TOF Mass Spectrometry Research Articles

Sensitivity to Enterocins of Thermophilic Campylobacter spp. from Different Poultry Species.

Campylobacter spp. have been isolated from various animals, including poultry. They are rapidly transmitted throughout broiler sheds by the fecal-oral route. A promising strategy to reduce Campylobacter spp. in poultry may be done due to the beneficial properties of probiotic bacteria and their bacteriocins. In this study, inhibition spectrum/activity of different enterocins was evaluated against Campylobacter spp. (isolated from different poultry) to indicate further practical use of enterocins. Enterocins are antimicrobial proteinaceous substances produced mostly by enterococci. Feces from broiler chickens (10), laying hens (47), ostriches (140), and ducks (40) were screened. Altogether, 23 strains were allotted to the species Campylobacter jejuni and Campylobacter coli using MALDI TOF mass spectrometry and confirmed by genotyping (PCR method). In the feces of ostriches, Campylobacter spp. were not confirmed. Campylobacter spp. isolated from different poultry showed resistance to nalidixic acid, ciprofloxacin, and ampicillin. Interestingly, strains demonstrating antibiotic resistance revealed sensitivity to at least one of the nine enterocins used (except C. coli Kc1, SZ3, and C. jejuni 1/D). Almost 52% strains were inhibited by Ent A (P). Enterocins can therefore be used to prevent or reduce Campylobacter spp.; it is a basis for practical use.

IDENTIFICATION OF YEASTS SPECIES COLLECTED FROM PATIENTS USING OF MALDI -TOF MASS SPECTROMETRY TECHNIQUE AND SEQUENCING

Background: Yeasts are important opportunistic pathogen in human, in which Candida spp. are the most common causative agents. This study was carried out on 121 yeast strains collected from 103 patients in Hue University Hospital and Hue Central Hospital from January 2013 to June 2014. Objective: To identify yeasts species from systemic mycoses and superficial mycoses. Methods: We applied MALDI - TOF Mass Spectrometry techniques, PCR and DNA sequencing to detect yeasts species. Results: There were 121 yeast strains collected, in which C.albicans 43.80%, C.tropicalis 17.36%, C.parapsilosis 11.75%, C.glabrata 7.44%, C.orthopsilosis 4.96%, C.metapsilosis 0.83%, C.krusei 3.31%, C.norvegensis 0.83%, C.guilliermondii 0.83%, C.digboiensis 2.48%, C.famata 1.65%, C.blankii 0.83%, C.mesorugosa 0.83%, Geotrichum capitatum 1.65%, Trichosporon asahii 1.65%. Conclusions: In our study, the prevalences of C.abicans, C.non albicans and other yeasts species were 43.80%, 47.90% and 3.30% respectively. We reported some rare species of Candida non albicans, including C. orthopsilosis, C.metapsilosis, C.norvegensis, C.digboiensis, C.blankii, and C.mesorugosa. Key words: Yeast, Candida spp., MALDI - TOF Mass Spectrometry

Study on the metabolites of isoalantolactone in vivo and in vitro by ultra performance liquid chromatography combined with Triple TOF mass spectrometry

Isoalantolactone (IAL) is an active sesquiterpene naturally present in many vegetables, condiments and medicinal plants. In this study, an efficient strategy was developed for the identification of metabolites following the in vivo metabolism and in vitro biotransformation of IAL with rat intestinal bacteria utilizing ultra performance liquid chromatography combined with Triple TOF mass spectrometry (UPLC-Triple TOF-MS/MS). As a result, 46 metabolites including 34 novel sulfur-containing products were identified. The results demonstrated that IAL could undergo general metabolic reactions, including oxidation, hydration, hydrogenation, demethylation, cysteine conjugation and N-acetylcysteine conjugation, but the most noticeable characteristic of IAL metabolism was the H2S addition to the double bond between C-11 and C-13 and subsequent reactions to produce a series of novel sulfur-containing dimers. This is the first study of IAL metabolism in vivo, whose results provide novel and useful data for a better understanding of the safety and efficacy of IAL.

23‐ and 27‐Membered Macrocyclic Diorganotin(IV) Bis‐dithiocarbamates: Synthesis, Spectroscopic Characterization, DFT Calculations, and Physicochemical Analysis as Anion Receptors

Four secondary diamines with aromatic spacer groups derived from α,α′‐dibromo‐p‐xylene and 4,4′‐bis(chloromethyl)‐1,1′‐biphenyl have been transformed into bis‐dithiocarbamate ligands and each combined with dimethyl‐, di‐n‐butyl‐, and diphenyltin(IV) dichloride to yield a total of 12 mononuclear diorganotin(IV) bis‐dithiocarbamate complexes with six‐coordinate metal coordination geometries and either 23‐ or 27‐membered macrocyclic structures. The products were fully characterized by IR and NMR (1H, 13C, 119Sn) spectroscopy and high‐resolution ESI+‐TOF mass spectrometry. A representative selection of the macrocyclic complexes was further examined by DFT computational studies and the relative binding energies of the cis and trans isomers of the different diorganotin complexes were calculated and compared. Additionally, four possible conformations of the trans isomers were analyzed. With the purpose of examining whether the complexes can be applied to the recognition of anions, five macrocycles were selected based on criteria that allowed the influence of the aromatic spacer group, the N substituent of the dithiocarbamate ligand, and the organic groups attached to the metal center on the recognition properties to be explored. Among the NMe4+X– salts (X– = F–, Cl–, Br–, I–, NO3–, HSO4–, and CH3COO–) examined, the diorganotin complexes were sensitive to fluoride and carboxylate anions.

Vortex Fluidic Device-Intensified Aqueous Two Phase Extraction of C-Phycocyanin from Spirulina maxima

A vortex fluidic device (VFD) is effective for downstream processing of C-phycocyanin (C-PC) from blue-green microalga Spirulina maxima using an aqueous two phase system (ATPS): polyethylene glycol (PEG) and aqueous potassium phosphate. The VFD-intensified ATPS continuous flow processing (VFD-ATPS) has spontaneous phase separation of the liquid exiting the 20 mm diameter tube inclined at 45° and rotating at 6550 rpm, for a 0.2 mL/min flow rate. This processing results in at least a 9.3-fold increase in phase demixing efficiency, 1.18-fold increase in C-PC purity, and 22% increase in yield compared to conventional ATPS methods. Multistage counter current distribution (CCD) using VFD-ATPS followed by removal of PEG and salt increased C-PC purity by 6-fold compared to the crude extract, with triple TOF mass spectrometry showing more than 95% sequence coverage for both subunits. This work establishes the utility of using VFD-intensified ATPS processing for protein purification and its potential as an effectiv...

Measurement of ion species produced due to bombardment of 450 eV N2+ ions with hydrocarbons-covered surface of tungsten: Formation of tungsten nitride

A laboratory experiment has been performed to study the ions that are produced due to collisions of 450eVN2+ ions with a hydrocarbons-covered surface of polycrystalline tungsten at room temperature. Using a TOF mass spectrometry technique, the product ions formed in these collisions have been detected, identified and analyzed. Different ion–surface reaction processes, namely, neutralization, reflection, surface induced dissociation, surface induced chemical reactions and desorption are observed and discussed. Apart from the presence of desorbed aliphatic hydrocarbon and other ions, the mass spectra obtained from the considered collisions show the formation and sputtering of tungsten nitride (WN). A layer of WN on tungsten surface is known to decrease the sputtering of bulk tungsten in fusion devices more effectively than when the tungsten is bombarded with other seeding gases (He, Ar). It is further noted that there is a negligible diffusion of N in the bulk tungsten at room temperature.

Proteomic analysis in the induction of nodular cluster cultures in the bromeliad Vriesea reitzii Leme and Costa

Nodular cluster cultures (NCs) are globular organogenic clumps with a high regenerative potential applied to the large-scale micropropagation of bromeliads. In the present work, we identified differentially expressed proteins involved in the induction of NCs from seeds and leaf explants of the Brazilian native bromeliad Vriesea reitzii. Those explants were inoculated into Murashige and Skoog (MS) liquid medium free of plant growth regulators (PGR). To promote the induction of NCs, the seeds were grown in MS medium supplemented with 4 μM α-naphthaleneacetic acid (NAA), and the leaf segments in MS medium supplemented with 4 μM NAA and 2 μM 6-benzylaminopurine (BAP). After 21 days in culture, samples of each type of explant were collected for histological analysis and protein extraction. Proteomic analysis was performed by two-dimensional (2D) electrophoresis and protein identification by MALDI-TOF–TOF mass spectrometry. Enhanced protein content and number of detected spots on cultures supplemented with PGR were observed as compared to the cultures maintained in PGR-free MS culture medium. Five differentially expressed proteins were identified during the induction of NCs: heat shock 22 kDa, chaperone protein dnaJ 50, S-adenosylmethionine synthase 3, UDP-arabinopyranose mutase 1, and 14-3-3-like protein E. Such proteins are involved in stress response, cell metabolism, and cell division. The ability to regulate the effects of stress conditions in which the explants were subjected shows the presence of competent tissues for the acquisition of the morphogenic route associated to the induction of NCs.

CHARACTERISTICS OF BIOLOGICAL AND MOLECULAR-GENETIC PROPERTIES OF LACTOBACILLUS FERMENTUM 90 TC-4 PROBIOTIC STRAIN

Confirmation of taxonomic position of Lactobacillus fermentum 90 TC-4 strain using phenotypic (classic microbiological, MALDI TOF mass-spectrometry) and genetic (16S rRNA gene segment sequencing and full genome sequencing) methods. Object of the study--Lactobacillus fermentum 90 TC-4 strains from various collections. Mass-spectrometric analysis was carried out using Autoflex MALDI TOF mass-spectrometer (Bruker Daltonics, Germany), study of biochemical properties of the strain was carried out using API 50 CHL strips (Biomerueux, France), "DNA-sorb B" kitwas used for isolation ofgenome DNA (CRIE, Moscow). Sequencing of the accumulated fragments of 16S rRNA gene was carried out using GenomeLab GeXP sequencing (Beckman Coulter, USA), full genome sequencing was carried out in MiSeq platform (Illumina). Assembly of genome and bioinformation analysis was carried out using BLAST program (www.blast.ncbi.nlm.nih.gov/blast.cgi), "CLC Bio Assembly" and genome server RAST (rast.nmpdr.org). L. fermentum 90 TC-4 strain was established to be contaminated by L. plantarum culture in a series of cases. As a result of identification of a pure culture of L. fermentum 90 TC-4 strain using a specter of high-technology methods, membership of the strain in L. fer- mentum species has been proven. Taxonomic status of L. fermentum 90 TC-4 strain was confirmed.

Noninvasive characterization of metabolites secreted in culture media by bovine embryos during in vitro production

Secreted molecules could be correlated with the potential of embryonic development. The development of new technologies, such as mass spectrometry (MS), has enabled analyzes in culture medium to favor the determination of embryos viability in order to improve embryo selection. To perform a non-invasive characterization of the secretome of in vitro produced embryos with different kinetics of cleavage and in different stages of development to obtain specific patterns based on embryonic phenotype through MALDI–TOF–MS. Bovine embryos were produced in vitro by standard protocols. The zygotes were transferred to individual culture medium and divided into two groups: Fast [4 cells-22 hours past the beginning of culture (hpc)] and Slow (2 cells-22 hpc). Culture media drops were collected at 22, 96 and 168 hpc. Analysis of embryonic secretome was made by MALDI–TOF–MS after extractions of the metabolites. Spectra were acquired in positive ionization mode. Univariate (Fold-change) and multivariate (Partial Least Squares Discriminants Analysis) analyses were performed by the online software Metaboanalyst. It was demonstrated that embryos with different kinetics have different spectrometric profiles during embryonic development. Moreover, secreted molecules in each developmental stage are differentially represented in embryos with different kinetics, and are related to specific pathways such as lipid and amino acids metabolism and cell proliferation. We propose that the analysis of culture media by MALDI–TOF–MS can be used for qualitative characterization of bovine embryos, allowing the identification of key molecules during in vitro culture.

Porphyrin-rhodamine conjugates as new materials with sensing ability

Conjugates based on porphyrin (porph) and rhodamine (rh) moieties linked by amide bonds, dyad 1 (porph-spacer-rh) and triad 2 (porph-spacer-rh-spacer-porph), were prepared respectively from precursor 1 and precursor 2. The sensing ability of precursor 1, dyad 1 and triad 2 towards the divalent metal ions Cu2+, Zn2+, Cd2+, Pb2+, Hg2+ and the monovalent Hg+ was explored in solution by absorption and fluorescence spectroscopy as well as in gas phase using matrix-assisted laser desorption/ionization (MALDI)-TOF mass spectrometry. In solution one-metal-per-ligand molar ratio was obtained with precursor 1 and triad 2, however, a two-metal-per-ligand molar ratio was observed with dyad 1. Additionally, the results obtained by MALDI-TOF-MS confirmed the gas-phase sensing abilities of dyad 1 towards all metal ions tested and of triad 2 towards Zn2+. Computational DFT calculations confirm the same stoichiometry.

Identification of in vivo and in vitro metabolites of 4,5-dimethoxycanthin-6-one by HPLC-Q-TOF-MS/MS.

4,5-Dimthexycanthin-6-one and 5-hydroxy-4-methoxycanthin-6-one are the main active ingredients of Picrasma quassioides, which is a widely used herbal medicine for the treatment of gastroenteritis, snakebite, infection and hypertension in China. In the present study, the in vitro metabolites of 4,5-dimethoxycanthin-6-one in rat, mouse, dog and human liver microsomes, as well as the in vivo metabolites in rat plasma and urine following a single oral dose of 4,5-dimethoxycanthin-6-one, were identified by high-performance liquid chromatography combined with triple TOF mass spectrometry (HPLC-TOF/MS/MS). The metabolites were elucidated based on an accurate mass measurement, the MS/MS fragmentation patterns, the retention times of the parent drug and its metabolites, and the relevant drug biotransformation rules. After incubation in liver mcrosomes for 50 min, 4,5-dimethoxycanthin-6-one produced 8 phase I metabolites including 2 mono-demethylated metabolites (M1, M2), 3 mono-hydroxylated metabolites (M3-M5), and 3 mono-demethylated and mono-hydroxylated metabolites (M6-M8) in rat and mouse liver microsomes, 7 phase I metabolites (without M7) in dog and human liver microsomes. After a single oral administration of 4,5-dimethoxycanthin-6-one to rats, there were 3 phase I metabolites (M1, M2 and M5) detected in rat plasma and 5 phase I metabolites (M1-M5) in rat urine. Phase II metabolites were not detected in rat plasma and urine. Among these metabolites, mono-demethylated metabolites (M1 and M2) were the major metabolites of 4,5-dimethoxycanthin-6-one, mono-hydroxylated metabolites (M3-M5) were the minor metabolites of 4,5-dimethoxycanthin-6-one.

Multidimensional Separation of Natural Products Using Liquid Chromatography Coupled to Hadamard Transform Ion Mobility Mass Spectrometry.

A high performance liquid chromatograph (HPLC)was interfaced to an atmospheric drift tube ion mobility time of flight mass spectrometry. The power of multidimensional separation was demonstrated using chili pepper extracts. The ambient pressure drift tube ion mobility provided high resolving powers up to 166 for the HPLC eluent. With implementation of Hadamard transform (HT), the duty cycle for the ion mobility drift tube was increased from less than 1% to 50%, and the ion transmission efficiency was improved by over 200 times compared with pulsed mode, improving signal to noise ratio 10 times. HT ion mobility and TOF mass spectrometry provide an additional dimension of separation for complex samples without increasing the analysis time compared with conventional HPLC. Graphical Abstract ᅟ.

Proteomic alteration of a thermo‐sensitive male sterility SP2S in rapeseed (Brassica napus) in response to mild temperature stress

AbstractWe conducted a proteomic comparison between thermo‐sensitive genic male sterility (TGMS) SP2S and its near‐isogenic line SP2F grown at 22°C. The proteomes at microsporocyte meiosis and uninucleate microspore stages were profiled using 2‐dimensional gel electrophoresis. Twenty‐five well‐reproducible spots (10 spots at microsporocyte meiosis stage and 15 spots at uninucleate microspore stage) containing 28 proteins were successfully identified by MALDI‐TOF/TOF mass spectrometry. An elongation factor EF‐2 at microsporocyte stage and 4 proteins (aconitate hydratase, triosephosphate isomerase, serine/arginine‐rich mRNA splicing factor and glutathione S‐transferase) at uninucleate microspore stage accumulated in SP2S, but more proteins were lost or reduced, including those associated with amino acid metabolism, photosynthesis, synthesis and degradation of protein, lipid metabolism, cytoskeleton, RNA modification, oxidoreductase and defence response. The dramatic decrease of tubulin, actin and Translationally controlled tumour protein (TCTP) crucial for microtubule and cell division and three enzymes for amino acid metabolite at early stage indicated a serious defect on the cytokinesis. They were important clues for us to search the TGMS genes and its interacting genes.

Improved mass resolution and mass accuracy in TOF-SIMS spectra and images using argon gas cluster ion beams.

The popularity of argon gas cluster ion beams (Ar-GCIB) as primary ion beams in time-of-flight secondary ion mass spectrometry (TOF-SIMS) has increased because the molecular ions of large organic- and biomolecules can be detected with less damage to the sample surfaces. However, Ar-GCIB is limited by poor mass resolution as well as poor mass accuracy. The inferior quality of the mass resolution in a TOF-SIMS spectrum obtained by using Ar-GCIB compared to the one obtained by a bismuth liquid metal cluster ion beam and others makes it difficult to identify unknown peaks because of the mass interference from the neighboring peaks. However, in this study, the authors demonstrate improved mass resolution in TOF-SIMS using Ar-GCIB through the delayed extraction of secondary ions, a method typically used in TOF mass spectrometry to increase mass resolution. As for poor mass accuracy, although mass calibration using internal peaks with low mass such as hydrogen and carbon is a common approach in TOF-SIMS, it is unsuited to the present study because of the disappearance of the low-mass peaks in the delayed extraction mode. To resolve this issue, external mass calibration, another regularly used method in TOF-MS, was adapted to enhance mass accuracy in the spectrum and image generated by TOF-SIMS using Ar-GCIB in the delayed extraction mode. By producing spectra analyses of a peptide mixture and bovine serum albumin protein digested with trypsin, along with image analyses of rat brain samples, the authors demonstrate for the first time the enhancement of mass resolution and mass accuracy for the purpose of analyzing large biomolecules in TOF-SIMS using Ar-GCIB through the use of delayed extraction and external mass calibration.

Identification of an antimicrobial peptide from large freshwater snail (Lymnaea stagnalis): activity against antibiotics resistant Staphylococcus epidermidis

Nowadays, antibiotic resistance in bacteria is a great public health problem of increasing magnitude due to quick evolution through mutation that has generated the urgency to find the effective solutions to address this problem. Aside the conventional antibiotics, antimicrobial peptides are a new class of antimicrobials is known to have the activity against a wide range of bacteria. An antimicrobial peptide was isolated and purified from the Lymnaea stagnalis, a fresh water large snail, using ultrafiltration and reversed phase liquid chromatography. The molecular mass of the peptide 2345 Da was determined using MALDI TOF mass spectrometry. This peptide is efficiently prevented the growth of Staphylococcus epidermidis that resistant to ampicillin and chloramphenicol antibiotics. The MIC value was 16 μg/mL and specifically damage to bacterial membranes. Hence, this reported peptide revealed an alternative candidate to controlling the Staphylococcal infections.

Selective cycloaddition of aryl azides to fullerene C60 in the presence of Cu(OTf)2

Cycloaddition of aryl azides to fullerene C60 in the presence of a stoichiometric amount of copper(II) trifluoromethanesulfonate selectively afforded the corresponding aziridinofullerenes whose structure was reliably proved by 1H and 13C NMR, MALDI TOF mass spectrometry, and IR spectroscopy.

Identification of antigenic proteins of lymphocystis disease virus (LCDV) by MALDI-TOF mass spectrometry

The antigenic proteins of Lymphocystis disease virus (LCDV) from tumors of olive flounder, Paralichthys olivaceus, are described following characterization by mass spectrometry. In SDS-PAGE, predominant protein bands were observed at 114, 88, 70, 54, 52, 47, 42 and 24 kDa. Western blot analysis showed that antisera reacted strongly at molecular weights of 114, 67 and 54 kDa, and reacted weakly at molecular weights of 74, 70, 36, 24 and 22 kDa. In the identification of LCDV antigenic proteins by matrix-assisted laser desorption ionization (MALDI) TOF mass spectrometry, 10 of 14 excised bands consisted mostly of proteins with amino acid sequences that matched LCDV-C (lymphocystis disease virus isolate China) ORFs. Strong antigens with molecular weights of 114, 67 and 54 kDa were identified as LDVICp236 (chromosome segregation ATPase), LDVICp033 (membrane bound metallopeptidase) and LDVICp157 (hypothetical protein), respectively. Minor antigens with molecular weights of 70, 36, 24 and 22 kDa proteins were identified as LDVICp160 (acetyl-coA hydrolase), LDVICp213 (hypothetical protein), LDVICp039 (hypothetical protein) and LDVICp213 (hypothetical protein). However, the major capsid protein (LDVICp043) did not react with the polyclonal antibody.

Identification of Bacillus strains by MALDI TOF MS using geometric approach.

Microorganism identification by MALDI TOF mass-spectrometry is based on the comparison of the mass spectrum of the studied organism with those of reference strains. It is a rapid and reliable method. However, commercial databases and programs are mostly designed for identification of clinically important strains and can be used only for particular mass spectrometer models. The need for open platforms and reference databases is obvious. In this study we describe a geometric approach for microorganism identification by mass spectra and demonstrate its capabilities by analyzing 24 strains belonging to the Bacillus pumilus group. This method is based on representing mass spectra as points on a multidimensional space, which allows us to use geometric distances to compare the spectra. Delimitation of microorganisms performed by geometric approach correlates well with the results of molecular phylogenetic analysis and clustering using Biotyper 3.1. All three methods used allowed us to reliably divide the strains into two groups corresponding to closely related species, Bacillus pumilus and Bacillus altitudinis. The method developed by us will be implemented in a Web interface designed for using open reference databases for microorganism identification. The data is available at http://www.bionet.nsc.ru/mbl/database/database.html.

Synthesis, structure and reactivity of iron(II) clathrochelates with terminal formyl (acetal) groups

3- and 4-Formylphenylboron-capped aliphatic iron(II) clathrochelates were obtained by direct template condensation of the corresponding α-dioxime with 3- and 4-formylphenylboronic acids on an iron(II) ion as a matrix under mild reaction conditions in methanol or acetonitrile media; with methanol as a solvent, such one-pot reactions gave also their acetals. The latters were converted to the formyl-terminated iron(II) clathrochelates by H+-catalyzed hydrolysis. The reactive formyl-terminated cage complexes obtained underwent their further H+-catalyzed condensation with isoniazid as a hydrazone component giving the hydrazonate cage complexes with vector apical substituents of this type. The formyl-terminated iron(II) hexachloroclathrochelate was synthesized by template reaction of dichloroglyoxime on an iron(II) ion with 3-formylphenylboronic acid. Nucleophilic substitution of its reactive chlorine atoms with thiolate ions generated in situ from para-mercaptobenzoic acid afforded hexasulfide iron(II) cage complex with six ribbed para-mercaptobenzoic substituents and formyl groups in its apical substituents. The iron(II) complexes synthesized were characterized using elemental analysis, MALDI–TOF mass spectrometry, IR, UV–Vis, 1H and 13C{1H} NMR spectroscopies and by X-ray crystallography (for three aliphatic macrobicyclic acetals).

Action of an endo-β-1,3(4)-glucanase on cellobiosyl unit structure in barley β-1,3:1,4-glucan.

β-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single β-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-β-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley β-1,3:1,4-glucan. To find the minor structure on which the endo-β-1,3(4)-glucanase acts, we prepared oligosaccharides from barley β-1,3:1,4-glucan by endo-β-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-β-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as β-Glc-1,3-β-Glc-1,4-β-Glc-1,3-β-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-β-1,3(4)-glucanase acts on the cellobiosyl units of barley β-1,3:1,4-glucan in an endo-manner.