Evaluation of the site specific protein glycation and antioxidant capacity of rare sugar-protein/peptide conjugates

Protein-sugar conjugates generated in nonenzymatic glycation of alpha-lactalbumin (LA) with rare sugars [D-allose (All) and D-psicose (Psi)] and alimentary sugars as controls [D-glucose (Glc) and D-fructose (Fru)] were qualitatively determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Mass spectra revealed that the extent of glycation at lysine residues on LA with D-aldose molecules was very much higher than that of glycation with d-ketose molecules. To identify the specific site of glycation, the peptide mapping was established from protease V8 digestion, using a combination of computational cutting of proteins and MALDI-TOF-MS. As compared to peptide mapping, three and seven glycation sites were located in the primary structure of LA-ketose and LA-aldose conjugates, respectively. On the other hand, the antioxidant activities of protein-sugar conjugates and their peptic hydrolysates were investigated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The antioxidant activities of proteins/peptides glycated with rare sugars were significantly higher than those modified with the control sugars. The results indicated that the glycation degree and position were not markedly different between rare sugar and corresponding control sugar, but the antioxidant properties of protein and its hydrolysate were significantly enhanced by modifying with rare sugar.     

Quantification of Aspergillus fumigatus and enteric bacteria in European compost and biochar

Although most potential human pathogens (PHPs) can be inactivated during composting, the risk that such substrates represent for human health remains largely unknown due to the shortage of information on presence and abundance of PHPs in finished composts. This study focused on the assessment of Salmonella spp., Listeria monocytogenes, Shiga toxin-producing Escherichia coli (STEC), and the opportunistic fungal pathogen Aspergillus fumigatus in different compost commodities. A total of fifteen European composts, made from different waste types and processes, were evaluated for the occurrence of the selected PHPs using molecular and traditional techniques. The analyses were extended to five biochar because of their growing application in agriculture, horticulture, floriculture, and private gardening.

Enteric bacteria were detected by molecular methods in eight out of fifteen composts; however, viable propagules were confirmed for L. monocytogenes only in two composts, and for STEC in three more composts. No bacterial pathogens were found in biochar. Living A. fumigatus was present in eleven composts and two biochars. None of the eighteen isolates contained single nucleotide polymorphisms (SNPs) relevant for resistance to azole fungicides. The role of compost and biochar as a source of PHPs in the environment and the risk for human health is discussed.     

Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates

The observation that the bitterest peptides from casein hydrolysates contain several proline residues led us to hypothesize that a proline-specific protease would be instrumental in debittering such peptides. To identify the desired proline-specific activity, a microbiological screening was carried out in which the chromogenic peptide benzyloxycarbonyl-glycine-proline-p-nitroanilide (Z-Gly-Pro-pNA) was used as the substrate. An Aspergillus niger (A. niger) strain was identified that produces an extracellular proline-specific protease with an acidic pH optimum. On the basis of sequence similarities, we conclude that the A. niger-derived enzyme probably belongs to the S28 family of clan SC of serine proteases rather than the S9 family to which prolyl oligopeptidases belong. Incubating the overexpressed and purified enzyme with bitter casein hydrolysates showed a major debittering effect. Reversed phase HPLC analysis revealed that this debittering effect is accompanied by a significant reduction of the number of hydrophobic peptides present.     

Isolation and characterization of an extracellular antimicrobial protein from Aspergillus oryzae

A 17 kDa antimicrobial protein was isolated from growth medium containing the filamentous fungus Aspergillus oryzae by extracting the supernatants from the culture media, ion exchange chromatography on CM-sepharose, and C18 reverse-phase high-performance liquid chromatography. This antimicrobial protein, which we considered to be an extracellular antimicrobial protein from A. oryzae (exAP-AO17), possessed antimicrobial activity but lacked hemolytic activity. The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus. To confirm that the protein acts as a regulation factor for extracellular secretion, we examined growth under varying conditions of N sources, C sources, ions, ambient pH, and stress. Various culture conditions were found to induce characteristic changes in the expression of protein synthesis as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly basic polypeptides were regulated by suppressing the ambient pH under acidic conditions and strongly induced under alkaline conditions, thus confirming that pH regulation is physiologically relevant. The expression of exAP-AO17 was upregulated by heat shock upon growth in the presence of NaCl. Automated Edman degradation showed that the N-terminal sequence of exAP-AO17 was NH 2-GLPGPAGAVGFAGKDQNM-. ExAP-AO17 showed partial sequence homology with a collagen belonging to the animal source. These results suggest that exAP-AO17 is an excellent candidate as a lead compound for the development of novel oral or other types of anti-infective agents.     

Protein glycation inhibitory and antioxidative activities of some plant extracts in vitro

The protein glycation inhibitory activity of aqueous ethanolic extracts from 25 plant tissues was evaluated in vitro using the model system of bovine serum albumin and fructose. The most bioactive plant tissue was Allium cepa (skin), followed by Illicium religiosum (bark and wood), Fagopyrum esculentum (hull), Origanum officinalis (leaf), Rosmarinus officinalis (leaf), Pyrus pyrifolia (bark),Acanthopanax senticosus (bark), Eugenia caryophllata (leaf), and Erigeron annuus (whole). The extracts with glycation inhibitory activity also showed antioxidative activity when a micellar linoleic acid peroxidation system was applied followed by 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization and 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assays. The glycation inhibitory activity was significantly correlated with the antioxidative potency of the extracts. The positive glycation inhibitory and antioxidative activities of these plants might suggest a possible role in targeting aging and diabetic complications.     

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in an aqueous solution: effect on association behavior and protein conformation

The effect of glycation with lactose on the association behavior and conformational state of bovine beta-lactoglobulin (beta-LG) was studied, using size exclusion chromatography, polyacrylamide gel electrophoresis, proteolytic susceptibility, and binding of a fluorescent probe. Two modification treatments were used, i.e., aqueous solution glycation and dry-way glycation. The results showed that the latter treatment did not significantly alter the nativelike behavior of the protein while the former treatment led to important structural changes. These changes resulted in a specific denatured beta-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation into a high molecular weight species, via noncovalent interactions. The association behavior of glycated beta-LG is discussed with respect to the known multistep denaturation/aggregation process of nonmodified beta-LG.     

Inhibitory effect of naturally occurring flavonoids on the formation of advanced glycation endproducts

The objective of this study was to investigate the inhibitory effect of naturally occurring flavonoids on individual stage of protein glycation in vitro using the model systems of delta-Gluconolactone assay (early stage), BSA-methylglyoxal assay (middle stage), BSA-glucose assay, and G.K. peptide-ribose assay (last stage). In the early stage of protein glycation, luteolin, qucertin, and rutin exhibited significant inhibitory activity on HbA1C formation (p < 0.01), which were more effective than that of aminoguanidine (AG, 10 mM), a well-known inhibitor for advanced glycation endproducts (AGEs). For the middle stage, luteolin and rutin developed more significant inhibitory effect on methylglyoxal-medicated protein modification, and the IC50's were 66.1 and 71.8 microM, respectively. In the last stage of glycation, luteolin was found to be potent inhibitors of both the AGEs formation and the subsequent cross-linking of proteins. In addition, phenyl-tert-butyl-nitron served as a spin-trapping agent, and electron spin resonance (ESR) was used to explore the possible mechanism of the inhibitory effect of flavonoids on glycation. The results indicated that protein glycation was accompanied by oxidative reactions, as the ESR spectra showed a clear-cut radical signal. Statistical analysis showed that inhibitory capability of flavonoids against protein glycation was remarkably related to the scavenging free radicals derived from glycoxidation process (r = 0.79, p < 0.01). Consequently, the inhibitory mechanism of flavonoids against glycation was, at least partly, due to their antioxidant properties.     

马立克氏病病毒MEQ蛋白对MDV增殖影响的研究

在马立克氏病病毒MDV致病机理的研究中，弄清致病基因meq与病毒增殖之间的关系及其分子机制是十分重要的基础，以重组反转录病毒（RCAS-meq)感染鸡胚成纤维细胞（CEF），使源于MDV强毒参考株（vMDV)GA株的meq基因表达于宿主细胞内，然后再用GA株感染这些细胞。通过利用MDV强毒株特异的抗pp38单克隆抗体所进行的“黑斑”试验以确定MDV的增殖水平，并与未接种RCAS-meq的CEF进行比较。研究结果发现，细胞内表达的meq基国产物可促进GA株于体外培养细胞中的感染与增殖（病毒斑数增多）。根据试验的结果，作者认为meq基因在感染细胞内的表达水平是MDV增殖以及进而能致病、致瘤的分子基础。     

Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities

Thirty-nine fungal metabolites 1-39, including two new alkaloids, 12β-hydroxy-13α-methoxyverruculogen TR-2 (6) and 3-hydroxyfumiquinazoline A (16), were isolated from the fermentation broth of Aspergillus fumigatus LN-4, an endophytic fungus isolated from the stem bark of Melia azedarach. Their structures were elucidated on the basis of detailed spectroscopic analysis (mass spectrometry and one- and two-dimensional NMR experiments) and by comparison of their NMR data with those reported in the literature. These isolated compounds were evaluated for in vitro antifungal activities against some phytopathogenic fungi, toxicity against brine shrimps, and antifeedant activities against armyworm larvae (Mythimna separata Walker). Among them, sixteen compounds showed potent antifungal activities against phytopathogenic fungi (Botrytis cinerea, Alternaria solani, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium solani, Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. vasinfectum, and Gibberella saubinettii), and four of them, 12β-hydroxy-13α-methoxyverruculogen TR-2 (6), fumitremorgin B (7), verruculogen (8), and helvolic acid (39), exhibited antifungal activities with MIC values of 6.25-50 μg/mL, which were comparable to the two positive controls carbendazim and hymexazol. In addition, of eighteen that exerted moderate lethality toward brine shrimps, compounds 7 and 8 both showed significant toxicities with median lethal concentration (LC(50)) values of 13.6 and 15.8 μg/mL, respectively. Furthermore, among nine metabolites that were found to possess antifeedant activity against armyworm larvae, compounds 7 and 8 gave the best activity with antifeedant indexes (AFI) of 50.0% and 55.0%, respectively. Structure-activity relationships of the metabolites were also discussed.     

A Prospective Study of Health Symptoms And Aspergillus fumigatus Spore Counts Near a Grass and Leaf Composting Facility

Aspergillus fumigatus (A. fumigatus), a fungus found in compost, is a respiratory allergen and can cause serious invasive disease in susceptible individuals. We conducted a study involving the collection of health symptom data and environmental monitoring data near a 40-acre grass and leaf composting facility. Analyses were based on symptom diary data from 63 individuals from the study area and 82 individuals from a reference area. Airborne A. fumigatus was not associated with increases in respiratory or irritative symptoms. Symptom incidence was associated with ragweed, ozone, temperature, and time since start of the study, although a tendency to report fewer symptoms as the study progressed may have confounded this result. Other features of the study design, including short-term spore count variability, lack of individual exposure data and gaps in the symptom diary data, complicated interpretation of the results. Although this study does not support an association between allergy and asthma symptom incidence and A. fumigatus spore levels, we could not assess the risk of unusual, but severe illnesses among very sensitive individuals.     

油茶籽饼粕中甲醇提取物抑制黄曲霉菌效果及成分分析

为了明确油茶籽饼粕中对黄曲霉菌有抑制作用的活性物质。该研究开展了油茶籽饼粕不同溶剂提取物对黄曲霉菌菌丝生长及产毒的影响研究。选用80%甲醇作为溶剂对油茶籽粕进行提取,提取液依次利用乙酸乙酯、饱和正丁醇进行梯度萃取,得到乙酸乙酯萃取相、正丁醇萃取相和水相,正丁醇萃取相对黄曲霉菌有较好的抑制效果,100mg/mL浓度效果最好,抑菌圈直径为22.00 mm,菌丝干质量相比对照减少了42.88%,黄曲霉毒素未被检出,而乙酸乙酯萃取相和水相对黄曲霉菌的抑制效果较弱,甚至无抑菌效果。采用柱层析、超高效液相色谱串联三重四级杆飞行时间质谱法对正丁醇萃取相中的抑菌物质进行纯化鉴定,分离出了3种黄酮苷类化合物:1)山奈酚-3-O-[β-D-吡喃葡萄糖苷-(1→3)-O-α-L-吡喃鼠李糖-(1→6)-O-β-D-吡喃半乳糖苷];2)山奈酚-3-O-[2-O-β-D-吡喃木糖基-6-O-α-L-吡喃鼠李糖]-β-D-吡喃葡萄糖苷;3)山奈酚-3-O-(6-反式-对-香豆酰基)-β-D-吡喃葡萄糖基-(1→3)-O-α-L-吡喃鼠李糖-(1→6)-O-β-D-吡喃半乳糖苷。以上研究结果为黄曲霉菌天然抑菌剂的研究和开发提供了参考,也拓宽了油茶副产物的加工利用途径。     

Studies on the physiological effect of humic acid

Many investigations have been reported on the physiological mechanisms of iron chlorosis induced by manganese. And several hypotheses such as “Oxidation reduction potential theory” (1) or “Chelation theory” (2) have been proposed concerning the interference of manganese on the absorption and translocation of iron in plants. However, from a comparative check of experimental results which had been done on various kinds of heavy metals inducing iron chlorosis, the authors have found that the degree of iron chlorosis did not strictly coincide with the order of oxidation reduction potential or stability constant of these chelate compounds. Also, CHINO et al. (3,4) reported that iron chlorosis induced by heavy metals did not coincide with the order of stability constant of these metal chelate compounds, but depended upon their inhibiting action on plant growth and iron absorption. But ASO et al. (5) reported previously that using root separating method they obtained higher iron absorption and better recovery of iron chlorosis of barley plants when iron and nitro-humic acid or EDTA were added simultaneously than when iron only was added. From these results, it was considered that the distribution and chemical form of iron in plants were affected by chelating action of nitro-humic acid or EDTA.     

Effects of protein and peptide addition on lipid oxidation in powder model system

The effect of protein and peptide addition on the oxidation of eicosapentaenoic acid ethyl ester (EPE) encapsulated by maltodextrin (MD) was investigated. The encapsulated lipid (powder lipid) was prepared in two steps, i.e., mixing of EPE with MD solutions (+/- protein and peptides) to produce emulsions and freeze-drying of the resultant emulsions. EPE oxidation in MD powder progressed more rapidly in the humid state [relative humidity (RH) = 70%] than in the dry state (RH = 10%). The addition of soy protein, soy peptide, and gelatin peptides improved the oxidation stability of EPE encapsulated by MD, and the inhibition of lipid oxidation by the protein and the peptides was more dramatic in the humid state. Especially, the oxidation of EPE was almost perfectly suppressed when the lipid was encapsulated with MD + soy peptide during storage in the humid state for 7 days. Several physical properties such as the lipid particle size of the emulsions, the fraction of nonencapsulated lipids, scanning electron microscopy images of powder lipids, and the mobility of the MD matrix were investigated to find the modification of encapsulation behavior by the addition of the protein and peptides, but no significant change was observed. On the other hand, the protein and peptides exhibited a strong radical scavenging activity in the powder systems as well as in the solution systems. These results suggest that a chemical mechanism such as radical scavenging ability plays an important role in the suppression of EPE oxidation in MD powder by soy proteins, soy peptides, and gelatin peptides.     

Iron availability from whey protein hydrogels: an in vitro study

The influence of whey protein hydrogel microstructure, filamentous versus particulate, on iron delivery was studied under different conditions, including simulated gastrointestinal conditions. Experiments were initially conducted to determine the impact of pH and enzymes on iron release. The results show that different iron release profiles can be obtained from filamentous and particulate gels. Particulate gels released more iron than filamentous gels at acidic pH, but the opposite was observed at alkaline pH. In the presence of pepsin at pH 1.2 or pancreatin at pH 7.5, both gel types showed increased protein hydrolysis, but only filamentous gels showed increased iron release, suggesting that matrix structure plays an important role in iron delivery. A dissolution test was carried out under gastrointestinal conditions to mimic the in vivo dissolution process. Filamentous gel released most of its iron during the intestinal phase of a simulated digestion, hence protecting iron during its transit in the gastric zone. Absorption of iron by the Caco-2 system, used to estimate intestinal absorption, revealed that filamentous gels favored intracellular iron absorption. These results suggest that filamentous gels show promise as matrices for transporting iron and promoting its absorption and therefore should be of major interest in the development of innovative functional foods.     

低温等离子快速提高糖基化花生分离蛋白溶解性及乳化性

为进一步提高花生蛋白的溶解特性,扩大花生蛋白在食品工业中的应用。采用低温等离子（Non-Thermal Plasma,NTP）诱导花生分离蛋白-葡聚糖（Peanut Protein Isolate-Dextran,PPI-Dex）湿法糖基化反应,研究NTP在处理0、0.5、1.5、2.0 、3.0 min的情况下,反应时间对花生分离蛋白与葡聚糖糖基化反应的影响。在低温等离子处理功率为70 W,反应液温度为60 ℃的状态下,随着NTP处理时间的延长,PPI-Dex的接枝度增加,在处理时间为1.5 min时,PPI-Dex接枝度达最大为21.62%,与超声波接枝PPI-Dex需要40 min,传统湿接枝需要24 h相比,缩短了接枝时间。PPI-Dex接枝后,接枝物溶解度和乳液稳定性显著增强,与未接枝相比,溶解度提高了22.28%。通过测定其分子量、氨基酸含量、红外图谱及表面疏水性变化分析NTP处理对花生分离蛋白结构影响。分析结果表明,NTP 处理1.5 min后,花生分离蛋白与葡聚糖发生糖基化反应形成偶联物,偶联物中羟基特征峰3 000～3 500 cm-1及1 000～1 260 cm-1的吸光度与未处理时相比增加,赖氨酸和苯丙氨酸相对含量显著降低（P<0.05）;同时,α-螺旋含量降低,β-折叠向β-转角转变,蛋白的有序结构被破坏,结构变松散,PPI构型向亲水型转变;接枝物的表面疏水性指数降低。花生分离蛋白与葡聚糖发生糖基化反应,反应位点可能为Lys和Phe。结果表明,低温等离子处理是一种快速促进蛋白与多糖接枝的有效方法。     

Studies of the binding of alpha-lactalbumin to immobilized peptide ligands.

The present work investigates the mechanism of binding of alpha-lactalbumin to the peptide ligand WHWRKR and its variants HWRKR and acetylated WHWRKR immobilized on a polymethacrylate chromatographic resin. The presence of two temperature-dependent binding mechanisms and one temperature-independent mechanism was demonstrated. Injections of different forms of alpha-lactalbumin (apo-alpha-lactalbumin, D87A mutant alpha-lactalbumin) displayed similar behaviors when compared to native alpha-lactalbumin, while lysozyme showed little or no binding to the WHWRKR and AcWHWRKR resins. An alternative process for isolation of alpha-lactalbumin from WPI was shown, using consecutive injections of WPI with limited elution.     

Studies on the effect of humic acid on availability of phosphorus

It is believed that some organic compounds form complexes with iron and aluminum and prevent the fixation of phosphate applied to soils.     

蛋白质体外进化建库技术研究

蛋白质体外进化技术是蛋白质工程发展的一个里程碑,也是改造蛋白质的一种有效工具。它不仅具有重要的应用价值,而且有助于蛋白质结构与功能的研究。通过蛋白质体外进化技术已成功地改造了许多蛋白质,有些已应用于工农业生产。体外进化技术分为两步：建库和筛选。本文主要对蛋白质体外进化策略及对体外随机突变技术、DNA重组技术、利用活细胞自身修复系统构建突变文库等几种定向进化突变文库建立技术进行了介绍与论述,同时还对蛋白质体外进化技术的应用及与其它学科结合的研究前景进行了分析,为获得具有改进功能或全新功能的蛋白质提供理论基础。     

Studies on N-terminal glycation of peptides in hypoallergenic infant formulas: quantification of alpha-N-(2-furoylmethyl) amino acids

To obtain information about the extent of the early Maillard reaction between the N-termini of peptides and lactose, alpha-N-(2-furoylmethyl) amino acids (FMAAs) were quantified together with epsilon-N-(2-furoylmethyl)lysine (furosine) in acid hydrolyzates of hypoallergenic infant formulas, conventional infant formulas, and human milk samples using RP-HPLC with UV-detection. FMAAs are formed during acid hydrolysis of peptide-bound N-terminal Amadori products (APs), and furosine is formed from the Amadori products of peptide-bound lysine. Unambiguous identification was achieved by means of LC/MS and UV-spectroscopy using independently prepared reference material. The extent of acid-induced conversion of APs to FMAAs was studied by RP-HPLC with chemiluminescent nitrogen detection (CLND). Depending on the corresponding alpha-N-lactulosyl amino acid, between 6.0% and 18.1% of FMAAs were formed during hydrolysis for 23 h at 110 degrees C in 8 N HCl. From epsilon-N-lactulosyllysine, 50% furosine is formed under these conditions. Whereas furosine was detectable in all assayed samples, five different FMAAs, alpha-FM-Lys, alpha-FM-Ala, alpha-FM-Val, alpha-FM-Ile, and alpha-FM-Leu, were exclusively detected in acid hydrolyzates of hypoallergenic infant formulas in amounts ranging from 35 to 396 mumol/100 g protein. Taking the conversion factors into account, modification of N-terminal amino acids in peptides by reducing carbohydrates was between 0.3% and 8.4%. This has to be considered within the discussion concerning the nutritional quality of peptide-containing foods.