WO1999007240A1

WO1999007240A1 - Glucosinolate-containing formulated foods

Info

Publication number: WO1999007240A1
Application number: PCT/GB1998/002370
Authority: WO
Inventors: Richard Francis Mithen
Original assignee: Plant Bioscience Limited
Priority date: 1997-08-12
Filing date: 1998-08-06
Publication date: 1999-02-18
Also published as: AU8640398A; EP1003386A1; GB9717104D0; CA2299703A1

Abstract

A formulated food, especially condiment mustard, including crucifer seed or a glucosinolate-containing extract thereof, said seed being other than mustard seed. The glucosinolate is generally able to induce Phase 2 detoxification enzymes in murine hepatoma cells in culture, indicative of likely health benefits such as prevention of cancer, and may be one or more methylthioalkyl, methylsulphinylalkyl, methylsulphinylalkenyl or methylsulphonylalkyl homologues of propyl, butyl or pentyl glucosinolates. Crucifer seed of Cherianthus cheiri, B. oleracea var Italica, Raphanus sativus, B. rupestris, B. villosa, a genotype of Iberis spp., a genotype of Alyssum, a fodder radish cultivar Crail or Neris, or a salad radish cultivar French Breakfast or Snowball may be employed.

Description

GLUCOSINOLATE-CONTAINING FORMULATED FOODS

The present invention relates to food products, especially formulated foods (made to a recipe of ingredients) . In particular, the invention provides food products, especially condiment mustard, which provide to the consumer an elevated level of certain isothiocyanates, in particular those which have been found to have health benefits.

Several recent research programmes indicate that isothiocyanates derived from the hydrolysis of 3- methylsulphinylpropyl and 4-methylsulphinylbutyl glucosinolates found in cruciferous vegetables and salad crops may be important in the human diet in preventing the development of cancer. Firstly, provision of cruciferous vegetables protects rodents against chemically induced carcinogenesis (Wattenberg, 1983; Negri et al, 1991).

Secondly, these isothiocyanates are potent inducers of Phase 2 detoxification enzymes in murine hepatoma cells in culture (Zhang et al, 1994; Rawfiq et al . , 1995), which are associated with reduced susceptibility of animals and their cell cultures to the toxic and neoplastic effects of carcinogens. Lastly, sulforaphane (the isothiocyanate derived from 4- methylsulphinylbutyl glucosinolate) blocks the formation of mammary tumours in Sprague-Dawley rats treated with 9,10- dimethyl-1, 2-benzanthracene (Zhang et al, 1994). This evidence is consistent with the lower tendency of people with high levels of fresh vegetables in their diet to develop cancer (Block et al . , 1992). 3-Methylsulphinylpropyl and 4-methylsulphinylbutyl glucosinolate are found in several cruciferous vegetable crops, but are most abundant in broccoli ( viz purple sprouting broccoli and calabrese) which lacks an active gene at the Gsl - alk locus { sensu Mithen et al . , 1995) which would convert these glucosinolates to their alkenyl homologues . In other Brassica oleracea vegetables these glucosinolates are minor components. They do not occur in B . rapa vegetables such as Chinese cabbage or turnip. The possible chemoprotective effects of these components has led to interest in the dietary intake of glucosinolates and isothiocyanates in broccoli and other cruciferous vegetables.

Estimates of dietary intake of glucosinolates in cooked vegetables of between 29.4 mg/day and 7.9 mg/day (Sones et al . , 1984) have been reported. The glucosinolates in these studies were mainly derived from cabbage and Brussels sprouts in which the majority of the glucosinolates do not have methylsulphinylalkyl side chains. Furthermore, upon cooking it is thought that up to 99.5% of the myrosinase (the enzyme that converts glucosinolates into isothiocyanate) activity is lost (Fenwick et al . , 1983). Thus it is unlikely that many Brassica vegetables are a major source of anticancer isothiocyanates .

Encouraging greater consumption of cruciferous vegetables or enhancing the levels of these specific glucosinolates in broccoli and other vegetables may provide a means to increase the dietary intake of these chemoprotectants . However, this may lead to the greater intake of other glucosinolates which also occur in vegetables which may have undesirable properties (Fenwick et al . , 1983).

O97/09889 identifies cruciferous sources of cancer chemoprotective agents rich in glucosinolates. It suggests use of particularly young sprouts of crucifers as a dietary means of reducing the level of carcinogens in mammals. The use of crucifer seeds as a food or drink supplement is also mentioned in passing as a possibility, the emphasis of the document being on use of crucifer sprouts, with ground crucifer seed flour or meal suggested for incorporation into breads, other baked goods, or health drinks or shakes, and solvent extracts suggested for preparation of soups, teas or other drinks and infusions. The document states that seeds can be used in many different foods such as salads, granolas, breads and other baked goods, among others.

The present invention has arisen in part from work showing that glucosinolates giving rise to isothiocyanates with desirable properties are found in high amounts in seeds of various particular crucifer species. It has been realised that these seeds may be used as sources of glucosinolates and isothiocyanates in the diet, especially in formulated foods to which whole or ground seed can simply be added. This is a much simpler approach than previously suggested ideas for breeding programmes to (try to) develop strains of vegetables such as broccoli with increased glucosinolate content.

Furthermore, the present inventors have realised that there exists in the diet a formulated food which is specifically "designed" to contain high levels of glucosinolates and whose very nature admits of ready addition of crucifer seed in order to provide increased levels of other, desirable glucosinolates. That food is condiment mustard, generally formulated using different combinations or proportions of water, mustard flour, usually wheat flour, and optionally sugar, salt and/or other additives to taste or as preservative .

Mustard is one of the oldest recorded spices, with records dating back to about 3000 B.C.. In terms of quantity, it is probably the most important spice on the world market . In value terms it is exceeded only by pepper. The total annual world trade for use in condiment is estimated as 150 kilotonnes, the main manufacturing centres being USA, UK, France, Germany and Japan. Four species of mustard have been grown for use as a spice, but today only Sinapis alba (white mustard) and Brassica juncea (brown or Indian mustard) are grown on a large scale in Canada and Europe .

Condiment mustards consist of two main types: "English mustard" , which includes p-hydroxybenzyl glucosinolate which is obtained from the seeds of Sinapis alba and contributes a "hot" flavour; and "French mustard", which includes a blend of p-hydroxybenzyl glucosinolate with propenyl glucosinolate obtained from the seeds of Brassica juncea contributing a "pungent" flavour. Some mustards contain 2 -propenyl glucosinolate derived from the roots of Armoracia lopathifolia (horseradish) . The addition of other glucosinolates and isothiocyanates to mustard provides a convenient and pleasant means of enhancing the level of specific isothiocyanates in the diet.

According to a first aspect of the present invention there is provided a formulated food including crucifer seed or a glucosinolate-containing extract thereof, said seed being other than mustard seed.

A "formulated food" is a product made to a recipe of ingredients. In other words, a "formulated food" according to the present invention is an edible formulation or composition including crucifer seed or a glucosinolate-containing extract thereof (as disclosed) as well as at least one other ingredient, generally several ingredients. Consumer laws in many countries require ingredients in formulated foods to be indicated on sales packaging. Such food products may be processed on an industrial scale. Suitability for human consumption is preferred in the present invention. The glucosinolate is generally a dietary glucosinolate, i.e. edible and non-toxic, particularly goitrogenic and may advantageously contribute to the taste characteristics of the food, preferably in a way that is pleasing to the consumer or a statistically significant proportion of consumers, such as a majority or at least about 60%. Commercial viability depends on a product being acceptable or desirable to sufficient people for a profit to be returned. This may of course alternatively be achieved by the glucosinolate making no or little perceptible difference to the taste characteristics of the food.

Preferred glucosinolates are functional in the sense of being capable of inducing Phase 2 detoxification enzymes in murine hepatoma cells in culture. It is standard in the art to use murine hepatoma cells such as Hepa lclc7 cells in determination of quinone reductase (QR) activity induced by a test agent . QR activity may be determined by means of measurement of formation of blue-brown reduced tetrazolium dye, e.g. using an optical scanner and analysis of absorbance . (Prochanska and Santamaria (1988); Prochaska et al . (1992))

Preferably, the glucosinolate in the seed or extract is methylthioalkyl, methylsulphinylalkyl, methylsulphinylalkenyl or methylsulphonylalkyl homologues of propyl, butyl or pentyl glucosinolates .

Most preferably, the glucosinolate in the seed or extract thereof is 3-methylsulphinylpropyl, 3-methylsulphonylpropyl , 4-methylsulphinylbutyl, 4-methylsulphinylbutenyl or 4- methylsulphonylbutyl glucosinolate. As discussed, the isothiocyanates corresponding to these glucosinolates are particularly implicated in protection against cancer.

However, other glucosinolates and isothiocyanates which have desirable properties for inclusion or elevation in the diet may be provided in a formulated food according to the present invention using appropriate crucifer seed.

Table 1 lists a number of wild and cultivated crucifers for which relative levels of various glucosinolates have been determined. Those identified as having a high level of a desirable glucosinolate are preferred for use in the invention (for instance, Cherianthus cheiri , B . oleracea var Italica or Raphanus sativus) . Daxenbichler et al . , 1991, have also reported the glucosinolate composition of seeds from various crucifers and those identified as having a high level of a desirable glucosinolate may be used advantageously in accordance with the present invention.

Seed of B . rupestris, B . villosa or genotypes of Iberis spp. may be employed as sources of 3-methylsulphinylpropyl glucosinolates (shown to have Phase 2 enzyme induction activity) and genotypes of Alyssum may be used to provide methylsulphinylpentyl glucosinolate .

Sources of 4-methylsulphinylbutenyl glucosinolate include the fodder radish cultivars Crail and Neris, and the salad radish cultivars French Breakfast and Snowball . These have greater than 75 μmoles g^"1 of 4-methylsulphinylbutenyl glucosinolate.

Seed employed in the present invention may contain about any of the following amounts of glucosinolate of interest, in ascending order of preference (μmoles g^"1) : 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90.

Seed may be ground, for example to a coarse or fine powder, in any suitable mill or grinder, preferably at ambient temperature.

In a most preferred embodiment of the present invention the formulated food is a condiment mustard including seed of a crucifer other than mustard or a glucosinolate-containing extract of such seed.

Mustard is a particularly appropriate foodstuff for addition of glucosinolate by means of other crucifer seed. For instance, it is not cooked so that high levels of isothiocyanates can be maintained, it naturally provides high levels of isothiocyanates, and its nature, flavour and consistency are such as to be compatible with addition of other crucifer seed and easy blending therewith.

Modifying mustard in accordance with the present invention provides an easy and convenient way of increasing the consumption of these minor but important plant metabolites in the diet. It is thought that a high- fat, high-meat and low fibre diet encourages the production of carcinogens, and this may be partially responsible for the relatively high incidence of cancer in affluent western societies . As mustard is usually consumed with food products which have these characteristics (including "fast food"), it is possible that the consumption of a modified mustard product as provided by the present invention could have a significant impact on health.

A condiment mustard formulation according to the invention may contain crucifer seed which w/w includes at least about 10%, preferably at least about 15%, more preferably at least about 20% more preferably at least about 25% and most preferably at least about 30% of crucifer seed other than mustard. The %w/w of non-mustard crucifer seed within the crucifer seed in the formulated food is preferably sufficient to induce Phase 2 detoxification enzymes, e.g. in murine hepatoma cells in culture (Zhang et al , 1994; Rawfiq et al , 1995). Experimental work using formulations containing crucifer seed including 30% w/w Crail radish seeds had demonstrated induction of Phase 2 enzymes in mouse hepatoma cells .

Crucifer seed other than mustard within a condiment mustard formulation in accordance with the present invention may be at least about 9%, or at least about 14%, or at least about 18%, or at least about 22%, or at least about 27% w/w of the condiment mustard formulation. Condiment mustard formulations may contain up to about 90% w/w seed.

A further aspect of the present invention provides a method of increasing glucosinolate content of a formulated food particularly condiment mustard, including addition to or inclusion in the food of seed from a crucifer other than mustard, or a glucosinolate-containing extract thereof. Addition of glucosinolate to a formulated food may follow grinding of the crucifer seed. The method may be preceded by a step of providing a glucosinolate-containing crucifer seed extract .

Suitably, a glucosinolate-containing crucifer seed extract may be provided by means of hot, e.g. boiling water, providing an extract that is safe for human consumption. Seed may be placed into hot water for a short time of the order of a few minutes, e.g. about 3 minutes, and may then be centrifuged with supernatent being collected and used, or stored for later usage. Other solvents may be used in preparation of a seed extract, including liquid carbon dioxide, ethanol, methanol and others. Preferably the solvent is non-toxic for human consumption. Where extraction is performed with a process that inactivates myrosinase (e.g. boiling), the formulated food may be supplemented with this enzyme. The inclusion of ground up seed may serve this purpose - for instance mustard meal in the condiment formulation. Another aspect of the invention provides the use of crucifer seed other than mustard seed, or an extract of such seed, as a source of glucosinolate in a formulated food, thereby increasing the glucosinolate content of the food. Thus, the invention also provides use of such seed or extract for increasing, enhancing or supplementing the glucosinolated content of the food, preferably condiment mustard as discussed.

A still further aspect of the present invention provides a method including identifying a crucifer species as having seed which contains a glucosinolate of interest, such as one implicated in protection against cancer as discussed herein, or selecting a crucifer species known to have seed which contains such a glucosinolate and formulating a composition for human consumption, particularly condiment mustard, including seed of the identified or selected crucifer species, or extract of such seed containing said glucosinolate. The seed may be ground as discussed. The composition may include one or more other ingredients, optionally including one or more ingredients of cruciferous origin such as mustard, cress or horseradish.

In another aspect, the invention provides a method of increasing glucosinolate and/or isothiocyanate content in an individual (human or animal) , including administration (generally self-administration by ingestion) of a composition including seed of a crucifer other than mustard, or a glucosinolate-containing extract of such seed, particularly a condiment mustard formulation. Evidence provides indication that this has a protective effect against cancer.

Preferred and advantageous glucosinolates, crucifers, formulated foods and compositions according to all aspects of the invention are as disclosed herein and discussed in relation to the first aspect.

Figure 1 shows HPLC chromatograms of desulphoglucosinolates extracted from (Figure 1A) mustard powder, (Figure IB) mustard powder with ground R . sativus seeds and (Figure 1C) mustard powder with ground C. cheiri seeds: 1: 3- methylsulphonylpropyl ; 2: 2 -propenyl; 3: 4- methylsulphinylbutenyl; 4: p-hydroxybenzyl; 5: 4-hydroxy-3- inolylmethyl; 6: benzyl (internal standard).

Figure 2 shows induction data with error bars showing standard deviation for each data point (squares) and cytotoxicity (diamonds) .

EXAMPLE 1

The glucosinolate content of the seed of a number of crucifers has been examined. Briefly, this was done by grinding seeds in a coffee grinder, extracting glucosinolates in hot methanol, converting to desulphoglucosinolates and analysing by HPLC, as described below.

The results are shown in Table 1.

Selected crucifer seeds were then ground and mixed with mustard powder to give a modified mustard glucosinolate profile.

Approximately 250 mg of dry mustard powder (Colman's Dry Mustard, Horseradish variety) was accurately weighed and added to 10 ml of hot 70% methanol. To other samples, 300 mg of ground crucifer seeds were also added as a source of Phase 2 enzyme inducing glucosinolates. Preliminary screening had identified seeds of specific species and cultivars to be good sources of particular glucosinolates. B . oleracea cv Italian Sprouting was used as a source of 4-methylsulphinylbutyl glucosinolate, Raphanus sativus cv Prinz Rotin was used as a source of 4-methylsulphinyl-3-butenyl glucosinolate, and

Chieranthus cheiri cv Bedders Mixed was used as a source of 3- methylsulphonylpropyl . 400 μl of 16 mM benzyl glucosinolate was added to the samples as an internal standard (previous studies had confirmed that this glucosinolate does not occur in the crucifer seeds or commercial mustard powder) . The samples were incubated for 10 minutes at 70 °C and centrifuged at 2000 rpm for 2 minutes. The supernatant was then added to sephadex columns to which sulphatase had been added as described by Heaney et al (1983) . Following overnight incubation, desulphoglucosinolates were eluted from the columns and analyzed by HPLC as described by Magrath et al (1993). Separation of 4-methylsulphinylbutyl and 2-propenyl glucosinolate was achieved through the use of 70% acetonitrile, and separation of benzyl and 4-methylthiobutyl glucosinolate with 20% acetonitrile.

Unmodified mustard powder contained two major glucosinolates, 2-propenyl and p-hydroxybenzyl glucosinolate and minor amounts of 4-hydroxy-3-indolylmethyl glucosinolate. Addition of ground crucifer seed resulted in the presence of the desired glucosinolates, but with no other significant changes to the glucosinolate profile (Figure 1; Table 2) . High levels of 3- methylsulphonylpropyl and 4-methylsulphinyl-3-butenyl glucosinolate were obtained from addition of C. cheiri and R . sativus seeds respectively, while the addition of B . oleracea seeds provided 4-methylsulphinylbutyl and smaller amounts of its precursor, 4-methylthiobutyl glucosinolate. Addition of R . sativus and B . oleracea seed also led to a small increase in the level of 4-hydroxy-3-indolylmethyl glucosinolate.

Fresh broccoli is estimated to contain 34.5 μmoles/100 g fresh wt of 4-methylsulphinylbutyl glucosinolate (unpublished data) . A 2 g portion of modified mustard could contain 85 μmoles of glucosinolates which induce detoxification enzymes. This would be equivalent of eating 246 g of fresh, raw broccoli. As it is likely that cooking or blanching prior to freezing results in degradation of up to 50% of the glucosinolates

(Sones et al, 1984) and loss of 99.5% of myrosinase activity (Fenwick et al . , 1984) considerably greater amounts of cooked or processed broccoli would need to be eaten to obtain equivalent levels of anticancer isothiocyanates.

Formulations containing crucifer seed of which 30% w/w was non-mustard, actually the fodder radish cultivar Crail , was tested in the cultured mouse hepatoma cell assay and shown to induce Phase 2 detoxification enzymes.

EXAMPLE 2

In trodu ction :

Mustard and radish were analysed for content and nature of glucosinolates as described in the report for the previous stage of the project. Certain glucosinolates have been proposed to have potential anticancer activity. This project was to determine: (1) if extracts from mustard and radish were able to induce the anticarcinogenic marker enzyme, quinone reductase, in Hepalclc7 cells; (2) the effect of storage on the activity in (1) .

Methods : Preparation of mustards :

Three mustards were received from Colmans and stored at 4°C. The three samples were: Ml = 30% radish and mustard mixture, M2 = mustard alone, M3 = radish alone. Each mustard was sampled and ground in liquid nitrogen, and then freeze dried. From the dried material, approx. 150 mg of each dried material was milled in a waring blender and 10 ml hot 70% methanol added. 100 μl of 16 mM GT was added as an internal standard and samples incubated at 70 °C for 20 minutes with occasional mixing and then centrifuged at 3000 rpm for 5 min. The supernatants were applied to Sephadex columns to which Sepharose had been added as described by Heaney (1993) . Following an overnight incubation at room temperature, desulpho-glucosinolates were eluted from the columns and analysed by HPLC according to Magrath et al . , (1993) . Separation of the individual glucosinolates was achieved through the use of 70% acetonitrile.

Induction of quinone reductase :

The method described by Tawfiq et al . , (1994) was followed with the following modifications. Each mustard was sampled and ground in liquid nitrogen, then freeze dried. Approx 500 mg of dried material was moistened with 2 ml of sterile water and homogenised (ultra turrax) for 10 sees. Each sample was left for one hour with occasional mixing to ensure hydrolysis of the glucosinolates. Hot 70% methanol (3 ml) was then added and each sample incubated at 70 °C for 15 min and centrifuged at 3000 rpm for 5 min. The supernatant was removed and concentrated under vacuum using a speedivac concentrator to approx l/5th that of the initial volume. The resulting concentrates were stored at -70 °C until tested in the quinone reductase (QR) assay. Testing in the QR assay was performed following the methods described by Prochaska and Santamaria (1988) . Each sample was tested over a range of concentrations up to 2.5 mg dry weight/ml.

Resul ts

The samples stored at +4'C were tested for ability to induce QR at time zero and after 6 months storage. This is the recommended storage temperature. The samples were analysed as described above across the concentration range 0.02 to 2.5 mg/ml . This was based on the results obtained from broccoli expts which indicated that this was the best range. No significant cytotoxicity was measured for the samples. The data for induction is shown in Table 3, also in Figure 2 along with the cytotoxicity data.

Discussion

The results show that the radish samples are better inducers of quinone reductase and therefore have higher potential anticancer activity than mustard. This activity shows a small increase after 6 months storage .

The mustard sample alone showed no significant activity in "fresh" samples. After storage the potential anticancer activity apparently increased. The mixture of radish and mustard was intermediate between the radish and mustard alone. This sample showed a slight decrease in activity with storage.

Overall, it can be noted that the potential anticancer activity of the samples did not decrease with storage time of 6 months .

References

All documents cited herein are incorporated by reference.

Block et al (1992). Nutrition and Cancer 18: 1-19. Fenwick et al (1983) . CRC Critical reviews in Food Science and Nutrition 18: 123-201.

Daxenbichler et al (1991) . Biochemistry Vol. 32 No. 8 pp .

2623-2638.

Heaney et al (1986) . Technical Bulletin, AFRC Food Research Institute, Norwich, U.K.

Magrath et al (1993). Plant Breeding 111, 55-72.

Magrath et al (1993). Plant Breeding 111, 65-73.

Mithen et al (1995). Heredity 74: 210-215.

Negri et al (1991) . Vegetable and Fruit consumption and cancer risk. Int. J. Cancer 48: 350-354.

Posner et al (1994). J. Medical Chemistry 37: 170-6.

Prochaska and Santamaria (1988). Anal. Biochem. 168: 328-336.

Prochaska et al (1992) Proc . Natl. Acad. Sci . USA 89: 2394-

2398. Sones et al (1983). J.Sc Food Agri. 35: 712-720. Tawfiq et al (1994) Eur. J. Cancer Prevention 3: 285-292. Tawfiq et al (1995). Carcinogenesis 16: 1191-1194. Wattenberg (1983). Cancer Research 43: 2448-2453. Zhang et al (1994). Proc . Natl. Acad. Sci . 91: 3147-3150.

Table 1. Glucosinolates in seeds of wild and cultivated crucifers .

Glucosinolate¹ Level²

Jiperis cv Fairy 2 -Propenyl Low umbellata mixed 3 -methylsulphinylpropyl Intermediate

3 -methylthiolpropyl High

Iberis cv Flash 2 -Propenyl Low umbellata 3 -methylsulphinylpropyl Intermediate

3 -methylthiolpropyl High

Cherianthus cv Bedders 3 - ethylsulphonylpropyl High cheiri * mixed

Cherianthus cv Orange 3 -methylsulphonylpropyl Low allioni bedder 4 -methylthiobutyl Low 4 -methylsulphinylbutyl Intermediate 4 -methylsulphonylbutyl Intermediate

Cherianthus unknown 3 -methylsulphonylproply Intermediate cheiri Intermediate

B . oleracea cv white 3 -methylsulphinylpropyl Low var i talica sprouting 4 -methylsulphinylbutyl Low

4 -methylthiobutyl Low

3 -indolylmethyl High

1-methoxy-3 -indolylmethyl High

B . oleracea cv early 3 -methylsulphinylpropyl Low var i talica purple 4 -methylsulphinylbutyl Low sprouting 4 -methylthiobutyl Low

3 -indolylmethyl High

1 -methoxy-3 - indolylmethyl High

B . oleracea cv Italian 4 -methylsulphinylbutyl Intermediate var sprouting 4 -methylthiobutyl Intermediate

Italica*

Raphanus cv Scarlet 4-methylsulphinylbutenyl Intermediate sativus Globe

Raphanus cv Long 4 -methylsulphinylbutenyl Intermediate sativus white icicle

Raphanus cv Prinz 4-methylsulphinylbutenyl High sativus* rotin

Raphanus cv French 4 -methylsulphinylbutenyl Intermediate sativus breakfast

Eruca 4-methylthiobutyl High sativa Malcolmia cv Finest 3 -methylsulphinylpropyl High mari tima mixed Hydroxypropyl High

Lepidium cv Fine Benzyl High sativum curled

Alyssum hydroxybenzyl High saxatile 5-methylsulphinylpentyl High

5-methylthiopentyl Low

Barbarea Phenylethyl High verna

^■""All samples contained low levels of 4 -hydroxy-3 -indolylmethyl glucosinolate .

²Initial studies did not quantify precise levels of glucosinolates, but approximate levels can be estimated from the hplc.

*These lines were used to develop the modified mustards as described herein.

Table 2. Composition of mustard powder.

Glucosinolates μmoles/g dry wt

Mustard Mustard + Mustard + Mustard

C. cheiri B. oleracea + R . sativus

2 -propenyl 34.4 13.6 15.9 14.6

3 -methylsulphonylpropyl 0 44.3 0 0

4 -methylsulphinylbutyl 0 0 20.1 0

4-methylsulphinyl-3-butenyl 0 0 0 42.6

4 -methylthiobutyl 0 0 12.8 0

p-hydroxybenzyl 53.8 26.3 26.9 30.7

4-hydroxy-3 -indolylmethyl 0.59 0.18 2.4 2.8

Table 3

Claims

1. Condiment mustard including crucifer seed or a glucosinolate-containing extract thereof, said seed being other than mustard seed.

2. Condiment mustard according to claim 1 wherein the seed or extract thereof contains glucosinolate able to induce Phase 2 detoxification enzymes in murine hepatoma cells in culture.

3. Condiment mustard according to claim 2 wherein the seed or extract thereof contains one or more methylthioalkyl, methylsulphinylalkyl, methylsulphinylalkenyl or methylsulphonylalkyl homologues of propyl, butyl or pentyl glucosinolates .

4. Condiment mustard according to claim 2 wherein the seed or extract thereof contains one or more glucosinolates selected from 3 -methylsulphinylpropyl, 3- methylsulphonylpropyl, 4 -methylsulphinylbutyl, 4- methylsulphinylbutenyl and 4-methylsulphonylbutyl glucosinolate .

5. Condiment mustard according to claim 4 wherein said crucifer seed is of Cherianthus cheiri , B . oleracea var

Italica or Raphanus sativus .

6. Condiment mustard according to claim 4 wherein said crucifer seed is of B . rupestris, B . villosa or a genotype of Iberis spp.

7. Condiment mustard according to claim 3 wherein said crucifer seed is of a genotype of Alyssum.

5 8. Condiment mustard according to claim 4 wherein said crucifer seed is of a fodder radish cultivar Crail or Neris, or a salad radish cultivar French Breakfast or Snowball .

9. Condiment mustard according to any preceding claim wherein said crucifer seed is ground.

10 10. Condiment mustard according to any preceding claim which contains crucifer seed which w/w includes at least about 10% of crucifer seed other than mustard.

11. Condiment mustard according to any preceding claim which is at least about 9% w/w said crucifer seed other than mustard

15 seed.

12. A method of increasing glucosinolate content of condiment mustard, the method including addition to or inclusion in the condiment mustard of seed from a crucifer other than mustard, or a glucosinolate-containing extract of such seed.

20 13. A method according to claim 12 which produces condiment mustard according to any of claims 2 to 11.

14. Use of crucifer seed or a glucosinolate-containing extract thereof, said seed being other than mustard seed, for increasing the glucosinolate content of condiment mustard.

15. Use according to claim 14 which results in condiment mustard according to any of claims 2 to 11.