Phenolic synthesis and phenylalanine ammonia-lyase activity in suspension cultures of Acer pseudoplatanus L.

Summary Phenolic metabolism is influenced by the levels of sucrose, nitrogen and 2,4 dichlorophenoxyacetic acid (2,4-D) in the growth medium. Chromatographic evidence suggests that the principle products are polymers of leucocyanin, (-) epicatechin and (+) catechin, constituting condensed tannins. Comparison of ethanolic cell extracts with extracts from plant organs shows that although these compounds are present in parts of the plant they are not the major phenolics.Cells maintained in a modified Heller's medium containing 9.0×10^–7 M 2,4-D produce increased levels of tannins from mid passage (day 12) onwards. The presence of 2,4-D at 9.0×10^–6 M supresses this response and increased initial sucrose levels cause the amount of tannins to be greater. At the period when tannin levels increase the standard medium is exhaused of its nitrogen sources, urea and nitrate. Increased initial nitrogen levels delay the beginning of increased tannin production and the addition of urea or 2,4-D to cultures already containing high levels of tannins causes the tannin content per gram fresh weight and per culture to decline. These results indicate an antagonism between tannin synthesis and nitrogen metabolism. The activity of phenylalanine ammonia-lyase EC 4.1.1.5. (PAL) estimated by a spectrophotometric method in acetone powders derived from Acer cells increases three to four fold at the onset of increased tannin synthesis and then declines sharply. The phase of high PAL activity correlates with the exhausion of the medium nitrogen sources.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid One of the authors (R.J.W.) was supported by a Science Research Council Studentship during the course of this work.     

Tannins as gibberellin antagonists in the synthesis of alpha-amylase and Acid phosphatase by barley seeds

The tannins chebulinic acid or tara tannin were added to an incubation system in which GA₃ induces enzyme synthesis in endosperm half seeds of barley (Hordeum vulgare L.). The activity of amylase and acid phosphatase in the incubation medium was reduced compared to the activity in the medium after incubation with GA₃ alone. When embryo half seeds of barley were incubated with chebulinic acid or tara tannin in the absence of added GA₃, the enzyme activity of the incubation medium was also reduced. The activity of preformed enzymes obtained from endosperm half seeds previously induced with GA₃ was not reduced by the addition of tannin. Comparisons were made of the amount of enzyme activity from breis of aleurone layers incubated with GA₃ in the presence and absence of tannins. The amounts of activity were relatively small and approximately equal in both cases, indicating that secretion from the aleurone was not blocked by the tannins. The reduction of enzyme activity caused by tannins in both endosperm and embryo half seeds could be completely reversed by the addition of GA₃.     

Immobilization of aminoacylase by adsorption to tannin immobilized on aminohexyl cellulose.

The immobilization of aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) was investigated by using tannin immobilized on aminohexyl cellulose. The most active immobilized aminoacylase was obtained when aminoacylase was adsorbed to the immobilized tannin in a weak alkaline medium containing sodium chloride and n-butanol at 37 degrees C. The activity of the immobilized tannin-aminoacylase complex per unit volume was five times higher than that of the DEAE-Sephadex-aminoacylase complex used for industrial production of L-amino acids in our plants. The half-life of the immobilized tannin-aminoacylase complex was 20 days under continuous operation at a high concentration of substrate; on the contrary, that of the DEAE-Sephadex-aminoacylase complex was 0.5 days.     

Effect of prolonged glucidic starvation on the contents of starch and tannins of Blechnum brasiliense L. gametophytes]

Some Blechnum brasiliense gametophytes have been maintained in darkness, in a purely mineral medium, for the purpose of investigating the effect of prolonged glucidic starving on the amount of tannins and starch that they contain. During the first two months the tannin contents undergoes a fast decrease. The starch variations are different : a transitory increase simultaneous with the fall in tannin; a decrease after the disappearance of 3/4 of the tannin contents; and lastly, a new amylogenese which precedes the prothallus necrosis. It seems that the gametophytes, in order to survive, degrade first their tannoidic reserves, and they utilize the starch that they contain only with difficulty. This behaviour can be explained by the distribution of the two metabolites; by the acid pH of the medium, which is not very propitious to the phosphorylases activity and lastly by the presence of chlorogenic acids, which are inhibitors of those enzymes.     

Tannins and saponin: Interaction in herbivore diets

Mice allowed to choose between diets containing tannin or saponin did not experience food intake depression, weight loss or high faeces weight to food weight ratios. Diets containing tannin produced all these effects and saponin diets resulted in weight losses and high faeces to food ratios. Mice provided with diets containing both tannin and saponin in predetermined proportions experienced weight losses similar to, or greater than, those of mice fed diets containing either toxin alone. Urinary glucuronide production by mice provided with a choice of tannin and saponin diets was less than that of mice feeding on diets containing either tannin or saponin alone. Simultaneous consumption of tannin and saponin (in the right proportions) may promote chemical interactions that inhibit the toxins' absorption from the intestinal tract. This type of interaction is likely to have influenced the evolution of herbivore feeding behaviour.     

Removal of tannin from cross-linked and open chain polymeric tannin substrates using heme peroxidases of Phanerochaete chrysosporium

Removal of tannin from different tannin substrates using heme peroxidases of Phanerochaete chrysosporium was studied. Complete removal of tannin components in spent tan liquor was observed after 24 h of incubation with peroxidases. Tannin in aqueous media containing tannic acid and condensed tannin substrates were removed by 65.70ǂ.97 and 52.43ǂ.83%. Chemical oxygen demand was reduced by 24.38ǂ.73, 33.22ǂ.20, and 58.94ǂ.07%, respectively, in spent tan liquor, tannic acid and condensed tannin substrates containing aqueous media.     

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

Effect of Serum Lipoproteins on Growth and Sterol Synthesis in Cultured Rat Brain Glial Cells

Cells dissociated from brains of 1-day-old rats were cultured in medium containing either lipoprotein-deficient serum (LPDS) or LPDS plus various lipoprotein fractions. Increases in number of cells and in DNA content served as a measure of cell growth. Cholesterol synthesis was measured from the incorporation of [14C]acetate into total nonsaponifiable lipids and digitonin-precipitable sterols, and from the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. The data indicated that cholesterol biosynthesis from acetate was reduced in cells cultured in medium containing either LPDS plus low-density lipoproteins (LDL), high-density lipoproteins (HDL), or total lipoproteins (LP) and that this reduction was accompanied by a reduction in the activity of the HMG CoA reductase and an increase in the esterified sterol content. The reduction in cholesterol synthesis from acetate was maximal in cells cultured in the presence of HDL, whereas the maximal reduction in the activity of HMG CoA reductase occurred in cells cultured in the presence of LP. The presence of LDL or LP in the culture medium enhanced the cell growth but the presence of HDL did not. Esterified sterol content was highest in cells cultured in the medium containing LPDS plus LP and was not detected in cells cultured in LPDS medium. It is inferred from these data that rat brain glial cells in culture are able to utilize cholesterol in lipoproteins, that the presence of LDL in the medium enhances cell growth, and that reduced cholesterol synthesis in the presence of lipoproteins may occur at the HMG CoA reductase step as well as at some other step(s).     

Production of tannase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> HA37 growing on tannic acid and Olive Mill Waste Waters

Production of tannase (tannin acyl hydrolase, EC 3.1.1.20) by Aspergillus nigerHA37 on a synthetic culture medium containing tannic acid at different concentrations has been studied. Maximal enzymatic activity increased according to the initial concentration of tannic acid; respectively 0.6, 0.9 and 1.5 enzyme activity units (EU) ml⁻¹ medium in the presence of 0.2%, 0.5% and 1% of tannic acid. Tannase production by A. niger HA37 on fourfold diluted olive mill waste waters (OMWW) as substrate, was between 0.37 and 0.65 EU ml⁻¹. Enzyme production on the diluted OMWW remained globally stable during more than 30 h. Growth of A. niger HA37 on OMWW was correlated with about 70% degradation of phenolic compounds present in the waste. This strain has therefore the capacity to degrade complex wastewaters which cause environmental damage to aquatic streams.     

Immobilization of Desulphovibrio desulphuricans: cell-associated hydrogenase in beaded matrices

The immobilization of the hydrogenase (cytochrome c₃ hydrogenase, hydrogen: ferricytochrome c₃ oxidoreductase, EC 1.12.2.1) activity associated with Desulphovibrio desulphuricans whole cells is described. The periplasmic hydrogenase was prevented from leaking from the cells by glutaraldehyde treatment. This modification left the hydrogenase activity of the cells unchanged. The resulting whole-cell preparation was then immobilized in beaded gels formed by either calcium alginate or low temperature radiation-polymerized polyacrylamide(s). The alginate matrix was used to study the effect of bead diameter and retention of hydrogenase activity after immobilization. Polyacrylamide matrices were used to study the effects of immobilization on hydrogenase activity vs. pH profiles and also matrix charge effects on enzymatic activity and long-term storage.     

Comparison of the nutritional value of low and medium tannin sorghum grains for pigs

The effect of sorghum grain of low (0.32%) and medium (0.94%) tannic acid content was studied in a growth experiment on Large White pigs. Medium tannin sorghum in pig diets had no significant adverse effect on growth, back fat thickness or feed conversion efficiency, but it was associated with a trend towards lower growth rate. All-sorghum diets, with either low or medium tannin, resulted in a significantly inferior feed conversion ratio compared to the wheat control. It was concluded that sorghum grain with up to about 0.94% tannic acid can be used as the main or sole grain in grower pig diets without the risk of any significant adverse effect on growth or back fat thickness.The production of medium tannin sorghum grain in bird-infested areas will depend on the extent to which pig producers would be prepared to use it in pig diets.     

Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts.

Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.     

Neutral Red Assay for Measurement of Quantitative Vero Cell Cytotoxicity

A neutral red assay involving Vero cells was used to quantitate the cytotoxic activity of verotoxins (VT) produced by Escherichia coli and to investigate changes in titer caused by altering the composition of the cell culture medium. Three variations on medium 199 were investigated: one involved supplementing the medium with 5% fetal bovine serum (FBS), a second was the use of serum-free (SF) medium that contained 5% bovine serum albumin and 5 μg of fibronectin per ml, and the third involved the use of 4% Ultroser G, a commercial serum replacement. The level of cytotoxicity varied markedly with the type of VT and with the medium that was used. For VT1, there was no difference in cytotoxicity between medium with 5% FBS and SF medium, but cytotoxicity was reduced more than 100-fold in medium containing Ultroser G compared with cytotoxicity in the other media. For VT2, VT2v, and VTe, there was a slight reduction in cytotoxicity in medium containing 4% Ultroser G and a more marked reduction in SF medium compared with cytotoxicity in medium containing 5% FBS.     

种子特征和结实量对啮齿动物取食和扩散种子行为的影响

为了深入了解啮齿动物在不同种子丰富度条件下对不同大小和单宁含量种子的觅食行为策略及其与植物种群更新的关系,在宁夏六盘山区的华北落叶松人工林,研究了不同大小和单宁含量[0%Tannin(T)、2%T、8%T和15%T]的人工种子在模拟结实小年和结实大年对啮齿动物取食和扩散行为的影响.结果表明: 啮齿动物消耗种子速度在结实小年更快,结实大年的种子消耗速度相对缓慢. 种子就地取食率(ISPR)在不同结实年份间无显著差异,扩散后取食率(PRAD)在结实小年显著高于结实大年,但前者的扩散后贮藏率(HRAD)显著低于后者;种子扩散后的取食距离(PDAD)和贮藏距离(HDAD)在结实小年均显著大于结实大年.在结实小年,大种子的PDAD和HDAD均大于小种子,前者在不同大小种子间均差异显著,而后者仅在2%T和15%T的不同大小种子间差异显著;在结实大年,除0%T外的其他单宁含量种子的PDAD和HDAD在不同大小种子间均差异显著.ISPR在中等单宁含量种子最大,高单宁含量种子最小;PRAD分别在结实小年的高单宁含量种子和结实大年的无单宁种子最大;不论在结实大年还是结实小年,HRAD均在高单宁含量种子最大,中等单宁含量种子最小.这说明结实大年可延缓啮齿动物对种子的消耗速率,提高种子的HRAD,但种子扩散距离减小;啮齿动物在结实大年和小年均表现出对大种子的扩散偏好,且大种子被扩散的距离更远;啮齿动物在不同结实年份均偏好于就地取食中等单宁含量种子,而扩散高单宁含量种子.     

Development and maturation of surghum seeds on detached panicles grown in vitro

Summary A procedure for culturing detached panicles of sorghum, Sorghum bicolor (L.) Moench, was developed to achieve flowering, fertilization, and subsequent seed development and maturation in vitro. Sixteen sorghum genotypes (five high and eleven low in tannin) were tested for their ability to develop normally in culture. Panicles collected one to two days before the initiation of anthesis were cultured in flasks containing liquid medium. Contamination and medium darkening were the major obstacles encountered. Up to 55% of the panicles cultured reached physiological maturity in vitro. The frequency of seed set ranged from 30 to 97% depending upon genotype and medium. Seed and glume color were normal. Seed produced in vitro resembled those grown in vivo and germinated well, but were smaller than normal (100 kernel weight reached 50 to 70% of the control). Grain polyphenols were synthesized in the cultured panicles. Seed of high tannin genotypes produced in vitro were lower in total phenols and tannins and higher in flavan-4-ols and the 3-deoxyanthocyanidin pigments than control seed. This technique can be used for harvesting late-maturing stocks and for various sorghum studies.     

Effect of methionine deprivation on S-adenosylmethionine decarboxylase of tumour cells

Transference of Walker carcinoma and TLX5 lymphoma from normal L-methionine-containing medium to medium containing limiting amounts of L-methionine, or L-homocysteine only, caused a 2-fold increase of S-adenosylmethionine decarboxylase activity. Kinetic analysis showed an increase in the V value of the enzyme from 22 to 53 pmol/min per mg protein in media containing only 0.1 mM L-homocysteine, without any alteration in the Km value (0.1 mM). The increase in enzyme activity does not result from (a) a reduction of the intracellular level of S-adenosylmethionine, since cycloleucine, an inhibitor of methionine adenosyltransferase, had no effect on enzyme activity; (b) an increase in intracellular adenosine 3',5' monophosphate (cyclic AMP), since high extracellular concentrations of N6-monobutyryl cyclic AMP had no effect on enzyme activity; (c) an alteration of polyamine levels, since addition of micromolar concentrations of exogenous putrescine, spermidine and spermine did not prevent the induction of S-adenosylmethionine decarboxylase activity in methionine-free media containing 0.1 mM L-homocysteine. The increased enzyme activity appears to be mainly due to enhanced stabilization, since the half-life was increased from 2.45 to 5.0 h in media containing only 0.1 mM L-homocysteine. Induction of enzyme activity is specific to the removal of L-methionine, since no increase occurred in the absence of L-serine or L-glycine, or both, or by reduction of the serum concentrations in the medium.     

Tannins determined by various methods as predictors of methane production reduction potential of plants by an in vitro rumen fermentation system

Relationships between chemical constituents, including values obtained with tannin assays (i.e., total phenols, total tannins, condensed tannins and tannin activity using a tannin bioassay) for plant materials (n = 17), and methane production parameters at 24 h of incubation in the in vitro Hohenheim gas method were established. The methane production reduction potential (MRP) was calculated by assuming net methane concentration for the control hay as 100%. The MRP of Bergenia crassifolia leaves and roots, and Peltiphyllum peltatum leaves, was >40%. Amongst the chemical constituents, neutral detergent fibre had a high correlation (r = 0.86) with methane concentration. There was negative relationship between total phenol, total tannins or tannin activity and methane concentration. However, a positive relationship existed between these tannin assays and the MRP, with r-values ranging from 0.54 to 0.79 (P<0.05). A very weak relationship (r = 0.09) occurred between condensed tannins and MRP. Similar results to those with MRP were obtained with the percent increase in methane on addition of polyethylene glycol. The highest correlations, 0.79 and 0.92 (P<0.001), were between tannin activity determined using the tannin bioassay and the MRP, or the percent increase in methane on addition of polyethylene glycol, respectively, suggesting that this tannin assay could be used to identify plants possessing antimethanogenic properties. Leaves of Rheum undulatum, Vaccinium vitis-idaea, B. crassifolia, Rhus typhina and P. peltatum, and roots of B. crassifolia have considerable potential (i.e., >25%) to decrease enteric methane production from ruminants.     

Effect of various culture conditions on shoot multiplication and GC–MS analysis of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> accessions for plumbagin production

Plumbago zeylanica, a pharmaceutically important medicinal plant, contains a wide range of phytocompounds. Culture parameters like carbon source, nitrogen source, and culture media are essential for the development and growth of explants. In this investigation, the influence of various carbon sources (sucrose, glucose, and fructose at 3% concentration), nitrogen source (ammonium nitrate, sodium nitrate, and potassium nitrate) and plant tissue culture media (MS medium, Gamborg’s B5 medium, White medium and Nitsch medium) on shoot multiplication of five different accessions was studied. Optimum growth of all five accessions was observed in MS media containing 3% sucrose and ammonium nitrate as a source of carbon and nitrogen. Out of five accessions, IC-524441 showed the highest shoot multiplication. Further, methanolic extracts of all accessions (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source) were prepared and comparison of extracts in DPPH assay indicated that accession number IC-524441 was the most effective free radical scavenging agent. Total phenolic, flavonoid and tannin content ranges were from 20 to 70 µg/ml, 40 to 100 µg/ml and 55 to 120 µg/ml, respectively, and the highest amount was found in accession number IC-524441. Sucrose and ammonium nitrate content may be responsible for increased antioxidant activity, flavonoids content, phenolic content, and tannin content in accession number IC-524441. GC–MS of ethyl acetate extract of all five accessions of P. zeylanica was conducted (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source). GC–MS analysis of the aerial part showed the presence of various phytocompounds, which include 1,4-naphthalenedione, 3-eicosene, 5-eicosene, phthalic acid, o-anisic acid, thioctic acid, 1-octadecene, 5-t-butyl-cycloheptene, 2-benzoyl-1,2-dihydro-1-isoquinolinecarbonitrile, octadecanal, silane, 3-methoxy-2-methyl-2-(1-phenyl-ethylamino)-propionic acid, and 1-nonadecene. Accession number IC-524441 contains the highest amount of plumbagin, i.e. 14.19?±?0.5 µg/ml as compared to the others.     

Stimulation of dehydrogenase synthesis by cadmium in a cadmium-resistant strain of Saccharomyces cerevisiae.

The activity of dehydrogenase in Saccharomyces cerevisiae was estimated by reduction of 2,3,5-triphenyltetrazolium chloride. By the adaptation of yeast to cadmium, the high activity of dehydrogenase was observed. Furthermore, the activity of dehydrogenase in Cd-resistant cells was increased by growing in medium containing CdSO4. However, the activity of dehydrogenase was inhibited by the addition of CdSO4 to the reaction mixture. The activity of dehydrogenase in Cd-sensitive cells was increased slightly by incubation with low concentrations of CdSO4. High activity of dehydrogenase in Cd-resistant cells was completely negated by the addition of cycloheximide to the incubation medium. The increase of dehydrogenase activity is due partly to de novo synthesis of protein.     

Isolation and characterization of proteolytic ruminal bacteria from sheep and goats fed the tannin-containing shrub legume Calliandra calothyrsus.

Tannins in forages complex with protein and reduce the availability of nitrogen to ruminants. Ruminal bacteria that ferment protein or peptides in the presence of tannins may benefit digestion of these diets. Bacteria from the rumina of sheep and goats fed Calliandra calothyrsus (3.6% N and 6% condensed tannin) were isolated on proteinaceous agar medium overlaid with either condensed (calliandra tannin) or hydrolyzable (tannic acid) tannin. Fifteen genotypes were identified, based on 16S ribosomal DNA-restriction fragment length polymorphism analysis, and all were proteolytic and fermented peptides to ammonia. Ten of the isolates grew to high optical density (OD) on carbohydrates (glucose, cellobiose, xylose, xylan, starch, and maltose), while the other isolates did not utilize or had low growth on these substrates. In pure culture, representative isolates were unable to ferment protein that was present in calliandra or had been complexed with tannin. One isolate, Lp1284, had high protease activity (80 U), a high specific growth rate (0.28), and a high rate of ammonia production (734 nmol/min/ml/OD unit) on Casamino Acids and Trypticase Peptone. Phylogenetic analysis of the 16S ribosomal DNA sequence showed that Lp1284 was related (97. 6%) to Clostridium botulinum NCTC 7273. Purified plant protein and casein also supported growth of Lp1284 and were fermented to ammonia. This is the first report of a proteolytic, ammonia-hyperproducing bacterium from the rumen. In conclusion, a diverse group of proteolytic and peptidolytic bacteria were present in the rumen, but the isolates could not digest protein that was complexed with condensed tannin.