Berberine is an isoquinoline alkaloid isolated from the traditional Chinese herb Coptis chinensis, which is widely used for its medicinal properties. Berberine has antidiarrheal, antibacterial, anti-inflammatory, antitumor, and hypoglycemic effects. It has good therapeutic effect on intestinal inflammation, diabetes, hypertension, and tumors (Fu et al., 2020; Huang et al., 2021; Patel, 2021). Specifically, the therapeutic effect of berberine on intestinal bacterial infection has been investigated for its potential use in clinical practice (Yu M. et al., 2020). Berberine helps in maintaining the intestinal health as it accumulates in the intestine easily and is beneficial in improving the imbalance in intestinal bacteria (Wu et al., 2020).

The intestinal microflora is a complex microbial system composed of a variety of microorganisms participating in numerous physiological processes of the body (Jin et al., 2019; Ma and Ma, 2019; Sun et al., 2020). The intestine harbors various microorganisms such as Lactobacillus, Bacillus, Enterobacter, Bifidobacterium, and Enterococcus. Initially, the gut microflora was thought to be closely related only to digestion and nutrient absorption; however, recent studies reported that it affects the body health by regulating metabolic diseases, such as obesity, diabetes, and cardiovascular diseases, as well as immune-related disorders (Schippa and Conte, 2014; Yadav and Jha, 2019). As the largest and most complex microecosystem of the body, intestinal microorganisms and their metabolites play an important role in animal health.

Limited studies have focused on the possible link between microbiome and function of gut microflora with berberine supplementation in weaned piglets. With the development of metagenomic high-throughput sequencing technology, it is possible to analyze a large number of microbial community species, abundance, and related biological information by performing total microbial DNA extraction from a specific environment and library construction (Fraher et al., 2012; Zhou et al., 2015; Quan et al., 2019). Thus, a large amount of information on non-culturable microbial flora can be obtained without the need of isolation and culture methods used in traditional microbial research (Guo et al., 2014; Walker et al., 2014). Currently, metagenomic sequencing technology has become an important tool to study intestinal environmental microorganisms. In the present study, this technique was used to characterize the microbial composition and function in the rectum of weaned pigs supplemented with berberine.

Early weaning of piglets can shorten the slaughter cycle of pigs and improve the reproductive performance of sows. However, incomplete development of intestinal microbiota in early weaned piglets may lead to intestinal irritability and reduced production performance (Moeser et al., 2017; Upadhaya and Kim, 2021). Antibiotics can improve the above conditions, but the problem of antibiotic residues needs to be addressed (Yang et al., 2019). Berberine, a traditional Chinese herbal extract, has strong antibacterial effect and is an effective antibiotic substitute (Xu et al., 2020; Zhu et al., 2021). However, few studies have focused on the effect of berberine on the gut microbiota in early weaned piglets. In this work, the macrogenomics and high-throughput sequencing techniques were used to investigate the effects of berberine on intestinal microbiome and function of early weaned piglets (weaning age: 21 days).

Metagenomic analysis involves the DNA extraction from all microorganisms in environmental samples directly followed by a metagenomic library construction and uses high-throughput sequencing technique to study the genetic composition and community functions of these microorganisms (Prayogo et al., 2020). Animal microbial population is distributed on the body surface, oral cavity, gastrointestinal tract, and reproductive tracts, but there are significant differences in the types and quantities of microorganisms based on dietary, species, sex, and age. The microbes in the gut are more numerous than the body’s other organs. The complexity of the environment and microorganisms results in less rigorous data analysis using traditional methods; however, the development of metagenomics and high-throughput sequencing technology has promoted the study of gut microbes considerably (Walker et al., 2014; Guo et al., 2021). In the present study, a major part of raw data (>99%) contributed to the clean data, and the Q20 (%) was more than 96% in the gut microbiomes of the control and berberine groups. A total of 68.7 and 68.3 billion bp clean reads were obtained by conducting metagenomic sequencing of the control and berberine groups, respectively. ANOSIM revealed that the control and berberine groups had significant differences at the genus and species levels of intestinal microorganisms in weaned piglets. These results suggest that the metagenomic sequencing data were reliable and sufficient to investigate the effects of berberine on the gut microbial diversity and function in weaned piglets.

Nutritional digestion and absorption, physiology, metabolism, intestinal barrier, immune function, and disease onset are affected by the composition, diversity, and functional changes in intestinal microflora (Chang and Martinez-Guryn, 2019; Liu et al., 2019). Accordingly, the stability of microecological environment plays a crucial role in animal health regulation. Berberine shows a broad-spectrum antibacterial effect against a variety of gram-positive, gram-negative, and drug-resistant bacteria (Yue et al., 2019; Jamshaid et al., 2020). The effects of berberine influence intestinal infectious disease development and body health through the inhibition of intestinal bacteria (Zhu et al., 2021). Studies have shown that berberine can directly regulate the structure of intestinal microbiota by reducing the number of intestinal microbes in a dose-dependent manner (Zhang et al., 2019). In metagenomics research, Chao1, ACE, Shannon, and Simpson indices are used to study the gut microbiome diversity. We found that dietary 0.1% berberine significantly decreased these indices at the species level compared with those in the control group, suggesting toward the efficacy of berberine in reducing the richness and diversity of intestinal microbiome. Similarly, research conducted by Zhang et al. (2019) revealed that berberine decreased the diversity and quantity of the intestinal microflora in db/db mice.

Berberine is not easily absorbed after oral administration; thus, it can maintain a high concentration in the gastrointestinal tract, providing the necessary conditions required for inhibiting the intestinal bacterial growth (Cheng et al., 2021). In addition, as an antibacterial drug, it can inhibit a variety of pathogenic bacteria and change the structure of intestinal microflora (Habtemariam, 2020; Yu C. et al., 2020; Cheng et al., 2021). The most common mechanism of berberine-mediated regulation of intestinal flora is to change the original dominant intestinal bacteria to maintain the microecological balance. Habtemariam (2020) suggested that the underlying mechanism for the multifunctional role of berberine was its regulation of gut microbiota. Zhang et al. (2012) showed that berberine with high-fat diet in rats could increase the abundance of Allobaculum and Blautia in the intestine. Dietary berberine also increased the abundance of beneficial bacteria including S. variabile, L. johnsonii, and P. distasonis, as shown in the present study. S. variabile improves gut mucosal immune response and inhibits food allergy in mice (Abdel-Gadir et al., 2019). L. johnsonii can promote growth, gut development, and intestinal microorganisms in pig, mice, and chicken, when used as a probiotic (Wang et al., 2017; He et al., 2019; Wang et al., 2020). The abundance of P. distasonis was negatively correlated with obesity, non-alcoholic fatty liver disease, diabetes, and other disease states, suggesting that it possibly plays a positive regulatory role in glucose and lipid metabolism (Wang et al., 2019). By contrast, dietary berberine decreased the abundance of P. copri, which leads to changes in microbiota metabolism and reduces interleukin-18 production. This aggravates the intestinal inflammation and may result in systemic autoimmunity (Ley, 2016).

Berberine can activate some signaling pathways and carbohydrate-related enzymes by improving intestinal microflora and health (Liao et al., 2020; Li et al., 2021). Findings from the present study revealed that berberine changed the structure, abundance, and function of gut microbiota in weaned piglets. Alterations in the gut microbiota lead to functional changes as well. Dietary berberine could markedly affect the CAZy activity of intestinal microflora. Similarly, Li et al. (2021) reported that berberine treatment affects the carbohydrate utilization by altering CAZy activity in the intestinal microflora. Alignment analysis based on KEGG database showed significantly enriched carbohydrate metabolism and environmental information processing pathways in the berberine group. Carbohydrate metabolism pathway mainly involves carbohydrate digestion to provide energy for microbial growth through fermentation in the large intestine, which leads to generation of volatile fatty acids and their derivatives to provide nutrition for the body (Tremaroli and Bäckhed, 2012). Environmental information processing pathway is related to the changes in intestinal microbiota (Arboleya et al., 2016).

In summary, there were microbial community and functional differences in the rectum of weaned piglets between the control and berberine groups. We demonstrated that berberine could improve the composition, abundance, structure, and function of gut microbiome in the weaned piglets. Our research might provide a novel scientific basis for the further development and application of berberine (such as replacing antibiotics) in the feed and food industries.

The raw sequencing data presented in the study are deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) repository, accession number PRJNA807368.

The animal study was reviewed and approved by the Animal Care and Use Committee of Anhui Science and Technology University.

HH and XB: data the collection and drafting the manuscript. HH, FZ, and XB: conceive and design the study. KX and KW: statistical analysis. FZ: critical revision of the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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