Antinutritional effects and ecological significance of dietary condensed tannins may not be due to binding and inhibiting digestive enzymes

The digestive enzymes alkaline phosphatase and 5-nucleotide phosphodiesterase, solubilized from bovine intestinal mucosa and purified to homogeneity, were found to be strongly inhibited in vitro by condensed tannins (proanthocyanidins) purified from sorghum seeds and from quebracho. Tannin inhibition was prevented and reversed by the detergent Triton X-100 (protein-binding agent), by soluble polyvinylpyrrolidone (tannin-binding agent), or by phosphatidylcholine (membrane component). When tested as a crude particulate membrane fraction more characteristic of their in vivo condition, both enzymes were inhibited much less than either purified enzyme at the same tannin concentration. Because the enzymes appear to be relatively insensitive to inhibition by tannin in conditions which mimic in vivo conditions, and because the proportion of the dietary tannin which is available to interact with these enzymes in the digestive tract is likely to be rather small, we suggest that the antinutritional effects and ecological significance of dietary tannins are not due to tannin inhibition of these or other digestive enzymes by direct binding to them.     

低浓度有机磷农药乐果促进普通小球藻生长的研究

以普通小球藻为试验对象，研究了有机磷农药乐果在低浓度（＜ＥＣ５０）时对其生长的影响．结果显示，普通小球藻的生长量、蛋白质含量、叶绿素含量、碱性磷酸酶活力等都有明显的增长．表明在低浓度时，乐果对普通小球藻的生长有明显的促进作用．     

Milk Alkaline Phosphatase Purification and Production of Polyclonal Antibodies

Purification was by electroelution from native polyacrylamide gels or by sequential use of three columns. Electroelution was faster and resulted in a higher yield (23.4 vs 1.6%) than column purification. The enzyme had a molecular mass of 187 kDa, and the isoelectric point ranged from 5.4 to 6.0. ALP purified by electroelution was used as the antigen to immunize rabbits for polyclonal antibody (PAb) production. Western blot analysis showed that PAbs cross-reacted with bovine milk and placenta ALP, but did not cross-react with ALP from calf or bovine intestinal mucosa, Escherichia coli or with other milk proteins.     

Impact of PEF treatment inhomogeneity such as electric field distribution, flow characteristics and temperature effects on the inactivation of E. coli and milk alkaline phosphatase

High intensity pulsed electric field (PEF) treatment was investigated focusing on the alteration of electric field distribution, flow characteristics and temperature distribution due to the modification of the treatment chamber. The aim was the improvement of the effectiveness of microbial inactivation of E. coli and to reduce the PEF impact on alkaline phosphatase (ALP) activity in raw milk. Mathematical simulation of the PEF process conditions considering different treatment chamber setups was performed prior to experimental verification. Finally the impact of the treatment chamber modifications on microbial inactivation and enzyme activity was determined experimentally. Using a continuous flow-through PEF system and a co-linear treatment chamber configuration the insertion of stainless steel and polypropylene grids was performed to alter the field strength distribution, increase the turbulence kinetic energy and improve the temperature homogeneity. The Finite Element Method (FEM) analysis showed an improved electric field strength distribution with increased average electric field strength and a reduced standard deviation along the center line of the treatment zone indicating a more homogenous electric field. The velocity profile was improved resulting in an increase of turbulence kinetic energy due to the insertion of the grids. As revealed by mathematical modeling, the temperature of the liquid was decreased, and formation of temperature peaks was avoided. Measured inactivation of heat sensitive alkaline phosphatase (ALP) was reduced from 78% residual activity to 92% after PEF treatment and it could be shown that thermal effects and temperature peaks have been the main reason for enzyme inactivation due to PEF. At the same time, an increase of microbial inactivation of 0.6 log–cycles could be determined experimentally due to the modification of the treatment chamber design.

Industrial relevance

The application of pulsed electric field as a non-thermal pasteurization technology requires an accurately defined treatment intensity followed by a predictable microbial inactivation. Unavoidable thermal effects occurring during PEF treatment due to ohmic heating have to be minimized to assure the retention of heat-sensitive nutrients and bioactive compounds. The presented investigations contribute to the fulfilment of these requirements for further successful industrial implementation of the PEF technology such as the selective inactivation or retention of enzyme activity in liquid food systems.     

新型染料配基对碱性磷酸酶的亲和纯化

设计合成了一种含有碱性磷酸酶抑制剂——对氨基苯磷酸的新型偶氮染料配基并将其偶联到凝胶Sepharose CL-6B 上,制备出新型的亲和层析介质,分离纯化小牛肠中的碱性磷酸酶。通过考察不同配基密度的层析介质对碱性磷酸酶的选择性,得出配基密度为4.55mg 配基(g 湿胶)-1的层析介质选择性高。用NaCl和Na2HPO4进行阶段洗脱,可使酶的纯化倍数一步达到65倍,活力回收率达到89%。通过测定不同配基浓度下酶的动力学,发现新合成的配基是碱性磷酸酶的竞争性抑制剂,其KI为3.0×10-3mol L-1。     

Molecular characterization of a gene that confers 2-deoxyglucose resistance in yeast

We have isolated a gene whose expression enables yeast cells to overcome the inhibition of growth produced by the presence of 2-deoxyglucose. The gene contains an open reading frame of 738 bp that may code for a protein of 27 100 Da. Cells carrying this gene contain high levels of a specific 2-deoxyglucose-6-phosphate phosphatase. The expression of this phosphatase is increased by the presence of 2-deoxyglucose and is constant along the growth curve. The sequence reported here has the GenBank accession number U03107.     

Deficiency in Tissue Non-Specific Alkaline Phosphatase Leads to Steatohepatitis in Mice Fed a High Fat Diet Similar to That Produced by a Methionine and Choline Deficient Diet

The liver expresses tissue-nonspecific alkaline phosphatase (TNAP), which may participate in the defense against bacterial components, in cell regulation as part of the purinome or in bile secretion, among other roles. We aimed to study the role of TNAP in the development of hepatosteatosis. TNAP^+/− haplodeficient and wild type (WT) mice were fed a control diet (containing 10% fat w/w) or the same diet deficient in methionine and choline (MCD diet). The MCD diet induced substantial weight loss together with hepatic steatosis and increased alanine aminotransferase (ALT) plasma levels, but no differences in IL-6, TNF, insulin or resistin. There were no substantial differences between TNAP^+/− and WT mice fed the MCD diet. In turn, TNAP^+/− mice receiving the control diet presented hepatic steatosis with alterations in metabolic parameters very similar to those induced by the MCD diet. Nevertheless, no weight loss, increased ALT plasma levels or hypoglycemia were observed. These mice also presented increased levels of liver TNF and systemic resistin and glucagon compared to WT mice. The phenotype of TNAP^+/− mice fed a standard diet was normal. In conclusion, TNAP haplodeficiency induces steatosis comparable to that produced by a MCD diet when fed a control diet.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

Effect of section of yam (Dioscorea cayenensis) tuber used in minisett on the biochemical properties of the resultant tuber

The effect of the section of yam (Dioscorea cayenensis) tuber used as planting material in minisett on the biochemical properties of the resultant tuber was studied. The biochemical parameters invertase (EC 3.2.1.26), acid phosphatase (EC 3.1.3.2), peroxidase (EC1.11.1.7), polyphenol oxidase (EC 1.10.3.1), total sugars and starch were evaluated in tubers resulting from field‐grown and tissue culture‐derived setts of the cultivars Roundleaf and Blackwiss yellow yams. No significant differences were observed between tubers resulting from field‐grown and tissue culture‐derived setts relative to the parameters studied. Invertase activity was found to be highest in tubers from middle‐derived setts, while tail‐derived setts had the lowest activity for both cultivars. Middle‐derived tubers were also found to have significantly higher levels of acid phosphatase activity and starch content. However, middle‐derived tubers had the lowest level of total sugars. Polyphenol oxidase and peroxidase were also found to be significantly higher in tubers from middle‐derived setts. These results suggest a higher rate of active starch formation in tubers from middle‐derived setts. © 2002 Society of Chemical Industry