• Asian-Australasian Journal of Animal Sciences
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• v.25 no.11
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• pp.1617-1626
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• 2012

The objective of this study was to evaluate the effects of adding essential oils to the diet of weaned pigs on performance, nutrient utilization, immune response and intestinal health. A total of 96 weaning pigs ($8.37{\pm}1.58$ kg) were allotted to one of three dietary treatments. The treatments consisted of an unsupplemented basal diet (negative control, NC) or similar diets supplemented with 0.01% of an essential oil product which contained 18% thymol and cinnamaldehyde (EOD) as well as a diet supplemented with 0.19% of an antibiotic mixture which provided 150 ppm chlortetracycline, 80 ppm colistin sulfate and 50 ppm kitasamycin (positive control, PC). Each treatment was provided to eight pens of pigs with four pigs per pen. Over the entire 35 d experiment, ADG and fecal score were improved (p<0.05) for pigs fed the PC and EOD compared with the NC. Dry matter and crude protein digestibility as well as lymphocyte proliferation for pigs fed the PC and EOD diets were increased significantly compared with NC (p<0.05). IGF-I levels in plasma were significantly increased (p<0.05) in pigs fed the PC diet compared with pigs fed the NC diet. Interleukin-6 concentration was lower (p<0.05) and the tumor necrosis factor-${\alpha}$ level was higher (p<0.05) in the plasma of pigs fed the EOD diet than the NC diet. Plasma total antioxidant capacity level increased (p<0.05) in pigs fed the EOD diet compared with pigs fed the NC. Villus height to crypt depth ratio in the jejunum was greater (p<0.05) in pigs fed the PC and EOD diets than the NC. The numbers of E. coli in the cecum, colon and rectum were reduced (p<0.05) in pigs fed the PC and EOD diets compared with the control. In the colon, the ratio of Lactobacilli to E. coli was increased (p<0.05) in pigs fed the EOD diet compared with NC diet. Total aerobe numbers in the rectum were decreased (p<0.05) in pigs fed the PC and EOD diets compared with the control. Collectively, these results indicate that blends of essential oils could be a candidate for use as an alternative to traditional antibiotics in weaning pig diets.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.9
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• pp.1330-1338
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• 2008

This study was conducted to investigate the effects of Acanthopanax senticosus extract (ASE) as a dietary additive on gut microflora in weaned piglets. A total of sixty pigs were weaned at 21 d of age (BW = $5.64{\pm}0.23kg$) and allocated on the basis of BW and litter to three dietary treatments in a randomized complete block design. The dietary treatments were: control group (basal diet), antibiotics group (basal diet+0.02% colistin), and ASE group (basal diet+0.1% ASE). On d 7, 14 and 28 after consuming the experimental diets, five piglets per group were sacrificed and then the contents from the jejunum, ileum and cecum were collected to determine changes in the microbial community by using a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique and estimating the contents of Lactobacillus and E. coli by in vitro culturing methods. The results showed that the ASE promoted the microflora diversity in the cecum. Enumeration of bacteria in the gut contents showed that the number of Lactobacillus increased (p<0.05), while that of E. coli decreased (p<0.05) when compared with the other 2 groups as the days of age progressed post-weaning. These findings suggested that the ASE, as a substitute for dietary antimicrobial products, could improve the development of the normal gut microflora and suppress bacterial pathogens, and effectively promote a healthy intestinal environment.

• Journal of Microbiology and Biotechnology
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• v.24 no.5
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• pp.619-628
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• 2014

To identify lipase LipA (PFL_0617) from Pseudomonas protegens Pf-5, a lipA deletion mutant (Pf0617) and a complementary strain (Pf0617lipA) were constructed, and their effects on the lipase production were examined. Pf0617 remarkably decreased its whole-cell lipase activity, whereas Pf0617lipA made its whole-cell lipase activity not only restore to wild-type level but also get a further increment. However, the deletion and overexpression of lipA did not affect the extracellular lipase activity. In addition, the unbroken whole cells of these strains were able to catalyze the hydrolysis of membrane-permeable p-nitrophenyl esters, but could not hydrolyze the membrane-impermeable olive oil. These results confirmed that LipA was an intracellular lipase and Pf-5 could also be used as a natural whole-cell biocatalyst. To evaluate the potential of Pf-5 as a whole-cell biocatalyst and separately characterize the whole-cell LipA, the properties of the whole-cell lipases from Pf-5 and Top10lipA were characterized. The results demonstrated that both Pf-5 and Top10lipA exhibited high tolerance to alkaline condition, high temperature, heavy metal ions, surfactants, and organic solvents. Taken together, lipA can realize functional expression in E. coli Top10, and Pf-5 and Top10lipA as whole-cell biocatalysts may have enormous potential in applications.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.8
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• pp.1131-1140
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• 2014

This research focused on the effects of different doses of Bacillus subtilis KN-42 on the growth performance, diarrhea incidence, faecal bacterial flora, and the relative number of Lactobacillus and Escherichia coli in faeces of weaned piglets to determine whether the strain can serve as a candidate antimicrobial growth promoter. A total of 360 piglets (initial body weight $7.14{\pm}0.63$ kg) weaned at $26{\pm}2$ days of age were randomly allotted to 5 treatment groups (4 pens per treatment with 18 pigs per pen) for a 28-day trial. Dietary treatments were basal diet without any antimicrobial (negative control; NC), basal diet supplemented with 120 mg/kg feed of neomycin sulfate (positive control; PC) and basal diet supplemented with $2{\times}10^9$ (L), $4{\times}10^9$ (M) and $20{\times}10^9$ (H) CFU/kg feed of B. subtilis KN-42. During the overall period, average daily gain and feed efficiency of piglets were higher in groups PC, M, and H than those in group NC (p<0.05), and all probiotics and antibiotics groups had a lower diarrhea index than group NC (p<0.05). The 16S rDNA gene-based methods were used to analyze faecal bacterial flora on day 28 of experiment. The result of denaturing gradient gel electrophoresis analysis showed that supplementation of B. subtilis KN-42 to the diet changed the bacterial communities, with a higher bacterial diversity and band number in group M than in the other four groups. Real-time polymerase chain reaction analysis showed that the relative number of Lactobacillus were higher in groups PC and H than in group NC (p<0.05), and the supplemented B. subtilis KN-42 to the diet also reduced the relative number of E. coli (p<0.05). These results suggest that dietary addition of B. subtilis KN-42 can improve the growth performance and gastrointestinal health of piglets.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.3
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• pp.411-416
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• 1999

Ninety-six, 35 day old, crossbred pigs, were fed either a basal diet based on com, soybean meal, fish meal and whey or one of three similar diets supplemented with either 0.5% organic acid or enzyme both alone and in combination. Neither organic acid nor enzyme produced any significant (p<0.05) improvements in daily gain or feed conversion. Organic acid, both alone and in combination with enzyme, significantly (p=0.04) decreased the pH in the lower colon. None of the treatments produced any effects on E. Coli or Lactobacillus numbers in any part of the gastrointestinal tract. Feeding enzyme increased trypsin (p=0.01), chymotrypsin (p=0.03) and amylase (p=0.08) levels in the jejunum. Chymotrypsin levels were higher (p=0.04) in the ileum of pigs fed enzyme. Serum glucose levels were lower (p=0.01) on day 14 in pigs fed enzyme either alone or in combination with acid. Under the conditions of this experiment (10% dietary whey, pigs weaned at 35 days of age), neither organic acid nor enzyme were effective in improving starter pig performance. Therefore, there would appear to be little justification for the routine inclusion of these products in diets fed to pigs weaned at 35 days or later.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.397-404
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• 2013

Paenibacillus xylanilyticus KJ-03 was isolated from soil samples obtained from a field with Amorphophallus konjac plants. A gene encoding xylanase was isolated from KJ-03 and cloned using a fosmid library. The xynA gene encodes xylanase; it consists of 1,035 bp and encodes 345 amino acids. The amino acid sequence deduced from the P. xylanilyticus KJ-03 xylanase showed 81% and 69% identities with those deduced from the P. polymyxa E681 and Paenibacillus sp. HPL-001 xylanases, respectively. The xynA gene comprises a single domain, consisting of a catalytic domain of the glycosyl hydrolase (GH) 10 family. The xynA gene was expressed in Escherichia coli BL21 (trxB), and the recombinant xylanase was purified by Niaffinity chromatography. The purified xylanase showed optimum activity with birchwood xylan as a substrate at $40^{\circ}C$ and pH 7.4. Treatment with $Mg^{2+}$ and $Li^+$ showed a slight decrease in XynA activity; however, treatment with 5 mM $Cu^{2+}$ completely inhibited its activity. The results of the thin layer chromatography analysis indicated that the major hydrolysis product was xylobiose and small amounts of xylose and xylotriose. XynA showed increased activity with oat spelt xylan and birchwood xylan, but showed only slight activity with locust bean gum.

• Journal of Microbiology and Biotechnology
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• v.31 no.8
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• pp.1154-1162
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• 2021

The transcriptional capacities of target genes are strongly influenced by promoters, whereas few studies have focused on the development of robust, high-performance and cross-species promoters for wide application in different bacteria. In this work, four novel promoters (P_k.rtufB, P_k.r1, P_k.r2, and P_k.r3) were predicted from Ketogulonicigenium robustum and their inconsistency in the -10 and -35 region nucleotide sequences indicated they were different promoters. Their activities were evaluated by using green fluorescent protein (gfp) as a reporter in different species of bacteria, including K. vulgare SPU B805, Pseudomonas putida KT2440, Paracoccus denitrificans PD1222, Bacillus licheniformis and Raoultella ornithinolytica, due to their importance in metabolic engineering. Our results showed that the four promoters had different activities, with P_k.r1 showing the strongest activity in almost all of the experimental bacteria. By comparison with the commonly used promoters of E. coli (tufB, lac, lacUV5), K. vulgare (Psdh, Psndh) and P. putida KT2440 (JE111411), the four promoters showed significant differences due to only 12.62% nucleotide similarities, and relatively higher ability in regulating target gene expression. Further validation experiments confirmed their ability in initiating the target minCD cassette because of the shape changes under the promoter regulation. The overexpression of sorbose dehydrogenase and cytochrome c551 by P_k.r1 and P_k.r2 resulted in a 22.75% enhancement of 2-KGA yield, indicating their potential for practical application in metabolic engineering. This study demonstrates an example of applying bioinformatics to find new biological components for gene operation and provides four novel promoters with broad suitability, which enriches the usable range of promoters to realize accurate regulation in different genetic backgrounds.

• Journal of Microbiology and Biotechnology
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• v.30 no.7
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• pp.996-1004
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• 2020

Various genetically engineered microorganisms have been developed for the removal of heavy metal contaminants. Metal biosorption by whole-cell biosorbents can be enhanced by overproduction of metal-binding proteins/peptides in the cytoplasm or on the cell surface. However, few studies have compared the biosorption capacity of whole cells expressing intracellular or surface-displayed metal-adsorbing proteins. In this study, several constructs were prepared for expressing intracellular and surface-displayed Ochrobactrum tritici 5bvl1 ChrB in Escherichia coli BL21(DE3) cells. E. coli cells expressing surface-displayed ChrB removed more Cr(VI) from aqueous solutions than cells with cytoplasmic ChrB under the same conditions. However, intracellular ChrB was less susceptible to variation in extracellular conditions (pH and ionic strength), and more effectively removed Cr(VI) from industrial wastewater than the surface-displayed ChrB at low pH (<3). An adsorption-desorption experiment demonstrated that compared with intracellular accumulation, cell-surface adsorption is reversible, which allows easy desorption of the adsorbed metal ions and regeneration of the bioadsorbent. In addition, an intrinsic ChrB protein fluorescence assay suggested that pH and salinity may influence the Cr(VI) adsorption capacity of ChrB-expressing E. coli cells by modulating the ChrB protein conformation. Although the characteristics of ChrB may not be universal for all metal-binding proteins, our study provides new insights into different engineering strategies for whole-cell biosorbents for removing heavy metals from industrial effluents.

• International Journal of Industrial Entomology and Biomaterials
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• v.8 no.1
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• pp.89-93
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• 2004

Expression of a fusion protein between B. thuringiensis crystal protein, Cry1Ac1 and a scorpion insect toxin (AaIT, Androctonus australis Hector insect toxin) in acrystalliferous B. thuringiensis strain (Cry-B strain) was examined. The cry 1Ac1 gene was cloned in B. thuringiensis-E coli shuttle vector, pHT3101, under the control of the native cry 1Ac1 gene promoter (pProAc) and a gene encoding AaIT was inserted in XhoI site in the middle of the cry 1Ac1 gene (pProAc-ScoR). B. thuringiensis Cry-B strain carrying pProAc-ScoR (PyoAc-ScoR/CB) produced an inclusion body of irregular shape and the expressed fusion protein is approximately 65 kDa in size. Sporulated cells and spore-crystal mixtures of ProAc-ScoR/CB had insecticidal activity against Plutella xylostella larvae, showing $LT_50$ of ProAc-ScoR/CB (22.59 hrs) lower than that of ProAc/CB (30.06 hrs) at $1{\times}{10^7} {CEU/cm^2}$. These results suggest that the fusion protein including a B. thuringiensis crystal protein and an AaIT may be functionally expressed in B. thupingiensis. Moreover, we verified the additive toxicity of AaIT, which is a new feasible candidate for insect control.

• BMB Reports
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• v.38 no.1
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• pp.41-48
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• 2005

In this study we developed the transposon-mediated shuttle vector 'Hanpvid', which composed of HaNPV (Heliothis armigera nuclear polyhedrosis virus) genomic DNA and a transposon cassette from Bacmid of Bac-to-Bac system. Hanpvid replicates in E. coli in the same way as Bacmid and retains infective function in cotton bollworm cells (Hz-AM1). Using Hanpvid we constructed a recombinant virus, which could infect Hz-AM1 cells and generate recombinant HaNPV (rHa-Bar) containing the barnase gene, a ribonuclease gene from Bacillus amyloliquefaciens. Since the expression vector carrying barnase gene cannot replicate in the absence of barstar, a specific inhibitor of barnase, we constructed a new cotton bollworm cell line (AM1-NB) using the marker rescue method. In AM1-NB barstar was integrated into the cellular chromosome to sustain the replication of rHa-Bar. To screen out recombinant HaNPV for potential use as biopesticide, Hz-AM1 and AM1-NB cell lines were infected with rHa-Bar, respectively. The results obtained indicate that Viral progenies in AM1-NB were 23 and 160 times greater than those in Hz-AM1 48 h and 72 h after infection, respectively. With additional insertion of the polyhedron gene from AcNPV (Autographa californica nuclear polyhedrosis virus) into the Hanpvid genome, rHa-Bar regained the polyhedron phenotype and its pest-killing rate greatly improved. Toxic analysis showed that the lethal dosages ($LD_{50}$) and the lethal time(s) ($LT_{50}$) of rHa-Bar were reduced by 20% and 30%, respectively, compared to wt-HaNPV in the third instar larvae of cotton bollworm. This study shows that in AM1-NB barnase can be effectively produced and used as pest-killing agent for the biological control of cotton pests.