Trehalose Supplementation Effects on Growth, Intestinal Morphology, Gut Bacteria, and Footpad Dermatitis of Broiler Chickens Reared at High Density.

This study aimed to measure the effects of trehalose (Tre) supplementation on the growth, intestinal morphology, gut bacteria, and footpad dermatitis (FPD) of broiler chickens reared at different stocking densities (SD). Four hundred newly hatched Ross 308 male chicks were randomly allocated to four groups of eight, following a 2 × 2 factorial arrangement in a randomized complete block design using two SDs (normal, 11; high, 14 birds/m2) and two diets: basal with and without 0.5% Tre. Tre supplementation was provided during the starter/grower phase, but not the finisher phase. Data were analyzed using a two-way analysis of variance. We observed no significant effects of SD or Tre, individually or combined, on body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) during the starter/grower period. However, high SD decreased both BWG (P < 0.001) and FI (P < 0.05), and increased FCR (P < 0.001), during the finisher period. Whereas Tre reduced FCR (P < 0.05) as a main effect, no combined effect was observed on FCR. Over the total period, high SD negatively affected BWG and FCR (P < 0.001), and Tre significantly reduced FCR, with its effect unaffected by SD. No significant effects of SD or Tre were observed on jejunal morphology. The ileal abundance of Clostridium perfringens (P > 0.05) was not affected by high SD but was significantly reduced by Tre. Neither high SD nor Tre altered Lactobacillus spp. counts; however, high SD increased FPD lesion scores, whereas Tre had no effect. The study showed that Tre supplementation during the starter/grower period improved FCR during the finisher period, possibly by decreasing the abundance of C. perfringens in broiler chickens.

Effect of trehalose supplementation in milk replacer on the incidence of diarrhea and fecal microbiota in preweaned calves.

Trehalose, a nonreducing disaccharide consisting of d-glucose with α,α-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n = 10]) based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.

Effect of trehalose supplementation on growth performance and intestinal morphology in broiler chickens.

Trehalose (Tre) is a natural disaccharide. A laboratory-scale investigation showed that Tre supplementation increased the growth rate in juvenile chicks, possibly via the improvement of innate intestinal immune responses. In this study, two trials were conducted to evaluate the growth-promoting effect of Tre supplementation in broiler chickens. In experiment-1, two thousand day-old male and female broiler chicks (Ross) were fed 0 (control), 0.25, 0.50, and 0.75% Tre-supplemented pellet-form diets from d 1-17, and subsequently, they were provided grower (d 18-30) and finisher (d 31-37) diets without Tre supplementation. Over the trial period, there was no significant difference in body weight (BW), feed intake and feed conversion ratio (FCR) between chickens in the control and Tre-fed groups. Tre treatment increased villus height (VH)/crypt depth (CD) ratio and villus surface in jejunum; decreased CD and increased VH/CD ratio in ileum on d 17, when these results were compared to the control group. In experiment-2, two hundred day-old female broiler chicks were fed an antibiotics-free and mash-form diet supplemented with 0.5% Tre from d 1-21, before being fed a non-supplemental diet until d 43. There was no difference in BW on d 21 between the control and Tre-0.5% groups; however, from d 22-43, Tre-0.5% group showed significantly higher BW gain and lower FCR compared to the control group. From these results, we suggest that Tre feeding can be beneficial for intestinal morphology and growth performance in broiler chickens. However, these outcomes did not occur in parallel owing to the different feeding conditions observed.

Time course of changes in antioxidant activity of milk from dairy cows fed a trehalose-supplemented diet.

This study was to investigate the time course of changes to the antioxidant activity of milk from cows fed a trehalose-supplemented diet, and to determine possible underlying mechanisms for observed changes. Six Holstein cows were used, and subjected to two experimental feeding periods consisting of a 1% trehalose-supplemented diet for 10 days, followed by a basal diet only (no trehalose) for 10 days. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities in milk were gradually increased during the trehalose supplementation period and were highest at the end of the second period. However, trehalose was not detected in the milk and plasma of dairy cows fed a diet supplemented with trehalose for 10 days, indicating that the increased antioxidant activity in the milk of trehalose-fed cows is not due to the direct transfer of trehalose to the milk. Plasma DPPH activities exhibited a similar time course to that seen for milk. Relative superoxide dismutase (SOD) activities in the rumen were higher 3 days after the end of trehalose supplementation than at any other time during the experimental periods. These results suggested that the improved antioxidant activity in milk and plasma of cows fed a trehalose-supplemented diet was due to improved ruminal relative SOD activity.

High hatching rates after cryopreservation of hydrated cysts of the brine shrimp A. franciscana.

Cysts of Artemia franciscana are known to be extremely tolerant to UV and ionizing radiation, hypoxia, dryness, osmotic pressure, and temperatures. However, when cysts are hydrated, their resistance to extreme environmental conditions is markedly reduced, and they subsequently enter a developmental sequence. The hatching rate of hydrated cysts declined when they were rapidly frozen after a short period of hydration but slow freezing improved hatching rates after 6-h hydration (1.4 g H2O per g dry wt). We observed that trehalose content in hydrated cysts was greatly reduced up to 6-h time. DSC analysis showed different thermal profiles at two cooling rates, suggesting the formation of a minuscule ice crystal inside the cells. High hatching rates can be obtained from highly hydrated cysts at slow cooling rate.

A novel glucanotransferase from a Bacillus circulans strain that produces a cyclomaltopentaose cyclized by an alpha-1,6-linkage.

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS-PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 degrees C, and stable from pH 4.5 to 9.0 at up to 35 degrees C. The addition of 1 mM Ca(2+) enhanced the thermal stability of the enzyme up to 40 degrees C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular alpha-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular alpha-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.

Construction and characterization of chimeric enzymes of kojibiose phosphorylase and trehalose phosphorylase from Thermoanaerobacter brockii.

Chimeric phosphorylases were constructed of the kojibiose phosphorylase (KP) gene and the trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii. Four chimeric enzymes had KP activity, and another had TP activity. Chimera V-III showed not TP, but KP activity, although only 125 amino acid residues in 785 residues of chimera V-III were from that of KP. Chimera V-III had 1% of the specific activity of the wild-type KP. Furthermore, the temperature profile and kinetic parameters of chimera V-III were remarkably changed as compared to those of the wild-type KP. The results of the molecular mass of chimera V-III using GPC (76,000 Da) strongly suggested that the chimera V-III protein exists as a monomer in solution, whereas wild-type KP and TP are hexamer and dimer structures, respectively. The result of the substrate specificity for phosphorolysis was that the chimera acted on nigerose, sophorose and laminaribiose, in addition to kojibiose. Furthermore, chimera V-III was also able to act on sophorose and laminaribiose in the absence of inorganic phosphate, and produced two trisaccharides, beta-D-glucosyl-(1-->6)-laminaribiose and laminaritriose, from laminaribiose.

Purification, characterization, and gene cloning of a novel maltosyltransferase from an Arthrobacter globiformis strain that produces an alternating alpha-1,4- and alpha-1,6-cyclic tetrasaccharide from starch.

A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50 degrees C and was stable from pH 5.0 to 9.0 and up to 30 degrees C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 45 degrees C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of > or =3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose --> 6(4)-O-alpha-maltosyl-maltotetraose + maltose and (ii) 6(4)-O-alpha-maltosyl-maltotetraose --> CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular alpha-1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to alpha-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.

Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis.

Random mutation by error-prone PCR was introduced into kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047. One thermostable mutant enzyme, D513N, was isolated. The D513N mutant enzyme showed an optimum temperature of 67.5-70 degrees C (the wild type, 65 degrees C), and thermostability up to 67.5 degrees C (the wild type, up to 60 degrees C). The half-lives of D513N were estimated to be 135 h at 60 degrees C, 110 min at 70 degrees C and 6 min at 75 degrees C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively.

Hyper expression of kojibiose phosphorylase gene and trehalose phosphorylase gene from Thermoanaerobacter brockii ATCC35047 in Bacillus subtilis and selaginose synthesis utilizing two phosphorylases.

The kojibiose phosphorylase (KP) gene and trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii ATCC35047 were intracellularly hyper-expressed under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be 2.1 g of KP and 4.9 g of TP per liter of medium. Selaginose, non-reducing trisaccharide, was synthesized from trehalose utilizing the recombinant KP and TP from B. subtilis. Selaginose was not hydrolyzed by salivary amylase, artificial gastric juice, pancreatic amylase, or small intestinal enzymes.

An enzymatically produced novel cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->} (cyclic maltosyl-(1-->6)-maltose), from starch.

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1-->4)- and alpha-(1-->6)-linkages. The cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages.

Cyclic tetrasaccharide-synthesizing enzymes from Arthrobacter globiformis A19.

A bacterial strain Arthrobacter globiformis A19 producing cyclic tetrasaccharide (CTS) was isolated from soil. The enzymes, 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT), involved in the synthesis of CTS were purified to homogeneity. The molecular and enzymatic properties of IMT from A. globiformis were similar to those of enzymes from Bacillus globisporus C11 and N75. Arthrobacter 6GT had a smaller molecular mass of 108 kDa and a higher optimum pH of 8.4 than the enzymes from strains of B. globisporus. The genes for IMT (ctsY) and 6GT (ctsZ) were cloned from the genome of A. globiformis A19. The two genes linked together in tandem and formed a gene cluster, ctsYZ. Both of the gene products showed similarities to alpha-glucosidases belonging to glycoside hydrolase family 31, and conserved two aspartic acids corresponding to the putative catalytic residues of the family enzymes. The enzymatic system for the production of CTS consisting of 6GT and IMT might be widespread among bacteria.

Cloning and sequencing of kojibiose phosphorylase gene from Thermoanaerobacter brockii ATCC35047.

A gene encoding kojibiose phosphorylase was cloned from Thermoanaerobacter brockii ATCC35047. The kojP gene encodes a polypeptide of 775 amino acid residues. The deduced amino acid sequence was homologous to those of trehalose phosphorylase from T. brockii and maltose phosphorylases from Bacillus sp. and Lactobacillus brevis with 35%, 29% and 28% identities, respectively. Kojibiose phosphorylase was efficiently overexpressed in Escherichia coli JM109. The DNA sequence of 3956 bp analyzed in this study contains three open reading frames (ORFs) downstream of kojP. The four ORFs, kojP, kojE, kojF, and kojG, form a gene cluster. The amino acid sequences deduced from kojE and kojF are similar to those of the N-terminal and C-terminal regions of a sugar-binding periplasmic protein from Thermoanaerobacter tengcongensis MB4. Furthermore, the amino acid sequence deduced from kojG is similar to that of a permease of the ABC-type sugar transport systems from T. tengcongensis MB4. Each of three amino acid substitutions, D362N, K614Q and E642Q, caused a complete loss of kojibiose phosphorylase activity. These results suggest that D362, K614 and E642 play an important role in catalysis. Another mutation, D459N, increased K(m) values for kojibiose (7-fold that for the wild type), beta-G1P (11-fold) and glucose (7-fold), whereas K(m) for inorganic phosphate was minimally affected by this mutation, suggesting that D459 may be involved in the binding to saccharides.

Purification and characterization of glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide in Bacillus globisporus C11.

Glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide (CTS; cyclo [-->6]-alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->)) from alpha-1,4-glucan were purified from Bacillus globisporus C11. The former was a 1,6-alpha-glucosyltransferase (6GT) catalyzing the a-1,6-transglucosylation of one glucosyl residue to the nonreducing end of maltooligosaccharides (MOS) to produce alpha-isomaltosyl-MOS from MOS. The latter was an isomaltosyl transferase (IMT) catalyzing alpha-1,3-, alpha-1,4-, and alpha,beta-1,1-intermolecular transglycosylation of isomaltosyl residues. When IMT catalyzed alpha-1,3-transglycosylation, alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS was produced from alpha-isomaltosyl-MOS. In addition, IMT catalyzed cyclization, and produced CTS from alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS by intramolecular transglycosylation. Therefore, the mechanism of CTS synthesis from MOS by the two enzymes seemed to follow three steps: 1) MOS-->alpha-isomaltosyl-->MOS (by 6GT), 2) alpha-isomaltosyl-MOS-->alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS (by IMT), and 3) alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS-->CTS + MOS (by IMT). The molecular mass of 6GT was estimated to be 137 kDa by SDS-PAGE. The optimum pH and temperature for 6GT were pH 6.0 and 45 degrees C, respectively. This enzyme was stable at from pH 5.5 to 10 and on being heated to 40 degrees C for 60 min. 6GT was strongly activated and stabilized by various divalent cations. The molecular mass of IMT was estimated to be 102 kDa by SDS-PAGE. The optimum pH and temperature for IMT were pH 6.0 and 50 degrees C, respectively. This enzyme was stable at from pH 4.5 to 9.0 and on being heated to 40 degrees C for 60 min. Divalent cations had no effect on the stability or activity of this enzyme.

Gene encoding a trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047.

A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047. The gene encodes a polypeptide of 774 amino acid residues. The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions. On the other hand, no homology was found between the T. brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa.