allosteric inhibitor to delay the reverse reaction from SECO to SDG; or iii) genetically modifying Bacillus cereus strain H62-L1.
Acknowlegements
The authors are grateful for the financial support offered by the National Science Council of Taiwan (NSC 95-2313-B-241-005-MY2).
Conflict of Interest and Disclosure Statement
The authors do not have any conflict of interest, and the authors have already disclosed any actual or potential conflicts of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence (bias) their work and related potential conflicts of interest including employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. Potential conflicts of interest should be disclosed at the earliest possible stage.
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References
[1] Imai T, Nomura M, Fukushima K. Evidence for involvement of the phenylpropanoid pathway in the biosynthesis of the norlignanagatharesinol. J Plant Physiol 2006; 163: 483-487.
[2] Eklund P, Hiilovaara-Teijo M, Kalapudas A, Kangas L, Lindholm A, Sjoholm R Sodervall M, Unkila M. 2005. US20050101541.
[3] Vaisey-Genser M, Morris DH. Flaxseed: Health, Nutrition and Functionality. Flax Council of Canada; 1977, p. 63-64. (Flax Council of Canada website.
www.flaxcouncil.ca)
[4] Clavel T, Lippman R, Gavini F, Dore J. Blaut M. Clostridium saccharogumia sp.
nov. and Lactonifactor longoviformis gen. nov., sp. nov., two novel human faecal bacteria involved in the conversion of the dietary phytoestrogen secoisolariciresinol diglucoside. Syst Appl Microbiol 2007; 30:16-26.
[5] Yan L, Yee JA, Li D, McGuire MH, Thompson LU. 1998. Dietary flaxseed supplementation and experimental metastasis of melanoma cells in mice. Cancer Lett 1998;124:181-186.
[6] Li D, Yee JA, Thompson LU, Yan L. Dietary supplementation with secoisolariciresinol diglycoside (SDG) reduces experimental metastasis of melanoma cells in mice. Cancer Lett 1999;142:91-96.
[7] Prasad K. Secoisolariciresinol diglucoside from flaxseed delays the development of type 2 diabetes in Zucker rat. J Lab Clin Med 2001;138: 32-39.
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[8] Pan A, Sun JQ, Chen YQ, Ye XW, Li HX, Yu ZJ, Wang YF, Gu WJ, Zhang XY, Chen XF, Demark-Wahnefried W, Liu Y, Lin X. Effects of a flaxseed derived lignan supplement in type 2 diabetic patients: A randomized, double-blind, crossover trial. P Los Clin Trials 2007;2:1-7.
[9] Zhang W, Wang X, Liu Y, Tian H, Flickinger B, Empie MW, Sung SZ. Dietary flaxseed lignan extract lowers plasma cholesterol and glucose concentrations in hypercholesterolaemic subjects. Br J Nutr 2008;99:1301-1309.
[10] Wang LQ. Mammalian phytoestrogens: enterodiol and enterolactone. J Chromatogr 2002;777: 289-309.
[11] Williams RL, Rutledge T. Recent phytoestrogen research. Chem. Industry, 1998;1:14-16.
[12] Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, Van der Saag PT, van der Burg B, Gustafsson JA. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinol 1998;139:4252-4263.
[13] Tan KP, Chen J, Ward WE, Thompson LU. Mammary gland morphogenesis is enhanced by exposure to flaxseed or its major lignan during suckling in rats. Exp Biol Med 2004;229:147-157.
[14] Yan L, Yee J, Li D, McGuire M, Thompson L. Dietary fsupplementation and experimental metastasis of melanoma cells in mice. Cancer Lett. 1998;124:181-186.
[15] Markaverich BM, Gregory RR, Alejandro MA, Clark JH, Johnson GA, Middleditch BS. Methyl p-hydroxyphenyllactate: an inhibitor of cell proliferation and
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an endogenous ligand for nuclear type-II binding sites. J Biol Chem 1988;263:
7203-7210.
[16] Benassayag C, Vitte C, Hassid J, Bogard C, Spyratos F, Martin, ME, Nunez EA.
Phytoestrogens and free fatty acids nutritional factors as modulators of the estrogen action in target cells. Proc Int Cancer Congr 1994;609-613.
[17] Jacobson HI, Bennett JA, Mizejewski GJ Inhibition of estrogen dependent breast cancer growth by a reaction product of α-fetoprotein and estradiol. Cancer Res 1990;50:415-420.
[18] Adlercreutz H, Mousavi Y, Clark J, Hockerstedt K, Hamalainen E,Wahala K, Makela T, Hase T. 1992. Dietary phytoestrogens and cancer: in vitro and in vivo studies. J Steroid Biochem 1992;41:331-337.
[19] Adlercreutz H, Bannwart C, Wähälä K, Mäkelä T, Brunow G, Hase T, Arosemena PJ, Kellis JT, Vickery LE. Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens. J Ster Biochem Molec Biol 1993;44:147-153.
[20] Westcott ND, Paton D. A complex containing lignan, phenolic and aliphatic substances from flax and process for preparing. 2001; U.S. Patent 6,264,853.
[21] Johnsson P, Kamal-Eldin A, Lundgren LN, Åman P. HPLC method for analysis of secoisolariciresinol diglucoside in flaxseeds. J Agric Food Chem 2000;48:5216-5219.
[22] Johnsson P, Peerlkamp N, Kamal-Eldin A, Andersson RE, Andersson R,
Accepted Manuscript
Lundgren LN, Åman P. Polymeric fractions containing phenol glucosides in flaxseed.
Food Chem 2002;76:207-212.
[23] Song YL, Kato N, Liu CX, Matsumiya Y, Kato H, Watanabe K. Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its £anking 23S rRNA. FEMS Microbiol Lett 2000;187:167-173.
[24] Kamal-Eldin A, Peerlkamp N, Johnsson P, Andresson RF, Lundrgen LN, Aman P.
An oilgomer from flaxseed composed of secoisolaricresinol diglucoside and 3-hydroxy-3-methyl glutaric acid residues. Phytochem 2001;58: 587-590.
[25] Struijs. K. The lignan macromolecule from flaxseed structure and bioconversion of lignans. The Netherlands: Wageningen Universiteit; Ph. D. Dissertation; 2008.
November 17, 2008. ISBN: 978-90-8585-247-6.
[26] Bokkenheuser VD, Shackleton CH, Winter J. Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans. J Biochem 1987;248:953-956.
[27] Clavel T, Borrmann D, Braune A, Dore J, Blaut M. Occurrence and activity of human intestinal bacteria involved in the conversion of dietary lignans. Anaerobe 2006a;12: 140-147.
[28] Clavel T, Henderson G, Alpert CA, Philippe C, Rigottier-Gois L, Dore J, Blaut M.
Intestinal bacterial communities that produce active estrogen-like compounds enterodiol and enterolactone in humans. Appl Environ Microbiol 2005;71: 6077-6085.
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[29] Clavel T, Henderson G, Engst W, Dore J, Blaut M. Phylogeny of human intestinal bacteria that activate the dietary lignans ecoisolariciresinol diglucoside. FEMS Microbiol Ecol 2006b;55:471-478.
[30] Jenab M, Thompson LU. The influence of flaxseed and lignans on colon carcinogenesis and beta-glucuronidase activity. Carcinogenesis 1996;17:1343-1348.
[31] Heinonen S, Nurmi T, Liukkonen K, et al. In vitro metabolism of plant lignans:
new precursors of mammalian lignans enterolactone and enterodiol. J Agric Food Chem 2001; 49:3178-3186.
[32] Kaufmann F, Wohlfarth G, Diekert G. O-demethylase from Acetobacterium dehalogenans—substrate specificity and function of the participating proteins. Eur J
Biochem 1998;253: 706-711.
[33] Day AJ, Canada FJ, Diaz JC, Kroon PA, Mclauchlan R, Faulds CB, Plumb GW, Morgan MR, Williamson G. Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase. FEBS Lett 2000;468:166-170.
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TABLE CAPTION
Table 1. Table 1. HPLC analysis of lignans present in the ethanolic extract from the alkaline hydrolysate of the defatted flaxseed flour before fermentation.a Table 2. The complicate lignan production kinetics for different substrates fermented by different phase of cells under different cultivation conditionsa
Table 3. Varying kinetic rates revealing the limited productivity of mammalian lignans due to the reversibility of the step from SDG to ENL
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Table 1. HPLC analysis of lignans present in the ethanolic extract from the alkaline hydrolysate of the defatted flaxseed flour before fermentation.a
CAG FAG SDG CA FA SECO END EBL
RT (min)
14.832 16.895 .
21.929 24.696 26.138 30.108 33.782 40.147
Content
aRT: retention time. NF: not found
CAG: coumaric acid glucoside. FAG: ferulic acid glucoside. SDG: secoisolariciresinol diglycoside. CA: coumaric acid. FA: ferulic acid. SECO: secoisolariciresinol. Data expressed in mean SD (n =6). END and ENL were absent before microbial transformation. The retention of END and ENL were obtained by spiking the authentic END and ENL in to the sample solution.
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Table 2. The complicate lignan production kinetics for different substrates fermented by different phase of cells under different cultivation conditionsa
Kinetic data for production rate (mg/L-h) Experiment/
substrate/culture
type CAG FAG SDG CA FA SECO
Exp 1/DFF fermented for 72 h. Exp 3: Resting cell + EEF, fermented for 72 h. All cultures were incubated at 37oC while striring at 150 rpm. Samplings were performed every 12 h.
Table 3. Varying kinetic rates revealing the limited productivity of mammalian
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lignans due to the reversibility of the step from SDG to ENL Biotransformation rate, g/L-h
bCalculated from Fig. 6. Substrate: authentic lignan mixture (each initial concentration was 27 mg/L).cCalculated from Fig. 8. Substrate EEA, incubated at 37oC for 72 h with a stirring rate at 150 rpm. The calculation was performed using the first kinetic order equations Eq. 1 and Eq.
3. For transit reaction, the first order kinetic equation dC/dt = k1C (approximately in form of
∆C/∆C = k1C) was used to calculate the kinetic parameters from the figures revealing linear correlation in each figure.
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FIGUTRE LEGEND
Fig. 1. The structure of main polyphenolics present in the desiccated defatted flaxseeds.
Fig. 2. The microbial biotransformation pathway from SDG to END and ENL (modified from Clavel et al, 2007).
Fig. 3. HPLC analysis of lignans present in the ethanolic extract of the alkaline hydrolysate of the defatted flaxseed flour (EEA) before fermentation. The EEA was filtered with 0.45 Micropore filter. An aliquot 20 L of the filtrate was subjected to HPLC analysis. The retention time (min) was: coumaric acid glucoside (CAG, 14.832). Ferulic acid glucoside (FAG, 16.895).
Secoisolariciresinol diglucoside (SDG, 21.929). Coumaric acid (CA, 24.696).
Ferulic acid (FA, 26.138). Secoisolariciresinol (SECO, 30.108). END and ENL were absent in the original sample. Their retentions time were obtained by spiking with authentic END and ENL. The retention times were33.782 min for enterodiol (END) and 40.147 min for enterolactone (ENL), respectively.
Fig. 4. Preliminary test on the release of lignans from the defatted flaxseed flours (DFF) by Bacillus cereus H62L-1.
To 2 mL of the DFF polymer solution, 8 mL tryptone broth and 0.5 mL of fresh growing culture were added and incubated at 37 oC for 360 h. The fermentation fluid was filtered with 0.45 Micropore filter. An aliquot 20 L of the filtrate was subjected to HPLC analysis.
Fig. 5. Effect of the resting cell- (5a) and the tryptone broth (5b) concentrations on the biotransformation of the authentic secoisolaricirecinol (SDG) by Bacillus cereus H62L-1 resting cells.
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Fig. 6. Higher biotransformation rate by Bacillus cereus H62L-1 resting cells found for the authentic lignans.
The initial concentration of authentic SDG, SECO, END, and ENL was 27 mg/L each. The authentic lignans were respectively fermented at 37oC with 20%
resting cells at a stirring speed 150 rpm for 36 h. The fermentation fluid was filtered with 0.45 Micropore filter. An aliquot 20 L of the filtrate was subjected to HPLC analysis.
Fig. 7. Sequential appearance of CA, FA, and SECO through deglucosylation of CAG, FAG, and SDG by Bacillus cereus H62L-1. Substrate, EEA. (Upper:
before fermentation. Lower: after fermented for 12, 24, and 36 h, respectively).
To 2 mL of the EAA polymer solution, 8 mL tryptone broth and 0.5 mL of fresh growing culture were added and incubated at 37 oC for 36 h. The fermentation fluid was filtered with 0.45 Micropore filter. An aliquot 20 L of the filtrate was subjected to HPLC analysis.
Fig. 8. Apparent reversibility found in the deglucosylation step for biotransforming secoisolaricirecinol (SDG) into secoisolariciresinol (SECO).
Fermentation condition: substrate EEA in 10 mL reaction mixture containing tryptone broth 15%, temperature, 37oC; stirring speed, 150 rpm; time period, 72 h. The fermentation fluid was filtered with 0.45 Micropore filter. An aliquot 20 L of the filtrate was subjected to HPLC analysis.
.
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Figure 1
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Figure 2
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Figure 3 HPLC Chromatogram
Minutes
0 5 10 15 20 25 30 35 40 45 50
0.000.020.040.060.080.100.120.14
Volts
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
CAG FAG SDG CA FA SECO END ENL
Volts
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Figure 4
Time ( day )
0 3 6 9 12 15
C o n ce n tr a ti o n ( m g / L )
0 20 40 60 80 100 120 140 160 180
CAG FAG SDG SECO
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Figure 5
Figure 5a
Time ( hour )
0 12 24 36 48
SDG concentration ( mg / L )
0 10 20 30 40
50 RC 0%
RC 5%
RC 10%
RC 15%
RC 20%
RC 25%
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Figure 5b
Time ( hour )
0 12 24 36 48
SDG concentration ( mg / L )
0 5 10 15
20 LB 0%
LB 15%
LB 35%
LB 55%
LB 75%
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Figure 6
Time ( hour )
0 6 12 18 24 30 36
Concentration ( mg / L )
0 5 10 15 20 25 30
35 SDG
SECO END ENL
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Figure 7
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Figure 8
Time ( hour )
0 12 24 36 48 60 72
C o n ce n tr a ti o n ( m g / L )
0 5 10 15 20 25
CAG CA FAG FA SDG SECO