**1. Introduction**

The intestinal tract is home to a large number of complex and diverse gut microbiota [1], such as different types of bacteria, viruses and fungi [2]. As a bridge between diet and host health, gut microbiota not only affects the digestion and absorption of nutrients in the diet, but also regulates the normal physiological functions and the occurrence of diseases in the host [3]. Recently, with the help of next-generation high-throughput sequencing technology, bioinformatics and metagenomics [4], researchers have verified that gut microbiota is vital to host health, and the disruption of gut microbiota has been shown to be associated with multiple diseases, including metabolic syndrome [5], obesity [6], tumor [7], diabetes [8], HIV [9], flu [10], fatigue [11], brain health [12], etc. At the same time, intestinal flora transplantation has shown promising application prospects in the treatment of diseases [13].

With their antibacterial and growth-promoting properties, antibiotics are widely used in disease treatment and daily production. However, abuse of antibiotics not only increases the antibiotic residue in foods and the resistance of disease-fighting microorganisms [14], but it also leads to drug-resistant genes being transmitted from livestock and microorganisms to humans [15]. Meanwhile, antibiotics can directly damage body health via disrupting the homeostasis of gut microbiota in the intestinal tract. For example, Cox et al. [16] found that mice treated with continuous low doses of penicillin could develop a higher body weight due to the disruption of the gut microbiota. Zhang et al. [2] reported

**Citation:** Sun, X.; Wang, Z.; Hu, X.; Zhao, C.; Zhang, X.; Zhang, H. Effect of an Antibacterial Polysaccharide Produced by *Chaetomium globosum* CGMCC 6882 on the Gut Microbiota of Mice. *Foods* **2021**, *10*, 1084. https:// doi.org/10.3390/foods10051084

Academic Editors: Jianhua Xie, Yanjun Zhang and Hansong Yu

Received: 29 March 2021 Accepted: 11 May 2021 Published: 13 May 2021

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that the gene expression and metabolic homeostasis of mice were affected by the administration of perfluorooctane sulfonate. Xu et al. [17] demonstrated that antibiotics could promote tumor initiation in mice by inducing gut microbiota dysbiosis. Therefore, looking for a new generation of safe, high-efficient, widely applicable and non-toxic antibiotics has been growing increasingly important.

As a kind of macromolecule connected by more than ten monosaccharides through a glycoside bond, the gut microbiota could convert polysaccharides into short-chain fatty acids (SCFAs), such as acetic, propionic and butyric acid, thus having a positive effect on gut microbiota and body health [18]. However, the effects of antibacterial polysaccharides on body health, especially the gut microbiota in the intestinal tract, are concealed and poorly understood. Herein, this work assayed the toxicity of an antibacterial polysaccharide (GCP) produced by *Chaetomium globosum* CGMCC 6882 [19] to Caco-2 cells. Secondly, the effects of GCP on the body weight and serum biochemistry of normal mice were detected. Finally, the influence of GCP on the gut microbiota of normal mice was assessed. We hope that this work could provide some help and guidance for the application of bacteriostatic polysaccharides.

#### **2. Materials and Methods**

#### *2.1. Preparation of GCP*

The preparation of GCP produced from *C. globosum* CGMCC 6882 was based on the methods reported in our previous work [20]. Briefly, fermentation liquid was filtered and centrifuged at 12,000× *g* for 30 min to remove mycelium and cells. The supernatant was de-proteinized by adding three volumes Sevag solution, then three volumes cold alcohol were added and it was kept at 4 ◦C overnight to precipitate GCP. The crude GCP re-dissolved in distilled water was de-pigmented with AB-8 macroporous resin (Beijing NuoqiYa Biotechnology Co., Ltd., Beijing, China) and then dialyzed for 48 h in distilled water. After this, GCP solution was filtered with a 0.22 μm filter and applied to a Sepharose CL-6B column (2.5 cm × 60 cm) for further purification, eluted with 0.1 mol/L NaCl solution at a flow rate of 0.6 mL/min, and the fraction was then collected. In the end, the purified GCP was lyophilized for further experiments.

#### *2.2. Cell Viability Assay*

The toxicity of GCP to Caco-2 cells (American Type Culture Collection, ATCC, HTB037) was measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-bromo diphenyltetrazolium (MTT) assay reported in our previous work [21] with some modifications. Dimethyl sulfoximine (DMSO), Dulbecco's modified Eagle medium (DMEM) and MTT were brought from Sigma-Aldrich (Shanghai, China). Meanwhile, fetal bovine serum, penicillin and streptomycin were brought from Sangon Biotech (Shanghai, China). The Caco-2 cells were cultured in DMEM containing 10% (*v*/*v*) fetal bovine serum, 100 U/mL penicillin and 100 μg/mL of streptomycin at 37 ◦C in a humidified 5% CO2 incubator (Series 8000 WJ, Thermo Fisher Seientific, Waltham, MA, USA). Before experiment, the dried GCP powder was dissolved in different concentrations of DMEM solution (100–600 μg/mL) and DMEM solution without GCP was used as the control. Briefly, Caco-2 cells were seeded into 96-well plates at a concentration of 2 × 104 cells/mL and incubated at 37 ◦C in 5% CO2 for 24 h before treatment.

Then, 100 μL GCP at different concentrations was added into wells and cultured for another 24 h. Afterwards, 20 μL of 5 mg/mL MTT was added. After 4 h of incubation, cell supernatant was discarded and 150 μL DMSO was added to dissolve the insoluble crystals in the cell. In the end, the absorbance of each well was recorded by a microplate reader (Bio-Rad Laboratories, Inc., Pleasanton, CA, USA) at 490 nm.

#### *2.3. Experimental Design and Samples Collection*

Specific pathogen free-male mice (20 ± 1 g) were purchased from the Laboratory Animal Center of Henan province (SCXK: 2017-0002; Zhengzhou, China). All mice were held in independent cages and kept in specific pathogen-free conditions at temperatures of 24 ± 1 ◦C, humidity of 60 ± 5%, and with a light to dark cycle of 12 h/12 h. During experiments, all mice were monitored every day, and the experiments were performed strictly according to the guidelines for the care and use of laboratory animals (Henan University of Technology, Zhengzhou, China). Forty mice were randomly divided into four groups (*n* = 10) after adaption for 7 days. One group was used as the normal control group (NC), and another three groups were designed as the experimental group and treated with 100 μg/mL GCP (low-dose group), 200 μg/mL GCP (middle-dose group) and 400 μg/mL GCP (high-dose group), respectively. Then, mice in the experimental groups were orally administered 0.5 mL GCP once a day, and mice in the normal control group were administered equal distilled water. The animal experiments lasted for 28 days and all mice were weighted weekly. During the whole experiment, all mice had free access to a basic diet and distilled water. At the end of experiment, all mice were killed after fasting for 12 h. Blood samples were collected from the orbit and centrifuged at 3000 r/min for 10 min to collect the serum. Meanwhile, the contents of the cecum were immediately collected in plastic tubes (1.5 mL) and stored at −80 ◦C for further analyses.

#### *2.4. Serum Biochemical Index Detection*

The levels of aspartate transaminase, alanine aminotransferase, total protein, albumin, globulin, urea, high-density lipoprotein and low-density lipoprotein, and the glucose concentration in the serum, were tested using the serum analyzer (BS-420, Shenzhen Mindray Biomedical Electronics Co., Ltd., Wuhan, China).
