Fermented Chinese Herbs Improve the Growth and Immunity of Growing Pigs through Regulating Colon Microbiota and Metabolites

Simple Summary Developing new antibiotic substitutes to promote pig growth and health, as well as new feed to improve feed utilization and reduce manure production during the breeding process, have become important ways to solve the current dilemma and promote the pig industry. In this study, we have documented that adding a 2% and 3% digestion-promoting fermented Chinese herbal formula to feed effectively promoted the growth of growing pigs, improved serum immune and antioxidant activities, ameliorated the structure of colonic microbiota, and altered the composition of metabolites in colon contents. In particular, adding a 2% digestion-promoting fermented Chinese herbal formula to feed effectively reduced opportunistic pathogens and increased potential probiotics in the colonic microbiota. This study provides experimental data for the application and feed development of digestion-promoting fermented Chinese herbs for pig production and expands the understanding of the impact of fermented Chinese herbs on the host’s gut microbiota and metabolism. Abstract (1) Background: the development of new antibiotic substitutes to promote pig growth and health has become an important way to solve the current dilemma and promote the pig industry. (2) Methods: to assess the effects of a fermented Chinese herbal (FCH) formula on the growth and immunity of growing pigs, 100 Duroc × Landrace × Yorshire three-way crossed growing pigs were randomly divided into control and treatment groups that were fed a basal diet, and a basal diet with 1% (group A), 2% (group B), and 3% (group C) FCH formulas, respectively. A sixty-day formal experiment was conducted, and their growth and serum indices, colonic microbiota, and metabolites were analyzed. (3) Results: the daily gain of growing pigs in groups A, B, and C increased by 7.93%, 17.68%, and 19.61%, respectively, and the feed-to-gain ratios decreased by 8.33%, 15.00%, and 14.58%, respectively. Serum immunity and antioxidant activities were significantly increased in all treatment groups. Particularly, adding a 2% FCH formula significantly changed the colon’s microbial structure; the Proteobacteria significantly increased and Firmicutes significantly decreased, and the metabolite composition in the colon’s contents significantly changed. (4) Conclusions: these results indicate that the FCH formula is a good feed additive for growing pigs, and the recommended addition ratio was 3%.


Introduction
Pork is the second most preferred meat for consumption globally because of its low relative price [1][2][3].In total, pork production is expected to grow by approximately 11 Mt Animals 2023, 13, 3867 2 of 18 by 2028 [3].Large-scale farming is an important method for the sustainable development of the pig industry because it reduces the labor costs of raising pigs [4].However, increased environmental and public concerns regarding manure management and welfare are expected to limit production growth [5,6].Developing new antibiotic substitutes to promote pig growth and health, as well as new feed to improve feed utilization and reduce manure production during the breeding process, have become important ways to solve the current dilemma and promote the pig industry [7][8][9][10][11][12].
Chinese herbs have been used to cure diseases for thousands of years.However, the bioactive ingredients in Chinese herbs are complex, and some natural Chinese herbal products cannot be directly absorbed by humans and animals.Moreover, the contents of most bioactive ingredients in Chinese herbs are low, and some natural products are toxic to humans and animals [24].Fermentation is a Chinese herb processing technique based on the action of microorganisms under proper temperature, humidity, and moisture conditions that enhances their original properties and produces new effects, expanding their scope of application to meet clinical demands [25].Furthermore, the fermentation of Chinese herbs can enhance Chinese herb bioactivities and decrease potential toxicities [24].Recently, fermented Chinese herbs (FCHs) have been shown to exhibit growth-promoting, immune-enhancing, and disease-resistant properties [26][27][28].
The intestinal microbiota plays an important role in host growth [29], immunity [30,31], development [32,33], metabolism [34,35], and health [36,37].Visconti et al. [38] estimated that the microbiome was involved in a dialogue between 71% of fecal and 15% of blood metabolites based on whole metagenome shotgun sequencing data in 1004 twins.Atarashi et al. [39] reported that the spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted T reg cell accumulation in mice colonic mucosa.They also found that the colonization of mice via a defined mix of Clostridium strains provided an environment rich in transforming growth factor-β, and affected Foxp3 + T reg number and function in the mouse colon [39].In promoting host growth, Qi et al. [40] reported that the body weight and lean mass of germ-free piglets were approximately 40% lower than those of normal piglets, and the deletion of the intestinal microbiota led to weakened muscle function and a reduction in myogenic regulatory proteins.Moreover, they also found that gut microbiota introduced into germfree piglets via fecal microbiota transplantation not only colonized the gut but also partially restored muscle growth and development [40].Furthermore, many probiotics that promote host growth or health have been isolated from intestinal microbiota, such as Lactobacillus strains [41,42] and Bacillus species [43,44].Notably, the therapeutic effects of Chinese herbs on various diseases are related to the regulation of intestinal microbiota [45][46][47][48].FCHs also promote growth and intestinal health and regulate the intestinal microbiota of weaned piglets [27,49] and chicks [50,51].Therefore, we speculated that the addition of digestion-promoting FCHs to feed could improve the digestion, absorption rate, and growth of pigs, and this effect may be related to gut microbiota and metabolism.To test this hypothesis, in this study, we analyzed the effect of a digestion-promoting FCH formula on growth, serum biochemical indicators, colon microbiota, and the metabolome of the colon contents of growing pigs.This study provides experimental data for the application and feed development of digestion-promoting FCHs for pig production.

Animals and Experimental Design
This animal study was approved by the Biomedical Research Ethics Committee of Hunan Agricultural University (approval number: Lunshenke 2023 No. 105) and conducted in accordance with its guidelines.
A total of 100 Duroc × Landrace × Yorshire three-way crossed growing pigs (25.75 ± 0.14 kg) were divided into four groups with five replicates per group and five pigs per replicate according to the principle of consistent average body weight and sex.Pigs in the control group were fed a basal diet (Table 1), and the diets in groups A, B, and C were provided a basal diet with 1%, 2%, and 3% FCHs, respectively.  The metabolizable energy datum was calculated, and the other nutrient components were actual measured values. 3ADF, acid detergent fiber. 4NDF, neutral detergent fiber.
The study lasted for 65 days, including a pre-feeding period of 5 days, and 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 ad libitum feeding environment.Immunization and disinfection were performed in strict accordance with pig farm management practices.

Determination of Growth Performance
The feed and remaining feed were weighed daily during the experiment to calculate the average daily feed intake (ADFI).The piglets with empty stomachs were weighed before feeding on the first and last mornings of the formal experiment.The average daily gain (ADG) and feed/gain ratio (F/G) were calculated based on the data collected in the experiment.

Colon Microbiota Analysis
The colon contents of the growing pigs were collected for 16S rRNA sequencing.The microbial DNA was extracted using a fecal genome DNA extraction kit (Tiangen, Beijing, China), and its concentration and purity were determined using a NanoDrop2000 ultramicro spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).The prokaryoticspecific primers of the 16S rRNA gene V3-V4 region were selected for PCR amplification of the colon contents [52].Novaseq6000 (Illumina, Shanghai, China) was used for sequencing.
The raw reads were merged using FLASH 1.2.11.Quality control was conducted using Trimmomatic 0.3.UCHIME 8.1 was used to remove the chimera sequences to obtain high-quality sequences.QIIME2 2020.6 was used to evaluate alpha diversity, beta diversity, and the taxonomy of the colon microbiota.

Non-Target Metabolomics Analysis
The colon content metabolomes were determined using the UHPLC-QTOF-MS method on the LC-QTOF platform from Biomarker Technology Co., Ltd.(Beijing, China).Chromatography was performed using a Xevo G2-XS QT high-resolution mass spectrometer (Waters, Milford, CT, USA) with a 1.8 µm 2.1 × 100 mm Acquity UPLC HSS T3 column (Waters, Milford, CT, USA).The raw data were obtained using MassLynx V4.2 (Waters, Milford, CT, USA).Progenesis QI (Waters, Milford, CT, USA) was used for the peak extraction and peak alignment of the raw metabolomic data.The theoretical fragments were identified based on the online METLIN database of the Progenesis QI version 4.0 software and the self-built database of Biomarker Technology (Beijing, China).The metabolic pathway enrichment analysis and functional annotation were performed using the Kyoto Encyclopedia of Genes and Genomes database (KEGG) (https://www.genome.jp/kegg/(accessed on 20 September 2023)).

Data Analysis
The data are presented as the mean ± standard error.Bartlett's homogeneity test of variance and a one-way ANOVA were conducted using SPSS Statistics 26.0 (IBM Corporation, Armonk, NY, USA).Waller-Duncan, LSD, and Tookey s-b post-hoc tests were used for multiple comparisons analysis.The statistical significance was set at p < 0.05.Histograms and boxplots were obtained using R 4.2.3 with the ggpubr package.Spearman's correlation coefficient was used to analyze the relationship between the colon microbiota and metabolites, and p < 0.05 and R > 0.60 was considered significant correlation.

Effects of FCHs on the Growth Performance and Serum Physiological and Biochemical Indicators of Growing Pigs
There was no significant difference in the IBW of growing pigs among the groups (F = 1.036, p = 0.398; Figure 1A).Although there was no significant difference in the ADFI of growing pigs among the groups during the experiment (F = 0.531, p = 0.662; Figure 1D), the ADGs of growing pigs in groups A, B, and C significantly increased by 7.93%, 17.68%, and 19.61%, respectively, compared with the controls.The FBWs and ADGs of groups B and C were significantly higher than those of the control (p < 0.05; Figure 1B,C).Moreover, the F/G ratios in groups A, B, and C decreased by 8.33%, 15.00%, and 14.58%, respectively, compared to the controls, and the F/G ratios in groups B and C were significantly lower than those in the control (F = 5.847, p < 0.05; Figure 1E).However, the FBW, ADG, and F/G were not significantly different between groups B and C (p > 0.05; Figure 1).The levels of serum TP, ALB, and HDL-Chol in all treatment groups were significantly increased compared to the controls (p < 0.01; Figure 2A,B,G), whereas the serum BUN, TG, LDL-Chol, GOT, and ALT levels were significantly decreased (p < 0.01; Figure 2C,D,F,H,I).However, the serum T-Chol levels were not significantly different (p > 0.05; Figure 2E).The levels of serum IgA, IgG, and IgM in all treatment groups were significantly higher than those in the control group (p < 0.05; Figure 2J-L).However, there was no significant difference in LZM activity (p = 0.278; Figure 2M).The activities of serum T-AOC, GSH-Px, and T-SOD in the treatment groups were significantly increased compared to those in the controls (p < 0.01; Figure 2N-P), whereas the MDA content was significantly decreased (p < 0.01; Figure 2Q).No significant difference was observed between groups B and C (p > 0.05; Figure 2N,P,Q), except for GSH-Px activity (p < 0.05; Figure 2O).cantly higher than those in the control group (p < 0.05; Figure 2J-L).However, there was no significant difference in LZM activity (p = 0.278; Figure 2M).The activities of serum T-AOC, GSH-Px, and T-SOD in the treatment groups were significantly increased compared to those in the controls (p < 0.01; Figure 2N-P), whereas the MDA content was significantly decreased (p < 0.01; Figure 2Q).No significant difference was observed between groups B and C (p > 0.05; Figure 2N,P,Q), except for GSH-Px activity (p < 0.05; Figure 2O).cantly higher than those in the control group (p < 0.05; Figure 2J-L).However, there was no significant difference in LZM activity (p = 0.278; Figure 2M).The activities of serum T-AOC, GSH-Px, and T-SOD in the treatment groups were significantly increased compared to those in the controls (p < 0.01; Figure 2N-P), whereas the MDA content was significantly decreased (p < 0.01; Figure 2Q).No significant difference was observed between groups B and C (p > 0.05; Figure 2N,P,Q), except for GSH-Px activity (p < 0.05; Figure 2O).

Effect of FCHs on the Colonic Microbiota of Growing Pigs
To assess the effect of FCHs on the colonic microbiota of growing pigs, the colonic microbiota in group B and the control group were analyzed.A total of 1,573,864 (157,386.4± 6517.70 per sample) effective merged tags were obtained from ten samples (five samples for each group).A total of 5453 amplicon sequence variants (ASVs) were detected.The rarefaction curve reached a plateau after sequencing more than 100,000 reads per sample, indicating sufficient sequencing data (Figure 3A).The abundance-based coverage estimators (ACE), Chao1, Shannon, and Simpson indices in group B were significantly elevated compared with those in the control group (p < 0.05: Figure 3B-E).The principal coordinates analysis indicated that the microbial structures in the colon were significantly different between the treatment and control groups (Figure 3E).Taxa with LDA > 3.0 and p < 0.05 were considered significant differences; the prefixes "f" and "g" represent the annotated family and genus levels, respectively.Pigs in the control group were fed a basal diet, and the diet of group B was the basal diet with 2% FCH.* p < 0.05; *** p < 0.001.

Discussion
Fermented feed is beneficial to the growth and production of pigs [53], and fermented feed additives have a positive effect on the intestinal microbiota of pigs by promoting the proliferation of beneficial bacteria and reducing the number of potentially harmful bacteria [54].The reduction in pH after the fermentation of compound Chinese herbs may be beneficial for inhibiting the growth and colonization of pathogens [55].Our results indicated that adding different doses of FCHs to the basal diet improved the growth performance and feed efficiency of growing pigs.The ADG of growing pigs increased with increasing FCH content, and the ADFI also showed an increasing trend, consistent with the results of Shi et al. [56].The improvement in growth performance may be due to the optimization of feed palatability and the improvement in intestinal enzyme activity and feed efficiency after the fermentation of Chinese herbs [57].
The serum TP, ALB, and BUN levels are used to indicate the body's protein metabolism and synthesis ability, which are closely related to the body's growth performance.Our results showed that adding 2% FCHs to the basal diet significantly increased serum TP and ALB and decreased the serum BUN concentrations, consistent with the results of Sun et al. [58].The main function of serum LDL-Chol is to transfer endogenous cholesterol to the extrahepatic tissues.The main function of serum HDL-Chol is cholesterol reverse transport, which can transport cholesterol from extrahepatic tissues to the liver for metabolism and is then excreted through the bile.The increase in LDL-Chol concentration increases the risk of coronary heart disease and directly promotes atherosclerosis formation [59].Our results showed that the levels of serum LDL-Chol and HDL-Chol in growing pigs in each treatment group were significantly improved compared to those in the control group.The GOT and ALT levels were negatively correlated with liver and cardiac functions.Elevated levels of these two enzymes reflect organ injury or disease, and a large number of intracellular enzymes enter the blood, resulting in elevated serum enzyme activities [60].Our results indicated that the serum GOT and ALT levels of growing pigs in each treatment group were significantly reduced, indicating that consumption of FCHs had a protective effect on the liver and heart.These results are consistent with the report by Parimoo et al. [61].
The serum antioxidant index is an important indicator of animal health [62].Dowarah et al. [63] proved that the improvement in the nutrient digestibility and growth performance of pigs was closely related to the improvement in antioxidant status.The use of Bacillus licheniformis to process Carthamus tinctorius L. can hydrolyze the glycosides to aglycogens, which significantly improves the body's ability to eliminate hydroxyl radicals, and inhibits the production of liver oxides, and the symptoms of erythrocyte hemolysis [64].T-SOD and GSH-Px are important antioxidant enzymes that help cells resist free radical damage [65].T-AOC represents the total antioxidant level composed of various antioxidant substances and enzymes, indicating its ability to resist oxidative damage caused by reactive oxygen species [66].MDA is naturally produced in vivo as a final product of membrane lipid peroxidation, and its elevated concentration indicates cell membrane damage [67].Moreover, the medicinal active substances of Chinese herbs can promote lymphocyte synthesis and improve immunoglobulin levels and phagocytic ability [68].Serum IgA, IgG, and IgM are important components of mammalian humoral immunity, which are the main immunoglobulins that protect the body against pathogen and virus invasion [69].Serum LZM is a natural "bacteriostatic agent" of the body.As a part of the immune defense mechanism in the body, it participates in a variety of nonspecific immune responses, cracking the peptidoglycan chemical bonds of bacterial cell walls and causing them to break and dissolve to achieve bactericidal and anti-inflammatory effects [70].Additionally, LZM exhibits immunomodulatory properties by regulating IgA production in the intestinal mucosa and serum of pigs [71].Our results showed that dietary supplementation with 2% or 3% FCHs significantly increased serum T-SOD, GSH-Px, T-AOC, IgA, IgG, and IgM levels, and significantly decreased the serum MDA concentration in growing pigs.These results indicate that adding FCHs to the basal diet of growing pigs improves serum antioxidant, biochemical, and immune indices, which is beneficial to the health of growing pigs.
A healthy intestinal microbiota is important for the intestinal absorption and development of the immune system of growing pigs [72].Bunte et al. [73] reported that feeding pigs with fermented feed improved the α-diversity of their gut microbiota.Wei et al. [74] reported that higher α-diversity is one of the indicators of improved growth performance in pigs.Our results showed that dietary supplementation with 2% FCHs significantly increased colonic microbiota richness and Shannon and ACE indices.Bacteroides in the intestinal tract can use polysaccharides, which can enhance the host's nutrient absorption capacity and help the formation of intestinal mucosa [75].Although previous studies have shown that obesity is associated with a high ratio of Firmicutes to Bacteroidetes [76], the interaction between host nutrient absorption and gut microbial composition is extremely complex and not directly related to the ratio of Firmicutes to Bacteroidetes [77].Rajput et al. [78] found that the IgA level was negatively correlated with the ratio of Firmicutes to Bacteroidetes.The IgA level was negatively correlated with the abundance of Firmicutes and positively correlated with the abundances of Bacteroidetes and Desulfobacterota, which indicates that a higher Firmicutes/Bacteroidetes ratio may be associated with poor mucosal humoral immunity.Otherwise, the decrease in the Firmicutes/Bacteroidetes ratio in the colonic contents increased the serum LZM level in growing pigs [79].Additionally, Bacteroidetes were more abundant in the intestines of pigs with higher feed efficiency [80,81].Our results showed that 2% FCH supplementation significantly increased the relative abundance of Bacteroidetes and decreased that of Firmicutes, which might be correlated to the significantly increased levels of serum IgA and LZM.
Prevotella is one of the most important genera in the gut of growing pigs [82] and is positively associated with the host's high cellulose intake, polysaccharide metabolism [83], feed efficiency [84], and body weight gain [85].Phascolarctobacterium is a gram negative anaerobic bacterium that degrades dietary fiber [86,87].Its decreased abundance is usually associated with inflammatory diseases, and its increased abundance is beneficial in reducing the occurrence of colitis [88].Quinella is a common propionate-producing bacterium in the gut that plays a role in host nutrient uptake and utilization [89].Sphaerochaeta is an intestinal short-chain fatty acid (SCFA)-producing bacterium that is highly enriched in genes involved in fermentation and carbohydrate metabolism [90].It produces a variety of carbohydrate enzymes (for example, β-xylosidases of the GH family process carbohydrates), rendering it an important contribution to lipid regulation in the body [91].Anaerovibrio, Fibrobacter, and Treponema process indigestible polysaccharides into SCFAs [92][93][94][95].Oscillospira participates in glucose synthesis and lipid metabolism in the host's body and its abundance is positively correlated with propionate production.It is a common core bacterium related to body health in animal intestinal microbiota [96,97].Parabacteroides is a gram negative bacterium that plays an important role in anti-inflammatory and lipid metabolism in the host [98].Pedersen et al. [99] found a higher relative abundance of Prevotella and Bacteroides in the gut of pigs with higher body weights.Our results showed that supplementation with 2% FCHs significantly increased the relative abundance of the cellulose-degrading and SCFAproducing bacteria Parabacteroides and Treponema in the colon microbiota of growing pigs.The correlation pattern between these bacteria and the gut-content metabolites was almost opposite to the pattern between the gut-content metabolites and multiple opportunistic pathogens, such as Escherichia_Shigella [100], Streptococcus [101,102], Terrisporobacter [103], and Clostridium sensu stricto1 [104].Dietary supplementation with 2% FCHs significantly reduced the relative abundance of these opportunistic pathogens.
Inflammation caused by Clostridium sensu stricto 1 reduces the SCFA content in the gut [105].The Eubacterium ruminantium group comprises potentially pro-inflammatory gut microbiota [106].Terrisporobacter produces trimethylamine-N-oxide, which induces intestinal oxidative stress and inflammation in the host [107] and is highly associated with sepsis [108].Our results indicated that adding 2% FCHs to the diet significantly reduced the relative abundance of these bacteria, indicating that FCHs inhibited the colonization of pro-inflammatory bacteria in the gut and may help the body resist inflammation.
SDA is a class of polyunsaturated fatty acids that are intermediates in the endogenous conversion of α-linolenic acid to eicosapentaenoic acid [109].SDA has many beneficial effects, such as regulating lipid metabolism [110] and demonstrating anti-inflammatory [111] and antitumoral effects [112].Wu et al. [113] reported that the intake of foods rich in SDA could reduce the production of PGE2, which showed a significant pro-inflammatory effect.Propionic acid, an SCFA, is derived from the intestinal anaerobic fermentation of dietary fiber and complex polysaccharides and has beneficial properties in different disease conditions, such as intestinal development and cardiometabolic diseases [114].SCFAs are common fiber degradation products that can be used as signal metabolites and energy substances for microorganisms to help the host stabilize metabolism and intestinal health [115].In experiments in apolipoprotein E-deficient (ApoE −/− ) mice with hypercholesterolemia and atherosclerosis susceptibility induced with a high-fat diet, Haghikia et al. [116] identified a novel regulatory pathway in which propionate acts as a prebiotic to selectively regulate the immune system in the gut, resulting in a reduction in the aortic atherosclerotic lesion area and the control of intestinal cholesterol homeostasis.In this study, supplementation with 2% FCHs significantly increased the colonic contents of SDA and propionic acid in growing pigs.

Conclusions
The addition of 2% or 3% FCHs significantly improved the growth performance, serum antioxidant, and immunity of growing pigs.In the colon, the dietary supplementation of 2% FCHs significantly increased the relative abundance of cellulose fermentation and SCFA-producing bacteria, decreased the relative abundance of opportunistic pathogens, and significantly increased the contents of SDA and propionic acid.These results indicated that FCHs were a good feed additive for growing pigs, and the recommended addition ratio was 3%.Further study is needed to investigate the effects of higher levels of FCHs on the growth and immunity of growing pigs.

Figure 3 .
Figure 3.Effect of fermented Chinese herbs (FCHs) on the colonic microbiota of growing pigs.(A) Rarefaction curve; (B) ASV number; (C) Shannon index; (D) ACE index; (E) principal co-ordinates analysis profile; (F) relative abundances of dominant phyla; (G) bar chart showing the linear discriminant analysis (LDA) results; (H) cladogram plot showing the LDA effect size (LEfSe) results.Taxa with LDA > 3.0 and p < 0.05 were considered significant differences; the prefixes "f" and "g" represent the annotated family and genus levels, respectively.Pigs in the control group were fed a basal diet, and the diet of group B was the basal diet with 2% FCH.* p < 0.05; *** p < 0.001.

Figure 4 .
Figure 4. Effect of fermented Chinese herbs on the metabolomics of the colonic contents of growing pigs.(A) The principal component analysis profile shows the difference in the metabolomes of the colonic contents between group B and the controls; (B) the volcano plot shows the different metabolites; (C) the bar chart shows the main differential metabolic pathways; (D) the bar chart shows the main differential metabolites; (E) the co-occurrence network shows the connections of the main metabolites and metabolic pathways; (F) the heatmap profile shows the collection between the main colonic microbes and metabolites.The enlarged heatmap profile is shown in Figure S3.Pigs in the control group were fed a basal diet, and the diet of group B was the basal diet with 2% FCH.* p < 0.05; ** p < 0.01; *** p < 0.001.

Table 1 .
Ingredient and nutrient components of the basal diet (air-dry basis) used in the study.