==== Front J Anim Sci Biotechnol J Anim Sci Biotechnol Journal of Animal Science and Biotechnology 1674-9782 2049-1891 BioMed Central London 37393326 888 10.1186/s40104-023-00888-3 Research Comparative effects of soy protein concentrate, enzyme-treated soybean meal, and fermented soybean meal replacing animal protein supplements in feeds on growth performance and intestinal health of nursery pigs Deng Zixiao 1 Duarte Marcos Elias 1 Kim So Young 2 Hwang Yunil 2 http://orcid.org/0000-0003-4591-1943 Kim Sung Woo sungwoo_kim@ncsu.edu 1 1 grid.40803.3f 0000 0001 2173 6074 Department of Animal Science, North Carolina State University, 116 Polk Hall, Campus Box 7621, Raleigh, NC 27695 USA 2 grid.480117.b 0000 0004 4649 0869 CJ Cheiljedang, Seoul, 04560 Korea 2 7 2023 2 7 2023 2023 14 899 1 2023 3 5 2023 © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Soy protein supplements, with high crude protein and less antinutritional factors, are produced from soybean meal by different processes. This study evaluated the comparative effects of various soy protein supplements replacing animal protein supplements in feeds on the intestinal immune status, intestinal oxidative stress, mucosa-associated microbiota, and growth performance of nursery pigs. Methods Sixty nursery pigs (6.6 ± 0.5 kg BW) were allotted to five treatments in a randomized complete block design with initial BW and sex as blocks. Pigs were fed for 39 d in 3 phases (P1, P2, and P3). Treatments were: Control (CON), basal diet with fish meal 4%, 2%, and 1%, poultry meal 10%, 8%, and 4%, and blood plasma 4%, 2%, and 1% for P1, P2, and P3, respectively; basal diet with soy protein concentrate (SPC), enzyme-treated soybean meal (ESB), fermented soybean meal with Lactobacillus (FSBL), and fermented soybean meal with Bacillus (FSBB), replacing 1/3, 2/3, and 3/3 of animal protein supplements for P1, P2, and P3, respectively. Data were analyzed using the MIXED procedure in SAS 9.4. Results The SPC did not affect the BW, ADG, and G:F, whereas it tended to reduce (P = 0.094) the ADFI and tended to increase (P = 0.091) crypt cell proliferation. The ESM did not affect BW, ADG, ADFI, and G:F, whereas tended to decrease (P = 0.098) protein carbonyl in jejunal mucosa. The FSBL decreased (P < 0.05) BW and ADG, increased (P < 0.05) TNF-α, and Klebsiella and tended to increase MDA (P = 0.065) and IgG (P = 0.089) in jejunal mucosa. The FSBB tended to increase (P = 0.073) TNF-α, increased (P < 0.05) Clostridium and decreased (P < 0.05) Achromobacter and alpha diversity of microbiota in jejunal mucosa. Conclusions Soy protein concentrate, enzyme-treated soybean meal, and fermented soybean meal with Bacillus could reduce the use of animal protein supplements up to 33% until 7 kg body weight, up to 67% from 7 to 11 kg body weight, and entirely from 11 kg body weight without affecting the intestinal health and the growth performance of nursery pigs. Fermented soybean meal with Lactobacillus, however, increased the immune reaction and oxidative stress in the intestine consequently reducing the growth performance. Keywords Enzyme-treated soybean meal Fermented soybean meal Intestinal health Nursery pigs Soy protein concentrate http://dx.doi.org/10.13039/100005825 National Institute of Food and Agriculture 02893 Kim Sung Woo http://dx.doi.org/10.13039/100009591 North Carolina Agricultural Foundation 660101 Kim Sung Woo CJ Cheiljedangissue-copyright-statement© Chinese Association of Animal Science and Veterinary Medicine 2023 ==== Body pmcBackground At weaning, the change from highly digestible sow milk to plant-based solid diets, living environment, and social hierarchy leads to the most stressful period for pigs [1, 2]. The stress commonly causes intestinal and immunological dysfunction leading to impaired health and growth of nursery pigs [3]. Animal proteins are usually added to the diet for nursery pigs to alleviate the weaning stress by stimulating feed intake and improving intestinal health due to the highly digestible protein and functional compounds [4, 5]. However, there are some concerns about using animal protein supplements in nursery diets, including the cost, availability, and safety issues [6]. Compared with animal proteins, soybean meal is a more affordable and steady protein source that is most widely used in the swine diet [7]. However, the use of soybean meal is limited especially for feeding nursery pigs due to its antinutritional factors including soy allergens, lectins, trypsin inhibitors, and flatulence-producing oligosaccharides [8]. These can impair the growth and intestinal health of pigs [9, 10]. Therefore, several methods have been attempted to eliminate the antinutritional factors in soybean meal. In order to overcome the limitations, such as antinutritional factors, soybean meal can be further processed. Soy protein concentrate is produced by ethanol extractions of soybean meal to remove soluble carbohydrates, reduce soy allergens, and keep a relatively high crude protein content than soybean meal [11, 12]. Enzyme treatment involves treating soybean meal with a proprietary mixture of enzymes to hydrolyze soluble carbohydrates and antinutritional factors [13]. The fermentation process involves treating soybean meal with different microorganisms to reduce antinutritional factors because of the enzymes secreted by microorganisms [8, 14]. Besides the reduction of antinutritional factors in soybean meal, the process of enzymatic hydrolysis and microbial fermentation also can increase the proportions of small peptides, which can improve the utilization of dietary proteins by nursery pigs when their endogenous protease secretion is limited [15–17]. Several studies have indicated that processed soybean products can improve the growth performance of nursery pigs compared to soybean meal [18–20]. In addition, studies have suggested that these processed soy products have the potential to partly replace the use of animal protein in the diets of nursery pigs [12, 21, 22]. Based on previous findings, it was hypothesized that soy protein concentrate has equal quality compared with enzyme-treated soy protein and fermented soybean meal to replace animal protein supplements in the diets of nursery pigs. To test the hypothesis, the objective of this study was to evaluate the comparative effects of soy protein concentrate, enzyme-treated soybean meal, and fermented soybean meal replacing animal protein supplements in feeds on intestinal immune status, intestinal oxidative stress, mucosa-associated microbiota, and growth performance of nursery pigs. Materials and methods The Institutional Animal Care and Use Committee of North Carolina State University (Raleigh, NC, USA) revised and approved the protocol used for this study. The animal experiment was conducted at the North Carolina State University Metabolism Educational Unit (Raleigh, NC, USA). Allergenic proteins in soy products The β-conglycinin ELISA Kit (BA-UBT001, Unibiotest, Wuhan, China) and Glycinin ELISA Kit (BA-UBT002, Unibiotest) were used to measure the content of β-conglycinin and glycinin in the soy protein supplements as previously described by Deng et al. [12]. The soy protein supplements included soybean meal (North Carolina State University Feed Mill Education Unit, Raleigh, NC, USA), soy protein concentrate (CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (Purina Animal Nutrition, Shoreview, MN, USA), and fermented soybean meal with Bacillus (CJ Bio, Seoul, Korea). The soy protein samples were ground and weighed (0.3 g) to mix with 30 mL sample extractant. The mixed samples were vigorously shaken for 16 h at 25 °C and then centrifuged at 4,000 × g for 5 min to get supernatant. Extracted samples and standards (50 µL) were added in the plate and antibody solution (50 µL) were added. The plate was gently shaken to mix these two reagents and incubated at 37 °C for 30 min. Next, the plate was washed by wash solution 4 times and 100 µL horseradish peroxidase (HRP) conjugate enzyme was added in each well to incubate at 37 °C for 30 min. The plate was washed then the mixture of chromogen solution A and B (100 µL) were added in each well to incubate at 37 °C for 15 min. At the end, 50 µL stop solution was added in each well and the plate was read within 15 min. The absorbance was measured by the spectrophotometer (Synergy HT, BioTek Instruments, Winooski, VT, USA) at 450 and 630 nm wavelengths. The concentrations of β-conglycinin and glycinin were expressed as mg/g (Table 1). Table 1 Concentration of soy antigens in soy products Item SBMa SPCa ESBa FSBLa FSBBa Glycinin, mg/g 112.6 ± 24.3 < 0.1 ± 0.0 0.2 ± 0.0 164.5 ±12.0 66.9 ± 13.6 β-conglycinin, mg/g 125.0 ± 10.2 0.1 ± 0.0 0.3 ± 0.0 137.9 ± 10.6 21.0 ± 0.1 aSBM: soybean meal, North Carolina State University Feed Mill Education Unit, Raleigh, NC, USA; SPC: X-Soy 200, CJ Selecta, Araguari, MG, Brazil; ESB: HP 300, Hamlet Protein Inc., Findlay, OH, USA; FSBL: FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA; FSBB: Soytide, CJ Bio, Jung-gu, Seoul, Korea Animal, design, and diets Sixty newly-weaned pigs (30 barrows and 30 gilts) weaned at 21 days of age with 6.6 ± 0.5 kg of initial body weight (BW) were allotted to four dietary treatments in a randomized complete block design. Sex and initial BW were considered as blocking criteria. Pigs were purchased from Kilpatrick Hog Farm (Magnolia, NC, USA) and individually housed in pens with a waterer and feeder. All pens (1.50 m × 0.74 m) were located in the same room. Pigs were fed for 39 d in 3 phases (P1 for 7 d [from wean to 7 kg BW]; P2 for 13 d [from 7 to 11 kg BW]; P3 for 19 d [from 11 to 25 kg BW]). Dietary treatments were control (CON): basal diet with fish meal (at 4%, 2%, and 1% for P1, P2, and P3 respectively), poultry meal (at 10%, 8%, and 4% for P1, P2, and P3 respectively), and blood plasma (at 4%, 2%, and 1% for P1, P2, and P3 respectively); SPC: basal diet with soy protein concentrate replacing animal protein supplements (at 1/3, 2/3, and 3/3 for P1, P2, and P3, respectively), ESB: basal diet with enzyme-treated soybean meal replacing animal protein supplements (at 1/3, 2/3, and 3/3 for P1, P2, and P3, respectively), FSBL: basal diet with fermented soybean meal with Lactobacillus replacing animal protein supplements (at 1/3, 2/3, and 3/3 for P1, P2, and P3, respectively); and FSBB: basal diet with fermented soybean meal with Bacillus replacing animal protein supplements (1/3, 2/3, and 3/3 for P1, P2, and P3, respectively). The replacement of protein supplements was gradually increased (1/3 to 3/3) with the progress of phase feeding (P1 to P3) based on the outcomes of our previous study [12]. All nutrients in the experimental diets met or were slightly higher than the requirement suggested by NRC [23]. An indigestible external marker (0.4% titanium dioxide) was mixed with all experimental diets and fed to pigs from d 32 to 39 of the experiment. The composition of the experimental diets are shown in Table 2. The Feed Mill Educational Unit of North Carolina State University (Raleigh, NC, USA) produced all experimental diets. Samples of treatment diets were ground and sent to the North Carolina Department of Agriculture and Consumer Services to analyze the nutritional compositions (Raleigh, NC, USA). Table 2 Composition of experimental diets for experiment Item Phase 1 Phase 2 Phase 3 CON SPCa ESBa FSBLa FSBBa CON SPC ESB FSBL FSBB CON SPC ESB FSBL FSBB Ingredientb, %  Corn, yellow 28.79 27.78 27.96 27.60 28.13 40.39 39.48 39.65 39.24 39.82 62.58 61.85 61.92 61.62 62.02  Whey permeate 24.00 24.00 24.00 24.00 24.00 15.00 15.00 15.00 15.00 15.00 5.00 5.00 5.00 5.00 5.00  Cookie meal 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 0.00 0.00 0.00 0.00 0.00  Soybean meal (48% CP) 16.00 16.00 16.00 16.00 16.00 19.00 19.00 19.00 19.00 19.00 23.00 23.00 23.00 23.00 23.00  Poultry meal 10.00 6.67 6.67 6.67 6.67 8.00 2.67 2.67 2.67 2.67 4.00 0.00 0.00 0.00 0.00  Fish meal (63% CP) 4.00 2.67 2.67 2.67 2.67 2.00 0.67 0.67 0.67 0.67 1.00 0.00 0.00 0.00 0.00  Blood plasma 4.00 2.67 2.67 2.67 2.67 2.00 0.67 0.67 0.67 0.67 1.00 0.00 0.00 0.00 0.00  X-Soy200 0.00 6.50 0.00 0.00 0.00  0.00 7.80 0.00 0.00 0.00  0.00 6.00 0.00 0.00 0.00   HP300 0.00 0.00 6.00 0.00 0.00  0.00 0.00 7.20 0.00 0.00  0.00 0.00 5.54 0.00 0.00   Fermex200 0.00 0.00 0.00 7.09 0.00  0.00 0.00 0.00 8.51 0.00  0.00 0.00 0.00 6.54 0.00   Soytide 0.00 0.00 0.00 0.00 6.62 0.00 0.00 0.00 0.00 7.95 0.00 0.00 0.00 0.00 6.11  Poultry fat 1.30 1.50 1.70 1.00 1.00 1.30 1.80 2.10 1.20 1.30 1.00 1.30 1.60 0.90 1.00  L-Lys HCl 0.52 0.53 0.54 0.54 0.54 0.51 0.52 0.53 0.53 0.53 0.42 0.43 0.43 0.43 0.43  L-Met 0.25 0.24 0.25 0.25 0.23 0.21 0.20 0.21 0.21 0.18 0.14 0.13 0.14 0.14 0.12  L-Thr 0.17 0.11 0.17 0.17 0.15 0.15 0.09 0.16 0.16 0.13 0.12 0.07 0.12 0.12 0.10  L-Trp 0.02 0.01 0.01 0.02 0.01 0.01 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.00  L-Val 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.02 0.01 0.00 0.00 0.00 0.00 0.00  Dicalcium phosphate 0.00 0.05 0.05 0.00 0.00 0.15 0.65 0.65 0.60 0.60 0.54 0.90 0.90 0.90 0.90  Limestone, ground 0.30 0.62 0.66 0.67 0.66 0.63 0.80 0.83 0.85 0.82 0.80 0.92 0.95 0.95 0.92  Zinc oxide 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.00 0.00 0.00 0.00 0.00  Salt 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22  Mineral premixc 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15  Vitamin premixd 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 Calculated composition (as-is)  Dry matter, % 91.3 91.4 91.2 91.0 91.0 90.7 90.8 90.6 90.4 90.4 89.7 89.8 89.6 89.4 89.4  ME, kcal/kg 3,454 3,454 3,449 3,452 3,449 3,435 3,437 3,435 3,436 3,437 3,373 3,373 3,374 3,373 3,376  Crude protein, % 24.6 24.4 23.8 23.8 24.1 22.5 21.9 21.2 21.2 21.5 20.9 20.5 19.9 19.9 20.1  SIDe Lys, % 1.50 1.50 1.50 1.50 1.50 1.35 1.35 1.35 1.35 1.35 1.23 1.23 1.23 1.23 1.23  SID Met+Cys, % 0.82 0.82 0.82 0.82 0.82 0.74 0.74 0.74 0.74 0.74 0.68 0.68 0.68 0.68 0.68  SID Trp, % 0.25 0.25 0.25 0.25 0.25 0.22 0.22 0.22 0.22 0.22 0.20 0.21 0.21 0.20 0.21  SID Thr, % 0.88 0.88 0.88 0.88 0.88 0.79 0.79 0.79 0.79 0.79 0.73 0.73 0.73 0.73 0.73  Ca, % 0.85 0.85 0.85 0.85 0.85 0.80 0.80 0.80 0.80 0.80 0.70 0.70 0.70 0.70 0.70  STTD Pf, % 0.50 0.45 0.45 0.45 0.45 0.40 0.40 0.40 0.40 0.40 0.33 0.33 0.33 0.33 0.33  Total P, % 0.73 0.67 0.66 0.65 0.65 0.63 0.62 0.62 0.60 0.61 0.58 0.57 0.57 0.56 0.56  Glycining, mg/g 18.0 18.0 18.0 29.7 22.4 21.4 21.4 21.4 35.4 26.7 25.9 25.9 25.9 36.7 30.0  β-Conglycininh, mg/g 20.0 20.0 20.0 29.8 21.4 23.8 23.8 23.8 35.5 25.4 28.8 28.8 28.8 37.8 30.0 Analyzed composition (as-is)  Dry matter, % 91.7 91.6 91.8 91.3 91.5 90.4 90.3 90.8 90.4 90.9 88.4 88.4 88.5 88.3 88.3  Crude protein, % 24.6 24.0 23.3 23.1 23.8 21.3 21.2 20.8 19.7 21.1 20.5 19.9 19.6 19.0 18.8  Neutral detergent fiber, % 8.1 7.9 7.8 7.6 7.6 9.1 8.6 8.6 8.3 8.2 9.8 9.6 9.0 8.8 9.4  Acid detergent fiber, % 3.0 3.2 3.3 3.0 3.5 3.5 3.9 3.7 3.7 3.7 4.1 3.9 4.0 4.0 4.1  Ca, % 0.77 0.78 0.86 0.80 0.81 0.76 0.74 0.79 0.77 0.79 0.68 0.67 0.73 0.74 0.62  Total P, % 0.71 0.66 0.66 0.65 0.66 0.62 0.62 0.62 0.58 0.60 0.57 0.58 0.55 0.53 0.54 aSPC, ESB, FSBL, and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively bThe nutritional composition of corn, soybean meal, fish meal, blood plasma, and poultry fat were from NRC [23]; the nutritional composition of poultry meal were from Rojas and Stein [24]; the nutritional composition of whey permeate, cookie meal, X-Soy200, HP300, Fermex200, and Soytide were from analyzed values (International Ingredient Corporation, Fenton, MO, USA; Darling Ingredients, Irving, TX, USA; CJ Selecta, Araguari, MG, Brazil; Hamlet Protein Inc., Findlay, OH, USA; Purina Animal Nutrition, Shoreview, SD, USA; CJ Bio, Seoul, Korea); the nutritional composition of all amino acids were from analyzed values (CJ Bio, Seoul, Korea); the nutritional composition of dicalcium phosphate, limestone, salt, mineral premix, and vitamin premix were from analyzed values (North Carolina State University Feed Mill Education Unit, Raleigh, NC, USA); the nutritional composition of zinc oxide was from analyzed values (Zinc Nacional, Monterrey, N.L., Mexico) cThe trace mineral premix provided per kilogram of complete diet: 33 mg of Mn as manganous oxide, 110 mg of Fe as ferrous sulfate, 110 mg of Zn as zinc sulfate, 16.5 mg of Cu as copper sulfate, 0.30 mg of I as ethylenediamine dihydroiodide, and 0.30 mg of Se as sodium selenite dThe vitamin premix provided per kilogram of complete diet: 6,614 IU of vitamin A as vitamin A acetate, 992 IU of vitamin D3, 19.8 IU of vitamin E, 2.64 mg of vitamin K as menadione sodium bisulfate, 0.03 mg of vitamin B12, 4.63 mg of riboflavin, 18.52 mg of D-pantothenic acid as calcium pantothenate, 24.96 mg of niacin, and 0.07 mg of biotin eSID Standardized ileal digestibility fSTTD P Standardized total tract digestible phosphorus gThe concentration of glycinin in the diets was calculated based on the analyzed glycinin of soy protein supplements hThe concentration of β-conglycinin in the diets was calculated based on the analyzed β-conglycinin of soy protein supplements Growth performance and fecal score The growth performance including average BW, ADG, ADFI, and G:F were calculated based on the BW and feed intake obtained on d 0, 7, 20, and 39. Fecal scores were evaluated daily by the same person considering the following scale: very hard and dry stool (score 1), firm stool (score 2), normal stool (score 3), loose stool (score 4), and watery stool with no shape (score 5) as described by Cheng et al. [25]. Sample collection At the end of the study, euthanasia of pigs was conducted by the penetration of a captive bolt to the head followed by exsanguination [26]. The entire gastrointestinal tract was removed to collect samples. Ileal digesta (50 cm anterior to the ileocecal junction to the end of the ileum) was collected and put on the ice, then stored at −20 °C for further analysis. To evaluate morphology, mid-jejunum segments (5 cm) were washed with 0.9% saline solution and then placed in a 50-mL falcon tube containing 10% buffered formaldehyde. Mid-jejunal mucosal samples were scraped off with a glass slide and collected in 2-mL tubes, then promptly frozen in liquid nitrogen. The mucosal samples were transferred to the freezer at −80 °C for further DNA extraction and intestinal oxidative stress and immune status analysis. Oxidative stress and immune status The mucosal samples were processed following Holanda and Kim [27]. One gram of jejunal mucosa was weighed and then suspended in 1 mL of phosphate-buffered saline (PBS) in a 5-mL tube. A tissue homogenizer (Tissuemiser; Thermo Fisher Scientific Inc, Waltham, MA, USA) was used to homogenize the suspended mucosal samples. The homogenized samples were transferred into a new tube and centrifuged (13,000 × g for 10 min), then six aliquots of the supernatant were pipetted off and kept at −80 °C. Colorimetric assays kits, commercially available, were used to measure the total protein, tumor necrosis factor-alpha (TNF-α), interleukin 8 (IL-8), immunoglobulin A (IgA), immunoglobulin G (IgG), malondialdehyde (MDA), and protein carbonyl following the instructions of the manufacturers. The OD value was measured using the spectrophotometer (Synergy HT, BioTek Instruments) and software (Gen5 Data Analysis Software, BioTek Instruments). According to the OD value of the respective standards, the concentration of each parameter was calculated. The total protein concentration was determined using the Pierce BCA Protein Assay Kit (#23225, Thermo Fisher Scientific) following the procedure described by Holanda et al. [28]. The mucosal supernatant was diluted (1:60) in PBS to provide the protein concentration within the proper range (20–2,000 g/mL). The samples and standards (25 µL) were pipetted in microplate well and then 200 µL working reagent was added to each well. The plate was incubated at 37 °C for 30 min. The absorbance was measured at 562 nm wavelength. The total protein concentration was used to further standardize the concentration of other measures in the mucosa. The concentration of TNF-α in the jejunal mucosa was determined using Porcine TNF--α DuoSet ELISA Kit (#DY690B, R&D Systems; Minneapolis, MN, USA) following the procedure described by Sun et al. [29]. The samples and standards (100 µL) were added in each well of the plate and then 100 µL detection antibody was added with 2 h incubation at room temperature. The Streptavidin-HRP (100 µL) and substrate solution (100 µL) were consecutively used in the analysis. Stop solution (50 µL) was used to stop the reaction. The absorbance was read at 450 nm wavelength and corrected at 570 nm wavelength. The concentration of TNF-α was reported as pg/mg of protein. The Porcine IL-8/CXCL8 DuoSet ELISA Kit (#DY535, R&D Systems) was used to determine the concentration of IL-8 in jejunal mucosa following the procedure described by Moita et al. [30]. The mucosal supernatant was diluted (1:5) using the reagent diluent provided in the kit to reach the proper working range. The samples and standards (100 µL) were added in each well of the plate and then 100 µL detection antibody was added with 2 h incubation at room temperature. The Streptavidin-HRP (100 µL) and substrate solution (100 µL) were consecutively used in the analysis. Stop solution (50 µL) was used to stop the reaction. The absorbance was read at 450 nm wavelength and corrected at 570 nm wavelength and the concentration of IL-8 was reported as pg/mg of protein. The Pig IgA ELISA Kit and Pig IgG ELISA Kit (E101-102 and E101-104, Bethyl Laboratories, Inc., Montgomery, TX, USA) were used to determine the concentration of IgA and IgG following the procedure described by Holanda et al. [28]. The PBS was used to dilute the mucosal supernatant (1:1,200 and 1:2,400, for IgA and IgG respectively) to reach the working range of the ELISA kits. The samples and standards were added in each and incubated at room temperature for 1 h. Then 100 µL anti-IgA detection antibody was added and incubated at room temperature for 1 h. The HRP solution (100 µL) and 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution (100 µL) were consecutively used in the analysis. Stop solution (100 µL) was used to stop the reaction. The absorbance was read at 450 nm wavelength and the concentration of IgA and IgG were reported as µg/mg of protein. The OxiSelect Protein Carbonyl ELISA Kit (#STA-310, Cell Biolabs, Inc.; San Diego, CA, USA) was used to determine the concentration of protein carbonyl in the jejunal mucosa as previously described by Duarte and Kim [31]. The mucosal supernatant was diluted with PBS to get 10 µg/mL of protein to provide the concentration of protein carbonyl within the standard range (0.375 to 7.5 nmol/mg protein). The diluted samples and standards were pipetted in each well and incubate at 4 °C overnight. After washing, 100 µL dinitrophenylhydrazine (DNPH) working solution was added and incubate at room temperature for 45 min. Next, the plate was washed and 200 µL blocking solution was added following incubation with 1.5 h at room temperature on an orbital shaker. The anti-DNP antibody and was used after washing and the plate was incubated at room temperature for 1 h. The HRP conjugated secondary antibody was consecutively used after washing and the plate was incubated at room temperature for 1 h. Then 100 µL substrate solution was added in each well after washing and incubate at room temperature for 5 min. The reaction was stopped by adding 100 µL stop solution. The absorbance was measured at 450 nm wavelength and the concentration of protein carbonyl was reported as nmol/mg of protein. The OxiSelect TBARS MDA Quantitation Assay Kit (#STA-330, Cell Biolabs) was used to determine the concentration of MDA in the jejunal mucosa as previously described by Cheng et al. [25]. The samples and standards (100 µL) were added in separate 2-mL microcentrifuge tubes and SDS lysis solution (100 µL) and thiobarbituric acid (TBA) reagent (250 µL) were mix with samples and standards. Next, the tubes were placed in water bath at 95 °C for 1 h. The cooled tubes were centrifuged at 3,000 × g for 15 min to get the supernatant. The absorbance was read at 532 nm wavelength and the concentration of MDA was reported as nmol/mg of protein. Intestinal morphology and crypt cell proliferation Mid-jejunal tissues of each pig were used to evaluate intestinal morphology and crypt cell proliferation. The tissues were kept in 10% buffered formaldehyde for 48 h for fixation. Two sections of fixed tissue (approximately 2 mm) were cut, placed in a cassette, and transferred to a 70% ethanol solution. The processed samples were sent to the North Carolina State University Histology Laboratory (College of Veterinary Medicine, Raleigh, NC, USA) for dehydration, embedment, and staining using a Ki-67 assay. The Biocare Intellipath Stainer (Biocare Medical, Pacheco, CA, USA) was used to process the automated Ki-67 staining. To get the proper working concentration, the primary monoclonal antibody of Ki-67 (#ACR325, Biocare Medical) was diluted (1:100), and then incubated for 30 min with processed slides at room temperature. For detection, Vector ImmPress Rabbit polymer was employed. Staining was carried out using chromogen diaminobenzidine (DAB). The microscope Olympus CX31 (Lumenera Corporation, Ottawa, Canada) and software (Infinity 2–2 digital CCD) were used to measure intestinal morphology (villus height, villus width, and crypt depth) at a magnification of 40× following the procedure described by Jang et al. [32]. Ten complete villi and crypts were chosen to represent the intestinal morphology of each pig. The villus length was measured from its top to its intersection with the crypt; the villus width was measured in the center of the villus; and the crypt depth was measured from its intersection with the villus to its bottom. The villus height was divided by the crypt depth to determine the villus height to crypt depth (VH:CD) ratio. The percentage of Ki-67 positive cells (the marker for proliferating cells in the crypt) was calculated using pictures of 10 complete crypts captured by the Olympus CX31 microscope at a magnification of 100× following the procedure described by Xu et al. [33]. The pictures were cropped and then uploaded to the software (Image JS) for analysis. All the procedures were conducted by the same person. Apparent ileal digestibility Ileal digesta were placed in the freeze dryer (24D 48, Virtis, Gardiner, NY, USA) for 48 h for drying. The concentration of titanium dioxide in feed and digesta was measured following the method previously reported by Myers et al. [34]. The dry matter (DM) in feed and digesta was measured following Passos et al. [35]. The gross energy (GE) content in feed and digesta was measured using the bomb calorimeter (Parr 6200, Parr instrument company, Moline, IL, USA). The LECO CN-2000 Nitrogen Analyzer (LECO Corporation, St. Joseph, MI, USA) was used to measure the crude protein (CP) in feed and digesta. The apparent ileal digestibility (AID) of nutrients was calculated with the formula previously reported by Chen et al. [26]: AID={1-[(TiO2diet/TiO2digesta)×(Nutrientdigesta/Nutrientdiet)]}×100 In which TiO2diet and TiO2digesta represented the measured titanium dioxide concentration of diet and digesta, respectively; Nutrientdigesta and Nutrientfeed represented the measured nutrient concentration of the digesta and diet, respectively. Relative abundance and diversity of jejunal mucosa-associated microbiota The QIAamp Fast DNA Stool Mini Kit (#51604, Qiagen, Germantown, MD, USA) was used to extract DNA from jejunal mucosas as previously described by Duarte and Kim [36]. The extracted DNA was sent to Diversigen Inc. (New Brighton, MN, USA) for shotgun metagenomic sequencing following their internal protocol. For BoosterShot (Diversigen: Shallow Sequencing, 5 million reads/sample), libraries were sequenced on an Illumina NovaSeq using paired-end 2 × 150 reads. The adaptor sequences were cut using Cutadapt, and the DNA sequences were screened based on quality (Q-Score < 30) and length (< 50). Using Bowtie2, host sequences were eliminated. The taxa tables were generated using the default parameters of the Kraken2 aligner (v2.0.8-beta). The database used was the “Standard” RefSeq database. The alignments were made at 97% identity against the reference genomes. In order to process further statistical analysis, the count data were converted to relative abundance as previously reported by Deng et al. [12] and Moita et al. [37]. The top 20 OTU data within each level were listed. Statistical analysis Data were analyzed by the UNIVARIATE procedures (SAS Inst. Inc., Cary, NC, USA) to test for homogeneity, normality, and outliers. The MIXED procedure (SAS 9.4, SAS Inc.) was used to test the pre-planned contrasts. Dietary treatments were the main effect which was considered a fixed effect. Initial BW and sex were considered blocks and were added in the model as random effects. The experimental unit was the pig that was fed and housed individually. The pre-planned contrasts (CON vs. SPC, CON vs. ESB, CON vs. FSBL, and CON vs. FSBB) were conducted to determine the effects of each soy protein supplement replacing animal protein supplements. A P value less than 0.05 was considered statistically significant, and a P value between 0.05 and 0.10 was considered a tendency. Results Allergenic proteins in soy products The concentrations of glycinin and β-conglycinin were reduced in soy protein concentrate, enzyme-treated soybean meal, and fermented soybean meal with Bacillus than soybean meal. However, the concentrate of glycinin and β-conglycinin in fermented soybean meal with Lactobacillus was greater than soybean meal. Compared to fermented soybean meal with Bacillus, soy protein concentrate and enzyme-treated soybean meal have lower concentration of glycinin and β-conglycinin (Table 1). Growth performance and fecal score In this study, the SPC tended to decrease (P = 0.094) ADFI on overall. The ESB did not affect BW, ADG, ADFI, and G:F. The FSBL decreased (P < 0.05) BW on d 7 and 39 and tended to decrease (P = 0.050) BW on d 20. The FSBL decreased (P < 0.05) ADG in P1, and overall and tended to decrease (P = 0.078) ADG in P3. The FSBL decreased (P < 0.05) ADFI in P1, and P2 and tended to decrease (P = 0.095) ADFI on overall. The FSBB did not affect BW, ADG, ADFI, and G:F (Table 3). Table 3 Growth performance of nursery pigs fed diets with SPC, enzyme-treated soy protein, and fermented soybean meal replacing animal protein supplementsa Item Treatment SEM P value CON SPCb ESBb FSBLb FSBBb CON vs. SPC CON vs. ESB CON vs. FSBL CON vs. FSBB BW, kg  d 0 6.6 6.6 6.6 6.6 6.6 0.3 0.862 0.914 0.948 0.931  d 7 7.3 7.1 7.1 6.8 7.2 0.2 0.394 0.370 0.035 0.592  d 20 12.0 11.3 11.5 11.0 11.6 0.4 0.177 0.389 0.050 0.414  d 39 26.1 24.6 25.0 24.1 25.3 0.8 0.122 0.230 0.038 0.381 ADG, g  Phase 1 (d 0 to 7) 95 66 65 26 77 25 0.349 0.337 0.029 0.561  Phase 2 (d 7 to 20) 363 324 333 322 340 21 0.194 0.325 0.168 0.439  Phase 3 (d 20 to 39) 744 702 698 691 722 27 0.159 0.125 0.078 0.456  Overall (d 0 to 39) 500 462 458 449 479 18 0.116 0.208 0.036 0.375 ADFI, g  Phase 1 (d 0 to 7) 141 116 118 92 131 16 0.212 0.260 0.017 0.605  Phase 2 (d 7 to 20) 494 438 456 410 463 29 0.170 0.359 0.043 0.452  Phase 3 (d 20 to 39) 1036 938 958 966 987 58 0.131 0.237 0.279 0.451  Overall (d 0 to 39) 695 624 641 624 659 36 0.094 0.215 0.095 0.395 G:F  Phase 1 (d 0 to 7) 0.60 0.69 0.58 0.57 0.63 0.10 0.475 0.824 0.880 0.829  Phase 2 (d 7 to 20) 0.74 0.74 0.73 0.79 0.74 0.03 0.985 0.902 0.148 0.875  Phase 3 (d 20 to 39) 0.72 0.76 0.73 0.72 0.75 0.02 0.287 0.888 0.979 0.457  Overall (d 0 to 39) 0.72 0.75 0.73 0.73 0.74 0.02 0.447 0.967 0.973 0.618 an = 12 bSPC, ESB, FSBL, and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Jung-gu, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively There were no differences among the treatments on the fecal score during the entire experimental period (Table 4). Table 4 Fecal score of nursery pigs fed diets with soy protein concentrate, enzyme processed soy protein, and fermented soybean meal replacing animal protein supplementsa Item Treatment SEM P value CON SPCb ESBb FSBLb FSBBb CON vs. SPC CON vs. ESB CON vs. FSBL CON vs. FSBB Fecal score  Phase 1 (d 0 to 7) 3.6 3.4 3.5 3.4 3.6 0.2 0.320 0.589 0.292 0.756  Phase 2 (d 7 to 20) 3.4 3.4 3.5 3.3 3.4 0.1 0.889 0.525 0.452 0.783  Phase 3 (d 20 to 39) 3.0 3.0 3.0 3.0 3.0 < 0.1 0.225 0.738 0.693 0.186 an = 12 bSPC, ESB, FSBL, and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Jung-gu, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively Immune status and oxidative stress The SPC did not affect the immune and oxidative stress parameters. The FSBL increased (P < 0.05) TNF-α content and tended to increase IgG (P = 0.089) and MDA (P = 0.065) content in the jejunal mucosa (Table 5). The FSBB tended to increase (P = 0.073) TNF-α content in the jejunal mucosa. The ESB tended to decrease (P = 0.098) the protein carbonyl content in the jejunal mucosa of weaned pigs compared to the CON treatment. Table 5 Oxidative stress and immune status of nursery pigs fed diets with soy protein concentrate, enzyme-treated soy protein, and fermented soybean meal replacing animal protein supplementsa Item Treatment SEM P value CON SPCb ESBb FSBLb FSBBb CON vs. SPC CON vs. ESB CON vs. FSBL CON vs. FSBB Jejunal mucosa IL-8c, pg/mg of protein 525 479 531 518 467 44 0.462 0.932 0.907 0.355 TNF-αd, pg/mg of protein 0.39 0.60 0.63 0.95 0.72 0.13 0.259 0.203 0.003 0.073 IgAe, µg/mg of protein 2.95 2.97 4.21 3.60 3.41 0.82 0.980 0.142 0.436 0.585 IgGf, µg/mg of protein 0.64 0.78 0.75 0.84 0.75 0.09 0.255 0.351 0.089 0.360 Protein carbonyl, nmol/mg of protein 1.93 1.59 1.38 1.56 1.60 0.34 0.290 0.098 0.247 0.300 MDAg, nmol/mg of protein 0.30 0.37 0.37 0.40 0.34 0.04 0.174 0.210 0.065 0.480 an = 12 bSPC, ESB, FSBL and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Jung-gu, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively cIL-8 Interleukin 8 dTNF-α Tumor necrosis factor alpha eIgA Immunoglobulin A fIgG Immunoglobulin G gMDA Malondialdehyde Intestinal morphology and crypt cell proliferation There were no differences in villus height, villus width, crypt depth, and VH:CD ratio among the treatments. The SPC tended to increase (P = 0.091) the ratio of Ki-67 positive cells to total cells compared to CON (Table 6). Table 6 Intestinal morphology of nursery pigs fed diets with soy protein concentrate, enzyme-treated soy protein, and fermented soybean meal replacing animal protein supplementsa Item Treatment SEM P value CON SPCb ESBb FSBLb FSBBb CON vs. SPC CON vs. ESB CON vs. FSBL CON vs. FSBB Villus height, µm 510 472 484 527 485 30 0.366 0.537 0.693 0.545 Villus width, µm 118 114 116 115 115 3 0.398 0.650 0.495 0.471 Crypt depth, µm 285 272 277 287 275 9 0.326 0.562 0.858 0.455 VH:CDc 1.84 1.73 1.76 1.84 1.78 0.13 0.522 0.645 0.984 0.725 Ki-67 positive, % 26.7 29.0 28.3 27.8 26.1 1.7 0.091 0.239 0.383 0.676 an = 12 bSPC, ESB, FSBL, and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Jung-gu, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively cVH:CD Villus height to crypt depth ratio Apparent Ileal digestibility The SPC, ESB, and FSBB did not affect the AID of DM, GE, and CP compared to CON. The FSBL increased (P < 0.05) AID of CP (Table 7). Table 7 Apparent ileal digestibility of nutrients of nursery pigs fed diets with soy protein concentrate, enzyme-treated soy protein, and fermented soybean meal replacing animal protein supplementsa Item Treatment SEM P value CON SPCb ESBb FSBLb FSBBb CON vs. SPC CON vs. ESB CON vs. FSBL CON vs. FSBB DMc, % 50.7 46.4 54.5 56.4 50.0 3.1 0.367 0.381 0.188 0.884 GEd, % 51.0 47.0 55.6 57.6 51.2 3.3 0.408 0.318 0.144 0.960 CPe, % 57.6 55.2 61.7 68.5 57.6 3.2 0.600 0.369 0.016 0.995 an = 12 bSPC, ESB, FSBL, and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Jung-gu, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively cDM Dry matter dGE Gross energy eCP Crude protein Relative abundance and diversity of jejunal mucosa-associated microbiota At the phylum level, the replacement of soy products did not affect the relative abundance of jejunal mucosa-associated microbiota (Table 8). Table 8 Relative abundance of jejunal mucosa-associated microbiota at the phylum level in nursery pigs fed diets with soy protein concentrate, enzyme-treated soy protein, and fermented soybean meal replacing animal protein supplementsa Item Treatment SEM P value CON SPCb ESBb FSBLb FSBBb CON vs. SPC CON vs. ESB CON vs. FSBL CON vs. FSBB Proteobacteria 44.52 42.89 43.99 42.05 41.40 2.55 0.435 0.802 0.240 0.140 Firmicutes 30.37 32.39 29.51 32.04 34.12 3.89 0.378 0.706 0.467 0.106 Actinobacteria 14.41 14.74 15.71 15.28 13.93 2.01 0.863 0.502 0.652 0.803 Bacteroidetes 3.89 3.10 3.25 3.57 3.53 0.39 0.398 0.650 0.495 0.471 Others 6.81 6.88 7.53 7.07 7.02 0.49 0.919 0.275 0.691 0.749 an = 12 bSPC, ESB, FSBL, and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Jung-gu, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively At the genus level, SPC tended to increase (P = 0.065) the relative abundance of Staphylococcus. The FSBL tended to increase (P = 0.078) the relative abundance of Klebsiella. The FSBB increased (P < 0.05) the relative abundance of Clostridium and decreased (P < 0.05) the relative abundance of Achromobacter. The FSBB tended to increase (P = 0.074) the relative abundance of Pseudomonas and tended to decrease (P = 0.078) the relative abundance of Nocardia (Table 9). Table 9 Relative abundance of jejunal mucosa-associated microbiota at the genus level in nursery pigs fed diets with soy protein concentrate, enzyme-treated soy protein, and fermented soybean meal replacing animal protein supplementsa Item Treatment SEM P value CON SPCb ESBb FSBLb FSBBb CON vs. SPC CON vs. ESB CON vs. FSBL CON vs. FSBB Clostridium 13.44 12.74 13.06 14.13 16.95 2.92 0.684 0.827 0.688 0.048 Staphylococcus 10.47 12.32 10.87 10.67 9.74 0.69 0.065 0.688 0.841 0.453 Sphingomonas 8.04 6.12 7.49 6.17 6.10 1.26 0.137 0.667 0.147 0.133 Streptomyces 6.53 8.08 8.40 7.56 7.75 1.41 0.371 0.282 0.550 0.479 Agrobacterium 4.84 5.19 3.69 2.93 5.01 1.32 0.843 0.520 0.287 0.923 Schlegelella 3.61 3.16 3.91 3.46 2.82 0.56 0.365 0.545 0.765 0.114 Pseudomonas 2.73 2.96 3.23 2.86 3.28 0.30 0.435 0.104 0.657 0.074 Pseudoalteromonas 2.72 2.84 2.69 3.07 3.01 0.22 0.695 0.930 0.274 0.364 Achromobacter 2.02 1.69 2.01 1.83 1.31 0.23 0.306 0.955 0.542 0.032 Klebsiella 1.95 2.02 1.76 2.77 1.67 0.37 0.862 0.679 0.078 0.550 Salmonella 1.88 1.81 1.96 1.88 1.08 0.21 0.816 0.774 0.995 0.802 Deinococcus 1.83 1.88 2.01 1.92 1.73 0.16 0.802 0.392 0.668 0.623 Xanthomonas 1.74 2.26 2.01 1.83 1.56 0.28 0.168 0.468 0.820 0.622 Bacillus 1.59 1.63 1.35 1.69 1.72 0.34 0.914 0.447 0.764 0.683 Micromonospora 1.26 0.84 1.33 1.04 0.92 0.31 0.125 0.787 0.416 0.205 Nocardia 1.23 1.34 0.90 1.15 0.67 0.22 0.725 0.298 0.788 0.078 Leptospira 1.22 0.81 1.27 0.96 0.58 0.43 0.346 0.911 0.534 0.144 Mycolicibacterium 1.09 0.74 1.05 0.96 1.08 0.16 0.101 0.843 0.522 0.975 Mycobacterium 0.98 0.88 0.93 1.10 0.84 0.15 0.599 0.790 0.519 0.457 Lelliottia 0.93 0.75 0.84 0.91 0.78 0.13 0.296 0.590 0.897 0.384 Others 29.91 29.93 29.25 31.14 30.69 1.11 0.925 0.727 0.579 0.599 an = 12 bSPC, ESB, FSBL, and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Jung-gu, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively At the species level, SPC tended to increase (P = 0.069) the relative abundance of Staphylococcus aureus. The FSBL tended to decrease (P = 0.096) the relative abundance of Sphingomonas sp. Cra20. The FSBB increased (P < 0.05) the relative abundance of Clostridium botulinum, and tended to decrease (P = 0.063) the relative abundance of Achromobacter spanius (Table 10). Table 10 Relative abundance of jejunal mucosa-associated microbiota at the species level in nursery pigs fed diets with soy protein concentrate, enzyme-treated soy protein, and fermented soybean meal replacing animal protein supplementsa Item Treatment SEM P value CON SPCb ESBb FSBLb FSBBb CON vs. SPC CON vs. ESB CON vs. FSBL CON vs. FSBB Clostridium botulinum 11.08 10.42 10.83 11.64 14.21 2.48 0.664 0.868 0.707 0.042 Staphylococcus aureus 10.34 12.18 10.75 10.53 9.52 0.70 0.069 0.675 0.843 0.410 Sphingomonas sp. AAP5 6.39 4.84 6.11 5.16 4.86 1.08 0.183 0.808 0.290 0.190 Agrobacterium fabrum 4.84 5.19 3.69 2.93 5.01 1.32 0.843 0.520 0.287 0.923 Schlegelella thermodepolymerans 3.61 3.16 3.91 3.46 2.82 0.56 0.365 0.545 0.765 0.114 Streptomyces platensis 2.04 2.14 2.49 1.90 1.73 0.49 0.792 0.253 0.731 0.440 Salmonella enterica 1.88 1.81 1.96 1.88 1.80 0.21 0.816 0.774 0.995 0.802 Deinococcus deserti 1.83 1.88 2.01 1.92 1.73 0.16 0.802 0.392 0.668 0.623 Achromobacter spanius 1.80 1.43 1.79 1.55 1.20 0.24 0.247 0.974 0.427 0.063 Streptomyces mobaraensis 1.74 1.80 2.57 1.88 1.80 0.54 0.909 0.137 0.788 0.910 Sphingomonas sp. Cra20 1.65 1.28 1.38 1.01 1.24 0.27 0.333 0.478 0.096 0.280 Pseudoalteromonas atlantica 1.55 1.37 1.35 1.91 1.78 0.17 0.474 0.427 0.151 0.354 Micromonospora auratinigra 1.26 0.84 1.33 1.04 0.92 0.31 0.125 0.787 0.416 0.205 Leptospira kmetyi 1.22 0.81 1.27 0.96 0.58 0.43 0.346 0.911 0.534 0.144 Pseudoalteromonas prydzensis 1.17 1.47 1.34 1.16 1.23 0.19 0.261 0.527 0.975 0.819 Pseudomonas stutzeri 1.04 0.86 0.92 0.86 1.07 0.11 0.238 0.432 0.256 0.860 Klebsiella pneumoniae 1.01 1.03 0.72 1.70 0.58 0.33 0.967 0.518 0.138 0.342 Lelliottia amnigena 0.93 0.75 0.84 0.91 0.78 0.13 0.296 0.590 0.897 0.384 Methylocaldum marinum 0.90 1.06 0.79 1.25 0.84 0.30 0.707 0.800 0.423 0.889 Klebsiella variicola 0.78 0.81 0.87 0.90 0.87 0.14 0.818 0.516 0.379 0.482 Others 42.96 44.86 43.08 45.46 45.43 1.26 0.554 0.862 0.171 0.600 an = 12 bSPC, ESB, FSBL, and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Jung-gu, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively At the alpha diversity, FSBB decreased (P < 0.05) Simpson index at the genus and species level (Table 11). There were no differences in SPC, ESB and FSBL compared to CON treatment on the alpha diversity of the mucosa-associated microbiota in jejunum. Table 11 Alpha diversity of jejunal mucosa-associated microbiota estimated with Chao1 richness, Shannon diversity, and Simpson diversity in nursery pigs fed diets with soy protein concentrate, enzyme-treated soy protein, and fermented soybean meal replacing animal protein supplementsa Item Treatment SEM P value CON SPCb ESBb FSBLb FSBBb CON vs. SPC CON vs. ESB CON vs. FSBL CON vs. FSBB Genus  Chao1 111.63 109.66 107.85 110.26 109.25 2.11 0.471 0.171 0.614 0.385  Shannon 3.64 3.61 3.60 3.69 3.58 0.03 0.480 0.320 0.288 0.113  Simpson 0.95 0.94 0.94 0.95 0.93 <0.01 0.566 0.571 0.727 0.005 Species  Chao1 165.96 164.01 164.33 164.17 167.29 2.19 0.428 0.507 0.466 0.589  Shannon 4.10 4.09 4.08 4.17 4.06 0.03 0.971 0.756 0.155 0.399  Simpson 0.96 0.96 0.96 0.96 0.95 <0.01 0.697 0.938 0.533 0.026 an = 12 bSPC, ESB, FSBL, and FSBB: basal diet with soy protein concentrate (X-Soy 200, CJ Selecta, Araguari, MG, Brazil), enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH, USA), fermented soybean meal with Lactobacillus (FerMex 200, Purina Animal Nutrition, Shoreview, SD, USA), and fermented soybean meal with Bacillus (Soytide, CJ Bio, Jung-gu, Seoul, Korea), replacing 1/3, 2/3, 3/3 of animal protein supplements in P1, P2, P3, respectively Discussion In this study, 1/3, 2/3, and 3/3 animal protein supplements in diets for nursery pigs were replaced by different soy protein supplements in phase 1, 2, and 3, separately. Soy protein concentrate, enzyme-treated soybean meal, and fermented soybean meal with Bacillus showed the potential to partly replace animal protein supplements without affecting growth performance, whereas fermented soybean meal with Lactobacillus replacing animal protein supplements in diets for nursery pigs impaired growth performance and intestinal health. The negative effects may be associated with the high level of allergenic proteins in fermented soybean meal with Lactobacillus. Antinutritional factors in soybean, such as trypsin inhibitors, lectins, and soy allergenic proteins, limit its use in nursery diets due to the adverse effects on growth performance of nursery pigs [38, 39]. In particular, glycinin and β-conglycinin are two soy antigens predominantly found in soybean meal resistant to heat processing, which can result in hypersensitive reaction and then impair growth performance of nursery pigs [40, 41]. Several antigenic epitopes were identified in glycinin and β-conglycinin [42–44]. In this study, ethanol extraction and enzymatic hydrolysis could efficiently reduce the concentration of allergenic proteins in soybean meal, which was in accordance with the previous studies [11, 12]. However, the fermentation of soybean meal remained a relatively higher concentration of allergenic proteins compared to ethanol extraction and enzymatic hydrolysis. In general, the quality of fermentation related to reducing the allergenicity in soybean meal can be mainly attributed to the fermentation process, which includes temperature, duration, pH, and microorganisms used in the process [8]. Bacillus subtilis and Lactobacillus plantarum, two commonly used bacterial strains for fermentation of soybean meal, were used in this study [45, 46]. Due to the different protease profiles and secretion abilities from these microorganisms, the fermented soybean meal may have varied contents of allergenic proteins, which could explain the variation between two fermented soybean meals used in this study [8]. Consequently, the relatively high allergenic proteins in fermented soybean meal can potentially impair growth performance of nursery pigs [47]. In this study, SPC partly replacing animal protein supplements in nursery diets did not affect the growth performance of pigs, which was in accordance with a previous study [12]. This could be associated with low allergenic proteins (less than 0.1 mg/g glycinin and 0.1 mg/g β-conglycinin) and balanced amino acid profile in soy protein concentrate. Even though the fermented soybean meal was indicated to a high digestible protein source, the growth performance of pigs in FSBL was impaired during the entire experiment [48]. Firstly, it can be explained by increased soy allergenic proteins in the diets. According to Friesen et al. [49], the inclusion of soybean meal containing high allergenic proteins in the diets of early-weaned pigs would decrease the growth performance. Secondly, it can be explained by the reduction of animal protein supplements in diets. Blood plasma has been indicated positive effects to stimulate the feed intake of nursery pigs [50]. Also, previous studies have shown that fish meal and poultry meal have the potential to stimulate feed intake of pigs [5, 51]. With the replacement of appetitive animal protein supplements by fermented soybean meal with Lactobacillus, the feed intake of nursery pigs can be negatively affected. In current study, no difference in fecal score were observed. Although, the allergenic proteins in soybean meal may result in diarrhea through interfering intestinal immune responses [52], the calculated concentration of allergenic proteins in soy protein concentrate and enzyme-treated soybean meal were similar to the control diet. Interestingly, fermented soybean meal with lactobacillus containing high allergenic proteins did not affect fecal score in this study. This result might be explained by the presence of Lactobacillus. Previous study has been indicated that Lactobacillus could improve function of the intestinal barrier then effectively prevent post-weaning diarrhea [53]. Even though the relatively higher allergenic proteins in FSBL, Lactobacillus may play some roles in preventing diarrhea. Intestinal immunity is one of the most essential factors in pigs that is highly responsible for growth performance [54]. In this study, pigs in FSBL increased the content of MDA, IgG, and TNF-α in jejunal mucosa compared to CON treatment. The MDA, one of the major products of lipid peroxidation, can be a direct indicator of lipid oxidative damage in the body [55]. Ma et al. [56] indicated that the weaned pigs fed diets containing low soy allergenic proteins decreased MDA concentration in serum, which could be contributed to the increased glutathione peroxidase (GSH-Px) activity. The IgG and TNF-α are also important biomarkers for pathogen infections and health status of the host [57, 58]. Pigs exposed to soy antigens increased the level of IgG due to the hypersensitivity reactions in pigs, which was highly associated with diarrhea [59–61]. The increased TNF-α in FSBL can also be explained by the high level of soy antigens. Peng et al. [62] suggested that soy antigens could increase the level of TNF-α through their impact on the expression of nuclear factor-kappa B (NF-κB), p38, and Jun N-terminal kinase. The FSBB also increased the level of TNF-α, which might be associated with relatively high level of antigens. The increased oxidative stress and immune response in the GI tract of nursery pigs indicated that soy antigens in fermented soybean meal severely impaired the intestinal integrity, then possibly resulted in the impaired growth performance [63]. Intestinal morphology reflects the nutrient digestion and absorption capacity of pigs [64]. Antinutritional factors in soybean meal, such as trypsin inhibitor (TI), have been shown to negatively affect the intestinal morphology of newly weaned pigs due to the interference with trypsin and chymotrypsin [9, 65]. In this study, no differences in intestinal morphology were observed among treatments, which indicated the different processes of soybean meal could efficiently remove the antinutritional factor in soybean meal. Feng et al. [66] showed that the fermentation of soybean meal with Bacillus subtilis could completely remove trypsin inhibitor in soybean meal. Enzyme treatment and ethanol extraction were indicated to partly remove TI in soybean meal [11, 67]. These findings are in accordance with the results of the current study. Nutrient digestibility is highly related to intestinal morphology. Due to the unchanged intestinal morphology among treatments, the nutrient digestibility was not affected in SPC, ESB, and FSBB. However, FSBL increased the crude protein digestibility, which can be attributed to the supplemental effect of Lactobacillus. Lactic acid and proteolytic enzymes produced by Lactobacillus can improve the nutrition digestibility in the GI tract [68]. Previous studies showed that the inclusion of Lactobacillus or Lactobacillus metabolites in nursery diets could enhance the crude protein digestibility of pigs, which was in accordance with the current study [69, 70]. It has been demonstrated that changing the protein source in the diets can modulate the microbiota composition of pigs because it changes the physicochemical conditions and the substrate availability in the intestine [12, 71]. In this study, FSBB increased the relative abundance of Clostridium and decreased the relative abundance of Achromobacter. Fermented soybean meal containing Bacillus and its metabolites may change the environment in the intestine, which could have promoted the growth of Firmicute bacteria including Clostridium, and correspondingly have reduced the growth of Proteobacteria including Achromobacter. Fermented soybean meal containing Lactobacillus did not affect the abundance of genera belonging to the Firmicutes, however, it increased the relative abundance of Klebsiella, a Proteobacteria that is considered a harmful bacteria causing different infections in animals and humans [72]. The increased relative abundance of Klebsiella can be associated with the increased intestinal immune response and oxidative stress of pigs in this study. In addition, FSBB decreased the alpha diversity of jejunal mucosa-associated microbiota in this study. The previous study showed that diet complexity could positively correlate with the microbiota diversity of pigs [73]. When all animal protein supplements were replaced by fermented soybean meal with Bacillus in P3, the diet complexity decreased, and then decreased the microbiota diversity. This result corresponded with the study previously reported by Deng et al. [12]. Conclusion Soy protein concentrate, enzyme-treated soybean meal, and fermented soybean meal with Bacillus could reduce the use of animal protein supplements up to 33% until 7 kg body weight, up to 67% from 7 to 11 kg body weight, and entirely from 11 kg body weight without affecting the intestinal health and the growth performance of nursery pigs. However, fermented soybean meal with Lactobacillus partly replacing animal protein supplements reduced the growth performance of nursery pigs, which was contributed by increasing the immune reaction and oxidative stress in the intestine. Abbreviations AA Amino acid ADFI Average daily feed intake ADG Average daily gain AID Apparent ileal digestibility BW Body weight CP Crude protein DM Dry matter DNA Deoxyribonucleic acid ELISA Enzyme-linked immunosorbent assay GE Gross energy GF Gain to feed ratio MDA Malondialdehyde IgA Immunoglobulin A IgG Immunoglobulin G IL-8 Interleukin-8 OUTs Operational taxonomic units PBS Phosphate-buffered saline SID Standard ileal digestibility TI Trypsin inhibitor TiO2 Titanium dioxide TNF-α Tumor necrosis factor-alph VH:CD Villus height to crypt depth ratio Acknowledgements The authors acknowledge Dr. Ki Beom Jang, Dr. Yi-Chi Cheng, Dr. Vitor H. C. Moita, Mr. Jonathan Baker, and Dr. Seokhwan Yoon for their technical supports. Authors’ contributions Conceptualization and design, SWK; methodology, SWK, ZD, and MED; formal analysis, ZD; investigation, SWK, ZD, and MED; data interpretation, SWK, ZD, and MED; writing-original draft preparation, SWK, ZD, and MED; writing-review and editing, SWK, ZD, and MED; supervision, SWK; funding acquisition, SWK. All authors have read and agreed to the published version of the manuscript. Funding This study was awarded and funded by USDA-NIFA Hatch Fund (#02636, Washington DC, USA), North Carolina Agricultural Foundation (#660101, Raleigh, NC, USA), and CJ CheilJedang (Seoul, Korea). Availability of data and materials All data generated or analyzed during this study are available from the corresponding author upon reasonable request. Declarations Ethics approval and consent to participate The Institutional Animal Care and Use Committee of North Carolina State University (Raleigh, NC, USA) revised and approved the protocol used for this study. Consent for publication Not applicable. Competing interests S. Y. Kim and Y. Hwang are employed by CJ Bio, Seoul, Korea. All other authors have no conflict of interest. ==== Refs References 1. Lallès J-P Boudry G Favier C Le Floc’h N Luron I Montagne L Gut function and dysfunction in young pigs: physiology Anim Res 2004 53 4 301 16 10.1051/animres:2004018 2. Kim SW Duarte ME Understanding intestinal health in nursery pigs and the relevant nutritional strategies Anim Biosci 2021 34 3 338 44 10.5713/ab.21.0010 33705620 3. Campbell JM, Crenshaw JD, Polo J. The biological stress of early weaned piglets. J Anim Sci Biotechnol. 2013;4:19. 10.1186/2049-1891-4-19. 4. Pierce JL Cromwell GL Lindemann MD Russell LE Weaver EM Effects of spray-dried animal plasma and immunoglobulins on performance of early weaned pigs J Anim Sci 2005 83 12 2876 85 10.2527/2005.83122876x 16282627 5. Kim SW Easter RA Nutritional value of fish meals in the diet for young pigs J Anim Sci 2001 79 7 1829 10.2527/2001.7971829x 11465370 6. Kim SW Less JF Wang L Yan T Kiron V Kaushik SJ Meeting global feed protein demand: challenge, opportunity, and strategy Annu Rev Anim Biosci 2019 7 1 221 43 10.1146/annurev-animal-030117-014838 30418803 7. Goldsmith PD. 5 - Economics of soybean production, marketing, and utilization. In: Johnson LA, White PJ, Richard Galloway R; editors. Soybeans. AOCS Press; 2008. p. 117–50. 10.1016/B978-1-893997-64-6.50008-1. 8. Mukherjee R Chakraborty R Dutta A Role of fermentation in improving nutritional quality of soybean meal — a review Asian-Australasian J Anim Sci 2015 29 11 1523 9 10.5713/ajas.15.0627 9. Dunsford BR Knabe DA Haensly WE Effect of dietary soybean meal on the microscopic anatomy of the small intestine in the early-weaned pig J Anim Sci 1989 67 7 1855 63 10.2527/jas1989.6771855x 2768128 10. Li DF, Nelssen JL, Reddy PG, Blecha F, Hancock JD, Allee GL, et al. Transient hypersensitivity to soybean meal in the early-weaned pig. J Anim Sci. 1990;68(6):1790–9. 10.2527/1990.6861790x. 11. Peisker M Manufacturing of soy protein concentrate for animal nutrition Feed Manuf Mediterr Reg Improv Saf From Feed to food 2001 54 i 103 7 12. Deng Z Duarte ME Jang KB Kim SW Soy protein concentrate replacing animal protein supplements and its impacts on intestinal immune status, intestinal oxidative stress status, nutrient digestibility, mucosa-associated microbiota, and growth performance of nursery pigs J Anim Sci 2022 10.1093/jas/skac255 35950990 13. Jiang HQ Gong LM Ma YX He YH Li DF Zhai HX Effect of stachyose supplementation on growth performance, nutrient digestibility and caecal fermentation characteristics in broilers Br Poult Sci 2006 47 4 516 22 10.1080/00071660600827708 16905479 14. Hong K-J Lee C-H Kim SW Aspergillus oryzae GB-107 fermentation improves nutritional quality of food soybeans and feed soybean meals J Med Food 2004 7 4 430 5 10.1089/jmf.2004.7.430 15671685 15. Kim SW van Heugten E Ji F Lee CH Mateo RD Fermented soybean meal as a vegetable protein source for nursery pigs: I. Effects on growth performance of nursery pigs J Anim Sci 2010 88 1 214 24 10.2527/jas.2009-1993 19783703 16. Frias J Song YS Martínez-Villaluenga C De Mejia EG Vidal-Valverde C Immunoreactivity and amino acid content of fermented soybean products J Agric Food Chem 2008 56 1 99 105 10.1021/jf072177j 18072744 17. Roh SG Carroll JA Kim SW Effects of fermented soybean meal on innate immunity-related gene expressions in nursery pigs acutely challenged with lipopolysaccharides Anim Sci J 2015 86 5 508 16 10.1111/asj.12319 25440755 18. Lenehan NA DeRouchey JM Goodband RD Tokach MD Dritz SS Nelssen JL Evaluation of soy protein concentrates in nursery pig diets J Anim Sci 2007 85 11 3013 21 10.2527/jas.2007-0071 17686901 19. Ma X Shang Q Hu J Liu H Brøkner C Piao X Effects of replacing soybean meal, soy protein concentrate, fermented soybean meal or fish meal with enzyme-treated soybean meal on growth performance, nutrient digestibility, antioxidant capacity, immunity and intestinal morphology in weaned pigs Livest Sci 2019 225 39 46 10.1016/j.livsci.2019.04.016 20. Feng J Liu X Xu ZR Lu YP Liu YY The effect of aspergillus oryzae fermented soybean meal on growth performance, digestibility of dietary components and activities of intestinal enzymes in weaned piglets Anim Feed Sci Technol 2007 134 3–4 295 303 10.1016/j.anifeedsci.2006.10.004 21. Sinn SM Gibbons WR Brown ML DeRouchey JM Levesque CL Evaluation of microbially enhanced soybean meal as an alternative to fishmeal in weaned pig diets Animal 2017 11 5 784 93 10.1017/S1751731116002020 27751197 22. Li R Hou GF Song ZH Zhao JF Fan ZY Hou D-X Nutritional value of enzyme-treated soybean meal, concentrated degossypolized cottonseed protein, dried porcine solubles and fish meal for 10- to -20 kg pigs Anim Feed Sci Technol 2019 252 23 33 10.1016/j.anifeedsci.2019.04.002 23. NRC. Nutrient requirements of swine. 11th rev. ed. Washington (DC): National Academies Press; 2012. 24. Rojas OJ Stein HH Concentration of digestible and metabolizable energy and digestibility of amino acids in chicken meal, poultry byproduct meal, hydrolyzed porcine intestines, a spent hen–soybean meal mixture, and conventional soybean meal fed to weanling pigs1 J Anim Sci 2013 91 7 3220 30 10.2527/jas.2013-6269 23798516 25. Cheng Y-C Duarte ME Kim SW Effects of Yarrowia lipolytica supplementation on growth performance, intestinal health and apparent ileal digestibility of diets fed to nursery pigs Anim Biosci 2022 35 4 605 13 10.5713/ab.21.0369 34727641 26. Chen H, Zhang S, Kim SW. Effects of supplemental xylanase on health of the small intestine in nursery pigs fed diets with corn distillers’ dried grains with solubles. J Anim Sci. 2020;98(6):skaa185. 10.1093/jas/skaa185. 27. Holanda DM Kim SW Investigation of the efficacy of mycotoxin-detoxifying additive on health and growth of newly-weaned pigs under deoxynivalenol challenges Anim Biosci 2021 34 3 405 16 10.5713/ajas.20.0567 33152208 28. Holanda DM Yiannikouris A Kim SW Investigation of the efficacy of a postbiotic yeast cell wall-based blend on newly-weaned pigs under a dietary challenge of multiple mycotoxins with emphasis on deoxynivalenol Toxins (Basel) 2020 12 8 504 10.3390/toxins12080504 32781569 29. Sun Y Duarte ME Kim SW Dietary inclusion of multispecies probiotics to reduce the severity of post-weaning diarrhea caused by Escherichia coli F18 + in pigs Anim Nutr 2021 7 2 326 33 10.1016/j.aninu.2020.08.012 34258420 30. Moita VHC Duarte ME da Silva SN Kim SW Supplemental effects of functional oils on the modulation of mucosa-associated microbiota, intestinal health, and growth performance of nursery pigs Animals 2021 11 6 1591 10.3390/ani11061591 34071448 31. Duarte ME Kim SW Phytobiotics from oregano extracts enhance the intestinal health and growth performance of pigs Antioxidants 2022 11 10 2066 10.3390/antiox11102066 36290789 32. Jang KB, Purvis JM, Kim SW. Dose–response and functional role of whey permeate as a source of lactose and milk oligosaccharides on intestinal health and growth of nursery pigs. J Anim Sci. 2021;99(1):skab008. 10.1093/jas/skab008. 33. Xu X Duarte ME Kim SW Postbiotic effects of Lactobacillus fermentate on intestinal health, mucosa-associated microbiota, and growth efficiency of nursery pigs challenged with F18 + Escherichia coli J Anim Sci 2022 100 8 kac210 10.1093/jas/skac210 34. Myers WD Ludden PA Nayigihugu V Hess BW Technical note: a procedure for the preparation and quantitative analysis of samples for titanium dioxide J Anim Sci 2004 82 1 179 83 10.2527/2004.821179x 14753360 35. Passos AA Park I Ferket P von Heimendahl E Kim SW Effect of dietary supplementation of xylanase on apparent ileal digestibility of nutrients, viscosity of digesta, and intestinal morphology of growing pigs fed corn and soybean meal based diet Anim Nutr Elsevier Ltd 2015 1 1 19 23 10.1016/j.aninu.2015.02.006 36. Duarte ME Kim SW Significance of mucosa-associated microbiota and its impacts on intestinal health of pigs challenged with F18 + E. coli Pathogens 2022 11 5 589 10.3390/pathogens11050589 35631110 37. Moita VHC, Duarte ME, Kim SW. Functional roles of xylanase enhancing intestinal health and growth performance of nursery pigs by reducing the digesta viscosity and modulating the mucosa-associated microbiota in the jejunum. J Anim Sci. 2022;100(5):skac116. 10.1093/jas/skac116. 38. Qin G ter Elst ER Bosch MW van der Poel AFB Thermal processing of whole soya beans: studies on the inactivation of antinutritional factors and effects on ileal digestibility in piglets Anim Feed Sci Technol 1996 57 4 313 24 10.1016/0377-8401(95)00863-2 39. Li DF Nelssen JL Reddy PG Blecha F Klemm R Goodband RD Interrelationship between hypersensitivity to soybean proteins and growth performance in early-weaned pigs J Anim Sci 1991 69 10 4062 9 10.2527/1991.69104062x 1778820 40. Sun P Li D Dong B Qiao S Ma X Effects of soybean glycinin on performance and immune function in early weaned pigs Arch Anim Nutr 2008 62 4 313 21 10.1080/17450390802066419 18763625 41. Hao Y Zhan Z Guo P Piao X Li D Soybean β-conglycinin-induced gut hypersensitivity reaction in a piglet model Arch Anim Nutr 2009 63 3 188 202 10.1080/17450390902860026 42. Taliercio E Kim SW Epitopes from two soybean glycinin subunits are antigenic in pigs J Sci Food Agric 2013 93 12 2927 32 10.1002/jsfa.6113 23426933 43. Taliercio E Kim SW Identification of a second major antigenic epitope in the α-subunit of soy β-conglycinin Food Agric Immunol 2014 25 3 311 21 10.1080/09540105.2013.791969 44. Fu C Jez JM Kerley MS Allee GL Krishnan HB Identification, characterization, epitope mapping, and three-dimensional modeling of the α-subunit of β-conglycinin of soybean, a potential allergen for young pigs J Agric Food Chem 2007 55 10 4014 20 10.1021/jf070211o 17439152 45. Han B-Z Rombouts FM Nout MJR A chinese fermented soybean food Int J Food Microbiol 2001 65 1–2 1 10 10.1016/S0168-1605(00)00523-7 11322691 46. Amadou I Le G-W Shi Y-H Jin S Reducing, radical scavenging, and chelation properties of fermented soy protein meal hydrolysate by lactobacillus plantarum LP6 Int J Food Prop 2011 14 3 654 65 10.1080/10942910903312502 47. Li DF Nelssen JL Reddy PG Blecha F Klemm RD Giesting DW Measuring suitability of soybean products for early-weaned pigs with immunological criteria J Anim Sci 1991 69 8 3299 10.2527/1991.6983299x 1894566 48. Jang KB Kim SW Evaluation of standardized ileal digestibility of amino acids in fermented soybean meal for nursery pigs using direct and difference procedures Anim Biosci 2023 36 2 275 83 10.5713/ab.22.0269 36108691 49. Friesen KG Goodband RD Nelssen JL Blecha F Reddy DN Reddy PG The effect of pre- and postweaning exposure to soybean meal on growth performance and on the immune response in the early-weaned pig2 J Anim Sci 1993 71 8 2089 98 10.2527/1993.7182089x 8376233 50. Van Dijk AJ Everts H Nabuurs MJA Margry RJCF Beynen AC Growth performance of weanling pigs fed spray-dried animal plasma: a review Livest Prod Sci 2001 68 2–3 263 74 10.1016/S0301-6226(00)00229-3 51. Keegan TP DeRouchey JM Nelssen JL Tokach MD Goodband RD Dritz SS The effects of poultry meal source and ash level on nursery pig performance J Anim Sci 2004 82 9 2750 6 10.2527/2004.8292750x 15446491 52. Sun P Li D Li Z Dong B Wang F Effects of glycinin on IgE-mediated increase of mast cell numbers and histamine release in the small intestine J Nutr Biochem 2008 19 9 627 33 10.1016/j.jnutbio.2007.08.007 18280135 53. Yang KM Jiang ZY Zheng CT Wang L Yang XF Effect of Lactobacillus plantarum on diarrhea and intestinal barrier function of young piglets challenged with enterotoxigenic Escherichia coli K881 J Anim Sci 2014 92 4 1496 503 10.2527/jas.2013-6619 24492550 54. Lee IK Kye YC Kim G Kim HW Gu MJ Umboh J Stress, nutrition, and intestinal immune responses in pigs — a review Asian-Australasian J Anim Sci 2016 29 8 1075 82 10.5713/ajas.16.0118 55. Pirinccioglu AG Gökalp D Pirinccioglu M Kizil G Kizil M Malondialdehyde (MDA) and protein carbonyl (PCO) levels as biomarkers of oxidative stress in subjects with familial hypercholesterolemia Clin Biochem 2010 43 15 1220 4 10.1016/j.clinbiochem.2010.07.022 20691171 56. Ma XK Shang QH Wang QQ Hu JX Piao XS Comparative effects of enzymolytic soybean meal and antibiotics in diets on growth performance, antioxidant capacity, immunity, and intestinal barrier function in weaned pigs Anim Feed Sci Technol 2019 248 47 58 10.1016/j.anifeedsci.2018.12.003 57. Zhu Y Österlundh I Hultén F Magnusson U Tumor necrosis factor-α, interleukin-6, serum amyloid A, haptoglobin, and cortisol concentrations in sows following intramammary inoculation of Escherichia coli Am J Vet Res 2004 65 10 1434 9 10.2460/ajvr.2004.65.1434 15524332 58. Chen Y Tibbs-Cortes LE Ashley C Putz AM Lim K-S Dyck MK The genetic basis of natural antibody titers of young healthy pigs and relationships with disease resilience BMC Genomics 2020 21 1 648 10.1186/s12864-020-06994-0 32962629 59. Huang Q Xu H Yu Z Gao P Liu S Inbred chinese Wuzhishan (WZS) minipig model for soybean glycinin and β-conglycinin allergy J Agric Food Chem 2010 58 8 5194 8 10.1021/jf904536v 20369852 60. Wu JJ Cao CM Meng TT Zhang Y Xu SL Feng S Bin Induction of immune responses and allergic reactions in piglets by injecting glycinin Ital J Anim Sci 2016 15 1 166 73 10.1080/1828051X.2016.1144488 61. Dréau D Lallès JP Philouze-Romé V Toullec R Salmon H Local and systemic immune responses to soybean protein ingestion in early-weaned pigs J Anim Sci 1994 72 8 2090 8 10.2527/1994.7282090x 7982839 62. Peng C Cao C He M Shu Y Tang X Wang Y Soybean glycinin- and β-conglycinin-induced intestinal damage in piglets via the p38/JNK/NF-κB signaling pathway J Agric Food Chem 2018 66 36 9534 41 10.1021/acs.jafc.8b03641 30139257 63. Vancamelbeke M Vermeire S The intestinal barrier: a fundamental role in health and disease Expert Rev Gastroenterol Hepatol 2017 11 9 821 34 10.1080/17474124.2017.1343143 28650209 64. Xiong X Yang HS Wang XC Hu Q Liu CX Wu X Effect of low dosage of chito-oligosaccharide supplementation on intestinal morphology, immune response, antioxidant capacity, and barrier function in weaned piglets J Anim Sci 2015 93 3 1089 10.2527/jas.2014-7851 26020885 65. Liener IE Implications of antinutritional components in soybean foods Crit Rev Food Sci Nutr 1994 34 1 31 67 10.1080/10408399409527649 8142044 66. Feng J Liu X Xu ZR Lu YP Liu YY Effect of fermented soybean meal on intestinal morphology and digestive enzyme activities in weaned piglets Dig Dis Sci 2007 52 8 1845 10.1007/s10620-006-9705-0 17410452 67. Cervantes-Pahm SK Stein HH Ileal digestibility of amino acids in conventional, fermented, and enzyme-treated soybean meal and in soy protein isolate, fish meal, and casein fed to weanling pigs J Anim Sci 2010 88 8 2674 83 10.2527/jas.2009-2677 20407072 68. Yu H Wang A Li X Qiao S Effect of viable Lactobacillus fermentum on the growth performance, nutrient digestibility and immunity of weaned pigs J Anim Feed Sci 2008 17 1 61 9 10.22358/jafs/66470/2008 69. Zhu X Wang L Zhang Z Ding L Hang S Combination of fiber-degrading enzymatic hydrolysis and lactobacilli fermentation enhances utilization of fiber and protein in rapeseed meal as revealed in simulated pig digestion and fermentation in vitro Anim Feed Sci Technol 2021 278 115001 10.1016/j.anifeedsci.2021.115001 70. Loh TC, Van Thu T, Foo HL, Bejo MH. Effects of different levels of metabolite combination produced by Lactobacillus plantarum on growth performance, diarrhoea, gut environment and digestibility of postweaning piglets. J Appl Anim Res. 2013;41(2):200–7. 10.1080/09712119.2012.741046. 71. Duarte ME Kim SW Intestinal microbiota and its interaction to intestinal health in nursery pigs Anim Nutr Elsevier 2022 8 1 169 84 10.1016/j.aninu.2021.05.001 72. Wyres KL Lam MMC Holt KE Population genomics of Klebsiella pneumoniae Nat Rev Microbiol 2020 18 6 344 59 10.1038/s41579-019-0315-1 32055025 73. Fouhse JM, Zijlstra RT, Willing BP. The role of gut microbiota in the health and disease of pigs. Anim Front. 2016;6(3):30–6. 10.2527/af.2016-0031.