High-yield cycloamylose production from sweet potato starch using Pseudomonas isoamylase and Thermus aquaticus 4-α-glucanotransferase

Food Science and Biotechnology - An optimal reaction condition for producing cycloamylose (CA) from sweet potato starch was investigated using a combination of isoamylase (from Pseudomonas sp.) and...     

Affinity purification of 4-α-glucanotransferase through formation of complex with insoluble amylose

4-α-Glucanotransferases (αGTases) are useful enzymes to modify starch structure using their unique hydrolysis pattern and transglycosylation activity. In spite of lacking of a carbohydrate binding module, a thermostable aGTase from Thermus thermophilus (TTαGT) bound to insoluble amylose, leading to simple recovery of the enzyme-amylose complex using centrifugation. At a preparative scale, the recovery yield was 40.3% of the subjected free enzyme via collection of the complex twice. The complex exhibited improved thermostability, which shifted the optimum temperature from 70 to 80°C and increased the half-life at 90°C by three-fold compared with the free enzyme. Texture profile analysis of the gels made of modified starch by either free TTαGT or the complex revealed that the complex with double dosage showed the performance similar to free TTαGT in the modification of starch. In conclusion, the purification method described here would be useful due to easy scale-up and simple process without chromatographic process.     

Identification of an acidic α-amylase from Alicyclobacillus sp. A4 and assessment of its application in the starch industry

An acidic α-amylase was purified from thermoacidophilic Alicyclobacillus sp. A4 by ion exchange chromatography with 22% recovery, and showed a molecular mass of 64 kDa by SDS-PAGE. Its amino acid sequence was determined by sequencing three internal peptides and the complete genome of strain A4, and shared highest identity (64%) with Alicyclobacillusacidocaldarius α-amylase. Compared with other reported α-amylases, the purified enzyme had some distinct characteristics. The optimal activity was found to occur at 75 °C and pH 4.2, similar to the glucamylase widely used in the starch industry. The enzyme was Ca²⁺ independent, and had strong ability to digest raw starch (96.71%) with commercial glucamylase in one step. These properties of the purified enzyme make up the deficiency of the commercial α-amylases currently used and avoid repeated adjustment of pH and temperature in double-enzymatic sugar-making process. The purified enzyme will be commercially valuable in the starch industry.     

Texture properties of rice cakes made of rice flours treated with 4-α-glucanotransferase and their relationship with structural characteristics

Rice flours were treated using a thermostable 4-α-glucanotransferase (GTase) from Thermus aquaticus for 1, 3, and 48 h. Molecular weight of modified rice starch rapidly decreased within 1 h of reaction, then slowed down. As the reaction proceeded, the proportions of short (<DP 11) and long (>DP 30) branched chains of modified starch increased, whereas the proportion of medium chains decreased. Rice cakes were prepared with native and GTase-treated rice flours (substitution at 5%) and kept for 3 and 21 h. Texture profile analysis indicated significantly increased hardness, adhesiveness, chewiness, and resilience in rice cakes containing treated rice flours. Sensory analysis revealed that the rice cakes containing 48 h-treated flour had significantly increased springiness, hardness, toughness, and adhesiveness during both storage periods. Meanwhile, it displayed the lowest starch-like attribute and crumbliness. These results suggested that the substitution for 48 h GTase-treated rice flour could retard the retrogradation of rice cakes.     

Complex formation of a 4-α-glucanotransferase using starch as a biocatalyst for starch modification

Food Science and Biotechnology - A 4-α-glucanotransferases from Thermus thermophilus (TTαGT) possesses an extra substrate binding site, leading to facile purification of the intact enzyme...     

Extraction of β-carotene produced from yeast Rhodosporidium sp. and its heat stability

This work determined the characteristics of β-carotene produced from Rhodosporidium sp. isolated from citrus fruits, and an effective extraction method was established. To extract β-carotene from the isolated Rhodosporidium sp., the cell walls were destroyed using dimethyl sulfoxide (DMSO) solution with and without the use of glass beads and a sonicator. Extracted β-carotene was identified by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) with a β-carotene standard. The yields of β-carotene extracted in DMSO, DMSO and glass beads, and DMSO with a sonicator were 3.371, 5.112, and 3.301 μg/mL, respectively. Isolated β-carotene was relatively heat stable, with 80% of the viable molecules remaining at 80°C.     

Purification, characterization, and production of β-mannanase from Bacillus subtilis TJ-102 and its application in gluco-mannooligosaccharides preparation

A novel β-mannanase-producing strain, Bacillus subtilis TJ-102, was identified and characterized. Response surface method was applied to improving and enhancing the enzyme production. The optimized media components were obtained: 45.25 g/L konjac, 9.29 g/L Na₂HPO₄·12H₂O, 2.60 g/L CaCO₃, 1.0 g/L (NH₄)₂SO₄, 0.3 g/L KH₂PO₄, 1.0 g/L NaCl, 1.0 g/L MgCl₂·6H₂O, and 0.01 g/L FeSO₄. Under these conditions, the β-mannanase activity could achieve 205.3 U/mL in a 7-L fermentor. Then, β-mannanase was 7.39-fold purified by salting out, ultrafiltration, anion-exchange, and size-exclusion preparative chromatography with a recovery of 21.41 % and a specificity of 125.36 U/mg proteins. β-Mannanase was stable below 65 °C and pH 5.0–8.0, which exhibited excellently enzymatic efficiency in the preparation of gluco-mannooligosaccharides (GMOS) by hydrolyzing konjac flour. The GMOS yield of 57.76 % has been achieved with 8.71 % of mannose and 14.49 % of glucose, demonstrating the potential use of β-mannanase in food industry.     

Expression and characterization of β-1,3-1,4-glucanase of Aspergillus usamii in Escherichia coli and its application in sourdough bread making

A β-1,3-1,4-glucanase gene (Auglu12A) from Aspergillus usamii was successfully expressed in Escherichia coli BL21(DE3). The recombinant enzyme, reAuglu12A was efficiently purified using the one-step nickel-nitrilotriacetic acid affinity chromatography. The specific activity of reAuglu12A was 694.8 U/mg, with an optimal temperature of 55°C and pH of 5.0. The reAuglu12A exhibited stability at temperatures up to 60°C and within the pH range of 4.0–5.5. The reAuglu12A hydrolytic activity was increased in the presence of metal ions, especially K⁺ and Na⁺, whereas it exhibited a K_m and V_max of 8.35 mg/mL and 1254.02 µmol/min/mg, respectively, toward barley β-glucan at pH 5.0 and 55°C. The addition of reAuglu12A significantly increased the specific volume (p < 0.05) and reduced crumb firmness and chewiness (p < 0.05) of wheat–barley sourdough bread during a 7-day storage period compared to the control. Overall, the quality of wheat–barley sourdough bread was improved after incorporation of reAuglu12A (especially at 3000 U/300 g). These changes were attributed to the synergistic effect of acidification by sourdough and its metabolites which provided a conducive environment for the optimal action of reAuglu12A in the degradation of β-glucans of barley flour in sourdough. This stabilized the dough structure, thereby enhancing the quality, texture, and shelf life of the bread. These findings suggest that reAuglu12A holds promise as a candidate for β-glucanase application in the baking industry.     

Purification and characterization of an alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 and its application in mannooligosaccharides preparation having prebiotic potential

An alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 was purified 38.96-fold to homogeneity with specific activity of 2,288.90 ± 27.80 U mg^?1 protein and final recovery of 8.92 ± 0.09 %. The purified β-mannanase was an extracellular monomeric protein with a molecular mass of 50 kDa on SDS–PAGE. The first 20 N-terminal amino acid sequence of mannanase enzyme was MVVKKLSSFILILLLVTSAL. The optimal temperature and pH for enzyme were 65 °C and 8.8, respectively. It was completely stable at 60 °C for 3 h and retained >50 ± 1.0 % activity at 70 °C up to 3 h. The β-mannanase was highly stable between pH 5–10 and retained >85 % of the initial activity for 3 h. The metal ions Ni⁺², Co⁺², Zn⁺² and Mg⁺² enhanced the enzyme activity. The enzyme remained stable after 3 h of preincubation with most of the tested organic solvents. According to substrate specificity study, the purified mannanase had high specificity to locust bean gum which was degraded mainly to mannooligosaccharides (MOS) like mannotriose, mannotetraose and mannopentose. These MOS enhanced the growth of Lactobacillus casei but inhibited the growth of Salmonella enterica indicating potential prebiotic properties. The properties of the purified β-mannanase from B. nealsonii PN-11 make this enzyme attractive for biotechnological applications.     

Acidic β-mannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application

The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035 U mg^–1 towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining > 60% of its maximum activity at pH 3.0–7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The K_m and V_max values were 5.6 and 4.8 mg mL^–1, and 2785 and 1608 μmol min^–1 mg^–1, respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96 mg g^–1 reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed.     

Occurrence of extended-spectrum β-lactamases among isolates of Salmonella enterica subsp. enterica from food-producing animals and food products,in Portugal

A total of 1120 Salmonella spp. isolates, recovered from poultry, swine and food products of animal origin (bovine, swine and poultry) over the period of 2009–2011, were investigated in order to determine their serotype, susceptibility to a panel of eleven antimicrobials (A, ampicillin; Ct, cefotaxime; Cp, ciprofloxacin; Tm, trimethoprim; Su, sulfamethoxazole; C, chloramphenicol; S, streptomycin; G, gentamicin; T, tetracycline; NA, nalidixic acid; Fl, florfenicol), and the presence of resistance determinants of extended-spectrum cephalosporins. Overall, Salmonella Enteritidis was the most common serotype in all three animal species. In 618 isolates of poultry, 32.8% comprised S. Enteritidis, 18.3% Salmonella Havana and 16.5% Salmonella Mbandaka; in 101 isolates of pigs, 21.8% comprised Salmonella Rissen and Salmonella Typhimurium, 10.9% Salmonella Derby and Salmonella London. Salmonella I 4,[5],12:i:- was the most common serotype recovered from pork and beef food products comprising 32.6% and 30% of isolates respectively, followed by S. Rissen (26% and 24%) and S. Typhimurium (18.2% and 19%), respectively. In poultry products, S. Enteritidis was the most frequent serotype (62.7%), followed by S. Mbandaka (10.2%) and S. Derby (8.5%). Susceptibility profiles differed according to the origin of the isolates. Five multidrug resistant isolates (0.45%) were further characterized as extended-spectrum β-lactamase (ESBL) producers. Polymerase chain reaction and sequencing of the amplicons confirmed the presence of bla_CTX-M-1 (n = 2), bla_CTX-M-14 (n = 1), bla_CTX-M-15 (n = 1) and bla_CTX-M-32 (n = 1); bla_SHV-12 and bla_TEM-1 genes were also detected in two isolates of S. I 4,[5],12:i:-. Four isolates, two S. Havana and two S. I 4,[5],12:i:-, carried class 1 integrons and in three, two S. I 4,[5],12:i:- and one S. Havana, ISEcp1 was identified associated to bla_CTX-M-1, bla_CTX-M-32 and bla_CTX-M-14 genes. Additionally, in one S. I 4,[5],12:i:- isolate, orf477 was identified linked to bla_CTX-M-32. No plasmid mediated quinolone resistance-encoding genes were detected. Here, we report for the first time the presence of bla_CTX-M genes in Salmonella enterica subsp enterica isolates recovered from poultry and food products of swine origin, in Portugal.     

Validation of a liquid chromatography–mass spectrometry method for determining the migration of primary aromatic amines from cooking utensils and its application to actual samples

Many cooking utensils are made of nylon, a material that may incorporate azodyes and where primary aromatic amines (PAAs) are the starting substances. Moreover, aromatic amines may also be present as technical impurities. Another source of PAAs could be aromatic isocyanates used as monomers in the production of polyurethanes. The aim of this work was to validate a simple LC–MS/MS method for the determination of eight primary aromatic amines (m-phenylenediamine, 2,6- and 2,4-toluenediamine, 1,5-diaminonaphthalene, aniline, 4,4′-diaminonaphenylether, 4,4′-methylenedianiline and 3,3′-dimethylbenzidine) in the aqueous food simulant 3% acetic acid (w/v). The detection limits calculated were adequate with respect to present legislation. The method was validated at four concentration levels (2, 5 10 and 20 µg kg^?1). Global internal reproducibility was in the range 5.6–21.4% (RSD_R) depending on the compound and concentration. Mean recoveries for all levels varied between 89 and 100%, depending on the amine. A total of 39 samples of cooking utensils were analyzed using the described method and the results obtained after the third migration test were not compliant in approximately half of the samples.     

Effects of low-shear modeled microgravity on a microbial community filtered through a 0.2-μm filter and its potential application in screening for novel microorganisms

The effects of low-shear modeled microgravity (LSMMG) on a microbial community filtered through a 0.2-μm filter were investigated, and the potential application of LSMMG in the screening of microorganisms was evaluated. Pond water was passed through a 0.2-μm filter and the filtrate inoculated into two kinds of media (Schneider's insect medium, and ten-times-diluted Schneider's insect [0.1-Sch] medium). The cultures were incubated under LSMMG and normal-gravity and the microbial cell growth rates compared. Cell growth rates, final cell concentrations, and substrate consumption rates were higher in the LSMMG culture than in the normal-gravity culture. The microbial communities obtained under the various culture conditions were subjected to denaturing gradient gel electrophoresis (DGGE), revealing three different groups of microorganisms: (i) microorganisms whose growth rates were increased by LSMMG; (ii) microorganisms whose growth rates were suppressed or inhibited by LSMMG; and (iii) microorganisms whose growth rates were not affected by LSMMG. Sequence analysis of the microorganisms whose growth rates were increased by LSMMG showed that some had high similarity with unculturable microorganisms. When these microorganisms that displayed similarity with unculturable microorganisms were cultivated on agar plates, some of the DGGE bands present in the LSMMG culture were also present. We show that it is possible to isolate and cultivate uncultured microorganisms by using combinations of LSMMG, normal-gravity, and agar plate culturing techniques.     

Physicochemical properties, α-amylase and α-glucosidase inhibitory effects of the polysaccharide from leaves of Morus alba L. under simulated gastro-intestinal digestion and its fermentation capability in vitro by human gut microbiota

The investigation aimed at determining the impact of sequential simulated digestion on the physicochemical properties and digestive enzymes inhibitory effects of the polysaccharides fraction (MLP-2) of Morus alba L. leaves as well as its in vitro fermentation behaviours. After artificial salivary, gastric and intestinal digestions, the chemical components and microstructure of MLP-2 were altered with significantly (P < 0.05) decreased molecular weight. The α-amylase and α-glucosidase inhibitory activities of MLP-2 were significantly (P < 0.05) improved throughout simulated digestion. MLP-2I, the intestinal digested fraction of MLP-2, could significantly (P < 0.05) decrease the pH value of fermented culture and increase the short-chain fatty acids (SCFA) concentrations, especially acetic, propionic and butyric acids. In conclusion, MLP-2 could be gradually degraded under simulated digestion with altered physicochemical properties and enhanced α-amylase and α-glucosidase inhibitory effects, and further utilised by human gut microbiota to decrease pH value and promote SCFA production.     

Hydrolytic degree and antioxidant activity of purified casein characterised by different haplotypes (αs1-, β- and k-casein) after enzymatic hydrolysis with pepsin and enzymatic extract from Pleurotus eryngii

The aim of this study was to estimate the hydrolytic degree and antioxidant activity of purified casein characterised by different haplotypes (α_s1-, β- and k-casein) after in vitro digestion with two different enzymatic systems: pepsin from porcine gastric mucosa (EP) and a crude enzymatic extract from the edible mushroom Pleurotus eryngii. The used enzymes showed a different mode of casein catalysis with a consequent production of peptides of different antioxidant activity. The CN haplotype significantly influenced peptides production; in fact, the amino acid substitutions caused by genetic polymorphisms at the α_s1-, β- and k-CN loci influenced the sites of enzymatic cleavage and therefore the produced peptides. The above is evidenced by the different antioxidant activity found in the hydrolysates depending on the used enzymatic system, the CN haplotype, and the CN haplotype × enzymatic treatment interaction. The findings of this study are a perspective for the production of specific foods that exert a biological effect in addition to the nutritional one.