
==== Front
BMC Vet Res
BMC Vet Res
BMC Veterinary Research
1746-6148
BioMed Central London

4133
10.1186/s12917-024-04133-5
Research
Effects of inactivated Lactobacillus rhamnosus on growth performance, serum indicators, and colonic microbiota and metabolism of weaned piglets
Shu Zhiheng 1
Zhang Junhao 1
Zhou Qingwen 1
Peng Yingjie 2
Huang Yuanhao 1
Zhou Yi 1
Zheng Jun 1
Zhao Manya 1
Hu Chao 546092186@qq.com

1
Lan Shile lans2016@hunau.edu.cn

1
1 https://ror.org/01dzed356 grid.257160.7 0000 0004 1761 0331 College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128 China
2 Guangdong Chuangzhan Bona Agricultural Technology Co., Ltd, Guangning, 526339 China
20 9 2024
20 9 2024
2024
20 42216 1 2024
11 6 2024
© The Author(s) 2024
2024
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

To assess the effects of inactivated Lactobacillus rhamnosus (ILR) on growth performance, serum biochemical indices, colonic microbiota, and metabolomics in weaned piglets, 120 piglets were randomly divided into five groups. Samples in the control group were fed a basal diet, while the experimental ILR1, ILR2, ILR3, and ILR4 groups were fed basal diets supplemented with 0.1%, 0.2%, 0.3%, and 0.4% ILR, respectively. The prefeeding period lasted for 5 days and was followed by a formal period of 28 days.

Results

Compared to the control, the average daily gain increased by 4.38%, 7.98%, 19.32%, and 18.80% for ILR1, ILR2, ILR3, and ILR4, respectively, and the ratio of feed to gain decreased by 0.63%, 3.80%, 12.66%, and 10.76%, respectively. Serum IgA, IgG, IgM, total antioxidant capacity, and glutathione peroxidase levels increased significantly in weaned piglets in the treatment groups. Addition of 0.3% ILR significantly increased the Shannon and Simpson indices of the colonic microbiota in weaned piglets and altered the microbiota composition. Changes in metabolic profiles were observed and were primarily related to the urea cycle, amino acid metabolism, and lipid metabolism.

Conclusion

ILR improved growth performance and serum immunological and biochemical indices and optimized the colonic microbiota structure and metabolism of weaned piglets.

Keywords

Weaned piglets
Inactivated lactobacillus rhamnosus
Colonic microbiota
Metabolome
Special Fund for the Construction of Innovative Provinces in Hunan Province2020NK2029 Hunan Provincial Natural Science Foundation2021JJ50130 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcBackground

The role of intestinal microbiota in host nutrition, growth, development, immunity, and health has been widely confirmed [1–3]. This extends the identification and functional study of probiotics in the gut microbiota [4, 5]. Common probiotics such as Lactobacillus and Bifidobacterium are typically consumed as active bacteria preparations [6]. Lactobacillus has been widely used in industry, medicine, and agriculture [7, 8]. Despite numerous reports supporting the health benefits of probiotics, the safety concerns resulting from the use of live bacteria remain controversial [9]. For example, horizontal gene transfer contributes to the spread of drug resistance in the gut microbiota [10–12]. Another important concern regarding the safety of live bacteria is the risk of translocation and subsequent bacteremia and septicemia [13]. Numerous cases of sepsis caused by Lactobacillus rhamnosus GG (LGG) have been reported clinically [14]. Probiotics may trigger an inflammatory response in highly susceptible individuals [15]. During breeding, the intestines of weaned piglets are weak, and the structure of the microbiota is incomplete, ultimately resulting in impaired antioxidant capacity and gastrointestinal function and leading to slow growth and even death [16]. Therefore, the addition of live bacteria to animal feed should be considered with caution. Furthermore, preservation conditions and quality control of live bacteria are difficult [17].

Inactivated lactic acid bacteria exhibit a beneficial nature similar to that of live bacteria [18, 19]. One study indicated that both live bacteria and heat-inactivated lactic acid bacteria reduced the aflatoxin content in PBS, and two inactivated L. rhamnosus (ILR) strains exhibited stronger adsorption capacities than did live bacteria [20]. Heat-inactivated lactic acid bacteria can release peptidoglycans, lipoteichoic acid, and extracellular polysaccharides (EPS) that all exert immunomodulatory effects [21]. Furthermore, heat-inactivated lactic acid bacteria still possess the ability to maintain the balance of intestinal microbiota [22, 23], and their products are easier to store and possess a longer expiration date [24].

L. rhamnosus regulates the gut microbiota and boosts immunity [25]. However, it has been reported that dietary supplementation with L. rhamnosus GG cannot prevent or reduce the adverse effects of E. coli F4 infection on the growth performance and health status of growing pigs, but it can decrease growth performance, increase diarrhea, and decrease serum immunoglobulin A (IgA) content [26]. Moreover, heat-killed L. rhamnosus improves growth performance and reduces diarrhea in growing pigs [27]. However, the effects of ILR on the structure and metabolism of the intestinal microbiota in weaned piglets have not been reported. Understanding the effects of ILR on the structure and metabolism of the intestinal microbiota in weaned piglets would help to systematically elucidate the mechanisms by which ILR promote growth and immunity. To provide a reference for the application of ILR in the production of weaned piglets, in this study the effects of ILR supplementation on growth performance, serum parameters, structure, and metabolism of the colonic microbiota in weaned piglets were analyzed.

Methods

Preparation of ILR

The L. rhamnosus strains used in this study were isolated from healthy pigs. Approximately 10.0 g of fresh fecal sample was weighed and transferred into a 250 mL sterile conical flask with 90 mL of sterile water and an appropriate amount of glass beads. After full shaking and mixing, 1 ml of mixed liquid was transferred to 100 mL of MRS medium and cultured at 37 °C for 48 h. After gradient dilution, 0.1 mL of the cultured medium was inoculated onto an MRS solid medium plate containing 1% CaCO3. After 48 h of culture at 37 °C, a single colony with obvious calcium lysosomes was picked and repeatedly purified on an MRS solid medium plate until the morphology of the bacteria was observed under a microscope. Morphological characteristics of the colonies were observed by optical microscopy and scanning electron microscopy. Gram staining was performed using the Gram Staining Kit (catalog number: G1060; Solarbio, Beijing, China). The V-P, nitrate reduction, and catalase tests were conducted according to the Handbook for the Identification of Common Bacterial Systems [28]. The 16 S rRNA gene was amplified using 27 F and 1492 R primers and sequenced as previously described [29]. Based on the morphology, Gram staining, catalase testing, and 16 S rRNA gene sequencing, the strain was identified as L. rhamnosus. The L. rhamnosus strain was cultured to 1 × 1010 CFU/ml in MRS medium, centrifuged at 4,000 g for 30 min to collect bacterial precipitate, then washed twice and suspended in distilled water, heated at 80℃ for 30 min, and freeze-dried. The determination of effective inactivation was to culture the freeze-dried sample at 37 °C for 48 h without colony formation.

Study design and measurement of growth indicators

This study was approved by the Biomedical Research Ethics Committee of Hunan Agricultural University (approval number: Lunshenke 2023 No. 127) and was conducted in accordance with its guidelines.

A total of 120 Duroc × Landrace × large hybrid weaned piglets with consistent body weights (8.31 ± 0.16 kg) were purchased from Dayuji Animal Husbandry Technolgy Co., Ltd (Beijing. China) and divided into five groups with six replicates per group and four pigs per replicate based on the principle of similar body weight and the same weight between males and females. Pigs in the control group (CON) were fed a basal diet (Table 1), and those in the ILR1, ILR2, ILR3, and ILR4 groups were fed basal diets supplemented with 0.1, 0.2, 0.3, and 0.4% ILR, respectively.

Table 1 Composition and nutrient levels of the basal diet (air-dry basis)

Items	Content (%)			
Ingredients		Calculated value of nutrients (%)		
Corn	56.00	Crude protein	19.00	
Soybean mead	12.00	Crude fiber	4.20	
Wheat bran	10.00	Lysine	1.35	
Extruded soybean	8.00	Threonine	0.76	
Fish meal	3.00	Methionine	0.46	
Whey powder	3.00	Calcium	0.70	
Sucrose	2.00	Total phosphorus	0.65	
Zeolite powder	1.00	STTDP	0.35	
CaHPO4	1.00			
Soybean oil	1.00			
Premix	3.00			
Total	100			
Premix provides 120 mg of Fe, 20 mg of Cu, 100 mg of Zn, 40 mg of Mn, 0.3 mg of Se, 0.5 mg of I, 0.2 mg of Co, 12,000 IU of VA, 2500 IU of VD3, 35 IU of VE, 4 mg of VK3, 1.5 mg of VB1, 4.5 mg of VB2, 3.5 mg of VB6, 0.05 mg of VB12, 50 mg of nicotinic acid, 2 mg of folic acid, 20 mg of pantothenic acid, and 0.4 mg of biotin per kilogram of feed. STTDP: standard total tract digestible phosphorus

A 28-day formal experiment after a 5-day pre-feeding was conducted at the Experimental Base of Chuangzhan Bona Agricultural Technology Co., Ltd. (Zhaoqing, Guangdong, China). During the experiment, the pigs were raised in the same feeding environment and were immunized and sterilized in strict accordance with the management methods of the pig farm. All fences were equipped with automatic feeders and drinking fountains for free access to feed and water.

On the 1st and 28th day of the formal experiment, the initial body weights (IBWs) and final body weights (FBWs) of the weaned piglets were measured, and daily feed consumption and surplus were recorded to calculate the average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F/G).

At the end of the experiment, six weaned piglets with body weights close to the average of the group were collected from each group, and blood samples were collected from the ear veins after fasting for 6 h. After standing for 30 min, the serum was obtained from blood by centrifugation at 4,000 rpm at 4 °C for 20 min and stored at − 80 °C. After blood collection, the piglets were euthanized in a commercial slaughterhouse (Zhaoqing, Guangzhou, China) by carbon dioxide asphyxiation with less than 2% oxygen (air replaced with carbon dioxide). Intestinal tissue was sampled immediately after dissection, and each intestinal segment was ligated. Colon contents were collected, subsequently transferred to liquid nitrogen for rapid freezing, and then transferred to a − 80℃ refrigerator for storage.

Determination of serum indicators

Serum total protein (TP), albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), blood urea nitrogen (BUN), lysozyme, total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), and malondialdehyde were measured using appropriate kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). IgA, immunoglobulin M (IgM), and immunoglobulin G (IgG) were measured using pig IgA ELISA kits (detection range: 0.146–37.5 µg/ml; catalog number: CSB-E13234p), IgM ELISA kits (detection range: 0.039–10 µg/ml; catalog number: CSB-E06805p), and IgG ELISA kits (detection range: 0.586–150 µg/ml; catalog number: CSB-E06804p), respectively (Cusabio, Wuhan, Hubei, China).

Microbiota composition analysis of colon contents

Colonic microbiome DNA was extracted using the TGuide S96 kit (TianGen, Beijing, China). The hypervariable V3-V4 region of the 16 S rDNA was amplified using primers 338 F and 806R as previously described with modifications [30]. Briefly, polymerase chain reactions (PCRs) were performed in duplicate with a 25-µl reaction mix containing 1 × PCR buffer, 0.25 U of Taq DNA polymerase (Transgen, Beijing), 0.2 mM of each deoxynucleoside triphosphate, 1.0 µM of each primer, and 10 ng microbial genomic DNA. The thermal cycling procedure consisted of an initial pre-denaturation step at 94 °C for 10 min that was followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 10 min. Subsequently, the PCR products were detected using 1.8% agarose gel electrophoresis and purified using an AxyPrep DNA gel extraction kit (Axygen, China). Sequencing was performed using a Sequel II sequencer (PacBio, Silicon Valley, CA, USA) (Biomarker Technologies, Beijing, China) [31]. Raw data were merged using FLASH version 1.2.11 The merged tags were quality controlled using Trimmomatic version 0.33. High-quality tags were obtained after removing chimeric sequences using UCHIME version 8.1 and were clustered into observed taxonomic units (OTUs) with 97% sequence similarity using USEARCH version 10.0. Each feature was annotated using the Silva rRNA database [32]. α-Diversity indices were calculated using Mothur 1.30. β-Diversity analysis was performed using QIIME2 [33] and visualized by component analysis (PCA).

Non-targeted metabolomes analysis of colon contents

The colon content samples were added to an extraction solution (methanol: acetonitrile: water = 2:2:1, interior label concentration of 2 mg/L) containing an interior label (1000:2), vortexed mixed for 30 s, then ground, and sonicated. After standing at − 20 °C for 1 h, the samples were centrifuged at 1,200 rpm for 15 min at 4 °C. The supernatant was transferred into an EP tube, dried in a vacuum concentrator, and re-dissolved in acetonitrile solution (1:1 acetonitrile: water). Subsequently, the supernatant samples were obtained by vortexing, ultrasonication, and re-centrifugation and then used for subsequent detection.

A non-targeted metabolomics assay was performed by Biomarker Biotechnology Co., LTD (Beijing, China). The LC-MS system consisted of an Acquity I-Class PLUS ultra-high performance liquid chromatography-mass spectrometer (Waters, Framing, Massachusetts, USA) in tandem with a Xevo G2-XS QT of high-resolution mass spectrometer (Waters) with an Acquity UPLC HSS T3 column (1.8 μm 2.1*100 mm; Waters). Raw data were collected using MassLynx V4.2, and peaks were extracted, aligned, and processed using Progenesis QI software. Material identification was carried out using Progenesis QI software with online METLIN, public, and Biomark self-built databases. Theoretical fragment identification was performed simultaneously. The mass deviation of the parent ion was within 100 ppm, and that of the fragment ion was within 50 ppm. The ropls R package was used for orthogonal partial least squares discriminant analysis (OPLS-DA), and permutation was used to test the reliability of the model. The identified metabolites were annotated using the KEGG, HMDB, and LIPID MAPS databases. The variable importance in projection (VIP) of the OPLS-DA model obtained from multivariate analysis was analyzed, and differential metabolites were identified for metabolic pathway analysis, combined with the p-values of univariate analysis.

Data analysis

Data were analyzed by one-way ANOVA using SPSS 20.0. Tukey’s test was used for multiple comparisons, and data are presented as means ± standard error. Histograms were plotted using the GraphPad Prism 6 software (GraphPad Prism Inc., USA). Spearman analysis was performed to reveal the correlation between gut microbial communities and the altered metabolites, and the R pheatmap package was used for visualization. Results were considered significant at P < 0.05.

Results

Effect of ILR on the growth performance of weaned piglets

There were no significant differences in IBW among the weaned piglets in any of the groups (P > 0.05; Fig. 1A), whereas the FBW of all treatment groups was significantly higher than that of the control group (P < 0.05; Fig. 1B). Compared to the control, the ADG of each experimental group significantly increased by 4.38%, 7.98%, 19.32%, and 18.80%, respectively (P < 0.05; Fig. 1C), and the F/G decreased by 0.63%, 3.80%, 12.66%, and 10.76%, respectively (Fig. 1E). Although the ADFI in each treatment group was significantly higher than that in the control group, there were no significant differences in ADFI among the treatment groups (Fig. 1D). These results revealed that the ILR3 and LIR4 groups exhibited the best weight gain and feed utilization of weaned piglets, but there was no significant difference between the groups (Fig. 1).

Fig. 1 Effect of inactivated Lactobacillus rhamnosus on the growth performance of weaned piglets. (A) initial body weight (IBM); (B) final body weight (FBW); (C) average daily gain (ADG); (D) average daily feed intake (ADFI); (E) feed conversion ratio (F/G). Different lowercase letters above the boxes indicate significant differences between the two datasets

Effect of ILR on serum biochemical indicators in weaned piglets

The serum TP of the weaned piglets treated with ILR was significantly higher than that of the control (P < 0.05), and the effects of ILR3 and ILR4 were the most obvious, whereas there was no significant difference between these two groups (P > 0.05; Fig. 2A). Serum albumin and HDL-C levels in the ILR2, ILR3, and ILR4 groups were significantly higher than those in the control group (P < 0.05), while serum LDL-C and triglyceride (TG) levels in each treatment group were significantly lower than those in the control group (P < 0.05; Fig. 2C and E). Serum T-CHO levels in the ILR2, ILR3, and ILR4 groups were significantly lower than those in the control group, and those in the ILR3 group decreased the most and were not significantly different from those in the ILR2 and ILR4 groups (Fig. 2F). BUN levels in the ILR3 and ILR4 groups were significantly lower than those in the control group (P < 0.05; Fig. 2G). There were no significant differences in serum ALT and AST levels between the treatment and control groups (P > 0.05; Fig. 2H and I).

Fig. 2 Effect of inactivated Lactobacillus rhamnosus on serum biochemical indicators in weaned piglets. (A) total protein (TP); (B) albumin (ALB); (C) low-density lipoprotein cholesterol (LDL-C); (D) high-density lipoprotein cholesterol (HDL-C); (E) TG; (F) total cholesterol (T-CHO); (G) blood urea nitrogen (BUN); (H) alanine aminotransferase (ALT); (I) aspartate aminotransferase (AST); (J) total antioxidant capacity (T-AOC); (K) glutathione peroxidase (GSH-Px); (L) malondialdehyde (MDA); (M) total superoxide dismutase (T-SOD); (N) immunoglobulin A (IgA); (O) immunoglobulin G (IgG); (P) immunoglobulin M (IgM); and (Q) lysozyme (LZM)

Serum T-AOC levels in the ILR addition group were significantly elevated compared to those in the control group (P < 0.05), and the ILR3 group exhibited the highest elevation of 74.08% (Fig. 2J). Serum GSH-Px levels in each treatment group were significantly higher than those in the control group (P < 0.05), and those in the ILR3 and ILR4 groups increased by 28.67% and 29.54%, respectively (Fig. 2K). The serum malondialdehyde level decreased significantly compared to that in the control (P < 0.05), with the ILR4 group exhibiting the greatest decrease, although there was no significant difference among the ILR2, ILR3, and ILR4 groups (P > 0.05; Fig. 2L). T-SOD activity was not significantly different among the groups (P > 0.05; Fig. 2M).

The activities of IgA, IgG, IgM, and lysozyme in weaned piglets fed ILR were significantly higher than those in the control (P < 0.05) and exhibited an increasing trend with increasing ILR supplementation (Fig. 2N-Q). However, these immune parameters were not significantly different between the ILR3 and ILR4 groups (P > 0.05; Fig. 2N-Q).

Effect of ILR on the colon microbiota structure of weaned piglets

Based on the results of growth performance and serum parameters, the control and ILR3 groups were selected to analyze the structure and metabolic profiles of the colonic microbiota in weaned piglets. In total, 4,521 OTUs were identified in both groups. Principal component analysis (PCA) demonstrated clear differences in colonic microbiota between the ILR3 and control groups (Fig. 3A). Although there were no significant differences in OTU number (P > 0.05; Fig. 3B), the Shannon and Simpson indices in the ILR3 group were significantly higher than those in the control (P < 0.05; Fig. 3C and D), thus indicating that ILR increased the α-diversity of colonic microbiota in weaned piglets.

Fig. 3 Effect of inactivated Lactobacillus rhamnosus on the colon microbiota structure of weaned piglets. (A) Principal component analysis profile; (B) Feature number; (C) Shannon index; (D) Simpson index; (E) Relative abundances of the top 10 phyla in the colon microbiota of weaned piglets; (F) Relative abundance of Firmicutes; (G) Relative abundance of Verrucomicrobiota; (H) Relative abundance of Spiroochaetota; (I) Relative abundance of Proteobacteria; (J) Relative abundance oof Fibrobacterota; (K) Relative abundance of Campylobacterota; (L) Relative abundance of Bacteroidota; (M) Relative abundance of Actinobacteriota; (N) Relative abundance of Patescibacteria; (O) Relative abundance of Desulfobacterota. ** P < 0.01; *** P < 0.001

Bacteroidetes and Firmicutes dominated the colonic microbiota of both groups (Fig. 3E). The relative abundances of Firmicutes, Verrucomicrobiota, Spirochaetota, Proteobacteria, Actinobacteria, and Patescibacteria in the ILR3 group were significantly lower than those in the control group (P < 0.05; Fig. 3F-I and M, and 3N), whereas those of Fibrobacterota, Campylobacterota, and Bacteroidetes were significantly higher than those in the control group (P < 0.05; Fig. 3J-L).

The relative abundances of Prevotella and Alloprevotella in Prevotellaceae and Phascolarctobacterium, Faecalibacterium, Lachnospira, and many unclassified genera in the ILR3 group were significantly increased compared to the control, and those of Streptococcus, Terrisporobacter, Treponema, Escherichia_Shigella, and many unclassified genera were significantly decreased (Fig. 4).

Fig. 4 LEfSe results indicated the significantly altered genera of colon microbiota in weaned piglets treated with inactivated Lactobacillus rhamnosus

Effect of ILR on the colon content metabolites in weaned piglets

The PCA results of the colonic content metabolites demonstrated a clear differentiation between the control and ILR treatments, thus indicating an altered metabolic profile in the colonic contents of weaned piglets (Fig. 5A). Based on a VIP value > 1 in the model and a p-value < 0.05 of univariate analysis, 314 metabolites were significantly upregulated and 402 metabolites were significantly downregulated in the ILR3 group compared to levels in the control group (Fig. 5B). In particular, in the ILR3 group, N-acetyl-L-glutamate (NAG), L-ornithine, cholic acid, chenodeoxycholic acid, 3,7-dihydroxy-5-cholestenoic acid, quinoline-4,8-diol, pyridoxamine phosphate, γ-linolenic acid, eicosapentaenoic acid, 2-polyprenyl-6-methoxyphenol, HSDC, 1-methylnicotinamide, D-xylono-1,5-lactone, and 3-indolepropionic acid were significantly up-regulated, and N-acetylornithine, cerebrosterol, coprocholic acid, 13(S)-HPODE, α-linolenic acid, L-tyrosine, N-butyl-N-(4-hydroxybutyl)nitrosamine, 9, 10-dihome, CMP-2-aminoethylphosphonate, creatinine aspartate, urobilinogen, I-urobilinogen, N-mononitrosopiperazine, and 4-hydroxy-2-nonenal-[Cys-Gly] conjugate were significantly down-regulated compared to levels in the control (Fig. 5C). Furthermore, metabolic pathway enrichment analysis demonstrated that the differences in metabolites between ILR3 and control groups were primarily related to amino acid metabolism, urea cycle, lipid metabolism, cofactor metabolism, and vitamin metabolism pathways (Fig. 5D).

Fig. 5 Effect of inactivated Lactobacillus rhamnosus on the metabolites of colon contents in weaned piglets. (A) OPLS-DA scores of all samples in the default mode; (B) Volcano plot indicates differential metabolites of colon contents in weaned piglets treated by inactivated L. rhamnosus; (C) Main significantly different metabolites of contents in weaned piglets treated with inactivated L. rhamnosus; (D) Classification diagram indicates the distribution of differential metabolites in KEGG pathways; (E) Heatmap indicates Spearman correlations between the main genera of colon microbiota and content metabolites in weaned piglets. * P < 0.05; ** P < 0.01

Spearman’s correlation analysis demonstrated that cholic acid was significantly negatively correlated with UCG_005, Terrisporobacter, and Streptococcus in the primary bile acid biosynthesis pathway (P < 0.05; Fig. 5E). Chenodeoxycholic acid was negatively correlated with UCG_005 and positively correlated with Alloprevotella. Coprocholic acid and 24OHC were significantly negatively correlated with Prevotella 9 and unclassified Lachnospiraceae and significantly positively correlated with Streptococcus. Moreover, Prevotella 9 was significantly negatively correlated with α-linolenic acid and positively correlated with NAG, eicosapentaenoic acid, L-ornithine, and 1-methylnicotinamide. Alloprevotella was significantly negatively correlated with N-mononitrosopiperazine, creatinine aspartate, 4-hydroxy-2-nonenal-[Cys-Gly] conjugate, and L-tyrosine and positively correlated with γ-linolenic acid, pyridoxamine phosphate, 3-indolepropionic acid, and SHCHC. Streptococcus was significantly and positively correlated with I-urobilinogen and significantly negatively correlated with 1-methylnicotinamide levels. Terrisporobacter was significantly positively correlated with n-butyl N-(4-hydroxybutyl) nitrosamine and negatively correlated with D-xylono-1,5-Lactone, NAG, eicosapentaenoic acid, 1-methylnicotinamide, quinoline-4,8-diol, and 2-polyprenyl-6-methoxyphenol (P < 0.05; Fig. 5E).

Discussion

Inactivated lactic acid bacteria possess excellent application prospects for animal production and are more economical and convenient to use than are live bacteria [24]. Adding heat-inactivated L. rhamnosus to the diet of growing pigs reduced diarrhea and improved growth performance [21]. In the present study, our results demonstrated that ILR supplementation significantly improved the growth performance of weaned piglets, and the best effect was achieved when the supplementation level was 0.3%.

Serum antioxidant enzyme activity is an important indicator of health and the ability to remove free radicals [34]. Lactic acid bacteria exert significant immunoregulatory effects, including the activation of macrophages, interferons, and IgA [35]. In this study, supplementation with ILR in the feed of weaned piglets significantly increased serum T-AOC and GSH-Px activity and decreased malondialdehyde levels. Generally, immunoglobulins are directly related to immune function, and lysozymes are involved in innate immunity. This study revealed that supplementation with ILR significantly increased serum IgA, IgG, IgM, and lysozyme levels in weaned piglets, and this is consistent with the effect of live bacteria on weaned piglets [36]. These results indicate that the improvement in the immune function and antioxidant capacity of weaned piglets by L. rhamnosus is not caused by its growth in the intestine to stimulate the development of the host intestinal immune system but by its cellular components. The specific cellular components that regulate host immunity and enhance antioxidant capacity require further investigation.

Serum TP, albumin, and BUN levels are related to the growth performance of weaned piglets and are often used as indicators of protein synthesis and metabolism [37]. Previous studies have revealed a strong negative correlation between the biological functions of diet and BUN [38]. In the present study, supplementation with 0.3% and 0.4% ILR significantly increased serum TP and albumin levels and reduced serum BUN levels, indicating that ILR supplementation promoted amino acid utilization and metabolism, and this was beneficial to the health and weight gain of weaned piglets. Serum T-CHO and TG levels reflect changes in the lipolysis rate [39]. The main function of LDL-C is to transport endogenous cholesterol, whereas HDL-C transfers cholesterol from peripheral tissues to the liver for metabolic decomposition [40]. In this study, compared to levels in the control group, serum T-CHO and TG levels in the 0.3% and 0.4% ILR supplementation groups were significantly decreased, whereas HDL-C and LDL-C levels were significantly increased and decreased, respectively, thus indicating that ILR supplementation promotes lipid and cholesterol metabolism in weaned piglets. This was consistent with the results of a previous study examining heat-killed L. rhamnosus in mice [41]. The increase in serum AST and ALT levels is likely related to liver injury and is a sensitive marker of hepatocyte damage [42]. Our results revealed that ILR supplementation did not alter serum AST and ALT levels in weaned piglets, thus indicating that ILR supplementation did not damage the liver or myocardial cells of weaned piglets.

In pigs, the gut microbiota is closely related to nutrient metabolism, immune regulation, and defense against pathogens [43]. This study is the first to report that ILR alters the structure and increases the α-diversity of the colonic microbiota in weaned piglets. Previous studies have revealed that increased levels of Firmicutes and decreased levels of Bacteroidetes contribute to obesity and excess body fat [44]. Bacteroidetes contain numerous probiotics and are rich in polysaccharide-degrading enzymes [45]. The ADG and serum levels of TP and BUN suggest that dietary ILR supplementation may contribute to growth and body fat reduction in weaned piglets, and this is consistent with the speculation of He et al. [46]. Proteobacteria are the microbial characteristics of intestinal inflammation [47]. Studies have also demonstrated that an increase in spirochetal levels may cause an inflammatory response in the body, ultimately leading to chronic gastritis in pigs and thereby affecting the digestive system [48]. Our results revealed that ILR supplementation significantly reduced the relative abundances of Proteobacteria and Spirochetes, thus indicating that ILR supplementation is beneficial to host health. Prevotella positively correlated with feed efficiency and growth performance [49]. This is likely due to the ability of Prevotella to ferment complex dietary polysaccharides [50], thereby promoting the uptake of monosaccharides by the host and conferring a growth performance advantage [51]. Simultaneously, large amounts of short-chain fatty acids (SCFA) are produced [52]. Our results indicate that ILR supplementation significantly increased the relative abundance of Prevotella in the colonic microbiota of weaned piglets. Furthermore, the addition of 0.3% ILR significantly increased the abundance of Phascolarctobacterium that can colonize the human gut, produce SCFA, and play a beneficial role [53]. SCFA are considered mediators of communication between the gut microbiota and the immune system and help to maintain an anti-inflammatory and pro-inflammatory balance [54]. An increase in an unclassified Lachnospiraceae bacterium also plays an important role in cellulose digestion [55]. The common diet used in this study contained certain complex dietary polysaccharides, and changes in the colonic microbiota likely improved digestion and absorption in growing pigs. This is likely one reason for the significant increase in the feed-to-gain ratio. Gut microbiota enriched in Prevotella also reduce cholesterol levels [56]. The reduction in serum cholesterol levels observed in the present study may be related to the high abundance of Prevotella. The most significant increase in relative abundance was Prevotella 9 that accounted for 5.22% in the ILR3 group and only 0.50% in the control group. Hung et al. [57] demonstrated that the abundance of Prevotella 9 in the feces of weaned piglets was negatively correlated with diarrhea and positively correlated with growth performance, whereas a decreased abundance of UCG-005 was positively correlated with diarrhea and negatively correlated with growth performance, and this is consistent with the results of this study. Alloprevotella can regulate intestinal inflammation and exhibit anti-inflammatory effects [58], and its relative abundance is inversely correlated with inflammation [59]. Prevotellaceae_NK3B31_group effectively alleviates intestinal inflammation, promotes intestinal nutrient absorption, and reduces immune rejection in autoimmune diseases [60]. Lachnospira can produce butyrate, and its low abundance is associated with constipation [61]. Faecalibacterium is one of the most common genera in the gut microbiota of healthy adults [62]. It can produce butyrate [63], peptides [64], and extracellular polymeric matrix [65], and other metabolites have been demonstrated to exhibit anti-inflammatory activities in animal models and in vitro experiments [66]. Our results revealed that the abundance of these bacteria increased after ILR supplementation, and this was related to the improvement in antioxidant and immune indices, thus indicating that ILR improve the intestinal microbiota of weaned piglets and enhance immunity.

Streptococcus is a major swine pathogen that leads to high economic losses in the pig industry and causes zoonotic infections such as meningitis and sepsis [67]. Therefore, effective treatment and prevention of Streptococcus suis infections are important in the pig industry. In this study, ILR supplementation significantly decreased Streptococcus numbers, thus indicating that ILR possess good disease-resistant potential. Moreover, the relative abundance of Terrisporobacter was significantly decreased in the ILR3 group. Terrisporobacter induces postoperative infection in patients [68], and its increased abundance may contribute to increased inflammation and oxidative stress [69]. Other bacteria prone to negative effects have been observed in reduced abundance such as Treponema, Christensenelleaceae R-7, and Escherichia-Shigella. The Christensenelleaceae R-7 group exhibited a high abundance of bacterial infections, ultimately resulting in loss of appetite and emotional anxiety that were negatively correlated with weight gain [70, 71]. Escherichia contains many pathological forms that cause diarrhea, dysentery, and parenteral infections, including urinary tract infections and meningitis [72]. Shigella is a major pathogen of bacillary dysentery worldwide and is commonly observed in cholera diarrhea or enterotoxigenic E. coli diarrhea [73]. Escherichia-Shigella is associated with significant dysregulation of the gut microbiota in patients with tuberculous meningitis [74]. Therefore, ILR optimizes the intestinal microbiota structure of weaned piglets and inhibits pathogenic microorganisms, thereby improving growth performance and immune function, and it possesses good prospects for the development of feed additives.

An important means by which gut microbes influence host health is through metabolites [75]. NAG induces essential allosteric activation of carbamyl phosphate synthetase I (CPS1), a key enzyme in the mammalian urea cycle [76]. NAG deficiency leads to urea cycle disease and carbamyl phosphate synthetase deficiency (CPS1D) [77]. L-ornithine is one of the products in the process of producing urea from the effect of L-arginine on L-arginine. It is a core part of the urea cycle and can remove excess nitrogen [78], promote lipid metabolism, activate the urea cycle, and stimulate urea synthesis [79, 80]. The increase in NAG and L-ornithine in this study suggests that ILR may promote the urea cycle and protein metabolism, maintain nitrogen equilibrium, and maintain health and nutritional status within the body. 1-Methylnicotinamide (MNA), a metabolite of vitamin B3, increases NO release from vascular endothelial cells and lowers blood pressure [81]. It also exerts antithrombotic and anti-inflammatory effects [82]. The increase in MNA observed in the present study may be related to positive changes in serum immune markers in the treatment group.

In the primary bile acid biosynthesis pathway, cerebrosterol and its intermediate product coprocholic acid were decreased in the ILR3 group, whereas its final products cholic acid and chenodeoxycholic acid were increased. Cerebrosterol is highly expressed in diseases such as Alzheimer’s disease and meningitis [83, 84]. A decrease in cerebrosterol levels indicates that more cholesterol is used to synthesize bile acids (BA). Cholic acid (CA) and chenodeoxycholic acid (CDCA) are the primary bile acids synthesized in the liver [85, 86]. BA plays an important role in lipid digestion and absorption and is an important regulator of the intestinal microbiota that is closely related to intestinal microbes and host health [87]. Therefore, ILR may promote fat digestion and absorption, regulate sterol metabolism, and improve feed efficiency by promoting CA and CDCA synthesis through the primary bile acid biosynthesis pathway. α-Linolenic acid (ALA) is an essential fatty acid belonging to the ω-3 series of polyunsaturated fatty acids (PUFAs). ALA can be metabolized into bioactive long-chain PUFAs such as eicosapentaenoic acid (EPA) in the human body [88]. However, EPA is not readily converted from the precursor ALA in humans and other mammals, as the enzyme activity involved in the conversion is weak [89]. In this study, ALA levels decreased and EPA levels increased in the ILR3 group, indicating that ILR can improve related enzyme activities and promote ALA metabolism, although more experimental evidence is needed. γ-Linolenic acid, an anti-inflammatory ω-6 PUFA [90], was increased in the ILR3 group. ω-6 and ω-3 PUFAs play key roles in a variety of biological functions and are essential for health [91]. Fermented rhamnose milk can participate in α-linolenic acid metabolism and arachidonic acid metabolism, promote fatty acid degradation, and regulate lipid metabolic homeostasis in rats [92]. This is consistent with our results with the addition of ILR.

Correlation analysis demonstrated that Terrisporobacter was significantly negatively correlated with ω-3 PUFA eicosapentaenoic acid. A previous study revealed that Terrisporobacter was significantly positively correlated with TC, TG, and LDL-C and negatively correlated with HDL-C and that reducing Terrisporobacter may increase the levels of unsaturated fatty acids and BAs [69]. Guo et al. [93] speculated that Terrisporobacter may be involved in the regulation of enzymes involved in BA metabolism or lipid biosynthesis, ultimately leading to higher lipid levels and dyslipidemia. Our results are consistent with these findings. Moreover, certain bioactive substances such as MMA and pyridoxamine phosphate (PMP) were upregulated in the ILR3 group. MMA also exerts anti-inflammatory effects [82]. PMP is a bioactive vitamin B6 [94]. Vitamin B6 exerts a positive effect on the development of immune organs, serum immunoglobulin content, and growth performance of weanling rabbits [95]. However, they are negatively correlated with Streptococcus and positively correlated with an unclassified genus (Prevotellaceae). Moreover, the toxic leukotoxin diol 9,10-dihome [96] was reduced by the addition of ILR and positively correlated with UCG_005. Overall, alterations in gut microbial composition and diversity produced by ILR supplementation may play an important role in the development of gut metabolism, ultimately benefiting the health of weaned piglets. Notably, due to limited experimental funds, we only compared the microbiota and metabolite compositions in the colon contents between the ILR3 and control groups. It is necessary to systematically study the effects of different concentrations of ILR on the composition of microbiota and metabolites in piglet colon contents.

Conclusions

Dietary supplementation with ILR can effectively improve the growth performance, serum antioxidant and immune indices, and composition and metabolic characteristics of the colonic microbiota of weaned piglets, and the 0.3% supplementation level was the best. The addition of 0.3% ILR increased the α-diversity and optimized the structure and metabolism of colonic microbiota, and this primarily affected amino acid metabolism, urea cycle, lipid metabolism, cofactor metabolism, and vitamin metabolism pathways. Therefore, the results of this study highlight the beneficial effects of ILR on the growth and health of weaned piglets and reveal the potential mechanism by which ILR improves the intestinal system.

Acknowledgements

The authors thank anonymous technicians at Guangdong Meilikang Bio-Science Ltd., China for assistance with data reanalysis and figure preparation.

Author contributions

Z.S., C.H. and S.L. conceived and designed the experiments; Z.S., J.H.Z. (Junhao Zhang), Q.Z., Y.P., Y.H., Y.Z., J.Z. (Jun Zheng), M.Z. and S.L. performed the experiments and collected the data; Z.S., J.H.Z. and S.L. performed the statistical analyses; Z.S. drafted the manuscript; C.H. supervised the experiments; C.H. and S.L. revised the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by the Special Fund for the Construction of Innovative Provinces in Hunan Province (grant number 2020NK2029) and the Hunan Provincial Natural Science Foundation (grant number 2021JJ50130).

Data availability

The raw sequences were deposited in the NCBI Sequence Read Archive database with accession number PRJNA1044105.

Declarations

Ethics approval and consent to participate

This animal study was approved by the Biomedical Research Ethics Committee of Hunan Agricultural University (approval number: Lunshenke 2023 No. 127) and conducted in accordance with its guidelines.

Consent for publication

Not applicable.

Competing interests

Yingjie Peng is an employee of Guangdong Chuangzhan Bona Agricultural Technology Co., Ltd., China.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Rowland I Gibson G Heinken A Scott K Swann J Thiele I Tuohy K Gut microbiota functions: metabolism of nutrients and other food components Eur J Nutr 2018 57 1 1 24 10.1007/s00394-017-1445-8 28393285
Rowland I, Gibson G, Heinken A, Scott K, Swann J, Thiele I, Tuohy K. Gut microbiota functions: metabolism of nutrients and other food components. Eur J Nutr. 2018;57(1):1–24. 10.1007/s00394-017-1445-8.28393285
2. Fan Y Pedersen O Gut microbiota in human metabolic health and disease Nat Rev Microbiol 2021 19 1 55 71 10.1038/s41579-020-0433-9 32887946
Fan Y, Pedersen O. Gut microbiota in human metabolic health and disease. Nat Rev Microbiol. 2021;19(1):55–71. 10.1038/s41579-020-0433-9.32887946
3. de Vos WM Tilg H Van Hul M Cani PD Gut microbiome and health: mechanistic insights Gut 2022 71 5 1020 32 10.1136/gutjnl-2021-326789 35105664
de Vos WM, Tilg H, Van Hul M, Cani PD. Gut microbiome and health: mechanistic insights. Gut. 2022;71(5):1020–32. 10.1136/gutjnl-2021-326789.35105664
4. Wang X Zhang P Zhang X Probiotics regulate gut microbiota: an effective method to improve immunity Molecules 2021 26 19 6076 10.3390/molecules26196076 34641619
Wang X, Zhang P, Zhang X. Probiotics regulate gut microbiota: an effective method to improve immunity. Molecules. 2021;26(19):6076. 10.3390/molecules26196076.34641619
5. Zhao Z Yang Q Zhou T Liu C Sun M Cui X Zhang X Anticancer potential of Bacillus coagulans MZY531 on mouse H22 hepatocellular carcinoma cells via anti-proliferation and apoptosis induction BMC Complement Med Th 2023 23 318 10.1186/s12906-023-04120-7
Zhao Z, Yang Q, Zhou T, Liu C, Sun M, Cui X, Zhang X. Anticancer potential of Bacillus coagulans MZY531 on mouse H22 hepatocellular carcinoma cells via anti-proliferation and apoptosis induction. BMC Complement Med Th. 2023;23:318. 10.1186/s12906-023-04120-7.
6. Kerry RG Patra JK Gouda S Park Y Shin HS Das G Benefaction of probiotics for human health: a review J Food Drug Anal 2018 26 927 39 10.1016/j.jfda.2018.01.002 29976412
Kerry RG, Patra JK, Gouda S, Park Y, Shin HS, Das G. Benefaction of probiotics for human health: a review. J Food Drug Anal. 2018;26:927–39. 10.1016/j.jfda.2018.01.002.29976412
7. Wang Y Wu J Lv M Shao Z Hungwe M Wang J Bai X Xie J Wang Y Geng W Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry Front Bioeng Biotechnol 2021 9 612285 10.3389/fbioe.2021.612285 34055755
Wang Y, Wu J, Lv M, Shao Z, Hungwe M, Wang J, Bai X, Xie J, Wang Y, Geng W. Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry. Front Bioeng Biotechnol. 2021;9:612285. 10.3389/fbioe.2021.612285.34055755
8. Deng Z Hou K Zhao J Wang H The probiotic properties of lactic acid bacteria and their applications in animal husbandry Curr Microbiol 2021 79 1 22 10.1007/s00284-021-02722-3 34905106
Deng Z, Hou K, Zhao J, Wang H. The probiotic properties of lactic acid bacteria and their applications in animal husbandry. Curr Microbiol. 2021;79(1):22. 10.1007/s00284-021-02722-3.34905106
9. Merenstein D Pot B Leyer G Ouwehand AC Preidis GA Elkins CA Hill C Lewis Z Shane AL Zmora N Petrova MI Collado MC Morelli L Montoya GA Szajewska H Tancredi DJ Sanders ME Emerging issues in probiotic safety: 2023 perspectives Gut Microbes 2023 15 1 2185034 10.1080/19490976.2023.2185034 36919522
Merenstein D, Pot B, Leyer G, Ouwehand AC, Preidis GA, Elkins CA, Hill C, Lewis Z, Shane AL, Zmora N, Petrova MI, Collado MC, Morelli L, Montoya GA, Szajewska H, Tancredi DJ, Sanders ME. Emerging issues in probiotic safety: 2023 perspectives. Gut Microbes. 2023;15(1):2185034. 10.1080/19490976.2023.2185034.36919522
10. Wassenaar TM Klein G Safety aspects and implications of regulation of probiotic bacteria in food and food supplements J Food Prot 2008 71 8 1734 41 10.4315/0362-028x-71.8.1734 18724773
Wassenaar TM, Klein G. Safety aspects and implications of regulation of probiotic bacteria in food and food supplements. J Food Prot. 2008;71(8):1734–41. 10.4315/0362-028x-71.8.1734.18724773
11. Mater DD Langella P Corthier G Flores M-J A probiotic Lactobacillus strain can acquire Vancomycin resistance during digestive transit in mice J Mol Microbiol Biotechnol 2008 14 1–3 123 7 10.1159/000106091 17957119
Mater DD, Langella P, Corthier G, Flores M-J. A probiotic Lactobacillus strain can acquire Vancomycin resistance during digestive transit in mice. J Mol Microbiol Biotechnol. 2008;14(1–3):123–7. 10.1159/000106091.17957119
12. Thumu SCR Halami PM Conjugal transfer of erm(B) and multiple tet genes from Lactobacillus spp. to bacterial pathogens in animal gut, in vitro and during food fermentation Food Res Int 2019 116 1066 75 10.1016/j.foodres.2018.09.046 30716890
Thumu SCR, Halami PM. Conjugal transfer of erm(B) and multiple tet genes from Lactobacillus spp. to bacterial pathogens in animal gut, in vitro and during food fermentation. Food Res Int. 2019;116:1066–75. 10.1016/j.foodres.2018.09.046.30716890
13. Liong MT Safety of probiotics: translocation and infection Nutr Rev 2008 66 4 192 202 10.1111/j.1753-4887.2008.00024.x 18366533
Liong MT. Safety of probiotics: translocation and infection. Nutr Rev. 2008;66(4):192–202. 10.1111/j.1753-4887.2008.00024.x.18366533
14. Chiang MC Chen CL Feng Y Chen CC Lien R Chiu CH Lactobacillus rhamnosus sepsis associated with probiotic therapy in an extremely preterm infant: Pathogenesis and a review for clinicians J Microbiol Immunol Infect 2021 54 4 575 80 10.1016/j.jmii.2020.03.029 32307246
Chiang MC, Chen CL, Feng Y, Chen CC, Lien R, Chiu CH. Lactobacillus rhamnosus sepsis associated with probiotic therapy in an extremely preterm infant: Pathogenesis and a review for clinicians. J Microbiol Immunol Infect. 2021;54(4):575–80. 10.1016/j.jmii.2020.03.029.32307246
15. Boyle RJ Robins-Browne RM Tang ML Probiotic use in clinical practice: what are the risks? Am J Clin Nutr 2006 83 6 1256 64 10.1093/ajcn/83.6.1256 16762934
Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: what are the risks? Am J Clin Nutr. 2006;83(6):1256–64. 10.1093/ajcn/83.6.1256.16762934
16. Tang X Xiong K Fang R Li M Weaning stress and intestinal health of piglets: a review Front Immunol 2022 13 1042778 10.3389/fimmu.2022.1042778 36505434
Tang X, Xiong K, Fang R, Li M. Weaning stress and intestinal health of piglets: a review. Front Immunol. 2022;13:1042778. 10.3389/fimmu.2022.1042778.36505434
17. Trush EA Poluektova EA Beniashvilli AG Shifrin OS Poluektov YM Ivashkin VT The evolution of human probiotics: challenges and prospects Probiotics Antimicrob Proteins 2020 12 4 1291 9 10.1007/s12602-019-09628-4 31907861
Trush EA, Poluektova EA, Beniashvilli AG, Shifrin OS, Poluektov YM, Ivashkin VT. The evolution of human probiotics: challenges and prospects. Probiotics Antimicrob Proteins. 2020;12(4):1291–9. 10.1007/s12602-019-09628-4.31907861
18. Hatab S Yue T Mohamad O Removal of patulin from apple juice using inactivated lactic acid bacteria J Appl Microbiol 2012 112 5 892 9 10.1111/j.1365-2672.2012.05279.x 22394257
Hatab S, Yue T, Mohamad O. Removal of patulin from apple juice using inactivated lactic acid bacteria. J Appl Microbiol. 2012;112(5):892–9. 10.1111/j.1365-2672.2012.05279.x.22394257
19. Aiba Y Ishikawa H Tokunaga M Komatsu Y Anti-helicobacter pylori activity of non-living, heat-killed form of lactobacilli including Lactobacillus johnsonii 1088 FEMS Microbiol Lett 2017 364 11 fnx102 10.1093/femsle/fnx102
Aiba Y, Ishikawa H, Tokunaga M, Komatsu Y. Anti-helicobacter pylori activity of non-living, heat-killed form of lactobacilli including Lactobacillus johnsonii 1088. FEMS Microbiol Lett. 2017;364(11):fnx102. 10.1093/femsle/fnx102.
20. Pierides M El-Nezami H Peltonen K Salminen S Ahokas J Ability of dairy strains of lactic acid bacteria to bind aflatoxin M1 in a food model J Food Prot 2000 63 5 645 50 10.4315/0362-028x-63.5.645 10826723
Pierides M, El-Nezami H, Peltonen K, Salminen S, Ahokas J. Ability of dairy strains of lactic acid bacteria to bind aflatoxin M1 in a food model. J Food Prot. 2000;63(5):645–50. 10.4315/0362-028x-63.5.645.10826723
21. Piqué N Berlanga M Miñana-Galbis D Health benefits of heat-killed (Tyndallized) probiotics: an overview Int J Mol Sci 2019 20 10 2534 10.3390/ijms20102534 31126033
Piqué N, Berlanga M, Miñana-Galbis D. Health benefits of heat-killed (Tyndallized) probiotics: an overview. Int J Mol Sci. 2019;20(10):2534. 10.3390/ijms20102534.31126033
22. Tan L Fu J Feng F Liu X Cui Z Li B Engineered probiotics biofilm enhances osseointegration via immunoregulation and anti-infection Sci Adv 2020 6 46 eaba5723 10.1126/sciadv.aba5723 33188012
Tan L, Fu J, Feng F, Liu X, Cui Z, Li B, et al. Engineered probiotics biofilm enhances osseointegration via immunoregulation and anti-infection. Sci Adv. 2020;6(46):eaba5723. 10.1126/sciadv.aba5723.33188012
23. Wang S Ahmadi S Nagpal R Jain S Mihra SP Kavanagh K Lipoteichoic acid from the cell wall of a heat killed Lactobacillus paracasei D3-5 ameliorates aging-related leaky gut, inflammation and improves physical and cognitive functions: from C. Elegans to mice Geroscience 2020 42 1 333 52 10.1007/s11357-019-00137-4 31814084
Wang S, Ahmadi S, Nagpal R, Jain S, Mihra SP, Kavanagh K, et al. Lipoteichoic acid from the cell wall of a heat killed Lactobacillus paracasei D3-5 ameliorates aging-related leaky gut, inflammation and improves physical and cognitive functions: from C. Elegans to mice. Geroscience. 2020;42(1):333–52. 10.1007/s11357-019-00137-4.31814084
24. Nataraj BH Ali SA Behare PV Yadav H Postbiotics-parabiotics: the new horizons in microbial biotherapy and functional foods Microb Cell Fact 2020 19 1 168 10.1186/s12934-020-01426-w 32819443
Nataraj BH, Ali SA, Behare PV, Yadav H. Postbiotics-parabiotics: the new horizons in microbial biotherapy and functional foods. Microb Cell Fact. 2020;19(1):168. 10.1186/s12934-020-01426-w.32819443
25. Capurso L Thirty years of Lactobacillus rhamnosus GG: a review J Clin Gastroenterol 2019 53 S1 10.1097/MCG.0000000000001170 30741841
Capurso L. Thirty years of Lactobacillus rhamnosus GG: a review. J Clin Gastroenterol. 2019;53:S1. 10.1097/MCG.0000000000001170.30741841
26. Trevisi P Casini L Coloretti F Mazzoni M Merialdi G Bosi P Dietary addition of Lactobacillus rhamnosus GG impairs the health of Escherichia coli F4-challenged piglets Animal 2011 5 9 1354 60 10.1017/S1751731111000462 22440280
Trevisi P, Casini L, Coloretti F, Mazzoni M, Merialdi G, Bosi P. Dietary addition of Lactobacillus rhamnosus GG impairs the health of Escherichia coli F4-challenged piglets. Animal. 2011;5(9):1354–60. 10.1017/S1751731111000462.22440280
27. Kang J Lee JJ Cho JH Choe J Kyong H Kim SH Effects of dietary inactivated probiotics on growth performance and immune responses of weaned pigs J Anim Sci Technol 2021 63 3 520 30 10.5187/jast.2021.e44 34189502
Kang J, Lee JJ, Cho JH, Choe J, Kyong H, Kim SH, et al. Effects of dietary inactivated probiotics on growth performance and immune responses of weaned pigs. J Anim Sci Technol. 2021;63(3):520–30. 10.5187/jast.2021.e44.34189502
28. Dong X Cai L Handbook for the identification of Common Bacterial systems 2001 Beijing Science
Dong X, Cai L. Handbook for the identification of Common Bacterial systems. Beijing: Science; 2001.
29. Choi WY Lee HY Complete nucleotide sequence of the 16S rRNA from Lactobacillus paracasei HS-05 isolated from women’s hands AMB Expr 2015 5 78 10.1186/s13568-015-0158-8
Choi WY, Lee HY. Complete nucleotide sequence of the 16S rRNA from Lactobacillus paracasei HS-05 isolated from women’s hands. AMB Expr. 2015;5:78. 10.1186/s13568-015-0158-8.
30. Xu Q Yang Z Chen S Zhu W Xiao S Liu J Wang H Lan S Effects of replacing dietary fish meal by soybean meal co-fermented using Bacillus subtilis and Enterococcus faecium on serum antioxidant indices and gut microbiota of crucian carp Carassius auratus Fishes 2022 7 54 10.3390/fishes7020054
Xu Q, Yang Z, Chen S, Zhu W, Xiao S, Liu J, Wang H, Lan S. Effects of replacing dietary fish meal by soybean meal co-fermented using Bacillus subtilis and Enterococcus faecium on serum antioxidant indices and gut microbiota of crucian carp Carassius auratus. Fishes. 2022;7:54. 10.3390/fishes7020054.
31. Huang Y Peng Y Yang Z Chen S Liu J Wang Z Wang G Lan S Effects of fermented bamboo shoot processing waste on growth performance, serum parameters, and gut microbiota of weaned piglets Animals 2022 12 2728 10.3390/ani12202728 36290114
Huang Y, Peng Y, Yang Z, Chen S, Liu J, Wang Z, Wang G, Lan S. Effects of fermented bamboo shoot processing waste on growth performance, serum parameters, and gut microbiota of weaned piglets. Animals. 2022;12:2728. 10.3390/ani12202728.36290114
32. Quast C Pruesse E Yilmaz P Gerken J Schweer T Yarza P Peplies J Glöckner FO The SILVA ribosomal RNA gene database project: improved data processing and web-based tools Nucleic Acids Res 2013 41 D590 6 10.1093/nar/gks1219 23193283
Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590–6. 10.1093/nar/gks1219.23193283
33. Bolyen E Rideout JR Dillon MR Bokulich NA Abnet CC Al-Ghalith GA Alexander H Alm EJ Arumugam M Asnicar F Bai Y Bisanz JE Bittinger K Brejnrod A Brislawn CJ Brown CT Callahan BJ Caraballo-Rodríguez AM Chase J Cope EK Da Silva R Diener C Dorrestein PC Douglas GM Durall DM Duvallet C Edwardson CF Ernst M Estaki M Fouquier J Gauglitz JM Gibbons SM Gibson DL Gonzalez A Gorlick K Guo J Hillmann B Holmes S Holste H Huttenhower C Huttley GA Janssen S Jarmusch AK Jiang L Kaehler BD Kang KB Keefe CR Keim P Kelley ST Knights D Koester I Kosciolek T Kreps J Langille MGI Lee J Ley R Liu YX Loftfield E Lozupone C Maher M Marotz C Martin BD McDonald D McIver LJ Melnik AV Metcalf JL Morgan SC Morton JT Naimey AT Navas-Molina JA Nothias LF Orchanian SB Pearson T Peoples SL Petras D Preuss ML Pruesse E Rasmussen LB Rivers A Robeson MS Rosenthal P Segata N Shaffer M Shiffer A Sinha R Song SJ Spear JR Swafford AD Thompson LR Torres PJ Trinh P Tripathi A Turnbaugh PJ Ul-Hasan S van der Hooft JJJ Vargas F Vázquez-Baeza Y Vogtmann E von Hippel M Walters W Wan Y Wang M Warren J Weber KC Williamson CHD Willis AD Xu ZZ Zaneveld JR Zhang Y Zhu Q Knight R, and Caporaso JG. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2 Nat Biotechnol 2019 37 852 7 10.1038/s41587-019-0209-9 31341288
Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, Bai Y, Bisanz JE, Bittinger K, Brejnrod A, Brislawn CJ, Brown CT, Callahan BJ, Caraballo-Rodríguez AM, Chase J, Cope EK, Da Silva R, Diener C, Dorrestein PC, Douglas GM, Durall DM, Duvallet C, Edwardson CF, Ernst M, Estaki M, Fouquier J, Gauglitz JM, Gibbons SM, Gibson DL, Gonzalez A, Gorlick K, Guo J, Hillmann B, Holmes S, Holste H, Huttenhower C, Huttley GA, Janssen S, Jarmusch AK, Jiang L, Kaehler BD, Kang KB, Keefe CR, Keim P, Kelley ST, Knights D, Koester I, Kosciolek T, Kreps J, Langille MGI, Lee J, Ley R, Liu YX, Loftfield E, Lozupone C, Maher M, Marotz C, Martin BD, McDonald D, McIver LJ, Melnik AV, Metcalf JL, Morgan SC, Morton JT, Naimey AT, Navas-Molina JA, Nothias LF, Orchanian SB, Pearson T, Peoples SL, Petras D, Preuss ML, Pruesse E, Rasmussen LB, Rivers A, Robeson MS, Rosenthal P, Segata N, Shaffer M, Shiffer A, Sinha R, Song SJ, Spear JR, Swafford AD, Thompson LR, Torres PJ, Trinh P, Tripathi A, Turnbaugh PJ, Ul-Hasan S, van der Hooft JJJ, Vargas F, Vázquez-Baeza Y, Vogtmann E, von Hippel M, Walters W, Wan Y, Wang M, Warren J, Weber KC, Williamson CHD, Willis AD, Xu ZZ, Zaneveld JR, Zhang Y, Zhu Q. Knight R, and Caporaso JG. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019;37:852–7. 10.1038/s41587-019-0209-9.31341288
34. Liu N Ma X Jiang X Effects of immobilized antimicrobial peptides on growth performance, serum biochemical index, inflammatory factors, intestinal morphology, and microbial community in weaning pigs Front Immunol 2022 13 872990 10.3389/fimmu.2022.872990 35422808
Liu N, Ma X, Jiang X. Effects of immobilized antimicrobial peptides on growth performance, serum biochemical index, inflammatory factors, intestinal morphology, and microbial community in weaning pigs. Front Immunol. 2022;13:872990. 10.3389/fimmu.2022.872990.35422808
35. Riaz Rajoka MS Shi J Zhu J Shao D Huang Q Yang H Capacity of lactic acid bacteria in immunity enhancement and cancer prevention Appl Microbiol Biotechnol 2017 101 1 35 45 10.1007/s00253-016-8005-7 27888334
Riaz Rajoka MS, Shi J, Zhu J, Shao D, Huang Q, Yang H, et al. Capacity of lactic acid bacteria in immunity enhancement and cancer prevention. Appl Microbiol Biotechnol. 2017;101(1):35–45. 10.1007/s00253-016-8005-7.27888334
36. Zhang L Xu YQ Liu HY Lai T Ma JL Wang JF Evaluation of Lactobacillus rhamnosus GG using an Escherichia coli K88 model of piglet diarrhoea: effects on diarrhoea incidence, faecal microflora and immune responses Vet Microbiol 2010 141 1–2 142 8 10.1016/j.vetmic.2009.09.003 19782483
Zhang L, Xu YQ, Liu HY, Lai T, Ma JL, Wang JF, et al. Evaluation of Lactobacillus rhamnosus GG using an Escherichia coli K88 model of piglet diarrhoea: effects on diarrhoea incidence, faecal microflora and immune responses. Vet Microbiol. 2010;141(1–2):142–8. 10.1016/j.vetmic.2009.09.003.19782483
37. Liu Y Huo B Chen Z Wang K Huang L Che L Effects of organic chromium yeast on performance, meat quality, and serum parameters of grow-finish pigs Biol Trace Elem Res 2023 201 3 1188 96 10.1007/s12011-022-03237-z 35524021
Liu Y, Huo B, Chen Z, Wang K, Huang L, Che L, et al. Effects of organic chromium yeast on performance, meat quality, and serum parameters of grow-finish pigs. Biol Trace Elem Res. 2023;201(3):1188–96. 10.1007/s12011-022-03237-z.35524021
38. Semeniuk W Grela ER Effect of a reduced protein content in the nutrition of growing-finishing pigs fed a restricted or ad libitum diet on nitrogen parameters in their blood and urine Med Weter Vet Med Sci Prac 2011 67 5 339 42
Semeniuk W, Grela ER. Effect of a reduced protein content in the nutrition of growing-finishing pigs fed a restricted or ad libitum diet on nitrogen parameters in their blood and urine. Med Weter Vet Med Sci Prac. 2011;67(5):339–42.
39. Li G Lee P Mori N Yamamoto I Arai T Long term intensive exercise training leads to a higher plasma malate/lactate dehydrogenase (M/L) ratio and increased level of lipid mobilization in horses Vet Res Commun 2012 36 2 149 55 10.1007/s11259-012-9515-0 22297553
Li G, Lee P, Mori N, Yamamoto I, Arai T. Long term intensive exercise training leads to a higher plasma malate/lactate dehydrogenase (M/L) ratio and increased level of lipid mobilization in horses. Vet Res Commun. 2012;36(2):149–55. 10.1007/s11259-012-9515-0.22297553
40. Chen Q Liu H Tan B Effects of dietary cholesterol level on growth performance, blood biochemical parameters and lipid metabolism of juvenile cobia (Rachycentron canadum) J Guangdong Ocean Univ 2016 36 1 35
Chen Q, Liu H, Tan B, et al. Effects of dietary cholesterol level on growth performance, blood biochemical parameters and lipid metabolism of juvenile cobia (Rachycentron canadum). J Guangdong Ocean Univ. 2016;36(1):35.
41. Arellano-García L Trepiana J Martínez JA Portillo MP Milton-Laskibar I Beneficial effects of viable and heat-inactivated Lactobacillus rhamnosus GG administration on oxidative stress and inflammation in diet-induced NAFLD in rats Antioxidants 2023 12 3 717 10.3390/antiox12030717 36978965
Arellano-García L, Trepiana J, Martínez JA, Portillo MP, Milton-Laskibar I. Beneficial effects of viable and heat-inactivated Lactobacillus rhamnosus GG administration on oxidative stress and inflammation in diet-induced NAFLD in rats. Antioxidants. 2023;12(3):717. 10.3390/antiox12030717.36978965
42. Huang X-J Choi Y-K Im H-S Yarimaga O Yoon E Kim H-S Aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) detection techniques Sensors 2006 6 7 756 10.3390/s6070756
Huang X-J, Choi Y-K, Im H-S, Yarimaga O, Yoon E, Kim H-S. Aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) detection techniques. Sensors. 2006;6(7):756. 10.3390/s6070756.
43. Vasquez R Oh JK Song JH Kang D-K Gut microbiome-produced metabolites in pigs: a review on their biological functions and the influence of probiotics J Anim Sci Technol 2022 64 4 671 95 10.5187/jast.2022.e58 35969697
Vasquez R, Oh JK, Song JH, Kang D-K. Gut microbiome-produced metabolites in pigs: a review on their biological functions and the influence of probiotics. J Anim Sci Technol. 2022;64(4):671–95. 10.5187/jast.2022.e58.35969697
44. Stojanov S Berlec A Štrukelj B The influence of probiotics on the Firmicutes/Bacteroidetes ratio in the treatment of obesity and inflammatory bowel disease Microorganisms 2020 8 11 1715 10.3390/microorganisms8111715 33139627
Stojanov S, Berlec A, Štrukelj B. The influence of probiotics on the Firmicutes/Bacteroidetes ratio in the treatment of obesity and inflammatory bowel disease. Microorganisms. 2020;8(11):1715. 10.3390/microorganisms8111715.33139627
45. Xu XX Dai M Lao F Chen F Hu X Liu Y Effect of glucoraphanin from broccoli seeds on lipid levels and gut microbiota in high-fat diet-fed mice J Func Foods 2020 68 103858 10.1016/j.jff.2020.103858
Xu XX, Dai M, Lao F, Chen F, Hu X, Liu Y, et al. Effect of glucoraphanin from broccoli seeds on lipid levels and gut microbiota in high-fat diet-fed mice. J Func Foods. 2020;68:103858. 10.1016/j.jff.2020.103858.
46. He W Gao Y Guo Z Yang Z Wang X Liu H Effects of fermented wheat bran and yeast culture on growth performance, immunity, and intestinal microflora in growing-finishing pigs J Anim Sci 2021 99 11 skab308 10.1093/jas/skab308 34687291
He W, Gao Y, Guo Z, Yang Z, Wang X, Liu H, et al. Effects of fermented wheat bran and yeast culture on growth performance, immunity, and intestinal microflora in growing-finishing pigs. J Anim Sci. 2021;99(11):skab308. 10.1093/jas/skab308.34687291
47. Shin NR Whon TW Bae JW Proteobacteria: microbial signature of dysbiosis in gut microbiota Trends Biotechnol 2015 33 9 496 503 10.1016/j.tibtech.2015.06.011 26210164
Shin NR, Whon TW, Bae JW. Proteobacteria: microbial signature of dysbiosis in gut microbiota. Trends Biotechnol. 2015;33(9):496–503. 10.1016/j.tibtech.2015.06.011.26210164
48. Mendes EN Queiroz DM Rocha GA Nogueira AM Carvalho AC Lage AP Histopathological study of porcine gastric mucosa with and without a spiral bacterium (Gastrospirillum suis) J Med Microbiol 1991 35 6 345 10.1099/00222615-35-6-345 1753392
Mendes EN, Queiroz DM, Rocha GA, Nogueira AM, Carvalho AC, Lage AP, et al. Histopathological study of porcine gastric mucosa with and without a spiral bacterium (Gastrospirillum suis). J Med Microbiol. 1991;35(6):345. 10.1099/00222615-35-6-345.1753392
49. Yang H Yang M Fang S Huang X He M Ke S Evaluating the profound effect of gut microbiome on host appetite in pigs BMC Microbiol 2018 18 1 215 10.1186/s12866-018-1364-8 30547751
Yang H, Yang M, Fang S, Huang X, He M, Ke S, et al. Evaluating the profound effect of gut microbiome on host appetite in pigs. BMC Microbiol. 2018;18(1):215. 10.1186/s12866-018-1364-8.30547751
50. Ellekilde M Selfjord E Larsen CS Jakesevic M Rune I Tranberg B Transfer of gut microbiota from lean and obese mice to antibiotic-treated mice Sci Rep 2014 4 5922 10.1038/srep05922 25082483
Ellekilde M, Selfjord E, Larsen CS, Jakesevic M, Rune I, Tranberg B, et al. Transfer of gut microbiota from lean and obese mice to antibiotic-treated mice. Sci Rep. 2014;4:5922. 10.1038/srep05922.25082483
51. Anguita M, Canibe N, Pérez JF, Jensen. 2006. Influence of the amount of dietary fiber on the available energy from hindgut fermentation in growing pigs: use of cannulated pigs and in vitro fermentation. J Anim Sci. 2006;84(10):2766. 10.2527/jas.2005-212.
52. Chen T Long W Zhang C Liu S Zhao L Hamaker BR Fiber-utilizing capacity varies in Prevotella- versus Bacteroides-dominated gut microbiota Sci Rep 2017 7 1 2594 10.1038/s41598-017-02995-4 28572676
Chen T, Long W, Zhang C, Liu S, Zhao L, Hamaker BR. Fiber-utilizing capacity varies in Prevotella- versus Bacteroides-dominated gut microbiota. Sci Rep. 2017;7(1):2594. 10.1038/s41598-017-02995-4.28572676
53. Wu F Guo X Zhang J Zhang M Ou Z Peng Y Phascolarctobacterium faecium abundant colonization in human gastrointestinal tract Exp Ther Med 2017 14 4 3122 6 10.3892/etm.2017.4878 28912861
Wu F, Guo X, Zhang J, Zhang M, Ou Z, Peng Y. Phascolarctobacterium faecium abundant colonization in human gastrointestinal tract. Exp Ther Med. 2017;14(4):3122–6. 10.3892/etm.2017.4878.28912861
54. Ratajczak W Rył A Mizerski A Walczakiewicz K Sipak O Laszczyńska M Immunomodulatory potential of gut microbiome-derived short-chain fatty acids (SCFAs) Acta Biochim 2019 66 1 1 12 10.18388/abp.2018_2648
Ratajczak W, Rył A, Mizerski A, Walczakiewicz K, Sipak O, Laszczyńska M. Immunomodulatory potential of gut microbiome-derived short-chain fatty acids (SCFAs). Acta Biochim. 2019;66(1):1–12. 10.18388/abp.2018_2648.
55. Wang J Fan H Han Y Zhao J Zhou Z Characterization of the microbial communities along the gastrointestinal tract of sheep by 454 pyrosequencing analysis Asian-Australas J Anim Sci 2017 30 1 100 10 10.5713/ajas.16.0166 27383798
Wang J, Fan H, Han Y, Zhao J, Zhou Z. Characterization of the microbial communities along the gastrointestinal tract of sheep by 454 pyrosequencing analysis. Asian-Australas J Anim Sci. 2017;30(1):100–10. 10.5713/ajas.16.0166.27383798
56. Roager HM Licht TR Poulsen SK Larsen TM Bahl MI Microbial enterotypes, inferred by the prevotella-to-bacteroides ratio, remained stable during a 6-month randomized controlled diet intervention with the new nordic diet Appl Environ Microbiol 2014 80 3 1142 10.1128/AEM.03549-13 24296500
Roager HM, Licht TR, Poulsen SK, Larsen TM, Bahl MI. Microbial enterotypes, inferred by the prevotella-to-bacteroides ratio, remained stable during a 6-month randomized controlled diet intervention with the new nordic diet. Appl Environ Microbiol. 2014;80(3):1142. 10.1128/AEM.03549-13.24296500
57. Hung DY Cheng YH Chen WJ Hua KF Pietruszka A Dybus A Bacillus licheniformis-fermented products reduce diarrhea incidence and alter the fecal microbiota community in weaning piglets Animals 2019 9 12 1145 10.3390/ani9121145 31847281
Hung DY, Cheng YH, Chen WJ, Hua KF, Pietruszka A, Dybus A, et al. Bacillus licheniformis-fermented products reduce diarrhea incidence and alter the fecal microbiota community in weaning piglets. Animals. 2019;9(12):1145. 10.3390/ani9121145.31847281
58. Ning K Lu K Chen Q Guo Z Du X Riaz F Epigallocatechin gallate protects mice against methionine-choline-deficient-diet-induced nonalcoholic steatohepatitis by improving gut microbiota to attenuate hepatic injury and regulate metabolism ACS Omega 2020 5 33 20800 9 10.1021/acsomega.0c01689 32875214
Ning K, Lu K, Chen Q, Guo Z, Du X, Riaz F, et al. Epigallocatechin gallate protects mice against methionine-choline-deficient-diet-induced nonalcoholic steatohepatitis by improving gut microbiota to attenuate hepatic injury and regulate metabolism. ACS Omega. 2020;5(33):20800–9. 10.1021/acsomega.0c01689.32875214
59. Wang J Wang P Li D Hu X Chen F Beneficial effects of ginger on prevention of obesity through modulation of gut microbiota in mice Eur J Nutr 2020 59 2 699 718 10.1007/s00394-019-01938-1 30859364
Wang J, Wang P, Li D, Hu X, Chen F. Beneficial effects of ginger on prevention of obesity through modulation of gut microbiota in mice. Eur J Nutr. 2020;59(2):699–718. 10.1007/s00394-019-01938-1.30859364
60. Li XL Zhang B Sun MJ Bao C-C Yuan B-Y Xie Q-F Wang L-J Wang M-X Mechanism of gut microbiota and Axl/SOCS3 in experimental autoimmune encephalomyelitis Biosci Rep 2019 39 7 BSR20190228 10.1042/BSR20190228 31221818
Li XL, Zhang B, Sun MJ, Bao C-C, Yuan B-Y, Xie Q-F, Wang L-J, Wang M-X. Mechanism of gut microbiota and Axl/SOCS3 in experimental autoimmune encephalomyelitis. Biosci Rep. 2019;39(7):BSR20190228. 10.1042/BSR20190228.31221818
61. Jalanka J Major G Murray K Singh G Nowak A Kurtz C The effect of psyllium husk on intestinal microbiota in constipated patients and healthy controls Int J Mol Sci 2019 20 2 433 10.3390/ijms20020433 30669509
Jalanka J, Major G, Murray K, Singh G, Nowak A, Kurtz C, et al. The effect of psyllium husk on intestinal microbiota in constipated patients and healthy controls. Int J Mol Sci. 2019;20(2):433. 10.3390/ijms20020433.30669509
62. Human Microbiome Project Consortium Structure, function and diversity of the healthy human microbiome Nature 2012 486 7402 207 14 10.1038/nature11234 22699609
Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature. 2012;486(7402):207–14. 10.1038/nature11234.22699609
63. Zhou L Zhang M Wang Y Dorfman RG Liu H Yu T Faecalibacterium prausnitzii produces butyrate to maintain Th17/Treg balance and to ameliorate colorectal colitis by inhibiting histone deacetylase 1 Inflamm Bowel Dis 2018 24 9 1926 40 10.1093/ibd/izy182 29796620
Zhou L, Zhang M, Wang Y, Dorfman RG, Liu H, Yu T, et al. Faecalibacterium prausnitzii produces butyrate to maintain Th17/Treg balance and to ameliorate colorectal colitis by inhibiting histone deacetylase 1. Inflamm Bowel Dis. 2018;24(9):1926–40. 10.1093/ibd/izy182.29796620
64. Quévrain E Maubert MA Michon C Chain F Marquant R Tailhades J Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn’s disease Gut 2016 65 3 415 25 10.1136/gutjnl-2014-307649 26045134
Quévrain E, Maubert MA, Michon C, Chain F, Marquant R, Tailhades J, et al. Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn’s disease. Gut. 2016;65(3):415–25. 10.1136/gutjnl-2014-307649.26045134
65. Rossi O Khan MT Schwarzer M Hudcovic T Srutkova D Duncan SH Faecalibacterium prausnitzii strain HTF-F and its extracellular polymeric matrix attenuate clinical parameters in DSS-induced colitis PLoS ONE 2015 10 4 e0123013 10.1371/journal.pone.0123013 25910186
Rossi O, Khan MT, Schwarzer M, Hudcovic T, Srutkova D, Duncan SH, et al. Faecalibacterium prausnitzii strain HTF-F and its extracellular polymeric matrix attenuate clinical parameters in DSS-induced colitis. PLoS ONE. 2015;10(4):e0123013. 10.1371/journal.pone.0123013.25910186
66. Martín R Bermúdez-Humarán LG Langella P Searching for the bacterial effector: the example of the multi-skilled commensal bacterium Faecalibacterium prausnitzii Front Microbiol 2018 9 346 10.3389/fmicb.2018.00346 29559959
Martín R, Bermúdez-Humarán LG, Langella P. Searching for the bacterial effector: the example of the multi-skilled commensal bacterium Faecalibacterium prausnitzii. Front Microbiol. 2018;9:346. 10.3389/fmicb.2018.00346.29559959
67. Seitz M Valentin-Weigand P Willenborg J Use of antibiotics and antimicrobial resistance in veterinary medicine as exemplified by the swine pathogen Streptococcus suis Curr Top Microbiol Immunol 2016 398 103 21 10.1007/82_2016_506 27738916
Seitz M, Valentin-Weigand P, Willenborg J. Use of antibiotics and antimicrobial resistance in veterinary medicine as exemplified by the swine pathogen Streptococcus suis. Curr Top Microbiol Immunol. 2016;398:103–21. 10.1007/82_2016_506.27738916
68. Shah M Ahmed Mir W Olowoyo O Chandna S Terrisporobacter glycolicus bacteremia in a patient with cirrhosis Consultant 2022 10.25270/con.2022.06.00009
Shah M, Ahmed Mir W, Olowoyo O, Chandna S. Terrisporobacter glycolicus bacteremia in a patient with cirrhosis. Consultant. 2022. 10.25270/con.2022.06.00009.
69. Cai C Zhang Z Morales M Wang Y Khafipour E Friel J Feeding practice influences gut microbiome composition in very low birth weight preterm infants and the association with oxidative stress: a prospective cohort study Free Radic Biol Med 2019 142 146 54 10.1016/j.freeradbiomed.2019.02.032 30851363
Cai C, Zhang Z, Morales M, Wang Y, Khafipour E, Friel J. Feeding practice influences gut microbiome composition in very low birth weight preterm infants and the association with oxidative stress: a prospective cohort study. Free Radic Biol Med. 2019;142:146–54. 10.1016/j.freeradbiomed.2019.02.032.30851363
70. Chen C Huang X Fang S Yang H He M Zhao Y Contribution of host genetics to the variation of microbial composition of cecum lumen and feces in pigs Front Microbiol 2018 9 2626 10.3389/fmicb.2018.02626 30429843
Chen C, Huang X, Fang S, Yang H, He M, Zhao Y, et al. Contribution of host genetics to the variation of microbial composition of cecum lumen and feces in pigs. Front Microbiol. 2018;9:2626. 10.3389/fmicb.2018.02626.30429843
71. Waters JL Ley RE The human gut bacteria Christensenellaceae are widespread, heritable, and associated with health BMC Biol 2019 17 1 83 10.1186/s12915-019-0699-4 31660948
Waters JL, Ley RE. The human gut bacteria Christensenellaceae are widespread, heritable, and associated with health. BMC Biol. 2019;17(1):83. 10.1186/s12915-019-0699-4.31660948
72. Kaper JB Nataro JP Mobley HL Pathogenic Escherichia coli Nat Rev Microbiol 2004 2 2 123 40 10.1038/nrmicro818 15040260
Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2(2):123–40. 10.1038/nrmicro818.15040260
73. Niyogi SK Shigellosis J Microbiol 2005 43 2 133 43 15880088
Niyogi SK, Shigellosis. J Microbiol. 2005;43(2):133–43.15880088
74. Li S Guo J Liu R Zhang F Wen S Liu Y Predominance of Escherichia-Shigella in gut microbiome and its potential correlation with elevated level of plasma tumor necrosis factor alpha in patients with tuberculous meningitis Microbiol Spectr 2022 10 6 e0192622 10.1128/spectrum.01926-22 36350161
Li S, Guo J, Liu R, Zhang F, Wen S, Liu Y, et al. Predominance of Escherichia-Shigella in gut microbiome and its potential correlation with elevated level of plasma tumor necrosis factor alpha in patients with tuberculous meningitis. Microbiol Spectr. 2022;10(6):e0192622. 10.1128/spectrum.01926-22.36350161
75. Sun X Jia Z Microbiome modulates intestinal homeostasis against inflammatory diseases Vet Immunol Immunopathol 2018 205 97 105 10.1016/j.vetimm.2018.10.014 30459007
Sun X, Jia Z. Microbiome modulates intestinal homeostasis against inflammatory diseases. Vet Immunol Immunopathol. 2018;205:97–105. 10.1016/j.vetimm.2018.10.014.30459007
76. Haskins N Panglao M Qu Q Majumdar H Cabrera-Luque J Morizono H Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods BMC Biochem 2008 9 24 10.1186/1471-2091-9-24 18801197
Haskins N, Panglao M, Qu Q, Majumdar H, Cabrera-Luque J, Morizono H, et al. Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods. BMC Biochem. 2008;9:24. 10.1186/1471-2091-9-24.18801197
77. Diez-Fernandez C Martínez AI Pekkala S Barcellona B Pérez-Arellano I Guadalajara AM Molecular characterization of carbamoyl-phosphate synthetase (CPS1) deficiency using human recombinant CPS1 as a key tool Hum Mutat 2013 34 8 1149 59 10.1002/humu.22349 23649895
Diez-Fernandez C, Martínez AI, Pekkala S, Barcellona B, Pérez-Arellano I, Guadalajara AM, et al. Molecular characterization of carbamoyl-phosphate synthetase (CPS1) deficiency using human recombinant CPS1 as a key tool. Hum Mutat. 2013;34(8):1149–59. 10.1002/humu.22349.23649895
78. Rodwell VW. Conversion of amino acids to specialized products. Rodwell VW, Bender DA, Botham KM, Kennelly PJ, Weil P, editors Harper’s Illustrated Biochemistry, 2018, 31e. McGraw Hill. https://accesspharmacy.mhmedical.com/content.aspx?bookid=2386&sectionid=187833183
79. Hayase K Yoshida A Role of ornithine in the N-acetylglutamate turnover in the liver of rats Biosci Biotechnol Biochem 1999 63 3 506 9 10.1271/bbb.63.506 10227137
Hayase K, Yoshida A. Role of ornithine in the N-acetylglutamate turnover in the liver of rats. Biosci Biotechnol Biochem. 1999;63(3):506–9. 10.1271/bbb.63.506.10227137
80. Sugino T Shirai T Kajimoto Y Kajimoto O L-ornithine supplementation attenuates physical fatigue in healthy volunteers by modulating lipid and amino acid metabolism Nutr Res 2008 28 11 738 43 10.1016/j.nutres.2008.08.008 19083482
Sugino T, Shirai T, Kajimoto Y, Kajimoto O. L-ornithine supplementation attenuates physical fatigue in healthy volunteers by modulating lipid and amino acid metabolism. Nutr Res. 2008;28(11):738–43. 10.1016/j.nutres.2008.08.008.19083482
81. Bartuś M Łomnicka M Kostogrys RB Kazmierczak P Watala C Slominska EM 1-Methylnicotinamide (MNA) prevents endothelial dysfunction in hypertriglyceridemic and diabetic rats Pharmacol Rep 2008 60 1 127 18276994
Bartuś M, Łomnicka M, Kostogrys RB, Kazmierczak P, Watala C, Slominska EM, et al. 1-Methylnicotinamide (MNA) prevents endothelial dysfunction in hypertriglyceridemic and diabetic rats. Pharmacol Rep. 2008;60(1):127.18276994
82. Brzozowski T Konturek PC Chlopicki S Sliwowski Z Pawlik M Ptak-Belowska A Therapeutic potential of 1-methylnicotinamide against acute gastric lesions induced by stress: role of endogenous prostacyclin and sensory nerves J Pharmacol Exp Ther 2008 326 1 105 16 10.1124/jpet.108.136457 18385449
Brzozowski T, Konturek PC, Chlopicki S, Sliwowski Z, Pawlik M, Ptak-Belowska A, et al. Therapeutic potential of 1-methylnicotinamide against acute gastric lesions induced by stress: role of endogenous prostacyclin and sensory nerves. J Pharmacol Exp Ther. 2008;326(1):105–16. 10.1124/jpet.108.136457.18385449
83. Leoni V Masterman T Mousavi FS Wretlind B Wahlund L-O Diczfalusy U Diagnostic use of cerebral and extracerebral oxysterols Clin Chem Lab Med 2004 42 2 186 91 10.1515/CCLM.2004.034 15061359
Leoni V, Masterman T, Mousavi FS, Wretlind B, Wahlund L-O, Diczfalusy U, et al. Diagnostic use of cerebral and extracerebral oxysterols. Clin Chem Lab Med. 2004;42(2):186–91. 10.1515/CCLM.2004.034.15061359
84. Lütjohann D von Bergmann K 24S-hydroxycholesterol: a marker of brain cholesterol metabolism Pharmacopsychiatry 2003 36 S102 6 10.1055/s-2003-43053 14574622
Lütjohann D, von Bergmann K. 24S-hydroxycholesterol: a marker of brain cholesterol metabolism. Pharmacopsychiatry. 2003;36:S102–6. 10.1055/s-2003-43053.14574622
85. Li J Li T Bile acid receptors link nutrient sensing to metabolic regulation Liver Res 2017 1 1 17 25 10.1016/j.livres.2017.04.001 29098111
Li J, Li T. Bile acid receptors link nutrient sensing to metabolic regulation. Liver Res. 2017;1(1):17–25. 10.1016/j.livres.2017.04.001.29098111
86. Šarenac TM Mikov M Bile acid synthesis: from nature to the chemical modification and synthesis and their applications as drugs and nutrients Front Pharmacol 2018 9 939 10.3389/fphar.2018.00939 30319399
Šarenac TM, Mikov M. Bile acid synthesis: from nature to the chemical modification and synthesis and their applications as drugs and nutrients. Front Pharmacol. 2018;9:939. 10.3389/fphar.2018.00939.30319399
87. Staley C Weingarden AR Khoruts A Sadowsky MJ Interaction of gut microbiota with bile acid metabolism and its influence on disease states Appl Microbiol Biotechnol 2017 101 1 47 64 10.1007/s00253-016-8006-6 27888332
Staley C, Weingarden AR, Khoruts A, Sadowsky MJ. Interaction of gut microbiota with bile acid metabolism and its influence on disease states. Appl Microbiol Biotechnol. 2017;101(1):47–64. 10.1007/s00253-016-8006-6.27888332
88. Stark AH Crawford MA Reifen R Update on alpha-linolenic acid Nutr Rev 2008 66 6 326 32 10.1111/j.1753-4887.2008.00040.x 18522621
Stark AH, Crawford MA, Reifen R. Update on alpha-linolenic acid. Nutr Rev. 2008;66(6):326–32. 10.1111/j.1753-4887.2008.00040.x.18522621
89. Sierra S Lara-Villoslada F Comalada M Olivares M Xaus J Dietary eicosapentaenoic acid and docosahexaenoic acid equally incorporate as decosahexaenoic acid but differ in inflammatory effects Nutrition 2008 24 3 245 54 10.1016/j.nut.2007.11.005 18312787
Sierra S, Lara-Villoslada F, Comalada M, Olivares M, Xaus J. Dietary eicosapentaenoic acid and docosahexaenoic acid equally incorporate as decosahexaenoic acid but differ in inflammatory effects. Nutrition. 2008;24(3):245–54. 10.1016/j.nut.2007.11.005.18312787
90. Kapoor R Huang YS Gamma linolenic acid: an antiinflammatory omega-6 fatty acid Curr Pharm Biotechnol 2006 7 6 531 4 10.2174/138920106779116874 17168669
Kapoor R, Huang YS. Gamma linolenic acid: an antiinflammatory omega-6 fatty acid. Curr Pharm Biotechnol. 2006;7(6):531–4. 10.2174/138920106779116874.17168669
91. Zamaria N Alteration of polyunsaturated fatty acid status and metabolism in health and disease Reprod Nutr Dev 2004 44 3 273 82 10.1051/rnd:2004034 15460166
Zamaria N. Alteration of polyunsaturated fatty acid status and metabolism in health and disease. Reprod Nutr Dev. 2004;44(3):273–82. 10.1051/rnd:2004034.15460166
92. Qu H Zong L Sang J Wa Y Chen D Huang Y Effect of Lactobacillus rhamnosus hsryfm 1301 fermented milk on lipid metabolism disorders in high-fat-diet rats Nutrients 2022 14 22 4850 10.3390/nu14224850 36432537
Qu H, Zong L, Sang J, Wa Y, Chen D, Huang Y, et al. Effect of Lactobacillus rhamnosus hsryfm 1301 fermented milk on lipid metabolism disorders in high-fat-diet rats. Nutrients. 2022;14(22):4850. 10.3390/nu14224850.36432537
93. Guo G Wu Y Liu Y Wang Z Xu G Wang X Exploring the causal effects of the gut microbiome on serum lipid levels: a two-sample mendelian randomization analysis Front Microbiol 2023 14 1113334 10.3389/fmicb.2023.1113334 36876057
Guo G, Wu Y, Liu Y, Wang Z, Xu G, Wang X, et al. Exploring the causal effects of the gut microbiome on serum lipid levels: a two-sample mendelian randomization analysis. Front Microbiol. 2023;14:1113334. 10.3389/fmicb.2023.1113334.36876057
94. Hamm MW Mehansho H Henderson LM Transport and metabolism of pyridoxamine and pyridoxamine phosphate in the small intestine of the rat J Nutr 1979 109 9 1552 9 10.1093/jn/109.9.1552 479950
Hamm MW, Mehansho H, Henderson LM. Transport and metabolism of pyridoxamine and pyridoxamine phosphate in the small intestine of the rat. J Nutr. 1979;109(9):1552–9. 10.1093/jn/109.9.1552.479950
95. Liu G Sun C Liu H Li F Zhu Y Li F Effects of dietary supplement of vitamin B6 on growth performance and non-specific immune response of weaned rex rabbits J Appl Anim Res 2018 46 1 1370 6 10.1080/09712119.2018.1512498
Liu G, Sun C, Liu H, Li F, Zhu Y, Li F. Effects of dietary supplement of vitamin B6 on growth performance and non-specific immune response of weaned rex rabbits. J Appl Anim Res. 2018;46(1):1370–6. 10.1080/09712119.2018.1512498.
96. Thompson DA Hammock BD Dihydroxyoctadecamonoenoate esters inhibit the neutrophil respiratory burst J Biosci 2007 32 2 279 10.1007/s12038-007-0028-x 17435320
Thompson DA, Hammock BD. Dihydroxyoctadecamonoenoate esters inhibit the neutrophil respiratory burst. J Biosci. 2007;32(2):279. 10.1007/s12038-007-0028-x.17435320
