Citrinin Determination in Red Fermented Rice Products by Optimized Extraction Method Coupled to Liquid Chromatography Tandem Mass Spectrometry (LC‐MS/MS)

A rapid and sensitive method was developed and validated for citrinin determination in red fermented rice products by liquid chromatography tandem mass spectrometry (LC‐MS/MS) under the selected reaction monitoring mode. Sample preparation was especially focused, and the quantitative methods of LC‐MS/MS and high‐performance liquid chromatography with fluorescence detection (HPLC‐FLD) were compared. In red fermented rice samples, the limit of detection was 1.0 μg/kg for LC‐MS/MS compared to 250 μg/kg for HPLC‐FLD, the limit of quantification was 3.0 μg/kg for LC‐MS/MS compared to 825 μg/kg for HPLC‐FLD. High correlation coefficient was obtained (R² = 0.999) within the linear range (0.1 to 100 μg/L) in the MS method. The recoveries ranging from 80.9% to 106.5% were obtained in different spiking concentrations. The average intra‐ and inter‐day accuracy ranged from 75.4% to 103.1%, and the intra‐ and inter‐day precisions were from 3.3% to 7.9%. The developed method was applied to 12 commercial red fermented rice products, and citrinin was found in 10 samples ranging from 0.14 to 44.24 mg/kg. Compared to traditional qualitative and quantitative methods, the newly developed LC‐MS/MS method for citrinin determination includes the merits of using a small amount of extraction solvent, simple preparation steps, and high sensitivity.     

Preparation and characterisation of glyceollin‐enriched soya bean protein using solid‐state fermentation

Glyceollins, stress‐induced phytoalexins from parent soya bean isoflavones, were elicited with Aspergillus oryzae. This solid‐state fermentation facilitated the conversion of isoflavone glycosides into aglycones and glyceollins, which could mainly enrich in soya bean protein isolate (SPI) during protein preparation due to protein–polyphenol interactions. Glyceollin‐enriched SPI exhibited less flavour volatiles, higher solubility, lower whiteness and higher antioxidant activity than unfermented SPI. Fermented SPI was more easily to be digested during in vitro pepsin–trypsin digestion. This may be attributed to partial degradation of protein, especially α′ and α subunits of β‐conglycinin and acidic subunit of glycinin. The antioxidant activity of digestive products derived from fermented SPI was obviously enhanced with increasing digestion time due to simultaneous release of antioxidant peptides and glyceollins involved in the interior of protein molecule. These results suggest an effective technique for producing a nutrient‐enhanced SPI as novel functional ingredients applied in food industry.     

Effects of pepsin hydrolysis on the soy β‐conglycinin aggregates formed by heat treatment at different pH

The effects of pepsin hydrolysis on the β‐conglycinin aggregates formed by heat treatment at different pH were investigated. Results showed that fibrils were still observed, whereas the random aggregates were easily to be digested in the simulated gastric fluid. Electrophoresis and molecular weight analysis indicated that large aggregates still existed after pepsin treatment for fibrils. Hydrolysis resulted in changes in the apparent viscosity (ηapp) of 6% fibril solutions. The ηapp at the shear rate range (0–30 s^?1) increased in the order of fibrils < fibrils with pepsin for 60 min < fibrils with pepsin for 30 min. Smaller peptide/fibril fragments were generated, and additional aggregates were reformed during the hydrolysis process, as evidenced by thioflavin T and atomic force microscopy images. The native β‐conglycinin hydrolysates comprised a mixture of polypeptides enriched in about 47 kDa. These findings would provide valuable information about effects of enzymatic hydrolysis on plant oligometric globulin aggregates.     

In vitro gastrointestinal digestion of the major peach allergen Pru p 3, a lipid transfer protein: Molecular characterization of the products and assessment of their IgE binding abilities

A simulated gastrointestinal digestion has been carried out on purified peach lipid transfer protein, one of the main allergens among the population of the Mediterranean area and the major allergen of peach allergic patients. The percentage of intact protein, after extensive digestion, measured by comparison with a non‐digestible peptide analogue used as internal standard, was found to be about one‐third of the original protein content. The peptides formed in digested fraction were characterized by means of LC/MS. The products of the digestion essentially derived from trypsin action, whereas the protein appeared to be resistant to pepsin and chymotrypsin. The identified peptides could be classified as low molecular weight and high molecular weight peptides. The latter consisted of the full protein, with the disulfide bridges still intact, deprived of the smaller peptides. The different digestion products, including the high and low molecular weight peptides, were purified by LC and assessed, together with the intact protein, by dot‐blot analysis with sera of allergic patients, allowing to estimate their potential allergenicity. The intact protein and the high molecular weight peptides were found to be recognized by patients' sera, whereas the small peptides were found to be not reactive.     

Viability of encapsulated Lactobacillus acidophilus (LA‐5) in UF cheese and its survival under in vitro simulated gastrointestinal conditions

In this study, the survival of Lactobacillus acidophilus (LA‐5) was monitored during storage for 60 days at (4 ± 1) °C and also under an in vitro gastrointestinal (GI) model. A significant increase in the number of LA‐5 was found subsequent to 30 days of storage but numbers were reduced following 60 days. While the survival of micro‐encapsulated Lactobacillus acidophilus (LA‐5) was enhanced during the storage and in vitro gastrointestinal simulation, the addition of inulin did not improve the viability of this micro‐organism. During the storage time, survival of L. acidophilus (LA‐5) remained up to 6 log (cfu/g) in the UF white cheese.     

High‐pressure microfluidisation pretreatment disaggregate peanut protein isolates to prepare antihypertensive peptide fractions

High‐pressure microfluidisation (HPM) pretreatment was applied to increase in vitro antihypertensive activity of peanut peptide fractions (PPF). The morphology of protein in aqueous dispersion revealed that peanut protein isolate (PPI) disaggregated at relatively low pressure (≤120 MPa) and re‐aggregated at relatively high pressures (150–210 MPa). The treated pressure of 120 MPa could lead to the most disaggregation of PPI. Small peptides contents, trichloroacetic acid‐nitrogen soluble index (TCA‐NSI) and degree of hydrolysis (DH) of peanut protein hydrolysates (PPH) all reached the highest at 120 MPa. Consequently, it possessed the highest angiotensin converting enzyme (ACE) and renin inhibitory activity. The highest surface hydrophobicity occurred at 120 MPa pretreatment samples. Thirty‐nine oligopeptides at 120 MPa pretreatment were identified by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight (UPLC‐Q‐TOF) mass spectrometer combined with Progenesis QI for Proteomics software compared with 29 and 35 at control and 210 MPa, respectively. This meant that disaggregation of PPI at 120 MPa resulted in the release of new hydrophobic peptide.     

Electrospray MS‐based characterization of β‐carbolines – mutagenic constituents of thermally processed meat

The β‐carbolines 1‐methyl‐9H‐pyrido [3,4‐b]indole and 9H‐pyrido[3,4b]indole have been implicated as having causative roles in a number of human diseases, such as Parkinson's disease and cancer. As they can be formed during the heating of protein‐rich food, a number of analytical methodologies have been proposed for their detection and quantification in foodstuff. For this purpose, LC‐MS and LC‐MS/MS have emerged as the most specific analytical methods, and the quantification is based on the occurrence of unusual ions, such as [M+H‐(H^?)]⁺ and [M+H‐2H]⁺. In this study, we have investigated the formation of these ions by accurate‐mass electrospray MS/MS and demonstrated that these ions are formed from gas‐phase ion‐molecule reactions between water vapor present in the collision cell and the protonated molecule of 1‐methyl‐9H‐pyrido [3,4‐b]indole and 9H‐pyrido[3,4b]indole. Although this reaction has been previously described for heterocyclic amine ions, it has been overlooked in the most of recent LC‐MS and LC‐MS/MS studies, and no complete data of the fragmentation are reported. Our results demonstrate that additional attention should be given with respect to eliminating water vapor residues in the mass spectrometer when analysis of β‐carbolines is performed, as this residue may affect the reliability in the results of quantification.     

Stability of antioxidant peptides from duck meat after post‐mortem ageing

The stability of antioxidant peptides from aged duck meat during processing and simulated gastrointestinal digestion was investigated. The antioxidant peptides preserved a high stability in the presence of diverse NaCl or upon various time heating. The antioxidant activities were strengthened by the addition of 4–8% glucose or by heating at 100 °C, whereas they were lost under alkaline conditions. During in vitro digestion, the antioxidant activities increased with pepsin treatment but then decreased following trypsin digestion. Pepsin hydrolysed peptides into short fragments and results in the increased exposure of internal hydrophobic amino acids. With further treatment by trypsin, peptides can be hydrolysed completely and more free amino acids were released, leading to the decline in surface hydrophobicity. These variations might be responsible for the change in antioxidant activity during in vitro digestion. The antioxidant peptides from aged duck with high stability can be used as functional food ingredients to improve human health.     

Identification by GeLC‐MS/MS of Trypsin Inhibitor in Sarcoplasmic Proteins of Three Tropical Fish and Characterization of Their Inhibitory Properties

Sarcoplasmic proteins from 3 fish species were fractionated by 50% to 70% ammonium sulfate precipitation. Lyophilized fractionated sarcoplasmic proteins of threadfin bream (TB‐SP), bigeye snapper (BS‐SP), and yellow croaker (YC‐SP) showed 80% to 92% trypsin inhibitory activity. Trypsin inhibitory activity staining gel electrophoresis revealed bands at 32, 33, 37, 45, 48, and 50 kDa for the 3 species, and a band at 95 kDa was observed for TB‐SP and YC‐SP. Alpha‐1‐antitrypsin with molecular mass of 45 to 50 kDa was identified in YC‐SP by gel‐based liquid chromatography‐tandem mass spectrometry (GeLC‐MS/MS). Other major protein bands appeared on trypsin activity staining included phosphorylase, glyceraldehyde‐3‐phosphate dehydrogenase, and creatine kinase with molecular mass of 95 and 35 to 40 kDa, respectively. But, these 3 proteins did not show true trypsin inhibitory activity. Trypsin inhibitory activity of fractionated sarcoplasmic proteins showed good stability, with >80% activity retained at 60 °C and up to 0.6 M NaCl. TB‐SP showed the highest inhibitory activity against autolysis of washed threadfin bream mince at 65 °C. Addition of 0.5% or 1% TB‐SP improved textural properties of threadfin bream surimi gels preincubated at 37 or 65 °C followed by heating at 90 °C. Therefore, TB‐SP could be a promising protein ingredient for enhancing surimi gel texture.     

Effect of in vitro‐simulated gastrointestinal digestion on the stability and antioxidant activity of blueberry polyphenols and their cellular antioxidant activity towards HepG2 cells

In this study we investigated the influence of in vitro‐simulated gastrointestinal digestion on the bioaccessibility and antioxidant activity of polyphenols from blueberries (Vaccinium spp.). Total phenolic, anthocyanin, and flavonoid content was determined, and extract and digesta compositions were analysed by HPLC‐ESI‐MS/MS. The phenolic compounds were relatively stable under a gastric environment, whereas polyphenols and anthocyanins were unstable under an intestinal environment. The bioaccessibility of polyphenol, anthocyanin, and flavonoid was greatly decreased after the intestinal digestion, and the recoveries were only 13.93%, 1.95%, and 15.68% (the IN sample), respectively. Polyphenolic profile alteration occurred during in vitro‐simulated gastrointestinal digestion. Changes of phenolic compound antioxidant activity during digestion correlated with polyphenol, flavonoid, and caffeic acid concentrations. Digested extract cellular antioxidant activity was lower than non‐digested extract activity (P < 0.05). Polyphenol dose–response correlations with cellular antioxidant activity were observed. These results indicated that in vitro‐simulated gastrointestinal digestion significantly impact polyphenols and their antioxidant activity.     

Iron‐encapsulated cold‐set whey protein isolate gel powder – Part 1: Optimisation of preparation conditions and in vitro evaluation

A central composite design with a quadratic model was used to investigate the effects of three independent variables involved in the synthesis of iron‐encapsulated cold‐set whey protein isolate gel (WPI) on encapsulation efficiency (EE) and L*, a*, b* colour characteristics. The optimal conditions for maximum EE with minimum colour alteration were determined as 6.8% WPI, 18.8 mM iron and pH 7. In an in vitro gastrointestinal assay, only about 28% of the encapsulated iron was released in the gastric condition (with pepsin at pH 1.2), compared to 95% in the intestinal condition (with pancreatin at pH 7.5).     

Development and Application of a Method for the Analysis of 9 Mycotoxins in Maize by HPLC‐MS/MS

A reliable and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁, and AFG₂), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEA), fumonisin B₁ (FB₁), and T2‐toxin in maize. The samples were first extracted using acetonitrile: water: acetic acid (79 : 20 : 1), and then further cleaned‐up using OASIS HLB cartridge. Optimum conditions for the extraction and chromatographic separation were investigated. The mean recoveries of mycotoxins in spiked maize ranged from 68.3% to 94.3%. Limits of detection and quantification ranged from 0.01 to 0.64 μg/kg and from 0.03 to 2.12 μg/kg, respectively. The LC‐MS/MS method has also been successfully applied to 60 maize samples, which were collected from Shaanxi Province of China. Twenty‐four of the total 60 samples (40%) were contaminated with at least 1 of these 9 mycotoxins. Occurrence of mycotoxins were 6.7%, 1.7%, 3.3%, 6.7%, 1.7%, 23.3%, and 3.3% for AFB₁, AFB₂, OTA, ZEA, DON, FB₁, and T2‐toxin, respectively. The results demonstrated that the procedure was suitable for the simultaneous determination of these mycotoxins in maize matrix.     

Phenolic composition,antioxidant and pancreatic lipase inhibitory activities of Chinese sumac (Rhus chinensis Mill.) fruits extracted by different solvents and interaction between myricetin‐3‐O‐rhamnoside and quercetin‐3‐O‐rhamnoside

This work was designed to investigate the phenolic composition, antioxidant and pancreatic lipase inhibitory activities of Rhus chinensis Mill. fruits extracted by different solvents, and to illustrate which major phenolic compounds were responsible for lipase inhibition and to evaluate their interactions. Results showed that R. chinensis Mill. fruits were rich in phenolics, which included 13 types identified and quantified by UHPLC‐ESI‐HRMS/MS. Among the identified phenolics, myricetin‐3‐O‐rhamnoside and quercetin‐3‐O‐rhamnoside were the most dominant detected in all extracts. Extracts with 80% methanol, 80% ethanol and 80% acetone exhibited strong antioxidant and pancreatic lipase inhibitory activities in vitro, and these activities were positively correlated with phenolic contents. Myricetin‐3‐O‐rhamnoside and quercetin‐3‐O‐ rhamnoside demonstrated good lipase inhibitory activities in a dose‐dependent manner and synergistically inhibited lipase. This work may provide insights into the potential uses of R. chinensis Mill. fruits in food and nutraceutical industries.     

Effects of particle size on physiochemical and in vitro digestion properties of durum wheat bran

In order to promote the potential health benefits of high fibre products, wheat bran is the main focus of the food industry. The physiochemical and in vitro digestion properties of wheat bran containing different particle size were investigated and compared against raw bran samples. Firstly, the bran sample containing superfine particles (11.63 μm) was hydrolyzed by the α-amylase, pepsin, and pancreatin enzymes separately using a single factor and orthogonal test. Secondly, optimized hydrolysis parameters of superfine particles were employed to measure in vitro digestibility of macronutrients in raw, coarse, medium, and superfine bran particles. The maximum degree of hydrolysis obtained via α-Amylase (concentration 10 mg mL⁻¹, pH 6.6, and time 12.5 min) was 55.71%; pepsin (concentration 50 mg mL⁻¹, pH 1.2, and time 9 h) was 82.10%; and pancreatin (concentration 100 mg mL⁻¹, pH 7.0 and time 12 h) was 84.71%, respectively. The highest in vitro digestibility rate of reducing sugar, protein, fat, and soluble fibre content was observed in superfine bran samples to 33.4%, 82.55%, 91.53%, and 21.66%, respectively.     

Proteome analysis of Pithecellobium dulce seeds using two‐dimensional gel electrophoresis and tandem mass spectrometry

BACKGROUND: Pithecellobium dulce Benth. belongs to the Leguminosae family, which contains several members that are important components of human diets owing to their high protein content and quality. In this study the seed proteins from P. dulce were separated and identified using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry respectively. RESULTS: The 2‐DE protein map revealed a total of 317 distinct protein spots, including a cluster of about 12 proteins located in the region of pI 5–6 with molecular masses of 55–97 kDa that accounted for more than 50% of the total proteins. Ninety‐six of the most abundant protein spots were analysed using nano liquid chromatography/tandem mass spectrometry (LC/MS/MS), from which 27 were successfully identified through the query of acquired tandem mass spectral data used in MASCOT searching against a custom legume protein database. A further four proteins from the highly abundant protein cluster were putatively identified using mass spectrometry‐driven BLAST (MS‐BLAST) homology searches. CONCLUSION: This research has generated a 2‐DE proteome reference map for P. dulce seeds and used LC/MS/MS to characterise the proteins. The identification of proteins from P. dulce was carried out using the sequence database successively for MASCOT and MS‐BLAST homology‐based searches. Copyright © 2009 Society of Chemical Industry     

Liquid Chromatography‐Diode Array and Electrospray High‐Accuracy Mass Spectrometry of Iso‐α‐Acids in DCHA‐Iso Standard and Beer

Excellent liquid chromatographic (LC) separation of cis/trans stereoisomers of iso‐α‐acids has been achieved with reversed‐phase sorbent XTerra MS C18. An isocratic alkaline mobile phase, consisting of a mixture of 5 mmol l^?1 ammonium acetate (pH 8)‐acetonitrile‐methanol, 62:21:17 (v/v/v), was used. In the DCHA‐Iso international calibration standard trans‐isoposthumulone was identified combining photo diode array (PDA) spectra and electrospray high‐accuracy mass spectrometric (MS) data. Moreover, the molecular mass of two degradation products resulting from the in‐solution storage of the DCHA‐Iso standard was determined. The presence of trans and cis isomers of isoposthumulone, isocohumulone, isoadhumulone and iso‐n‐humulone in beer samples was confirmed. The trans isomers of iso‐α‐acids showed characteristic and reproducibly slightly different ultraviolet absorbance spectra with respect to cis isomers.     

Effect of digestive enzymes on the activity of camel‐milk insulin

This study investigated the effect of digestive enzymes on the activity of camel‐milk insulin. The digestion was performed using the sequential action of pepsin and pancreatin. Proteolysis degree was estimated using the O‐phthaldialdehyde method. Insulin concentration was determined using an enzyme‐linked immunosorbent assay (ELISA). Results revealed that milk proteins were partially digested by pepsin alone and the degradation was increased during the pepsin–pancreatin digestion as compared to control. Insulin lost its activity after 30 min of pepsin digestion, and it was not detected by ELISA. This study strongly suggests that insulin is not responsible for the antidiabetic action of camel's milk.     

Antioxidant activities of pepsin hydrolysates of water‐ and salt‐soluble protein extracted from pork hams

The objective of this study was to evaluate the antioxidant activities of pepsin‐digested water‐soluble protein (WSP) and salt‐soluble protein (SSP) extracted from pork ham. WSP and SSP were hydrolysed by pepsin for 2–10 h with 2‐h increments. The protein hydrolysates by pepsin were analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and reducing power of the hydrolysates was measured. In addition, antioxidant activity of the hydrolysates was determined using linoleic acid emulsion system. Protein bands with molecular weight higher than 7 kDa in WSP and SSP were completely hydrolysed by pepsin after 2 h of digestion time. WSP hydrolysates (WSPH) and SSP hydrolysates (SSPH) had higher ferric reducing power than controls (WSP and SSP without pepsin digestion). Reducing powers of WSPH were higher than those of SSPH, regardless of digestion time. The oxidative activity of linoleic acid was predominantly inhibited by the addition of WSPH and SSPH, especially 0.5% protein hydrolysate. These results indicate that several functional peptides of pork protein digested by pepsin might have antioxidant activity, and thus they may be used as an antioxidant agent in muscle food system.     

Effects of light exposure on chlorophyll,sugars and vitamin C content of fresh‐cut celery (Apium graveolens var. dulce) petioles

Effects of light exposure (24 μmol m^?2 s^?1) on fresh‐cut celery nutritional quality were evaluated during 8‐day storage at 7 °C using darkness as control. Light exposure preserved 47% chlorophyll a and 48% chlorophyll b contents more than darkness at the end of storage. Sucrose, reducing sugar and glucose contents in light‐stored petioles were 17%, 25% and 67% higher than those in dark‐stored petioles after 8‐day storage, respectively, thus resulting in higher total soluble solids content in light condition. Moreover, l ‐ascorbic acid content increased at 2‐day storage in light condition and was 46% more than in darkness at the end of storage. The fresh weight loss significantly increased in all petioles, and this increase was markedly accelerated by light exposure with a maximum of 1.43% at the end of storage. Dry matter content was induced more by light exposure than by darkness at 2‐day storage.     

Enzymatic hydrolysis of by‐products from the fish‐filleting industry; chemical characterisation and nutritional evaluation

Fish frames without heads from Atlantic cod and Atlantic salmon were proteolysed with the industrial enzymes neutrase®, alcalase® and pepsin for 1, 15, 30, 45, 60, 90 and 120 min. After 120 min of hydrolysis, salmon treated with alcalase and cod treated with pepsin yielded significantly (p < 0.05) higher protein recoveries (67.6 and 64% respectively) as compared to salmon treated with neutrase or pepsin and cod treated with neutrase or alcalase (53–62%). To minimise bitterness in the fish hydrolysates, kojizyme™ was added after 120 min of pre‐hydrolysis with alcalase, and the hydrolysis was run for additional times of 120, 240, 360, 480, 600 and 720 min. Protein recovery did not change significantly during the hydrolysis with kojizyme, but the degree of hydrolysis increased significantly (p < 0.01) in both the cod and salmon hydrolysates. A hydrolysate from cod treated with alcalase (150 min) followed by treatment with kojizyme (510 min) was produced. The final hydrolysate was freeze‐dried to a fish protein hydrolysate (FPH) and chemically characterised. The nutritional value of the FPH was established in an experiment with rats. Inclusion of 10% FPH‐N showed significantly (p < 0.05) higher nutritional value as compared to rats fed higher inclusion levels of FPH. © 2000 Society of Chemical Industry