Abstract/Summary

Brassica vegetables such as broccoli and red cabbage have been associated with positive health effects due to their phytochemical content. More specifically, isothiocyanates, the hydrolysis products of glucosinolates that are present in Brassica, have been shown to exert potential anti carcinogenic effects and antimicrobial activities against various bacterial and fungal pathogens. The overall aims of this work was to: (i) increase our understanding of how the cooking preparation methods affect the synthesis of the bioactive glucosinolate hydrolysis products in broccoli, super broccoli and a glucoraphanin-rich variety red cabbage, (ii) investigate the antimicrobial activity of broccoli extracts against potentially pathogenic Gram positive and Gram negative bacteria and study the mechanisms of action using a large library of E.coli K-12 single gene deletion mutants, and (iii) develop a sulforaphane-rich broccoli soup formulation with good organoleptic characteristics based on a consumer evaluation test and carry out a dietary intervention study with subjects ileostomy patients, i.e. patients who have their large intestine surgically removed and their wastes flow through the end of the small intestine into a special bag (stoma bag), with the aim to evaluate the absorption of sulforaphane by measuring the sulforaphane concentration in the collected ileal fluids. The results indicated that cooking broccoli, super broccoli and red cabbage in vacuum sealed bags (sous vide) prevented leaching of glucosinolates into boiling water. However, very little hydrolysis of the glucosinolates took place most likely due to the inactivation of myrosinase and the epithiospecifier protein (ESP). The addition of myrosinase sources, i.e. horseradish, mustard seed, rocket and watercress during the cooling stage of the cooked broccoli and red cabbage resulted in a significant increase by one to ten-fold for most isothiocyanates with very little amounts of their nitrile analogues being produced, indicating the absence of ESP in most cases in the myrosinase sources used. A wider range of hydrolysis products were detected in red cabbage including sulforaphane nitrile, iberin nitrile, erucin nitrile, iberverin nitrile, 1-cyano,2,3-epithiopropane and l-cyano-2-hydroxy-3-butene, formed by hydrolysis of various glucosinolates. The proposed cooking strategy can be used in home cooking as well as industrial cooking to increase the levels of the bioactive isothiocyanates. The antimicrobial assays demonstrated that broccoli extracts had a wide spectrum of strong inhibition against both Gram positive and Gram negative bacteria and that there was a significant positive correlation between antimicrobial activity and sulforaphane concentration. Several pathogens that were resistant to ampicillin, such as Salmonella Typhimurium DTI 04 TID, Escherichia coli 0157:H7 VT and E. coli K-12, and to tetracycline, such as Salmonella hadar, were sensitive to sulforaphane. The results from assessing the antimicrobial activity of pure sulforaphane against the E. coli K-12 mutant strains indicated that the deletion of genes such as acrR, ToIR, hfq, which are associated with antimicrobial resistance, increased the sensitivity to sulforaphane. Several genes were also identified that are associated with membrane integrity, such as BolA and Sip, whereas others such as malS, milE, to metabolic functions and cell wall recycling. Interestingly, some genes identified in this study, such as moeA, fdnH, lehB, and ycdB, are known to be important for microbial resistance to various metals, although sulforaphane has not been previously shown to be related to metals. Overall, the study suggests that sulforaphane, due to its strong antibacterial activity, could be used as a natural antibacterial agent for food and medical applications. The results also indicate the possibility to increase the antimicrobial effects of broccoli by reducing the temperature of the thermal treatment during cooking or through the addition of myrosinase sources. The developed soup formulations had good organoleptic properties and got satisfactory scores by the consumer panel for overall liking followed by liking of appearance, taste and texture. The best soup formulation was prepared in two ways, one was a standard formulation, and one had increased sulforaphane content through the addition of myrosinase containing mustard seeds during the cooling stage of the cooked soup. Analysis of the ileal fluids indicated that although the ileal fluids collected after feeding with sulforaphane-rich broccoli soup had a higher sulforaphane content, for both types of soups the concentration of sulforaphane was extremely low. Overall, the results demonstrated that more than 98% of sulforaphane was absorbed rapidly, i.e. within 4 hours after feeding. Sulforaphane was also extracted from the ileal fluid and re-constituted at a similar concertation to that found in the ileal fluid, and tested for its antimicrobial activity against E. coli. No inhibitory activity was observed from this test, which was expected taking into account the high minimum inhibitory concentration (MIC); however, the high initial sulforaphane concentration in the broccoli soups and the high microbial inhibitory activity obtained in the in vitro studies suggest that sulforaphane might be exerting its antimicrobial effects during its transit through the stomach and the small intestine, particularly against bacteria associated with small intestinal bacterial overgrowth (SmO) disorder and potentially Helicobacler pylori.