Effect of Dietary Supplementation of Enteric Avian-Origin Lactobacillus casei-Fermented Soybean Meal on the Growth Performance and Intestinal Health of Broiler Chickens

Abstract

The bacterial strain is key to fermentation, and the intestinal tract in livestock and poultry is a resource bank of good natural strains. The objective of this study was to evaluate the effect of soybean meal fermented using Lactobacillus casei, isolated from healthy broiler intestines with excellent organic acid production, on the intestinal health and growth performance of broilers. A total of 120 Arbor Acre male broiler chickens aged 21 days were fed until 42 days of age. These chickens were randomly divided into four groups with five replicates per group. Each replicate contained six broiler chickens. The specific groups were the control group (basal diet), the low-dose fermented soybean meal (FSBM) additive group (FSBML, basal diet + 0.2 kg/t FSBM), the middle-dose FSBM additive group (FSBMM, basal diet + 2 kg/t FSBM), and the high-dose FSBM additive group (FSBMH, basal diet + 5 kg/t FSBM). The results demonstrated a significant increase in the average daily feed intake (ADFI) and average daily gain (ADG) of the FSBMH group (p < 0.05). The FSBMH group displayed a significantly increased villus height (VH) to crypt depth (CD) ratio (VH/CD) for the duodenum (p < 0.05) and rectum (p < 0.05). The examination of the ileal mucosa showed that the FSBMH group (p < 0.05) had significantly higher levels of glutathione (GSH) activity, as well as higher relative mRNA expression of ZO-1, ZO-2, Occludin, IL-4, IL-6, MCP-1, TNF-α, IFN-α, IFN-β, and IFN-γ. However, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were significantly lower in the FSBMH group (p < 0.05). The FSBMH group also showed higher levels of Nitriliruptoraceae and Ruminococcaceae. In conclusion, the addition of 5 kg/t FSBM to diets had an ameliorative effect on broiler growth performance and intestinal health.

1. Introduction

Soybean meal (SBM) is extensively utilized in poultry and pig feed due to its well-balanced amino acid (AA) composition and high protein content [1]. In recent years, fermentation has been increasingly used to enhance the utilization of SBM and mitigate anti-nutritional factors. Several studies have demonstrated that the fermentation of SBM by the microbiome leads to an increase in peptide and protein levels, enhancing the uptake of nutrients in avian and swine species [2,3]. It has been observed that the fermentation of feed by Lactobacillus plantarum results in the production of significant amounts of lactic acid and D-amino acid (DAA) [4]. The pH value of the feed is reduced by lactic acid, which inhibits the growth of pathogenic bacteria and prevents them from attaching to the intestinal wall [5]. After the enzymatic oxidation of DAA by D-amino acid oxidase, it effectively reduces the colonization of intestinal pathogens and enhances the secretion of immunoglobulin A within the intestinal mucosa, strengthening the immune response of the intestinal mucosa and promoting gastrointestinal homeostasis [6]. Additionally, fermented feed contains a significant amount of probiotics. These live probiotics can colonize the gut, and even after they die, their flagellin can still attach and occupy the intestinal space [7]. Inactivated probiotics can improve gut health by stimulating the function of the epithelial barrier, producing antimicrobial substances and limiting the access of pathogenic microorganisms to nutrient resources [8]. Some studies have also revealed that inactivated Lactobacillus and its metabolites have bacteriostatic, growth-promoting, barrier-preserving, immune-modulating, and anti-inflammatory effects, which are beneficial for poultry health [9,10]. Based on the aforementioned research, the effectiveness of fermented feeds dependents not only on the degree of pre-digestion, but also on the specific microbial strains used.

Numerous studies have validated the advantages of fermented feeds for animals, and their utilization has been increasingly widespread. Chen et al. found that the combination of Bacillus subtilis and Saccharomyces cerevisiae fermentation in feed significantly increases the body weight gain and feed intake of broilers [11]. The inclusion of Aspergillus oryzae fermented soybean meal (FSBM) in broiler diets led to a significant increase in average daily feed intake (ADFI) and average daily gain (ADG), while also increasing jejunal villus height (VH) and decreasing jejunal crypt depth (CD) [12,13]. However, the fermentation of feed with lactobacillus and yeast did not improve the growth performance of yellow-feathered broilers [14]. By adding 15% feed fermented with Lactobacillus and Bacillus subtilis, there was a significant increase in the concentration of monounsaturated fatty acids and a decrease in the concentration of saturated fatty acids in the breast muscle of broilers, thereby improving the quality of chicken meat [15]. Consequently, it can be inferred that the efficacy of fermented feeds varies depending on the microbial strains used.

Currently, the microbial strains used in fermented feeds typically include probiotics intended for human use and strains isolated from plants, among others. However, the animal gastrointestinal tract is abundant with microorganisms, which presents a potential natural repository of strains for fermentation purposes. Relevant research suggests that Lactobacillus isolated from the ileal and cecal digesta of pigs, when utilized in the fermentation of liquid feed, results in a higher concentration of lactic acid and enhanced antimicrobial activity against Salmonella spp. [16]. Additionally, Lactobacillus strains isolated from poultry exhibit a greater affinity for adhesion to the epithelial cells of chickens [17]. Lactobacillus plantarum, isolated from the cecum of Tibetan chickens, exhibits enhanced tolerance to strong acids and bile salts, thereby facilitating its colonization in the gastrointestinal tract [18]. Consequently, the indigenous microbiota present in the animals’ digestive system can be considered as promising microbial strains for the manufacturing of fermented feeds.

Consistent with the aforementioned report, our laboratory has successfully isolated a Lactobacillus casei strain from the intestinal tract of healthy broilers for feed fermentation, yielding promising outcomes in the context of broiler feeding [19]. This study aims to investigate the effects of SBM fermented by Lactobacillus casei C37d72 and the metabolites produced during fermentation on the growth performance and intestinal health of broilers. The findings are anticipated to offer a practical foundation for utilizing endogenous gut bacteria as strains in feed fermentation. Consequently, we conducted a refined selection of C37d72 from a collection of strains, focusing on its metabolic traits such as alterations in pH levels, oligopeptide yield, organic acid content, and partial DAA concentrations.

2. Materials and Methods

All experimental Arbor Acre broiler chickens were fed following “The Guidelines for the Care of Laboratory Animals” issued by the Ministry of Science and Technology of the PRC and approved by the Animal Experimentation Committee of South China Agricultural University (Guangzhou, China, Project number SYXK 20220136).

2.1. Preparation of Fermented Soybean Meal

A slightly modified version of the previously described protocol was used to prepare FSBM [19]. Five strains of Lactobacillus casei (C37m39, C37d72, C80b18, C80c26, C80d20) used in this study were pre-screened by our laboratory and stored at −80 °C. The strains were placed in de Man, Rogosa, and Sharpe (Huankai Microbiology Technology Co., Ltd., Guangzhou, China) liquid medium and shaken at 37 °C and 180 rpm for 12 h. The fermentation material ratio was corn meal, bran, and SBM (46%) = 1:1:3, which were powdered and provided by Huayang Feedstuff Co. (Foshan, China), and the water content was 40%. Then, the fermentation material was sterilized at 120 °C for 20 min, and after cooling, 1 × 10⁵ cfu/g Lactobacillus casei strain was added to the fermentation material in a sterile environment. After anaerobic fermentation in a bacterial incubator (Yiheng Scientific Instrument Co., Shanghai, China) at 37 °C for 15 days, the total bacterial count reached 1 × 10¹⁰ cfu/g. Then, High-Performance Liquid Chromatography (HPLC, Agilent Technologies, Inc., Model 1260, Santa Clara, CA, USA) was used to detect fermented lactic acid (LA), acetic acid (HAc), small peptides, D-glutamic acid (D-Glu), D-alanine (D-Ala), and D-aspartate (D-Asp). The reagents were purchased from MACKLIN Biochemical Technology Corporation (Shanghai, China). To eliminate the possibility of further fermentation during the experimental process and to mitigate the impact of viable bacteria on the results, the FSBM was sterilized at 100 °C for 1 h to terminate bacterial activity, followed by drying at 65 °C, grinding, and subsequent storage for use.

2.2. Experimental Birds and Diets

A total of 120 male broilers (Guangdong Qingnong New Agricultural Technology Co., Ltd., Qingyuan, China) aged 21 days were fed until 42 days of age. These chickens were randomly divided by body weight into four groups with five replicates per group. Each replicate contained six broiler chickens. Each broiler was housed in a single cage. The experimental groups consisted of the control group (basal diet), the low-dose FSBM additive group (FSBML, basal diet + 0.2 kg/t FSBM), the middle-dose FSBM additive group (FSBMM, basal diet + 2 kg/t FSBM), and the high-dose FSBM additive group (FSBMH, basal diet + 5 kg/t FSBM). The broilers had ad libitum access to feed and water. The temperature was maintained at 25 ± 1 °C, humidity between 50 and 60%, and 24 h of light exposure. The test diet was formulated based on National Research Council guidance (NRC, 1994) [20] and combined with the Arbor Acre broiler feeding manual (NY/T 33-2004 Chicken feeding standard). The composition and nutrient levels of the basal rations are shown in

Table 1. Ingredient composition and nutrient levels of the experimental diet.

Items	Finisher Diet (22 d–42 d)
Ingredients (%)	Control	FSBML	FSBMM	FSBMH
Corn	65.80	65.78	65.68	65.49
Soybean meal (46%)	23.40	23.40	23.35	23.28
Corn gluten powder	5.00	5.00	4.99	4.98
Soybean Oil	2.10	2.10	2.10	2.09
FSBM	0	0.02	0.20	0.50
Limestone	1.20	1.20	1.20	1.19
Calcium hydrogen phosphate	1.00	1.00	1.00	1.00
Salt	0.17	0.17	0.16	0.16
Propionic acid antifungal agent 4	0.05	0.05	0.05	0.05
Premix of trace elements 1	0.10	0.10	0.10	0.10
Vitamin premix 2	0.03	0.03	0.03	0.03
L-Lysine	0.36	0.36	0.36	0.36
DL Methionine	0.15	0.15	0.14	0.14
L-Threonine	0.03	0.03	0.03	0.03
Choline chloride	0.10	0.10	0.10	0.10
Antioxidants	0.01	0.01	0.01	0.01
Heat-resistant phytase 4	0.01	0.01	0.01	0.01
AB enzyme complex 3	0.01	0.01	0.01	0.01
Sodium humate	0.21	0.21	0.21	0.21
Mannanase (K302) 4	0.01	0.01	0.01	0.01
Sodium bicarbonate	0.16	0.16	0.16	0.15
Zeolite 4	0.10	0.10	0.10	0.10
Total	100.00	100.00	100.00	100.00
Nutrient content 5
Metabolizable energy (kcal/kg)	3050.00	3051.00	3056.00	3066.00
Crude protein (%)	19.19	19.19	19.24	19.33
Calcium (%)	0.76	0.76	0.76	0.76
Available phosphorus (%)	0.49	0.49	0.49	0.49
Lysine (%)	1.10	1.10	1.10	1.10
Methionine (%)	0.41	0.41	0.41	0.41
Methionine + cystine (%)	0.84	0.84	0.84	0.84
Threonine (%)	0.79	0.79	0.79	0.79
Tryptophan (%)	0.22	0.22	0.22	0.22

Note: ¹ Trace element premix (mg/kg feed): Mn 100, Fe 80, Cu 8, Zn 75, Se 0.15, I 0.35. ² Vitamin premix (provided per kilogram of feed): Vitamin A 12500 IU, Vitamin B1 2 mg, Vitamin B2 6 mg, Vitamin B12 0.025 mg, Vitamin D3 2500 IU, Vitamin E 30 IU, Vitamin K3 2.65 mg, pantothenic acid 12 mg, biotin 0.0325 mg, niacin 50 mg, folic acid 1.25 mg. ³ AB enzyme complex: Acid protease, neutral proteases, xylanase, cellulase, glucoamylase. ⁴ Heat-resistant phytase, Mannanase (K302) and Zeolite provided by SUNHY Co. (Wuhan, China), Propionic acid antifungal agent provided by KEMIN (China) Technology Co. (Guangzhou, China). ⁵ Metabolizable energy values were calculated; others were measured values.

. The experimental diet was provided by Huayang Feedstuff Co. (Foshan, China).

2.3. Performance and Sample Collection

The Arbor Acre broilers were fed from aged 21 d and reached 42 d at the end of the experiment. Subsequently, the ADFI, ADG, and feed conversion ratio (FCR) of each chicken were calculated and the daily mortality rates recorded. The FSBMM group exhibited no significant variation in ADG and displayed a higher FCR relative to the control group. Consequently, we opted to investigate the underlying mechanisms of FSBM’s impact on broiler growth performance using the FSBML and FSBMH groups, and no samples were collected from the FSBMM group for this study. Fifteen healthy broilers close to the sample mean were selected from each group (control, FSBML, and FSBMH) for sampling. Three chickens were randomly sampled per replicate for blood collection via the wing veins. The obtained blood was processed by centrifugation at 4 °C at 3000 rpm for 15 min to separate the serum, which was then aliquoted and stored at −20 °C. Following blood collection, the birds were euthanized via cervical dislocation. Subsequently, the weights of vital organs were recorded, including the liver, abdominal adipose tissue, spleen, breast muscles and thigh muscles, while the breast muscles and thigh muscles were stored at 4 °C and the meat color was measured. Mucosal samples and contents of the duodenum, ileum, and cecum were scraped, snap-frozen in liquid nitrogen, and stored at −80 °C until further analysis.

2.4. Meat Color Measurements

Meat color attributes, including luminance (L*), redness (a*), and yellowness (b*), were measured using the CIELAB trichromatic system (CIE, International Commission on Illumination, Vienna, Austria). Before using a high-quality computerized colorimeter (model 431 NR20XE, manufactured by Shenzhen SUNSHI TECHNOLOGY CO., Ltd., Shenzhen, China), zero calibration and white calibration were carried out, followed by 2 measurements from the middle and 3 measurements from the corners of the left breast and thigh muscle samples.

2.5. Morphometric Analysis of the Intestine

At the conclusion of the experiment, 15 broilers randomly selected from each treatment group were sacrificed. During the necropsy, the intestine was meticulously divided into three segments: duodenum, ileum, and rectum. The intestinal segments were rinsed with isotonic solution twice to remove the contents. Then, a 2 cm segment was cut from the center of each intestinal segment, fixed in a 10% buffered formalin solution at pH 7.2, and stored overnight. Subsequently, the intestinal tissues were embedded in paraffin, processed, and sectioned into slices with a thickness of 5 µm, followed by staining with hematoxylin and eosin. For each section, five regions of the villi (distance from the crypt opening to the villus terminus) and crypts (from the crypt-villus junction to the base of the crypt) were randomly selected. The intestinal morphology was then observed and assessed using a light microscope (Nikon, Tokyo, Japan). The VH and CD were measured using ImageJ 6.0 software (National Institutes of Health, Bethesda, MD, USA), and the ratio of villi height to crypt depth (VH/CD) was calculated.

2.6. Oxidative Damage and Immune Barrier in the Intestine

The ileal mucosa was isolated on an ice tray and homogenized with cold saline with a weight-to-volume ratio of 1:9. The homogenate was centrifuged at 4 °C and the supernatant was collected for assay. Commercial kits (Nanjing Jiancheng, Nanjing, China) were employed to assess the total antioxidant capacity (T-AOC), reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), oxidized glutathione (GSSG), and serum uric acid (UA). The serum lipopolysaccharide (LPS) level was determined using a commercial kit (Enzyme, Shanghai, China). The mucosal protein concentration was measured employing a BCA protein concentration assay kit (Thermo, Wilmington, NC, USA). All measurements followed the manufacturer’s instructions.

2.7. RNA Extraction and Polymerase Chain Reaction (PCR) Amplification

RNA from cryopreserved ileum was extracted according to the Hipure Universal RNA Mini Kit (R4130-02, Magen Biotechnology Co., Ltd., Guangzhou, China), and its concentration was determined using a spectrophotometer (NanoDrop Products, Wilmington, DE, USA). The M-MLV enzyme (Promega, Madison, WI, USA) and primers were used to convert 2 µg RNA into cDNA. Then, quantitative reverse transcription PCR (qRT-PCR) was performed on a QuantStudio 3 Flex real-time PCR Detection System (7300HT, App-plied Biosystems, Waltham, MA, USA) combined with SYBR Green Master Mix (Q711, Vazyme, China) and primers [21]. The total volume of the reaction was 20 μL:0.5 μL of each primer, 2 μL cDNA, 10 μL SYBR Green Master Mix, and 7 μL double-distilled H₂O. The qRT-PCR program was as follows: 95 °C denaturation for 30 s, 95 °C denaturation for 15 s, and 60 °C annealing for 10 s and 72 °C extension for 30 s, 40 cycles. Melt curve analysis was then conducted to determine the reaction specificity. Each sample was prepared in triplicate. We used three common housekeeping genes (GAPDH, 28S rRNA, and β-Actin) to analyze the control samples, and the results showed the same trend for all three housekeeping genes. Therefore, we used β-Actin as the common housekeeping gene in the research. Refer to

Table 2. Fluorescent quantitative PCR primers.

Genes	Sequence Type-Probe/Primer Sequence	Tm	Product Length	Accession Number
β-Actin	forward 5′-CTGTGCCCATCTATGAAGGCTA-3′	60 °C	139 bp	NM_205518.2
	reverse 5′-ATTTCTCTCTCGGCTGTGGTG-3′
ZO-1	forward 5′-TCATCCTTACCGCCGCATAT-3′	60 °C	206 bp	XM_046925214.1
	reverse 5′-GTTGACTGCTCGTACTCCCT-3′
ZO-2	forward 5′-AGTCCACCTCCAGCATTCAA-3′	60 °C	165 bp	XM_046934796.1
	reverse 5′-CACAGAAACAGGTGGTGGTG-3′
Claudin-1	forward 5′-TGGAGGATGACCAGGTGAAG-3′	60 °C	137 bp	NM_001013611.2
	reverse 5′-TGTGAAAGGGTCATAGAAGG-3′
Claudin-2	forward 5′-CGCTCGTATCTCTTGCTTGG-3′	60 °C	185 bp	NM_001277622.1
	reverse 5′-AGAGTATGGCTGTGACGAGG-3′
IL-1β	forward 5′-GCTTCATCTTCTACCGCCTG-3′	60 °C	161 bp	XM_046931582.1
	reverse 5′-ACTTAGCTTGTAGGTGGCGA-3′
IL-2	forward 5′-CAAGAGTCTTACGGGTCTAAATCAC-3′	60 °C	100 bp	NM_204153.2
	reverse 5′-GTTGGTCAGTTCATGGAGAAAATC-3′
IL-4	forward 5′-GTGCCCACGCTGTGCTTAC-3′	60 °C	82 bp	NM_001007079.2
	reverse 5′-AGGAAACCTCTCCCTGGATGTC-3′
IL-6	forward 5′-AAATCCCTCCTCGCCAATCT-3′	60 °C	106 bp	NM_204628.2
	reverse 5′-CCCTCACGGTCTTCTCCATAAA-3′
IL-8	forward 5′-ATTCAAGATGTGAAGCTGAC-3′	60 °C	301 bp	NM_205498.2
	reverse 5′-AGGATCTGCAATTAACATGAGG-3′
IL-10	forward 5′-CGCTGTCACCGCTTCTTCA-3′	60 °C	88 bp	NM_001004414.4
	reverse 5′-TCCCGTTCTCATCCATCTTCTC-3′
IFN-α	forward 5′-TTCAGCTGCCTCCACACCTT-3′	60 °C	101 bp	XM_046936231.1
	reverse 5′-TTGTGGATGTGCAGGAACCA-3′
IFN-β	forward 5′-CAGCTCTCACCACCACCTTCTC-3′	60 °C	100 bp	NM_001024836.2
	reverse 5′-GGAGGTGGAGCCGTATTCTG-3′
IFN-γ	forward 5′-GACAAGTCAAAGCCGCACA-3′	60 °C	127 bp	NM_205149.2
	reverse 5′-TCAAGTCGTTCATCGGGAGC-3′
TNF-α	forward 5′-TGTTCTATGACCGCCCAGTT-3′	60 °C	164 bp	XM_046927265.1
	reverse 5′-AGCATCAACGCAAAAGGGAA-3′
Occludin	forward 5′-CTTCAGGTGTTTCTCTTCCTCCTC-3′	60 °C	131 bp	XM_040680624.2
	reverse 5′-CTGTGGTTTCATGGCTGGA-3′
NF-κB	forward 5′-GAAGGAATCGTACCGGGAACA-3′	60 °C	131 bp	XM_046915553.1
	reverse 5′-CTCAGAGGGCCTTGTGACAGTAA-3′

Note: Zonula occludens protein 1 (ZO-1), Zonula occludens protein 2 (ZO-2), Interleukin 1β (IL-1β), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 6 (IL-6), Interleukin 8 (IL-8), Interleukin 10 (IL-10), Interferon α (IFN-α), Interferon β (IFN-β), Interferon γ (IFN-γ), Tumor necrosis factor α (TNF-α), Nuclear factor kappa B (NF-κB).

for the primers used in this experiment and their sizes. Using β-Actin as a housekeeping target gene and the mean value of Ct as an internal control, the relative expression of the target gene was calculated using the 2^−ΔΔCt method.

2.8. Microbiological Analysis of the Cecum Microbiota

In a previous study [19], the microbial DNA was extracted from cecal contents using the HiPure Stool DNA Mini Kit (Magen Biotechnology Co., Ltd., Guangzhou, China). Subsequently, its quality and concentration were assessed utilizing a NanoDrop 2000 UV-visible spectrophotometer (Thermo Scientific, Wilmington, NC, USA) and analyzed through electrophoresis on 1% agarose gel for verification. Furthermore, primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGT-WTCTAAT-3′) were used to amplify the V3–V4 hypervariable region of the bacterial 16S rRNA gene. The AxyPrep DNA gel extraction kit (Axygen Biosciences, Union City, CA, USA) was used for 2% agarose gel purification, and 16S rRNA gene sequencing was performed using the Novogene Bioinformatics Technology Ltd. MiSeq kit (Beijing, China). The protocol was performed on the Illumina MiSeq platform (San Diego, CA, USA) for 2 × 250 bp double-end sequencing.

Furthermore, orthogonal polynomial comparisons were used to perform linear and quadratic analyses of increasing FSBM doses. Alpha diversity (Shannon, Simpson, Chao1, and ACE) and beta diversity analyses (PCoA) were performed using QIIME2 software (version 1.9.1). The linear discriminant analysis effect size (LEfSe) method was used to detect taxonomic units rich in between-group differences (LDA score > 3.0).

2.9. Statistics

The data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test for multiple comparisons using SPSS 26.0 (IBM, Armonk, NY, USA). GraphPad Prism 8.0 (San Diego, CA, USA) was utilized for visualization purposes. The alpha diversity index and relative species abundance of the gut microbial communities were analyzed using the Kruskal-Wallis test. LEfSe was used to identify microorganisms of different taxa among strains using default parameters. All results are presented as the mean ± SEM, and statistical significance was determined at a threshold of p < 0.05.

3. Results

3.1. Comparison and Screening of Fermentation Effects of Different Intestinal Probiotics in Chickens

As can be seen in Figure 1, the final pH of the five strains was basically the same after 15 d of fermentation (Figure 1a). In terms of organic acid production, C37d72, C80b18, and C80d20 were more capable of lactic and acetic acid production (Figure 1b,c). In terms of small peptide content, the small peptide content of FSBM was measured on the 15th day, and the strains with high small peptide production rates were C37d72, C37m39, and C80d20, with the C37d72 strain having the greatest ability to produce small peptides (Figure 1d). When detecting the changes in microbial-derived D-amino acids in the five FSBM strains, the results responded to basically the same content of D-Ala on the 15th day, and C37d72 produced higher levels of D-Glu and D-Asp (Figure 1e,f). In conclusion, C37d72 was selected as the FSBM strain in this experiment.

3.2. Effect of Incorporating Varying Levels of Fermented Soybean Meal into Diets on the Growth Performance of Broiler Chickens

As shown in

Table 3. Effect of incorporating varying levels of fermented soybean meal into diets on the growth performance of broiler chickens.

Items	Control 2	FSBML 2	FSBMM 2	FSBMH 2	SEM	p-Value
Initial BW 1 (g)	451.61	449.48	449.30	448.51	4.03	0.994
Final BW 1 (g)	1468.11 b	1539.88 ab	1517.75 ab	1626.02 a	19.28	0.036
ADFI 1 (g/d)	97.61 b	109.97 a	107.61 a	111.25 a	1.41	0.002
ADG 1 (g/d)	48.37 b	51.90 ab	50.96 b	56.06 a	0.82	0.011
FCR 1 (g/g)	2.04	2.06	2.13	2.05	0.47	0.741

^a,b Means with different superscripts in each row are statistically significantly different (p < 0.05). The values are presented as the mean (n = 30). ¹ BW, body weight; ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio. ² Control, basal diet; FSBML, basal diet + 0.2 kg/t FSBM; FSBMM, basal diet + 2 kg/t FSBM; FSBMH, basal diet + 5 kg/t FSBM.

, the average initial body weight (BW) of the broilers in the four groups was similar. In contrast, the final BW of the broilers in the FSBMH group was significantly higher than in the control group (p < 0.05). However, the final BW of the FSBML and FSBMM groups was not significantly different from that of the control group. Both ADFI (p < 0.05) and ADG (p < 0.05) were significantly higher in the FSBML and FSBMH groups. The FSBMM group exhibited a significant increase in ADFI (p < 0.05), but showed no impact on ADG. However, FSBM did not improve the FCR of the broilers. These results suggest that FSBM can improve ADFI and ADG in broilers.

3.3. Effect of Incorporating Varying Levels of Fermented Soybean Meal into Diets on the Slaughter Performance and Meat Color of Broiler Chickens

The results in

Table 4. Effect of incorporating varying levels of fermented soybean meal into diets on the slaughter performance and meat color of broiler chickens.

Items	Control 1	FSBML 1	FSBMH 1	SEM	p-Value
Liver percentage/%	2.02	1.91	2.06	0.39	0.278
Spleen percentage/%	0.25	0.24	0.21	0.15	0.485
Abdominal fat percentage/%	0.94 c	1.21 b	1.65 a	0.65	0.000
Breast muscle percentage/%	13.58	14.01	13.33	0.16	0.226
Thigh muscle percentage/%	15.33 ab	15.78 a	14.84 b	0.16	0.043
Breast muscle luminance /L*	50.52	50.985	50.98	0.36	0.838
Breast muscle redness/a*	7.21	6.85	7.53	0.20	0.376
Breast muscle yellowness/b*	10.27	10.44	10.31	0.24	0.956
Thigh muscle luminance /L*	57.3	56.53	56.01	0.42	0.462
Thigh muscle redness/a*	11.82 b	13.62 a	13.99 a	0.32	0.011
Thigh muscle yellowness/b*	11.81	11.98	11.09	0.25	0.303

^a–c Means with different superscripts in each row are statistically significantly different (p < 0.05). The values are presented as the mean (n = 15). L*, Luminance, a*, redness, b*, yellowness. The percentages of abdominal fat, breast muscle, and thigh muscle were calculated relative to the weight of eviscerated carcass; organ index  =  (organ weight in grams)/(live weight in grams) × 100. ¹ Control, basal diet; FSBML, basal diet + 0.2 kg/t FSBM; FSBMH, basal diet + 5 kg/t FSBM.

show that there is no difference between the percentage of liver, spleen, breast muscle, thigh muscle, and color of breast muscle in the different doses of the FSBM treatment group and the control group. However, the percentage of abdominal fat (p < 0.05) and thigh muscle redness (p < 0.05) were both significantly higher in the FSBML and FSBMH groups. These findings suggest that FSBM enhances thigh muscle redness and the accumulation of abdominal fat in broilers.

3.4. Effect of Incorporating Varying Levels of Fermented Soybean Meal into Diets on the Intestinal Morphology of Broiler Chickens

The histological and morphological characteristics of the duodenum, ileum, and rectum in all groups of broilers are shown in Figure 2. The intestines exhibited a normal and organized structure. This study revealed that the FSBMH group had significantly higher VH/CD of the duodenum and rectum than the control group (p < 0.05, Figure 2c,i). However, VH/CD in the ileum was not statistically significant (Figure 2f). At the same time, VH and CD of the duodenum, ileum, and rectum in the FSBMH group were not statistically significant (Figure 2a,b,d,e,g,h). Additionally, the FSBML group did not affect the development of any intestinal segments. These results showed that the FSBMH group can improve the development of the intestinal villus and promote the maturation of intestinal cells in broilers.

3.5. Effect of Fermented Soybean Meal on Oxidative Damage in the Ileal Intestine of Broiler Chickens

An examination of the ileal mucosa was conducted to elucidate the impact of FSBM on intestinal oxidative damage in broilers (Figure 3). Compared to the control group, the results demonstrated a significant decrease in SOD activity in both the FSBML group and the FSBMH group (p < 0.05, Figure 3b). Additionally, there was a notable increase in GSH content in the FSBMH group, while GSH-Px activity significantly decreased in this same group (p < 0.05, Figure 3c,d). Furthermore, the FSBM group did not exhibit any significant impact on the levels of MDA, ROS, CAT, GSSG, or T-AOC in the ileal mucosa (Figure 3a,e,f,g,h). These findings suggest that the FSBMH treatment enhanced the intestinal redox status of broilers.

3.6. Effect of Fermented Soybean Meal on the Integrity of the Ileal Intestinal Barrier in Broiler Chickens

The impact of FSBM on the ileal mucosa of broiler chickens was investigated by examining the intestinal barrier function (Figure 4). The results in Figure 4 indicate a significant increase in the relative mRNA expression of ZO-1 and Occludin in both the FSBML and FSBMH groups (p < 0.05, Figure 4a,e). There was a significant increase in the relative mRNA expression of ZO-2 in the FSBMH group (p < 0.05, Figure 4b). Moreover, there was a significant decrease in the relative mRNA expression of Claudin-2 (p < 0.05, Figure 4d). However, the relative mRNA expression of Claudin-1 and serum LPS content did not change in the FSBM group (Figure 4c,f). The research results show that the addition of FSBM enhances the intestinal barrier of broiler chickens.

3.7. Effect of Fermented Soybean Meal on Intestinal Inflammation and Immune Factor Expression in Broiler Chickens

As can be seen from the results in Figure 5, the ileal mucosa was examined to determine the effect of FSBM on intestinal inflammation in broilers. This study found that the mRNA expression of IL-1β, IL-10, IFN-γ, and NF-κB was significantly increased in the FSBML group (p < 0.05, Figure 5a,f,k,l). Similarly, the mRNA expression of IL-10, MCP-1, IFN-α, and IFN-β was significantly increased in the FSBMH group (p < 0.05, Figure 5f–j). Moreover, the mRNA expression of IL-4 and IL-6 was significantly increased in both FSBML and FSBMH groups (p < 0.05, Figure 5c,d). However, FSBM did not affect IL-2 and IL-8 (Figure 5b,e).

3.8. Effect of Fermented Soybean Meal on the Cecal Microbiota of Broiler Chickens

According to the alpha diversity analysis, there were no significant differences in the Shannon index, Simpson index, Chao1 index, or ACE index between the control group and FSBMH group (Figure 6a–d). A principal coordinate analysis (PCoA) using weighted UniFrac distances showed that the newly formed microbial communities in the FSBMH group did not exhibit significant clustering (Figure 6e).

As can be seen from the results in Figure 7, at the phylum level, Firmicutes, Proteobacteria, Bacteroidota, and Campylobacterota were the dominant phyla in two groups (Figure 7a). At the class level, Bacilli, Gammaproteobacteria, Bacteroidia, and Clotridia were the most dominant bacterial classes (Figure 7b). On the other hand, the dominant orders included Lactobacillales and Bacteroidales, whereas Burkholderiales was endemic to the FSBMH group, and Mycoplasmatales was endemic to the control group (Figure 7c). Interestingly, the dominant families were Lactobacillale and Enterococcaceac, whereas Burkholderiaceae was endemic to the FSBMH group, and Mycoplasmataceae was endemic to the control group (Figure 7d). Meanwhile, Ligilactobacillus, Lactobacillus, and Enterococcus were the dominant genera; Ralstonia was endemic to the FSBM group; and Ureaplasma was endemic to the control group (Figure 7e). Furthermore, Lactobacillus_aviarius, Enterococcus_cecorum, and Lactobacillus_salivarius were the dominant species, but Ralstonia_pickettii was endemic to the FSBM group (Figure 7f).

The application of LDA-based LEfSe analysis enabled the identification of differently represented microbial groups between the control and FSBMH groups. The microbial composition of the control group exhibited a higher abundance of genera and orders, such as Mycoplasmaceaes and Peptostreptococcaceae. Conversely, the FSBMH group demonstrated elevated levels of the order and genus Nitriliruptoraceae, as well as the genus Ruminococcaceae (Figure 8a,b).

4. Discussion

Microbial fermentation has been proven as a way to improve SBM utilization [22]. Replacing SBM with FSBM in the diet leads to a significant increase in feed intake and body weight gain in broilers [23]. The supplementation of the base diet with varying doses of corn-soybean-meal-fermented feed significantly increased the VH/CD in laying hens’ duodenum and jejunum [24]. Therefore, the dietary incorporation of FSBM enhances intestinal development and broiler growth performance.

4.1. Comparison and Screening of Fermentation Effects of Different Intestinal Probiotics in Chickens

The fermentation process generates a multitude of metabolites, including organic acids, enzymes, and probiotics, all of which have been demonstrated to exert critical functions in the production of livestock and poultry [25]. Organic acids (lactic acid, acetic acid, propionic acid, citric acid, etc.) lower the pH value of feed and the gastrointestinal tract, thereby promoting the growth of beneficial bacteria or inhibiting the proliferation of pathogenic microorganisms, which, in turn, enhances the growth performance of poultry and livestock [26]. The inoculation fermentation of chicken cecum contents produced more short-chain fatty acids to inhibit the growth of Escherichia coli [27]. In this study, Lactobacillus casei produced substantial amounts of lactic acid and acetic acid, which may be one of the contributing factors. Furthermore, research indicates that Lactobacillus secretes enzymes involved in DAA metabolism, which aids in enhancing the flavor of the fermentation and suppressing the synthesis of bacterial biofilms [28]. DAAs also serve as substrates recognized by neutrophils via G-protein-coupled receptors, thus participating in mucosal defense mechanisms and enhancing intestinal health [6]. In this study, DAAs produced by Lactobacillus casei may also function in broilers through this mechanism.

4.2. Effect of Incorporating Varying Levels of Fermented Soybean Meal into Diets on the Growth Performance of Broiler Chickens

Numerous studies have demonstrated that the supplementation of fermented feeds can augment the growth performance of animals [29]. In this study, FSBM significantly improved the ADFI and ADG of broilers. Relevant studies showed that the addition of 5% fermented feed led to a significant improvement in the ADG and ADFI of broilers [15]. Feeding rapeseed meal fermented by Lactobacillus fermentum to broilers improved weight gain and FCR [30]. The growth performance of Ross broilers was significantly improved by supplementation with varying doses of Saccharomyces cerevisiae cell wall extracts [31]. Therefore, adding 5 kg/t of FSBM resulted in better growth performance in broilers.

4.3. Effect of Incorporating Varying Levels of Fermented Soybean Meal into Diets on the Slaughter Performance and Meat Color of Broiler Chickens

Slaughter performance indicates the animal’s ability to convert feed into muscle and fat, with muscle and abdominal fat being the main indicators. Both fermented ginkgo biloba and fermented citrus by-products significantly increased thigh muscle percentage in broilers [32,33]. In this study, adding 0.2 kg/t and 5 kg/t of FSBM to the diet resulted in a significant increase in the percentage of thigh muscle in the broilers. Furthermore, the weight of abdominal fat in the broilers increased gradually with age, while the abundance of Ruminococcus also increased [34]. The FSBMH group exhibited an increased abundance of Ruminococcaceae, potentially contributing to abdominal fat deposition. This suggests that supplementing feed with 5 kg/t of FSBM enhances muscle accretion.

4.4. Effect of Incorporating Varying Levels of Fermented Soybean Meal into Diets on the Intestinal Morphology of Broiler Chickens

The morphology of the intestine reflects the health and nutrient absorption capacity of the animal’s intestine, with VH/CD being an important indicator of intestinal nutrient absorption [35]. In this study, the addition of FSBM to the basal diet improved the VH/CD in the duodenum and rectum, but had no significant effect on VH and CD. Relevant studies found that feeding with Lactobacillus salivarius fermented feed significantly improved the VH in the duodenum of hens, and also enhanced the VH/CD in both the duodenum and ileum [36]. Feeding with a mixed microbial strain fermented feed significantly reduced the CD in the cecum of Xue Feng Black-bone chickens, but had no effect on the VH or the VH/CD [37]. In this study, adding FSBM to the basal feed may enhance digestion and the absorption of nutrients.

4.5. Effect of Fermented Soybean Meal on Oxidative Damage in the Ileal Intestine of Broiler Chickens

The production of free radicals in animals is balanced with their antioxidant system, which is essential for maintaining good health [38]. SOD, GSH, and GSH-Px as antioxidant enzymes can remove excess ROS in the body, thereby reducing the tissue damage caused by ROS [39]. In this study, the addition of 5 kg/t of FSBM significantly increased the GSH content and decreased the SOD and GSH-Px activities of the ileal mucosa. Simultaneously, the fermentation of soybean meal with Lactobacillus casei resulted in the production of DAAs, including D-Glu, D-Ala, and D-Asp. These DAAs enter the intestine, interacting with D-amino acid oxidase secreted by intestinal goblet cells, leading to the generation of H₂O₂ [40]. This H₂O₂ combated intestinal pathogens, regulated the balance of the intestinal microbiota, and ultimately diminished the oxidative stress exerted by microorganisms on the intestinal tract [41]. Therefore, we speculate that the intestine requires lower levels of SOD and GSH-Px activity, and the addition of 5 kg/t of FSBM to the diet may improve the intestinal redox status.

4.6. Effect of Fermented Soybean Meal on the Integrity of the Ileal Intestinal Barrier in Broiler Chickens

Improving intestinal tight junction proteins effectively prevents the paracellular transport of intestinal toxins, bacteria, and inflammatory mediators, thereby maintaining the integrity of the intestinal epithelial barrier [42]. Our results revealed that adding FSBM increased the relative mRNA expression of ileum tight junction proteins ZO-1 and Occludin in broilers. Similarly, the addition of Lactobacillus plantarum and Bacillus polymyxa to the base diet was able to regulate ileal-associated barrier gene expression in broilers [43]. Some studies found that supplementation with CSFBP fermented feed can increase the relative mRNA expression levels of Claudin, Occludin, and ZO-1 in the jejunum of broilers [44]. The results showed that adding FSBM in the base diet improved the intestinal barrier function.

4.7. Effect of Fermented Soybean Meal on Intestinal Inflammation and Immune Factor Expression in Broiler Chickens

Further study of intestinal inflammation in the ileum of broilers is needed. In this study, the addition of 5 kg/t of FSBM significantly upregulated the relative mRNA expression levels of IL-6, IFN-α, and IFN-β in the ileal mucosa of broilers. The fermentation of soybean meal with Bacillus subtilis and Lactobacillus strains significantly upregulated the mRNA expression levels of IL-6, IL-10, and NF-κB in the jejunum of broilers [45]. This may be due to the FSBM and the fermentation-produced DAAs regulating the composition and diversity of the intestinal microbiota, although further research is required [46]. Moreover, the addition of 5 kg/t of FSBM also significantly upregulated the relative mRNA expression levels of IL-4. The fermentation of wheat bran by Lactobacillus plantarum and Bacillus subtilis significantly enhanced IL-4 contents in broiler serum and enhanced the anti-inflammatory capacity [47].

4.8. Effect of Fermented Soybean Meal on the Cecal Microbiota of Broiler Chickens

Bacterial diversity at the species level was supported by adding FSBM to the meal fed to broilers. FSBM affected the intestinal flora, which was associated with intestinal health [48]. Healthy and economically efficient broilers can be produced by regulating the cecal microflora through diet [49,50,51]. Incorporating organic acids into feed had been shown to enhance broiler growth performance, while their synergistic combination with dietary fiber can further augment the humoral immune response [52]. The impact of FSBM on the cecal microbiota of broilers was investigated using 16S rRNA gene sequencing in this experiment. The results showed that FSBM changed the beta diversity of the cecum microorganisms in broiler chickens. This proved that adding fermented feed to the basal diet can better maintain the balance of the intestinal flora in broilers, thus protecting the intestinal health [53,54]. Nitriliruptoraceae belongs to the Actinobacteria phylum and harbors homologs of genes that are indispensable for the biosynthesis of glutamine, glutamate, and proline. The genes of tyrosine kinase receptor A and tyrosine kinase receptor H in Nitriliruptoraceae played a role in potassium uptake and high sodium/hydrogen reverse transporter activity under high-salt conditions [55,56]. Meanwhile, Ruminococcaceae are a group of anaerobic bacteria of the strict phylum firmicutes present in the mucosal biofilm of the colon in healthy individuals [57]. Ruminococci was crucial for gut health, producing short-chain fatty acids like butyrate, which provided energy and carbon for colon cells [58]. The enrichment of Rumenococcaceae in the FSBMH group conferred advantageous effects on broiler intestinal health.

5. Conclusions

In conclusion, the fermentation of soybean meal by Lactobacillus casei C37d72 produced 170 mg/g lactic acid and 0.5–2 mg/g DAA. Supplementation of 5 kg/t FSBM enhanced the ADG and ADFI, as well as the VH/CD in the duodenum and rectum of broilers. The FSBM also concurrently upregulated the mRNA expression levels of tight junction proteins and interleukins, regulating the redox status of the ileal mucosa.

This study can provide a basis for utilizing animal intestinal microorganisms as strains in the production of fermented feeds and offer new evidence for the role of dead bacteria and fermentation metabolites in enhancing the growth performance of animals. Fermented soybean meal can increase the ADG and intestinal health of broilers and improve the redness of thigh muscle, thereby reducing the use of therapeutic medications, increasing the profitability for farmers, and providing consumers with higher quality meat. However, the broilers and diet formulations used in this study are more adapted to the environment of South China, and the effectiveness of their application in other regions remains to be investigated.