Production and metabolism of lignans by the human faecal flora

Lignans have, until recently, been found only in plants. Enterolactone and enterodiol are the major lignans present in the urine of humans and have a potential physiological protective role against cancer. It has been shown that these compounds can be formed in vitro by human faecal flora and that enterodiol is oxidized to enterolactone by bacteria that are present in stools at a concentration of up to 10(3)/g. It was also possible to produce both of these lignans in vitro from linseeds and from secoisolariciresinol, a precursor present in linseed, by bacteria present in stools, at a concentration of between 10(3) and 10(4)/g. Enterolactone was produced from matairesinol, a more abundant plant lignan than secoisolariciresinol, after incubation with a mixed faecal flora under both aerobic and anaerobic conditions. In each case conversion was dependent on the presence of viable bacteria. These findings indicate that a number of different pathways operate to produce enterolactone and enterodiol depending on the ingested dietary precursor.     

In vitro metabolism of flax lignans by ruminal and faecal microbiota of dairy cows

Aims: To determine the in vitro conversion of plant lignans from two flax products (hull and seed) into the mammalian lignans, enterolactone and enterodiol, by bovine ruminal and faecal microbiota. Methods and Results: Flax seeds and hulls were incubated in vitro over a 96-h time course with ruminal or faecal inoculum. Plant lignans in flax seeds and hulls averaged 9·2 and 32·0 nmol mg⁻¹, respectively. The highest net production of enterodiol at 72 and 96 h of incubation was obtained with flax hulls incubated with faecal microbiota. There was no difference in net production of enterodiol between flax products within the first 24 h of incubation. In general, net production of enterolactone over the 96-h time course was significantly higher for flax products incubated with ruminal than with faecal microbiota. Net production of enterolactone at 72 and 96 h of incubation was greater for flax hulls than flax seeds. Conclusions: Results of the present experiment suggest that, of the metabolites studied, the main mammalian lignan metabolite produced from flax hulls and seeds by ruminal microbiota is enterolactone while faecal microbiota leads mainly to the net production of enterodiol. Significance and Impact of the Study: This research will improve the understanding of the metabolic pathway of mammalian lignans in dairy cows, in order to enable targeted manipulation of their quantities in milk.     

A review of the bacterial flora of teleosts and elasmobranchs, including methods for its analysis

Techniques for the quantitative and qualitative analysis of the commensal bacterial flora of fish are reviewed. The literature concerning the commensal bacterial flora of fish indicates that teleost skin often features 10²–0⁷ organisms per cm² of skin, representing the following taxa, in descending numerical order, Achromobacter, Pseudomonas, Flavo-bacterium and Cytophaga species also coryneform organisms. Actively feeding teleosts appear to have an alimentary canal bacterial flora similar to that of the skin and gills, often there are from 10³–10⁸ organisms per 1.0 g, wet weight, of tissue. According to the limited data available for elasmobranchs Gram–positive bacteria are the predominant component of the bacterial flora on the skin of sharks, though the bacterial flora of North Sea skate ( Raja spp.) is reported as being similar to that of teleosts. The commensal flora of fish, particularly of teleosts, is related to their environment and is influenced by environmental changes that affect the quality of the water. Regular monitoring of the water of commercial fish ponds and aquaria for bacterial quality, temperature, dissolved oxygen concentration and pH, combined with a base–line knowledge of the water's quality, may provide an advanced indication of the presence of bacteria potentially able to produce diseased conditions, and factors that will enhance their growth.     

Uptake and metabolism of enterolactone and enterodiol by human colon epithelial cells

The enterolignans enterolactone and enterodiol are phytoestrogens that are formed from plant lignans by microorganisms in the human colon. Enterolignans circulate in plasma as conjugates. We hypothesized that conjugation of enterolignans takes place in colon epithelial cells, and studied the time course of uptake and metabolism of enterolactone and enterodiol in three human colon epithelial cell lines. In addition, the conjugates were identified by mass spectrometry with accurate mass measurement (LC/QTOFMS/MS). Intracellular levels of conjugated enterolactone and enterodiol in HT29 cells rose immediately after starting the exposure. This was accompanied by a rapid decrease in free enterolactone and enterodiol in the exposure medium of HT29 and (un)differentiated CaCo-2 but not of CCD841CoTr cells. Conjugation and excretion of enterolactone and enterodiol was complete within 8 h, except for enterodiol in CaCo-2 cells ( approximately 48 h). Enterolactone appears to be more rapidly metabolized and/or excreted than enterodiol, and also the appearance of conjugated enterolactone in medium is less affected by the presence of enterodiol than vice versa. Total (free plus conjugated) enterolignan concentrations remained constant throughout the experiments. Three conjugates were identified in exposure medium of HT29 cells: enterolactone-sulfate, enterolactone-glucuronide, and enterodiol-glucuronide.Taken together, our data suggest that phase II metabolism of enterolactone and enterodiol already may take place during uptake in the colon and that colon epithelial cells may be responsible for this metabolism.     

Starch utilization by the human large intestinal microflora

High levels (2–565 units/g) of amylase activity were observed in human faeces. Over 92% of amylase activity in faeces obtained from healthy persons was extracellular, whereas only about 9% of activity was associated with particulate material and washed cells. Bacterial cell-bound amylases were considerably more efficient in breaking down starch, however, than were the soluble enzymes which occurred in cell-free faecal supernatant fluids. Cell population densities of anaerobic starch-hydrolysing bacteria in the stools of ten persons ranged from 1.1 × 10¹⁰ to 3.3 × 10¹²/g of faeces. Identification of 120 starch-hydrolysing colonies isolated from the stools of six subjects showed that the predominant amylolytic bacteria belonged to the genera Bifidobacterium, Bacteroides, Fusobacterium and Butyrivibrio. Mixed populations of gut bacteria rapidly fermented starch with the production of volatile fatty acids and organic acids. Lactate was observed to be a major, though transient intermediate during starch fermentation by these cultures. Approximately 60% of starch utilized was converted to volatile fatty acids, which in the human colon would be potentially available for absorption.     

Relation between Aeromonas and faecal coliforms in fresh waters

A possible correlation between the presence of mesophilic aeromonads and the number of faecal coliforms present in three fresh-water habitats subject to differing levels of faecal pollution was investigated. Concentration of Aeromonas spp. between 10²–10⁹ cfu/100 ml and faecal coliforms of between 9–10⁷ cfu/100 ml were found in the waters. In water free from faecal pollution there was no correlation but in polluted waters there was a significant relationship between the numbers of aeromonads, faecal coliform and the concentration of organic matter measured by biological oxygen demand.     

Biosurfactant production by Pseudomonas aeruginosaGS3 from molasses

Patterns of long-chain faecal fatty acids were studied by gas–liquid chromatography in 55 newborn infants in a neonatal intensive care unit. Decreased fractions of fatty acid C16 : 1⁹ and increased fractions of C16 : 0 and C17 : 0 were associated with the occurrence of abdominal distension. Decreased fractions of C16 : 1⁹ and C18 : 2^9,1 were associated with diarrhoea. Flatulence was found in infants who had relatively smaller amounts of fatty acids C17 : 0D and C15 : 0 in their faecal samples. The differences in the patterns of faecal fatty acids are due to differences in bacterial flora. The results support the hypothesis that the initial intestinal colonization plays a role in the later gastrointestinal signs of newborn infants.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.     

Liquid chromatography method for plant and mammalian lignans in human urine

Recently new mammalian lignan precursors were identified but no analysis methods are available for assay of those compounds in human urine. Previously published methods were developed for GC-MS about only two plant lignans were included. Consequently, a method for HPLC equipped with a coulometric electrode array detector was developed to measure plant and mammalian lignans in human urine. The plant lignans, secoisolariciresinol (Seco), matairesinol (Mat), lariciresinol (Lar), pinoresinol (Pin), syringaresinol (Syr) and isolariciresinol (IsoL) were included into the new method together with two mammalian lignans, enterolactone (Enl) and enterodiol (End). Validation of the method demonstrated that it could be applied to normal urine containing low amounts of plant lignans and moderate amounts of mammalian lignans, but the method was also applicable for samples from study subjects in supplementation studies, i.e. sample with very high concentrations of mammalian lignans. The method was found to be a useful tool for studies on plant lignan intake and the activity of micro flora in the metabolism of plant lignans.     

Nitrogen metabolism by the microbial flora of the rabbit caecum

The dense microbial flora of the rabbit caecum consisted chiefly of bacteria (10¹¹/g) with small numbers of yeast cells (10⁶/g). Using strictly anaerobic techniques, 23% of the direct microscopic cell count was cultivated and 55% of the cultivatable bacteria utilized ammonia as the sole source of nitrogen. Ureolytic bacteria were isolated from the caecal lumen and mucosa and were identified as Bacteroides vulgatus, Clostridium clostridiiforme, Bacillus spp. and Staphylococcus spp. Ammonia assimilation by the bacterial flora of the caecum was by incorporation into α-oxoglutarate catalysed by NADPH-linked glutamate dehydrogenase.     

An HPLC procedure for the quantification of anhydrosecoisolariciresinol. Application to the evaluation of flax lignan content

Plant lignans are natural products resulting from the phenylpropanoid metabolic pathway. Some of these compounds have phytoestrogen properties and may protect humans against hormone-dependent cancers such as breast cancer. Secoisolariciresinol, usually in glycosidic form, is the major lignan in flaxseed (Linum usitatissimum L.), and the main precursor of the mammalian lignans (enterodiol, enterolactone) known for their beneficial effects on human health. The quantification of secoisolariciresinol requires a preliminary acid hydrolysis, necessary for the release of lignans from their complex form and aglycone from the glycosylated derivatives. This step partially converts secoisolariciresinol into its anhydrous form: anhydrosecoisolariciresinol. For this reason, we have developed an HPLC quantification method of secoisolariciresinol from flaxseed through its derived form obtained by a total acid hydrolysis. These conditions allow a simplification of the HPLC procedure and allow complete transformation of secoisolariciresinol into its anhydrous form. Using this method, the lignan level in L. usitatissimum seeds was determined to be about 6 mg g^–1 DW. Furthermore, levels of anhydrosecoisolariciresinol were also determined in the different organs of the whole plant, in particular the leaves, stems, roots and fruits. Seeds and fruits accumulated the highest levels of lignans.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes'milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 × 10² Enterobacteriaceae/ml, 3.9 × 10² coliforms/ml and 2.0 × 10² faecal coliforms/ml, whereas the respective mean counts were 6.2 × 10³/ml, 5.4 × 10³/ml and 1.3 × 10³/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.     

Urinary excretion of lignans after administration of isolated plant lignans to rats: the effect of single dose and ten-day exposures

The difference in urinary excretion of mammalian and plant lignans in rats was determined after oral administration of equivalent doses (25 mg/kg of body weight) of 7-hydroxymatairesinol (HMR), lariciresinol (LAR), matairesinol (MR), and secoisolariciresinol (SECO). Twenty-four hours-urine samples were collected after a single dose and after administration of one dose/day for 10 days. Eight lignans were analysed in urine extracts using a high-performance liquid chromatography-tandem mass spectrometry method showing good sensitivity and repeatability. After a single dose of HMR, LAR, MR, and SECO, the main metabolites were 7-hydroxyenterolactone (HENL), cyclolariciresinol (CLAR), enterolactone (ENL), and enterodiol (END), respectively, but after 10-day exposure ENL was the main metabolite of all the tested lignans, showing a considerably higher excretion than after a single dose. Metabolic transformations of plant lignans into each other could also be observed.     

Concentration of the mammalian lignans enterolactone and enterodiol in milk of cows fed diets containing different concentrations of whole flaxseed

A total of 32 lactating Holstein cows with mean body weight of 622 kg (s.e. = 24) were allotted, at week 25 of lactation, to eight groups of four cows blocked for similar days in milk. The objective of the experiment was to determine the effect of feeding four dietary concentrations (0, 50, 100 or 150 g/kg of dry matter) of whole flaxseed, which contains the plant lignan precursor secoisolariciresinol diglucoside (SDG), on concentrations of two mammalian lignans (enterodiol and enterolactone) in milk. The effects of the four diets on feed intake, milk production, milk composition and digestion were also studied. Cows within each block were assigned to one of the four isonitrogenous and isoenergetic total mixed diets and the experiment was carried out from week 25 to 29 of lactation. Diets were fed for ad libitum intake. Enterolactone was the mammalian lignan, of the two metabolites studied, detected in the milk of cows and its concentration in milk tended (P = 0.08) to increase linearly with higher intake of SDG in the diet. Feed intake, milk yield and milk composition were similar among diets. Milk fatty acid profile was slightly improved by feeding flaxseed, as shown by higher concentrations of fatty acids (e.g. n-3) recognized as being beneficial for human health. Those results suggest that feeding of whole flaxseed may result in changes in milk fatty acid composition and enterolactone content, which offer benefits for consumers.     

Performance and egg quality of laying hens fed flaxseed: highlights on n-3 fatty acids,cholesterol, lignans and isoflavones

Flaxseed is a rich source of α-linolenic acid and phytoestrogens, mainly lignans, whose metabolites (enterodiol and enterolactone) can affect estrogen functions. The present study evaluated the influence of dietary flaxseed supplementation on reproductive performance and egg characteristics (fatty acids, cholesterol, lignans and isoflavones) of 40 Hy-Line hens (20/group) fed for 23 weeks a control diet or the same diet supplemented with 10% of extruded flaxseed. The flaxseed diet had approximately three times the content of lignans (2608.54 ng/g) as the control diet, mainly secoisolariciresinol diglucoside (1534.24 v. 494.72 ng/g). When compared with the control group, hens fed flaxseed showed a similar deposition rate (72.0% v. 73.9%) and egg yield. Furthermore, there was no effect of flaxseed on the main chemical composition of the egg and on its cholesterol content. Estradiol was higher in the plasma of the control group (1419.00 v. 1077.01 pg/ml) probably due to the effect of flaxseed on phytoestrogen metabolites. The plasma lignans were higher in hens fed flaxseed, whereas isoflavones were lower, mainly due to the lower equol value (50.52 v. 71.01 ng/ml). A similar trend was shown in eggs: the flaxseed group had higher level of enterodiol and enterolactone, whereas the equol was lower (198.31 v. 142.02 ng/g yolk). Secoisolariciresinol was the main lignan in eggs of the flaxseed group and its concentration was three times higher then control eggs. Flaxseed also improved the n-3 long-chain polyunsaturated fatty acids of eggs (3.25 v. 0.92 mg/g egg), mainly DHA, however, its oxidative status (thiobarbituric reactive substances) was negatively affected. In conclusion, 10% dietary flaxseed did not affect the productive performance of hens or the yolk cholesterol concentration, whereas the lignans and n-3 polyunsaturated fatty acid content of eggs improved. Further details on the competition between the different dietary phytoestrogens and their metabolites (estrogen, equol, enterodiol and enterolactone) should be investigated.     

Hydrolysis of iodothyronine glucuronides by obligately anaerobic bacteria isolated from human faecal flora

Abstract 35 bacterial strains isolated from the human faecal flora were screened for hydrolysis of the glucuronides of 3,3',5-triiodothyronine and 3,3'-diiodothyronine. Two Gram-positive obligately anaerobic strains possessed glucuronidase activity. These strains probably belong to the genus Eubacterium , but ethanol was produced in high concentrations during glucose fermentation, which makes final classification difficult. Considering the number of bacteria in the intestinal flora (> 10⁸/ml) and the biliary excretion of iodothyronine conjugates, the strains must be able to hydrolyse a major part of the total daily intestinal supply of these iodothyronine metabolites. The study extends previous observations with faecal suspensions of human and rat origin [24]. The relevance of bacterial β-glucuronidase activity for a possible enterohepatic circulation of iodothyronines is discussed.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Interaction between the effects of bacteria and dry storage on the opening and water relations of cut rose flowers

W.G. VAN DOORN AND K. D'HONT. 1994. Flowering stems of four rose cultivars (Sonia, Madelon, Jacaranda and Frisco) were placed in aqueous suspensions of bacteria at 10⁴ and 10⁸ colony-forming units (cfu) ml^-1 for 24 h at 5C, then stored dry or held in water for 24 h at 8C and subsequently placed in vase-water at 20C. The effects of these treatments on vase-water uptake were similar to the effects on flower opening. In Sonia and Madelon roses flower opening was negatively affected both by 10⁸ cfu ml^-1 of bacteria and by dry storage. No effect was found at 10⁴ cfu ml^-1, but this concentration had a detrimental effect on flower opening when combined with dry storage. Although flower development in Jacaranda roses was not affected by the bacteria treatments it was inhibited by dry storage. This inhibition was progressively greater when the stems had previously been pulse-treated with a larger number of bacteria. Flower opening in Frisco roses was not affected by even the highest concentration of bacteria, nor by the period of dry storage. It is concluded that placing flowers in water containing bacteria (up to 10⁸ cfu ml^-1) may not always have a negative effect on flower development in cut rose flowers but, together with the effects of dry storage, the presence of even a low number of exogenous bacteria (10⁴ cfu ml^-1) inhibits the development in several cultivars. Such bacterial counts are nearly always found in samples of water used for standing roses during distribution to the consumers.     

Mammalian lignans: possible involvement in endogenous digitalis activity

Lignans are natural products, some of which were recently discovered in animal urines, semen and blood plasma. We investigated the actions of animal lignans obtained by total synthesis or extracted from urines of pregnant women on Na+, K+-ATPase in human red cells and human and guinea-pig heart cell membranes. Some of the tested lignans (enterolactone, prestegane B and 3-O-methyl enterolactone) inhibited Na+, K+-pump activity in human red cells with IC50 ranging from 5 to 9 X 10(-4) M. The IC50 for ouabain (7 X 10(-7) M) was not modified by addition of lignans. Enterolactone inhibited Na+, K+-ATPase activity in human and guinea pig heart membranes. It also displaced [3H]-ouabain binding from human heart with IC50 = 1.5 X 10(-4) M. The apparent dissociation rate constants (kd) of [3H]-ouabain were not different in presence of digoxin or enterolactone. Enterolactone exhibited a poor cross reactivity against antidigoxin antibodies. The aglycones of the lignans studied here were slight inhibitors of the Na+, K+-ATPase. However, we cannot exclude that a glycosyl- (and/or butenolide-) derivative of enterolactone could be one "endogenous ouabain-like" factor.