DFA III production from inulin with inulin fructotransferase in ultrafiltration membrane bioreactor

An ultrafiltration membrane bioreactor was used for the production of DFA III from enzymatic conversion of inulin. Compared with the traditional batch reactor, the productivity and purity of DFA III could be markedly enhanced and product inhibition was removed and IFTase could be continuously used for six runs in the UF membrane bioreactor. When the substrate concentration was 100 g/L, the concentration of DFA III was about 78.4 g/L, while the productivity and purity of DFA III could attain about 2385 and 92%, respectively.     

Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1

A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequence was determined. The cloned fragment contained a 1353 bp open reading frame. The initiation codon was estimated to be an unusual codon, GTG. The gene encoded a signal peptide (40 amino acid residues) for secretion. The molecular mass of the native enzyme was calculated as 43,400 Da from the sequencing data. The deduced amino acid sequence of the enzyme had 74.0 % homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacter sp. H65-7. It also had 45.1% homology with that of inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. The enzyme produced in the culture supernatant of an Escherichia coli clone was purified to the electrophoretically homogeneous stage. The N-terminal amino acid sequence of the cloned enzyme secreted in the broth was the same as that of the native enzyme from A. globiformis C11-1. Therefore, on this enzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.     

Cloning and extracellular expression of inulin fructotransferase from Arthrobacter aurescens SK 8.001 in E. coli

BACKGROUND: Difructose anhydride (DFA) III is a natural and low‐calorie sweetener. It stimulates the absorption of calcium and other minerals. Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin hydrolysis to DFA III, is considered to be the most promising enzyme for the production of DFA III. RESULTS: IFTase gene from Arthrobacter aurescens SK 8.001 was cloned and sequenced. Transformant with native IFTase signal peptide was a useful system for extracellular over‐expression of IFTase, and its extracellular IFTase activity reached 81.0 U mL^?1. This value was 4.1‐fold of that obtained with A. aurescens SK 8.001 for IFTase production. The recombinant IFTase was purified to electrophoretical homogeneity and characterized. The enzyme showed maximum activity at pH 6.0 and 55 °C, and retained 81.3% of its initial activity after incubation at 60 °C for 4 h. CONCLUSION: IFTase gene from A. aurescens SK 8.001 was cloned, sequenced and over‐expressed in E. coli. IFTase was reported for the first time to be over‐expressed extracellularly. The recombinant IFTase was purified and characterized, and shown to be a good candidate for potential application in DFA III production. Copyright © 2011 Society of Chemical Industry     

One-pot conversion of levan prepared from Serratia levanicum NN to difructose anhydride IV by Arthrobacter nicotinovorans levan fructotransferase

The newly established difructose anhydride IV (DFA IV) production system is comprised of the effective production of levan from sucrose by Serratia levanicum NN, the conversion of the levan into DFA IV by levan fructotransferase from Arthrobacter nicotinovorans GS-9, which is highly expressed in an Escherichiacoli transformant, and a practical purification step. The chemical properties of DFA IV were also investigated.     

Synbiotic potential of fresh cream cheese supplemented with inulin and Lactobacillus paracasei in co-culture with Streptococcus thermophilus

The influence of the addition of Lactobacillus paracasei and Streptococcus thermophilus on the fructan content at the beginning and at the end of storage at 4 ± 1 °C of a potentially synbiotic fresh cream cheese manufactured with inulin was investigated. Three cheese-making trials were prepared, all supplemented with a lactic culture of S. thermophilus (T1, T2 and T3). L. paracasei subsp. paracasei was added in T1 and T2. Inulin was added in T2 and the fructan content was measured after 1 and 21 days of storage. Samples of T2 possessed similar mean concentrations of fructans after 1 and 21 days of storage, 7.32% and 7.27%, respectively, and no significant difference was observed. These results indicated that the metabolism of starter and probiotic bacteria did not degrade the fructans present in those cheeses. Additionally, synbiotic cheeses possessed a fructan content higher than 7 g per 100 g, sufficient to confer prebiotic potential during the entire storage period of these products.     

O(2)-stable membrane-bound [NiFe]hydrogenase from a newly isolated Citrobacter sp. S-77

Hydrogenases are of great interest due to their potential use in H(2)-based technology. However, most hydrogenases are highly sensitive to O(2), which have been the major bottleneck in hydrogenase studies. Here we report an O(2)-stable membrane-bound [NiFe]hydrogenase (MBH) purified from a newly isolated strain, S-77. According to the 16S rRNA gene sequence and phylogenetic analysis of the strain S-77, it belongs to the genus of Citrobacter. In vitro experiments using the cytoplasmic membrane of strain S-77 suggested that a cytochrome b acts as the physiological electron acceptor of the MBH. The purified MBH was composed of a dimer of heterodimers, consisting of two distinct subunits with the molecular weights of 58.5 and 38.5?kDa. The enzyme showed a specific activity for H(2)-oxidation of 661U/mg, which is 35-fold greater than that for H(2)-production of 18.7U/mg. Notably, the MBH showed a remarkable O(2)-stability, maintaining almost 95% of its original activity even after incubation for 30?h in air at 4°C. These results suggest that the O(2)-stable MBH may play an important role in the H(2)-metabolic pathway under the aerobic conditions of Citrobacter sp. S-77. This is the first report of the purification and biochemical characterization of an O(2)-stable MBH from the genus of Citrobacter.     

Purification and characterization of an extracellular lipase from Mucor hiemalis f. corticola isolated from soil

We have screened 39 microfungi isolates originated from soil in terms of lipolytic activity. Out of all screened, a novel strain of Mucor hiemalis f. corticola was determined to have the highest lipase activity. The extracellular lipase was produced in response to 2% glucose and 2.1% peptone. The lipase was purified 12.63-folds with a final yield of 27.7% through following purification steps; ammonium sulfate precipitation, dialysis, gel filtration column chromatography and ion exchange chromatography, respectively. MALDI-TOF MS analysis revealed 31% amino-acid identity to a known lipase from Rhizomucor miehei species. The molecular weight of the lipase was determined as 46kDa using SDS-PAGE and analytical gel filtration. Optimal pH and temperature of the lipase were determined as 7.0 and 40°C, respectively. The enzyme activity was observed to be stable at the pH range of 7.0-9.0. Thermostability assays demonstrated that the lipase was stable up to 50°C for 60min. The lipase was more stable in ethanol and methanol than other organic solvents tested. Furthermore, the activity of the lipase was slightly enhanced by SDS and PMSF. In the presence of p-NPP as substrate, K(m) and V(max) values of the lipase were calculated by Hanes-Woolf plot as 1.327mM and 91.11μmol/min, respectively.     

Fusarium sibiricum sp. nov, a novel type A trichothecene-producing Fusarium from northern Asia closely related to F. sporotrichioides and F. langsethiae

Production of type A trichothecenes has been reported in the closely related species Fusarium langsethiae and F. sporotrichioides. Here, we characterized a collection of Fusarium isolates from Siberia and the Russian Far East (hereafter Asian isolates) that produce high levels of the type A trichothecene T-2 toxin and are similar in morphology to the type A trichothecene-producing F. langsethiae, and to F. poae which often produces the type B trichothecene nivalenol. The Asian isolates possess unique macroscopic and microscopic characters and have a unique TG repeat in the nuclear ribosomal intergenic spacer (IGS rDNA) region. In Asian isolates, the TRI1-TRI16 locus, which determines type A versus type B trichothecene production in other species, is more similar in organization and sequence to the TRI1-TRI16 locus in F. sporotrichioides and F. langsethiae than to that in F. poae. Phylogenetic analysis of the TRI1 and TRI16 gene coding regions indicates that the genes in the Asian isolates are more closely related to those of F. sporotrichioides than F. langsethiae. Phylogenetic analysis of the beta-tubulin, translation elongation factor, RNA polymerase II and phosphate permease gene sequences resolved the Asian isolates into a well-supported sister lineage to F. sporotrichioides, with F. langsethiae forming a sister lineage to F. sporotrichioides and the Asian isolates. The Asian isolates are conspecific with Norwegian isolate IBT 9959 based on morphological and molecular analyses. In addition, the European F. langsethiae isolates from Finland and Russia were resolved into two distinct subgroups based on analyses of translation elongation factor and IGS rDNA sequences. Nucleotide polymorphisms within the IGS rDNA were used to design PCR primers that successfully differentiated the Asian isolates from F. sporotrichioides and F. langsethiae. Based on these data, we formally propose that the Asian isolates together with Norwegian isolate IBT 9959 comprise a novel phylogenetic species, F. sibiricum, while the two subgroups of F. langsethiae only represent intraspecific groups.     

Hydrolysis of fructans from Agave tequilana Weber var. azul during the cooking step in a traditional tequila elaboration process

In traditional tequila production, the heads of Agave tequilana Weber var. azul are cooked in brick ovens to hydrolyze the fructan content and release fermentable sugars. The juice generated during cooking (known as “cooking honey”) was collected periodically in a tequila distillery and characterized to study the efficiency of fructan hydrolysis. The complex structure of fructans from A. tequilana was confirmed. The generation of 5-(hydroxymethyl)-furfural, an increase in absorbance and °Brix, and a decrease in pH and apparent average degree of polymerization of fructans during cooking were observed. The conversion of fructans in the flowing honey increased gradually from 20% at the onset of cooking to 98% after 25.5 h, where fructose represented more than 80% of the total carbohydrates. The proportion of non-hydrolyzed fructans in the cooking honey collected before this time resulted in a total ethanol loss of 6% in the tequila distillery investigated.     

Antimicrobial activity of catechol isolated from Diospyros kaki Thunb. roots and its derivatives toward intestinal bacteria

The growth-inhibiting activities of the methanol extract of Diospyros kaki Thunb. roots were examined on the growth of Bifidobacterium breve, B. longum, Clostridium difficile, Clostridium perfringes, Escherichia coli, and Lactobacillus casei. In addition, the biologically active component of D. kaki roots was purified using silica gel column chromatography and HPLC. The active component was characterised as catechol by spectroscopic analyses. The antimicrobial activity of the isolated catechol varied according to the dose and bacterial strains tested. Catechol significantly (++++) inhibited the growth of C. perfringens at 2.0 mg/disc, and moderately (++) inhibited its growth at 0.25 mg/disc. At a dose of 5.0 mg/disc, catechol significantly inhibited the growth of C. difficile and moderately inhibited the growth of E. coli. However, this isolate did not inhibit the growth of bifidobacteria and lactobacilli. When various functional groups were added to the catechol, selective growth-inhibiting activity against harmful intestinal bacteria was observed in response to treatment with low concentrations. Taken together, these findings indicate that D. kaki root-isolated catechol and its derivatives (4-nitrocatechol, 4-tert-butylcatechol, tetrabromocatechol) could be useful as preventive agents against diseases caused by harmful intestinal bacteria.     

Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer,against Listeria monocytogenes

Enterococcus faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on Listeria monocytogenes in Munster cheese, a red smear soft cheese. The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. A brine solution inoculated at 10⁵ CFU of E. faecium WHE 81 per mL was sprayed on the cheese surface during the first smearing operation. On day 7, smearing of cheese samples with a brine solution at 10² CFU of L. monocytogenes per mL yielded initial cell counts of approximately 50 CFU g⁻¹ of the pathogen on the cheese surface. Although, in some instances, L. monocytogenes could survive (<50 CFU g⁻¹) in the presence of E. faecium WHE 81, it was unable to initiate growth. In control samples however, L. monocytogenes counts often exceeded 10⁴ CFU g⁻¹. In other respects, E. faecium WHE 81, which naturally existed in Munster cheese, did not adversely impact on the ripening process.     

Phylogenetic analyses and toxigenic profiles of Fusarium equiseti and Fusarium acuminatum isolated from cereals from Southern Europe

Fusarium equiseti and Fusarium acuminatum are toxigenic species that contaminate cereal crops from diverse climatic regions. They are common in Spanish cereals. The information available on their phylogenetics and toxigenic profiles is, however, insufficient to assist risk evaluation. In this work, phylogenetic analyses were performed using partial sequences of the translation elongation factor gene (EF-1α) of F. equiseti and F. acuminatum strains isolated from barley and wheat from Spain and other countries. The Northern and Southern European F. equiseti strains largely separated into two phylogenetically distinct clusters. This suggests the existence of two distinct populations within this species, explaining its presence in these regions of markedly different climate. Production of type A and B trichothecenes by the Spanish strains, examined in wheat cultures using a multitoxin analytical method, indicated that F. equiseti could produce deoxynivalenol and nivalenol and other trichothecenes, at concentrations that might represent a significant risk of toxin contamination for Southern European cereals. F. acuminatum showed low intraspecific genetic variability and 58% of the strains could produce deoxynivalenol at low level. Neither species was found to produce T-2 or HT-2 toxins. The present results provide important phylogenetic and toxigenic information essential for the accurate prediction of toxigenic risk.     

Production and characterization of ectoine by Marinococcus sp. ECT1 isolated from a high-salinity environment

A halophilic bacterium isolated from a salt environment in southern Taiwan was identified as a Marinococcus sp. ECT1. This bacterium could synthesize and accumulate intracellular ectoine as a compatible solute capable of resisting osmotic stress in a hyper-osmotic environment. This study also developed a semi-synthesized medium (YAMS medium), capable of facilitating the growth of this Marinococcus sp. ECT1 with 600 mg/L crude ectoine production. Moreover, Marinococcus sp. ECT1 was grown on YAMS medium containing different initial yeast extract concentrations (C(YE)) (0 to 60 g/L) to demonstrate how C(YE) affects crude ectoine production. While the maximum cell concentration was increased by 23-fold when the C(YE) was 40 g/L, the maximum crude ectoine production reached 2.5 g/L when C(YE) was 40 g/L. In addition to demonstrating the success of the fermentation strategy of ectoine in increasing the production and production yield, experimental results further demonstrated that the fermentation medium of ectoine is highly promising for commercialization. Furthermore, the molecular weight and chemical structure of ectoine were identified and characterized by FAB-MS and (1)H-NMR.     

Tyramine production among lactic acid bacteria and other species isolated from kimchi

Lactic acid bacteria (LAB) are naturally found in fermented vegetable products. The ability of 230 kimchi bacterial isolates was investigated to produce tyramine by biochemical and genetic methods. The production of tyramine was determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The presence of the gene encoding the corresponding tyrosine decarboxylase was also determined by PCR assay. After the production of tyramine was confirmed by chromatographic and molecular methods, the bacterial isolates producing the amine were identified by 16S rRNA gene sequence and species-specific PCR analyses. Only a small proportion of the bacterial isolates (14/230 isolates) decarboxylated tyrosine in vitro. All of the 14 bacterial isolates that produced tyramine were shown to possess the tdc gene, indicating that a positive correlation existed between the production of tyramine and the presence of the corresponding decarboxylase gene. The 14 isolates included three LAB species and one other species: Lactobacillus brevis (six), Lactobacillus curvatus (four), Leuconostoc mesenteroides (two), and Staphylococcus hominis (two). This study demonstrated that only a small proportion of LAB and other microbiota growing in kimchi had the ability to produce tyramine.     

Properties and safety aspects of Enterococcus faecium strains isolated from Chungkukjang, a fermented soy product

Enterococcus faecium isolated from Chungkukjang, a Korean traditional fermented soybean food was studied for their functional characteristics as potential new starter culture and safety. Microbiological analysis of ripened Chungkukjang revealed the presence of an enterococcal population in numbers of up to 6 log CFU per g. Seven isolates with higher activity were selected for further study and the strains were identified as E. faecium. The E. faecium strains showed resistance against simulated gastrointestinal conditions such as acidic environment and the presence of bile salts. These strains also showed bile salt hydrolase activity but neither hemolytic activity nor virulence determinant such as gelE and efaAfm. All strains were susceptible to glycopeptides and lacked potential as vancomycin-resistant enterococci (VRE). Two strains, S2C10 and S2C11, showed inhibited the viability of Listeria monocytogenes in vitro. The ability was probably due to the production of bacteriocin. The lipase activity influenced the stability, while either acidic condition or high temperature did not play a significant role in the activity of the antimicrobial substances. The strains also produced thermostable listericidal antimicrobial substance. For this reason, the strains could be used as selected starters or protective cultures in soybean fermented food production.