A modified method for the detection of microbial proteases on agar plates using tannic acid

In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.     

Effect of phytic acid,tannic acid and pectin on fasting iron bioavailability both in the presence and absence of calcium

ObjectiveTo determine the effect of phytic acid, tannic acid and pectin on fasting non-heme iron bioavailability in both the presence and absence of calcium.Research methodsTwenty-eight apparently healthy adult females participated in two iron absorption studies using radioactive iron isotopes (⁵⁹Fe and ⁵⁵Fe). One group received 5 mg of iron (as FeSO₄) alone (control), together with 10 mg of phytic acid, 100 mg of tannic acid and 250 mg of pectin (study A), on different days. The second group received the same iron doses and compounds as the other group, plus 800 mg of calcium (CaCl₂) (study B). The compounds were administered after an overnight fast, and no food or beverages were consumed for the following 3 h. Iron status and circulating radioactivity were measured in venous blood samples.ResultsThe geometric means of iron bioavailability (range ± 1SD) for iron alone, iron with phytic acid, iron with tannic acid, and iron with citrus pectin were 25.0% (11.9–52.0); 18.9% (9.9–35.8); 16.8% (8.7–32.3); and 21.1% (10.2–43.9), respectively (repeated-measures ANOVA, p < 0.02 (Dunnett's post hoc: control vs tannic acid p < 0.05). When 800 mg of calcium was added (study B), iron bioavailability was 16.7% (10.1–27.5); 13.2% (7.1–24.6); 14.8% (8.8–25.1); and 12.6% (5.5–28.8), respectively (repeated-measures ANOVA, NS).ConclusionsTannic acid decreases the fasting bioavailability of non-heme iron, however this effect did not exist in the presence of calcium. No effect was observed by phytic acid or citrus pectin on fasting non-heme iron bioavailability in both the presence and absence of calcium.     

The influence of addition of gallic acid, tannic acid, or quebracho tannins to alfalfa hay on in vitro rumen fermentation and microbial protein synthesis

Tannins may reduce rumen degradability of protein, increase the proportion of feed protein reaching the lower digestive tract for enzymatic digestion and thereby increase the efficiency of protein utilization. The objective was to assess the effects of different types and levels of tannins on rumen in vitro gas production and its kinetics, in vitro true degradability (IVTD) and rumen degradability of protein (IVRDP), and microbial protein synthesis by incubating alfalfa (Medicago sativa L.) hay in buffered rumen fluid. Alfalfa was incubated in buffered rumen fluid with and without the addition of different levels of gallic acid (GA), quebracho tannin (QT), or tannic acid (TA). Tannins at the lower inclusion levels had minimal effects on fermentation products compared to the higher levels. Addition of QT and TA reduced ammonium-N (NH₄⁺-N) concentration. Addition of QT at 20, 40, and 60 g/kg DM decreased NH₄⁺-N by 2, 7, and 12% compared with control whereas addition of TA reduced NH₄⁺-N by 5, 6, and 12% when added at 20, 40 and 60 g/kg DM, respectively. In experiment 2, addition of QT at 50, 100, and 150 g/kg DM, resulted in reduction of NH₄⁺-N by 12, 30, and 51%, respectively, compared with the control. Addition of TA at 50, 100, and 150 g/kd DM reduced NH₄⁺-N by 14, 26, and 47% compared with control. Inclusion of QT at 50, 100, 150 DM reduced IVRDP by 13, 30, and 36% compared with control whereas at these levels of inclusion, TA resulted in reduction of IVRDP by 14, 25, and 48%. Rate of gas production decreased (P<0.001), while asymptotic gas production increased (P<0.0001) with increasing level of GA and TA. Quebracho tannin decreased (P<0.0001) both the rate and asymptotic of gas production. Gallic acid had a positive effect on fermentation as indicated by increased gas production and total short chain fatty acids (SCFA) production. Quebracho tannin decreased 24 h gas production, IVTD, and total SCFA production. Acetate to propionate ratio increased with the addition of GA and but decreased when QT was added. Addition of tannins did not markedly increase total purines but numerical values tended to be higher in the presence of tannins compared with the control. Efficiency of microbial growth was lower in the presence of GA and unaltered by TA, but higher in the presence of QT compared with the control. The effect of tannins on rumen fermentation and protein degradation varied with type and level of tannins. In vivo studies will be conducted to validate the in vitro results.     

Enzymatic synthesis of gallic acid from tannic acid with an inducible hydrolase of Enterobacter spp

Gallic acid acts as a precursor molecule to synthesize various tannin molecules. These are plant polyphenols and were proved to be good anti-oxidant, anti-cancerous, anti-inflammatory, anti-microbial compounds. In order to fully exploit prominent biological activities of specific tannins and to develop tannin-based new medicines, it is necessary to obtain their pure preparations with an aim of high yield and specificity. In the present study, gallic acid is synthesized by the hydrolysis of tannic acid using a microbial based transformation process. The microorganism was isolated and identified. The ability of the isolated microorganism to covert tannic acid into gallic acid was determined by HPLC and enzyme production.

• Highlights

• The present investigation signifies the role of Enterobacter spp. in various processes:

• ??To synthesize gallic acid (a precursor for food oxidant such as propyl gallate) and a bacteriostatic antibiotic (trimethoprim).

• ??To protect the environment from tannery’s discharge through the process of biodegradation.

• ??To reduce the toxicity of tannins in animal feed.

     

Ruthenium red staining and tannic acid fixation of dental basement membrane

Summary Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat non-striated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.These studies were supported by INSERM, grant n 537785 and DGRST     

The antioxidant effect of tannic acid on the in vitro copper-mediated formation of free radicals

Tannic acid (TA) has well-described antimutagenic and antioxidant activities. The antioxidant activity of TA has been previously attributed to its capacity to form a complex with iron ions, interfering with the Fenton reaction [Biochim. Biophys. Acta 1472, 1999, 142]. In this work, we observed that TA inhibits, in the micromolar range, in vitro Cu(II) plus ascorbate-mediated hydroxyl radical (*OH) formation (determined as 2-deoxyribose degradation) and oxygen uptake, as well as copper-mediated ascorbate oxidation and ascorbate radical formation (quantified in EPR studies). The effect of TA against 2-deoxyribose degradation was three orders of magnitude higher than classic *OH scavengers, but was similar to several other metal chelators. Moreover, the inhibitory effectiveness of TA, by the four techniques used herein, was inversely proportional to the Cu(II) concentration in the media. These results and the observation of copper-induced changes in the UV spectra of TA are indications that the antioxidant activity of TA relates to its copper chelating ability. Thus, copper ions complexed to TA are less capable of inducing ascorbate oxidation, inhibiting the sequence of reactions that lead to 2-deoxyribose degradation. On the other hand, the efficiency of TA against 2-deoxyribose degradation declined considerably with increasing concentrations of the *OH detector molecule, 2-deoxyribose, suggesting that the copper-TA complex also possesses an *OH trapping activity.     

Production profiles of phenolics from fungal tannic acid biodegradation in submerged and solid-state fermentation

Potent antioxidant phenolics are derived from tannin biodegradation. Understanding of biodegradation pathways through the identification of the intermediates molecules of great value like tannins is important to pursuit the production of bioactive monomers. Biodegradation of tannins remains poorly understood due to their chemical complexity and reactivity. Tannic acid biodegradation by Aspergillus niger GH1 in submerged fermentation (SF) and solid state fermentation (SSF) was evaluated by liquid chromatography coupled to mass spectrometry (LC–MS). Both cultures were kinetically monitored for the biodegradation profiles during 72 h. Differences in tannic acid composition were evidenced and the consumption of substrate and identification of biodegradation intermediates were achieved. The mechanism of tannic acid degradation by A. niger GH1 is by degradation of high molecular weight gallotannins and highly polymerized tannins to small molecules like gallic acid, digalloyl glucose and trigalloyl glucose. Important differences on time of substrate uptake and product release were revealed.     

Effects of tannic acid and its related compounds on food mutagens or hydrogen peroxide-induced DNA strands breaks in human lymphocytes

The effect of tannic acid (TA), gallic acid (GA), propyl gallate (PA) and ellagic acid (EA) on DNA damage in human lymphocytes induced by food mutagens [3-amino-1-methyl-5H-pyrido (4,3-b) indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimadazo (4,5-b) pyridine (PhIP) or H₂O₂ was evaluated by using single-cell electrophoresis (comet assay). The toxicity of these tested compounds (0.1–100 μg/ml) on lymphocytes was not found. These compounds did not cause DNA strand breaks at lower concentrations of 0.1–10 μg/ml. At a concentration of 100 μg/ml, TA and GA exhibited slight DNA damage, whereas PA and EA showed no DNA strand breaks. TA and its related compounds decreased the DNA strand breaks induced by Trp-P-2, PhIP or H₂O₂ at concentrations of 0.1–10 μg/ml. DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycoslase (FPG)] were used to examine the levels of oxidised pyrimidines and purines in human lymphocytes induced by H₂O₂. All the compounds at 10 μg/ml can reduce the level of FPG sensitive sites. However, only EA inhibited the formation of EndoIII sensitive sites. The results indicated that these compounds can enhance lymphocytes resistance towards DNA strand breaks induced by food mutagens or H₂O₂ in vitro.     

两种微生物法测定食品中叶酸含量的比较

比较酪乳酸杆菌(Lactobacillus casei,L.casei ATCC 7469)、粪链球菌(Streptococcus faecalis,S.aecalisATCC 8043)测定强化食品中叶酸的含量,结果表明,S.faecalis比L.casei检测时限短1d,实验操作容易把握,分别对两种菌检测结果重现性进行比较,P＞0.05,差异无统计学意义,两种方法实验结果比较,P＞0.05,差异无统计学意义.     

Impact of tannic acid on blood pressure,oxidative stress and urinary parameters in L-NNA-induced hypertensive rats

Hypertension is a major health problem with increasing prevalence around the world. Tannic acid is water-soluble polyphenol that is present in tea, green tea, coffee, red wine, nuts, fruits and many plant foods. It has been reported to serve as an antioxidant or a pro-oxidant depending on the type of cells and its concentration. The purpose of our study was to evaluate the effect of tannic acid on systolic blood pressure, oxidative stress and some urinary parameters in the rat model of essential hypertension. Blood pressures of all rats were measured using the tail-cuff method. The nitric oxide synthase inhibitor N (omega)-nitro-L-arginine was administered orally at a dose of 0.5 g/l/day for 15 days to rats in order to create an animal model of hypertension. Tannic acid was intraperitoneally injected at a dose of 50 mg/kg for 15 days. Superoxide dismutase, catalase activity and the concentration of malondialdehyde (MDA) were determined in blood plasma and homogenates of heart, liver and kidney. In order to evaluate renal functions, urine pH, urine volume, urine creatine, uric acid, and urea nitrogen values were measured. Compared with the hypertension group, a decrease in MDA concentrations of heart tissue (p < 0.01), urea nitrogen values (p < 0.01) and urine volumes (p < 0.001) were established in hypertension + tannic acid group. There was also a decrease in blood pressure values (20th and 30th days) of this group, but there was no a statistical difference according to hypertension group. The findings of our research show the effect of tannic acid in lowering blood pressure in hypertensive rats.     

单宁酸对布氏田鼠抗氧化水平和肝脏自噬相关基因表达的影响

单宁为植物中广泛存在的酚类植物次生代谢物,具有抗氧化功能,但是否影响动物细胞的自噬功能,仍不清楚.本研究对4周龄雌雄性布氏田鼠饲喂高、低浓度单宁酸溶液9周后,分别用酶联免疫吸附法和荧光定量PCR法测定血浆抗氧化水平和肝脏细胞自噬相关基因表达的变化.结果 显示,单宁酸对布氏田鼠的体重增长率和肝脏指数均无显著影响.高剂量组...     

Regions of putative acetylcholine receptors at synaptic contacts between neurons maintained in culture and subsequently fixed in solutions containing tannic acid

Summary Spinal cord neurons from 9-day chick embryos were maintained in culture for up to 35 days and then fixed in 4% cacodylate-buffered glutaraldehyde containing 2% tannic acid. After about 15 days in culture a small percentage of the synaptic specializations present were characterized by striking electron-dense striations averaging 15 nm in width, oriented perpendicular to the postsynaptic membrane. These structures increased in frequency with time in culture (to a maximum of about 10% of all synapses in the oldest cultures); they were asymmetrical, protruding approximately 8 nm into the synaptic cleft, and more deeply (approximately 15–18 nm), into the postsynaptic cytoplasm. On the basis of earlier work by Sealock (1980) they are interpreted as concentrations of acetylcholine receptors.Similar membrane differentiations were also seen associated with active-zone areas of a few presynaptic membranes, and the possibility that these represent presynaptic acetylcholine receptors is discussed. Additional observations reported are (1) the presence of striations resembling those seen at the postsynaptic membrane in the membranes of some postsynaptic vesicles, and (2) filamentous links between the striations and cytoskeletal elements of the postsynaptic cell.     

Development and industrialisation of enzymatic shrink-resist process based on modified proteases for wool machine washability

There is currently considerable interest in the use of enzymes to achieve a variety of finishing effects on wool, but it is apparent that the extent of fibre degradation by enzymes is of major concern during their commercial application. Proteolytic enzymes are known to penetrate and degrade the internal wool structure during processing, causing fibre damage, rather than limiting the degradation to the cuticle cells. The ability to be able to control the exact location of proteolytic attack on wool protein structures will lead to the successful development of enzymatic treatments for achieving a variety of finishing effects for wool-containing products. This present work describes the modification of proteases so that enzymatic modification of wool fibres is restricted to the cuticle scales of the fibres.

Bulk trials have demonstrated that novel modifications of the enzyme enable the reaction of the enzyme with wool to be controlled, so that less degradation of the wool occurs than in similar treatments with the native protease. An anti-felting effect has been achieved without any significant weight loss being caused by the modified protease during the treatment. This novel enzymatic process leads to environmentally friendly production of machine washable wool.     

A method for the simultaneous alignment of three or more amino acid sequences

Summary We describe an algorithm for the concurrent comparison of three or more amino acid sequences. The basis of the approach is a progressive evaluation of selected segments from each sequence. Only a small subset of all possible segments from each sequence is compared, and a minimum of information is retained for the trace-back of the alignment. As a result, this method has the advantage of being both rapid and minimally consumptive of computer memory when constructing an alignment. This being the case, there are no practical limits on the length of sequences that may be aligned. A computer program for the alignment of three sequences is described, and this method is compared with two three-sequence extensions of the Needleman and Wunsch variety, including a recently published approach. In addition, we have made simultaneous alignments of sets of four and five sequences with this selected-segment method.     

Differential effect of tannic acid on two tree-feeding Lepidoptera: implications for theories of plant anti-herbivore chemistry

Summary Feeding efficiencies of ultimate instar larvae of two polyphagous tree-feeding Lepidoptera, Malacosoma disstria (Lasiocampidae) and Orgyia leucostigma (Liparidae), were measured on artificial diets containing from 0% to 8% tannic acid. Relative growth rate (RGR) of O. leucostigma was not affected by up to 8% tannic acid, suggesting that O. leucostigma has evolved an effective counteradaptation to hydrolyzable tannins. In contrast, as little as 0.5% tannic acid caused a significant reduction in RGR of M. disstria, due both to reduced efficiency of conversion of digested food (ECD) and reduced relative consumption rate (RCR), and caused a significant increase in mortality during the pupal stage. Moreover, when reared from hatching on tannin-containing diets, no M. disstria larvae survived past the fourth instar.Although tannins are commonly referred to as digestibility-reducing substances, tannic acid did not reduce the ability of M. disstria or O. leucostigma larvae to digest either the whole diet or nitrogen contained in the diet. For M. disstria, tannic acid acts as a toxin and a feeding deterrent, but not as a digestibility-reducing substance. Growing evidence that tannins commonly act as toxins warrants a reassessment of their role in anti-herbivore chemistry.     

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). at pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.     

A sensitive inhibition chemiluminescence method for the determination of trace tannic acid using the reaction of luminol-hydrogen peroxide catalysed by tetrasulphonated manganese phthalocyanine.

A novel CL method for the determination of tannic acid (TA) was established based on TA inhibition of the chemiluminescence (CL) emission of the luminol-hydrogen peroxide CL system catalysed by tetrasulphonated manganese phthalocyanine (MnTSPc) under alkaline conditions. The peak height is proportional to the natural logarithm of concentration of TA in the range 1.0 x 10(-7)-3.0 x 10(-10) mol/L with a detection limit of 5.6 x 10(-11) mol/L and the relative standard deviation was 2.5% for the determination of 1.0 x 10(-8) mol/L TA (n = 11). The proposed method has been successfully applied to the determination of TA in Chinese gall and industrial wastewater.     

单宁酸对高原鼠兔和根田鼠生殖激素的影响

高原鼠兔和根田鼠是生存于青藏高原的小哺乳动物,它们所取食的高原植物含有多种次生化合物。为了证明食物中的次生化合物对高原鼠兔和根田鼠繁殖相关激素的影响,探讨它们与取食植物间的协同进化,分别对两种动物灌服0、5、10 和20 mg/ kg BW 剂量单宁酸1 d、3 d 及5 d 后,测定其下丘脑促性腺激素释放激素、血浆中睾酮和血浆雌二醇的含量。结果表明,单宁酸对高原鼠兔的LD₅₀ 为112 ±27.72 mg/ kg BW,对根田鼠的LD₅₀ 为117 ± 17.37 mg/ kg BW;单宁酸可以使高原鼠兔和根田鼠血浆睾酮和雌二醇水平升高,但对下丘脑促性腺激素释放激素水平没有明显的影响。说明单宁酸可能会促进这两种动物的性成熟,有利于它们的繁殖。     

Genotoxicities of nitropyrenes and their modulation by apigenin, tannic acid, ellagic acid and indole-3-carbinol in the Salmonella and CHO systems

Four naturally occurring compounds, indole-3-carbinol (I3C), apigenin (Api), ellagic acid (EA) and tannic acid (TA), were tested for their inhibitory effects against 1-nitropyrene- (1-NP) or 1,6-dinitropyrene (1,6-DNP)-induced genotoxicity in Salmonella tester strains and Chinese hamster ovary (CHO) cells. Api and TA strongly inhibited the bacterial mutagenesis induced by nitropyrenes, while 13C and EA had little or no effect. For example, in TA98, 0.2 μmole Api resulted in 48% and 56% inhibition of the mutagenicity induced by 4 nmole 1-NP and 0.035 nmole 1,6-DNP, respectively. With an equal dose, expected, a good correlation was observed between the antimutagenicity of nitropyrenes and their inhibitory effect on nitroreductase activity. This indicated that one of the possible antimutagenic mechanisms of Api or TA was to inactivate the metabolism of nitropyrenes. Two biological end-points, cytotoxicity and sister-chromatid exchange (SCEs), were used to screen the antigenotoxic effects of these compounds in CHO cells. At the sub-cytotoxic dose, 13C, Api and TA all protected against the cytotoxicity induced by 1-NP and 1,6-DNP, but only TA and Api gave a significant reduction of the frequency of SCEs. Moreover, this reduction was found to be highly dose-dependent.     

Optical resolution of unusual amino acids using microbial proteases

We have developed novel enzymatic methods for the optical resolution of unusual amino acids. In this work, we tried two microbial proteases, available inexpensively in a crude state, from Aspergillus oryzae and from Bacillus subtilis. The enantioselective hydrolysis of the methyl esters of the N-benzyloxycarbonyl (Z) derivatives of a number of amino acids, both aliphatic and aromatic, was examined using these microbial proteases. The enantiomeric purities of the resolved Z-amino acids were determined accurately by methods based on the reversed-phase HPLC separation of diastereomeric derivatives or the HPLC separation of enantiomeric derivatives on chiral stationary phases. In general, B. subtilis protease yielded better results than A. oryzae protease. Using the former protease, the amino acids bearing aliphatic side chains were resolved with good to excellent enantioselectivities and reasonable hydrolysis rates. The speed of hydrolysis was reduced significantly when the length of the side chain was longer than 5 carbon atoms. Phenylalanine, halogenated phenylalanines, and phenylalanine homologs were also resolved, generally with high enantiomeric purities, though the hydrolysis rates were not always reasonably fast. In all the cases examined, the L -enantiomers were preferentially hydrolyzed as in the lipase-catalyzed enantioselective hydrolysis reported previously. © 1992 Wiley-Liss, Inc.