Relationship of equol production between children aged 5-7 years and their mothers.

PURPOSE: The factors responsible for the production of isoflavone metabolites have not yet been identified. We aimed to examine the relationships of equol production between mother and child in a birth cohort in Japan. METHODS: Subjects were a part of the participants in a longitudinal study on pregnant women and their offspring. When children were 5-7 years old, mothers and children were asked to reply to a questionnaire on lifestyles and a 3-day child's dietary record. Mothers and children were given a bar-shaped soy snack (Soyjoy®) daily on two consecutive days (soy challenge). The snack contained 14 mg of overall soy isoflavones as the sum of aglycones and the glucosides for mothers and 7.5 mg for children. On the morning of day 0 and 3, they were asked to mail their first-void urines. Urinary isoflavone metabolites of 159 mother-child pairs were measured by a high-performance liquid chromatography method. RESULTS: Equol producers were 35.5 % among mothers and 13.8 % among children. Equol producer status of a child was neither associated with dietary intake nor with urinary levels of daidzein and genistein. After multiple adjustments for potential confounders, the estimated relative risk of equol producer was 2.75 (95 % confidence interval 1.00, 7.52) among children whose mother was an equol producer, compared with children whose mother was a non-producer. CONCLUSION: Child's equol production was associated with the mother's equol producer status. The effects of maternal factors on child's equol production should be studied further.

Isolation and characterization of novel S-equol-producing bacteria from brines of stinky tofu, a traditional fermented soy food in Taiwan.

Six strains capable of transforming daidzein to S-equol were isolated from the fermented brines of stinky tofu purchased in Taiwan. Daidzein was completely converted into S-equol within 24h of incubation in five strains. All the strains were gram-positive, rod-shaped, obligately anaerobic, non-motile, and non-spore-forming. In a phylogenetic analysis based on 16S rRNA gene sequences, the strains distributed into three groups in the family Coriobacteriaceae. SNR40-432 (Group I) showed 98.6% 16S rRNA gene similarity and 48-49% DNA-DNA relatedness with Paraeggerthella hongkongensis HKU10(T), suggesting the possibility that SNR40-432 represents a new species in the genus Paraeggerthella. SNR48-44 (Group II) and SNR44-10, SNR45-571, SNR46-41, SNR48-350 (Group III) showed a maximum of 92.2 and 92.1% 16S rRNA gene similarities with Eggerthella sinensis HKU14(T) and Eggerthella lenta JCM9979(T), respectively, which denotes that each group may represent a novel genus and species in the family Coriobacteriaceae. This is the first report isolating equol-producing bacteria from food.

Identification of a novel dihydrodaidzein racemase essential for biosynthesis of equol from daidzein in Lactococcus sp. strain 20-92.

Equol is metabolized from daidzein, a soy isoflavone, by the gut microflora. In this study, we identified a novel dihydrodaidzein racemase (L-DDRC) that is involved in equol biosynthesis in a lactic acid bacterium, Lactococcus sp. strain 20-92, and confirmed that histidine-tagged recombinant L-DDRC (L-DDRC-His) was able to convert both the (R)- and (S)-enantiomers of dihydrodaidzein to the racemate. Moreover, we showed that recombinant L-DDRC-His was essential for in vitro equol production from daidzein by a recombinant enzyme mixture and that efficient in vitro equol production from daidzein was possible using at least four enzymes, including L-DDRC. We also proposed a model of the metabolic pathway from daidzein to equol in Lactococcus strain 20-92.

Discovery of an S-equol rich food stinky tofu, a traditional fermented soy product in Taiwan.

A recent epidemiological study showed that daily intake of mg quantities of S-equol is required for health-promoting effects in menopausal women. However, the maximum equol content in food was reported to be approximately 130 µg/100 g in egg yolk. The objective of this study was to find a high equol-containing food. We measured the equol content of 33 egg yolks and 21 fermented soybean foods. Equol was detected in 28 egg yolks at the maximum content of 43 µg/100 g. In the fermented soybean foods, equol was detected only in stinky tofu. We examined 16 stinky tofu samples purchased during different seasons and the average equol content was 1.39 mg/100 g, ranging from 0.34 to 2.68 mg/100 g. Equol was present in stinky tofu as the S-enantiomeric form and as an aglycon type. This is the first report demonstrating that stinky tofu contains high levels of S-equol, which may exert beneficial effects in menopausal women.

Identification of two novel reductases involved in equol biosynthesis in Lactococcus strain 20-92.

Lactococcus strain 20-92 is a bacterium that produces equol directly from daidzein under anaerobic conditions. In this study, we reveal that the transcription of the gene encoding daidzein reductase in Lactococcus strain 20-92 (L-DZNR), which is responsible for the first stage of the biosynthesis of equol from daidzein, is regulated by the presence of daidzein. We analyzed the sequence surrounding the L-DZNR gene and found six novel genes, termed orf-US4, orf-US3, orf-US2, orf-US1, orf-DS1 and orf-DS2. These genes were expressed in Escherichia coli, and the resulting gene products were assayed for dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR) activity. The results showed that orf-US2 and orf-US3 encoded DHDR and THDR, respectively. DHDR in Lactococcus strain 20-92 (L-DHDR) was similar to the 3-oxoacyl-acyl-carrier-protein reductases of several bacteria and belonged to the short chain dehydrogenase/reductase family. THDR in Lactococcus strain 20-92 (L-THDR) was similar to several putative fumarate reductase/succinate dehydrogenase flavoprotein domain proteins. L-DHDR required NAD(P)H for its activity, whereas L-THDR required neither NADPH nor NADH. Thus, we succeeded in identifying two novel enzymes that are related to the second and third stages of the biosynthetic pathway that converts daidzein to equol.

Cloning and expression of a novel NADP(H)-dependent daidzein reductase, an enzyme involved in the metabolism of daidzein, from equol-producing Lactococcus strain 20-92.

Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species.

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.