Alpha-amylase inhibitor from local common bean selection: Effect on growth and development of Corcyra cephalonica

Cultivars of common bean (Phaseolus vulgaris L.) from the Kalpa valley of the Kinnaur district were screened for alpha-amylase inhibitor activity against porcine pancreatic α-amylase and significant differences were found amongst them. The inhibitor protein was partially purified from Triloki cultivar by ammonium sulphate precipitation and Sephadex G-100 gel filtration chromatography. Inhibitor protein was non-competitive, heat labile and retained 55.51 per cent activity at 70 °C. It had two pH optima of 5.0 and 7.6. Inhibitor protein inhibited amylases of 3rd -4th instar larval extracts of Corcyra cephalonica Stainton, the rice moth. C. cephalonica larvae (3rd -4th instar) were fed on wheat flour mixed with 0.013 percent (w/w) of partially purified α-amylase inhibitor protein. The growth of larvae in the treatment was significantly reduced. Treated larvae showed 100 percent mortality after 11 days. In the untreated control, no larval mortality was observed and adults emerged after 45 days. The inhibitor protein was effective at very low concentrations and could be exploited for control of this grain storage pest.     

Total determination of residual flutolanil and its metabolites in livestock products and seafood using liquid chromatography-tandem mass spectrometry

ABSTRACT

We have developed a simple and sensitive LC-MS/MS analytical method for the determination of residual flutolanil and its principal metabolites, including α,α,α-trifluoro-3′-hydroxy-o-toluanilide (M-4) and its conjugates, in livestock and seafood products. Both flutolanil and its metabolites contain the 2-(trifluoromethyl)benzoic acid (2-TFMBA) moiety. In this method, flutolanil and its metabolites are converted to 2-TFMBA by hydrolysis. The method involves direct hydrolysis with sodium hydroxide at 200°C, acidification, partitioning into a mixture of ethyl acetate-n-hexane (1:9, v/v), clean-up using a strong anion exchange cartridge (InertSep SAX), and then quantification using LC-MS/MS. The optimal conditions for the complete hydrolysis of flutolanil to 2-TFMBA are an incubation time of 6 h and a temperature of 200°C. The developed method was evaluated using seven types of food: bovine samples of muscle, fat, liver and milk, as well as egg, eel, and freshwater clam. Samples were spiked both at 0.01 mg/kg and at the Japanese maximum residue limit (MRL) established for each food type. The validation results show excellent recoveries (88–107%) and precision (< 10%) for flutolanil and M-4. The limit of quantification (S/N ≥ 10) of the developed method is 0.01 mg/kg. The developed method is applicable to the definition of residual flutolanil for animal-based food commodities and MRLs established by the Codex Alimentarius, and will be useful for the regulatory monitoring of residual flutolanil and its metabolites in food products.     

天然产物α-葡萄糖苷酶抑制剂筛选研究进展

近年来糖尿病发病率在世界范围内呈上升趋势,已成为一种严重慢性病,α–葡萄糖苷酶抑制剂因能延缓人体对葡萄糖吸收,故应用于临床能有效降低餐后血糖。该文对α–葡萄糖苷酶抑制剂来源、制备方法、筛选方法及抑制机理等方面研究现状进行综述。     

银耳多糖对淀粉消化酶的抑制作用及其机理研究

目的:研究银耳多糖对胰α-淀粉酶和α-葡萄糖苷酶的抑制作用及机制。方法:以干银耳为原料,分别采用碱法提取、酶法脱蛋白和柱层析分离,得到总糖含量为92.45%的银耳多糖（Tremella fuciformis polysaccharide,TP）,采用可见光分光光度法分析了TP对胰α-淀粉酶和α-葡萄糖苷酶的抑制作用,采用荧光光谱法和圆二色谱法表征了TP对该两种酶结构的影响。结果:TP能抑制该两种酶的活性,其对胰α-淀粉酶的抑制作用明显高于α-葡萄糖苷酶,对该两种酶的半抑制浓度（IC₅₀）分别为7.6835和16.9306 mg/mL。TP通过与该两种淀粉消化酶发生相互作用抑制其活性。TP与胰α-淀粉酶相互作用明显,可静态猝灭此酶,改变其二级结构;TP与α-葡萄糖苷酶相互作用微弱,不能改变其二级结构。结论:TP通过与淀粉消化酶发生相互作用抑制其活性。     

L-阿拉伯糖对α-葡萄糖苷酶抑制活性的体外试验研究

目的探讨L-阿拉伯糖对小肠α-葡萄糖苷酶的抑制作用及与阿卡波糖的联用效应。方法利用体外实验,提取大鼠小肠粘膜上清液为α-葡萄糖苷酶粗酶液,分别以终浓度为60 mg/ml蔗糖、20 mg/ml麦芽糖/α-糊精为底物,建立最佳抑制反应体系,测定L-阿拉伯糖对α-葡萄糖苷酶抑制活性(IC50)及抑制作用类型;采用4×3析因设计,研究L-阿拉伯糖与阿卡波糖联用效果。结果以蔗糖、麦芽糖、α-糊精为底物时,L-阿拉伯糖对小肠α-葡萄糖苷酶活性均有一定抑制作用,但选择性较高地抑制蔗糖酶活性,当L-阿拉伯糖添加量为0.5%蔗糖浓度时酶活性抑制百分率>50%,最高酶活性抑制百分率约93%,且有良好剂量-反应关系,IC50为0.164 mg/ml,抑制类型为反竞争性抑制(Ki,0.558 mg/ml);与阿卡波糖联用二者有交互作用,尤其以蔗糖为底物时联用效果较明显,联合应用可能提高了抑制活性。结论 L-阿拉伯糖有抑制α-葡萄糖苷酶活性作用,尤其对蔗糖酶有良好的选择性抑制;在α-葡萄糖苷酶抑制作用方面,其与阿卡波糖有一定联用效果,L-阿拉伯糖在含糖食品中可能有较良好的实际应用前景。     

Sources, properties and suitability of new thermostable enzymes in food processing

Investigations concerning recombinant a-amylases from Pyrococcus woesei and thermostable a-glucosidase from Thermus thermophilus indicate their suitability for starch processing. Furthermore, the study of recombinant ss-galactosidase from Pyrococcus woesei suitable for purpose of low lactose milk and whey production are also presented. The activity of this enzyme in a wide pH range of 4.3-6.6 and high thermostability suggests that it can be used for processing of dairy products at temperatures which restrict microbial growth during a long operating time of continuous-flow reactor with an immobilized enzyme system. Preparation of recombinant a-amylase and ss-galactosidase was facilitated by cloning and expression of genes from Pyrococcus woesei in Escherichia coli host. Satisfactory level of recombinant enzymes purification was achieved by thermal precipitation of native proteins originated from Escherichia coli. The obtained a-amylase has maximal activity at pH 5.6 and 93 degrees C. The half-life of this preparation (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. An advantageous attribute of recombinant a -amylase is independence of its activity and stability on calcium salt. a-Glucosidase from Thermus thermophilus also not require metal ions for stability and retained about 80% of maximal activity at pH range 5.8-6.9. Thus, this enzyme can be used together with recombinant a-amylase.     

α-淀粉酶产生菌的分离、筛选及鉴定

通过富集培养、平板筛选、摇瓶复筛从40 ℃的温泉底泥中分离筛选出一株产α-淀粉酶菌株，通过菌株的生理生化试验、16S rRNA基因序列遗传分析对该菌株进行鉴定。结果表明，菌株FA4被鉴定为苏云芽孢杆菌（Bacillus thuringiensis），该菌株具有较强的热稳定性，在55 ℃下仍能生长。     

Alpha-Amylase from Persimmon Honey: Purification and Characterization

The α-amylase was extracted from pure persimmon honey and purified by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies. Molecular weight of purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively suggested that the purified enzyme was a monomer. Optimum pH of the enzyme was 6.0?7.0 and optimum temperature 40°C. The enzyme was extremely inactivated at pH was higher than 7.0 or lower than 5.0. Heat inactivation occurred at 40°C. This enzyme activated by Ca ²⁺ , Mn²⁺, PCMB, and DTNB, but inhibited by Ba²⁺, Fe³⁺, Hg²⁺, Mg²⁺, and iodoacetic acid. The purified enzyme was of α?-type by TLC analysis. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α?-1,4-linkages and with increasing percentage of α?-1,6-linkages in substrate similar to the results from commercially available honey.     

Differential biochemical and kinetic properties of α-amylases from Rhyzopertha dominica (F.) progenies reared on wheat varieties differing in α-amylase inhibitory activity

The present study was accomplished to gain insights into the biochemical and kinetic properties of Rhyzopertha dominica’s α-amylase isoforms (named RdA70, RdA79, and RdA90) expressed in progenies reared in wheat varieties differing in α-amylase inhibitory activity. An inverse relationship was observed between the progenies’ amylase activity and the wheat inhibitory activity. Wheat samples with a high and low infestation (named HI-Borlaug and LI-Villa Juarez, respectively) were chosen to simplify the study. The progenies amylases were isolated by hydrophobic interaction chromatography, while the wheat samples were analyzed in α-amylase inhibitory activity by size exclusion chromatography. The isoforms RdA70 and RdA90 from LI-Villa Juarez progeny showed higher enzyme activities (73.8 and 43.4%, respectively) than those from HI-Borlaug. When the amylase isoforms were tested in susceptibility to inhibition by the inhibitory albumins, those from LI-Villa Juarez were more susceptible than those of the HI-Borlaug. Determination of the kinetic parameters revealed that RdA70 from the HI-Borlaug progeny showed 3.0-fold less starch affinity than that from the LI-Villa Juarez (K_m of 12.3 ± 1.8 versus 4.0 ± 0.3). The rest of the α-amylases did not show the same pattern as RdA70 in the HI-Bourlag as that their starch affinity was further reduced (RdA79 K_m = 23.1 ± 4.3, RdA90 K_m = 17.1 ± 2.9). Estimation of IC₅₀ values confirmed the high sensitivity of the three α-amylases of the LI-Villa Juarez progeny to wheat α-amylase inhibitors. The inhibitor constant K_i was the lowest for RdA70 in the LI-Villa Juarez progeny indicating the inhibitor’ tight binding to that isoenzyme. These results suggest that R. dominica uses RdA70 to bind large amounts of wheat α-amylase inhibitors than RdA79 and RdA90 as a physiological defense mechanism.     

Fatty Acid Composition,Total Sugar Content and Anti-Diabetic Activity of Methanol and Water Extracts of Nine Different Fruit Tree Leaves Collected from Mediterranean Region of Turkey

In this research, the total sugar content, fatty acid compositions, and α-amylase and α-glucosidase inhibitory activity of methanolic and water extracts of nine different fruit tree leaves were determined. α-amylase and α-glycosidase inhibitory activity were determined by using Caraway-Somogyi iodine/potassium iodide and 4-nitrophenyl-α-D-glucopyranoside as substrate, respectively. The total sugar content of the nine different fruit tree leaves varies from 281.02 mg GE/g (glucose equivalents) to 643.96 mg GE/g. Methanolic extract from avocado leaves had the strongest in α-amylase and α-glucosidase inhibitory activity, 69.21 and 96.26%, respectively. Fatty acid composition of nine fruit tree leaves was characterized by gas chromatography and 24 components were identified. Among the tested fruit tree leaves, the main component was linolenic acid (49.09%). The level of essential fatty acids are over 50% in mulberry, grape, and loquat leaves. Polyunsaturated fatty acids were the major group of fatty acids present in oils of mulberry, fig, pomegranate, grape, and loquat leaves. Therefore, these oils can be considered as a good source of polyunsaturated fatty acids. Furthermore, avocado can be regarded as a new source for diabetic therapies.     

The nutraceutical value of grain legumes: characterisation of bioactives and antinutritionals related to diabesity management

Beyond the nutritional value, legumes and particularly common beans are found in several dietary supplements used to treat diabesity (diabetes and obesity). These products contain not only inhibitors of carbohydrate-hydrolising enzymes (α-amylase, α-glucosidase), but also antinutritional factors that can cause adverse effects on human health. In the present research, twenty-two accessions of grain legumes were screened for bioactive (α-amylase and α-glucosidase inhibitors) and antinutritional (lectins, flatulence-producing sugars, trypsin inhibitors) phytochemicals. Results showed that four accessions had high α-amylase inhibiting activities (AI > 30%), and particularly the common bean ‘Great Northern’ resulted of interest for its high carbo-blocker activity (AI = 42.6 ± 0.5%), absence of lectins, low amounts of flatulence-producing oligosaccharides (2.5 ± 0.2 g/100 g DW) and low anti-trypsin activity (22.5 ± 4.3 trypsin inhibiting unit/mg DW). The knowledge offered from this work provides leads to the ultimate goal of developing new, more effective and safer dietary supplements for diabesity management.     

几种原花青素的降血糖作用及与常见食品原料的结合研究

以4种不同来源的原花青素为研究对象,利用高通量淀粉浊度法测定其对α-淀粉酶和α-葡萄糖苷酶活力的抑制效果,研究其与常见食物原料之间的相互作用,为进一步开发降血糖主食提供理论依据。结果表明:葡萄籽与高粱麸皮原花青素对α-淀粉酶和α-葡萄糖苷酶活力抑制效果相对较好,蔓越莓与苹果原花青素对α-淀粉酶和α-葡萄糖苷酶活力抑制效果相对较差;食物中的主要成分与原花青素结合后会影响其对酶活力的抑制效果,常见食物中糯米、散装大米、高粱米等与葡萄籽原花青素结合明显,降低其对碳水化合物消化酶活力抑制效果,但玉米淀粉、木薯粉、马铃薯粉、生粉、米粉、小西米、小黄米、玉米片、红薯粉、黑米及黑豆与葡萄籽原花青素之间几乎不结合,是适宜发挥原花青素对碳水化合物消化酶活力抑制作用的食品原料。     

生物法合成α-酮戊二酸及其衍生物的研究进展

α-酮戊二酸是三羧酸循环的中间代谢产物,参与氨基酸、维生素和有机酸的合成及能量代谢,具有广泛的应用前景。该研究从α-酮戊二酸及其衍生物的应用、生物合成途径及代谢调控、生物法合成策略等方面综述了生物法合成α-酮戊二酸及其衍生物的研究进展,旨在为生物法合成α-酮戊二酸及其衍生物以及生产菌株的代谢工程研究提供参考。     

Characterization of metabolic pathways for biosynthesis of the flavor compound 3-methylbutanal by Lactococcus lactis

3-Methylbutanal is a key volatile compound that imparts a nutty flavor to Cheddar cheese. Lactococcus lactis has been successfully applied as a starter to increase the level of 3-methylbutanal produced during the ripening of cheese. However, the mechanism of action and genetic diversity of this bacterium for 3-methylbutanal biosynthesis remains unclear. In this study, we investigated the association between the L. lactis genotype and phenotype in the biosynthesis of 3-methylbutanal via both direct and indirect pathways. Fourteen strains of L. lactis were screened for the capacity to produce 3-methylbutanal, and strain 408 (>140 μM) produced the highest among all tested strains, which exhibited both α-keto acid decarboxylase and α-ketoacid dehydrogenase activities. Furthermore, the results of a sodium meta-arsenite inhibition experiment showed that the 3-methylbutanal–producing capacities of each strain declined to various degrees. The kdcA gene, which encodes the direct pathway component α-ketoacid decarboxylase, was detected in 4 of the 14 strains, of which only strain 408 contained the full-length gene. We then characterized the genes associated with the indirect pathway by detecting the expression levels of the pdh gene cluster, ack, and pta, which were expressed at relatively higher levels in a high-yield strain than in a low-yield strain. As a result, these L. lactis strains were divided into 3 categories according to gene diversity, gene expression, and 3-methylbutanal production. The results of this study refine our knowledge of the genetic determinants of 3-methylbutanal biosynthesis in L. lactis and explain the effect of both synthesis pathways on 3-methylbutanal production.