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Recombinant porcine epidermal growth factor-secreting Lactococcus lactis promotes the growth performance of early-weaned piglets.

ABSTRACT: BACKGROUND: Epidermal growth factor (EGF) is an important growth factor in regulation of cell proliferation, differentiation, survival and apoptosis. Studies showed that food-grade Lactococcus lactis (L. lactis) and NICE expression system have superior performance in exogenous protein expression. This study aimed to construct and express porcine EGF (pEGF), and use L. lactis as vehicle for producing and delivering pEGF. Furthermore, investigating biological activity of pEGF and exploring applications feasibility of combination effects of L. lactis and pEGF on early weaned piglets' production. RESULTS: A recombinant Lactococcus lactis which produced and secreted pEGF at 1000 ng/ml in culture supernatant was generated. Secreted pEGF was a fully biologically active protein, as demonstrated by its capacity to stimulate L929 mouse fibroblast cell line proliferation in vitro. For in vivo study, forty piglets were randomly allocated to control, antibiotic control, empty vector-expressing L. lactis (LL-EV) and pEGF-secreting L. lactis (LL-pEGF). After 14 d of rearing, final body weight and average daily gain in LL-pEGF were greater (P < 0.05, 8.95 vs. 8.37 kg, 206.1 vs. 157.7 g/day, respectively) than those in control, but no significant differences between LL-pEGF, LL-EV and antibiotic control. Overall period average daily feed intake was higher in LL-pEGF, LL-EV and antibiotic control than in control (P < 0.05, 252.9, 255.6, 250.0, 207.3 g/day, respectively). No significant difference was observed on ADFI/ADG. LL-pEGF increased villous height in the duodenum, jejunum and ileum than in control and LL-EV (P < 0.05). Sucrase in the 3 intestinal segments, aminopeptidase A in the duodenum and Jejunum, aminopeptidase N and dipeptidase IV in the duodenum in LL-pEGF were higher than those in control (P < 0.05). Furthermore, Escherichia coli and Enterococcus counts decreased in the ileum and Lactobacillus increased in the ileum and cecum digesta in LL-pEGF compare with the control (P < 0.05). Lactobacillus increased in the cecum in LL-EV compared with control and antibiotic control (P < 0.05). CONCLUSION: We have generated a recombinant Lactococcus lactis which produced and secreted fully biologically active porcine EGF. Oral administration of pEGF-secreting L. lactis had beneficial effects on intestinal health and performance of early-weaned piglets.

SUBMITTER: Wang D

PROVIDER: S-EPMC4236833 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Project description:Small-scale studies investigating the relationship between pigs' intestinal microbiota and growth performance have generated inconsistent results. We hypothesized that on farms under favorable environmental conditions (e.g., promoting sow nest-building behavior, high colostrum production, low incidence of diseases and minimal use of antimicrobials), the piglet gut microbiota may develop toward a population that promotes growth and reduces pathogenic bacteria. Using 16S rRNA gene amplicon sequencing, we sampled and profiled the fecal microbiota from 170 individual piglets throughout suckling and postweaning periods (in total 670 samples) to track gut microbiota development and its potential association with growth. During the suckling period, the dominant genera were Lactobacillus and Bacteroides, the latter being gradually replaced by Clostridium sensu scricto 1 as piglets aged. The gut microbiota during the nursery stage, not the suckling period, predicted the average daily growth (ADG) of piglets. The relative abundances of SCFA-producing genera, in particular Faecalibacterium, Megasphaera, Mitsuokella, and Subdoligranulum, significantly correlated with high ADG of weaned piglets. In addition, the succession of the gut microbiota in high-ADG piglets occurred faster and stabilized sooner upon weaning, whereas the gut microbiota of low-ADG piglets continued to mature after weaning. Overall, our findings suggest that weaning is the major driver of gut microbiota variation in piglets with different levels of overall growth performance. This calls for further research to verify if promotion of specific gut microbiota, identified here at weaning transition, is beneficial for piglet growth. IMPORTANCE The relationship between pigs' intestinal microbiota and growth performance is of great importance for improving piglets' health and reducing antimicrobial use. We found that gut microbiota variation is significantly associated with growth during weaning and the early nursery period. Importantly, transitions toward a mature gut microbiota enriched with fiber-degrading bacteria mostly complete upon weaning in piglets with better growth. Postponing the weaning age may therefore favor the development of fiber degrading gut bacteria, conferring the necessary capacity to digest and harvest solid postweaning feed. The bacterial taxa associated with piglet growth identified herein hold potential to improve piglet growth and health.

Project description:Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisin-controlled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granule-associated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.

Project description:The present study aimed to investigate the effects of dietary zinc sources on the growth performance and gut health of weaned piglets. In total, 96 Duroc × Landrace × Yorkshire (DLY) weaned piglets with an initial average body weight of 8.81±0.42kg were divided into four groups, with six replicates per treatment and four pigs per replicate. The dietary treatment groups were as follows: (1) control group, basal diet; (2) zinc sulphate (ZnSO4) group, basal diet +100mg/kg ZnSO4; (3) glycine zinc (Gly-Zn) group, basal diet +100mg/kg Gly-Zn and (4) zinc lactate group, and basal diet +100mg/kg zinc lactate. The whole trial lasted for 28days. Decreased F/G was noted in the Gly-Zn and zinc lactate groups (p<0.05). The zinc lactate group had a lower diarrhea rate than the control group (p<0.05). Moreover, the ZnSO4, Gly-Zn, and zinc lactate groups had significantly higher apparent total tract digestibility of dry matter (DM), crude protein (CP), ether extract (EE), crude ash, and zinc than the control group (p<0.05). The Gly-Zn and zinc lactate groups had higher jejunal villus height and a higher villus height:crypt depth ratio than the control group (p<0.05). In addition, the ZnSO4, Gly-Zn and zinc lactate groups had a significantly lower mRNA expression level of jejunal ZRT/IRT-like protein 4 (ZIP4) and higher mRNA expression level of jejunal interleukin-1β (IL-1β) than the control group (p<0.05). The mRNA expression level of jejunal zinc transporter 2 (ZNT2) was higher and that of jejunal Bcl-2-associated X protein (Bax) was lower in the Gly-Zn and zinc lactate groups than in the control group (p<0.05). Moreover, the zinc lactate group had a higher count of Lactobacillus spp. in the cecal digesta and higher mRNA expression levels of jejunal occludin and mucin 2 (MUC2) than the control group (p<0.05). In conclusion, dietary supplementation with 100mg/kg ZnSO4, Gly-Zn, or zinc lactate could improve the growth performance and gut barrier function of weaned piglets. Dietary supplementation with organic zinc, particularly zinc lactate, had the best effect.

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Recombinant porcine epidermal growth factor-secreting Lactococcus lactis promotes the growth performance of early-weaned piglets.

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