Effects of lactobacillus plantarumZJ316 on pig growth and pork quality
- Cheng Suo†1,
- Yeshi Yin†2Email author,
- Xiaona Wang1,
- Xiuyu Lou1,
- Dafeng Song1,
- Xin Wang2 and
- Qing Gu1Email author
© Suo et al.; licensee BioMed Central Ltd. 2012
Received: 30 December 2011
Accepted: 14 June 2012
Published: 25 June 2012
Lactobacillus plantarum is a plant-associated bacterial species but it has also been found in human, mouse and porcine gastrointestinal tracts. It can ferment a broad spectrum of plant carbohydrates; it is tolerant of bile salts and low pH, and it has antagonistic potential against intestinal pathogens. However, experiments reporting the use of L. plantarum as a probiotic are limited. In this study, the effects of L. plantarum ZJ316 isolated from infant fecal samples on pig growth and pork quality were investigated.
One hundred and fifty newly weaned pigs were selected randomly and divided into five groups. Group 1 was fed a diet supplemented with the antibiotic mequindox; Groups 2, 3 and 4 were fed a diet supplemented with L. plantarum and no antibiotic; and Group 5 was fed a mixture of mequindox and L. plantarum. After a 60 days initial treatment, samples were collected for evaluation. The results showed that, the L. plantarum ZJ316 has probiotic effects on pig growth and that these effects are dose dependent. The effects of a dose of 1 × 109 CFU/d were more pronounced than those of a dose of 5 × 109 CFU/d or 1 × 1010 CFU/d. In Group 2 (1 × 109 CFU/d), the diarrhea (p = 0.000) and mortality rates (p = 0.448) were lower than in antibiotic-treated pigs (Group 1), and the daily weight gain (p = 0.001) and food conversion ratios were better (p = 0.005). Improved pork quality was associated with Lactobacillus treatment. pH (45 min, p = 0.020), hardness (p = 0.000), stickiness (p = 0.044), chewiness (p = 0.000), gumminess (p = 0.000) and restoring force (p = 0.004) were all significantly improved in Lactobacillus-treated pigs (Group 2). Although we found that L. plantarum exerted probiotic effects on pig growth and pork quality, the mechanisms underlying its action require further study. Polymerase chain reaction-denaturing gradient gel electrophoresis results showed that the gut bacterial communities in Lactobacillus- and antibiotic-treated pigs were very similar and the quantity of L. plantarum ZJ316 was below the detection limits of DGGE-band sequencing. The concentration of short-chain fatty acids in Lactobacillus- and antibiotic-treated fecal samples were not significantly different (p = 0.086). However, the villus height of ilea (p = 0.003), jejuna (p = 0.000) and duodena (p = 0.036) were found to be significantly improved by Lactobacillus treatment.
L. plantarum ZJ316 was found to have probiotic effects, improving pig growth and pork quality. The probiotic mechanism might not involve L. plantarum colonization and alteration of the gut bacterial community. Rather, it might be related to the inhibition of the growth of opportunistic pathogens and promotion of increased villus height.
KeywordsProbiotics Lactobacillus plantarum Pig Pork quality
Weaning stress can destroy the balance of intestinal microbiota in young mammals. Such periods of stress may allow opportunistic pathogens to multiply and cause gastrointestinal (GI) disorders . To promote growth and reduce the incidence of diarrhea, sub-therapeutic antibiotics have been widely used in pig diets . However, this procedure has public health consequences because of the high risk of the development of antimicrobial resistance among pathogenic bacteria, which may be then transferred to humans [3, 4]. It is therefore necessary to find ways to replace antibiotics in pig feeding strategies.
Several members of Lactobacillus, which is part of the normal mucosal microbiota of pigs, have been found to be good probiotics [5, 6]. Lactobacillus and similar bacteria, as well as their metabolites, can control pathogens, such as Escherichia coli[7, 8], Salmonella typhimurium, and others. Selected strains of L. plantarum possess properties that make them promising candidates for probiotics in feed additives . However, experiments reporting the use of L. plantarum as a probiotic are limited. Pieper et al. determined whether a single administration of L. plantarum DSMZ 8862/8866 either before or at the time of weaning can affect the intestinal microbiota of pigs. The results showed that L. plantarum DSMZ 8862/8866 has positive results on GI health . The effects were found to be better when the probiotics were administered at the time of weaning than when they were administered before weaning.
Due to the high mortality of pigs after removing antibiotics at weaning (unpublished data), most farmers are reluctant to accept this option . In this study, we determined whether L. plantarum could replace commonly used antibiotics. We also investigated the effects of L. plantarum ZJ316 on pig growth, pork quality, gut morphology and gut microbiota.
Inhibitory effects of L. PlantarumZJ316 culture supernatants
Inhibition spectrum of culture supernatants of L. plantarum ZJ316
Lactobacillus plantarum ZJ316
Effects of L. PlantarumZJ316 on pig growth
Effects of different treatments on pig growth parameters, mortality and prevalence of diarrhea #
Food intake (g/day)
974.21 ± 5.51a
911.42 ± 34.50ab
864.81 ± 43.78b
901.39 ± 44.51ab
860.80 ± 19.73b
Daily gain (g/day)
454.90 ± 18.14a
520.30 ± 9.29b
470.20 ± 13.27a
477.50 ± 13.87a
469.30 ± 10.31a
Food conversion ratio
2.23 ± 0.08a
1.76 ± 0.12b
1.85 ± 0.15b
1.89 ± 0.04b
1.83 ± 0.02b
10.00 ± 10.00 a
3.33 ± 3.33a
3.33 ± 3.33 a
6.67 ± 6.67 a
3.33 ± 3.33a
7.06 ± 0.66 a
2.17 ± 0.10 b
2.28 ± 0.20b
3.39 ± 0.78 bc
4.28 ± 0.22c
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
Parameters used for evaluation of pork quality #
Longissimus muscle color
39.06 ± 1.13ab
41.58 ± 1.71a
35.39 ± 0.96b
5.10 ± 0.92a
5.95 ± 0.11a
4.48 ± 0.70a
1.88 ± 0.29a
2.42 ± 0.30a
1.57 ± 0.41a
55.72 ± 1.04ab
52.47 ± 2.51a
59.97 ± 0.93b
49.60 ± 5.03a
45.82 ± 3.39ab
39.08 ± 0.40b
10.93 ± 0.92a
11.16 ± 0.85a
9.11 ± 0.61a
3.73 ± 0.99a
3.18 ± 0.57a
2.07 ± 0.06a
47.04 ± 4.64a
50.66 ± 3.14ab
56.86 ± 0.36b
6.25 ± 0.01a
6.38 ± 0.03b
6.39 ± 0.04b
5.82 ± 0.04a
5.84 ± 0.02a
5.83 ± 0.03a
Drip loss (%)
5.95 ± 0.94a
5.70 ± 1.01a
3.28 ± 0.42a
2.80 ± 0.69 ab
1.70 ± 0.29b
3.02 ± 0.12a
3.25 ± 0.12a
3.25 ± 0.12a
209.43 ± 16.50a
138.47 ± 6.70b
75.03 ± 7.27c
-4.43 ± 0.39a
-6.88 ± 1.30b
-2.82 ± 0.38a
1.01 ± 0.02a
0.96 ± 0.03a
1.09 ± 0.08a
152.14 ± 13.50a
93.51 ± 8.57b
61.35 ± 5.45c
153.96 ± 10.27a
97.80 ± 10.35b
55.59 ± 7.61c
0.73 ± 0.01a
0.71 ± 0.02a
0.73 ± 0.01a
0.46 ± 0.03a
0.35 ± 0.02b
0.42 ± 0.02a
Histologic alterations of intestinal ileal mucosa
The effects of Lactobacillus on gut villus height (μm) and crypt depth (μm) #
Villus height (VH)
479.81 ± 19.07a
557.92 ± 19.99b
532.83 ± 15.34b
Crypt depth (CD)
263.91 ± 12.56a
226.01 ± 13.24b
202.92 ± 7.46b
1.89 ± 0.09a
2.71 ± 0.17b
2.74 ± 0.13b
Villus height (VH)
411.18 ± 14.67a
504.54 ± 14.06b
534.72 ± 13.49b
Crypt depth (CD)
280.13 ± 14.89a
264.14 ± 15.83a
222.80 ± 10.54b
1.64 ± 0.14a
2.07 ± 0.11b
2.61 ± 0.17c
Villus height (VH)
410.10 ± 11.24a
453.35 ± 13.17b
576.80 ± 17.85c
Crypt depth (CD)
221.23 ± 10.11a
212.45 ± 10.79a
217.98 ± 10.60a
2.00 ± 0.13a
2.28 ± 0.12a
2.88 ± 0.19b
Changes in the gut bacterial community due to different treatments
Identification of PCR-DGGE bands using cloning and sequencing
Eothenomys smithi HEG193, Streptococcus hyointestinalis
Aerococcus urinaeequi, Weissella paramesenteroides
Enterobacteriaceae bacterium, Streptococcus hyointestinalis, Blautia glucerasea
Anaerococcus tetradius, Clostridium sp.
Rumen bacterium NK4B114, Clostridium cadaveris, Lactobacillus gallinarum
Lachnospira multipara, Bacterium YE62, Acetivibrio ethanolgignens
Eubacterium eligens, Clostridiales bacterium
Streptococcus gallolyticus subsp.
Rumen bacterium NK4B114
Lachnospiraceae bacterium, Blautia glucerasea
Anaerostipes butyraticus, Gemmiger formicilis, Ruminococcus obeum
Peptoniphilus sp., Faecalibacterium prausnitzii
Eubacterium hadrum, Bacterium YE257
Peptococcus sp., Anaerococcus tetradius
Lactobacillus acidophilus, Ruminococcus sp., Lactobacillus amylolyticus
Clostridium sordellii, Lachnospira multipara
Rumen bacterium NK4B114, Streptococcus gallolyticus subsp.
Clostridium nexile, Klebsiella sp., Clostridiales bacterium
Eothenomys smithii, Lactobacillus gasseri, Clostridium cadaveris
Actinomycetales bacterium, Ruminococcus obeum, Lactobacillus vaginalis
Blautia glucerasea, Bacterium YE257
Streptococcus gallolyticus subsp.
Clostridiales bacterium Clostridium sordellii
Peptostreptococcus sp., Staphylococcus sp., Eubacterium sp.
Changes in the concentration of short-chained fatty acids (SCFAs) in pig fecal samples
Concentration of short-chain fatty acids in pig fecal samples #
8.45 ± 1.13
11.68 ± 0.39
0.48 ± 0.04
0.44 ± 0.05
0.35 ± 0.07
0.48 ± 0.09
0.52 ± 0.07
0.53 ± 0.01
7.66 ± 1.80
10.87 ± 2.71
1.76 ± 0.92
4.33 ± 2.33
2.47 ± 0.47
3.04 ± 0.15
24.92 ± 1.01
28.57 ± 4.96
1.88 ± 0.60
3.49 ± 2.18
48.49 ± 5.38
63.43 ± 3.79
The inhibitory effects of mequindox on L. PlantarumZJ316
Results of this study showed that L. plantarum ZJ316, isolated from infant fecal samples, had a good inhibitory effect on some pathogenic bacteria in vitro. And L. plantarum ZJ316 can significantly improve pig growth at a dose of 1 × 109 CFU/d (Group 2). The probiotic effects of L. plantarum ZJ316 are consistent with those observed in previous studies. Foo et al. reported that the effects of feeding Lactobacillus species I-UL4 and their metabolites to weaned rats can improve growth [13, 14]. Thanh et al. showed that metabolites produced by L. plantarum RS5, RI11, RG14 and RG11 strains can improve chicken growth .
Although the effects of probiotics usage in pigs are not always consistent, beneficial effects have been documented [16–18]. The mode of action of a given probiotic may include modulation of host microflora [19, 20], modifications of the morphology of the intestinal epithelium , regulation of the host immunity system , and the concentration of gut SCFAs, such as acetate . In this study, we investigated the change in gut bacterial community, morphology of ileal mucosa, and the concentration of gut SCFAs due to treatment with L. plantarum ZJ316.
As shown in Figure 1, the bacterial communities in antibiotic- and Lactobacillus-treated groups (Groups 1, 2-1 and 2-2) were very similar. These results showed that the community of gut bacteria was not significantly altered by treatment with L. plantarum ZJ316. Some previous researches have shown similar results. Ohashi et al. showed only a slight change in bacterial communities attributable to orally administered Lactobacillus casei strain Shirota (LCS) . Su et al. also reported no remarkable changes in the overall microbial community in the hind gut after orally administrated L. sobrius S1 . In this study, there are three possible explanations for explain this phenomenon. The first is that the pigs may have developed a stable microbiota after weaning, and this microbiota may be hard to change. Although the porcine GI tract harbors a highly diverse microbial ecosystem , Konstantinov et al. reported that once the gut microbiota has matured, it can remain stable for a long time . Second, probiotics, known widely as beneficial bacteria and yeasts, assist in the restoration of normal levels of beneficial microorganisms without destroying the bacterial communities of the GI tract. Third, the ability of this Lactobacillus to colonize the gut epithelium may be low, because the bacterial species used in this experiment was isolated from infant fecal samples. DGGE sequencing results verified this. 34 prominent DGGE bands were extracted for sequencing and the results are listed in Table 5. We can see that, although five bands maybe related to Lactobacillus species, no L. plantarum was found in any of them. Dunne et al. reported that permanent persistence of an allochthonous strain in the host GI microbiota was virtually impossible . L. plantarum is not the predominant Lactobacillus in pigs. L. sobrius was found to be the most dominant species of Lactobacillus in pigs in both pre- and post-weaning [27, 29, 30]. Previous results have also demonstrated that ingested probiotic strains do not become established members of the normal microbiota but persist only for a short time [31–34]. There is also evidence that common probiotic strains differ in their degree of persistence [31, 35].
Villi are important components of the digestive tract. They are involved in the absorption of nutrients from the small intestine. The condition of intestinal villi and epithelial cells on the apical surface of the villi is known to be a reliable indicator of the enteral nutrient absorption of feed ingredients in chickens  and pigs . However, during post weaning, pigs commonly suffered morphologic atrophy and crypt hyperplasia, which can limit the absorption of voluntary feed intake and weight gain after weaning [38, 39]. One characteristic of an effective probiotic is to increase the villus height. SCFAs were considered to be the main factors for stimulating the development of intestinal mucosa . Although the concentration of SCFAs was not significantly different between antibiotic- and Lactobacillus-treated groups (Group 1 and Group 2) in this study (Table 6), the villus height and crypt depth both improved after treatment with L. plantarum ZJ316. There may be other mechanisms by which probiotics can improve intestinal epithelial and villi. It has been established that the effects of probiotic bacteria may also result from soluble factors that can alter epithelial permeability . Two soluble proteins, p40 and p75, were purified from the culture supernatant of Lactobacillus rhamnosus GG (LGG), can prevented TNF-induced apoptosis and intestinal barrier disruption in colonic epithelial cells . The same group also reported that the probiotic LGG can prevent cytokine-induced apoptosis in colon cells through activation the pathways of anti-apoptotic Akt and protein kinase B, and inactivation of the pathway of pro-aptoptic p38 mitogen-activated protein kinase .
The effect of L. plantarum ZJ316 on pig growth was found to be dose-dependent. The effects of a dose of 1 × 109 CFU/d were more pronounced than those of doses of 5 × 109 CFU/d and 1 × 1010 CFU/d. This may be related to cross-talk between the probiotics and the host’s immune system. Besides these anti-infective properties, probiotics also act upon the immune and inflammatory response. Probiotic supplementation can enhance SIgA production in both rodents and humans . This appears to be a paradox. Probiotics and nonpathogenic commensals can boost the overall SIgA antibody response, while SIgA can trigger intestinal exclusion and subsequent elimination [45–48]. A high dose of probiotics may induce a strong immune and stress response. Rodrigues et al. reported that germ-free mice colonized with S. boulardii displayed more pronounced anti-S. boulardii IgA expression than un-colonized mice . This dose-dependent effect of probiotics has also been observed by other researchers. A change in levels of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in the blood due can be attributed to the administration of Bifidobacterium animalis subsp lactis (BB-12) and Lactobacillus paracasei subsp paracasei (CRL-431) was dependent on a specific dose of 108 for LDL and 109 for HDL . A daily dose of Lactobacillus rhamnosus GR-1 plus Lactobacillus fermentum RC-14 at 1.6 × 109 has a better success of restoring and maintaining a normal vaginal flora than doses of 8 × 108 and 6 × 109. There may be some other mechanisms that may explain this phenomenon. In another study, they found that low bacteria/DC (dendritic cells) ratio better regulates the effects on DCs in vitro, suggesting that different intracellular signaling pathways become activated when bacteria are present at high doses . Considering the dose-dependent nature of the effects of L. plantarum ZJ316, this may be the reason why mixed treatment group (Group 5, 105 g mequindox and 1 × 1010 L. plantarum ZJ316 CFU/d) experienced a better probiotic effect than the same dose of antibiotic and probiotic administered alone (Group 1 and Group 4). As shown in Table 2, although the differences between Groups 4 and 5 were not significant, the parameters used for evaluating growth performance had better values for Group 5 than for Groups 1 and 4. These values were similar to those of the lower dose group (Group 3, 5 × 109 L. plantarum ZJ316 CFU/d). As shown in Figure 2, mequindox inhibited the growth of L. plantarum ZJ316. This shows that mixtures of mequindox and L. plantarum ZJ316 may affect the effective dose of L. plantarum ZJ316. This may be why the effects of mixture group more similar to lower dose group (Group 3). Other researches also showed that antibiotics can affect the effects of bacteria-derived probiotics, and should be separated from antibiotics by at least two hours .
Here, treatment with L. plantarum also improved pork quality. Significant improvements were observed in Group 2 with regards to hardness, stickiness, chewiness, gumminess, and restoring force. This may meet the nutritional needs of human consumers. Until now, reports on the effects of probioitcs on pork quality have been rare; however, further study is needed.
The mechanism whereby L. plantarum ZJ316 improved pig growth and pork quality may not be through L. plantarum colonization and alteration of the gut bacterial community. The promotion of pig growth and pork quality may rather be related to the metabolites inhibiting the growth of opportunistic pathogens and increasing the villus height.
Animals and experimental design
Composition of the diet used in this study
Crude protein, %
Soy bean meal
Digestible energy (Mcal/kg)
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 . 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 . 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 .
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 . 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 DcodeTM 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. . 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. . 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 PrevailTM 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.
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.
This research was kindly supported by the National Natural Science Foundation of China (No. 31071513, No. 30970108, No. 31100097), the Natural Science Foundation of Zhejiang Province (No. Z3110399), and the Program for Science and Technology from Zhejiang Province (No. 2007 C12037). We would also like to thank Mr. Thomas Ryan Withers of Marshall University for editing this paper. We would especially like to thank the anonymous reviewers for their constructive suggestions and critiques.
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