Isolation of Bacillus cereus and its probiotic effect on growth performance, antioxidant capacity, and intestinal barrier protection of broilers

Abstract

Probiotics are effective for improving poultry health. Probiotic Bacillus cereus strains are widely used to improve animal health by stimulating the immune system. In this study, we obtained a B. cereus 13 (BC13) strain that functions in acid, high-temperature, and bile salt resistance. It also degrades starch, cellulose, and other proteins. To better understand the probiotic effects of BC13, we added the strain to the diet of broilers and observed its effects. We found that BC13 significantly improved the growth performance of broilers. The levels of total antioxidant capacity, superoxide dismutase, and glutathione peroxidase were increased, and the concentration of malondialdehyde was reduced by BC13. Supplementation with BC13 enhanced immune function by increasing the levels of secretory immunoglobulin A (sIgA) in the jejunum mucosa; IgA, IgM, and IgG in the serum; mRNA levels of Zo-1, claudin and occludin of the jejunal mucosa; and increased villus height/crypt depth of the jejunum. Furthermore, BC13 improved the composition of intestinal microbes, especially at the genus level of Akkermansia. The addition of BC13 increased the levels of acetic, butyric, valeric, and propionic acids. These results emphasise the potential of BC13 as a probiotic dietary supplement to improve the antioxidant capacity, intestinal barrier function, and gut microbial composition to enhance body health.

Introduction

Sustainable poultry production is crucial for meeting the protein requirements of a growing population. Antibiotics are widely used for the prevention and treatment of animal diseases and for growth promotion, and their consumption is increasing worldwide (Kovalakova et al., 2020). However, the continuous use of antibiotics has led to antimicrobial resistance, which poses a threat to public health security (Huan et al., 2020). In recent years, owing to the high demand for antibiotics in poultry, the use of probiotics has increased steadily (Jha et al., 2020). Probiotics are considered an alternative solution to antibiotics because they form an effective protective barrier to prevent excessive colonisation of pathogenic bacteria in the digestive system (He et al., 2023). Bifidobacterium, Lactococcus, Lactobacillus, Bacillus, Streptococcus, and yeasts, such as Candida, are the main genera of probiotic microorganisms commonly used in poultry (Jha et al., 2020).

Bacillus is a mature provider of proteases and lipases (Shan et al., 2023), and many probiotic food supplements and therapeutic products containing or composed of Bacillus strains/species have become available for human consumption (Li et al., 2019). Bacillus cereus strains have been reported to stimulate the immune system and synthesise different antimicrobials (Lee et al., 2019). There are several B. cereus stains are used in probiotic products, such as B. cereus strain GM in Biovicerin, B. cereus (CECT 953) in Esporafeed Plus, B. cereus CIP5832b (ATCC 14893) in Paciflor C10, B. cereus var vietnami in Subtyl (Hoa et al., 2000), and B. cereus var toyoi (NCIMB-40112/CNCM-1012) in Toyocerin (Hong et al., 2005). And B. cereus var toyoi is one of the products authorised by the European Union for use as a feed additive for sows and piglets (Scharek-Tedin et al., 2013).

The gastrointestinal tract is the largest immunological organ in the body and plays a key role in regulating immune homeostasis (Takiishi et al., 2017). The gut is the most extensive interface between internal and external environments (Di Vincenzo et al., 2024). It contains a vast commensal microbial community, including bacteria, viruses, archaea, yeasts, and fungi, that colonise the bowel (Tropini et al., 2017). In recent years, the gut microbiota has been reported to play a vital role in immune regulation, lipid and bile acid metabolism, and neuroregulation (Ma et al., 2022). Intestinal microbiome dysregulation refers to changes in the composition of the gut microbiome associated with functional changes in the microbial transcriptome, proteome, or metabolome (Zeng et al., 2017). The addition of probiotics to feed can change the gut histomorphology, improve immune function, and alleviate intestinal disorders (Shen et al., 2024). It has been reported that the probiotic B. cereus can improve the gut microbiota and intestinal barrier function and inhibit inflammation; therefore, it has been widely used to improve health (Sheng et al., 2021).

In the current study, we isolated strain B. cereus 13 (BC13) with potential as a probiotic and explored its role in the growth performance, immune function, colonic VFAs, intestinal morphology, and gut microbiota of Arbor Acres (AA) broilers.

Materials and methods

Observation of colony morphology

BC13 was isolated from the soil around commercial broiler farms. Briefly, the bacterial solution was diluted to an appropriate multiple, applied to Luria broth (LB) solid medium, and incubated at 37 °C in an incubator overnight. The size, colour, shape, edge integrity, and transparency of the colonies were recorded.

16S rDNA gene sequence analysis

A DNA extraction kit (A29790; Invitrogen, Waltham, MA, USA) was used to extract genomic DNA of the isolated strain, and the DNA was sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing, and the results were entered into the NCBI nucleic acid database for sequence comparison.

Gram staining

First, the bacteria were smeared and fixed, and crystal violet staining was performed on the fixed culture. After 1 min, the crystal violet stain was removed and the cells were rinsed with water. The iodine solution was then applied for 30 s. The iodine solution was removed and rinsed with running water. A few drops of 95 % alcohol were added and the mixture was gently shaken for decolourisation. After 20 s, the samples were washed with water and moisture was absorbed. After staining with a diluted safranin solution for 1 min, the samples were rinsed with distilled water. Finally, the samples were dried and subjected to microscopic examination.

Growth characteristics

A single colony of BC13 was selected and cultured in LB medium with shaking at 37 °C, then inoculate the fresh bacterial suspension in LB liquid medium. The number of bacteria was measured at 600 nm every 30 min using an Infinite 200 Pro microplate reader (Tecan, Switzerland).

High-temperature resistance

After quantifying the bacteria using a turbidity meter, the fresh bacterial suspension was placed in a water bath at different temperatures (37, 70, 80, 90, and 100 °C) for 0, 3, 5, 10, and 15 min. The number of viable bacteria was calculated using the plate-counting method.

Bile salt tolerance

Bacteria were incubated in LB medium with concentrations of porcine bile salts (0.0, 0.1, 0.2, 0.3, and 0.4 %), and incubated at 37 °C for 1, 2, 3, and 4 h. The number of viable bacteria was then calculated using the plate counting method.

Acid resistance

Bacteria were incubated in LB medium at different pH values (2.0, 3.0, 4.0, and 5.0) and incubated at 37 °C for 1, 2, 3, and 4 h. The number of viable bacteria were calculated using the plate counting method.

Ability of hydrolysis starch

Soluble starch agar (CN230550; Chinook, China) was used. A drop of bacterial solution was placed onto an agar plate and cultured at 37 °C for 24 h. A few drops of Lugo's iodine solution (CN230370; Chinook) were then added to the colony. After 3 min, a transparent circle is observed.

Ability of degrading protein

Casein agar (CN230555; Chinook) was also used. After cultured a drop of bacteria solution at 30 °C for 24 h, observed the size of the transparent circle.

Detection and quantitation of cellulase

Congo red sodium carboxymethyl cellulose screening medium (CN230510; Chinook) was used. After culturing a drop of bacterial solution at 30 °C for 24 h, it was stained with 1 mg/mL Congo Red for 30 min. The waste liquid was discarded, the formation of transparent circles was observed, and the cellulose degradation ability of the strain preliminarily determined based on the ratio of the diameter of the transparent circle to the diameter of the colony.

Antibiotic sensitivity assay

Antibiotic sensitivity was determined using drug-sensitive paper tablets (Hangzhou Microbial Reagent Co Ltd., China). A bacterial suspension was prepared with a 0.5 McPherson turbidity standard using broth. The bacterial suspension was inoculated onto the MH agar plates within 15 min of preparation. Sterile tweezers were used to attach the paper sheet to the surface of the agar and were gently pressed. It was then placed in a 37 °C incubator for 16–18 h. The diameters of the antibacterial zones were measured using a ruler. Each tablet was tested in triplicate.

Experimental animal and sample collection

All experimental procedures were approved by the Animal Protection and Utilization Organization Committee. One-day-old AA broilers (n = 240) were randomly divided into two treatment groups: blank control (Con; basic diet) and BC13 treatment (BC13; basic diet + 0.5 mL bacterial solution, 1 × 10⁹ cells/mL). The nutritional values of the broiler feed are shown in Table 3. Each treatment had 6 replicates with 20 chickens per replicate. The weight and feed intake of AA broilers were recorded every week, and the average daily gain, average daily feed intake, and feed weight ratio were calculated based on the daily weight change and feed intake.

Table 3.

Nutritional value of broiler feed.

Nutritional value	1–21d (%)	22–42d (%)
Water	≤ 14	≤ 14
Crude protein	≥ 20	≥ 19
Calcium	0.6–1.20	0.6–1.20
Available phosphorus	≥ 0.5	≥ 0.4
NaCl	0.2–0.8	0.2–0.8
Crude fibre	≤ 6.0	≤ 6.0
Crude ash	≤ 8.0	≤ 8.0
Methionine+cystine	≥ 0.82	≥ 0.71

On days 21 and 42, seven broiler chickens were euthanised in each group. The serum was separated and stored at -80 °C, as well as the liver and caecal contents. Jejunal segments were fixed in 4 % paraformaldehyde for the morphological assessment, and the jejunum mucosa was stored at -80 °C for later analysis.

Morphological analysis of jejunum

The intestinal histological sections were stained with haematoxylin and eosin (H&E) and observed under an inverted microscope. The lengths and depths of the intestinal villi were measured using an image analysis program.

Capacity of anti-oxidation in serum and liver

Serum and liver concentrations of total antioxidant capacity (T-AOC), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were determined using commercially available kits (Jiancheng, China).

RT-qPCR

Total RNA was separated from the jejunal mucosa using TRIzol reagent (Takara, China). RNA was reverse transcribed into cDNA using PrimeScript™ RT Reagent Kit (Takara). All qRT-PCRs were performed and analysed using a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR Green PCR Master Mix (Takara,). The qPCR primers for mRNA encoding tight junction proteins (ZO-1, claudin, and occludin) are presented in Table 1. The levels of relative mRNA were assessed using the 2^-ΔΔCT method. β-actin was used as an internal standard for data analysis and normalisation.

Table 1.

Primers for qPCR.

Gene	Primer sequence
zo-1	Forward: 5′-AAGCTCAGCCTCAGCCAAAT-3′
	Reverse: 5′-GGGGAAAGGTAAGGGACTGC-3′
claudin1	Forward: 5′-GGTGTACGACTCGCTGCTTA-3′
	Reverse: 5′-CTTCATGCACTTCATGCCCG-3′
occludin	Forward: 5′-GCGGTTACTACTACAGCCCC-3′
	Reverse: 5′-TAGCGCCAGATCTTACTGCG-3′
β-actin	Forward: 5′-GCCAACAGAGAGAAGATGACAC-3′
	Reverse: 5′-GTAACACCATCACCAGAGTCCA-3′

Determinants of immunoglobulin levels

Levels of immune-related indicators of the jejunum mucosa, namely secretory immunoglobulin A (sIg)A, and IgA, IgG, and IgM in the serum, were tested using ELISA kits per the manufacturer's instructions (Jiancheng, China).

Short-chain fatty acid analysis

Short-chain fatty acid (SCFA) levels are commonly analysed using gas chromatography–mass spectrometry (GC-MS). They primarily include valeric, isovaleric, caproic, acetic, propionic, butyric, and isobutyric acids. The caecal content (0.5 g) was weighed and sent to Sangon Biotech for GC analysis.

DNA extraction, 16S rRNA sequencing, and statistical analysis

Total DNA was extracted using the QIAamp DNA Mini Kit (#51304; Qiagen, Germany) and quantified using a Nanodrop spectrophotometre (Thermo Fisher Scientific, Waltham, MA, USA). The 16S rRNA V3-V4 region was amplified using the specific primers 341F (CCTACGGGRSGCAGCAG) and 806R (GGACTACV VGGGTATCTAATC). The plate was sealed and PCR performed in a thermal instrument (GeneAmp™ PCR System 9700 Fast Thermal Cycler; Applied Biosystems, Foster City, CA, USA) using the following program: 1 cycle of denaturing at 95 °C for 3 min, first 5 cycles of denaturing at 95 °C for 30 s, annealing at 45 °C for 30 s, elongation at 72 °C for 30 s, 20 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 30 s, and a final extension at 72 °C for 5 min. The quality of PCR products was assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies Inc., Santa Clara, CA, USA). The PCR products were sequenced using an Illumina MiSeq system (Illumina Inc., San Diego, CA, USA).

After sequencing, two short Illumina readings were assembled using the PEAR software (version 0.9.8). The effective tags were clustered into operational taxonomic units (OTUs) of ≥97 % similarity using Usearch software (version 11.0.667). OTU sequences were taxonomically classified by blasting against the RDP database. The α-diversity indices (Chao1, Simpson, and Shannon indices) were quantified in terms of OTU richness. All α-diversity indices were calculated with Mothur software (version 3.8.31). The within-sample α-diversity was calculated by the t-test for the two groups. β-diversity evaluates differences in the microbiome among samples and is normally combined with dimensional reduction methods such as principal coordinate analysis (PCoA). These analyses were visualised using the R vegan package (version 2.5-6).

Linear discriminant analysis (LDA) coupled with effect size (LEfSe) was performed using the LEfSe tool, and the P-value was determined by Metastats analysis using the stats R package to discriminate bacterial taxa with significantly different abundances. Only colonies that showed a P-value < 0.05 and a log LDA score >2 were included. Statistical significance was set at P < 0.05 was considered significant.

Statistical analysis

The reported results were statistically evaluated using a paired Student's t-test, and comparisons between more than two groups were performed using ANOVA. The reported values are expressed as the standard error of the mean (SEM). Data were analysed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). The levels of significance were set at ns > 0.05, *P < 0.05, and **P < 0.01. Each experiment was performed in at least three replicates.

Results

Identification of BC13

Morphology and 16s rDNA detection were used to identify the bacteria. The colony morphology on LB agar plates was mainly milky-white, round, raised, and opaque, with extended edges and a rough surface resembling white wax (Fig. 1A). Gram staining revealed a Gram-positive rod-shaped bacterium with a square end approximately 2–5 μm in length (Fig. 1B). From the growth curve, we found that the bacteria were in the logarithmic growth phase from 2–8 h, in the plateau phase from 8–14 h, and then began the logarithmic growth phase again (Fig. 1C). The 16S rRNA sequences were submitted to BLAST in the NCBI database and the most closely related species was B. cereus (Fig. 1D). This strain was named BC13.

Fig. 1.

Identification of BC13. (A) Colon morphology of BC13. (B) Gram staining of BC13, bar = 25 μm. (C) Growth curve of BC13. (D) Phylogenetic tree of BC13.

Temperature, bile salts, and pH resistance of BC13

When probiotics are added to feed at high temperatures, their heat resistance must be considered. As shown in Fig. 2A, the survival rate BC13 was approximately 75 % after heat treatment at 70 °C for 15 min, 50 % at 80 °C, and 25 % at 90 °C. This indicates that BC13 exhibited good heat resistance.

Fig. 2.

Probiotic properties of BC13. (A) Effect of temperature on growth of BC13. (B) Ability of bile salt tolerance of BC13. (C) Survival rate of BC13 at various pH levels. Data are expressed as mean ± SEM, n = 3. (D) Disintegrating starch ability of BC13. (E) Degrading protein ability of BC13. (F) Degrading cellulose ability of BC13. Data are expressed as mean; n = 3.

The intestine is the main site where probiotics function; therefore, probiotics need to tolerate stomach acid and bile salts. As shown in Fig. 2B, when BC13 was treated with 0.1 % bile salt for 1 h, the survival rate was 75 %, which dropped to 25 % after 2 h, suggesting that this strain has common bile salt tolerance. At pH 4 and 5, BC13 survived as expected. Although the survival rate showed a decreasing trend at pH 3, the bacterial survival rate still exceeded 50 % after 4 h. At pH2, the survival rate of BC13 cells was approximately 50 % after 2 h of treatment (Fig. 2C), indicating that BC13 has good acid resistance.

In this study, we examined the function of BC13 in the hydrolysis of starch, cellulose, and proteins. As shown in Fig. 2D, the diameter of the transparent circle (T), colony (C), and T/C were 1.1, 0.50, and 2.20, respectively, indicating that BC13 has a pretty ability to degrade starch. When T/C was 2.42 (Fig. 2E), the function of BC13 in degrading proteins was ideal. In the detection of cellulose degradation function, T/C was 0.90 (Fig. 2F).

Antibiotic sensitivity of BC13

With the widespread application of probiotics, safety issues have gradually attracted attention and antibiotic resistance is a possible threat to health (Li, et al., 2020). In the present study, 20 antibiotics were used to determine bacterial sensitivity (Table 2). The results showed that BC13 was sensitive to 18 antibiotics, moderately sensitive to penicillin, and resistant only to polyxin B.

Table 2.

Drug sensitivity tests.

Drug category	Drug name	Drug content	Zone (mm)	Sensitivity
Nitrofuran	Furazolidone	300 μg	25	S
Amphenicols	Chloroamphenicol	30 μg	22	S
Cephalosporins	Cefradine	30 μg	30	S
	Cefoperazone	75 μg	30	S
Glycopeptides	Vancomycin	30 μg	24	S
Tetracyclines	Tetracycline	30 μg	25	S
	Doxycycline	30 μg	26	S
Penicillins	Penicillin	10 U	19	M
	Piperacillin	100 μg	36	S
	Ampicillin	10 μg	27	S
Lincosamides	Clindamycin	2 μg	30	S
Quinolones	Ciprofloxacin	5 μg	37	S
	Norfloxacin	10 μg	29	S
	Ofloxacin	5 μg	29	S
Polypeptides	Polymyxin B	300 IU	11	R
Macrolides	Midecamycinum	30 μg	33	S
	Erythromycin	15 μg	24	S
Aminoglycosides	Kanamycin	30 μg	29	S
	Neomycin	30 μg	25	S
	Gentamicin	10 μg	26	S

A circle diameter <15 mm was resistant, 16–20 mm was medium sensitive, and diameter >20 mm was sensitive.

Effects of BC13 on growth performance of AA broilers

Feed conversion ratio (FCR) is the ratio of feed intake to body weight gain, with lower values indicating better feed efficiency (Yi et al., 2018). The effects of BC13 supplementation on the growth performance of AA broilers are shown in Table 4. There was no significant difference in the initial weight of AA broilers between groups. Compared to the Con group, broiler chickens fed BC13 showed better growth performance. In phase 1 (1–21 days), BC13 significantly increased average daily gain (ADG) and FCR (P < 0.05). In phase 2 (22–42 days), BC13 supplementation remarkably improved FCR (P < 0.05) but slightly enhanced (P > 0.05) ADG. For all phases, the BC13 group showed significantly improved growth performance in ADG and FCR.

Table 4.

Effects of BC13 on growth performance of AA broilers.

Items	Con	BC13	SEM	P
Initial weight (g)	48.10	47.67	1.01	0.692
Weight of 21 day (g)	749.36a	759.29b	3.33	0.041
Weight of 42 day (g)	2532.26a	2590.35b	20.33	0.046
Phase (1–21d)
ADG (g)	33.39a	33.89b	0.13	0.019
ADFI (g)	52.81	52.57	0.38	0.565
FCR (F/G)	1.58a	1.55b	0.01	0.027
Phase (22–42 d)
ADG (g)	84.90	87.19	0.99	0.082
ADFI (g)	175.09	175.17	1.80	0.967
FCR(F/G)	2.06a	2.01b	0.01	0.007
All phase (1–42 d)
ADG (g)	59.15	60.54	0.48	0.044
ADFI (g)	113.95	113.87	0.91	0.934
FCR (F/G)	1.93a	1.88b	0.01	0.003

ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio; means within a row with different letters indicate significant differences (P < 0.05).

Antioxidant indicators of liver and serum

To prevent excessive reactive oxygen species (ROS) accumulation, the body employs various antioxidant mechanisms that inhibit ROS production. In this study, we tested antioxidant-related indicators in the liver and serum, and the results were shown in Fig. 3. Compared to the Con group, the levels of liver T-AOC, SOD, and GSH-Px increased significantly on days 21 and 42, whereas the MDA content in the BC13 group was significantly lower (Fig. 3A–D). The changes in serum antioxidant indices in the BC13 group were similar to those in the liver (Fig. 3E–H), which also showed a significant increase in antioxidant levels. These results showed that BC13 significantly improved the antioxidant capacity in AA broilers.

Fig. 3.

Effect of BC13 on antioxidant indexes. Levels of (A) T-AOC, (B) MDA, (C) SOD, and (D) GSH-Px in liver. Levels of (E) T-AOC, (F) MDA, (G) SOD, and (H) GSH-PX in serum. Data are expressed as mean ± SEM; n = 6; ns > 0.05; **P < 0.01; *P < 0.05.

Morphological analysis of jejunum

The tissue structure of the jejunum in AA broilers is shown in Fig. 4. The jejuna of both the Con and BC13 groups were integral and composed of slender villi and complete crypts (Fig. 4A). Compared to the Con group, supplementation with BC13 significantly increased the villus height (V) of the jejunum (Fig. 4B) at days 21 and 42, but had no significant effect on jejunum crypt depth (C) (Fig. 4C). There was no significant difference in the V/C ratio of broilers between the Con and BC13 groups on day 21. However, on day 42, the V/C ratio in the BC13 group was significantly higher than that in the Con group (Fig. 4D).

Fig. 4.

Effect of BC13 on jejunal morphology. (A) Intestinal morphology. (B) Villus height in jejunum. (C) Crypt depth of jejunum. (D) Ratio of villus height to crypt depth (V/C ratio). Data are expressed as mean ± SEM; n = 6; ns > 0.05; **P < 0.01; *P < 0.05.

Effects of BC13 on jejunum mucosa tight junction and immunoglobulins

Tight junctions play an important role in the intestinal barrier function. In this study, we examined the mRNA levels of three tight junction-related proteins (Zo-1, claudin, and occludin). As shown in Fig. 5A–C, compared to the Con group, the mRNA levels of Zo-1, claudin and occludin were markedly enhanced when AA broilers were fed BC13 at both days 21 and 42, indicating that BC13 significantly improved the protective effect on the intestinal barrier.

Fig. 5.

Effect of BC13 on jejunal mucosa tight junctions and immunoglobulins. Fold change of mRNA expression of (A) zo-1, (B) claudin, and (C) occludin were determined using qRT-PCR (n = 6). Levels of immunoglobulin (D) sIgA in jejunum mucosa, (E) IgA, (F) IgM, and (G) IgG in serum were determined using ELISA. Data are expressed as mean ± SEM; n = 6; ns > 0.05; **P < 0.01; *P < 0.05.

The effects of BC13 on the levels of immunoglobulins (sIgA in jejunal mucosa and IgA, IgM, and IgG in serum) are illustrated in Fig. 5D–G. Compared to the Con group, the BC13 group showed significantly higher levels of sIgA, IgA, and IgM at days 21 and 42. IgG levels increased significantly on day 42 and only slightly on day 21. Overall, BC13 significantly enhanced the immune capacity in AA broilers.

Microbial composition analysis

To evaluate the effects of BC13 on the intestinal microbial composition in AA broilers, 16S rDNA sequencing was used to analyse the caecum chime samples. Compared to the Con group, the Chao, Shannon, and Simpson indices in the BC13 group were significantly different (Fig. 6A). PCA showed that the caecal community composition of the Con and BC13 groups was significantly different (PC1 = 48.55 % and PC2 = 28.26 %) (Fig. 6B). According to the common strains analysed using the Venn diagram, there were 1862 common strains between these two groups (Fig. 6C), and BC13 treatment enhanced species abundance in the caecum microbiota composition. Statistical analysis of the top 15 intestinal flora abundances at the genus level are shown in Fig. 6D; Alistipes, Limosilactobacillus, Clostridiales, Ruminococcaceae, and Lactobacillus were the dominant species in the caecal flora of 42-day-old broilers. Compared to the Con group, the addition of BC13 increased the relative abundances of Akkermansia and Intestinimonas.

Fig. 6.

Modulation of BC13 on caecum microbiota composition. (A) Comparison of α-diversity (index Chao, Shannon, and Shannoneven) based on genus profiles. (B) PCA of caecum microbiota. (C) Venn diagram between Con and BC13 group. (D) Relative abundance of top 15 genus in caecum. Data are shown as mean. (E) LEfSe analysis of caecum microbiota; n = 7–8.

Differences in bacterial taxa were identified by LEFSe using an LDA score threshold of >2.0; g_unclassified_Ruminococcaeae, g_Akkermansia, f_Akkermansiaceae, p_Verrucomicrobia, o_Verrucomicrobiaies, c_Verrucomicrobiae, g_Paludicola, g_Intestinimonas, and g_Neglecta were significantly enriched in the BC13 group, and c_Bacilli, f_Lactobacillaceae, o_Lactobacillales, and g_Limosilactobacillus were dominant in the Con group (Fig. 6E). At the genus level, using P < 0.05, we found that BC13 dramatically increased the abundance of g_Akkermansia, g_Paludicola, and g_unclassified_Ruminococcaeae (Fig. 6F).

Concentrations of SCFAs in colonic content

The effects of BC13 on SCFA levels in the colonic content in AA broilers are shown in Fig. 7. The levels of acetic, butyric, valeric, and propionic acids were markedly elevated in the BC13 group compared to those in the Con group at both days 21 and 42 (P < 0.05). The isobutyric acid levels were markedly elevated compared to those in the Con group at 21 days (P < 0.05), whereas BC13 only slightly increased. The isobutyric acid content was higher than that in the Con group at day 42. There were no significant differences in the levels of isovaleric and caproic acids between the Con and BC13 groups at either days 21 or 42 (P > 0.05).

Fig. 7.

Effects of BC13 on colonic levels of SCFAs. (A) SCFA levels in colonic contents of 21-day-old broilers. (B) SCFA levels in colonic contents of 42-day-old broilers. Data are expressed as mean ± SEM; n = 6; ns > 0.05; **P < 0.01; *P < 0.05.

Discussion

Probiotics are described as “live microbes when administered in adequate quantities, confer health benefits on host organisms” according to the Food and Agriculture Organization (FAO) and World Health Organization (WHO) (Gasbarrini et al., 2016). They are widely used in human, animal husbandry, plant, and environmental remediation to prevent and treat diseases (Cui, et al., 2019). Probiotic B. cereus strains stimulate the immune system and synthesise different antimicrobials (Lee, Kim and Paik, 2019), but the regulatory mechanisms remain unclear. In the current study, we isolated the B. cereus strain BC13 and examined its probiotic properties and mechanisms of action.

Because feed accounts for approximately 70 % of the total cost in the poultry industry, improving feed efficiency is an important goal for poultry production (Wen et al., 2018). BC13 had the ability to degrade starch, cellulose, and protein and helped the body absorb the hydrolysed products (Fig. 2D–F). Therefore, the addition of BC13 significantly improved the growth performance of broilers (Table 4). The villi of the small intestine are the main components of human digestion and absorption, and their shape reflects the absorptive function of the small intestine. The surface area of the small intestine is significantly increased by the presence of villi and microvilli, which increase the intestinal surface area by 30–600 times (Kiela and Ghishan, 2016). Villus height and crypt depth are key morphological indices that reflect the digestive and absorption functions of the small intestine. The larger the V/C value, the better the intestinal development and the ability to digest and absorb nutrients (Wang et al., 2021a). The addition of probiotics, such as Bacillus subtilis and Lactobacillus spp. is beneficial for improving the intestinal structure (Zhu et al., 2022). In the present study, we found that dietary BC13 supplementation improved jejunal villus height, crypt depth, and V/C ratio (Fig. 4). These findings suggest that the addition of BC13 can effectively improve intestinal morphology, increase intestinal surface area, promote the absorption of nutrients, and reduce the ratio of feed to meat in AA broilers, indicating that BC13 has the potential to become a probiotic.

Oxidative stress is a state of imbalance in the production and elimination of ROS, which increases the number of free radicals (Hussain et al., 2016). Excessive ROS production causes various types of damage to the body, including protein damage, DNA damage, lipid peroxidation, and cell function damage (Guo et al., 2021). MDA, a free radical, has been used as a biomarker to measure oxidative stress in biological samples from patients with various diseases (Chen et al., 2015). As shown in Fig. 3B and F, compared to the Con group, MDA levels in the group BC13 were significantly reduced, indicating that BC13 can reduce the levels of oxidative stress. T-AOC plays a vital role in the antioxidant defense system. This refers to the overall ability of a biological sample to resist oxidative stress by neutralising ROS and other harmful free radicals (Silvestrini et al., 2023). As shown in Fig. 3A and E, BC13 significantly increased T-AOC levels, suggesting that BC13 improved the antioxidant capacity. First-line defense antioxidants, including SOD, catalase, and GSH-Px, are indispensable in the overall defense strategy of antioxidants, especially when it comes to the superoxide anion free radical (*O2) that is continuously produced in normal body metabolism (Wang et al., 2019). We found that BC13 significantly increased the levels of SOD and GSH-Px (Fig. 3C, D, G, and H). Overall, BC13 significantly enhanced antioxidant levels in the body.

The intestinal barrier is an important line of defence for maintaining homeostasis of the intestinal microenvironment, mainly through the interaction of mechanical, chemical, immune, and microbial barriers (Camilleri et al., 2012; Konig et al., 2016). Tight junctions are intercellular junctions that are major components of the epithelial barrier. It is mainly composed of several transmembrane and cytoplasmic proteins such as occludin, claudin, and ZOs (Chelakkot et al., 2018). In this study, we tested the mRNA expression of zo-1, claudin, and occludin (Fig. 5A–C), and found that BC13 significantly increased their levels, indicating that BC13 can improve the tight junctions of the jejunal mucosa. Probiotics can induce IgA circulation, strengthen and maintain immune surveillance of the parenteral mucosa, and promote the maturation of immune mechanisms (Wang et al., 2021b). sIgA from the plasma cells participates in the immune defense mechanism of the intestinal barrier (Wells et al., 2017). In the present study, BC13 increased sIgA levels which modulated intestinal immunity (Fig. 5D). This phenomenon has been shown with respect to S. boulardii, which could increase intestinal sIgA levels (Gou et al., 2022). In our study, BC13 also enhanced the levels of IgM, IgG, and IgA in the serum (Fig. 5E and F), leading to increased protection of the body from invasion by pathogenic microorganisms (Megha and Mohanan, 2021). This shows that BC13 can improve the immune function in the body.

The gut microbiota is a key component of the gut ecosystem and plays a vital role in human health, including barrier action against pathogens, formation and maturation of immunity, regulation of metabolic intake, and absorption of nutrients and drugs (Nogal et al., 2021). In the present study, BC13 altered the intestinal microbial composition in AA broilers and increased the relative abundances of Akkermansia and Intestinimonas (Fig. 6E). At the genus level, using P < 0.05, we found that BC13 dramatically increased the abundance of g_Akkermansia, g_Paludicola, and g_unclassified_Ruminococcaeae (Fig. 6F). Akkermansia muciniphila (A. muciniphila), a bacterium rich in the intestinal mucus layer, has been widely studied for its multiple functions and beneficial roles in systemic metabolism, immunity, intestinal barrier, tumours, and other diseases, and is considered a promising next-generation probiotic (Nogal, Valdes, and Menni, 2021; Zhu et al., 2023). These results indicated that BC13 treatment increased the content of beneficial bacteria in the intestinal tract. SCFAs are small organic carboxylic acids with carbon atomic numbers <7 and are mainly produced by the fermentation of dietary fibre (Xiao et al., 2022). A large number of commensal microbes inhabiting in the gastrointestinal lumen send signals to the immune and endocrine systems of the host by secreting metabolites such as SCFAs, polyamines, and amino acids (Xiao et al., 2022). In our study, we found that BC13 significantly increased SCFA levels, especially acetic, butyric, valeric, and propionic acids, suggesting that BC13 enhances collective immunity by increasing the concentration of SCFAs.

Conclusions

In conclusion, we obtained strain BC13 that exhibited acid, high-temperature, and bile salt resistance. It degraded starch, cellulose, and proteins, and improved the growth performance, antioxidant capacity, intestinal immunity, and gut microbiota of AA broilers. In general, BC13 has the potential as a probiotic, which can improve the immunity of broilers by improving the gut microbiota (Fig. 8).

Fig. 8.

Graphical representation of how BC13 improves growth performance, immune function, and intestinal health of AA broilers.

Ethicals approval

All animal experiments were performed in accordance with the Ethical Principles in Animal Research and were approved by the Ethics Committee of the Institute of Animal science and Veterinary Medicine, Shandong Academy of Agricultural Sciences (approval number: IASVM-2023-011).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.