similarity and compared to database annotations for species identification. The OTUs were then annotated, compared, and matched to the SILVA138 database using the MOTHUR method [24]. Taxonomic information was obtained, and the community composition of the sample was determined at each classification level (kingdom, phylum, class, order, family, genus, and species). To establish the phylogenetic relationship of the OTUs, MUSCLE software (Version 3.8.31) was used for rapid multi-sequence alignment [25]. The data from each sample were standardized through homogenization, with the sample containing the least amount of data used as the reference. Data analysis based on the standardized data was performed using the Quantitative Insights Into Microbial Ecology (QIIME) software (Version 4.0.3). The alpha and beta diversity of the samples were analyzed using QIIME’s internal Perl script. Differences between groups were analyzed using the rank sum test in IBM SPSS Statistics 25 software. UniFrac distance was calculated using QIIME software, and the principal coordinate analysis (PCoA) diagram was generated using R software (Version 4.0.3). The difference in flora between groups was analyzed using the Linear Discriminant Analysis Effect Size (LEfSe) software (Version 2.7) with an LDA score set to 4. Finally, correlation analysis based on the Spearman coefficient was performed using R (Version 4.0.3).

2.5. Statistical Analysis

The experimental data were analyzed using IBM SPSS Statistics 25 for single-factor ANOVA analysis and the Tukey method for multiple comparisons. p < 0.05 indicates significant differences, and 0.05 < p < 0.10 shows a significant trend.

3. Results

3.1. Effects of Plectasin and Monensin on Rumen Fermentation Characteristics

Compared to the CON and MON groups, the PLE group exhibited an increase in 24-h total gas production (p = 0.001) (Table 2), while rumen pH decreased (p = 0.008). Although the molar proportion of acetate and valerate did not show any differences (p > 0.05) in the three groups, there was a trend for higher VFA in PLE and lower VFA in MON relative to the CON treatment (p = 0.091). In the MON group, the proportion of isobutyrate was lower than that in the PLE and CON groups (p = 0.001). In the PLE group, the proportion of propionate was lower than those in the MON and CON groups (p < 0.001). The proportion of butyrate, isovalerate (including isovalerate plus 2-methylbutyrate), and the acetate/propionate ratio were higher in the PLE group compared to the other two groups (p < 0.001).

3.2. Effects of Plectasin and Monensin on Rumen Bacterial Communities

A total of 1,029,804 pairs of reads were obtained from all 15 samples. After quality control, a total of 971, 850 clean reads were generated, with each sample producing at least 64,450 clean reads and an average of 64,790 clean reads (Supplementary Table S1). Using Usearch to cluster reads at a similarity level of 97.0%, a total of 471 OTUs were obtained, and there was no unique OTU in the MON treatment or PLE treatment (Figure 1A). The dilution curve showed a flat trend (Supplementary Figure S1), indicating that the sequencing depth met the requirements for downstream analysis.
To investigate the effects of the three treatments on rumen bacteria from sheep, the alpha diversity of the rumen bacteria was assessed (Figure 1B). The results showed that there was no difference in the rumen bacterial ACE index between the PLE and CON groups. However, the CON group had higher levels compared to the MON group (p = 0.018). Both CON and PLE groups had higher Chao1 index levels compared to the MON group (p = 0.018). Furthermore, the Simpson index of the MON group was higher than the CON group (p = 0.021) and the Shannon index of the PLE group was higher (Figure 1C) than that of the MON group (p = 0.018). To assess the phylogenetic relationship between OTUs, UniFrac distances were calculated. PCoA based on weighted UniFrac distances revealed a separation among the three treatments (p = 0.001), while there was a partial overlap between the CON and the PLE groups (Figure 1C).
The effective tags were annotated through the 16S SILVA database, and the statistical analysis annotated the composition of each sample community at different levels (phylum, class, order, family, genus, specifications). The phylogenetic tree of genus-level species showed that >50% of the genus-level species belonged to Firmicutes in the annotated genus-level species (Supplementary Figure S2). The annotation results of relative abundance TOP10 were analyzed, uncultured bacterium and less than 0.5% of species in the sample were merged into another species. At the phylum level, the rumen composition of the three treatments was similar, and the dominant phyla of the three groups were Bacteroidetes, Firmicutes, and Proteobacteria, accounting for more than 90%.
The relative abundance of bacterial phyla was altered differently among the treatments (Table 3). The relative abundance of Bacteroidetes and Patescibacteria was lower (p < 0.001) in the MON group compared to the CON and the PLE groups, while the relative abundances of Cyanobacteria and Fibrobacteres were higher than those of the CON group. Additionally, the relative abundances of Firmicutes, Kiritimatiellaeota, and Patescibacteria in the PLE group were higher compared to the CON and MON groups (p < 0.05), while the relative abundances of Proteobacteria, Spirochaetes, and Synergistes were reduced (p < 0.05). At the genus level, MON and PLE showed different effects on some bacterial genera. Compared with the CON group, the relative abundances of Anaerovibrio and Desulfovibrio were increased in the PLE group (Table 4), but the relative abundance of Prevotella was decreased in the MON and PLE groups (p < 0.05). The relative abundance of Bacteroides showed differences between the different treatment groups (p < 0.01), with the highest observed in the MON group, followed by the CON group, and the lowest observed in the PLE group. The relative abundance of Succinivibrio, Treponema, Fretibacterium, and Butyrivibrio in the PLE group were lower than that in the MON group (p < 0.01). On the contrary, the relative abundance of Barnesiella in the PLE group was higher (p < 0.05) compared to the other two groups.
LEfSe was used to screen the signature rumen microbial taxa in the in vitro fermentation fluid of three treatments of Hu sheep with LDA score = 4, and the results of the evolutionary tree were shown (Figure 2). These rumen microbial taxa in in vitro fermentation fluid of Hu sheep were statistically different between the different treatments. Furthermore, these taxa in each group were not unique to the individual treatments, but instead, these taxa were significantly elevated in relative abundance within the listed treatment group. This study identified six distinct bacterial taxa in the MON group, including Proteobacteria, Succinivibrionaceae, Gammaproteobacteria, Aeromonadales, Succinivibrio, and one species within that genus. The CON group had six distinct bacterial taxa. The PLE group had nineteen bacterial taxa.

3.3. Correlations Between the Relative Abundance of the Signature Rumen Microbial Flora and Fermentation Parameters

In this study, we combined the differential flora and LEfSe analysis results to screen out 9 annotated biomarkers from the family and genus level out of the 31 different microbial flora, and their correlation with key VFA was analyzed (Figure 3). Two biomarkers were identified in the MON group, both belonging to the phylum Proteobacteria. These biomarkers showed positive correlations with propionate and pH in the in vitro fermentation fluid of Hu sheep while showing negative correlations with isobutyrate, butyrate, isovalerate, acetate/propionate, and gas production. A total of six biomarkers were identified in the PLE group, among which only Rikenellaceae, Rikenellaceae_RC9_gut_group, and Lachnospiraceae were positively correlated with isobutyrate, butyrate, isovalerate, TVFA, acetate/propionate, and gas production. However, they negatively correlated with propionate and pH.

4. Discussion

The impact of monensin on rumen microbiota in sheep has been extensively studied [2]. In this study, the addition of monensin caused decreases in the molar proportion of butyrate and isobutyrate but an increase in the molar proportion of propionate, these results are consistent with Polizel [26]. However, the addition of plectasin had the opposite effect compared to monensin. The impact of Plectasin on the rumen microbiota of sheep is rarely reported. Therefore, we performed 16 S rDNA sequencing on rumen fermentation fluid microbes to reveal these effects of different additives. Studies have shown a positive correlation between the relative abundance of Succinivibrio in the rumen and the propionate, as well as a negative correlation with the butyrate. Members of the Succinivibrio genus [27] produce succinate as their primary fermentation product. The propionate increased with succinate production, as succinate can be converted into propionate and used as an end product of rumen fermentation [28]. Bacteria that convert succinate to propionate are resistant to the effects of ionophores, which explains the increase in the molar proportion of propionate in the MON group [29]. Previous studies have shown that Succinivibrio can reduce methane production by competing for H2 with methanogens [30]. This may be the main reason for the decrease in gas production after the addition of monensin in this study. Meanwhile, since propionate is the main precursor for glucose synthesis in ruminants [31], a higher propionate production can provide more substrates for gluconeogenesis in the liver [32]. Unlike monensin, the increase in butyrate content and the decrease in propionate content in the PLE group may be due to the specificity of plectasin towards Succininivibrio. Although the abundance of Butyrivibrio in the PLE group was lower compared to the MON group, the relative abundance ratio of Butyrivibrio and Succininivibrio in the PLE group (0.31/4.71) was much higher than that in the MON group (0.58/16.89). The functional characteristics of a small number of Butyrivibrio can also have an impact on ecosystem functioning [33]. On the other hand, in the present study, it was observed that the abundance of Lachnospiraceae in the rumen is positively correlated with the butyrate and negatively correlated with the propionate, and the Lachnospira species is known to synthesize butyrate [34]. Previous studies have shown that the proliferation of Lactobacillus can lower local pH, as they produce a large amount of lactic acid [35,36], which may explain the decrease in pH in the PLE group. Furthermore, the decrease in the abundance of succinate and propionate producers and the increase in other butyrate producers in the PLE group may further explain the higher molar proportion of butyrate compared to the MON group. This study showed that the addition of plectasin can affect the change change of rumen fermentation mode to butyrate fermentation mode, which also indicates that plectasin may have advantages in helping the development of rumen epithelium in young ruminants [37]. However, since these are in vitro tests, these results cannot fully reflect the changes in fermentation parameters in animals, and further in vivo experiments are needed to verify them.
Antibiotics and antimicrobial peptides can inhibit symbiotic bacteria in the gastrointestinal tract, which is beneficial for animal production [38]. Studies have shown that both monensin and plectasin had impacts on the diversity and richness of rumen bacteria. The Chao1 and Ace indices revealed that the species richness of rumen bacteria was lower in the MON group compared to the CON group. Furthermore, a difference in the microbial Shannon and Simpson indices was observed between the three treatment groups, indicating that adding plectasin increased the diversity and richness of rumen bacteria, which improved their activity and function in rumen fermentation. Studies have shown that the higher alpha diversity indicated a more diverse and complex microbial composition in one rumen [39], which could enhance the resistance to the environment and the adaptability of the host [40]. However, monensin has the opposite effect to plectasin. The PCoA based on UniFrac distance indicates differences in the bacterial community composition among the three treatment groups. In the process of regulating bacteria, monensin accumulates as ionophores in the cell membranes. Due to its high affinity for sodium ions, it acts as antiporters of transporter protein by increasing the influx of sodium and protons [41], depleting the active sodium/potassium pumps with consequent hydropic degeneration and cell death [42]. Therefore, ionophore resistance is mainly correlated with differences in cell envelope structure [41]. However, PLE, a small peptide substance, can directly bind to the precursor lipid II of bacterial cell walls, inhibiting the synthesis of bacterial cell walls and leading to bacterial death [43]. The different mechanisms of action between the two may be the main reason for the differences in changes in the rumen bacterial community composition of Hu sheep. In this study, plectasin demonstrates a remarkable potency, eliciting significant effects at very small concentrations, in stark contrast to monensin, which necessitates higher concentrations to achieve comparable efficacy. This disparity underscores the imperative for further experimental investigations employing a broader range of concentrations to elucidate the potential for more pronounced differential impacts between these two compounds on the rumen bacterial community composition.
The 16S rDNA gene sequencing revealed that among the top 10 relative abundance phyla, Bacteroidetes, Firmicutes, and Proteobacteria were the dominant phyla in all treatment groups, with a relative abundance sum of up to 94%. Prevotella was the dominant genus, which is consistent with previous research results in Hu sheep [44]. Both the MON and PLE groups decreased the relative abundance of Bacteroidetes. In contrast, the relative abundance of Firmicutes increased in the PLE group. These results illustrate that both MON and PLE alter the composition of what has been described as the core microbiome in the rumen bacterial microbiome. The core microbiome is mainly composed of Firmicutes and Bacteroidetes [45]; Bacteroidetes are the primary degraders of non-structural carbohydrates and non-fibrous polysaccharides in the plant cell wall, while Firmicutes primarily act on the degradation of structural carbohydrates [46]. Therefore, plectasin may influence rumen fermentation by altering the composition of the core microbiome. Proteobacteria is a major phylum of Gram-negative bacteria, which produces lipopolysaccharide (LPS) endotoxin that can enter the blood, reduce the number of intestinal barrier cells, and increase intestinal permeability, leading to a chronic inflammatory response [47]. In the present study, the relative abundance of Proteobacteria in the MON group increased compared to the CON and PLE groups, implying that monensin is less active against Gram-negative bacteria. The increase in these Gram-negative bacteria will cause these bacteria to release a large amount of LPS after lysis.
The in vitro fermentation fluid of Hu-sheep-added monensin increased the relative abundance of Gram-negative bacteria such as Succinivibrio and Bacteroides. Pure culture experiments using rumen bacteria have shown that low concentrations of ion carrier antibiotics such as MON can inhibit the proliferation of Ruminococcus and Butyrivibrio [9]. In this study, this effect was not observed. However, it was found that plectasin had stronger activity against Ruminococcus and Butyrivibrio compared to monensin, which may be attributed to the fact that although Butyrivibrio is Gram-negative, its cell wall has a very thin layer of Gram-positive peptidoglycan, and Ruminococcus belongs to Gram-positive bacteria, which is consistent with previous reports [14]. Interestingly, the addition of plectasin also reduced the relative abundance of Prevotella and Bacteroides. The antimicrobial activity of plectasin might involve a specific interaction with the surface of the target microorganism; further work remains for exploring the detailed relationship between PLE, Lipid II, and divalent cations [48]. When ion carriers such as monensin and salinomycin are used as feed additives in ruminants, they can regulate the diversity and richness of rumen microorganisms to reduce gas emissions and alter rumen fermentation patterns [49]. Meanwhile, after adding monensin and plectasin to the study, the diversity and richness of rumen microorganisms and fermentation parameters of the in vitro fermentation fluid notably changed compared to that of the CON group.
LEfSe analysis identified 31 different microbial flora in the in vitro rumen fermentation fluid to elucidate the key differences between different treatments. As differences in rumen microbes may impact the phenotypes of hosts [50], Pearson correlation analysis was conducted to explore the relationships between rumen fermentation characteristics and bacteria shifts. The analysis revealed that the high abundance of Succinivibrio in the MON group may be responsible for altering the fermentation mode. These findings are consistent with the observations of Shen et al. [2]. Conversely, in the PLE group, the results showed decreased propionate and a low abundance of Succinivibrio. It was found that Lachnospiraceae and Rikenellaceae are likely to be influenced by or contribute to these changes in the fermentation characteristics. Previous studies have demonstrated that a high abundance of Rikenellaceae_RC9_gut_group was found in the rumen microbiota of yaks and Tan sheep [51], and this specific group has been shown to affect intramuscular fat synthesis by regulating the production of butyrate [52]. This may be related to the Rikenellaceae_RC9_gut_group showing a negative correlation with propionate and a positive correlation with isobutyrate, butyrate, and isovalerate.

5. Conclusions

Our results indicate that plectasin has the advantage of promoting in vitro VFA production compared to monensin. In particular, plectasin exhibited activity against propionate-producer Succinivibrio and decreased the molar proportion of propionate. Meanwhile, plectasin also increased the molar proportions of butyrate, isobutyrate, and acetate/propionate compared to monensin.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation10110542/s1, Figure S1: All sample dilution curve; Figure S2: The phylogenetic tree of genus-level species; Table S1: Statistics of sample sequencing data processing results.

Author Contributions

Conceptualisation, Methodology, Data curation, Writing—original draft, Writing—review and editing, Q.L.; Formal analysis, Data curation, B.Z.; Conceptualisation, Writing—review, F.L.; Supervision, Conceptualisation and Resources, Z.M. and X.W.; Funding acquisition, L.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Cattle and sheep Industry Development Research Institute of Linxia (KJJC-LX-2023-06), Science and Technology Program of Gansu Province [22YF7WA011], and Science and Technology Program of Gansu Province [24CXNA032].

Institutional Review Board Statement

It is confirmed that this study followed the recommendations of the Biological Studies Animal Care and Use Committee of Gansu Province, China (2005–2012). The experiment was approved by the animal care committee of Lanzhou University on 2 March 2018 (Protocol number: LZU 201803002), and it was conducted according to their established guidelines. All efforts were made to minimize animal suffering.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors thank Animal Husbandry and Veterinary Station in Minqin for their help with the feeding trial.

Conflicts of Interest

The authors declare no real or perceived conflicts of interest.

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Fermentation 10 00542 g001
Figure 1. Effects of treatment on bacterial diversity of in vitro fermentation. (A) UpSetR figure of rumen OTUs of various varieties. (B) Alpha diversity comparison of rumen microbes. (C) Principal coordinate analysis based on weighted UniFrac distance. * p < 0.05; ** p < 0.01, ns p > 0.05.
Figure 1. Effects of treatment on bacterial diversity of in vitro fermentation. (A) UpSetR figure of rumen OTUs of various varieties. (B) Alpha diversity comparison of rumen microbes. (C) Principal coordinate analysis based on weighted UniFrac distance. * p < 0.05; ** p < 0.01, ns p > 0.05.
Fermentation 10 00542 g001
Fermentation 10 00542 g002
Figure 2. Biomarkers and in vitro fermentation traits of Hu sheep. Linear discriminant analysis effect size (LEfSe) analysis.
Figure 2. Biomarkers and in vitro fermentation traits of Hu sheep. Linear discriminant analysis effect size (LEfSe) analysis.
Fermentation 10 00542 g002
Fermentation 10 00542 g003
Figure 3. Correlation analysis between biomarkers and rumen (VFA). * p < 0.05; ** p < 0.01.
Figure 3. Correlation analysis between biomarkers and rumen (VFA). * p < 0.05; ** p < 0.01.
Fermentation 10 00542 g003
Table 1. Ingredient and proximate composition of basal substrate for in vitro fermentation.
Table 1. Ingredient and proximate composition of basal substrate for in vitro fermentation.
ItemsContents
Ingredient, % of DM
Alfalfa hay25.00
Corn straw25.00
Corn17.70
Corn bran16.80
Soybean meal3.50
Cottonseed meal3.50
Corn germ meal5.90
Limestone0.50
NaCl0.30
Expanded urea0.25
Lamb premix 10.25
Molasses premix1.30
Total100
Proximate composition, % of DM
Dry matter (DM)89.75
Crude protein (CP)12.16
Neutral detergent fiber (NDF)40.83
Acid detergent fiber (ADF)27.51
1: A kilogram of premix included Fe 25 mg, Zn 40 mg, Cu 8 mg, Mn 40 mg, I 0.3 mg, Se 0.2 mg, Co 0.1 mg, Vitamin A 940 IU, Vitamin D 111 IU, and Vitamin E 20 IU.
Table 2. Effects of treatment on the fermentation parameters of rumen fluid during in vitro fermentation.
Table 2. Effects of treatment on the fermentation parameters of rumen fluid during in vitro fermentation.
ItemsControlMONPLESEMp-Value
Gas production, mL217 b186 b338 a21.2960.001
Ruminal pH6.14 a6.18 a5.99 b0.0290.008
TVFA (mmol/L)50.7234.4962.745.3930.091
Acetate (mol %)50.0848.1048.410.3960.081
Propionate (mol %)21.65 b28.96 a18.31 c1.228<0.001
Iso-butyrate (mol %)1.08 a0.95 b1.18 a0.0310.001
Butyrate (mol %)20.82 b16.30 c25.16 a1.053<0.001
Iso-valerate 1 (mol %)3.62 b3.07 b4.42 a0.1790.001
Valerate (mol %)2.762.622.520.0570.243
Acetate/Propionate2.32 b1.66 c2.66 a0.117<0.001
a–c Means within a row with different superscripts differ (p < 0.05). 1 The Iso-valerate including isovalerate plus 2-methylbutyrate.
Table 3. Effects of treatment on relative abundance of bacterial phyla in vitro fermentation, %.
Table 3. Effects of treatment on relative abundance of bacterial phyla in vitro fermentation, %.
ItemsCONMONPLESEMp-Value
Bacteroidetes66.74 a53.98 c60.58 b1.624<0.001
Firmicutes18.81 b18.26 b26.42 a1.3970.014
Proteobacteria9.21 b20.37 a5.26 c1.805<0.001
Kiritimatiellaeota1.61 b2.59 b4.28 a0.3660.002
Cyanobacteria1.37 bc2.27 a1.80 ab0.1240.003
Spirochaetes0.72 a0.95 a0.18 b0.095<0.001
Patescibacteria0.54 b0.16 c0.75 a0.069<0.001
Synergistetes0.41 ab0.56 a0.26 bc0.0410.004
Fibrobacteres0.18 b0.50 a0.03 b0.059<0.001
Lentisphaerae0.120.150.140.0090.615
Others0.260.220.310.0170.136
a–c Means within a row with different superscripts differ (p < 0.05).
Table 4. Effects of treatment on relative abundance of bacterial genus in vitro fermentation, % 1.
Table 4. Effects of treatment on relative abundance of bacterial genus in vitro fermentation, % 1.
ItemsCONMONPLESEMp-Value
Bacteroidetes
Prevotella50.04 a41.65 b40.49 b1.4880.005
Paraprevotella0.700.540.600.0330.107
Bacteroides0.82 b1.12 a0.65 c0.057<0.001
Barnesiella0.46 b0.83 b1.73 a0.1910.008
Firmicutes
Succinivibrio8.42 b16.89 a4.71 b1.460<0.001
Succiniclasticum3.313.543.310.2150.481
Ruminococcus2.58 b3.96 a2.77 b0.183<0.001
Butyrivibrio0.48 ab0.58 a0.31 b0.0400.011
Lactobacillus0.920.631.240.5860.075
Spirochaetes
Treponema0.82 a1.13 a0.13 b0.123<0.001
Synergistetes
Fretibacterium0.47 a0.55 a0.23 b0.0480.006
Proteobacteria
Desulfovibrio0.27 b0.33 ab0.55 a0.0470.021
Anaerovibrio0.35 c0.65 a0.52 b0.034<0.001
Others27.48 b24.88 b36.42 a1.459<0.001
1 Only the abundant taxa with the average relative abundance of at least one treatment > 0.1% were presented in the table. a–c Means within a row with different superscripts differ (p < 0.05).
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Li, Q.; Zhu, B.; Li, F.; Ma, Z.; Guo, L.; Weng, X. Revealing the Potential Advantages of Plectasin Through In Vitro Rumen Fermentation Analysis. Fermentation 2024, 10, 542. https://doi.org/10.3390/fermentation10110542

AMA Style

Li Q, Zhu B, Li F, Ma Z, Guo L, Weng X. Revealing the Potential Advantages of Plectasin Through In Vitro Rumen Fermentation Analysis. Fermentation. 2024; 10(11):542. https://doi.org/10.3390/fermentation10110542

Chicago/Turabian Style

Li, Qinwu, Baozhen Zhu, Fei Li, Zhiyuan Ma, Long Guo, and Xiuxiu Weng. 2024. "Revealing the Potential Advantages of Plectasin Through In Vitro Rumen Fermentation Analysis" Fermentation 10, no. 11: 542. https://doi.org/10.3390/fermentation10110542

APA Style

Li, Q., Zhu, B., Li, F., Ma, Z., Guo, L., & Weng, X. (2024). Revealing the Potential Advantages of Plectasin Through In Vitro Rumen Fermentation Analysis. Fermentation, 10(11), 542. https://doi.org/10.3390/fermentation10110542

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