1. Introduction
As common probiotics, lactic acid bacteria can maintain the balance of the intestinal microbiota and competitively eliminate pathogens [
1,
2] as well as improve growth performance, feed conversion efficiency, nutrient utilization, and gut health [
3]. They can change the intestinal environment by producing antibacterial substances and reducing the pH value, thus obtaining advantages in intestinal colonization. For example,
Lactobacillus lactis produces nisin and
Lactobacillus acidophilus produces bacteriostatic proteins, thus acquiring colonization advantages in the gut. These bacteriocins inhibit and eliminate intestinal pathogens [
4,
5]. The bacteriocin produced by
Lactobacillus E50–52 can significantly reduce the number of
Salmonella in animals [
6].
Lactobacillus rhamnosus secretes antibacterial substances with inhibitory activities [
5] and has a broad-spectrum antibacterial activity against pathogenic bacteria [
7]. Scharek et al. [
8] found that the intestinal mucosal immune function of weaned piglets was significantly enhanced after feeding them with lactic acid bacteria. Lactic acid bacteria can increase the expression and secretion of β-defensins to prevent the growth and reproduction of pathogenic microorganisms [
9]. Studies have demonstrated that the isolated, species-specific strain Pediococcus acidilactici FT28 possessed potential in vitro probiotic properties. More importantly, compared with a piglet control, it showed potential capabilities such as higher nutrient digestibility, hemato-biochemical parameters, and antioxidant status [
10]. In another study, lactic acid bacteria isolated from homologous animals played a more stable prebiotic role [
11].
In the intestines of pigs,
Firmicutes,
Bacteroidetes, and
Proteobacteria account for approximately 90% of the total intestinal microbiota, but their distribution patterns are different [
12]. The main factors causing intestinal microbiota fluctuations may be related to the animal species, diets, and additive contents [
12]. The feeding environment is an essential factor affecting the colonization of the intestinal microbiota. In China, a diverse range of pig resources can be observed across the country. In Anhui in particular, we found a variety of high-quality local pig breeds, including the Yuexi Black Pig, HuoShou Black Pig, and six Taihu White Pigs. These breeds display extraordinary traits such as a high tolerance to rough feeding and strong stress resistance. It has been hypothesized that these characteristics might be intricately linked to their gut microbiota [
13,
14]. However, the specific composition and functions of their gut microbiota remain unexplored.
In summary, this study used high-throughput sequencing to analyze the composition and function of microorganisms in the intestinal tract of Anhui local pig breeds. With traditional isolation and cultivation techniques, we isolated lactic acid bacteria from the intestinal contents of superior breeds of pigs and tested their stress resistance and bacteriostatic ability. The results attained from this study establish a foundation for the future creation of microecological preparations for pigs.
2. Materials and Methods
2.1. Samples
Intestinal content samples of three pig breeds (Yuexi Black Pig, HuoShou Black Pig, and Taihu LiuBai Pig) were collected, with at least three samples from each pig, totaling 15 samples. The samples were placed in a dry refrigerator after collection and transported to the laboratory. The control strain Enterococcus faecium A21 was isolated and preserved by our laboratory. Escherichia coli CMCC44102 and Staphylococcus aureus ATCC29523 were used as indicator strains.
2.2. 16S rRNA Amplicon Sequencing
The DNA extraction was performed according to the QIAamp Fast DNA Stool Mini Kit instructions using the forward primer 338F (5′-ACTCCTACGGGAGGCAGCAGCAGCAG-3′) and the reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′) for the PCR amplification of the V3–V4 variable regions. A QIAquick Gel Extraction Kit was used for PCR product purification and after the amplified product was detected with agarose gel, the cut gel was recovered and purified. The Shanghai Meiji Company was entrusted with the Miseq PE300 platform sequencing.
2.3. Metagenomic Sequencing of Intestinal Contents of Local Pigs from the Anhui Province
After extracting genomic DNA, Covaris M220 IIIumina HiSeq sequencing was commissioned using fastp [
15] to cut the linker sequences at both ends of the original sequencing data. We used a sliding window with a size of 5 bp starting from both ends of the sequence so that the average mass of bases in the window was more than 20 bp. Finally, sequences with a length above 40 bp and an average base mass above 15 bp were retained. The QC sequence was aligned to the pigs’ reference genomic sequence of Sscrofa11.1 (GCA 000003025.6), retaining the sequence without the alignment. Sequences with a removed host genome were assembled using MEGAHIT, preserving contigs longer than 1000 bp for prediction and gene annotation in open reading frames. Kraken2 [
16] was employed to classify the sequence and assembled configs of the deactivated host genome, and Bayesian-based Bracken [
17] was used to estimate the abundance of individual species in each sample based on the classification results of kraken2. The gene function was annotated using eggNOG-mapper [
18] and the eggNOG database [
19]. The system function information, chemical function information, and gene function information were analyzed using the KEGG database. The TPM was calculated using Salmon [
20] to estimate the gene abundance.
2.4. Isolation and Purification of Lactic Acid Bacteria
We weighed 0.1 g of pig intestinal content, diluted it to an appropriate concentration with sterilized PBS, and evenly coated it onto an MRS solid medium containing 1% CaCO
3. Subsequently, the sample was placed into a 37 °C incubator and after 24 h, the colony growth and bacterial morphology were observed. Single colonies with neatly edged and milky white calcium-dissolving rings were selected, cultured on MRS agar plates for 24–48 h, continuously purified, and cultured for two to three passages, and then placed in MRS broth for proliferation. After drying, Gram staining was performed; bacteria with purple cells were classified as Gram-positive bacteria, whereas those with red cells were classified as Gram-negative bacteria. The isolated bacteria were extracted following the instructions of the bacterial genome extraction kit and the 16S sequence was amplified. The recovered fragment gel products were subsequently submitted to Beijing Tsingke Biotech Co., Ltd. (Beijing, China). for sequencing. The sequences were then subjected to a BLAST comparison and analysis [
21].
2.5. Determination of Acid-Production Capacity and Growth Curve
Isolated lactic acid bacteria were activated in MRS broth two to three times, inoculated into 5 mL of an MRS medium at an inoculum volume of 1:100, and incubated at 37 °C for 24 h. The pH value was measured and recorded using a pH meter every 2 h, and a pH value change curve was generated according to the results. The strains with a low pH value in the above acid-production test were selected for activation and a seeding volume of 1:100 was inserted into the MRS liquid medium and incubated at 37 °C for 24 h. Subsequently, the OD600nm value was measured every 2 h and the absorbance was calibrated using blank MRS as the control; the growth curve was generated with the measured OD value as the ordinate.
2.6. Acid-Resistance Determination
We adjusted the MRS broth medium with hydrochloric acid to pH 2.0 and pH 3.0, autoclaved it, and set it aside. After activating the screened strain two to three times, it was inoculated into 5 mL of liquid MRS medium at 1% and incubated in a 37 °C incubator for 24 h. We set the MRS broth with the unadjusted pH as a negative control. Absorbance at OD
600nm was determined after 0 and 4 h, and the diluted sample was evenly coated on the MRS plates. The survival rate was calculated according to the plate-counting method using the following equation:
where N1 is the number of viable bacteria (cfu/mL) cultured for 0 h in the MRS liquid medium at pH 2.0 and pH 3.0, respectively, and N2 is the number of viable bacteria after 4 h of culture.
2.7. Determination of Bile Salt Resistance
The strain was placed into 5 mL of an MRS broth medium at an inoculation volume of 1:100. After standing at 37 °C for 24 h, the bile salt content of the MRS broth was adjusted to 0.1% and 0.3% using porcine bile salt and the MRS broth without bile salt regulation was set as a negative control. Samples were taken after standing the culture for 0 and 4 h, and the survival rate was calculated using the plate-counting method [
21].
2.8. Trypsin Tolerance Measurement
We adjusted the concentration of the MRS liquid medium with a trypsin solution to 1 mg/mL, pipetted 1 mL of the bacterial solution, centrifuged it at 8000 r/min for 3 min, and carefully washed it two to three times with a PBS buffer. The bacterial cells were suspended in 1 mL PBS, inoculated into 5 mL of MRS broth at 1%, and cultured at 37 °C for 24 h; MRS broth without trypsin regulation was used as a negative control. Samples were taken and the MRS plates were evenly coated after standing the culture for 0 and 4 h. The survival rate was calculated using the plate-counting method [
22].
2.9. Surface Hydrophobicity
Referring to the microbial adhesive hydrocarbon compound method (BATH) [
23], the screened lactic acid bacteria were activated and centrifuged at 8000 r/min for 10 min, then the supernatant was removed. The resuspended bacteria were washed with a PBS buffer and the operation was repeated two to three times. Subsequently, we resuspended the bacterial cells in 0.1 M of a KNO
3 solution and adjusted its absorbance to OD
600nm = 0.5 ± 0.02 (A0) under OD
600nm. We then added 1 mL of xylene and 3 mL of the KNO
3 resuspension solution and allowed the mixture stand for 15 min. After shaking for 60 s and standing for 15 min until the solution showed aqueous phase stratification, the aqueous phase was slowly absorbed to determine absorbance (A1) at OD
600nm. Hydrophobicity was determined as follows:
2.10. Self-Aggregation Capability
According to the method of Xu et al. [
24], we placed 2 mL of bacterial suspension in a centrifuge tube, followed by shaking for 15 s and standing at 37 °C for 2 h. Subsequently, 1 mL of the supernatant was removed, and absorbance (B1) at OD
600nm was determined. Self-aggregation was determined using the following equation:
2.11. Co-Agglutination of Lactic Acid Bacteria with Pathogenic Bacteria
Referring to the steps described in Collado et al. [
25], the lactic acid bacteria were obtained by preliminary screening and the concentration of the bacterial solution was adjusted to 1.0 × 10
8 (cfu/mL). Subsequently, we aspirated 1.5 mL of lactic acid bacteria and mixed it with a causative bacteria suspension, then added 3 mL of the PBS dilution to mix. After the three groups of bacteria were thoroughly shaken for 20 s, they were placed in a temperature box at 37 °C and incubated for 2 h. We then slowly pipetted 500 μL of the supernatant and determined the absorbance values of C1 (mixed bacterial solution), C2 (lactic acid bacteria single bacterial solution), and C3 (pathogenic bacteria single bacterial solution) at OD
600nm [
26]. Co-agglutination was calculated using the following equation:
2.12. In Vitro Bacteriostatic Activity Assay
The antibacterial activity of lactic acid bacteria against the index bacteria Escherichia coli CMCC44102 and Staphylococcus aureus ATCC25923 was determined by agar perforation diffusion. The isolated lactic acid bacteria were inoculated at 1:100 into the MRS broth; after 24 h, the mixture was centrifuged at 6000× g for 15 min and the supernatant was removed. The LB solid medium was prepared with 0.1% indicator bacteria; holes were punched using an 8 mm sterile punch and a fresh culture solution as well as the supernatant of the 100 μL isolate were added. The plate was placed into an incubator at 37 °C and cultured; subsequently, we determined the diameter of the inhibition circle.
2.13. Determination of Inhibition of Pathogenic Bacteria
After standing at 37 °C for 24 h in MRS broth, L. johnsonii L5 and L. reuteri L8 were centrifuged at 6000 r/min for 10 min. The supernatant was collected and filtered. After activation, E. coli CMCC44102 was inoculated at a 1% ratio to the supernatant of L. johnsonii L5 and L. reuteri L8; the LB medium was set as the control group. After 16 h, 10 μL of the culture was dropped onto the center of the LB solid medium and incubated at 37 °C for 6 h. Subsequently, the movement diameter of E. coli was measured and the experiment was repeated three times.
2.14. Data Analysis
SPSS 26 data analysis software was used for the data processing and significance analysis. The test results were expressed as the mean ± standard deviation. Different lower case letters indicated a significant difference (p < 0.05) and the same letter or no letter indicated no significant difference (p > 0.05).
4. Discussion
In this study, the intestinal contents of Anhui local pigs of different breeds were analyzed using 16S rDNA sequencing to determine the composition and diversity of the intestinal bacteria. The main functions included cell wall membrane envelope biogenesis, defense mechanisms, replication recombination and repair, signal transduction mechanisms, and the translation ribosomal structure and biogenesis. The contribution of lactic acid bacteria in the YueHei Pig group was almost not reflected, whereas the lactic acid bacteria in the HuoShou Pig group contributed most significantly to the functional annotation.
As an important probiotic, lactic acid bacteria must overcome the adverse environment of the gastrointestinal tract when entering the body via ingestion to ensure survival. The hyperpermeable environment caused by high levels of gastric acid and bile salts in the gastrointestinal tract is the primary problem faced by bacteria in this habitat. A pH of 3.0 is one of the most important criteria for probiotic tolerance and screening [
27]. The lactic acid bacteria screened in this trial had a low survival rate at pH 2.0, but the survival rate of
L. reuteri L8 and
L. johnsonii L5 at pH 2.0 reached 60%, showing strong acid resistance. Studies have shown that most lactic acid bacteria exhibit good viability at pH 3.0 and low viability at pH 2.0 [
28].
The tolerance of lactic acid bacteria to small intestinal transport is critical for their colonization and metabolism. The bile salt concentration is one of the important factors affecting the colonization of microorganisms in the intestine and most lactobacilli play a probiotic role due to their bile salt tolerance and stable presence in the intestine [
29,
30]. The greater the bile salt tolerance of
Lactobacillus, the more conducive it is to colonizing the intestine and exerting physiological activity. Lactic acid bacteria from different sources have different bile salt tolerances [
31]. Zhang Li et al. [
32], regarding the tolerance of
Lactobacillus bile salts isolated from yak milk, found that the tolerance of different strains to different bile salts increased over time at a concentration of 0.3% bile salts. In addition, animal-derived
Lactobacillus bile salts are better tolerated than those from other sources; possibly, animal-derived strains that have adapted to the gastrointestinal environment exhibit better probiotic properties [
33]. In this study, lactic acid bacteria isolated from the intestinal contents of HuoShou pigs were subjected to different bile salt concentrations and a trypsin tolerance assay.
Lactobacillus johnsonii L5 and
L. reuteri L8 retained a viable bacterial count of 1.0 × 10
8 cfu/mL at a bile salt concentration of 0.3% and a trypsin level of 1.0%, showing a higher tolerance to these conditions.
Due to some differences among strains, the adhesion performance of lactic acid bacteria in the intestine also varies [
34]. In this study, the adhesion capacity was determined using the BATH method and a self-polymerization test; strains L2, L5, L8, and L17 showed a good performance. The hydrophobicity of L2, L5, and L8 was as high as 70%. When co-agglutinated with the pathogenic bacteria
E. coli CMCC44102 and
S. aureus ATCC25923,
L. johnsonii L5 and
L. reuteri L8 had a higher agglutination effect and co-agglutination with
S. aureus ATCC25923 was more obvious. The combination of
L. johnsonii L5 and
L. reuteri L8 showed a more significant hydrophobicity and agglutination of pathogenic bacteria. Highly hydrophobic lactic acid bacteria exhibit a higher affinity with the intestinal mucosa, thus rendering a greater prospect of colonization, a quintessential prerequisite for lactic acid bacteria to function as probiotics. Furthermore, the enhanced agglutination of pathogenic bacteria is advantageous in thwarting the diffusion and subsequent infections of pathogenic microorganisms within the intestinal tract [
35]. Safety is a prerequisite for the clinical use of probiotics. During the screening process, the strain should be isolated from healthy animals [
36]. Studies have shown that some lactobacilli can secrete bacteriocins that inhibit the growth of gastrointestinal pathogens [
37]. In this test,
E. coli CMCC44102 and
S. aureus ATCC25923 were selected as indicator strains and the isolated lactic acid bacteria were subjected to the Oxford cup antibacterial test. The inhibition diameters of strains L2, L5, L8, and L17 reached more than 16 cm, indicating a strong bacteriostatic activity.
In summary, combined with the traditional isolation and culturing, two strains of Lactobacillus johnsonii with strong stress resistance and probiotic effects were successfully screened from the intestinal contents of the HuoShou Pig. This has important guiding significance for the development and production of lactic acid bacteria resources with local-breed pigs.