Effects of lactobacillus plantarumZJ316 on pig growth and pork quality

Inhibitory effects of L. PlantarumZJ316 culture supernatants

Effects of L. PlantarumZJ316 on pig growth

The effects of L. plantarum ZJ316 on growth was more pronounced in Group 2 than in Groups 3, 4, or 5. Only pigs in groups 1 and 2 were selected for further analysis.

Effects of L. PlantarumZJ316 on pork quality

Histologic alterations of intestinal ileal mucosa

Changes in the gut bacterial community due to different treatments

DGGE profiles of PCR products of the 16 S rRNA gene V3 region from pig fecal, ileal mucosa and cecal contents revealed that the overall bacterial community was not significantly changed by Lactobacillus treatment (Group 2-1 and 2-2) compared to antibiotic-treated (Group 1). Before starting this experiment, the bacterial community of pig fecal samples were randomly distributed and varied due to individual animal differences (Figure 1A). The patterns of DGGE bands in fecal, cecal and ileal samples were similar between antibiotic- and Lactobacillus-treated groups (Figures 1B, 1C and 1D). To obtain an objective interpretation of the electrophoretic patterns of the different treatment groups, the samples were subjected to a numerical analysis based on the Dice similarity coefficient, then used for cluster analysis. As shown in Figure 1, the cluster analysis results indicated that the bacterial community in fecal, cecal and ileal samples did not show major changes between antibiotic and Lactobacillus treatment. Samples taken from different treatment groups were distributed randomly in the cluster tree.

Changes in the concentration of short-chained fatty acids (SCFAs) in pig fecal samples

The inhibitory effects of mequindox on L. PlantarumZJ316

As shown in Figure 2, the growth of L. plantarum ZJ316 was affected by adding 50 μg/ml, 100 μg/ml, 250 μg/ml, 500 μg/ml and 750 μg/ml of mequindox, and the inhibitory effects were found to be dose-dependent. The high concentration of mequindox had strong inhibitory effect on the growth of L. plantarum ZJ316.

Animals and experimental design

All pigs used in these experiments were treated according to the current regulation of laboratory animal management in China and approved by the laboratory animal care and usage committee, Zhejiang Academy of Agricultural Sciences.

Isolation and identification of L. PlantarumZJ316

Lactobacillus was isolated from an infant fecal samples using Man–Rogosa–Sharpe (MRS) medium (Hopebio, Qingdao, China). In brief, fecal suspension was spread on MRS agar plates and cultured overnight at 37 °C under anaerobic conditions (MACS1000 Anaerobic Cabinet, Don Whitley, U.K.). Then a single clone was randomly selected and was passed more than 10 times on MRS plates. Morphology observation and Gram staining verified clonal purity. After that, purified bacteria was used for biochemical and molecule identification. The biochemical test results are listed in Additional file 1 Table S1. They showed that these bacteria belong to Lactobacillus. For molecular identification, 16 S rDNA primer 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) were used in this study. BLAST results showed that this Lactobacillus was more closely related to Lactobacillus plantarum (99% max identity and 96% query coverage), and was therefore named Lactobacillus plantarum ZJ316. The 16 S rDNA sequence has been submitted to NCBI with accession number [GenBank: JN126052].

Antibacterial activity of L. PlantarumZJ316 culture supernatants

The inhibitory effects of the raw extraction of L. plantarum ZJ316 culture supernatants were evaluated on 17 bacteria strains (Table 1) using agar plates. An Oxford cup test was selected for this study [54]. In brief, 10 ml of semi-solid medium containing 300 μl of indicator bacteria, such as E. coli, was poured onto each agar plate. Then Oxford cups were put on the agar plate. After that, fermented liquid or other activated liquid, such as antibiotic solution, was added to each Oxford cup. After overnight culture, the diameter of the inhibition zone was measured and used as an indicator. In order to reduce possible effects of low pH of the culture supernatants on E. coli and Salmonella, we adjusted the pH to 6.0 using 1 mol/L NaOH. In addition, the possible inhibitory effects of equivalent acetic acid (200 mM/L) and lactic acid (15 mM/L) were assessed using the solutions of these two acids with pH 3.5.

Data and sample collection

At the end of the study (day 95), average daily feed intakes (ADFI), average daily weight gain (ADG), food conversion ratio (FCR), mortality and diarrheal rate were calculated for evaluating the effects of different treatments on pig growth. The quantity of food provided and food left over was recorded every day, and the ADFI was calculated as follows: (given feed weight – residual feed weight)/(number of pigs × number of days). The ADG was calculated as follows: (average pig weight at the end – average pig weight at the beginning)/(number of days). The FCR was calculated as follows: (ADG/ADFI). The numbers of dead and diarrheal pigs were recorded every day and the ratios were calculated as follows: (number of dead pigs)/(total number of pigs) and (number of diarrheal pigs)/(total number of pigs). Nine pigs were selected for slaughter at the end of the experiment; and then muscle samples were collected for pork quality evaluation, ileal mucosa samples were collected for morphology observation, and ileal mucosa and cecal contents were collected for bacterial community analysis.

Evaluation of pork quality

After 60 d of exposure to antibiotics and Lactobacillus, three pigs from Group 1, six pigs from Group 2 (three selected immediately after the cessation of the addition of Lactobacillus, and three selected one week after halting Lactobacillus) were randomly selected for evaluation of pork quality. After slaughter, muscle samples from the Longissimus thoracis (LT, located between the 12th and 13th ribs) and fillet were collected for evaluation of the pork quality. A reflectance spectrophotometer (Minolta CM-2002; Osaka, Japan) was used to measure the color at the surface of a 2-cm-thick steak of Longissimus thoracis muscle after exposure to air for two hours. The parameters registered were L* (lightness), a* (redness), and b* (yellowness). Each value was the mean of 10 determinations per sample on the same slice, avoiding areas with excess fat.

Muscle pH was measured at 45 min and at 24 hours (starting points from the minute the muscles were removed from the corpse) using a portable pH meter equipped with a glass electrode (Hanna HI 8424, Hanna Instruments, Eibar, Spain). To measure the drip loss, samples were placed on a supporting mesh in a sealed plastic container with no contact between sample and container. Three pigs were selected for sample collection and three muscle samples were collected from each pig. After a storage period of 24 hours and 48 hours at 4 °C, the samples were taken out of the container, dabbed lightly onto filter paper, and weighed again. Drip loss was expressed as a percentage of the initial weight based on Honikel [55]. Marbling scores were calculated according to the NPCC 1999.

Texture profile analysis (TPA) was measured using a TA-XT2 Texture Analyser (Stable Micro Systems, Godalming, U.K.) equipped with a 25 kg load cell. The Texture Expert computer program (version 1.20, Stable Micro Systems) was used for data collection and calculations. Before TPA analysis, samples were vacuum-packaged (DZD-400/2 S, Jiangsu Tengtong Packing Machinery Co., Ltd), placed in a cooler (4 °C, wind velocity of 0.5 m/s) for 7 days, and then frozen on stainless steel trays at -20 °C. At the beginning of TPA analysis, the samples were fast-thawed in tap water (4 h). Then the vacuum was broken and the samples were wrapped in aluminum foil and cooked at 200 °C in a double-plate grill (Sammic GRS-5) until the internal temperature reached 72 °C. After cooking, steaks were placed in a vacuum bag and immediately immersed in an ice bath to stop further cooking. In this study, hardness, stickiness, springiness, chewiness, gumminess, cohesiveness, and restoring force were determined as described by Bourne [56].

Morphologic observation of gut ileal mucosa

After animals were killed, ileal tissues (approximately 10 cm anterior to the ileo-cecal junction) were harvested and cut into 1.5 cm × 1.5 cm × 0.3 cm pieces for sectioning. The pieces were fixed in 10% formalin after washing with PBS. Then, the formalin-fixed samples were dehydrated in ethanol, cleared with xylene, and embedded in paraffin wax. Sections (6 μm thick) were stained with hematoxylin and eosin and observed using a light microscope (Leica, Germany).

For measurement of villus height and crypt depth, 10 villi and crypts were selected per section using Leica MZ16A software. The villus height was measured from the villus tip to the bottom, not including the intestinal crypt. The crypt depth was measured from the crypt tip to the bottom. An average of 10 villi and crypts per section was expressed as a mean villus height and crypt depth for each pig.

Analysis of the gut bacterial community

Ileal mucosa (approximately 10 cm anterior to the ileo-cecal junction) and cecal contents (5-10 g) were collected after slaughter. Fresh fecal samples were collected at the beginning and end of the experiment, corresponding to 35, 95 and 102 days of age. Some gut samples collected from Group 1, Group 2-1 and Group 2-2 were used for bacterial genomic DNA extraction. In this study, a bead-beating method was used as previously described [57]. The concentration of extracted DNA was determined using a NanoDrop ND-2000 (NanoDrop Technologies, U.S.), and its integrity and size were checked by agar gel electrophoresis (1.0%). High-quality DNA was then used for polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis. Primer 341 F (5′-ATT ACC GCG GCT GCT GG-3′) and 534R with GC clips (5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-3′) against the V3 region of the 16 S rRNA genes were used in this study. The PCR program included 20 touchdown cycles (65 °C-55 °C); followed by 5 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min followed by extension at 72 °C for 10 min. Reconditioning PCR was performed before DGGE. DGGE was then performed on a Dcode^TM universal detection system (BIO-RAD Laboratories Inc, U.S.) with 8% polyacrylamide gels (ratio of acrylamide to bisacrylamide, 37.5:1) at 60 °C. The gels were electrophoresed at 200 V for 4 h, and then stained with SYBR GREEN І. The bands were visualized and analyzed with Quantity One software (Version 4.6.1; BIO-RAD Laboratories Inc, U.S.) using a match tolerance of 2%.

DGGE band sequencing

DGGE band sequencing was carried out according to Li et al. [58]. The stable bands in the DGGE gels verified as single bands were excised and eluted in 30 μl TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0). The supernatant after centrifugation (12,000 rpm, 5 min, 4 °C) was used for 16 S rDNA-V3 amplification with the V3 primers 341 F and 534R without GC-clamp using the same program as Li et al. [58]. The amplification 16 S rDNA-V3 segments were cloned into a PMD18-T vector after being purified with a Biospin Gel Extraction Kit (Bioer Technology co., Ltd., Japan). The positive recombinants were screened on 5-bromo-4-chloro-3-indolyl-b-D- galactopyranoside (X-Gal), isopro-pyl-b-D-thiogalactopyranoside (IPTG) and ampicillin indicator plates by color-based recombinant selection. Positive clones were selected for sequencing using an ABI 3730 DNA Sequencer (U.S.) with M13 primer at the Beijing Genomics Institute (BGI, China). In all, 34 DGGE bands were sequenced and most closed sequences were identified using a BLAST search.

Short-chain fatty acids (SCFAs) analysis

Fecal samples (0.5 g) were dissolved into 10 ml phosphate buffered saline (PBS), vortexed, and then centrifuged at 12,000 × g for 10 minutes at 4 °C. Then the supernatants were filtered through a 0.45 μm membrane filter for HPLC detection. In this study, the levels of acetic, butyric, citric, formic, isovaleric, lactic, malic, propionic, and tartaric acids were investigated. A Prevail^TM Organic Acid column (250 mm × 4.6 mm) was used with the detection conditions: temperature, 40 °C; wavelength, 217 nm; pressure, 0.1-4,000 psi.

The inhibitory effects of mequindox on L. PlantarumZJ316

An Oxford cup test was executed as described above, and the inhibitory effects of 50 μg/ml, 100 μg/ml, 250 μg/ml, 500 μg/ml and 750 μg/ml of mequindox were evaluated.

Statistical analysis

To analyze growth, each pen from each group was considered a single replicate. For meat color, pH value, drop loss and marbling score, each pig from each group was considered a single replicate. For meat TPA analysis, three samples were collected from each pig and used as replicates. For gut villus height and crypt depth analysis, 30 values were collected from each group and used as replicates. All of the above data were statistically analyzed using the One-Way ANOVA program included in the statistical software package SPSS 13.0 (IBM, U.S.). Least-significant difference (LSD) was selected for post hoc multiple comparisons. For fatty acid analysis, fecal samples were collected from pigs that had been selected for meat quality evaluation and were considered as replicate. They were then analyzed using the means program included in the statistical software package SPSS 13.0 (IBM, U.S.). All values were presented as mean ± standard error. p < 0.05 was considered significant.