Glycosylation,amino acid analysis and kinetic properties of a major Kunitz-type trypsin inhibitor from Acacia victoriae Bentham seeds

An Acacia victoriae trypsin inhibitor (AvTI) was purified from the seeds of prickly wattle (A. victoriae Bentham) by salt precipitation, ion-exchange and gel filtration chromatography, and its degree of glycosylation, amino acid composition, and kinetic properties were determined. Gel electrophoresis revealed at least four glycoprotein bands in the crude extract, salt-precipitated and ion-exchange protein fractions, while the purified AvTI showed only one band and a degree of glycosylation of 2.06%. Glutamate (13.3%), aspartate (10.3%), leucine (7.62%) and lysine (7.01%) were the major amino acids in AvTI while the contents of sulphur-containing amino acids, cysteine (1.38%) and methionine (0.75%), as well as of tryptophan (1.17%) were low. Its dissociation constant (K_i) for the inhibition of bovine trypsin was found to be 1.06 × 10⁻⁸ M, indicating a high affinity between AvTI and this enzyme, and its role as a competitive inhibitor was confirmed by a double reciprocal plot. These results complement our earlier studies which indicated the presence of three isoforms of this Kunitz-type trypsin inhibitor in prickly wattle seed.     

In vitro and in vivo antifungal properties of cysteine proteinase inhibitor from green kiwifruit

BACKGROUND: Higher plants possess several mechanisms of defense against plant pathogens. Proteins actively synthesized in response to those stresses are called defense‐related proteins which, among others, include certain protease inhibitors. It is of particular relevance to investigate plant natural defense mechanisms for pathogen control which include cystatins—specific inhibitors of cysteine proteases. RESULTS: In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. Immuno‐tissue print results indicated that CPI is most abundant in the outer layer of pericarp, near the peel, and the inner most part of the pulp—sites where it could act as a natural barrier against pathogens entering the fruit. The purified protein (15 µmol L^?1) showed antifungal activity against two phytopathogenic fungi (Alternaria radicina and Botrytis cinerea) by inhibiting fungal spore germination. In vivo, CPI (10 µmol L^?1) was able to prevent artificial infection of apple and carrot with spore suspension of B. cinerea and A. radicina, respectively. It also exerted activity on both intracellular and fermentation fluid proteinases. CONCLUSION: Identification and characterization of plant defense molecules is the first step towards creation of improved methods for pathogen control based on naturally occurring molecules. Copyright © 2012 Society of Chemical Industry     

Extraction,purification and properties of trypsin inhibitor from Thai mung bean (Vigna radiata (L.) R. Wilczek)

A trypsin inhibitor from mung bean (Vigna radiata (L.) R. Wilczek) seeds grown in Thailand was extracted and characterised. The optimal extraction medium was attained by shaking the defatted mung been seed powder in distilled water (P < 0.05). The extraction time affected the inhibitor recovery significantly (P < 0.05). The extraction time of 2 h was optimum for the recovery of the trypsin inhibitor from mung bean seeds. The trypsin inhibitor from mung bean seeds was purified by heat-treatment at 90 °C for 10 min, followed by ammonium sulphate precipitation with 30–65% saturation and gel filtration on Sephadex G-50. It was purified to 13.51-fold with a yield of 30.25%. Molecular weight distribution and inhibitory activity staining showed that the purified trypsin inhibitor had a molecular weight of 14 kDa. However, the purified inhibitor had no activity under reducing condition (βME). The purified inhibitor was heat stable up to 50 min at 90 °C. The inhibitory activity was retained over a wide pH range. NaCl, at 0–3% concentration, did not influence the inhibitory activity of the purified trypsin inhibitor from mung bean seeds.     

Milk from cows fed a diet with a high forage:concentrate ratio improves inflammatory state,oxidative stress,and mitochondrial function in rats

Excessive energy intake may evoke complex biochemical processes characterized by inflammation, oxidative stress, and impairment of mitochondrial function that represent the main factors underlying noncommunicable diseases. Because cow milk is widely used for human nutrition and in food industry processing, the nutritional quality of milk is of special interest with respect to human health. In our study, we analyzed milk produced by dairy cows fed a diet characterized by a high forage:concentrate ratio (high forage milk, HFM). In view of the low n-6:n-3 ratio and high content of conjugated linoleic acid of HFM, we studied the effects of this milk on lipid metabolism, inflammation, mitochondrial function, and oxidative stress in a rat model. To this end, we supplemented for 4 wk the diet of male Wistar rats with HFM and with an isocaloric amount (82 kJ, 22 mL/d) of milk obtained from cows fed a diet with low forage:concentrate ratio, and analyzed the metabolic parameters of the animals. Our results indicate that HFM may positively affect lipid metabolism, leptin:adiponectin ratio, inflammation, mitochondrial function, and oxidative stress, providing the first evidence of the beneficial effects of HFM on rat metabolism.     

Short communication: Roles of outer membrane protein W on survival,cellular morphology,and biofilm formation of Cronobacter sakazakii in response to oxidative stress

Cronobacter species are a group of opportunistic food-borne pathogens that cause rare but severe infections in neonates. Tolerance to environmental stress in Cronobacter is known; however, factors involved in oxidative stress are undefined. In this study, Cronobacter sakazakii survival, cellular morphology, and biofilm formation in response to oxidative stress were evaluated between the wild type (WT) and an outer membrane protein W (OmpW) mutant. The survival rates of ΔOmpW strain after treatment with 1.0 and 1.5 mM hydrogen peroxide were significantly reduced compared with those of WT. Morphological changes, including cell membrane damage and cell fragmentation, in ΔOmpW were more predominant than those in WT. By crystal violet staining, we also observed increased biomass in ΔOmpW biofilms as compared with WT following treatment with 0.5 and 1.0 mM H₂O₂. Biofilms using scanning electron microscopy and confocal laser scanning microscopy further confirmed the structural changes of biofilms between WT and ΔOmpW in response to oxidative stress. The current findings show that OmpW contributed to survival of planktonic cells under oxidative stress and the deletion of OmpW facilitated the biofilm formation in C. sakazakii to adapt to oxidative stress.     

响应面法优化纤维素酶辅助提取西蓝花萝卜硫素工艺研究

为探究酶解法制备萝卜籽萝卜硫素的最佳工艺及其消化特性。采用红萝卜种子为原料,以萝卜硫素得率为指标,通过单因素实验和响应面优化试验得出最佳酶解工艺条件。进一步采用体外模拟胃肠消化模型,探究萝卜籽乙酸乙酯提取物萝卜硫素含量及其抗氧化活性的变化规律。得到萝卜籽制备萝卜硫素的最佳酶解条件为：酶解时间22 min,酶解温度40 ℃,V_C添加量0.8 mg/g,在此最佳工艺条件下,萝卜硫素得率为2.11±0.02 mg/g,与预测值（2.14 mg/g）接近,相对误差为1.4%,酶解工艺切实可行。体外消化实验结果表明,胃肠消化液中萝卜硫素保留率分别为79.64%,76.22%;与未消化提取物比较,胃消化液中ABTS⁺、DPPH、OH三种自由基清除率均显著降低（P<0.05）,分别为54.23%、33.90%、15.07%,而肠消化液与胃消化液中自由基清除率均无显著性差异（P>0.05）。本研究丰富了酶法制备萝卜硫素方面的研究,将为萝卜籽开发及高附加值利用提供科学依据。

     

Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: a comparative study

The distribution of virulence properties in 142 strains of Aeromonas isolated from diarrhoeic patients, food and surface water in Italy and identified by biochemical and molecular methods was investigated. The virulence properties studied were the presence of genes for the aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act); and cytotoxicity for Vero cells and adhesion on Hep-2 cells. A. hydrophila and A. caviae were the species most commonly isolated from clinical and environmental samples (9/30; 30.0% and 5/27; 18.5%, respectively) while mesophilic A. salmonicida was most common in food samples (19/80; 23.7%). Out of 142 strains, 86 (60.6%) were positive for at least one of the virulence properties. All the toxin genes were present in 4/18 (22.3%) of clinical strains. Most of the food isolates (54/55; 98.2%) were cytotoxic and most of the environmental strains (12/13; 92.3%) were adhesive. The aerA gene was present in most toxigenic strains (72/86; 83.7%), irrespective of their origin. The growth temperature affected the expression of cytotoxicity and adhesivity. Aeromonas strains from food and surface water frequently had toxin gene patterns similar to those of clinical strains and expressed virulence properties at human body temperature. These findings indicate that aeromonads have the potential to cause human illness and confirm the role of food and water as vehicles for Aeromonas diseases.