Triple extraction

1 mL of aqueous supernatant containing the glucosinolate hydrolysis product was added to 2 mL DCM, the tube tightly capped, and shaken vigorously for 2 minutes. Then the tube was centrifuged at 1500 x g for 3

minutes to separate the phases. The lower phase containing the DCM was removed carefully using a glass Pasteur pipette and filtered into a 2 mL GC vial for analysis. All of the solvent was carefully removed leaving the aqueous phase behind. A further 2 mL DCM was added and the whole process repeated twice more (in triplicate).

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2.4 References

(1) Stepper, J.; Shastri, S.; Loo, T. S.; Preston, J. C.; Novak, P.; Man, P.; Moore, C. H.; Havlícek, V.; Patchett, M. L.; Norris, G. E. Cysteine S- glycosylation, a new post-translational modification found in glycopeptide bacteriocins. FEBS Lett 2011,585, 645-650.

(2) Zhou, J. S.; Shu, Q.; Rutherfurd, K. J.; Prasad, J.; Birtles, M. J.; Gopal, P. K.; Gill, H. S. Safety assessment of potential probiotic lactic acid bacterial strains Lactobacillus rhamnosus HN001, Lb. acidophilus HN017, and Bifidobacterium lactis HN019 in BALB/c mice. Int J Food Microbiol 2000, 56, 87-96.

(3) Schillinger, U.; Lücke, F. K. Antibacterial activity of Lactobacillus sake

isolated from meat. Appl Environ Microbiol 1989,55, 1901-1906.

(4) Kelly, W. J.; Asmundson, R. V.; Huang, C. M. Isolation and characterization of bacteriocin-producing lactic acid bacteria from ready-to-eat food products. Int J Food Microbiol 1996,33, 209-218.

(5) Blodgett, R. Most probable number from serial dilutions. In Bacteriological Analytical Manual F.U.S.F.a.D. Administration: 2006; pp 1-15.

(6) Oblinger, L.; Koburger, J. A. Understanding and teaching the most probable number technique. J. Milk Food Technol 1975,38, 540-545.

(7) Hanahan, D. Studies on transformations of Escherichia coli with plasmids. J Mol Biol 1985,165, 557-580.

(8) Hanahan, D. DNA cloning: A practical approach. In Techniques for transformation of E. coli; Glover, D. M., Ed.; IRL Press: Oxford, United

Kingdom, 1985; 109-135.

(9) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970,227, 680-685.

(10) Wilkinson, A. P.; Rhodes, J. C.; Fenwick, G. R. Determination of myrosinase (thioglucosidase glucohydrolase) activity by a spectrophotmetric coupled enzyme assay. Anal Biochem 1984, 139, 284-

291.

(11) Shevchenko, A.; Jensen, O. N.; Podtelejnikov, A. V.; Sagliocco, F.; Wilm, M.; Vorm, O.; Mortensen, P.; Shevchenko, A.; Boucherie, H.; Mann, M. Linking genome and proteome by mass spectrometry: large-scale

identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci U S A 1996,93, 14440-14445.

(12) Song, L.; Iori, R.; Thornalley, P. J. Purification of major glucosinolates from Brassicaceae seeds and preparation of isothiocyanate and amine metabolites. J Sci Food Agric 2006,86, 1271-1280.

(13) Rochfort, S.; Caridi, D.; Stinton, M.; Trenerry, V. C.; Jones, R. The isolation and purification of glucoraphanin from broccoli seeds by solid phase extraction and preparative high performance liquid chromatography. J Chromatogr A 2006,1120, 205-210.

(14) Fahey, J. W.; Zhang, Y.; Talalay, P. Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc Natl Acad Sci U S A 1997,94, 10367-10372.

(15) Song, L.; Morrison, J. J.; Botting, N. P.; Thornalley, P. J. Analysis of glucosinolates, isothiocyanates, and amine degradation products in vegetable extracts and blood plasma by LC-MS/MS. Anal Biochem 2005, 347, 234-243.

(16) Bialecki, J. B.; Ruzicka, J.; Weisbecker, C. S.; Haribal, M.; Attygalle, A. B. Collision-induced dissociation mass spectra of glucosinolate anions. J Mass Spectrom 2010,45, 272-283.

(17) Chiang, W. C. K.; Pusateri, D. J.; Leitz, R. E. A. Gas chromatography mass spectrometry method for the determination of sulforaphane and sulforaphane nitrile in broccoli. J Agric Food Chem 1998,46, 1018-1021.

(18) Clarke, D. B. Glucosinolates, structures and analysis in food. Anal Methods

2010,2, 301-416.

(19) Lu, M.; Hashimoto, K.; Uda, Y. Rat intestinal microbiota digest desulfosinigrin to form allyl cyanide and 1-cyano-2,3-epithiopropane.

Food Res Int 2011,44, 1023-1028.

(20) Matusheski, N. V.; Wallig, M. A.; Juvik, J. A.; Klein, B. P.; Kushad, M. M.; Jeffery, E. H. Preparative HPLC Method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea. J Agric Food Chem 2001,49, 1867-1872.

(21) McLafferty, F. W. Mass spectrometric analysis. Aliphatic nitriles. Anal Chem 1962,34, 26-30.

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(22) Prestera, T.; Fahey, J. W.; Holtzclaw, W. D.; Abeygunawardana, C.; Kachinski, J. L.; Talalay, P. Comprehensive chromatographic and spectroscopic methods for the separation and identification of intact glucosinolates. Anal Biochem 1996,239, 168-179.

(23) Spencer, G. F.; Daxenbichler, M. E. Gas Chromatography-Mass Spectrometry of nitriles, isothiocyanates and oxazolidinethiones derived from cruciferous glucosinolate. J Sci Food Agric 1980,31, 359-367.

(24) Jin, Y.; Wang, M.; Rosen, R. T.; Ho, C. T. Thermal degradation of sulforaphane in aqueous solution. J Agric Food Chem 1999, 47, 3121-

3123.

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THREE

3.0 Separation and characterisation of glucosinolate-

containing broccoli material

Broccoli contains a number of glucosinolates with the type and concentration varying depending on the plant tissue and the age of the plant. Estimates vary but the total glucosinolate content has been determined to be low at less than 15 μmoles per gram of dry mass and the major glucosinolate, glucoraphanin, at ~7.1 μmoles per gram1-5. Broccoli seed is known to contain concentrated amounts of glucoraphanin (45-114 μmoles per gram), but equally, broccoli sprouts are known to contain high levels of glucosinolates and not much else6,7. A source of glucosinolates was sought, preferably high in glucoraphanin the precursor of sulforaphane. It needed to be a vegetable crucifer that one could include in the diet. Finally, it needed to be in sufficient quantity to enable large-scale extraction generating enough material to allow multiple experiments while maintaining the same glucosinolate profile and quality throughout.