Proteomic analysis of the distribution of the major seed allergens in wild,landrace, ancestral,and modern soybean genotypes

BACKGROUND: A comparative analysis of seed allergens from various soybean genotypes is crucial for identifying and eliminating potential allergens. We have investigated the distribution of three major allergens (Gly m Bd 60K, Gly m Bd 30K and Gly m Bd 28K) in wild, landrace, ancestral and modern soybean genotypes. RESULTS: Gly m Bd 60K allergens consist of α subunits of β‐conglycinin and G2 subunits of glycinin. In wild genotypes, α subunits of β‐conglycinin separated into six to seven protein spots whereas five to seven spots were observed in the landraces. All genotypes of modern and ancestral groups showed 3–5 protein spots of α subunits of β‐conglycinin. All genotypes showed eight spots of glycinin G2 subunits except one ancestral genotype which had seven spots. Two protein spots were detected for Gly m Bd 30K in 14 genotypes but one spot was detected in two wild genotypes. Two protein spots were detected for Gly m Bd 28K in all genotypes. CONCLUSION: Considerable heterogeneity of the α subunit of β‐conglycinin distribution exists among these 16 soybean genotypes. Significant proteomic variation was observed between different soybean groups rather than among genotypes in the same group. This investigation would be valuable to researchers working with soybean and nutrition. Copyright © 2007 Society of Chemical Industry     

Two Novel Bioactive Peptides from Antarctic Krill with Dual Angiotensin Converting Enzyme and Dipeptidyl Peptidase IV Inhibitory Activities

Inhibition of dipeptidyl peptidase IV (DPP‐IV) and angiotensin converting enzyme (ACE) are considered useful in managing 2 often associated conditions: diabetes and hypertension. In this study, corolase PP was used to hydrolyze Antarctic krill protein. The hydrolysate (AKH) was isolated by ultrafiltration and purified by size‐exclusion chromatography, ion exchange chromatography and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) sequentially. The in vitro inhibitory activities of all AKHs and several fractions obtained against ACE and DPP‐IV were assessed. Two peptides, purified with dual‐strength inhibitory activity against ACE and DPP‐IV, were identified by TOF‐MS/MS. Results indicated that not all fractions exhibited dual inhibitory activities of ACE and DPP‐IV. The purified peptide Lys‐Val‐Glu‐Pro‐Leu‐Pro had half‐maximal inhibitory concentrations (IC₅₀) of 0.93±0.05 and 0.73±0.04 mg/mL against ACE and DPP‐IV, respectively. The other peptide Pro‐Ala‐Leu had IC₅₀ values of 0.64±0.05 and 0.88±0.03 mg/mL against ACE and DPP‐IV, respectively. This study firstly reported the sequences of dual bioactive peptides from Antarctic krill proteins, further provided new insights into the bioactive peptides responsible for the ACE and DPP‐IV inhibitory activities from the Antarctic krill protein hydrolysate to manage hypertension and diabetes.     

Comparison and Characterization of Compounds with Antioxidant Activity in Lycium barbarum Using High‐Performance Thin Layer Chromatography Coupled with DPPH Bioautography and Tandem Mass Spectrometry

Methanol extracts from 50 batches of Lycium barbarum (L. barbarum, wolfberry) in China were compared and characterized using high‐performance thin‐layer chromatography coupled with 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) bioautography (HPTLC‐DPPH) and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF‐MS/MS), respectively. Results showed that similar components occupying the major antioxidant activity existed in L. barbarum collected from different origins. However, the average antioxidant capacities of methanol extracts of L. barbarum collected in Ningxia were significantly higher than those of Qinghai, Xinjiang, Inner Mongolia, and Gansu, which may contribute to rational use of L. barbarum in China. Furthermore, the chemical structure of compound with the highest antioxidant capacity was tentatively identified as 2‐O‐β‐d ‐glucopyranosyl‐l ‐ascorbic acid using ESI‐Q‐TOF‐MS/MS analysis, which possessed high potentials to be used as an antioxidant biomarker for the quality control of L. barbarum. Results are helpful for the bioactivity‐based quality control of L. barbarum, and beneficial for the improvement of their performance in functional/health foods area, suggesting that HPTLC‐DPPH bioautography with ESI‐Q‐TOF‐MS/MS could be used as a routine approach for quality control of antioxidant components in L. barbarum.     

Application of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a monitoring tool for in‐house brewer's yeast contamination: a proof of concept

Contamination of brewer's pitching yeast cultures with wild‐type yeasts or bacteria is unwanted as it can corrupt the fermentation outcome and causes huge economic losses for the brewing industry. The applicability of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a fast tool to monitor the purity of brewer's yeast cultures was investigated. This proof of concept was examined for a brewer's yeast strain contaminated with wild‐type yeast and for bottled beer produced by fermentation with that particular contaminated brewer's yeast strain. The data demonstrated that MALDI‐TOF MS is very suitable to discriminate between brewing and non‐brewing yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

Impact of the synbiotic combination of Lactobacillus casei shirota and oligofructose-enriched inulin on the fecal volatile metabolite profile in healthy subjects

Scope: Hypothesis‐driven approaches have mainly focused on the quantification of SCFAs as mediators of beneficial effects of synbiotics. However, the emergence of metabolite profiling strategies allows to evaluate the colonic metabolism from a top‐down approach. In the present study, we evaluated the impact of a synbiotic combination on fecal metabolite profiles. Methods and results: A synbiotic combination (Lactobacillus casei Shirota cells+oligofructose‐enriched inulin) was evaluated in nine healthy volunteers. Before the start, during and after 4‐wk treatment, fecal samples were obtained. GC‐MS technology was applied to analyze the volatile metabolites. Application of a Type III test revealed that the metabolite profiles from the three conditions were significantly different. We identified three volatile organic compounds, acetate, dimethyl trisulfide and ethyl benzene, which were significantly affected. The acetate levels increased, whereas the dimethyl trisulfide levels decreased during and after the intervention. For ethyl benzene only an effect during the synbiotic intervention period was observed. Conclusion: We report a detailed analysis of the influence of L. casei Shirota combined with oligofructose‐enriched inulin on fermentation metabolites. Our results indicated a stimulation of saccharolytic fermentation and, importantly, a reduction of potentially toxic protein fermentation metabolites dimethyl trisulfide and ethyl benzene attended these effects.     

Bioavailability study of a polyphenol‐enriched extract from Hibiscus sabdariffa in rats and associated antioxidant status

The aqueous extracts of Hibiscus sabdariffa have been commonly used in folk medicine. Nevertheless, the compounds or metabolites responsible for its healthy effects have not yet been identified. The major metabolites present in rat plasma after acute ingestion of a polyphenol‐enriched Hibiscus sabdariffa extract were characterized and quantified in order to study their bioavailability. The antioxidant status of the plasma samples was also measured through several complementary antioxidant techniques. High‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry (HPLC‐ESI‐TOF‐MS) was used for the bioavailability study. The antioxidant status was measured by ferric reducing ability of plasma method, thiobarbituric acid reactive substances assay, and superoxide dismutase activity assay. Seventeen polyphenols and metabolites have been detected and quantified. Eleven of these compounds were metabolites. Although phenolic acids were found in plasma without any modification in their structures, most flavonols were found as quercetin or kaempferol glucuronide conjugates. Flavonol glucuronide conjugates, which show longer half‐life elimination values, are proposed to contribute to the observed lipid peroxidation inhibitory activity in the cellular membranes. By contrast, phenolic acids appear to exert their antioxidant activity through ferric ion reduction and superoxide scavenging at shorter times. We propose that flavonol‐conjugated forms (quercetin and kaempferol) may be the compounds responsible for the observed antioxidant effects and contribute to the healthy effects of H. sabdariffa polyphenolic extract.     

Rapid and accurate bacterial identification in probiotics and yoghurts by MALDI-TOF mass spectrometry

Probiotic food is manufactured by adding probiotic strains simultaneously with starter cultures in fermentation tanks. Here, we investigate the accuracy and feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for bacterial identification at the species level in probiotic food and yoghurts. Probiotic food and yoghurts were cultured in Columbia and Lactobacillus specific agar and tested by quantitative real-time PCR (qPCR) for the detection and quantification of Lactobacillus sp. Bacterial identification was performed by MALDI-TOF analysis and by amplification and sequencing of tuf and 16S rDNA genes. We tested 13 probiotic food and yoghurts and we identified by qPCR that they presented 10(6) to 10(7) copies of Lactobacillus spp. DNA/g. All products contained very large numbers of living bacteria varying from 10(6) to 10(9) colony forming units/g. These bacteria were identified as Lactobacillus casei, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus delbrueckii, and Streptococcus thermophilus. MALDI-TOF MS presented 92% specificity compared to the molecular assays. In one product we found L. lactis, instead of Bifidus spp. which was mentioned on the label and for another L. delbrueckii and S. thermophilus instead of Bifidus spp. MALDI-TOF MS allows a rapid and accurate bacterial identification at the species level in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified. Practical Application: MALDI-TOF MS is rapid and specific for the identification of bacteria in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified.     

HPLC–DAD–ESI–MS Analysis of Phenolic Compounds During Ripening in Exocarp and Mesocarp of Tomato Fruit

Identification of phenolic compounds was done by means of liquid chromatography (HPLC) coupled to mass spectrometry (MS) using the electrospray ionization interface (ESI). Quantification of phenolic compounds was carried out by using HPLC with diode array detector (DAD) in exocarp and mesocarp of tomato fruit at 6 different ripeness stages (mature‐green, breakers, turning, pink, light‐red, and red). Several phenolic compounds were identified including chlorogenic acid, caffeic acid, p‐coumaric acid, ferulic acid, and rutin and some combined phenolic acids were tentatively identified, mainly glycosides, such as caffeoyl hexose I, caffeoyl hexose II, caffeoylquinic acid isomer, dicaffeoylquinic acid, p‐coumaroyl hexose I, p‐coumaroyl hexose II, feruloyl hexose I, feruloyl hexose II, siringyl hexose, and caffeoyl deoxyhexose hexose. Fruit exocarp had higher quantities of total soluble phenolics (TSP) compared to mesocarp. During ripening, TSP increased in both exocarp and mesocarp, mainly in exocarp. While rutin increased, chlorogenic acid decreased in both tissues: exocarp and mesocarp.     

Identification and proteomic analysis of a novel gossypol-degrading fungal strain

BACKGROUND: Cottonseed meal, an important source of feed raw materials, has limited use in the feed industry because of the presence of the highly toxic gossypol. The aim of the current work was to isolate the gossypol‐degrading fungus from a soil microcosm and investigate the proteins involved in gossypol degradation. RESULTS: A fungal strain, AN‐1, that uses gossypol as its sole carbon source was isolated and identified as Aspergillus niger. A large number of intracellular proteins were detected using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but no significant difference was observed between the glucose‐containing and gossypol‐containing mycelium extracts. Two‐dimensional gel electrophoresis results showed that the protein spots were concentrated in the 25.0–66.2 kDa range and distributed in different pI gradients. PDQuest software showed that 51 protein spots in the gels were differentially expressed. Of these, 20 differential protein spots, including six special spots expressed in gossypol, were analyzed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. CONCLUSION: The fungus AN‐1 biodegraded gossypol and the proteomic analysis results indicate that some proteins were involved in the gossypol biodegradation during fungus survival, using gossypol as its sole carbon source. Copyright © 2011 Society of Chemical Industry     

Molecular cloning,structural analysis and mass spectrometric identification of native dioscorins of various yam species

BACKGROUND: Dioscorins are the major storage proteins of yam tubers. However, the molecular nature of their heterogeneity in tubers has not been fully elucidated. In this study the authors isolated the dioscorin gene families of Dioscorea japonica and Dioscorea pseudojaponica, performed matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and elucidated which dioscorin isoforms are the major constituents in tubers. RESULTS: The dioscorin gene families of D. japonica (Dj‐dioA1‐Dj‐dioA4, Dj‐dioB1 and Dj‐dioB2) and D. pseudojaponica (Dp‐dioA1‐Dp‐dioA5 and Dp‐dioB1) were cloned from cDNA libraries of yam tubers. The dioscorins isolated from Dioscorea alata (Da‐dioscorins), D. japonica (Dj‐dioscorins) and D. pseudojaponica (Dp‐dioscorins) were mainly monomers, with a few dimers. The monomers contained one intramolecular disulfide bond (Cys²⁸‐Cys¹⁸⁷) and belonged to Class A dioscorins with two cysteine residues. The dimers consisted of Class B dioscorins with one intermolecular disulfide bond (Cys⁴⁰‐Cys⁴⁰). Results of MALDI‐TOF‐MS revealed that the Da‐dioscorins were mainly encoded by Da‐dioA2, Da‐dioA3 and Da‐dioA4. The majority of the Dj‐dioscorins were encoded by Dj‐dioA1, Dj‐dioA2, Dj‐dioA3 and Dj‐dioB2. The Dp‐dioscorins mainly comprised proteins encoded by Dp‐dioA1, Dp‐dioA3, Dp‐dioA4, Dp‐dioB1 and Dp‐dioB2. CONCLUSION: Determination of the constituents of dioscorin isoforms in yam tubers provides a basis for future studies of their physiological and biomedical functions. © 2012 Society of Chemical Industry     

Chemical Profiling Using Uplc Q‐Tof/Ms and Antioxidant Activities of Fortunella Fruits

The fruits of Fortunella Swingle are widely consumed as fresh fruits and traditional medicine in China. China is the origin center and has the largest cultivated area of the genus Fortunella. In this study, the chemical compositions of ethanol extracts of the major Fortunella cultivated types including Fortunella japonica Swingle, Fortunella margarita Swingle, Fortunella crassifolia Swingle 1 (Lanshang) and Fortunella crassifolia Swingle 2 (Liuyang) were determined using ultra performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (UPLC Q‐TOF/MS) method, and their antioxidant activities were evaluated. 12 compounds were identified and 5 compounds were tentatively characterized. The results showed that the chemical compositions of the ethanol extracts of 4 Fortunella cultivated types were largely the same. 3′, 5′‐di‐C‐glucopyranosylphloretin was the predominant flavonoid in Fortunella fruits, and Fortunella margarita Swingle had higher contents of flavonoids than other species. In addition, the data demonstrated high antioxidant activities of Fortunella fruits. The developed method could be available to rapidly analyze the chemical compounds in Fortunella fruits and its products. This study will provide information for further quality assessment and utilization of Fortunella resources.