Vitamins B1 and B2 contents in cultivated mushrooms

Mushrooms have long been treated as a delicacy. Nowadays however, many researchers consider them to be nutraceutical foods, which has stimulated new and existing Brazilian producers to search for more productive techniques and to introduce other species. The objective of this study was to determine the vitamin B₁ and B₂ contents in mushrooms. The main species of mushroom cultivated in Brazil and analysed in this study are: Agaricus bisporus (white button mushroom and portobello), Lentinula edodes (shiitake) and Pleorotus spp. (shimeji and oyster mushroom). The methodology employed used acid hydrolysis followed by enzymatic hydrolysis and separation of the vitamins by high performance liquid chromatography using a C₁₈ reverse phase column and fluorescence detector. The results obtained for thiamine (vitamin B₁) were from 0.004 to 0.08 mg/100 g and for riboflavin (vitamin B₂), from 0.04 to 0.3 mg/100 g.     

Detection of Aspergillus spp. and aflatoxin B1 in rice in India

Twelve hundred rice samples consisting of paddy (675) and milled rice (525) were collected from 20 states across India. These samples were assessed for Aspergillus spp. infection on selective medium and aflatoxin B₁ (AFB1) by indirect competitive ELISA. In this investigation, Aspergillus flavus contamination dominated in all the seed samples. The other major contaminants were Aspergillus niger, Aspergillus ochraceus and Aspergillus parasiticus. Out of 1200 rice samples, 67.8% showed AFB1 ranging from 0.1 to 308.0 μg/kg. All the paddy samples from Chattishgarh, Meghalaya and Tamil Nadu showed AFB1 contamination. Milled rice grains from different states showed below the permissible levels of AFB1 (average 0.5–3.5 μg/kg). Eighty-two percent of samples from open storage that were exposed to rain showed AFB1 contamination followed by one-year-old seed. Out of 1200 samples, 2% showed AFB1 contamination above the permissible limits (>30 μg/kg). This is the first comprehensive report of aflatoxin contamination in rice across 20 states in India.     

Further data on the levels of emerging Fusarium mycotoxins enniatins (A,A1, B,B1), beauvericin and fusaproliferin in breakfast and infant cereals from Morocco

Sixty-eight samples of cereals products, including breakfast cereals (n = 48) and infant cereals (n = 20), purchased from supermarkets and pharmacies in Rabat-Salé area from Morocco were analysed for the determination of six emerging mycotoxins: four enniatins ENs (ENA, ENA1, ENB and ENB1), beauvericin (BEA) and fusaproliferin (FUS). Samples were extracted with a mixture of acetonitrile:water (85:15, v/v), using an Ultra-Turrax® homogeniser. Mycotoxins were then identified and quantified by liquid chromatography (LC) with diode array detection (DAD). Positive samples were confirmed by LC–MS/MS.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

Characterisation of monoclonal antibody against aflatoxin B1 produced in hybridoma 2C12 and its single-chain variable fragment expressed in recombinant Escherichia coli

An anti-aflatoxin B₁ monoclonal antibody (anti-AFB₁ mAb) from the hybridoma 2C12 was established and its inhibition concentration fifty (IC₅₀) for AFB₁ and relative cross-reactivities (CRs) to other mycotoxins were estimated to be 8 ng/mL and less than 4% compared with AFB₁ by a competitive direct enzyme-linked immunosorbent assay. For production of anti-AFB₁ single-chain variable fragment (anti-AFB₁ scFv) in recombinant Escherichia coli, its scFv-coding genes were cloned from the hybridoma 2C12. The anti-AFB₁ scFv formed inclusion bodies in the cytoplasm of E. coli required in vitro refolding process and hence recovered to retain binding activity successfully. Surface plasmon resonance analysis resulted that anti-AFB₁ scFv possessed 1.16 × 10⁻⁷ M of equilibrium dissociation constant (K_D), which was about 17 times higher than the parental anti-AFB₁ mAb of 6.95 × 10⁻⁹ M.     

Triglycerides constituted of short and medium chain fatty acids and dicarboxylic acids in Momordica charantia, as well as capric acid,inhibit PGE2 production in RAW264.7 macrophages

This study was aimed to identify compounds in bitter gourd (Momordica charantia) ethyl acetate extract (EAE), which inhibit lipopolysaccharide-induced prostaglandin E₂ (PGE₂) production in RAW264.7 cells. Bitter gourd EAE was partitioned between n-hexane and methanol/H₂O (90/10). The hexane fraction was further separated by repeated silica gel chromatographies, and a reverse phase (RP) C18 chromatography. Fraction RP-10 showed the highest inhibition effect on PGE₂ production (Max inhibition = 96%, IC₅₀ = 2.3 μg/ml) and was identified to be triglycerides constituted of short and medium chain fatty acids by ¹H NMR, IR and H–HCOSY, and dicarboxylic acids by GC/MS. Fatty acids with 3–20 carbons were tested for the inhibitory activity, and capric acid exhibited the highest effect (Max inhibition = 99%, IC₅₀ = 6.5 μM). In conclusion, triglycerides composed of short and medium chain fatty acids and dicarboxylic acids in bitter gourd inhibit PGE₂ production, and capric acid is the most potent inhibitor among the fatty acids.     

Degradation study of enniatins by liquid chromatography–triple quadrupole linear ion trap mass spectrometry

Enniatins A, A₁, B and B₁ (ENs) are mycotoxins produced by Fusarium spp. and are normal contaminants of cereals and derivate products. In this study, the stability of ENs was evaluated during food processing by simulation of pasta cooking. Thermal treatments at different incubation times (5, 10 and 15 min) and different pH (4, 7 and 10) were applied in an aqueous system and pasta resembling system (PRS). The concentrations of the targeted mycotoxins were determined using liquid chromatography coupled to tandem mass spectrometry. High percentages of ENs reduction (81–100%) were evidenced in the PRS after the treatments at 5, 10 and 15 min of incubation. In contrast to the PRS, an important reduction of the ENs was obtained in the aqueous system after 15 min of incubation (82–100%). In general, no significant differences were observed between acid, neutral and basic solutions. Finally, several ENs degradation products were identified using the technique of liquid chromatography–triple quadrupole linear ion trap mass spectrometry.     

Purification and characterization of a novel glucoamylase from Fusarium solani

Thermostable enzymes are currently being investigated to improve industrial processes of starch saccharification. A novel glucoamylase was purified to electrophoretic homogeneity from the culture supernatant of Fusarium solani on a fast protein liquid chromatographic system (FPLC). The recovery of glucoamylase after gel filtration on FPLC was 31.8% with 26.2-fold increase in specific activity. The enzyme had a molecular mass of 40 kDa by SDS-PAGE and 41 kDa by gel filtration. The glucoamylase exhibited optimum activity at pH 4.5. The K_cat and K_m were 441/min and 1.9 mg/ml, respectively, for soluble starch, specificity constant (K_cat/K_m) was 232. The enzyme was thermally stable at 50 °C and retained 79% activity after 60 min at this temperature. The half-life of the enzyme was 26 min at 60°C. The enzyme was slightly stimulated by Cu²⁺ and Mg²⁺ and strongly inhibited by Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe³⁺.     

Purification and identification of polysaccharide derived from Chlorella pyrenoidosa

The conditions for extracting and purifying polysaccharides from Chlorella pyrenoidosa, including intensity and duration of ultrasound, the temperature and incubation time, and ethanol concentration, were investigated through an orthogonal design of L₁₆(4⁵) in this work. High performance liquid chromatography (HPLC) and gas chromatography (GC) were used to characterize the compounds in C. pyrenoidosa. The highest yield of 44.8 g kg⁻¹ was achieved at 400 W of ultrasound for 800 s and then followed by incubation in water bath at 100 °C for 4 h in 80% ethanol. Two polysaccharide fractions (S1 and S2) were separated from the extracts of C. pyrenoidosa using Sepharose 4B column chromatography. The average molecular weights (M_w) of S1 and S2 were 81,877 Da and 1749 Da, respectively. Gas chromatographic (GC) traces of the hydrolyzed polysaccharides showed that most of the majority of monosaccharide in both fractions was mannose (78.0% and 76.5% of relative mass from S1 and S2, respectively) with low levels of glucose (13.2% and 8.4% of relative mass from S1 and S2, respectively). The Fourier-transform infrared spectra (FT-IR) of S1 and S2 revealed typical characteristics of polysaccharides. Both samples had the characteristics of hydroxyl groups, weak C–H band and α-pyranoses; however, only S2 had a carboxyl group.     

Effects of menthol stereoisomers on the growth,sporulation and fumonisin B1 production of Fusarium verticillioides

Menthol is a naturally occurring cyclic terpene alcohol of plant origin from the Lamiaceae family. It has three chiral centres, implying eight possible different stereoisomers, which in turn define four pairs of enantiomers. This is the first work that reports on the stereoselective antifungal and antitoxigenic activities of the menthol stereoisomers on Fusarium verticillioides, with the (−)-menthol and (+)-menthol enantiomers found to be the most active inhibitors of fungal growth and sporulation. The results obtained suggest the importance of the presence of these substituents in the equatorial positions of menthol stereoisomers in the antifungal activity. The stereoisomer (−)-menthol, followed by (+)-menthol, were the most active compounds in the inhibition of fumonisin B₁ (FB₁) biosynthesis. The different antitoxigenic activities of (−)-menthol and (+)-menthol revealed that the molecular requirements to affect the FB₁ production were dependent not only on the presence of the substituents in the equatorial positions, but also on their spatial arrangements.     

Dioscin: A synergistic tyrosinase inhibitor from the roots of Smilax china

In our study, we investigated the inhibition of mushroom tyrosinase in Smilax china. A methanol (MeOH) extract of S. china was partitioned into hexane, ethyl acetate (EtOAc) and water. Of the three fractions, EtOAc extract showed the strongest inhibition of tyrosinase activity with l-tyrosine or l-DOPA as a substrate. Two compounds were isolated from a final active fraction by activity-guided column chromatography. These compounds were identified as dioscin and oxyresveratrol by comparing their mass, ¹H- and ¹³C-NMR spectral data with those reported in the literature. Dioscin showed little inhibition activity of tyrosinase, whereas oxyresveratrol, a known tyrosinase inhibitor, showed a strong tyrosinase inhibitory activity. We discovered that a mixture of oxyresveratrol and dioscin (IC₅₀ = 5.1 and 5.7 μg/ml) highly increased the inhibition of tyrosinase activity with l-tyrosine or l-DOPA as the substrate as compared to either oxyresveratrol (IC₅₀ = 7.8 and 10.9 μg/ml) or dioscin (IC₅₀ > 100 and 100 μg/ml) alone.     

A new cytotoxic cyclic pentadepsipeptide,neo-N-methylsansalvamide produced by Fusarium solani KCCM90040, isolated from potato

A new cytotoxic cyclic pentadepsipeptide, neo-N-methylsansalvamide, was produced by Fusarium solani KCCM90040, which was isolated from potato in Korea. The species of Fusarium was identified as F. solani by examining its morphological characteristics and by ITS-5.8rDNA sequence analysis. This compound was analysed for C₃₃H₅₂N₄O₆ by electrospray ionisation mass spectrometry and combined structural analysis. The one and two-dimensional NMR and the absolute configurations of amino acids spectral data allowed the resolution of five subunits linked in the following order: (S)-2-hydroxy-4-methylpentanoic acid, N-methyl-l-leucine, l-valine, l-leucine, and l-phenylalanine. The in vitro cytotoxic effect of the purified cyclic pentadepsipeptide was evaluated to establish the possibility that it would be a biohazard if present in foods. The concentrations of purified cyclic pentadepsipeptide required to inhibit cell growth in vitro by 50% for A549 (lung cancer), SK-OV-3 (ovarian cancer), SK-MEL-2 (skin melanoma), and MES-SA (uterine sarcoma) cell lines were 10.7 ± 0.15, 11.2 ± 1.23, 10.0 ± 0.53, and 14.0 ± 0.74 M, respectively (mean ± SE).     

The effects of pre-treatment with vitamin B6 on memory retrieval in rats

The effects of pre-treatment with vitamin B₆ on memory retrieval in adult male Wistar rats were evaluated using a step-through passive avoidance task. The rats were divided into three groups of 10 each. All animals were fed standard rodent chow. Vitamin B₆ (50 or 100 mg/kg) was administered intraperitoneally (i.p.) every other day for 1 month before training was initiated. Three retention tests were performed to assess the memory of the rats. Vitamin B₆ (100 mg/kg) significantly increased the step-through latency of the passive avoidance response compared with the control in the first retention test of the passive avoidance paradigm (p < 0.05). In addition, vitamin B₆ at 100 mg/kg significantly increased memory retrieval in the second and third retention tests conducted 2 days and 1 week after training, respectively, compared with the control (p < 0.05). These results indicate that pre-treatment with vitamin B₆ potentially enhances memory retrieval.     

Hollow fibre-based liquid phase microextraction combined with high-performance liquid chromatography for the analysis of flavonoids in Echinophora platyloba DC. and Mentha piperita

A simple, inexpensive and efficient three phase hollow fibre liquid phase microextraction (HF-LPME) technique combined with HPLC was used for the simultaneous determination of flavonoids in Echinophora platyloba DC. and Mentha piperita. Different factors affecting the HF-LPME procedure were investigated and optimised. The optimised extraction conditions were as follows: 1-octanol as an organic solvent, pH_donor = 2, pH_acceptor = 9.75, stirring rate of 1000 rpm, extraction time of 80 min, without addition of salt. Under these conditions, the enrichment factors ranged between 146 and 311. The values of intra and inter-day relative standard deviations (RSD) were in the range of 3.18–6.00% and 7.25–11.00%, respectively. The limits of detection (LODs) ranged between 0.5 and 7.0 ng mL⁻¹. Among the investigated flavonoids quercetin was found in E. platyloba DC. and luteolin was found in M. piperita. Concentration of quercetin and luteolin was 0.015 and 0.025 mg g⁻¹ respectively.     

Association studies on the porcine RETN, UCP1, UCP3 and ADRB3 genes polymorphism with fatness traits

Searching for effects of candidate gene polymorphisms on fatness traits is an important goal for pig industry. In this study we evaluated polymorphism of four porcine genes involved in energy metabolism (RETN, UCP1, UCP3 and ADRB3). Moreover, their association with fat deposition traits was analyzed in two breeds (Polish Landrace, Polish Large White) and a Polish synthetic line (L990). Altogether, five SNPs were identified, including two novel ones in the 5′-flanking region of the RETN gene and a novel missense substitution in the UCP3. Distribution of these polymorphisms in the studied five breeds and the synthetic line was not uniform. Two of the analyzed SNPs: g.−178G > A in the RETN and g.946C > T in the UCP3 gene revealed a significant association with abdominal fat weight or backfat thickness. Such associations were not observed for the UCP1 or ADRB3 gene polymorphisms. Our study showed that polymorphisms of the UCP3 and RETN genes are potentially associated with porcine fatness traits.     

Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.)

Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical characteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K_M and V_max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min⁻¹ for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min⁻¹ for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a temperature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione ≈ ascorbic acid > l-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa.     

Study on ginsenosides in different parts and ages of Panax quinquefolius L.

The contents of 12 ginsenosides (Rg₁, Re, F₁₁, Rf, Rg₂, Rh₁, Rb₁, Rc, Rb₂, Rb₃, Rd, Rh₂) in different parts and ages of Panax quinquefolius L. (American ginseng) were quantified by high pressure microwave-assisted extraction (HPMAE) high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD). The analytical method was established and analytical performances were evaluated. The chemical marker of American ginseng F₁₁ was detected, and Rf which is the chemical marker of Asian ginseng was not found. Rare ginsenoside Rh₁, Rg₂ and Rh₂ were also studied in this experiment. The total contents of these 12 ginsenosides in the different parts of 5-year-old American ginseng follow this order: leaf > root-hair > rhizome > root > stem. Therefore, compared with the other parts of American ginseng, the leaf is a better available source of ginsenosides. The contents of ginsenosides in root and leaf also change with age.     

Tungsten permanent chemical modifier with co-injection of Pd(NO3)2 + Mg(NO3)2 for direct determination of Pb in vinegar by graphite furnace atomic absorption spectrometry

A tungsten carbide coating on the integrated platform of a transversely heated graphite atomizer (THGA^®) used together with Pd(NO₃)₂ + Mg(NO₃)₂ as modifier is proposed for the direct determination of lead in vinegar by graphite furnace atomic absorption spectrometry. The optimized heating program (temperature, ramp time, hold time) of atomizer involved drying stage (110 °C, 5 s, 30 s; 130 °C, 5 s, 30 s), pyrolysis stage (1000 °C, 15 s, 30 s), atomization stage (1800 °C, 0 s, 5 s) and clean-out stage (2450 °C, 1 s, 3 s). For 10 μL of vinegar delivered into the atomizer and calibration using working standard solutions (2.5–20.0 μg L⁻¹ Pb) in 0.2% (v/v) HNO₃, analytical curve with good linear correlation (r = 0.9992) was established. The characteristic mass was 40 pg Pb and the lifetime of the tube was around 730 firings. The limit of detection (LOD) was 0.4 μg L⁻¹ and the relative standard deviations (n = 12) were typically <8% for a sample containing 25 μg L⁻¹ Pb. Accuracy of the proposed method was checked after direct analysis of 23 vinegar samples. A paired t-test showed that results were in agreement at 95% confidence level with those obtained for acid-digested vinegar samples. The Pb levels varied from 2.8 to 32.4 μg L⁻¹. Accuracy was also checked by means of addition/recovery tests and recovered values varied from 90% to 110%. Additionally, two certified reference materials were analyzed and results were in agreement with certified values at a 95% confidence level.     

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M₁ (AFM₁), named as 2C9, was selected by semi-solid HAT medium. It exhibited high affinity for AFM₁ of 1.74 × 10⁹ L/mol and no cross-reactivity to aflatoxin B₁, B₂, G₁ and G₂. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM₁ in milk and infant milk products. Assays were performed in the AFM₁-BSA coated (0.0625 μg/mL) ELISA format in which the antibody was diluted 1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method.     

Composition of flavonoids and phenolic acids in Glycin tomentella Hayata cultivated in various soils

A high-performance liquid chromatography (HPLC) method was developed to simultaneously separate 12 phenolic acids and 21 flavonoids. The separation was performed with a C₁₈ column and a binary gradient solvent system consisting of methanol and water with 9% glacial acetic acid. The peak resolutions (Rs) were 0.51–12.41 and separation factors (α) were all higher than 1. The method was used to survey these phenolic components in Glycin tomentella Hayata. Diadzein was the major flavonoid in the leaves and roots, and gentisic acid and ferulic acid were the major phenolic acids in the leaves. Amounts of phenolic acids and flavonoids in the leaves from varied soil cultivations were in the order: loam > sand > red loamy sand. Flavonoid amounts in the roots also showed the same trend; however, phenolic acid amounts were low and did not present significant differences. Overall, the roots had much higher phenolic contents than the leaves.