Bacillus velezensis CML532 supplementation improved growth performance, protein digestion, and gut health in broilers fed with corn-miscellaneous meal-based low-protein diets

Abstract

The poultry industry faces challenges due to its high reliance on imported soybean meal (SBM) and the rising costs of conventional protein ingredients, prompting interest in using miscellaneous meals as alternative protein sources. However, low-protein diets formulated with these meals often impair broiler growth performance and protein digestion. In the present study, the effects of Bacillus velezensis CML532 supplementation on growth performance, protein digestion, and gut health were investigated in broilers fed corn-miscellaneous meal-based diets without SBM during the grower and finisher phases. A total of 252 one-d-old healthy male Shengze 901 plus broiler chicks (initial body weight 43.76 ± 0.61 g) were randomly assigned to 3 dietary treatments, each comprising 6 replicates of 14 birds: control group, corn-miscellaneous meal-based normal-protein diet (CM); corn-miscellaneous meal-based low-protein diet (CML) group; and CML diet with supplemental 5 × 10⁹ colony-forming unit (CFU)/kg B. velezensis CML532 (CMLB) group. The experiment lasted 42 d. The results showed that, compared with the CM group, the lower crude protein level (−2%) in the CML group significantly increased feed conversion ratio (FCR), and decreased half-eviscerated rate, eviscerated rate, and breast muscle rate (P < 0.05). Meanwhile, the mRNA expression of calcium sensing receptor (CaSR) and cationic amino acid transporter 1 (CAT1), as well as the concentration of anti-inflammatory factor interleukin-10 (IL-10), were significantly downregulated (P < 0.05). Compared with the CML group, B. velezensis supplementation significantly decreased FCR, improved slaughter traits, increased apparent protein and amino acid digestibility (P < 0.05), and reduced serum uric acid levels (P < 0.001). Notably, growth performance in the CMLB group in terms of final body weight and FCR of the whole growth period was comparable to that in the CM group (P > 0.05). In the jejunum, the CMLB group significantly increased the expression of intestinal epithelial barrier proteins, and elevated the activities of α-amylase and chymotrypsin in the digesta compared with both the CM and CML groups (P < 0.05). Furthermore, the mRNA expression levels of amino acid sensing receptors CaSR, taste receptor type 1 member 1 (T1R1), G protein-coupled receptor class C group 6 member A (GPRC6A), and transporter CAT1 were markedly upregulated in the CMLB group (P < 0.05). Ileal microbial analysis revealed a decreased relative abundance of uncultured_bacterium_g_Lactobacillus in the CML group, whereas B. velezensis supplementation increased the enrichment of Coriobacteriaceae_bacterium_CHKCI002 and B. velezensis, accompanied by enhanced His metabolism and biosynthesis pathways. These results indicated that B. velezensis CML532 supplementation improved protein digestion and utilization by enhancing digestive enzyme activity and modulating gut microbial composition, thereby promoting growth performance and intestinal health in broilers fed with low-protein corn-miscellaneous meal-based diets to levels comparable with those fed normal protein diets.

1. Introduction

The rapid expansion of the poultry industry has intensified the demand for sustainable and cost-effective protein feed ingredients. In China, the feed industry relies heavily on imported protein sources such as soybean and faces increasing challenges due to the rising costs of conventional protein ingredients like soybean meal (SBM) (Martens et al., 2012). Consequently, considerable attention has been directed toward identifying alternative protein sources, such as miscellaneous meals, and developing strategies to reduce dietary crude protein (CP) levels without compromising performance (Liu et al., 2017, 2024). However, broiler chickens fed low-protein diets formulated with unconventional protein feed ingredients based on the ideal essential amino acid (EAA) pattern established by our laboratory have exhibited suboptimal growth performance, especially in terms of feed conversion efficiency (unpublished data).

Miscellaneous meals primarily refer to non-SBM agricultural by-products generated during the processing of oil seeds and other feed stuffs, after the extraction of their main nutritional components, such as rapeseed meal, peanut meal, and cottonseed meal (Olukomaiya et al., 2019). These ingredients have received growing interest as an alternative to SBM due to their lower cost and greater availability (Wang et al., 2024; Zhang et al., 2022). Despite these advantages, the application of miscellaneous meals has been limited by their imbalanced amino acid (AA) profiles, high fiber content, and presence of anti-nutritional factors, all of which can impair nutrient absorption, protein digestibility, and intestinal health (Adedokun et al., 2008; Kim et al., 2012). These challenges are particularly pronounced when miscellaneous meals completely replace SBM, which negatively impacts growth performance and increases metabolic stress in broilers. Recent advances in the utilization of alternative protein have demonstrated that double-low rapeseed meal (low erucic acid and glucosinolate) can substitute 6%–10% of SBM at 7%–14% dietary inclusion. Similarly, low-gossypol cottonseed meal may replace 2%–10% of SBM when incorporated at 2.5%–10.5%, and peanut meal demonstrates higher substitution potential at 15% SBM replacement with 15% dietary inclusion (Ministry of Agriculture and Rural Affairs of the People’s Republic of China, 2021). Moreover, Payvastegan et al. (2017) observed a linear decrease in body weight gain (BWG) from 1 to 42 d of age as canola meal (a double-low rapeseed meal bred in Canada) progressively replaced SBM from 0 to 30% in nutritionally balanced diets.

Probiotics have been widely used as feed additives to enhance nutrient utilization and support gut health in broilers. Their beneficial effects have been attributed to multiple mechanisms, including the production of digestive enzymes, modulation of gut microbiota, and reinforcement of intestinal barrier function (Pan et al., 2022; Zhang et al., 2019). Konieczka et al. (2019) noted that the negative effects of partially replacing SBM with 25% rapeseed meal, narrow-leaved lupin, and distillers dried grains with solubles on microbial activity and diversity were alleviated by the administration of a liquid multimicrobial probiotic preparation. Among probiotic species, Bacillus subtilis has been extensively studied in poultry nutrition due to its ability to form heat-stable spores and secrete digestive enzymes (Dicandia et al., 2022; Nadler et al., 2018). In addition, Bacillus pumilus and Bacillus licheniformis have been recognized for their exceptional environmental resilience and antimicrobial activity (Ongena and Jacques, 2008; Samon et al., 2022). Recently, Bacillus velezensis has attracted increasing interest owing to its broad enzymatic repertoire (proteases, cellulases, and phytases), and remarkable tolerance to bile salts and high temperatures (Li et al., 2023; Soni et al., 2021). Moreover, B. velezensis strains have been shown to suppress pathogenic microbes and synthesize bioactive compounds, such as bacilysin and bacillomycin D (Rabbee et al., 2019), highlighting their potential to enhance the intestinal environment.

These characteristics suggest that B. velezensis may serve as a promising candidate for enhancing protein digestibility and growth performance in broilers, especially under low-protein diets. In our laboratory, a strain of B. velezensis CML532 was isolated from the cecum of the Shanzhongxian rooster, a native chicken breed known for its adaptability and stress tolerance (Zhu et al., 2024). In vitro assays revealed the potential probiotic properties, including thermal stability, enzymatic activity, and resistance to pathogenic bacteria. Whole-genome sequencing further revealed the presence of genes related to AA transport and metabolic pathways, supporting its potential application in enhancing protein utilization in poultry diets.

This study was conducted to evaluate the effects of B. velezensis CML532 supplementation in broiler diets with reduced CP levels (2% lower than normal), in which SBM was entirely replaced by concentrated cottonseed protein, rapeseed meal, and peanut meal. The findings are expected to provide a more comprehensive understanding of the role of B. velezensis CML532 in facilitating the utilization efficiency of miscellaneous protein feed stuffs and supporting dietary CP reduction strategies in broiler nutrition.

2. Materials and methods

2.1. Animal ethics statement

All animal handling and management procedures adhered to Chinese animal welfare regulations and received approval from the Animal Welfare Committee of China Agricultural University (approval No. Aw40704202-1-3).

2.2. Trial design and animal management

The experiment involved 252 healthy 1-d-old male Shengze 901 plus broiler chicks (initial body weight [IBW] 43.76 ± 0.61 g), which were randomly divided into 3 treatment groups, each with 6 replicates of 14 birds. The treatments were as follows: control group, corn-miscellaneous meal-based normal-protein diet (CM); corn-miscellaneous meal-based low-protein diet (CML) group; and CML diet with supplemental 5 × 10⁹ colony-forming unit (CFU)/kg B. velezensis CML532 (CMLB) group. In this study, the corn-miscellaneous meal-based diet refers to a formulation in which SBM was entirely replaced by alternative protein ingredients, including concentrated cottonseed protein, rapeseed meal, and peanut meal (collectively categorized as miscellaneous meals). The experiment spanned 42 d and was divided into starter (1-14 d), grower (15-28 d), and finisher (29-42 d) phases. The composition and nutritional levels of the basal diets were formulated based on the China National Feeding Standard of Chicken NY/T 33-2004, 2004, as detailed in Table 1.

Table 1.

Composition and nutrient levels of basal diets (%, air-dried basis)¹.

Items	1–14 d		15–28 d		29–42 d
	CM	CML	CM	CML	CM	CML
Ingredients
Corn (7.8%)	46.00	53.62	54.48	58.72	56.05	60.70
Wheat flour	10.00	10.00	10.00	10.00	10.00	10.00
Corn gluten meal (60%)	8.68	6.98	8.82	4.82	10.00	6.00
Soybean meal (43%)	5.46
Concentrated cottonseed protein	10.00	8.54	9.10	7.00	6.01	3.52
Rapeseed meal	5.00	5.00	5.00	5.00	5.00	5.00
Peanut meal (47.8%)	5.00	5.00	5.00	5.00	5.00	5.00
Soybean oil	3.55	2.80	1.75	2.35	2.80	3.30
Limestone powder	1.48	1.51	1.40	1.40	1.31	1.32
Dicalcium phosphate	1.46	1.50	1.24	1.25	1.09	1.11
Sodium chloride	0.18	0.13	0.06	0.10	0.15	0.14
Choline chloride (50%)	0.16	0.19	0.20	0.19	0.22	0.22
Mineral premix2	0.20	0.20	0.20	0.20	0.20	0.20
Vitamin premix3	0.02	0.02	0.02	0.02	0.02	0.02
L-Lys hydrochloride	0.60	0.80	0.64	0.72	0.58	0.67
DL-Met	0.10	0.18	0.05	0.15		0.07
L-Cys		0.02		0.02		0.04
L-Thr	0.09	0.20	0.10	0.19	0.07	0.17
L-Try	0.04	0.08	0.06	0.06	0.04	0.05
L-Arg		0.22		0.16		0.20
L-Ile	0.14	0.29	0.15	0.27	0.11	0.25
L-Leu	0.14	0.42	0.05	0.41	0.09	0.45
L-Ser	0.14	0.29	0.14	0.27	0.11	0.24
L-His	0.01	0.10	0.04	0.09	0.03	0.09
L-Val	0.11	0.24	0.13	0.21	0.08	0.18
Gly	0.07	0.17	0.10	0.15	0.07	0.13
Phytase4	0.02	0.02	0.02	0.02	0.02	0.02
Potassium carbonate	0.60	0.75	0.39	0.44	0.23	0.28
Sodium bicarbonate	0.25	0.25	0.35	0.28	0.22	0.14
Titanium dioxide	0.50	0.50	0.50	0.50	0.50	0.50
Total	100.00	100.00	100.00	100.00	100.00	100.00
Nutrients
ME5, MJ/kg	13.00	13.00	13.23	13.23	13.40	13.40
CP5	23.00	21.00	20.02	18.34	20.00	18.00
Ca6	1.05	1.05	0.94	0.94	0.86	0.86
Non-phytate phosphorus6	0.35	0.35	0.31	0.31	0.28	0.28
Digestible Lys5	1.07	1.07	0.99	0.95	0.90	0.90
Digestible Met5	0.47	0.50	0.32	0.29	0.36	0.35
Digestible Cys5	0.35	0.32	0.30	0.26	0.31	0.30
Digestible Met + Cys5	0.82	0.82	0.62	0.55	0.67	0.65
Digestible Thr5	0.71	0.71	0.58	0.55	0.62	0.61
Digestible Arg5	1.38	1.35	1.15	1.06	1.07	1.06
Digestible His5	0.51	0.51	0.41	0.34	0.45	0.44
Digestible Ile5	0.89	0.89	0.60	0.53	0.78	0.77
Digestible Leu5	2.04	2.04	1.81	1.63	1.93	1.92
Digestible Trp5	0.22	0.22	0.18	0.17	0.18	0.17
Digestible Val5	0.89	0.89	0.77	0.68	0.78	0.77
Digestible Gly5	0.71	0.71	0.58	0.57	0.61	0.60
Digestible Ser5	1.00	1.00	0.77	0.67	0.88	0.88
Digestible Gly + Ser5	1.71	1.71	1.34	1.24	1.49	1.48

CP = crude protein; ME = metabolizable energy.

¹CM, corn-miscellaneous meal-based normal-protein control group; CML, corn-miscellaneous meal-based low-protein group.

²Trace mineral elements per kg of diet are as follows: copper, 16 mg; iron, 80 mg; zinc, 110 mg; manganese, 120 mg; iodine, 1.5 mg; selenium, 0.3 mg; cobalt, 0.5 mg.

³Vitamins per kg of diet are as follows: vitamin A, 10,000 IU; vitamin D₃, 2400 IU; vitamin E, 20 IU; vitamin K₃, 2 mg; vitamin B₁, 1.6 mg; vitamin B₂, 6.4 mg; vitamin B₆, 2.4 mg; vitamin B₁₂, 0.02 mg; niacinamide, 30 mg; D-pantothenic acid, 9.2 mg; folic acid, 1 mg; biotin, 0.1 mg.

⁴Heat-tolerant phytase (≥10,000 U/g) supplied by Beijing Xindayang Technology Development Co., Ltd. (Beijing, China).

⁵The values are analyzed based on the actual measurements of the experimental diets during the grower phase (15–28 d), and calculated according to the Chinese Chicken Feeding Standard (2004) for the starter (1–14 d) and finisher phases (29–42 d).

⁶The value is calculated based on the Chinese Chicken Feeding Standard (2004).

The study was conducted at the Poultry Experimental Base of China Agricultural University in Zhuozhou (Hebei, China), and the broilers were reared in single-layer cages. The temperature was set at 33 °C during the first week, and gradually decreased by 3 °C each week until reaching 24 °C. Throughout the whole period, broilers had free access to crumble-pellet feed and water, with light in accordance with the broiler standard, which consisted of a 24-h photoperiod for the first 2 d, followed by a step-wise reduction to 23 h (d 3), 22 h (d 4-5), 21 h (d 6-7), and finally 20 h from d 8 to 31. The photoperiod was subsequently extended back to 24 h towards the end of the rearing cycle.

2.3. Sample collection

On the 28th d of the experiment, one broiler close to the average body weight (BW) was chosen from each replicate. Wing vein blood was sampled and centrifuged at 2000 × g under 4 °C for 15 min, and then stored in a −80 °C refrigerator for biochemical analysis. Subsequently, the broilers were stunned electrically and euthanized by exsanguination. The immune organs (liver, spleen, thymus, and bursa of fabricius) were extracted and weighed for organ index determination. About 1 cm of jejunal tissue and jejunal chyme were collected for subsequent determination of gene expression levels and digestive enzyme activity. Ileal and cecal chyme were obtained for B. velezensis quantification and microbial composition analysis.

2.4. Growth and slaughter trait measurements

After a 12-h feed withdrawal period, the BW and feed consumption of broilers were accurately recorded to calculate BWG, feed intake (FI), and feed conversion ratio (FCR) for the phases of 0-14 d, 15- 28 d, and 29-42 d. On d 42, one broiler near the average BW was randomly selected from each replicate, sacrificed via exsanguination, and defeathered for slaughter traits assessment. Carcass characteristics like carcass rate, eviscerated rate, half-eviscerated rate, wing rate, thigh rate, breast muscle rate, and abdominal fat rate were determined following the methods specified in NY/T 823-2020 (Ministry of Agriculture of the People's Republic of China, 2020).

2.5. Immunological organ indexes

The liver, spleen, thymus, and bursa were weighed, and the organ index was determined as follows:

Organ index (g/kg) = Organ weight (g)/BW (kg).

2.6. Serum biochemistry assay

Serum levels of total protein (TP; A045-4-2), albumin (ALB; A028-2-1), uric acid (UA; C012-2-1), blood urea nitrogen (BUN; C013-2-1), D-lactic acid (D-LA; A019-3-1), and diamine oxidase (DAO; A088-3-1) were analyzed using kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The optical density (OD) for each assay was read using a microplate reader (Synergy 4, Bio-Tek Instruments Inc., Winooski, VT, USA).

2.7. Apparent nutrient digestibility measurement

The apparent digestibility of nutrients was determined using the exogenous indicator method. The excreta samples were collected on d 25 and 26. After spraying 10% hydrochloric acid on the surface to fix nitrogen and mixing well, the excreta samples were dried in an oven (model 101A-2B, Shanghai Huyue Ming Scientific Instrument Co., Ltd., Shanghai, China) at 65 °C until a constant weight was achieved, then crushed and passed through a 40-mesh sieve (aperture size 0.425 mm). Gross energy (GE), CP, and AA contents in both diets and excreta samples were analyzed as follows. GE was measured by oxygen bomb calorimetry using a 6400 automatic isoperibol calorimeter (Parr Instrument Co., Moline, IL, USA) in accordance with the method of 9831 (ISO, 1998). CP was analyzed via a Kjeldahl nitrogen analyzer (KT200, FOSS Analytical A/S, Hillerød, Denmark) following the GB/T 6432-2018 (China National Standard, 2018). AA composition was analyzed on an amino-acid analyzer (A300; membraPure GmbH, Hennigsdorf, Germany) after sample hydrolysis and pre-column derivatization as described by the method of Cohen (2000). Metabolizable energy (ME) in the diets and apparent nutrient digestibility were measured as follows:

Apparent nutrient digestibility (%) = [1 – (TiO₂ content in diets/TiO₂ content in excreta) × (Nutrient content in excreta/Nutrient content in diets)] × 100;

ME in diets (MJ/kg) = GE of feed × Apparent energy digestibility.

2.8. Detection of digestive enzyme activity in jejunal digesta

The obtained samples of jejunal chyme were accurately weighed and diluted with normal saline at a 1:9 (w/v) ratio to make a 10% tissue homogenate. The supernatant was collected for the measurement of α-amylase (C016-1-1), trypsin (A080-2-2), and chymotrypsin (A080-3-1) activities, following centrifugation at 1000 × g for 10 min at 4 °C. The specific test method was performed following the instructions provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The OD value for each assay was read using a microplate reader (Synergy 4).

2.9. Intestinal mRNA expression analysis

Total RNA was obtained from jejunal samples by Trizol (Cat. No. 9109, Takara Bio Inc., Kusatsu, Shiga, Japan), and its concentration was measured with NanoDrop 2000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). After complementary DNA‌ (cDNA) was generated by reverse transcription with the PrimeScript RT reagent kit (RR047A, Takara Bio Inc., Kusatsu, Shiga, Japan), the target gene expression levels were quantified by TB Green Premix Ex Taq kit (RR420A, Takara Bio Inc., Kusatsu, Shiga, Japan) with Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific Inc., Foster City, CA, USA), and β-actin served as an internal reference. The 2^−ΔΔCt method was used to calculate the relative expression levels of the target genes, with the specific primer sequences listed in Table S1. The sampling setup and reaction procedure were presented in Tables S2 and S3.

2.10. Quantification of inflammatory cytokines in the jejunum

Jejunal segments were homogenized and centrifuged at 4 °C, and the resulting supernatants were collected for subsequent analysis following the manufacturers' protocols. Total protein in the tissue extracts was determined using a bicinchoninic acid (BCA) protein assay kit (P0012S, Beyotime Biotech Inc., Shanghai, China). Concentrations of tumor necrosis factor-α (TNF-α; TOPEL03104), interleukin (IL)-6 (TOPEL03137), and IL-10 (TOPEL303344) in the jejunal supernatants were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Beijing Biotopped Technology Co., Ltd., Beijing, China) according to the manufacturers’ instructions. Cytokine contents were normalized to total tissue protein, and are expressed as pg cytokine per mg protein.

2.11. Quantification of B. velezensis

After extracting the total genomic DNA from the jejunum, ileum, and cecum contents using commercial DNA extraction kit (CW2091S, Jiangsu Cowin Biotech Co., Ltd., Taizhou, Jiangsu, China), real-time quantitative PCR (RT-qPCR) was performed with TB Green Premix Ex Taq kit (RR420A, Takara Bio Inc., Kusatsu, Shiga, Japan). The primers targeting total bacteria and B. velezensis were used based on previously validated sequences (Guo et al., 2022; Wu et al., 2024). Specifically, the bmmA gene, which is conserved across B. velezensis strains, located between the position 1,465,100 and 1,466,230 of the B. velezensis CML532 genome. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and are listed in Table S1. Standard curves were generated using plasmids containing 16S rRNA genes, and bacterial gene copy numbers were quantified based on these curves. The relative abundance of B. velezensis was determined by the ratio of its gene copy number to that of total bacteria. Gene copy numbers were calculated using the formula:

Gene copy (number/μL) = [DNA concentration (ng/μL) × 6.0233 × 10²³ copies/mol]/[DNA size (bp) × 660 (g/mol) × 10⁹].

2.12. Ileal microbial 16S rRNA gene sequencing analysis

Microbial genomic DNA from the ileum was extracted using the FastPure Stool DNA Isolation kit (T10–100, Shanghai Majorbio Yuhua Bio-Pharm Technology Co., Ltd., Shanghai, China), and the V3–V4 region of the bacterial 16S rRNA gene was amplified with universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Following magnetic bead purification, the amplified products were fluorescently quantified with the Qubit 4.0 (Thermo Fisher Scientific Inc., Waltham, MA, USA). High-throughput sequencing was conducted on the Illumina Nextseq2000 platform (Illumina Inc., San Diego, CA, USA) by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). All raw sequencing data were deposited in the SRA database of National Center for Biotechnology Information (NCBI) under accession number PRJNA1211260 (16S rRNA).

2.13. Statistical analysis

All experimental data were statistically analyzed using SPSS 29.0 single factor ANOVA, followed by multiple comparisons with Duncan's method. The statistical model used in this study is shown below. P < 0.05 indicates a notable difference, and 0.05 < P < 0.10 suggests a tendency of difference. Graphical representation of the data was performed using GraphPad Prism 9.5.1.

Y_ij = μ + α_i + ε_ij,

where Y_ij is the j-th observation in the i-th group; μ is the overall mean; α_i is the effect of the i-th treatment; ε_ij is the residual error associated with Y_ij.

3. Results

3.1. Growth performance

The effects of B. velezensis supplementation on growth performance of broiler chickens are presented in Table 2. In comparison to the CM group, the CML group displayed significantly reduced final body weight (FBW) during the grower and finisher phases (P < 0.05), as well as a marked decrease in BWG and an increase in FCR over the whole experimental period (P < 0.05). However, the addition of B. velezensis notably improved the growth and development of broilers, as reflected by significantly increased FBW during the grower and finisher phases (P < 0.05) relative to the CML group. In the CMLB group, BWG was significantly increased (P = 0.016), and FCR decreased (P < 0.001) throughout the whole period compared to the CML group, showing comparable performance to the CM group.

Table 2.

Effects of Bacillusvelezensis CML532 supplementation on growth performance of broilers fed with corn-miscellaneous meal diets.

Items	Groups1			SEM	P-value
	CM	CML	CMLB
Starter phase (1-14 d)
IBW, kg	0.044	0.044	0.044	0.0001	0.345
FBW, kg	0.394	0.377	0.388	0.0037	0.144
BWG, kg/bird	0.351	0.333	0.345	0.0037	0.129
FI, kg/bird	0.445	0.432	0.442	0.0044	0.473
FCR	1.27c	1.30a	1.28b	0.004	0.001
Grower phase (15-28 d)
FBW, kg	1.36a	1.31b	1.37a	0.011	0.029
BWG, kg/bird	0.967ab	0.932b	0.983a	0.0083	0.029
FI, kg/bird	1.40	1.38	1.43	0.011	0.261
FCR	1.45b	1.48a	1.45b	0.006	0.032
Finisher phase (29-42 d)
FBW, kg	2.72a	2.60b	2.78a	0.027	0.014
BWG, kg/bird	1.39	1.29	1.41	0.023	0.088
FI, kg/bird	2.32	2.32	2.39	0.036	0.706
FCR	1.67b	1.80a	1.70b	0.016	<0.001
Whole period (1-42 d)
BWG, kg/bird	2.71a	2.56b	2.73a	0.029	0.016
FI, kg/bird	4.17	4.13	4.26	0.038	0.406
FCR	1.54b	1.62a	1.56b	0.009	<0.001

IBW = initial body weight; FBW = final body weight; BWG = body weight gain; FI = feed intake; FCR = feed conversion ratio; SEM = standard error of the means.

Within a row, means without a common superscript letter differ at P < 0.05, n = 6.

¹CM, corn-miscellaneous meal-based normal-protein control group; CML, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group.

3.2. Immune organ indexes and slaughter traits

The liver index in both CML and CMLB groups was significantly higher than that of CM group at the finisher phase (P = 0.049, Table 3). To further evaluate the impact of B. velezensis on slaughter traits, parameters including carcass rate, breast muscle rate, and abdominal fat rate were analyzed (Table 4). Compared to the CM group, the CML group showed significant reductions in half-eviscerated rate, eviscerated rate, and breast muscle rate (P < 0.05), indicating that reducing dietary CP levels adversely affected carcass characteristics. In contrast, supplementation with B. velezensis in the CMLB group significantly increased these parameters relative to the CML group (P < 0.05), restoring them to levels comparable to those observed in broilers fed normal-protein diets (CM group).

Table 3.

Effects of Bacillusvelezensis CML532 supplementation on immune organ indexes of broilers fed with corn-miscellaneous meal diets (g/kg).

Items	Groups1			SEM	P-value
	CM	CML	CMLB
Starter phase (1-14 d)
Liver	33.9	36.4	36.3	0.69	0.274
Spleen	0.848	0.919	0.884	0.0495	0.857
Thymus	4.12	4.90	4.46	0.222	0.380
Bursa of fabricius	1.84	1.70	1.48	0.101	0.362
Grower phase (15-28 d)
Liver	31.6	32.9	32.4	0.463	0.542
Spleen	0.833	0.901	0.821	0.0449	0.760
Thymus	4.87	4.89	4.86	0.150	0.996
Bursa of fabricius	1.64	1.71	1.59	0.102	0.906
Finisher phase (29-42 d)
Liver	20.3b	23.8a	23.3a	0.67	0.049
Spleen	0.850	0.819	0.661	0.0532	0.317
Thymus	3.75	3.47	3.12	0.180	0.374
Bursa of fabricius	0.817	0.912	0.905	0.0386	0.566

SEM = standard error of the means.

Within a row, means without a common superscript letter differ at P < 0.05, n = 6.

¹CM, corn-miscellaneous meal-based normal-protein control group; CML, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group.

Table 4.

Effects of Bacillusvelezensis CML532 supplementation on slaughter traits of broilers fed with corn-miscellaneous meal diets (%).

Items	Groups1			SEM	P-value
	CM	CML	CMLB
Carcass rate	95.4	95.1	95.1	0.17	0.706
Half-eviscerated rate	91.5a	89.9b	91.2a	0.24	0.005
Eviscerated rate	79.0a	77.4b	79.0a	0.23	<0.001
Wing rate	7.64	8.65	7.94	0.182	0.056
Breast muscle rate	29.6a	28.2b	29.6a	0.25	0.024
Thigh rate	21.7	22.4	22.3	0.24	0.487
Abdominal fat rate	2.58	2.40	2.54	0.095	0.758

SEM = standard error of the means.

Within a row, means without a common superscript letter differ at P < 0.05, n = 6.

¹CM, corn-miscellaneous meal-based normal-protein control group; CML, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group.

3.3. Intestinal barrier and immune function

Real-time quantitative-PCR analysis demonstrated that the CMLB group significantly upregulated the gene expression levels of intestinal epithelial tight-junction proteins ZO-1 and claudin-1 compared to the CML group (P < 0.05). Specifically, claudin-1 expression in the CMLB group was also significantly higher than that in the CM group (P < 0.05, Fig. 1D–G). Furthermore, the mRNA and protein levels of anti-inflammatory cytokine IL-10 were markedly downregulated in the CML group relative to the CM group (P < 0.05), and IL-10 concentration showed an increasing trend in the CMLB group compared to the CML group (P = 0.069, Fig. 1H–N). However, no statistical differences were observed in the serum levels of D-LA and DAO among the treatment groups (Fig. 1B and C).

Fig. 1.

Effects of Bacillus velezensis CML532 supplementation on intestinal epithelial barrier and immune function in broilers. Wing vein blood was collected from broilers at 28 d (A) to determine serum levels of D-LA (B) and DAO (C). Jejunal mRNA expression levels of mucin2 (D), ZO-1 (E), claudin-1 (F), occludin (G), TNF-α (H), IL-6 (I), IL-10 (J), and NF-κB (K) were quantified by real-time quantitative PCR. Protein concentrations of TNF-α (L), IL-6 (M), and IL-10 (N) in the jejunum. D-LA = D-lactic acid; DAO = diamine oxidase. CM, corn-miscellaneous meal-based normal-protein control group; CM, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group. ∗ P < 0.05, ∗∗ P < 0.01; n = 6.

3.4. Protein digestibility and utilization

As shown in Table 5 and Table 6, the serum BUN levels and apparent total tract digestibility of His, Ile, Leu, and Val in the CML group were significantly decreased compared to the CM group (P < 0.05). In comparison to the CM group, the CMLB group displayed a significant reduction in UA and BUN contents (P < 0.05), whereas TP and ALB levels showed no significant differences. Notably, the apparent total tract digestibility of CP, Met, Arg, and Gly of broilers in the CMLB group was significantly higher than that in both CM and CML groups (P < 0.01), and apparent His, Ile, and Val digestibility was markedly increased compared to the CML group (P < 0.01).

Table 5.

Effects of Bacillusvelezensis CML532 supplementation on serum biochemical indexes of broilers fed with corn-miscellaneous meal diets.

Items	Groups1			SEM	P-value
	CM	CML	CMLB
TP, mg/mL	32.1	30.6	33.3	0.73	0.321
ALB, g/L	14.0	14.0	14.2	0.23	0.939
UA, μmol/L	469a	456a	310b	19.6	<0.001
BUN, mmol/L	12.3a	10.0b	8.91b	0.508	0.013

TP = total protein; ALB = albumin; UA = uric acid; BUN = blood urea nitrogen; SEM = standard error of the means.

Within a row, means without a common superscript letter differ at P < 0.05, n = 6.

¹CM, corn-miscellaneous meal-based normal-protein control group; CML, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group.

Table 6.

Effects of Bacillusvelezensis CML532 supplementation on apparent nutrient digestibility of broilers fed with corn-miscellaneous meal diets (%).

Items	Groups1			SEM	P-value
	CM	CML	CMLB
CP	54.1b	56.5b	61.4a	0.87	<0.001
GE	77.9	77.7	78.6	0.18	0.103
Lys	88.8	88.1	89.0	0.22	0.204
Met	89.3b	88.3b	91.1a	0.34	<0.001
Cys	82.7	80.5	80.2	0.53	0.100
Thr	82.8	82.8	83.3	0.54	0.904
Arg	94.4b	94.1b	95.1a	0.13	0.001
His	86.3a	83.6c	84.7b	0.32	<0.001
Ile	84.9a	83.7b	85.2a	0.21	0.003
Leu	90.4a	89.6b	90.0ab	0.12	0.035
Trp	89.3	87.3	89.0	0.56	0.315
Val	83.4a	81.7b	83.5a	0.25	<0.001
Gly	66.3b	67.0b	72.3a	0.73	<0.001
Ser	87.1	85.8	86.8	0.38	0.375

CP = crude protein; GE = gross energy; SEM = standard error of the means.

Within a row, means without a common superscript letter differ at P < 0.05, n = 6.

¹CM, corn-miscellaneous meal-based normal-protein control group; CML, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group.

Efficient nutrient digestion and absorption in broilers primarily rely on endogenous digestive enzymes and AA sensing and transport systems. In this study, the activities of α-amylase, trypsin, and chymotrypsin in the jejunal chyme of chickens were analyzed (Table 7). The addition of B. velezensis significantly increased α-amylase activity (P = 0.017), and chymotrypsin activity showed an increasing trend in the CMLB group compared to the CML group (P = 0.057), while trypsin activity was unaffected. In terms of AA sensing receptors and transporters (Fig. 2), the CML group exhibited a significant reduction in the mRNA expression level of AA transporter CAT1 compared to the CM group (P < 0.01). However, CAT1 expression in the CMLB group was significantly higher than that in the CML group (P = 0.039). The mRNA level of EAAT3 was significantly elevated in the CMLB group compared to the CM group (P = 0.012). Additionally, lower dietary CP levels markedly decreased the expression of AA sensing receptor CaSR relative to the CM group (P < 0.01), whereas the supplementation of B. velezensis reduced these adverse effects, bringing CaSR expression to a comparable level as that of the CM group. Furthermore, the mRNA expression levels of T1R1 and GPRC6A were significantly upregulated in the CMLB group relative to the CML group (P < 0.05).

Table 7.

Effects of Bacillusvelezensis CML532 supplementation on digestive enzyme activity in jejunal chyme of broilers fed with corn-miscellaneous meal diets (U/mg prot).

Items	Groups1			SEM	P-value
	CM	CML	CMLB
α-Amylase	14.3b	13.9b	21.8a	1.36	0.017
Trypsin	5161	5009	5293	166.9	0.806
Chymotrypsin	9.36	8.63	12.63	0.749	0.057

SEM = standard error of the means; prot = protein.

Within a row, means without a common superscript letter differ at P < 0.05, n = 6.

¹CM, corn-miscellaneous meal-based normal-protein control group; CML, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group.

Fig. 2.

Effects of Bacillus velezensis CML532 supplementation on amino acid (AA) sensing and transport in jejunum of broilers. Dietary protein absorption and utilization are regulated by multiple AA sensing receptors and transporters in broiler intestinal epithelial cells (A). The gene expression levels of transporters PepT1 (B), CAT1 (C), EAAT3 (D), B^0,+AT (E), B⁰AT (F), LAT1 (G), and sensing receptors GPR142 (H), CaSR (I), T1R1 (J), GPRC6A (K) were detected. CM, corn-miscellaneous meal-based normal-protein control group; CM, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group. ∗ P < 0.05, ∗∗ P < 0.01; n = 6.

3.5. Ileal microbial diversity and metabolic function

To further confirm the colonization of B. velezensis in different intestinal segments, RT-qPCR was conducted on jejunal, ileal, and cecal digesta samples using B. velezensis specific primers. As shown in Fig. 3A, supplementation with B. velezensis CML532 did not alter its relative abundance in the jejunum or cecum of the CMLB group compared to the CML group. However, in the ileum, the CMLB group displayed a markedly higher relative abundance of B. velezensis than both the CM and CML groups (P < 0.001). Based on these findings, the ileal microbiota was chosen for subsequent diversity analysis.

Fig. 3.

Effects of Bacillus velezensis CML532 supplementation on ileal microbial diversity in broilers. (A) Relative abundance of B. velezensis in jejunal, ileal, and cecal digesta, as determined by real-time quantitative PCR. (B) Rarefaction curves of samples. (C) Sample coverage rates. (D–F) Chao1 index, abundance-based coverage estimator (ACE) index, and observed_species index in α-diversity. (G) Venn diagram of optical transform units (OTUs). (H) β-diversity analysis using non-metric multidimensional scaling (NMDS) based on weighted_normalized_unifrac distances. (I) Relative abundances of Firmicutes, Bacteroidota, and the Firmicutes/Bacteroidota ratio. (J) Species composition at the phylum level. CM, corn-miscellaneous meal-based normal-protein control group; CML, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; n = 6.

As illustrated in Fig. 3B, rarefaction curves plateaued at approximately 50,000 sequences, indicating sufficient sequencing depth, with coverage exceeding 99.9% (Fig. 3C). It was confirmed that the sequencing depth was adequate to represent the microbial composition of broiler ileal chyme for further analysis.

According to the statistical analysis results of α-diversity indexes (Fig. 3D–F), B. velezensis significantly increased the Chao1 index in the CMLB group compared to the CML group (P < 0.05), and the abundance-based coverage estimator (ACE) index showed an increasing trend (P = 0.077), which were both significantly higher than those in the CM group (P < 0.05). Venn and non-metric multidimensional scaling (NMDS) analysis indicated a distinct difference in the ileal microbial community structure among the treatment groups (Fig. 3G and H). At the phylum level, the relative abundances of Firmicutes, Actinobacteria, Proteobacteria, Bacteroidota, Campilobacteria, and Cyanobacteria were identified (Fig. 3J), and the addition of B. velezensis CML532 did not cause a significant variation in the relative abundances of Firmicutes and Bacteroidota, which are critical for maintaining intestinal homeostasis in broilers (Fig. 3I).

As shown in Fig. 4A, the top 10 genera in relative abundance included Lactobacillus, Streptococcus, Candidatus_Arthromitus, Enterococcus, Romboutsia, Staphylococcus, Bacillus, Christensenellaceae_R-7_group, and Corynebacterium. Compared to the CM group, the relative abundance of uncultured_bacterium within Lactobacillus was significantly decreased in the CML group (P < 0.05), but increased in the CMLB group relative to the CML group (Fig. 4D). Additionally, the relative abundance of Coriobacteriaceae_bacterium_CHKCI002 in the CMLB group was significantly higher than that observed in both CM and CML groups (P < 0.05, Fig. 4C). Interestingly, linear discriminant analysis effect size (LEfSe) analysis identified B. velezensis as a specific biomarker bacterium (Fig. 4H), which also exhibited a higher relative abundance in the CMLB group (P < 0.05, Fig. 4B). Correlation analysis between B. velezensis abundance and host metabolic pathways predicted by PICRUSt2 revealed positive associations with His metabolism, biosynthesis, and mineral absorption (P < 0.10, Fig. 4E–G). To further elucidate the role of B. velezensis in broiler growth performance and AA metabolism, Spearman analysis was conducted (Fig. 4I), which showed significant positive correlations with chymotrypsin activity and apparent protein digestibility (P < 0.01). Moreover, during the whole period, BWG was positively correlated with FI, AA transporter CAT1, and AA sensing receptors CaSR and T1R1 mRNA expression (P < 0.05). Conversely, FCR was negatively correlated with CAT1 and CaSR (P < 0.05), while apparent protein digestibility exhibited a strong positive correlation with α-amylase activity (P < 0.01).

Fig. 4.

Effects of Bacillus velezensis CML532 supplementation on amino acid metabolism pathways in broiler ileal microbiota. (A) Composition of the top 20 species in abundance at the genus level. (B–D) Relative abundance of B. velezensis,Coriobacteriaceae_bacterium_CHKCI002, uncultured_bacterium_g_Lactobacillus. (E–G) Spearman correlation analysis between B. velezensis abundance and mineral absorption, His metabolism, and His biosynthesis. (H) Differential biomarker species identified between CML and CMLB groups using linear discriminant analysis (LDA) effect size (LEfSe) analysis. (I) Correlation heatmap depicting associations between B. velezensis and parameters related to growth performance and AA metabolism. CM, corn-miscellaneous meal-based normal-protein control group; CML, corn-miscellaneous meal-based low-protein group; CMLB, corn-miscellaneous meal-based low-protein with supplemental B. velezensis CML532 group. FBW = final body weight; BWG = body weight gain; FI = feed intake; FCR = feed conversion ratio. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; n = 6.

4. Discussion

Compared to SBM-based diets, miscellaneous meal diets typically contain an imbalanced AA profile and higher levels of anti-nutritional factors, including non-starch polysaccharides, phytates, and protease inhibitors, which have been reported to impair protein digestion and nutrient absorption in broilers (Kim et al., 2012). Although increasing evidence has demonstrated that Bacillus fermentation enhanced the nutritional properties of miscellaneous meal by significantly increasing its CP content, dry matter retention, and optimizing AA composition (Yang et al., 2016), there is a lack of research on the direct effects and underlying mechanisms of Bacillus supplementation in miscellaneous meal-based diets. Liu et al. (2017) found that reducing dietary CP level from 22.5% to 20.5% by decreasing the proportion of miscellaneous meals from rapeseed and peanut, resulted in a notable decrease in BWG of broilers from 1 to 21 d of age. Similarly, this results showed that broilers fed the low-CP diet without SBM (CML group) exhibited significantly lower BWG and higher FCR throughout the whole period compared with those fed the normal-CP diet without SBM (CM group). However, dietary supplementation with B. velezensis CML532 (CMLB group) effectively alleviated these negative effects, probably due to the production of digestive enzymes and modulation of gut microbiota composition, as previously suggested by Zhu et al. (2024). Moreover, the observed improvements in half-eviscerated rate, eviscerated rate, and breast muscle rate in CMLB group further supported the beneficial impact of B. velezensis in promoting broiler growth performance and carcass traits, achieving levels comparable with those of normal protein diets.

Incomplete protein digestion fosters the proliferation of pathogenic microorganisms and the accumulation of toxic metabolites (Dallas et al., 2017), consequently impairing the intestinal barrier and triggering persistent adaptive immune responses and hepatic edema (Chopyk and Grakoui, 2020). In this study, a significantly increased liver index and a notable reduction in IL-10 content were observed in the CML group compared with the CM group. However, no significant differences were detected in serum D-LA and DAO levels, which are commonly recognized as indicators of intestinal epithelial barrier integrity (Chen et al., 2017). Given that IL-10 is a key anti-inflammatory cytokine involved in limiting excessive immune activation and maintaining mucosal homeostasis (Bedke et al., 2019; Zhang and Kuchroo, 2019), its partial restoration in the CMLB group suggested a modulatory effect of B. velezensis CML532 on intestinal immune balance, although no significant improvement was noted in liver index. Tight junction proteins such as ZO-1, claudin-1, and occludin are critical structural components of the intestinal epithelial barrier and have been reported to prevent the translocation of luminal toxins and pathogens (Wallez and Huber, 2008; Schlegel and Waschke, 2010). In the current study, the expression levels of ZO-1 and claudin-1 were significantly upregulated in broilers fed with miscellaneous meal-based diets supplemented with B. velezensis CML532, indicating that the probiotic conferred beneficial effects on intestinal barrier integrity.

Whole-genome sequencing of B. velezensis CML532 conducted by Zhu et al. (2024) revealed that AA transport, metabolism, and biosynthesis were among the most enriched pathways identified in the clusters of orthologous groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. In line with these findings, the study showed that compared with the CML group, broilers in the CMLB group exhibited significantly reduced serum UA and BUN levels, along with elevated apparent CP and AA digestibility. UA and BUN concentrations are well-known items for evaluating AA balance and nitrogen metabolism in animals. As the end product of protein and nucleic acid catabolism, UA serves as a reliable marker for evaluating protein utilization efficiency (Donsbough et al., 2010), while reduced serum BUN concentrations have been associated with improved protein synthesis and nitrogen retention (Huang et al., 2012). Therefore, the observed decrease in both UA and BUN levels suggested that B. velezensis CML532 supplementation enhanced nitrogen utilization and protein synthesis, thereby contributing to improved growth and carcass traits. Interestingly, the CML group exhibited an increasing trend in protein digestibility and significantly reduced BUN levels compared to the CM group. This could be attributed to the inclusion of crystal AAs and the reduced proportion of miscellaneous meals, which lowered the levels of anti-nutritional factors and improved nitrogen utilization.

During the starter growth phase, broilers possess immature digestive systems with limited gastric acid secretion and digestive enzyme production (Zhang et al., 2022). Anti-nutritional factors in miscellaneous meals have been shown to bind dietary proteins, impair protein digestion and absorption, and even disrupt the AAs balance. Therefore, enzyme-producing probiotics have been proposed as a potential nutritional strategy to enhance dietary nutrient utilization (Li et al., 2023). As previously reported, B. velezensis CML532 promoted the production of amylase, protease, and cellulase in vitro (Zhu et al., 2024), and increased the activity of α-amylase and chymotrypsin in the digesta in vivo. Enzymes such as proteases and amylases have been demonstrated to improve growth performance and nutrient digestibility by degrading complex feed components (Gadde et al., 2017; Park et al., 2020). This further supported the secretion of digestive enzymes by B. velezensis CML532, which likely contributed to the improved nutrient release and utilization from miscellaneous meals, thereby enabling the growth performance of broilers fed a low-protein diet to be equivalent to that of conventional protein levels.

Amino acids in the gastrointestinal tract are recognized by surface-localized AA sensors, which activate intracellular signaling pathways to regulate gastrointestinal functions like appetite and motility (Daly et al., 2013). The intestinal AA transporters not only mediate AA absorption, but also maintain AA homeostasis in response to luminal concentration changes (Tolhurst et al., 2011). In this study, the jejunum of broilers in the CMLB group exhibited significantly upregulated expression of AA sensing receptors, including CaSR, GPRC6A, and GPR142, along with increased expression of CAT1. These findings further demonstrate that B. velezensis CML532 enhanced AA sensing and transport capacity, thereby promoting protein utilization. A positive correlation was also observed between the abundance of B. velezensis and His metabolism and biosynthesis pathways in the digesta. As an EAA in poultry, His has been explored to regulate protein synthesis and muscle development by suppressing intestinal inflammation, and improving growth performance (Hu et al., 2017). Notably, CaSR is known to sense various AAs, particularly those with aromatic side chains or positive charges, such as Arg, Lys, and His. Among them, His has been found to stimulate CaSR to modulate downstream signaling pathways by increasing its sensitivity to extracellular Ca²⁺ (Leech and Habener, 2003). CAT1 has also been reported to transport Lys, Arg, and His (Shima et al., 2006).

The gut microbiota plays a crucial role in regulating immune function and growth performance in broilers (Kim et al., 2021). Previous studies demonstrated that B. velezensis CML532 positively impacted the gut environment by inhibiting harmful bacteria such as Clostridium perfringens (Zhu et al., 2024). Similarly, in this study, B. velezensis CML532 appeared to exert positive effects by increasing beneficial bacteria. The α- and β-diversity analysis revealed that B. velezensis altered microbial diversity and community structure in the ileum of broilers. The dominant bacterial phyla in the poultry intestine include Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes (Waite and Taylor, 2015). Among these, Firmicutes are involved in maintaining intestinal homeostasis and immune regulation (Colston and Jackson, 2016), and are more efficient in nutrient transport than Bacteroidetes, contributing to improved BWG in broilers (Zhu et al., 2021b). Although previous studies reported that in response to stress, the ratio of Firmicutes to Bacteroidetes levels in birds was significantly increased (Liu et al., 2020; Zhou et al., 2022), B. velezensis did not induce such changes, suggesting it does not disrupt microbial balance. At the species level, the relative abundance of beneficial bacteria like uncultured_bacterium_g_Lactobacillus was significantly decreased in the CML group, while B. velezensis, Coriobacteriaceae_bacterium_CHKCI002, and uncultured_bacterium_g_Lactobacillus were increased in the CMLB group. Coriobacteriaceae has been associated with host energy metabolism (Claus et al., 2011), while Lactobacillus, a key lactic acid bacterium, promotes intestinal motility and correlates positively with broiler growth (Zhu et al., 2021a). Probiotics composed of Bacillus strains have been reported to increase lactic acid bacteria, which support gut health by reducing pH levels to inhibit pathogen colonization (Thanh Lam and Jamikorn, 2017; Sugiharto et al., 2018). These results suggested that the optimized gut microbiota composition induced by B. velezensis CML532 could improve gut health and growth performance of chickens under low-CP diets containing miscellaneous protein sources.

5. Conclusions

In summary, B. velezensis CML532 supplementation significantly improved broiler growth performance and slaughter traits in corn-miscellaneous meal-based low-protein diets by enhancing protein and AA digestibility, ultimately achieving performance levels comparable to those of normal-protein diets. This efficacy was likely due to the direct production of digestive enzymes by B. velezensis, and its beneficial influence on ileal microbial diversity and biological processes, such as His metabolism. Additionally, the enhanced intestinal epithelial barrier integrity by B. velezensis also improved gut health and benefited broiler growth performance (Fig. S1).

Credit Author Statement

Jinping Wang: Writing – original draft, Data curation, Conceptualization. Xiaodan Zhang: Formal analysis. Caiwei Luo: Methodology. Mi Wang: Funding acquisition. Xinzhi Wang: Funding acquisition. Yuming Guo: Writing – review & editing, Supervision, Resources.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mi Wang and Xinzhi Wang are currently employed by Shenyang Boeing Feed Co. Yuming Guo is an Associate Editor for Animal Nutrition and was not involved the editorial review or the decision to publish this article.

Appendix A. Supplementary data

The following is the Supplementary data to this article.