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。     

啤酒中的双乙酰

双乙酰是啤酒中一个主要的风味物质。它是由前体物质α-乙酰乳酸在酵母细胞外经非酶氧化脱羧作用形成，然后渗透到细胞内经双乙酰还原酶的作用而消除。控制双乙酰含量的途径可以减少α-乙酰乳酸的形成或加速双乙酰的还原。生产控制措施，主要应加强酵母质量的管理并保证麦汁组成合理。     

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.     

糙米多酚对淀粉消化酶的抑制作用及机理

本研究将糙米多酚(brown rice polyphenols,BRP)经过提取和C₁₈固相柱纯化后,研究了BRP对α-淀粉酶和α-葡萄糖苷酶的抑制作用及其机理。液相色谱-质谱联用技术(UPLC-DAD-ESI-Q-TOF-MS)分析结果表明,BRP中大部分多酚以糖苷的形式存在,包括1种酚醛、2种酚酸酰胺、4种酚酸糖苷、6种酚酸、6种酚酸酯和8种黄酮糖苷。BRP对α-葡萄糖苷酶显示出剂量依赖型酶抑制活性,当浓度为2 mg/mL时,抑制率最高可达到39.37%,而BRP对α-淀粉酶无明显抑制作用。此外,荧光光谱和热力学研究结果表明,BRP能与α-葡萄糖苷酶发生疏水相互作用,对α-葡萄糖苷酶的内源荧光具有动态猝灭作用。以上结果表明,BRP主要通过与α-葡萄糖苷酶发生疏水相互作用抑制α-葡萄糖苷酶的活性,从而延缓碳水化合物的水解。本文为改善餐后血糖提供一定的参考。     

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.     

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.     

老山芹降血糖功能成分提取及活性研究

为增加老山芹资源的开发利用价值,以老山芹为原料,采用不同溶剂(不同浓度乙醇溶液、甲醇、乙酸乙酯)超声波辅助提取老山芹活性成分,以α-葡萄糖苷酶抑制率和α-淀粉酶抑制率为指标,确定适宜的提取溶剂,并通过单因素和响应面优化试验确定可保留老山芹降血糖功能成分的最佳提取条件。结果表明:适宜的提取溶剂为80%乙醇溶液,优化后的提取条件为超声温度49 ℃,超声时间114.5 min,料液比1:15.3 (g/mL),在此条件下得到老山芹提取液对α-葡萄糖苷酶抑制率为69.52%±1.13%,α-淀粉酶抑制率为56.38%±0.96%,二者的IC₅₀分别为0.169、0.339 mg/mL。老山芹可以作为具有降血糖功能的抑制α-葡萄糖苷酶和α-淀粉酶的物质来源。     

激活剂在棉型织物α-淀粉酶退浆中的应用

采用耐高温α-淀粉酶及烧碱对涤棉混纺织物进行退浆处理,对比了不同激活剂对酶退浆效果的影响。并以白度、毛效、断裂强力等为测试指标,对碱退浆、酶退浆的效果进行评判与分析。     

竹叶椒乙醇提取物对α-葡萄糖苷酶的抑制作用及其机理研究

目的:研究竹叶椒乙醇提取物对α-糖苷酶的抑制作用。方法:本实验采用体外抑制模型评价竹叶椒乙醇提取物对α-葡萄糖苷酶（酵母菌来源、小鼠小肠来源）和α-淀粉酶的抑制活性。并采用Lineweaver-Burk双倒数法研究α-葡萄糖苷酶的动力学性质。结果:竹叶椒乙醇提取物对α-葡萄糖苷酶（酵母菌来源）的半数抑制浓度（IC50）为（0.660±0.145）mg·m L-1,对小鼠小肠内α-葡萄糖苷酶半数抑制浓度（1.944±0.078）mg·m L-1,α-淀粉酶的半数抑制浓度为（1.185±0.132）mg·m L-1。动力学研究表明,竹叶椒乙醇提取物对α-葡萄糖苷酶的抑制作用为典型非竞争性抑制。结论:竹叶椒乙醇提取物对α-糖苷酶活性的抑制效果显著,具有很好的开发利用价值。      

α-淀粉酶对小麦麸皮淀粉的酶解作用

用α-淀粉酶酶解小麦麸皮中淀粉,以酶解后小麦麸皮中淀粉残留量为考察指标,研究酶解反应过程中加水量、加酶量、反应时间及反应温度四个因素对酶解效果影响。实验结果表明:耐高温α-淀粉酶酶解小麦麸皮淀粉较好工艺条件为:用水量120ml,加酶量0.08g,反应时间25min,反应温度95℃;酶解后淀粉含量由186.0mg/g降至5.0mg/g以下。     

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.     

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.     

Effects of exogenous amylolytic or fibrolytic enzymes inclusion on in vitro fermentation of lactating dairy cow diets in a dual-flow continuous-culture system

The objective of this study was to determine the effects of including exogenous amylolytic or fibrolytic enzymes in a diet for high-producing dairy cows on in vitro ruminal fermentation. Eight dual-flow continuous-culture fermentors were used in a replicated 4 × 4 Latin square. The treatments were control (CON), a xylanase and glucanase mixture (T1), an α-amylase mixture (T2), or a xylanase, glucanase, and α-amylase mixture (T3). Treatments were included at a rate of 0.008% of diet dry matter (DM) for T1 and T2 and at 0.02% for T3. All treatments replaced the equivalent amount of soybean meal in the diet compared with CON. All diets were balanced to have the same nutrient composition [30.2% neutral detergent fiber (NDF), 16.1% crude protein (CP), and 30% starch; DM basis], and fermentors were fed 106 g/d divided into 2 feedings. At each feeding, T2 was pipetted into the respective fermentor and an equivalent amount of deionized water was added to each fermentor to eliminate potential variation. Experimental periods were 10 d (7 d for adaptation and 3 d for sample collection). Composite samples of daily effluent were collected and analyzed for volatile fatty acids (VFA), NH₃-N, and lactate concentrations, degradability of DM, organic matter, NDF, CP, and starch, and flow and metabolism of N. Samples of fermentor contents were collected from each fermentor at 0, 1, 2, 4, 6, and 8 h after feeding to determine kinetics of pH, NH₃-N, lactate, and VFA concentrations over time. All data were analyzed using PROC GLIMMIX of SAS (SAS Institute Inc.), and the repeated variable of time was included for kinetics measurements. Treatment did not affect mean pH, degradability, N flow and metabolism, or the concentrations of VFA, NH₃-N, or lactate in the effluent samples. Treatment did not affect pH, acetate:propionate ratio, or the concentrations of lactate, NH₃-N, total VFA, acetate, propionate, butyrate, isobutyrate, valerate, or caproate. However, the concentration of total VFA tended to change at each time point depending upon the treatment, and T2 tended to have a greater proportion of 2-methylbutyrate and isovalerate than CON, T1, or T3. As 2-methylbutyrate and isovalerate are branched-chain VFA that are synthesized from branched-chain amino acids, T2 may have an increased fermentation of branched-chain amino acids or decreased uptake by fibrolytic microorganisms. Although we did not observe changes in N metabolism due to the enzymes, there could be changes in microbial populations that utilize branched-chain VFA. Overall, the tested enzymes did not improve in vitro ruminal fermentation in the diet of high-producing dairy cows.