Dietary supplementation with Bacillus mixture modifies the intestinal ecosystem of weaned piglets in an overall beneficial way

Abstract

Aims

This study was conducted to investigate the effects of dietary supplementation with a mixture of Bacillus, which serves as an alternative of antibiotics on the intestinal ecosystem of weaned piglets.

Methods and Results

We randomly assigned 120 piglets to three groups: a control group (a basal diet), a probiotics group (a basal diet supplemented with 4 × 10⁹ CFU per gram Bacillus licheniformis‐Bacillus subtilis mixture; BLS mix), and an antibiotics group (a basal diet supplemented with 0·04 kg t⁻¹ virginiamycin, 0·2 kg t⁻¹ colistin and 3000 mg kg⁻¹ zinc oxide). All groups had five replicates with eight piglets per replicate. On days 7, 21 and 42 of the trial, intestine tissue and digesta samples were collected to determine intestinal morphology, gut microbiota and bacterial metabolite composition, and the expression of genes related to the gut barrier function and inflammatory status. The results showed that the BLS mix decreased the jejunum crypt depth, while increased the ileum villus height and the jejunum and ileum villus height to crypt depth ratio. The BLS mix increased Simpson’s diversity index in the gut microbiota and the relative abundances of o_Bacteroidetes and f_Ruminococcaceae, but decreased the relative abundances of Blautia and Clostridium. Dietary BLS mix supplementation also modified the concentration of several bacterial metabolites compared to the control group. In addition, BLS mix upregulated the expression level of E‐cadherin in the colon and pro‐inflammatory cytokines and TLR‐4 in ileum and colon. Lastly, Spearman’s rank‐order correlation revealed a potential link between alterations in gut microbiota and health parameters of the weaned piglets.

Conclusion

These findings suggest that dietary BLS mix supplementation modifies the gut ecosystem in weaned piglets. The potential advantages of such modifications in terms of intestinal health are discussed.

Significance and Impact of the Study

Weaning is the most important transition period of piglet growth and development. This study showed that dietary supplementation of a probiotic mixture of Bacillus, an effective alternative of antibiotics, was beneficial in improving the intestinal ecosystem of weaned piglets.

Bacillus, gut microbiota and metabolites, intestinal morphology, probiotics, weaned piglets

Introduction

In commercial swine production, early weaning may affect the intestinal development, digestive, and immune functions in weaned piglets, which leads to reduced feed intake and growth inhibition (Campbell et al. 2013). Antibiotics are commonly used to control the incidence of infectious diseases and improve the growth in weaned piglets (Kim et al. 2012). However, many countries banned antibiotics in the livestock production due to the increasing resistance of pathogens and antibiotic residues in food (Allen et al. 2014). Therefore, effective antibiotic alternatives are urgently needed to reduce the dependence of the animal industry on antibiotics. Previous studies have focused on the development of novel alternatives to antibiotics, including probiotics (Hu et al. 2018), prebiotics (Tran et al. 2018), and synbiotics (Zhang et al. 2018). Among these alternatives, probiotics have a higher potential to act for pathogen exclusion (Azad et al. 2018).

Previous studies indicated that Bacillus spp. possess anti‐oxidant capacity against pathogens and immuno‐modulatory abilities (Elisashvili et al. 2019). Bacillus spp. also produces various digestive enzymes and stimulates peristalsis of the intestine, thereby improving nutrient digestion (Hilker et al. 2008; Giang et al. 2011). Presently, Bacillus spp. are widely used as commercialized probiotic products for humans and animals (Cutting 2011). A previous study showed that a mixture of Bacillus subtilis and Bacillus licheniformis can improve the growth and immunological status of farmed tilapia (Abarike et al. 2018). Moreover dietary supplementation with B. subtilis DSM32315 played a beneficial role in maintaining the intestinal barrier function and microflora balance of weaned piglets, in association with the improved growth performance (Tang et al. 2019). Dietary supplementation with B. licheniformis and B. subtilis complex in growing‐finishing pigs increased the digestibility and faecal Lactobacillus counts and decreased the faecal NH₃ and total mercaptan emissions (Lan and Kim 2019). Decreased ammonia concentration in faeces is considered as beneficial since this metabolite is known as a polluting substance and acts as an inhibitor of colonocyte mitochondrial energy metabolism.

However, the B. licheniformis and B. subtilis mixture (BLS mix), as with other strains of Bacillus, have little been studied as probiotics for weaned piglets. Furthermore, the effects of BLS mix on the intestinal epithelium morphology, microflora composition and metabolic activity is still remained unknown in weaned piglets. Our previous study indicated that dietary supplementation with B. subtilis (4 × 10⁹ CFU per gram) can decrease the severity of diarrhoea rate and improve the body weight gain of weaned piglets (Wang et al. 2019b). Thus, we hypothesized that dietary BLS mix supplementation as alternative of antibiotics will improve the intestinal ecosystem with associated beneficial effects on the development and health of weaned piglets. Therefore, the present study was aimed to investigate the effects of dietary BLS mix supplementation as an alternative of antibiotics on the intestinal epithelium morphology, microbiota communities and bacterial metabolites in weaned piglets.

Material and methods

Ethical approval

The experimental design and procedures used in this study were reviewed and approved by the Animal Care and Use Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (ISA‐2017‐016). The animal experiments and sample collection strictly followed the relevant guidelines.

Experimental design and dietary treatments

A total of 120 healthy crossbred piglets (Landrace × Large white, 7·00 ± 0·50 kg body weight) were weaned at 25 days of age and fed a corn and soybean meal‐based diet. After 3 days of adaptation, the piglets were randomly assigned to one of three groups: a control group (a basal diet), a probiotics group (a basal diet supplemented with 4 × 10⁹ CFU per gram BLS mix), and an antibiotic group (a basal diet supplemented with 0·04 kg t⁻¹ virginiamycin, 0·2 kg t⁻¹ colistin and 3000 mg kg⁻¹ zinc oxide). All groups had five replicates with eight piglets per replicate. The addition amount of the probiotics in diets was the recommended dose by the manufacturer (Evonik Degussa (China) Co. Ltd, Beijing, China). The composition and nutrient levels of the basal diet met the nutritional requirements for nursey piglets established by the National Research Council (2012), which are shown in Table S1. The experiments lasted for 42 days.

The probiotic BLS mix was produced by fermentation; the conditions were 200 rev min⁻¹ stirring speed and 350 l h⁻¹ throughput. The fermentation product was filtered with an organic ceramic membrane filtration system and mixed with mineral adsorbent at a ratio of 1 : 1. The final product was a dry powder that was counted by the plate counting method to determine the viable number and then mixed in the diet.

Sample collection and preparation

On days 7, 21 and 42 of the trial and 12 h after the last feeding, one piglet from each replicate (n = 5 per group) close to average weight were slaughtered by electric shock (120 V, 200 Hz). The intestinal contents from each colon (10 cm from the posterior to the ileocecal valve) were collected and stored at −20°C for analyses of the short‐chain fatty acids (SCFAs), indoles, skatoles, bioamines and the composition of the microbiota. Samples of the jejunum, ileum and colon tissue (approximately 2 cm) were collected, washed with cold physiological saline, immediately frozen in liquid nitrogen, and stored at −80°C for further analyses. The jejunum and ileum from all piglets were fixed with 4% paraformaldehyde‐PBS for overnight, and then dehydrated and embedded in paraffin.

Intestinal histological examination

The intestinal histological analysis was performed on paraformaldehyde‐fixed intestinal segments (from jejunum and ileum) that were sectioned (5 µm) and stained with haematoxylin and eosin. Intestinal histology was determined using a light microscope (Leica, Wetzlar, Germany) with Leica Application suit image analysis software (Leica). From each intestinal sample, villus height (from the tip of the villus to the mouth of crypt), and crypt depth (from the mouth of the crypt to the base) were measured at 10 visual fields. The villus height to crypt depth ratio was calculated.

16S sequencing and bioinformatics analysis

Microbial genomic DNA was extracted from all samples (n = 5) using a HiPure Stool DNA Kit (Magen, Guangzhou, China) following the manufacturer’s instructions. A multiplexed amplicon library covering V3–V4 region of the 16S rDNA gene was PCR‐amplified with optimized primer sets for the Illumina HiSeq 2500 sequencing instrument (Illumina, San Diego, CA). Each paired‐end read was then spliced using the flash (Magoc and Salzberg 2011) software (ver. 1.2.1) to obtain original spliced sequence (Raw contigs). Raw tags were mass filtered using a Trimmomatic software (ver. 0.33) to obtain high‐quality clean data. All chimeric sequences were removed by Uchime (Edgar et al. 2011) (ver. 4.2). The chimera‐free sequences were processed with a standard qiime 1.91 pipeline (Bokulich et al. 2013) and clustered into operational taxonomic units at a 97% similarity threshold using an ‘Open‐Reference’ approach. The raw Illumina pair‐end read data for all samples have been deposited in NCBI Sequence Read Archive database with accession number PRJNA589726.

Alpha diversity was analysed by Chao1, Shannon, and Simpson indices (Chao and Lee 1992). To decipher the differences in microbiota structure between groups, LEfSe (linear discriminant analysis effect size) was performed, and the cladogram was graphed with default parameters (Segata et al. 2011). To probe the microbial metabolism and predict metagenome functional content from the marker genes, PICRUSt was used to explore differences in the KEGG pathway between groups (Langille et al. 2013). Spearman correlation coefficients were calculated for the correlation between gut health and the change in microbiota to establish suitable microbial compositions for better gut health.

Bacterial metabolites in colonic contents

The colonic contents were homogenized and centrifuged at 1000 g for 15 min, as described previously (Kong et al. 2016). The intestinal SCFAs, including straight‐chain fatty acids (acetate, propionate, butyrate and pentanoate) and branched‐chain fatty acids (BCFA; isobutyrate and isopentanoate) were detected by gas chromatography, as described previously (Zhou et al. 2014). The bioamines, including putrescine, tryptamine, tyramine, spermidine and spermine were measured by high‐performance liquid chromatography, as described previously (Xu et al. 2014). Indoles and skatoles were analysed as in (Kong et al. 2016).

Analysis of gene expression related to gut health

Gene expression was determined by real‐time polymerase chain reaction (RT‐PCR), as described by (Su et al. 2018). Briefly, total RNA was isolated from colonic tissues using TRIzol (Invitrogen, Carlsbad, CA) and reverse transcribed with a Prime Script RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). RT‐PCR was conducted with primers of the target genes (Table S2), as well as the reference gene β‐actin, and fluoresce was monitored by the SYBR Green detection kit (Thermo Fisher Scientific, Waltham, MA) in a 7900 Fast Real‐Time PCR System (Applied Biosystems, Foster City, CA). The RT‐PCR conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 5 s and annealing at 60°C for 30 s. Relative gene expression was calculated by the 2^−ΔΔCt method (Schmittgen and Livak 2008).

Statistical analysis

Intestinal morphology index, colonic metabolite and the genes expression were analysed with a one‐way analysis of variance using spss 17.0 software (SPSS, Inc., Chicago, IL). The data are presented as means ± SE and P < 0·05 indicates statistical significance. The alpha diversity indices, relative species abundances and overall composition of gut microbiota were analysed using the Kruskal‐Wallis test. Spearman correlation coefficient was used to assess the relationships between health parameters and the relative abundances of genera. LEfSe was used to identify different taxa microbes using default parameters.

Results

Effect of BLS mix on intestinal morphology of weaned piglets

The intestinal morphology data are summarized in Table 1 and Fig. 1. On day 7 of the trial, dietary supplementation with antibiotics or BLS mix decreased the crypt depth (P < 0·05) and increased the ratio of villus height to crypt depth in the jejunum compared with the control group (P < 0·05). On days 7 and 42 of the trial, the villus height and the ratio of villus height to crypt depth in the ileum were increased in the antibiotics and BLS mix groups compared with the control group (P < 0·05). Interestingly, an increase in the crypt depth was observed in the BLS mix group relative to the antibiotics group on day 21 of the trial (P < 0·05) in the ileum.

Table 1

Effect of dietary supplementation with BLS mix on intestinal morphology in weaned piglets

Item	Jejunum			Ileum
	Control group	Antibiotics group	Probiotics group	Control group	Antibiotics group	Probiotics group
Day 7 of the trial
VH (µm)	334·42 ± 6·77	370·64 ± 9·29	421·88 ± 12·83	304·69 ± 19·15b	386·02 ± 40·10a	380·09 ± 21·48a
CD (µm)	291·98 ± 31·40a	218·00 ± 10·30b	246·73 ± 7·63ab	171·76 ± 4·10	198·66 ± 19·75	181·13 ± 12·14
VH/CD	1·20 ± 0·13b	1·71 ± 0·05a	1·71 ± 0·01a	1·78 ± 0·14b	1·94 ± 0·06a	2·12 ± 0·15a
Day 21 of the trial
VH (µm)	357·82 ± 15·15	440·79 ± 51·64	452·42 ± 16·00	362·35 ± 7·42	389·18 ± 32·53	414·61 ± 26·22
CD (µm)	260·30 ± 19·98	269·95 ± 38·28	315·32 ± 16·74	174·85 ± 3·86b	188·91 ± 8·06b	226·93 ± 11·06a
VH/CD	1·41 ± 0·13	1·67 ± 0·10	1·45 ± 0·09	2·08 ± 0·06	2·09 ± 0·25	1·84 ± 0·12
Day 42 of the trial
VH (µm)	408·59 ± 4·41	476·16 ± 19·93	439·19 ± 11·75	338·67 ± 7·07b	385·19 ± 21·46a	453·39 ± 60·52a
CD (µm)	296·57 ± 16·46	332·11 ± 22·76	263·13 ± 10·27	206·36 ± 11·20	175·23 ± 16·39	198·35 ± 23·65
VH/CD	1·39 ± 0·06	1·45 ± 0·07	1·68 ± 0·08	1·67 ± 0·13b	2·23 ± 0·13a	2·27 ± 0·05a

The data are presented as means ± SE (n = 5). Values in the same row with different letters are statistically significant (P < 0·05). VH, villus height; CD, crypt depth; and VH/CD, villus height to crypt depth ratio.

Figure 1

Morphology of jejunum and ileum of weaned piglets from control, antibiotic, and probiotics groups on days 7 (a), 21 (b) and 42 (c) of the trial (n = 5). Intestinal morphology was tested by H.E. staining (100×).

Effect of BLS mix on microbiota diversity of weaned piglets

The V3–V4 region of the microbial 16S rDNA sequencing generated 843 766 high‐quality reads, with an average of 56 251 reads (ranges 45 822–67 947) per sample. Alpha diversity was measured to detect the diversity and structure of colonic microbial communities in the different treatment groups (Fig. 2). The Simpson index did not differ throughout the experimental period (P > 0·05). However, compared to the control group, the Chao1 index of the BLS mix group was lower on day 7 of the trial (P < 0·05), whereas the Shannon index was higher on day 21 of the trial (P < 0·05). Furthermore, the BLS mix supplementation trended to decrease the Chao 1 and increase the Shannon index compared with the antibiotics group on days 7 and 21 of the trial, respectively. The results indicate that dietary supplementation with BLS mix increased the colonic microbial diversity of the piglets.

Figure 2

Alpha diversity of the colonic bacterial community of weaned piglets with different treatments on days 7 (a), 21 (b) and 42 (c) of the trial (n = 5). Control (), a control group (a basal diet); antibiotics (), an antibiotic group (a basal diet supplemented with 0·04 kg t⁻¹ virginiamycin, 0·2 kg t⁻¹ colistin and 3000 mg kg⁻¹ zinc oxide); and probiotics (), a probiotics group (a basal diet supplemented with 4 × 10⁹ CFU per gram BLS mix).

Effect of BLS mix on microbial communities of weaned piglets

Taxonomic classification of the microbial composition of the colonic contents revealed that Firmicutes, Bacteroidetes and Tenericutes were the most dominant bacterial phyla for the whole trial period (Fig. 3a). At the genus level, Lactobacillus, Ruminococcaceae, Clostridiales and Clostridiaceae were the most dominant strains (Fig. 3b). On days 7 and 21 of the trial, Lactobacillus (regardless of treatment) was the dominant strain, but on day 42 of the trial, Ruminococcaceae was the dominant strain in the BLS mix and control groups.

Figure 3

Colonic microbiota composition of weaned piglets with different treatments (n = 5). Microbial community bar plot at the phylum (a) and genus (b) levels. Control, a control group (a basal diet); antibiotics, an antibiotic group (a basal diet supplemented with 0·04 kg t⁻¹ virginiamycin, 0·2 kg t⁻¹ colistin and 3000 mg kg⁻¹ zinc oxide); and probiotics, a probiotics group (a basal diet supplemented with 4 × 10⁹ CFU per gram BLS mix) (a: Day 7: (  Chlamydiae;  TM7; other;  Spirochaetes;  Cyanobacteria;  Proteobacteria;  Actinobacteria;  Tenericutes;  Bacteroidetes;  Firmicutes; a: Day 21:  Chlamydiae;  Euryarchaeota;  TM7; other;  Spirochaetes;  Actinobacteria;  Proteobacteria;  Tenericutes;  Bacteroidetes;  Firmicutes; a: Day 42:  Fibrobacteres;  TM7;  Cyanobacteria; other;  Actinobacteria;  Proteobacteria;  Spirochaetes;  Tenericutes;  Bacteroidetes;  Firmicutes; b: Day 7:  Bacteroidales;  Ruminococcus;  Tremblayales;  RF39;  Clostridiaceae;  Prevotella; other;  Ruminococcaceae;  Clostridiales;  Lactobacillus;  S24.7;  Coriobacteriaceae;  Oscillospira; p.75.a5;  Lachnospira;  Lachnospiraceae;  Phascolarctobacterium;  Collinsella;  Roseburia;  SMB53; b: Day 21:  Ruminococcus;  S24.7;  RF39;  Tremblayales;  Clostridiaceae;  Prevotella; other;  Clostridiales;  Ruminococcaceae;  Lactobacillus; p.75.a5;  Lachnospiraceae;  Coriobacteriaceae;  Lachnospira;  Oscillospira;  Phascolarctobacterium;  SMB53;  Roseburia;  Streptococcus;  Bacteroidales; b: day 42:  Clostridiaceae;  Lachnospiraceae;  Lachnospira;  Streptococcus;  S24.7;  Prevotella; other;  Lactobacillus;  Clostridiales;  Ruminococcaceae;  Ruminococcus;  RF39;  Clostridium;  Bacteroidales;  Tremblayales;  Oscillospira;  Prevotella;  Megasphaera;  Roseburia;  Treponema;  coprococcus).

The differences in the microbial communities at the phylum and genus levels are shown in Fig. 4. On day 7 of the trial, the relative abundance of Ruminococcaceae was higher in the BLS mix group than in the control group (P < 0·05), and the relative abundance of Blautia was lower in the antibiotics and BLS mix groups than in the control group (P < 0·05). However, the BLS mix group had an increased abundance of Ruminococcaceae compared with the antibiotics group but not significantly (Fig. 4a). On day 21 of the trial, antibiotics led to an increase inTenericutes compared to their relative abundances in the control group (P < 0·05), while the BLS mix increased (P < 0·05) the abundance of Bacteroidale and decreased (P> 0·05) the abundances of Tenericutes compared with the antibiotics group. Furthermore, the BLS mix led to an increase in the abundance of Bacteroidales compared with the antibiotics and control groups (P < 0·05; Fig. 4b). On day 42 of the trial, the relative abundance of Prevotella was lower in the BLS mix group (P < 0·05), and the relative abundance of Clostridium was lower in the antibiotics and BLS mix groups, when compared the control group (P < 0·05); antibiotics led to a decrease in Lactobacillus and an increase in RF39 compared to their relative abundances in the control group (P < 0·05; Fig. 4c).

Figure 4

Comparison of the colonic microbial community in weaned piglets from control, antibiotic and probiotics groups on days 7 (a), 21 (b), and 42 (c) of the trial (n = 5). *P < 0·05, and **P < 0·01. Control (), a control group (a basal diet); antibiotics (), an antibiotic group (a basal diet supplemented with 0·04 kg t⁻¹ virginiamycin, 0·2 kg t⁻¹ colistin and 3000 mg kg⁻¹ zinc oxide); probiotics (), a probiotic group (a basal diet supplemented with 4 × 10⁹ CFU per gram BLS mix).

Effect of BLS mix on microbial function of weaned piglets

The LefSe analysis was performed to confirm the different effects of BLS mix on intestinal microbiota in piglets (Fig. 5). Significant differences in the colonic microbiota were found among the control, antibiotic and BLS mix groups for entire trial period. These results confirmed a significant enrichment of Ruminococcaceae in the BLS mix group on day 7 of the trial (Fig. 5a). On day 21 of the trial, Clostridia was the most abundant in the antibiotics group, and Elusimicrobia, Sphaerocheatales and Enterobacteriales were the most abundant in the BLS mix group (Fig. 5b). On day 42 of the trial, Clostridium,  Epsilonproteobacteria and Campylobacterales were enriched in the control group, while Chloroflexi, Streptomycetaceae and Sva0853 were enriched in the antibiotics group (Fig. 5c).

Figure 5

LefSe analysis of the colonic microbial community in weaned piglets from control, antibiotic and probiotics groups on days 7 (a), 21 (b) and 42 (c) of the trial (n = 5). Control (), a control group (a basal diet); antibiotics (), an antibiotic group (a basal diet supplemented with 0·04 kg t⁻¹ virginiamycin, 0·2 kg t⁻¹ colistin and 3000 mg kg⁻¹ zinc oxide); probiotics (), a probiotic group (a basal diet supplemented with 4 × 10⁹ CFU per gram BLS mix).

The PICRUSt algorithm was performed to assess the functional differences by plotting different pathways against the KEGG database (Fig. 6). On day 7 of the trial, pathways were enriched by the intestinal microbiota of the BLS mix group via African trypanosomiasis and ether lipid metabolism. On day 21 of the trial, pathways were enriched by the intestinal microbiota of the BLS mix group via terpenoids and polyketides, cofactors and vitamins, amino acid metabolism and carbohydrate metabolism. On day 42 of the trial, pathways were enriched by the intestinal microbiota of the BLS mix group via carbohydrates (such as pyruvate, citrate cycle, propanoate and butanoate), lipids, amino acid metabolism (such as lysine, tyrosine, cysteine and methionine), cofactors and vitamins, and glycan biosynthesis and metabolism.

Figure 6

PICRUSt and KEGG analysis of the colonic microbial community in weaned piglets from control, antibiotic and probiotics groups on days 7 (a), 21 (b), and 42 (c) of the trial, respectively. Control, a control group (a basal diet); Antibiotics, an antibiotic group (a basal diet supplemented with 0·04 kg t⁻¹ virginiamycin, 0·2 kg t⁻¹ colistin and 3000 mg kg⁻¹ zinc oxide); probiotics, a probiotic group (a basal diet supplemented with 4 × 10⁹ CFU per gram BLS mix).

Effect of BLS mix on gut metabolites of weaned piglets

As shown in Table S3, the concentrations of SCFA and bioamines in the colonic contents of the three treatment groups did not differ on day 7 of the trial (P > 0·05). The SCFAs concentrations on day 21 of the trial also did not differ (Table 2). On day 42 of the trial, the concentrations of isobutyrate and isovalerate were decreased in the BLS mix group compared with the control group (P < 0·05), while the concentration of acetate was increased (P < 0·05) in the BLS mix group compared with the control and antibiotics groups (Table 2). The bioamines concentration of the colonic contents is shown in Table 2. On day 21 of the trial, the concentration of spermine was increased in the BLS mix group compared with the control and antibiotics groups (P < 0·05). The skatoles concentration was increased in the antibiotics group compared with the control and BLS mix groups (P < 0·05). However, the concentration of skatoles was decreased in the antibiotics and BLS mix groups compared with the control group on day 42 of the trial (P < 0·05).

Table 2

Effect of dietary supplementation with BLS mix on gut metabolites in weaned piglets

Items	Day 21 of the trial			Day 42 of the trial
	Control group	Antibiotics group	Probiotics group	Control group	Antibiotics group	Probiotics group
Short‐chain fatty acids (mg g−1)
Acetate	3·06 ± 0·05	3·15 ± 0·17	2·94 ± 0·15	2·23 ± 0·20b	2·30 ± 0·22b	3·06 ± 0·08a
Propionate	1·61 ± 0·05	1·79 ± 0·12	1·46 ± 0·10	1·09 ± 0·16	0·97 ± 0·13	1·25 ± 0·06
Butyrate	1·13 ± 0·09	1·08 ± 0·04	0·97 ± 0·09	0·87 ± 0·12	0·64 ± 0·13	0·85 ± 0·13
Isobutyrate	0·10 ± 0·01	0·10 ± 0·01	0·11 ± 0·01	0·13 ± 0·03a	0·09 ± 0·02ab	0·07 ± 0·00b
Valerate	0·21 ± 0·03	0·19 ± 0·02	0·17 ± 0·03	0·22 ± 0·07	0·15 ± 0·04	0·12 ± 0·01
Isovalerate	0·17 ± 0·01	0·18 ± 0·01	0·20 ± 0·03	0·24 ± 0·07a	0·17 ± 0·05ab	0·11 ± 0·01b
Bioamines (μg g−1)
Phenylethylamine	0·07 ± 0·05	0·04 ± 0·03	0·34 ± 0·14	0·04 ± 0·04	0·17 ± 0·06	0·08 ± 0·06
Putrescine	2·15 ± 0·73	4·70 ± 3·05	2·13 ± 1·21	15·44 ± 1·60	13·98 ± 3·80	14·88 ± 1·43
Spermidine	3·78 ± 1·06	4·53 ± 0·93	2·65 ± 0·43	21·23 ± 1·38	20·83 ± 3·96	17·86 ± 1·88
Spermine	2·44 ± 1·48b	1·40 ± 0·49b	6·22 ± 0·47a	4·01 ± 0·50	5·01 ± 1·17	4·33 ± 0·36
Tryptamine	0·13 ± 0·03	0·20 ± 0·18	0·05 ± 0·03	1·38 ± 0·34	1·29 ± 0·36	1·30 ± 0·29
Tyramine	0·37 ± 0·09	0·87 ± 0·52	0·31 ± 0·09	3·09 ± 0·54	3·07 ± 1·84	3·12 ± 1·24
Indoles	2·66 ± 0·48	2·21 ± 0·32	4·37 ± 0·87	3·70 ± 0·90	3·78 ± 0·95	3·54 ± 1·21
Skatoles	6·82 ± 0·75b	11·58 ± 1·68a	4·61 ± 0·85b	11·59 ± 3·50a	5·26 ± 1·00b	2·73 ± 0·74b

The data are presented as means ± SE (n = 5). Values in the same row with different letters are statistically significant (P < 0·05).

Effect of BLS mix on intestinal health‐related genes of weaned piglets

On day 7 of the trial, dietary supplementation with BLS mix increased the mRNA levels of interleukin (IL)‐2, IL‐6, IL‐1β and toll‐like receptor (TLR)‐4 (P < 0·05) and decreased the level of tumor necrosis factor (TNF)‐α (P < 0·05) in the ileum compared with the antibiotics group. Dietary BLS mix supplementation also increased the level of IL‐2 in the jejunum compared with the antibiotics group (P < 0·05) (Table S4). On day 21 of the trial, dietary BLS mix supplementation increased the level of TLR‐4 (P < 0·05) and decreased the level of TNF‐α (P < 0·05) in the ileum compared with the antibiotics group (Table S4). On day 42 of the trial, an increase in the level of TLR‐4 in the ileum was observed in the BLS mix group relative to the control group (P < 0·05) (Table S4).

The mRNA levels in the colon contents of piglets from different dietary treatments are shown in Table 3. Compared to the control group, dietary supplementation with BLS mix increased the levels of IL‐6, IL‐1β and TLR‐4 on day 7 of the trial, and the levels of E‐cadherin and IL‐1β on day 42 of the trial (P < 0·05). Dietary supplementation with antibiotics increased the mRNA levels of E‐cadherin, IL‐2, IFN‐α, TLR‐4 and TNF‐α on day 21 of the trial.

Table 3

Effect of dietary supplementation with BLS mix on intestinal health‐related genes in weaned piglets

Items	Day 7 of the trial			Day 21 of the trial			Day 42 of the trial
	Control group	Antibiotics group	Probiotics group	Control group	Antibiotics group	Probiotics group	Control group	Antibiotics group	Probiotics group
IL‐1β	1·00 ± 0·06b	1·59 ± 0·79ab	2·56 ± 0·25a	1·00 ± 0·25	1·76 ± 0·31	1·48 ± 0·10	1·00 ± 0·15b	3·35 ± 0·37a	3·04 ± 0·40a
IL‐2	1·00 ± 0·12	1·23 ± 0·39	1·27 ± 0·04	1·00 ± 0·31b	2·25 ± 0·42a	1·25 ± 0·24b	1·00 ± 0·18	3·74 ± 1·84	1·08 ± 0·15
IL‐6	1·00 ± 0·10b	1·10 ± 0·10b	1·63 ± 0·15a	1·00 ± 0·07	1·00 ± 0·08	1·30 ± 0·08	1·00 ± 0·14	0·99 ± 0·05	1·21 ± 0·13
IL‐10	1·00 ± 0·17	0·99 ± 0·25	1·47 ± 0·12	1·00 ± 0·24	1·42 ± 0·18	0·94 ± 0·07	1·00 ± 0·05	2·11 ± 0·44	1·58 ± 0·16
IFN‐α	1·02 ± 0·12	1·15 ± 0·36	1·19 ± 0·04	1·00 ± 0·17b	1·41 ± 0·28ab	1·54 ± 0·12a	1·00 ± 0·19	1·13 ± 0·31	0·87 ± 0·10
TLR‐4	1·00 ± 0·06b	1·10 ± 0·24b	2·42 ± 0·62a	1·00 ± 0·15b	1·91 ± 0·24a	1·35 ± 0·16ab	1·00 ± 0·14	1·56 ± 0·17	1·57 ± 0·12
TNF‐α	1·00 ± 0·13	1·16 ± 0·41	1·97 ± 0·22	1·00 ± 0·04b	2·09 ± 0·64a	2·25 ± 0·30a	1·00 ± 0·05	2·48 ± 0·24	2·54 ± 0·30
E‐cadherin	1·00 ± 0·08	1·19 ± 0·23	1·22 ± 0·03	1·00 ± 0·10b	1·63 ± 0·12a	1·27 ± 0·14ab	1·00 ± 0·14b	1·70 ± 0·16a	1·63 ± 0·16a
ZO‐1	1·00 ± 0·08	1·22 ± 0·13	1·95 ± 0·29	1·00 ± 0·02	1·54 ± 0·20	1·06 ± 0·09	1·00 ± 0·14	1·31 ± 0·18	1·09 ± 0·09

Data are presented as means ± SE (n = 5). Values in the same row with different superscripts differ significantly (P < 0·05). IFN‐α, interferon‐alpha; IL, interleukin; TLR, toll‐like receptor; TNF‐α, tumor necrosis factor‐alpha; ZO‐1, zonula occludens‐1.

Relationship between gut microbiota, metabolites and intestinal health‐related genes

The Spearman’s rank‐order correlation analysis was performed to evaluate the potential link between alterations in gut microbiota and health parameters of the weaned piglets (Fig. 7). The genus Prevotella was positively correlated with spermidine and spermine (P < 0·01) and negatively correlated with acetate and propionate (P < 0·05). The phylum Bacteroidetes and genus CF231 were positively correlated with tryptamine, spermidine and spermine (P < 0·01) but negatively correlated with acetate (P < 0·01). The genus Anaerovibrio was positively correlated with spermine but negatively correlated with acetate, propionate, and butyrate. The order Bacteroidetes was positively correlated with spermine (P < 0·05). The phylum Treponema was positively correlated with tyramine, tryptamine, putrescine and spermidine but negatively correlated with skatoles (P < 0·05). The family Erysipelotrichaceae was positively correlated with spermidine, TLR‐4, and IL‐1β (P < 0·05). The genus Clostridium was positively correlated with spermidine and negatively correlated with acetate (P < 0·05). The order Clostridiales was positively correlated with Occludin (P < 0·05). The phylum Firmicutes was negatively correlated with spermidine (P < 0·05). The family Ruminococcaceae was positively correlated with Occludin and TLR‐4 (P < 0·05) but negatively correlated with tyramine, tryptamine and putrescine (P < 0·05). The order RF39 and phylum Tenericutes were negatively correlated with tyramine, tryptamine, and putrescine (P < 0·05). The genus Lactobacillus was positively correlated with acetate (P < 0·05) but negatively correlated with spermidine, E‐cadherin, and IL‐1β (P < 0·05). The genus Blautia was negatively correlated with IL‐1β (P < 0·05).

Figure 7

Correlations between the microbiota, health parameters and colonic metabolite concentrations in weaned piglets.

Discussion

Bacillus spp. possess pathogen exclusion, anti‐oxidant and immuno‐modulatory abilities (Elisashvili et al. 2019). The results of the present study clearly show that dietary BLS mix supplementation modifies several important aspects of the characteristics of the intestinal ecosystem.

The intestinal mucosal barrier is an important modulator of intestinal homeostasis, and intestinal morphology reflects mucosal integrity and injury (Blikslager et al. 2007). The villus height and crypt depth play crucial role for nutrients absorption and digestion, and protect from the pathogenic infection (Yin et al. 2019). Previous studies have shown that Bacillus spp. supplementation in diets can improve the nutrients absorption and digestibility by increasing the villus height and villus height to crypt depth ratio (Li et al. 2019). Our results showed that dietary BLS mix supplementation decreased crypt depth in the jejunum, increased villus height in the ileum, and changed the ratio of villus height to crypt depth in the jejunum and ileum, suggesting an improved nutrient digestibility and absorption capacity of weaned piglets. Recent studies suggested that the gut physiological barrier is formed by epithelial cells, tight junction proteins and intestinal secretions (Suzuki 2013; Yan and Ajuwon 2017), in which E‐cadherin, and Occludin are indicators for tight junction assembly, stability, and barrier function (Zihni et al. 2016). The results of the present showed that the BLS mix upregulated the expression of E‐cadherin in the colon, suggesting that the BLS mix reinforces the integrity of the intestinal mucosa.

Alpha diversity reflects the species diversity in a single sample by incorporating indices for species richness (Chao and Ace) and diversity (Shannon and Simpson) (DeSantis et al. 2006). Our findings are consistent with those of (Wang et al. 2019a), who found that probiotics increase the Simpson’s diversity index of the microbial ecosystem in piglets. Firmicutes and Bacteroidetes are regarded as the main microbiota phyla in the pig gut, regardless of probiotic or antibiotic use (Wang et al. 2019a). The present study showed that dietary supplementation with BLS mix increased the relative abundances of Bacteroidale and Ruminococcaceae and decreased the abundances of Blautia and Clostridium. Bacteroidales can degrade proteins and carbohydrates (Thomas et al. 2011) and activate the host’s immune system (Mazmanian et al. 2008). Clostridium is closely related to protein fermentation and can increase the risk of diarrhea (Rist et al. 2014). The lower abundance of Clostridium in the probiotics group may explain our previous finding that dietary supplementation with B. subtilis decreased the diarrhoea rate in the piglets (Wang et al. 2019b), which is also consistent with the antibiotics group. Ruminococcaceae ferments cellulose and hemicellulose and produces SCFAs for energy production (Biddle et al. 2013) and the regulation of gene expression in the colonocytes. These findings suggest that the BLS mix may regulate gut community composition and improve gut health.

PICRUSt (Langille et al. 2013) can be used to investigate the functional differences in microbiota to determine the metabolic alterations caused by antibiotics or probiotics (Wang et al. 2019a). In this study, dietary BLS mix supplementation enriched the metabolic pathways involved in cofactors/vitamin and carbohydrate metabolism on day 21 of the trial, as well as the metabolic pathways involved in carbohydrate, lipid, amino acid metabolism, synthesis of cofactors, vitamins, and glycan, metabolism on day 42 of the trial. Vitamins and cofactors are notably crucial for the bioconversion of nutrients to energy, and for maintaining homeostasis in different tissues (Upston et al. 2003; Jorde and Grimnes 2011; Hu et al. 2016; Sharma et al. 2019). The glycan biosynthesis and metabolism pathway is important for carbohydrate metabolism (Hu et al. 2016; Varki 2017). (Gao et al. 2017) showed that feed‐additive probiotics accelerated intestinal microbiota maturation. Therefore, our findings suggest that dietary BLS mix supplementation might accelerate intestinal microbiota maturation by the enrichment of important metabolic pathways.

The SCFAs produced by colonic microbes via the fermentation of indigestible fibre are important for gut integrity, glucose homeostasis and immune function (Morrison and Preston 2016; Yin et al. 2018a). In our present study, acetate and propionate were the major SCFAs in the colon, which was consistent with previous findings in pregnant Huanjiang mini‐pigs (Kong et al. 2016). Acetate can inhibit pathogenic bacteria, while butyrate acts as a major source of energy for colonic epithelial cells (Morrison and Preston 2016). BCFAs are produced by microbes through the deamination and decarboxylation of amino acids (Mukherji et al. 2003; Le Roy et al. 2013). In the present study, dietary BLS mix supplementation increased the acetate concentration and decreased the concentrations of isobutyrate and isovalerate. BCFAs are indicators of the extent of protein fermentation in the intestinal content, thus suggesting that BLS mix supplementation decreases the capacity of the microbiota for the degradation of amino acids.

The colonic microbiota catabolizes nitrogenous compounds to putrefactive catabolites, such as bioamines, indoles and skatoles (Kong et al. 2016). Our results showed that dietary BLS mix supplementation increased the spermine concentration and decreased the skatoles concentration in colon. Spermine is a polyamine, an important component for bacterial growth, and can increase the reactive oxygen species production and DNA damage in colonocytes when present in excess (Blachier et al. 2017). Furthermore, higher skatoles concentration might be toxic to gut health (Yin et al. 2018b). Thus, decreasing the concentration of skatoles in the colon by supplementing a BLS mix in piglets’ diet may exert beneficial effects on gut health.

Cytokines play crucial roles in the regulation of immune function, inflammatory responses, and the barrier integrity of the gut (Andrews et al. 2018). LPS‐mediated induction of the TLR‐4 signaling pathway results in the activation of nuclear factor κB (NF‐κB), and therefore, the expression of pro‐ and anti‐inflammatory cytokines. The piglets challenged with LPS upregulated their gene expression of pro‐inflammatory cytokines in the jejunal mucosa, including TNF‐α, IL‐1β, IL‐6, IFN‐γ and IL‐8 (Cao et al. 2018). Our findings showed that dietary BLS mix supplementation increased the levels of pro‐inflammatory cytokine and TLR‐4 in the ileum and colon, suggesting that the BLS mix might activate the intestinal immune response. (Shi et al. 2019) showed that moderate activation of the TLR‐4 signalling pathway promoted repair of the intestinal epithelium in the DSS‐induced colitis model.

In conclusion, dietary supplementation with BLS mix impacts the intestinal ecosystem with overall beneficial effects in weaned piglets in terms of microbiota composition and metabolic activity, as well as in terms of the effects recorded on the intestinal mucosa. Therefore, the present study indicates that dietary probiotics mixture would be an effective alternative of antibiotics.

Acknowledgements

This study was jointly supported by the National Key Research and Development Project (2017YFD0500503), National Natural Science Foundation of China (31772613 and 31572421), and STS regional key project of the Chinese Academy of Sciences (KFJ‐STS‐QYZD‐052). The authors thank the staff and postgraduate students of Hunan Provincial Key Laboratory of Animal Nutritional Physiology and Metabolic Process for collecting samples, and technicians from CAS Key Laboratory of Agro‐ecological Processes in Subtropical Region for providing technical assistance. We would like to thank Editage (www.editage.cn) for English language editing.

Authors' contributions

X.D.W., Z.L.T., W.M.Z. and M.A.K.A. performed the experiments. X.D.W. and Z.L.T. performed the statistical analyses and wrote the manuscript. Z.B.W. and X.F.K. contributed to experimental concepts and design, provided scientific direction and finalized the manuscript with the help of F.B. All authors read and approved the final manuscript.

Conflict of interest

Author Wenming Zhang was employed by the company Evonik Degussa (China). The remaining 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.

Author notes

Xiaodan Wang and Zhilong Tian contributed equally to this manuscript.