阅读说明：本技术 含异硫氰酸酯的十字花科产物及其制备方法 (Brassicaceae products containing isothiocyanates and methods of making the same ) 是由 M·A·奥古斯丁 N·希费劳特雷费 于 2018-09-27 设计创作，主要内容包括：本发明涉及由十字花科(Brassicaceae)材料生产含异硫氰酸酯的产物的方法和用于该方法的乳酸菌。本发明还涉及通过这样的方法由十字花科材料生产的含异硫氰酸酯的产物。(The present invention relates to a method for producing isothiocyanate-containing products from cruciferous (Brassicaceae) materials and lactic acid bacteria used in the method. The invention also relates to isothiocyanate-containing products produced from cruciferous material by such methods.)

1.A method of producing an isothiocyanate containing product from cruciferous (Brassicaceae) material, comprising:

i) pre-treating cruciferous material to increase the accessibility of myrosinase to glucosinolates;

ii) fermenting the material obtained from step i) with lactic acid bacteria to form said isothiocyanate containing product.

2. The method of claim 1, wherein the pre-processing comprises one or more of:

i) heating;

ii) impregnation;

iii) microwave treatment;

iv) exposure to high frequency sound waves (ultrasound), or

v) pulsed electric field treatment;

wherein the temperature of the cruciferous material does not exceed about 75 ℃ during the pretreatment.

3. The method of claim 1 or claim 2, wherein the pretreatment reduces epidermal specific sulfur protein (ESP) activity while maintaining endogenous myrosinase activity.

4. A method according to any one of claims 1 to 3, wherein the pre-treatment comprises heating and impregnating the cruciferous material, and wherein the temperature of the cruciferous material does not exceed about 75 ℃ during the pre-treatment.

5. The method of claim 4, wherein heating occurs before impregnation or wherein heating and impregnation occur simultaneously.

6. The process of any one of claims 1 to 5, wherein pre-treating comprises heating the cruciferous material to a temperature of about 50 ℃ to about 70 ℃ prior to steeping.

7. A method according to any one of claims 1 to 6, wherein the cruciferous material is heated in a sealed package.

8. A method according to any one of claims 2 to 7, wherein the cruciferous material is impregnated such that at least about 80% of the cruciferous material has a dimension of about 2mm or less.

9. The method of any one of claims 1-8, wherein the isothiocyanate containing product comprises at least about 10 times more isothiocyanate than impregnated cruciferous material.

10. The method of any one of claims 1 to 9, wherein the isothiocyanate containing product comprises at least about 2-fold the expected maximum yield of isothiocyanate, based on extractable glucosinolate content.

11. The method of any one of claims 1 to 10, further comprising acidifying the isothiocyanate containing product from step ii) to a pH of about 4.4 or less.

12. The method according to any one of claims 1 to 11, wherein the lactic acid bacteria are selected from one or more genera selected from: lactobacillus, Leuconostoc, Pediococcus, lactococcus, Streptococcus, Aerococcus, Carnobacterium, enterococcus, Oenococcus, Bacillus, Tetragenococcus, Rogococcus and Weissella.

13. The method of claim 12, wherein the lactic acid bacteria are selected from one or more of the following: leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus brevis, lactococcus lactis, Pediococcus pentosaceus, Lactobacillus rhamnosus and Pediococcus acidilactici.

14. The method according to claim 12 or 13, wherein the lactic acid bacteria are isolated from broccoli and/or the lactic acid bacteria lack myrosinase activity.

15. The method according to any one of claims 12 to 14, wherein the lactic acid bacteria are selected from the group consisting of:

i) leuconostoc mesenteroides;

ii) Lactobacillus plantarum;

iii) Lactobacillus pentosus;

iv) lactobacillus rhamnosus;

v) a combination of i) and ii);

vi) a combination of i), ii), and iii); and

vii) combinations of i), ii) and iv).

16. The method of claim 15, wherein the leuconostoc mesenteroides is BF1 deposited at the australian national institute of metrology and research on day 9 and 25 in 2017 as V17/021729 and/or BF2 deposited at the australian national institute of metrology and research on day 9 and 25 in 2017 as V17/021730.

17. The method according to any one of claims 12 to 15, wherein the lactic acid bacteria are selected from one or more or all of the following:

i) b1, deposited at the national institute for metrological and hyperthyroidism in australia as V17/021731 on 25/9/2017;

ii) B2, deposited at the national institute for metrological hyperthyroidism in australia on 25/9/2017 at V17/021732;

iii) B3, deposited at the national institute for metrological hyperthyroidism in australia as V17/021733, 25/9/2017;

iv) B4 deposited as V17/021734 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017; and

v) 9/25 days 2017 as V17/021735 in B5 of the national Mediterranean institute of Canton, Australia.

18. The method of any one of claims 1 to 17, wherein fermentation is carried out for about 10 hours to about 17 days.

19. The method of any one of claims 1 to 18, wherein fermentation is carried out for about 10 to about 24 hours.

20. The method of any one of claims 1 to 19, wherein fermentation is at about 22 ℃ to about 34 ℃.

21. The method of any one of claims 1 to 20, wherein fermentation is at about 30 ℃.

22. The method of any one of claims 1 to 21, wherein fermentation, if present, reduces the number of one or more or all of: coli (e.coli), salmonella, and listeria.

23. The method of any one of claims 1 to 22, wherein the isothiocyanate is stable for at least 4 weeks, or at least 8 weeks, or at least 12 weeks, when stored at about 4 ℃ to about 25 ℃.

24. The method of any one of claims 1 to 23, wherein the isothiocyanate containing product has one or more or all of the following characteristics:

i) tolerant to yeast, mold, and/or coliform growth for at least 4 weeks, or at least 8 weeks, or at least 12 weeks when stored at about 4 ℃ to about 25 ℃;

ii) comprises a concentration of at least 10⁸Lactic acid bacteria in CFU/g; and

iii) including isothiocyanate bioactive derivatives.

25. The method of any one of claims 1-24, wherein the glucosinolate is selected from one or more of the following: glucoraphanin (4-methylsulfinylbutyl), sinigrin (2-propenyl), glucogenuin (gluconapocin) (3-butenyl), glucobrassin (gluconasicanacin) (4-pentenyl), pregungin (progoitrin) (2(R) -2-hydroxy-3-butenyl), epirubicin (2(S) -2-hydroxy-3-butenyl), 2-hydroxy-4-pentenylthio (gluconologenin) (2-hydroxy-4-pentenyl), glucooirin (3-methylthiopropyl), sesamoside (4-methylthioerucyl), 4-methylthio-3-butenylthio (4-methylthio-3-butenyl), Crocin (glucoraphenin) (3-methylsulfinylpropyl), glucoraphanin (glucoraphanin) (4-methylsulfinyl-3-butenyl), glucolamin (glucolysin) (5-methylsulfinylpentenyl), glucoerucin (glucoerylysin) (3-methylsulfonylbutyl, 4-mercaptobutyl), glucobrassicin (glucobrassicin) (3-indolylmethyl), 4-hydroxybrassinoside (4-hydroxybrassicin) (4-hydroxy-3-indolylmethyl), 4-methoxybrassinoglucosin (4-methoxyglucopyranoside) (4-methoxy-3-indolylmethyl), neobrassinoside (neobrassinosteroid) (1-methoxy-3-indolylmethyl), glucoraphanin (glucopyranoside) (benzylropiolin) (benzyl), and watercresin (2-phenylethyl).

26. The method of claim 25, wherein the glucosinolate is selected from one or both of glucoraphanin (4-methylsulfinylbutyl) and glucoraphanin.

27. The method of any one of claims 1-26, wherein the isothiocyanate is sulforaphane.

28. The method of claim 24, wherein the isothiocyanate bioactive derivative is selected from one or more or all of the following: iberin, allyl isothiocyanate, indole-3-methanol, methoxy-indole-3-methanol, pro-ascorbate and neoascorbate.

29. The method of any one of claims 1-28, wherein the isothiocyanate is sulforaphane and the isothiocyanate containing product comprises at least 150mg/kg dw, at least 200mg/kg dw, at least 300mg/kg dw, at least 400mg/kg dw, or at least 450mg/kg dw, or at least 500mg/kg dw, or at least 550mg/kg dw, or at least 600mg/kg dw, or at least 650mg/kg dw, or at least 700mg/kg dw, or at least 1000mg/kg dw, or at least 2000mg/kg dw, or at least 3000mg/kg dw, or at least 4000mg/kg dw, or at least 5000mg/kg dw, or at least 6000mg/kg dw, or at least 7000mg/kg dw of isothiocyanate.

30. The method of claim 27, wherein the isothiocyanate containing product comprises from about 2000mg/kg dw to about 4000mg/kg dw of isothiocyanate.

31. The method of any one of claims 1 to 30, wherein the crucifer is selected from the group consisting of Brassica oleracea (Brassica oleracea), basilica Brassica (Brassica baleraca), isosbeia Brassica (Brassica carinata), long mustard (Brassica elongate), mediterranean Brassica (Brassica fruulosa), mustard (Brassica haliionis), Brassica juncea (Brassica juncea), Brassica napus (Brassica napus), black mustard (Brassica nigra), pinus tabulaeformis (Brassica pervirilia), turnip (Brassica rapa), brown mustard (Brassica rupestris), mustard (Brassica raps), and asia mustard (Brassica toumuefii).

32. The method of claim 31, wherein the Brassicaceae is Brassica oleracea (Brassica oleracea).

33. A method according to any one of claims 1 to 32, wherein the cruciferous material is selected from one or more of the following: leaves, stems, flowers, florets, seeds and roots.

34. A process for preparing an isothiocyanate containing product from cruciferous material comprising:

i) pre-treating cruciferous material to increase the accessibility of myrosinase to glucosinolates; and

ii) acidifying the material obtained from step i) to form an isothiocyanate containing product.

35. The method of claim 34, wherein acidifying comprises lowering the pH to about 4.4 or below.

36. A process for preparing an isothiocyanate containing product from broccoli material, the process comprising fermenting the material with the lactic acid bacteria leuconostoc mesenteroides and/or lactobacillus plantarum to form the isothiocyanate containing product, wherein the process optionally comprises pre-treating the broccoli material to increase the accessibility of myrosinase to glucosinolates.

37. A process for preparing an isothiocyanate containing product from cruciferous material, the process comprising fermenting the material with the lactic acid bacteria Leuconostoc mesenteroides and/or Lactobacillus plantarum isolated from broccoli to form the isothiocyanate containing product, wherein the process optionally comprises pre-treating the cruciferous material to increase the accessibility of myrosinase to glucosinolates.

38. The method of claim 36 or claim 37, wherein the leuconostoc mesenteroides is BF1 deposited at the australian national institute of metrological and hyperthyroidism at 9 month 25 day 2017 as V17/021729 and/or BF2 deposited at the australian national institute of metrological and hyperthyroidism at 9 month 25 day 2017 as V17/021730.

39. The method of claim 36 or claim 37, wherein the lactic acid bacteria are selected from one or more or all of the following:

i) b1, deposited at the national institute for metrological and hyperthyroidism in australia as V17/021731 on 25/9/2017;

ii) B2, deposited at the national institute for metrological hyperthyroidism in australia on 25/9/2017 at V17/021732;

iii) B3, deposited at the national institute for metrological hyperthyroidism in australia as V17/021733, 25/9/2017;

iv) B4 deposited as V17/021734 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017; and

v) and B5, deposited at the national institute for metrological hyperthyroidism in australia as V17/021735 on 25.9.2017.

40. The method of any one of claims 1 to 39, wherein after fermentation or acidification, the isothiocyanate containing product is post-treated to inactivate microorganisms.

41. The method of claim 40, wherein the isothiocyanate containing product is post-treated with high pressure treatment or thermal treatment.

42. An isolated lactic acid bacterial strain selected from the group consisting of:

i) BF1, deposited as V17/021729 at 9/25.2017 at the national institute of Mediterranean research, Australia;

ii) BF2 deposited as V17/021730 at 9, 25.2017 at the national Mediterranean Kangh, Australia;

iii) B1, deposited at the national institute for metrological hyperthyroidism in australia as V17/021731, 25/9/2017;

iv) B2 deposited as V17/021732 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017;

v) B3 deposited at the national institute for metrological and hyperthyroidism in australia as V17/021733 on 25/9/2017;

vi) B4, deposited at the national institute for metrological hyperthyroidism in australia as V17/021734 on 25/9/2017; and

vii)2017, 9, 25, B5 at the national institute for metrological hyperthyroidism in australia as V17/021735.

43. An isolated Leuconostoc mesenteroides strain comprising genomic DNA which when cleaved with SmaI and/or NotI gives the same SmaI and/or NotI fingerprinting as BF1 or BF2 or an isolated Lactobacillus plantarum strain comprising genomic DNA which when cleaved with SmaI and/or NotI gives the same SmaI and/or NotI fingerprinting as B1, B2, B3, B4 or B5.

44. An isolated leuconostoc mesenteroides strain comprising one or more or all of the polymorphisms listed in table 18 or 19 other than ATCC 8293; or an isolated lactobacillus plantarum strain comprising one or more or all of the polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014.

45. A starter culture for the production of an isothiocyanate containing product or a probiotic comprising lactic acid bacteria selected from one or more or all of the following:

i) BF1, deposited as V17/021729 at 9/25.2017 at the national institute of Mediterranean research, Australia;

ii) BF2 deposited as V17/021730 at 9, 25.2017 at the national Mediterranean Kangh, Australia;

iii) B1, deposited at the national institute for metrological hyperthyroidism in australia as V17/021731, 25/9/2017;

iv) B2 deposited as V17/021732 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017;

v) B3 deposited at the national institute for metrological and hyperthyroidism in australia as V17/021733 on 25/9/2017;

vi) B4, deposited at the national institute for metrological hyperthyroidism in australia as V17/021734 on 25/9/2017; and

vii)2017, 9, 25, B5 at the national institute for metrological hyperthyroidism in australia as V17/021735.

46. The starter culture of claim 44, wherein the starter culture comprises a concentration of at least about 10⁸cfu/mL of lactic acid bacteria.

47. A probiotic composition comprising lactic acid bacteria selected from one or more or all of the following:

i) BF1, deposited as V17/021729 at 9/25.2017 at the national institute of Mediterranean research, Australia;

ii) BF2 deposited as V17/021730 at 9, 25.2017 at the national Mediterranean Kangh, Australia;

iii) B1, deposited at the national institute for metrological hyperthyroidism in australia as V17/021731, 25/9/2017;

iv) B2 deposited as V17/021732 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017;

v) B3 deposited at the national institute for metrological and hyperthyroidism in australia as V17/021733 on 25/9/2017;

vi) B4, deposited at the national institute for metrological hyperthyroidism in australia as V17/021734 on 25/9/2017; and

vii)2017, 9, 25, B5 at the national institute for metrological hyperthyroidism in australia as V17/021735.

48. An isothiocyanate-containing product obtained or obtainable by the method of any one of claims 1 to 41.

49. An isothiocyanate containing cruciferous product comprising at least about 10 times more isothiocyanate than the impregnated cruciferous material.

50. An isothiocyanate containing cruciferous product comprising at least about 2 times the expected maximum yield of isothiocyanate, based on extractable glucosinolate content.

51. The isothiocyanate containing product of claim 48 or claim 49, wherein the isothiocyanate containing product comprises at least 150mg/kg dw, at least 200mg/kg dw, at least 300mg/kg dw, at least 400mg/kg dw, or at least 450mg/kg dw, or at least 500mg/kg dw, or at least 550mg/kg dw, or at least 600mg/kg dw, or at least 650mg/kg dw, or at least 700mg/kg dw, or at least 1000mg/kg dw, or at least 2000mg/kg dw, or at least 3000mg/kg dw, or at least 4000mg/kg dw, or at least 5000mg/kg dw, or at least 6000mg/kg dw, or at least 7000mg/kg dw of sulforaphane.

52. The isothiocyanate-containing product of any one of claims 47-50, wherein the isothiocyanate-containing product has one or more or all of the following characteristics:

i) stable for at least 4 weeks, or at least 8 weeks, or at least 12 weeks when stored at about 4 ℃ to about 25 ℃;

ii) is resistant to yeast, mold and/or coliform growth for at least 4 weeks, or at least 8 weeks, or at least 12 weeks when stored at about 4 ℃ to about 25 ℃; and

iii) comprises at least 10⁷CFU/g Leuconostoc mesenteroides and/or Lactobacillus plantarum.

53. The isothiocyanate-containing product of any one of claims 47-51, wherein the product is selected from the group consisting of: nutraceuticals, supplements and food ingredients.

54. The isothiocyanate-containing product of any one of claims 47-52, wherein the product is a probiotic.

55. The isothiocyanate-containing product of any one of claims 47-53, wherein the product is in powder form.

Technical Field

The present invention relates to a method for producing an isothiocyanate-containing product from a cruciferous material and a lactic acid bacterium used for the method. The invention also relates to isothiocyanate containing products produced from cruciferous (Brassicaceae) material by such methods.

Background

Members of the cruciferae family are rich in glucosinolates, which can be converted to isothiocyanates by myrosinase, which have been noted to have beneficial effects on some types of cancer (Moktari et al, 2017; Capuano et al, 2017; Kim and Park et al, 2016). For example, sulforaphane has been found to reduce hepatic glucose production and improve glucose control in obese patients with type 2 diabetes (Axelsson et al, 2017). However, many members of the cruciferae family are highly perishable after harvest and the quality and quantity of nutrients rapidly decline if the product is not stored well.

Crucifers are often treated to increase the shelf life which can lead to nutrient loss. The main methods for extending shelf life are heat treatment, freezing, modified atmosphere storage and the addition of chemical preservatives, which also cause undesirable changes in the chemical composition.

These methods can result in the loss of glucosinolates or reduce the ability of myrosinase to convert glucosinolates into isothiocyanates. For example, conventional broccoli treatment/preservation involves blanching to inactivate mass degrading enzymes (e.g., lipoxygenase) prior to freezing. Peroxidase inactivation is often used as an indicator of the adequacy of blanching. Conditions which inactivate peroxidase lead to inactivation of myrosinase, so that the resulting product is free from isothiocyanates (Dosz and Jeffery, 2013).

Thus, there remains a need for improved methods of producing cruciferous products that include plant nutrients (e.g., isothiocyanates).

Disclosure of Invention

The present inventors have developed a process for preparing isothiocyanate containing products from cruciferous material.

In one aspect, the present invention provides a process for preparing an isothiocyanate containing product from cruciferous material comprising:

i) pre-treating cruciferous material to increase the accessibility of myrosinase to glucosinolates;

ii) fermenting the material obtained from step i) with lactic acid bacteria to form an isothiocyanate containing product.

In one embodiment, the pretreatment comprises one or more of:

i) heating;

ii) impregnation;

iii) microwave treatment;

iv) exposure to high frequency sound waves (ultrasound); or

v) pulsed electric field treatment

Wherein the temperature of the cruciferous material does not exceed about 75 ℃ during the pretreatment.

In one embodiment, the pretreatment reduces epidermal specific sulfur protein (ESP) activity while maintaining endogenous myrosinase activity.

In one embodiment, the pretreatment comprises heating and impregnating the cruciferous material, and wherein the temperature of the cruciferous material does not exceed about 75 ℃ during the pretreatment. In one embodiment, the heating occurs before the impregnation or wherein the heating and the impregnation occur simultaneously. In one embodiment, the pretreatment comprises heating the cruciferous material to a temperature of about 50 ℃ to about 70 ℃ prior to impregnation. In one embodiment, cruciferous material is impregnated such that at least about 80% of the cruciferous material has a size of about 2mm or less. In one embodiment, the cruciferous material is heated in a sealed package.

In one embodiment, the isothiocyanate containing product comprises at least about 10 times more isothiocyanate than impregnated cruciferous material.

In one embodiment, the isothiocyanate containing product comprises at least about 12 times more isothiocyanate than impregnated cruciferous material.

In one embodiment, the isothiocyanate containing product comprises at least about 14 times more isothiocyanate than impregnated cruciferous material.

In one embodiment, the isothiocyanate containing product comprises at least about 16 times more isothiocyanate than impregnated cruciferous material.

In one embodiment, the isothiocyanate containing product comprises at least about 2 times the expected maximum yield of isothiocyanate, based on extractable glucosinolate content.

In one embodiment, the lactic acid bacteria are isolated from broccoli and/or the lactic acid bacteria lack myrosinase activity.

In one aspect, the present invention provides a process for preparing an isothiocyanate containing product from cruciferous material comprising:

i) pre-treating cruciferous material to increase the accessibility of myrosinase to glucosinolates; and

ii) acidifying the material obtained from step i) to form an isothiocyanate containing product.

In one aspect, the invention provides a process for preparing an isothiocyanate containing product from broccoli material, the process comprising fermenting the material with the lactic acid bacteria leuconostoc mesenteroides and/or lactobacillus plantarum to form the isothiocyanate containing product, wherein the process optionally comprises pre-treating the broccoli material to increase the accessibility of myrosinase to glucosinolates.

In one aspect, the invention provides a process for preparing an isothiocyanate containing product from cruciferous material, the process comprising fermenting the material with the lactic acid bacteria leuconostoc mesenteroides and/or lactobacillus plantarum isolated from broccoli to form the isothiocyanate containing product, wherein the process optionally comprises pre-treating the cruciferous material to increase the accessibility of myrosinase to glucosinolates.

In one aspect, the present invention provides an isolated lactic acid bacterial strain selected from the group consisting of:

i) BF1, deposited as V17/021729 at 9/25.2017 at the national institute of Mediterranean research, Australia; and

ii) BF2, deposited as V17/021730 at the national Mediterranean Kangh of Australia on 25/9/2017.

In one aspect, the present invention provides an isolated lactic acid bacterial strain selected from the group consisting of:

i) BF1, deposited as V17/021729 at 9/25.2017 at the national institute of Mediterranean research, Australia;

ii) BF2 deposited as V17/021730 at 9, 25.2017 at the national Mediterranean Kangh, Australia;

iii) B1, deposited at the national institute for metrological hyperthyroidism in australia as V17/021731, 25/9/2017;

iv) B2 deposited as V17/021732 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017;

v) B3 deposited at the national institute for metrological and hyperthyroidism in australia as V17/021733 on 25/9/2017;

vi) B4, deposited at the national institute for metrological hyperthyroidism in australia as V17/021734 on 25/9/2017; and

vii)2017, 9, 25, B5 at the national institute for metrological hyperthyroidism in australia as V17/021735.

In one aspect, the invention provides a starter culture for the production of an isothiocyanate containing product or a probiotic comprising a lactic acid bacterium selected from one or more or all of the following:

i) BF1, deposited as V17/021729 at 9/25.2017 at the national institute of Mediterranean research, Australia;

ii) BF2 deposited as V17/021730 at 9, 25.2017 at the national Mediterranean Kangh, Australia;

iii) B1, deposited at the national institute for metrological hyperthyroidism in australia as V17/021731, 25/9/2017;

iv) B2 deposited as V17/021732 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017;

v) B3 deposited at the national institute for metrological and hyperthyroidism in australia as V17/021733 on 25/9/2017;

vi) B4, deposited at the national institute for metrological hyperthyroidism in australia as V17/021734 on 25/9/2017; and

vii)2017, 9, 25, B5 at the national institute for metrological hyperthyroidism in australia as V17/021735.

In one embodiment, the starter culture comprises a concentration of at least about 10⁸cfu/mL of lactic acid bacteria.

In one aspect, the present invention provides a probiotic composition comprising lactic acid bacteria selected from one or more or all of the following:

i) BF1, deposited as V17/021729 at 9/25.2017 at the national institute of Mediterranean research, Australia;

ii) BF2 deposited as V17/021730 at 9, 25.2017 at the national Mediterranean Kangh, Australia;

iii) B1, deposited at the national institute for metrological hyperthyroidism in australia as V17/021731, 25/9/2017;

iv) B2 deposited as V17/021732 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017;

v) B3 deposited at the national institute for metrological and hyperthyroidism in australia as V17/021733 on 25/9/2017;

vi) B4, deposited at the national institute for metrological hyperthyroidism in australia as V17/021734 on 25/9/2017; and

vii)2017, 9, 25, B5 at the national institute for metrological hyperthyroidism in australia as V17/021735.

In one aspect, the invention provides an isothiocyanate containing product obtained by the methods described herein.

In one aspect, the invention provides an isothiocyanate containing product obtainable by the method described herein.

In one aspect, the invention provides an isothiocyanate containing cruciferous product comprising at least about 10 times more isothiocyanate than the impregnated cruciferous material.

In one aspect, the invention provides an isothiocyanate containing cruciferous product comprising from about 10 to about 16 times more isothiocyanate than impregnated cruciferous material.

In one aspect, the invention provides an isothiocyanate containing cruciferous product comprising at least about 2 times the expected maximum yield of isothiocyanate, based on extractable glucosinolate content.

In one aspect, the invention provides an isothiocyanate containing cruciferous product comprising from about 2-fold to about 4-fold the expected maximum yield of isothiocyanate, based on extractable glucosinolate content.

In one aspect, the invention provides an isothiocyanate containing cruciferous product comprising at least 150mg/kg dw of isothiocyanate.

In one embodiment, the invention provides an isothiocyanate containing product comprising at least 150mg/kgdw, at least 200mg/kg dw, at least 300mg/kg dw, at least 400mg/kg dw, or at least 450mg/kg dw, or at least 500mg/kg dw, or at least 550mg/kg dw, or at least 600mg/kg dw, or at least 650mg/kg dw, or at least 700mg/kg dw, or at least 1000mg/kg dw, or at least 2000mg/kg dw, or at least 3000mg/kg dw, or at least 4000mg/kg dw, or at least 5000mg/kg dw, or at least 6000mg/kg dw, or at least 7000mg/kg sulforaphane.

In one embodiment, the isothiocyanate containing product comprises leuconostoc mesenteroides and/or lactobacillus plantarum.

In one embodiment, the isothiocyanate containing product has one or more or all of the following characteristics:

i) stable for at least 4 weeks, or at least 8 weeks, or at least 12 weeks when stored at about 4 ℃ to about 25 ℃;

ii) is resistant to yeast, mold and/or coliform growth for at least 4 weeks, or at least 8 weeks, or at least 12 weeks when stored at about 4 ℃ to about 25 ℃; and

iii) comprises at least 10⁷CFU/g Leuconostoc mesenteroides and/or Lactobacillus plantarum.

Unless specifically stated otherwise, any embodiment herein should be mutatis mutandis to any other embodiment. For example, as will be appreciated by those skilled in the art, the examples of lactic acid bacteria outlined above for the method of the invention are equally applicable to the product of the invention.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as exemplary only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of matter shall be taken to include one or more (i.e., one or more) of those steps, compositions of matter, groups of steps or group of matter.

The invention is described below by way of the following non-limiting examples and with reference to the accompanying drawings.

Drawings

FIG. 1.A) shows the pathway of hydrolysis of glucoraphanin to sulforaphane and sulforaphonitrile. B) The effect of impregnation and fermentation on sulforaphane content (mg/kg, DW) in broccoli puree is shown. C) The effect of fermentation on lactic acid bacteria count (logCFU/gm) during storage of broccoli puree is shown.

FIG. 2.A) shows the effect of fermentation on sulforaphane stability in broccoli puree stored at 4 ℃ and 25 ℃ (RT). B) The effect of heat treatment conditions on the conversion of sulforaphane to sulforaphane in the broccoli matrix.

FIG. 3.A) shows the total phenolic content (mg GAE/100g DW) of the starting material broccoli at 25 ℃ and 4 ℃ respectively and its variation during fermentation and storage. B) ORAC (oxygen radical absorbance capacity) antioxidant capacity (. mu.mol TE/g DW) of the starting material broccoli at 25 ℃ and 4 ℃ and its changes during fermentation and storage are shown, respectively.

Figure 4 shows the fermentation time taken to reach a pH of 4.4 or below for different combinations of lactic acid bacterial strains.

FIG. 5.A) shows the sulforaphane yield (μmol/kg DW) under different heat treatment conditions of broccoli with sealed pouches. B) The sulforaphane yields (μmol/kg DW) under different heat treatment conditions of broccoli directly immersed in water are shown.

FIG. 6 shows the comparative effect of the combined effect of impregnation, pre-heating and fermentation and only impregnation and pre-heating and impregnation, pre-heating and chemical acidification on sulforaphane yield (μmol/kg DW) after processing and during storage at 4 ℃ and 25 ℃. The samples were pre-treated in sealed packages at 65 ℃ for 3 minutes.

FIG. 7 shows the effect of fermentation and storage on sulforaphane content. The glucoraphanin content was reduced in the fermented samples stored at 25 ℃ and 4 ℃ compared to the original samples.

FIG. 8, PLS-DA score plot showing the difference in polyphenol metabolite profiles of raw and fermented broccoli puree.

FIG. 9 important features distinguishing between PLS-DA identified fermented and non-fermented samples. The boxes on the right represent the relative concentrations of the metabolites in each group.

FIG. 10 shows the effect of lactic acid fermentation on Broccoli puree metabolite profiles based on non-targeted LC-MS analysis. This indicates that fermentation releases bound phytochemicals such as polyphenol glycosides and glucosinolates and enhances their biological acceptability.

Figure 11 shows volcano plots indicating metabolites with significant (p < 0.05) fold changes after fermentation based on non-targeted LC-MS analysis. The first 50 metabolites with significant fold changes and their individual fold changes are listed in table 8.

FIG. 12 shows the effect of lactic acid fermentation on broccoli polyphenols based on non-targeted LC-MS analysis. A6.6 fold change was observed in chlorogenic acid (2.4-15.8. mu.g/mg), a 23.8 fold increase was observed in sinapic acid (3.6-86.6. mu.g/mg, a 10.5 fold increase was observed in kaempferol (12.7-134.6. mu.g/mg) and a 0.48 fold decrease was observed in p-coumaric acid.

FIG. 13 shows SmaI and NotI restriction of BF1 and BF2 genomic DNAs obtained by pulsed field gel electrophoresis.

Detailed Description

General techniques and definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be deemed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., enzymes, fermentation, inoculation, etc.).

The term "and/or", e.g., "X and/or Y", is to be understood as "X and Y" or "X or Y" and should be taken as providing explicit support for both meanings or for one of the meanings.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein, unless otherwise specified to the contrary, the term "about" means +/-10%, more preferably +/-5%, even more preferably +/-1% of the specified value.

An "allele" refers to a particular form of a genetic sequence (e.g., a gene) within a cell, individual plant, or population that differs from other forms of the same gene in the sequence of at least one (usually more than one) variant site within the same gene sequence. The sequences at these variant sites that differ between different alleles are referred to as "variants", "polymorphisms", or "mutations".

Cruciferae family

One skilled in the art will appreciate that the methods described herein are applicable to the production of isothiocyanate containing products from any cruciferous material that includes glucosinolates. As used herein, "brassicaceae" refers to members of the family brassicaceae (family), commonly known as mustard (musard), crucifer (cruicifer), or cabbage (cabbagage). One skilled in the art will appreciate that the material may be from more than one cruciferae (Brassicaceae).

In one embodiment, the Brassicaceae family is selected from the genera Brassica (genus Brassica) or Cardamine (Cardamine). In one embodiment, the brassica is selected from the group consisting of: barrialia brassicae (Brassica baserica), Italiana mustard (Brassica carinata), Brassica juncea (Brassica jungate), Brassica juncea (Brassica juncea), Brassica juncea (Brassica napus), Brassica nigra (Brassica nigra), Brassica oleracea (Brassica oleracea), Brassica juncea (Brassica juncea), Brassica napus (Brassica napus), Brassica nigra (Brassica nigra), Brassica oleracea (Brassica oleracea), Brassica juncea (Brassica perviridis), Brassica rapa (Brassica rapa), Brassica nigra (Brassica rupestris), Brassica juncea (Brassica septicemia), and Brassica oleracea (Brassica teniformis).

In one embodiment, the Brassica (Brassica) is Brassica oleracea (Brassica oleracea).

In one embodiment, the brassica is selected from the group consisting of: brassica oleracea var oleracea (wild cabbage)), Brassica oleracea var capitis (cabbage), Brassica oleracea (cabbage), Brassica rapapocyna (cabbage), Brassica rapa subcapios (cabbage), Brassica napus (cabbage), Brassica rapa (cabbage), Brassica oleracea (cabbage), Brassica oleracea (cabbage coat), Brassica oleracea (cabbage coat), Brassica oleracea (cabbage coat), Brassica oleracea (cabbage coat), Brassica oleracea (kali (cabbage coat), Brassica oleracea (coat), Brassica oleracea (kali), Brassica oleracea) and Brassica oleracea) can be used for ornamental purposes of the food, cabbage (cabbage, cabbage (kale, cabbage) and kale, cabbage (, Cabbage (Brassica oleracea) variety of Brassica oleracea (Brassica oleracea), stem (Brassica oleracea), portuguese (Brassica oleracea) variety of Brassica oleracea (Brassica oleracea), stem (kohlrabi), broccoli (broccoli oleracea) variety of Brassica oleracea (btrussell), stem (kohlrabi), broccoli (broccoli), cauliflower (cauliflower, roman cauliflower, broccoli) (Brassica oleracea), and broccoli (Brassica oleracea), cauliflower (cauliflower, broccoli) variety of Brassica oleracea (Brassica oleracea), and broccoli (Brassica oleracea) variety of Brassica oleracea variety of the same.

In one embodiment, the brassica is cabbage, italian variety (broccoli).

In one embodiment, the cruciferous plant is selected from the group consisting of: cardamine hirsute (brassica), Cardamine hirsute (castercress), Cardamine hirsute (bellidicus), Cardamine alba (bellidiflora), Cardamine Sinorhiza Sinicae (charlock), Horseradish (radish) (Armoracia rusticana), Caragana clarkii (kaladar) cabbage (kalanchoe), Arabidopsis thaliana (Thlaspi arvense), Raphanus sativus (radish), Cardamine sativus (raphanus sativus), Brassica juncea (arundinacea) (mustard sativa), Arabidopsis thaliana (mustard sativa) (carrot sativa), Pasteurella (mustard), Cardamine sativa (mustard) and Crassamica sativa) (mustard green), and Crassia japonica (crassima), Capsella sativa (mustard) and Crassaria italica (mustard) containing (aca) and Pastalaria italica (mustard) White mustard (Sinapis alba (white mustard)), menhaden (whitlow grass), wild mustard (wild radish) (Raphanus rapana (wildraddish)), Isatis tinctoria (woad), and watercress (Indian cress) (nasturtium microphyllum (yellow crook)).

In one embodiment, the cruciferae family has high levels of one or more glucosinolates. In one embodiment, the cruciferae family has been selectively propagated to have high levels of one or more glucosinolates. In one embodiment, the "high levels" of glucosinolates in the corresponding cruciferae family may include higher levels of glucosinolates than shown in table 2 of Verkerk et al (2009). In one embodiment, the high level of glucosinolates is a level of glucosinolates greater than 3400 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a level of glucosinolates greater than 4000 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a level of glucosinolates of greater than 5000 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a glucosinolate level of greater than 8000 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a level of glucosinolates greater than 10,000 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a level of glucosinolates greater than 12,000 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a level of glucosinolates greater than 15,000 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a level of glucosinolates greater than 18,000 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a level of glucosinolates greater than 20,000 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a level of glucosinolates greater than 25,000 μmol/kg dry weight. In one embodiment, the high level of glucosinolates is a level of glucosinolates greater than 30,000 μmol/kg dry weight. In one embodiment, the Brassicaceae family has been genetically modified or subjected to biotic or abiotic stress to have high levels of one or more glucosinolates. One skilled in the art will appreciate that cruciferae may be modified by any method known to one skilled in the art.

In one embodiment, the glucosinolate is glucoraphanin (4-methylsulfinylbutyl). In one embodiment, the glucosinolate is canola glucosinolate (3-indolylmethyl).

As used herein, "cruciferous material" refers to any part of the brassicaceae family, which includes glucosinolates, including, but not limited to, leaves, stems, flowers, florets, seeds, and roots, or mixtures thereof.

One skilled in the art will appreciate that the methods described herein are applicable to different volumes of cruciferous material, such as, but not limited to, at least 30kg, or at least 50kg, or at least 80kg, or at least 100kg, or at least 1,000kg, or at least 2,000kg, or at least 5,000kg, or at least 8,000kg, or at least 10,000kg, or at least 15,000kg, or at least 20,000 kg.

In one embodiment, the cruciferous material has been washed. As used herein, "washing" removes visible soil and contaminants. In one embodiment, the cruciferous material has been sterilized. As used herein, "disinfection" refers to the reduction of pathogens on cruciferous material.

In one embodiment, cruciferous plants are mixed with other plant material. In one embodiment, the other plant material is a vegetable or fruit material. In one embodiment, the vegetable is carrot or beet.

Glucosinolates

As used herein, "glucosinolate" refers to a secondary metabolite found at least in the brassicaceae family that shares a chemical structure consisting of a β -D-glucopyranose residue linked via a sulfur atom to a (Z) -N-hydroxamic acid sulfate, and a variable R group derived from an amino acid as described by Halkier et al (2006). Examples of glucosinolates are provided in Halkier et al (2006) and Agerbirk et al (2012). Hydrolysis of glucosinolates can produce isothiocyanates, nitriles, episulfide nitriles, thiocyanates, and oxazolidine-2-thiones (FIG. 1A). Many glucosinolates play a role in the defense mechanism of plants against pests.

Glucosinolates are stored in the storage sites of cruciferae. As used herein, a "storage site" is a site in the brassicaceae family where glucosinolates are present and myrosinase is not present.

As used herein, "myrosinase (myrosinase)" also referred to as "thioglucosidase", "myrosinase (sinigrinase)" or "glucoraphaninase (sinigrinase)" refers to a family of enzymes involved in the plant defense mechanism that cleave sulfur-linked glucose (EC 3.2.1.147). Myrosinase catalyzes the hydrolysis of glucosinolates, resulting in the production of isothiocyanates. Myrosinase is sometimes stored as a granule of myrosinase in the vacuole of a particular heteromorphic cell called a myrosinase cell, but has also been reported in the proteome or vacuole, and as a cytoplasmic enzyme that tends to bind to the membrane. Thus, in one embodiment, myrosinase is stored in a black mustard seed cell of the Brassicaceae family.

In one embodiment, the pretreatment as described herein improves the accessibility of myrosinase to glucosinolates. As used herein, "improving access" or "access improved" refers to increasing the availability of glucosinolates to myrosinase to allow for the production of isothiocyanates. In one embodiment, access is improved by releasing glucosinolates from glucosinolate storage sites. In one embodiment, the glucosinolate storage sites are mechanically disrupted (i.e., by impregnation) or enzymatically degraded. In one embodiment, glucosinolates are released from the glucosinolate storage sites by the activity of one or more polysaccharide degrading enzymes (e.g., cellulases, hemicellulases, pectinases and/or glycosidases). In one embodiment, access is improved by allowing myrosinase to enter the glucosinolate storage site. In one embodiment, access is improved by releasing myrosinase from the black mustard daughter cells. In one embodiment, about 10% to about 90% of the glucosinolates are released from the glucosinolate storage site. In one embodiment, about 20% to about 80% of the glucosinolates are released from the glucosinolate storage site. In one embodiment, about 30% to about 70% of the glucosinolates are released from the glucosinolate storage site. In one embodiment, about 40% to about 60% of the glucosinolates are released from the glucosinolate storage site. In one embodiment, about 45% to about 55% of the glucosinolates are released from the glucosinolate storage site. In one embodiment, about 10% of the glucosinolates are released from the glucosinolate storage sites. In one embodiment, about 20% of the glucosinolates are released from the glucosinolate storage sites. In one embodiment, about 30% of the glucosinolates are released from the glucosinolate storage sites. In one embodiment, about 40% of the glucosinolates are released from the glucosinolate storage sites. In one embodiment, about 50% of the glucosinolates are released from the glucosinolate storage sites. In one embodiment, about 60% of the glucosinolates are released from the glucosinolate storage sites. In one embodiment, about 70% of the glucosinolates are released from the glucosinolate storage sites. In one embodiment, about 80% of the glucosinolates are released from the glucosinolate storage sites. In one embodiment, about 90% of the glucosinolates are released from the glucosinolate storage sites.

In one embodiment, the cruciferous material includes one or more glucosinolates selected from aliphatic, indole, or aromatic glucosinolates.

In one embodiment, the aliphatic glucosinolate is selected from one or more of the following: glucoraphanin (4-methylsulfinylbutyl or 4-methylsulfinylbutenyl thioglucoside (glucoraphanin)), sinigrin (sinigrin) (2-propenyl), glucogenuinacin (glucogenuinin) (3-butenyl), brassica glucosides (glucobrassicanacin) (4-pentenyl), pregungrin (progoitrin) (2(R) -2-hydroxy-3-butenyl), epidotril (epidotrin) (2(S) -2-hydroxy-3-butenyl), 2-hydroxy-4-pentenylthio (glucoronao 1eiferin) (2-hydroxy-4-pentenyl), glucoibervirin (3-methylthiopropyl), sesamolin (glucoerucin) (4-methylthiobutyl), 4-methylthio-3-butenyl thioglycoside (4-methylthio-3-butenyl), Chikungunya (glucooberin) (3-methylsulfinylpropyl), glucoraphanin (glucoraphanin) (4-methylsulfinyl-3-butenyl), glucomustine (glucolysin) (5-methylsulfinylpentenyl), and glucomustardsin (glucoerylysin) (3-methylsulfonylbutyl, 4-mercaptobutyl).

In one embodiment, the indole glucosinolate is selected from one or more of the following: canola glucosinolates (3-indolylmethyl), 4-hydroxy canola glucosinolates (4-hydroxy-3-indolylmethyl), 4-methoxy canola glucosinolates (4-hydroxy-3-indolylmethyl), and novel canola glucosinolates (1-methoxy-3-indolylmethyl).

In one embodiment, the indole glucosinolate is one or more of: tropane glucosinolate (benzyl) and watercresin (2-phenylethyl).

In one embodiment, the cruciferous material comprises one or more glucosinolates selected from the group consisting of benzyl glucosinolate, allyl glucosinolate, and 4-methylsulfinylbutyl. In one embodiment, the glucosinolate is glucoraphanin (4-methylsulfinylbutyl). In one embodiment, the glucosinolate is canola glucosinolate (3-indolylmethyl).

In one embodiment, the pretreatment as described herein increases the extractable glucosinolate content as compared to the extractable glucosinolate content of the cruciferous material prior to the pretreatment.

As used herein, "extractable glucosinolate content" refers to the level of glucosinolates accessible in cruciferous material for conversion to isothiocyanates. The expected maximum yield from 1 mole of glucosinolate isothiocyanate, excluding conversion to nitrile and other compounds, is1 mole of isothiocyanate (1 mole of glucosinolate can be converted to a maximum of 1 mole of isothiocyanate, 1 mole of glucose and 1 mole of sulfate ion). Thus, in one embodiment, the extractable sulforaphane content of a commercial broccoli cultivar is 3400 μmol sulforaphane/kg dw, and the expected maximum yield of sulforaphane for a commercial broccoli cultivar is 3400 μmol sulforaphane/kg dw.

Isothiocyanates

As used herein, "isothiocyanate" refers to a sulfur-containing phytochemical having the general structure R-N ═ C ═ S, which is the product of the activity of myrosinase on glucosinolates and biologically active derivatives thereof. In one embodiment, the isothiocyanate is sulforaphane (1-isothiocyano-4-methylsulfinylbutane). In one embodiment, the isothiocyanate is isothiocyanate (3-isothiocyanato-1-propene). In one embodiment, the isothiocyanate is benzyl isothiocyanate. In one embodiment, the isothiocyanate is phenethylisothiocyanate. In one embodiment, the isothiocyanate is 3-butenyl isothiocyanate. In one embodiment, the isothiocyanate is 5-vinyl-1, 3-oxazolidine-2-thione. In one embodiment, the isothiocyanate is 3- (methylthio) propyl isothiocyanate. In one embodiment, the isothiocyanate is 3- (methylsulfinyl) -propyl isothiocyanate. In one embodiment, the isothiocyanate is 4- (methylthio) -butyl isothiocyanate. In one embodiment, the isothiocyanate is 1-methoxyindole-3-carbinol isothiocyanate. In one embodiment, the isothiocyanate is 2-phenylethyl isothiocyanate. In one embodiment, the isothiocyanate is iberin.

In one embodiment, the isothiocyanate containing product further comprises one or more isothiocyanate bioactive derivatives or oligomers thereof. In one embodiment, the isothiocyanate bioactive derivative is a derivative of any isothiocyanate described herein. In one embodiment, the isothiocyanate biologically active derivative is a derivative of sulforaphane. In one embodiment, the isothiocyanate bioactive derivative is iberin. In one embodiment, the biologically active derivative of an isothiocyanate is allyl isothiocyanate. In one embodiment, the isothiocyanate bioactive derivative is indole-3-carbinol. In one embodiment, the isothiocyanate bioactive derivative is methoxy-indole-3-carbinol. In one embodiment, the isothiocyanate bioactive derivative is procarbazine. In one embodiment, the isothiocyanate bioactive derivative is neoascorbyl.

Pretreatment of

As used herein, "pre-treatment" or "pre-treating" releases or aids in the release of glucosinolates from glucosinolate storage sites and/or allows myrosinase to enter glucosinolate storage sites in cruciferous material. In one embodiment, the pretreatment increases exposure of glucosinolates to myrosinase, thereby allowing myrosinase to convert the glucosinolates into isothiocyanates.

In one embodiment, the pretreatment reduces epidermal specific sulfur protein (ESP) while maintaining endogenous myrosinase activity. As used herein, "epidermis-specific sulfur protein" or "ESP" refers to a protein that directs myrosinase activity to produce nitriles and away from isothiocyanate production. Decreasing or inhibiting ESP production (mRNA or protein) or activity may increase isothiocyanate production.

As used herein, "reducing epidermal specific sulfur proteins" refers to reducing the protein production or activity of an ESP. In one embodiment, reducing the ESP comprises inactivating (e.g., denaturing) the ESP at an elevated temperature. In one embodiment, the ESP is denatured at a temperature of about 50 ℃ to about 80 ℃.

As used herein, "maintaining endogenous myrosinase activity" means not significantly reducing myrosinase activity compared to an untreated control. In one embodiment, the endogenous myrosinase activity is not reduced by about 5% or more. In one embodiment, the endogenous myrosinase activity is not reduced by about 10% or more. In one embodiment, the endogenous myrosinase activity is not reduced by about 15% or more. In one embodiment, the endogenous myrosinase activity is not reduced by about 20% or more. In one embodiment, the endogenous myrosinase activity is not reduced by about 30% or more. In one embodiment, the endogenous myrosinase activity is not reduced by about 40% or more. In one embodiment, the endogenous myrosinase activity is not reduced by about 50% or more.

In one embodiment, the pretreatment comprises one or more of: i) heating; ii) impregnation; iii) microwave treatment; iv) exposure to high frequency sound waves (ultrasound), or v) pulsed electric field treatment, wherein the temperature of the cruciferous material does not exceed about 75 ℃ during the pretreatment.

In one embodiment, the cruciferous material is heated in a fuel-based heating system, an electrical-based heating system (i.e., oven or ohmic heating), radio frequency heating, autoclaving, or steam-based heating system (indirect or direct application of steam). In one embodiment, cruciferous material is heated in a sealed package (e.g., in a retort pouch). In one embodiment, the cruciferous material is heated in an oven, a water bath, a bioreactor, a furnace, a water blancher, or a steam blancher. In one embodiment, cruciferous material is heated via high pressure thermal heating. In one embodiment, the cruciferous material is heated via ohmic heating. In one embodiment, the cruciferous material is heated via radio frequency. In one embodiment, cruciferous material is blanched in water. In one embodiment, cruciferous material is heated via autoclaving. In one embodiment, cruciferous material is placed in a sealed package for autoclaving.

In one embodiment, the pretreatment comprises heating the cruciferous material to a temperature in the range of about 50 ℃ to about 70 ℃. In one embodiment, the pretreatment comprises heating the cruciferous material to a temperature in the range of about 50 ℃ to about 65 ℃. In one embodiment, the pretreatment comprises heating the cruciferous material to a temperature in the range of about 50 ℃ to about 60 ℃. In one embodiment, heating comprises heating the cruciferous material to a temperature in the range of about 55 ℃ to about 70 ℃. In one embodiment, heating comprises heating the cruciferous material to a temperature in the range of about 60 ℃ to about 70 ℃. In one embodiment, heating comprises heating the cruciferous material to about 65 ℃ to about 70 ℃. In one embodiment, the cruciferous material is heated for about 30 seconds. In one embodiment, the cruciferous material is heated for about 1 minute. In one embodiment, the cruciferous material is heated for about 2 minutes. In one embodiment, the cruciferous material is heated for about 3 minutes. In one embodiment, the cruciferous material is heated for about 4 minutes. In one embodiment, the cruciferous material is heated for about 5 minutes.

In one embodiment, cruciferous material is heated in a sealed package at about 60 ℃ for about 1 minute. In one embodiment, cruciferous material is heated in a sealed package at about 60 ℃ for about 2 minutes. In one embodiment, the cruciferous material is heated in a sealed package at about 60 ℃ for about 3 minutes. In one embodiment, the cruciferous material is heated in a sealed package at about 65 ℃ for about 4 minutes. In one embodiment, the cruciferous material is heated in a sealed package at about 65 ℃ for about 1 minute. In one embodiment, the cruciferous material is heated in a sealed package at about 65 ℃ for about 2 minutes. In one embodiment, the cruciferous material is heated in a sealed package at about 65 ℃ for about 3 minutes. In one embodiment, the cruciferous material is heated in a sealed package at about 65 ℃ for about 4 minutes.

In one embodiment, cruciferous material is heated in water at about 60 ℃ for about 1 minute. In one embodiment, cruciferous material is heated in water at about 60 ℃ for about 2 minutes.

In one embodiment, the heating comprises steaming the cruciferous material. In one embodiment, the pretreatment comprises steaming the cruciferous material. In one embodiment, the cruciferous material is steamed to a temperature of about 50 ℃ to about 70 ℃. In one embodiment, the cruciferous material is steamed to a temperature of about 60 ℃ to about 70 ℃. In one embodiment, the cruciferous material is steamed for at least about 30 seconds. In one embodiment, the cruciferous material is steamed for at least about 1 minute. In one embodiment, the cruciferous material is steamed for at least about 2 minutes. In one embodiment, the cruciferous material is steamed for at least about 3 minutes. In one embodiment, the cruciferous material is steamed for at least about 4 minutes. In one embodiment, the cruciferous material is steamed for at least about 5 minutes.

In one embodiment, the pretreatment comprises impregnating cruciferous material. As used herein, "maceration," "maceration," and "maceration" refer to the breaking of cruciferous material into smaller pieces. In one embodiment, maceration comprises breaking down at least about 30% to about 90% of the cells of the cruciferous material to allow myrosinase to access its substrate, glucosinolates. In one embodiment, maceration comprises disaggregating at least about 40% to about 90% of the cells of the cruciferous material. In one embodiment, maceration comprises disaggregating at least about 50% to about 90% of the cells of the cruciferous material. In one embodiment, maceration comprises disaggregating at least about 60% to about 90% of the cells of the cruciferous material. In one embodiment, maceration comprises disaggregating at least about 70% to about 90% of the cells of the cruciferous material. As will be understood by those skilled in the art, disrupting cells includes disrupting the cell wall and disrupting compartmentalization of intracellular organelles.

In one embodiment, the cruciferous material is impregnated with a blender, grinder, or pulverizer. In one embodiment, cruciferous material is impregnated such that at least about 80% of the cruciferous material has a size of about 2mm or less. In one embodiment, cruciferous material is impregnated such that at least about 80% of the cruciferous material has a size of about 1mm or less. In one embodiment, cruciferous material is impregnated such that at least about 80% of the cruciferous material has a size of about 0.5mm or less. In one embodiment, cruciferous material is impregnated such that at least about 80% of the cruciferous material has a size of about 0.25mm or less. In one embodiment, cruciferous material is impregnated such that at least about 80% of the cruciferous material has a size of about 0.1mm or less. In one embodiment, cruciferous material is impregnated such that at least about 80% of the cruciferous material has a size of about 0.05mm or less. In one embodiment, cruciferous material is impregnated such that at least about 80% of the cruciferous material has a size of about 0.025mm or less. In one embodiment, cruciferous material is impregnated such that at least about 80% of the cruciferous material has a size of about 0.01mm or less. In one embodiment, cruciferous material is impregnated such that about 50% to about 90% of the cruciferous material has a size of about 2mm or less. In one embodiment, cruciferous material is impregnated such that about 60% to about 80% of the cruciferous material has a size of about 2mm or less. In one embodiment, cruciferous material is impregnated such that about 50% to about 90% of the cruciferous material has a size of about 1mm or less. In one embodiment, cruciferous material is impregnated such that about 60% to about 80% of the cruciferous material has a size of about 1mm or less. In one embodiment, the cruciferous material is heated to a temperature of about 50 ℃ to about 70 ℃ during impregnation. In one embodiment, the cruciferous material is heated to a temperature of about 55 ℃ to about 70 ℃ during impregnation. In one embodiment, the cruciferous material is heated to a temperature of about 60 ℃ to about 70 ℃ during impregnation. In one embodiment, the cruciferous material is heated to a temperature of about 65 ℃ to about 70 ℃ during impregnation.

In one embodiment, the pretreatment comprises heating and impregnating the cruciferous material. In one embodiment, the pretreatment produces a mud. As used herein, "puree" refers to cruciferous material mixed with a creamy or liquid consistency.

Those skilled in the art will appreciate that "microwave" or "microwaving" heats a substance (e.g., a cruciferous material) by passing microwaves through the substance. In one embodiment, the pretreatment comprises microwave treatment of cruciferous material. In one embodiment, the cruciferous material is pretreated in consumer or industrial microwaves. In one embodiment, the industrial microwave is a continuous microwave system, such as, but not limited to, MIP 11 industrial microwave continuous cooking (ferrite microwave technology). In one embodiment, the pretreatment comprises microwave treatment of cruciferous material. In one embodiment, cruciferous material is microwaved at about 0.9 to about 2.45 GHz. In one embodiment, the cruciferous material is microwaved for at least about 30 seconds, or at least about 1 minute, or at least about 2 minutes, or at least 3 minutes.

In one embodiment, the pretreatment comprises exposing the cruciferous material to low to medium frequency ultrasound. In one embodiment, the pretreatment comprises exposing the cruciferous material to thermal ultrasound (low to medium frequency ultrasound with heat of about 30 ℃ to about 60 ℃). In one embodiment, the ultrasound is generated using an industrial scale ultrasound processor. In one embodiment, the ultrasonic processor is a continuous or batch ultrasonic processor. In one embodiment, the ultrasound processor is such as, but not limited to, UIP500hd or UIP4000(Hielscher, ultrasound technology). In one embodiment, the frequency of the ultrasound is from about 20kHz to about 600 kHz. In one embodiment, the cruciferous material is exposed to sound waves for at least about 30 seconds, or at least about 1 minute, or at least about 2 minutes, or at least about 3 minutes, or about 5 minutes.

In one embodiment, the pretreatment comprises exposing the crucifer material to a pulsed electric field treatment. Pulsed electric field processing is a non-thermal processing technique that involves the application of short, high voltage pulses. The pulse-induced electroporation of the cells of cruciferous plant material promotes the accessibility of myrosinase to glucosinolates. In one embodiment, the pulsed electric field treatment heats cruciferous material to a temperature of about 40 ℃ to about 70 ℃. In one embodiment, the pulsed electric field treatment heats cruciferous material to a temperature of about 50 ℃ to about 70 ℃. In one embodiment, the pulsed electric field treatment heats cruciferous material to a temperature of about 60 ℃ to about 70 ℃. In one embodiment, the pulsed electric field treatment comprises treating the crucifer material with a voltage pulse of about 20 to about 80 kV. In one embodiment, the pretreatment converts about 10% to about 90% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 20% to about 80% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 30% to about 70% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 40% to about 60% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 10% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 20% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 30% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 40% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 50% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 60% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 70% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 80% of the glucosinolates to isothiocyanates. In one embodiment, the pretreatment converts about 90% of the glucosinolates to isothiocyanates.

Fermentation of

One skilled in the art will appreciate that the fermentation process described herein may include the use of any lactic acid bacteria. As used herein, "fermentation" refers to the biochemical breakdown of cruciferous material by lactic acid bacteria. In one embodiment, the fermentation with lactic acid bacteria is performed using the addition of exogenous lactic acid bacteria. As used herein, "lactic acid bacteria" or "lactic acid bacteria" are bacteria that produce lactic acid as an end product of carbohydrate fermentation and may include, but are not limited to, bacteria from the following: lactobacillus, Leuconostoc, Pediococcus, lactococcus, Streptococcus, Aerococcus, Carnobacterium, enterococcus, Oenococcus, Bacillus, Tetragenococcus, Rogococcus and Weissella. In one embodiment, the lactic acid bacteria comprise myrosinase activity. In one embodiment, the lactic acid bacteria are from the genus leuconostoc. In one embodiment, the lactic acid bacteria are from the genus lactobacillus.

In one embodiment, the lactic acid bacteria are selected from one or more of the following: lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus rhamnosus, Lactobacillus pentosus, Lactobacillus brevis, lactococcus lactis, Pediococcus pentosaceus and Pediococcus acidilactici.

In one embodiment, the lactic acid bacteria are isolated from the Brassicaceae family. In one embodiment, the lactic acid bacteria are isolated from cabbage. In one embodiment, the lactic acid bacteria are isolated from broccoli. In one embodiment, the lactic acid bacteria are isolated from broccoli leaves. In one embodiment, the lactic acid bacteria are isolated from broccoli stems. In one embodiment, the lactic acid bacteria are isolated from broccoli puree. In one embodiment, the lactic acid bacteria are isolated from broccoli australia.

In one embodiment, the lactic acid bacterium lacks myrosinase activity.

In one embodiment, the lactic acid bacterium is lactobacillus.

In one embodiment, the lactic acid bacteria are selected from: i) leuconostoc mesenteroides; ii) Lactobacillus plantarum; iii) Lactobacillus pentosus; iv) lactobacillus rhamnosus; v) a combination of i) and ii); vi) a combination of i), ii), and iii); and vii) combinations of i), ii) and iv).

In one embodiment, the lactic acid bacterium is leuconostoc mesenteroides. In one embodiment, leuconostoc mesenteroides is ATCC 8293. In one embodiment, the leuconostoc mesenteroides is BF1 and/or BF 2. In one embodiment, leuconostoc mesenteroides lacks myrosinase activity.

In one embodiment, the lactic acid bacterium is lactobacillus plantarum. In one embodiment, the lactobacillus plantarum lacks myrosinase activity.

In one embodiment, about 50% of the lactic acid bacteria are leuconostoc mesenteroides and about 50% of the lactic acid bacteria are lactobacillus.

In one embodiment, about 50% of the lactic acid bacteria are leuconostoc mesenteroides and about 50% of the lactic acid bacteria are lactobacillus plantarum.

In one embodiment, the lactobacillus plantarum is selected from one or more or all of B1, B2, B3, B4 and B5. In one embodiment, the lactobacillus plantarum is B1. In one embodiment, the lactobacillus plantarum is B2. In one embodiment, the lactobacillus plantarum is B3. In one embodiment, the lactobacillus plantarum is B4. In one embodiment, the lactobacillus plantarum is B5.

In one embodiment, the fermentation occurs in the presence of at least 2, or at least 3, or at least 4, or at least 5, or at least 6 lactic acid bacteria strains selected from BF1, BF2, B1, B2, B3, B4, and B5.

In one embodiment, the lactic acid bacterium is a recombinant bacterium modified to produce a high level of myrosinase activity compared to a control bacterium lacking the modification. The skilled person will understand that the recombinant lactic acid bacterium is produced by any technique known to the skilled person.

In one embodiment, the lactic acid bacteria are subjected to a stress, such as, but not limited to, heat stress, cold stress, sub-lethal ultrasound (e.g., about 20 to about 2000MHz), high voltage, dynamic high voltage, or pulsed electric field, to increase myrosinase activity and the activity of the polysaccharide degrading enzyme as compared to a control lactic acid bacteria that is not subjected to stress. In one embodiment, the heat stress comprises heating the bacteria to greater than about 40 ℃ to about 75 ℃. In one embodiment, the heat stress comprises heating the bacteria to greater than about 45 ℃ to about 65 ℃. In one embodiment, the heat stress comprises heating the bacteria to greater than about 45 ℃ to about 55 ℃. In one embodiment, the cold stress comprises reducing the bacteria to a temperature of about 0 ℃ to about 8 ℃. In one embodiment, the cold stress comprises reducing the bacteria to a temperature of about 2 ℃ to about 6 ℃. In one embodiment, the cold stress comprises reducing the bacteria to a temperature of about 4 ℃.

In one embodiment, at least about 10 is used⁵CFU/g of the lactic acid bacteria described herein inoculate cruciferous material. In one embodiment, at least about 10 is used⁶CFU/g of the lactic acid bacteria described herein inoculate cruciferous material. In one embodiment, at least about 10 is used⁷CFU/g of the lactic acid bacteria described herein inoculate cruciferous material. In one embodiment, at least about 10 is used⁸CFU/g of the lactic acid bacteria described herein inoculate cruciferous material. In one embodiment, the cruciferous material has been pretreated.

In one embodiment, the fermentation is conducted at about 20 ℃ to about 34 ℃. In one embodiment, the fermentation is conducted at about 22 ℃ to about 34 ℃. In one embodiment, the fermentation is conducted at about 24 ℃ to about 34 ℃. In one embodiment, the fermentation is conducted at about 24 ℃ to about 30 ℃. In one embodiment, the fermentation is conducted at about 34 ℃ to about 34 ℃. In one embodiment, the fermentation is conducted at about 25 ℃. In one embodiment, the fermentation is conducted at about 30 ℃. In one embodiment, the fermentation is conducted at about 34 ℃.

In one embodiment, the fermentation is conducted for about 8 hours to about 17 days. In one embodiment, the fermentation is conducted for about 8 hours to about 14 days. In one embodiment, the fermentation is conducted for about 8 hours to about 7 days. In one embodiment, the fermentation is conducted for about 8 hours to about 5 days. In one embodiment, the fermentation is conducted for about 8 hours to about 4 days. In one embodiment, the fermentation is conducted for about 8 hours to about 3 days. In one embodiment, the fermentation is conducted for about 8 hours to about 30 hours. In one embodiment, the fermentation is conducted for about 8 to about 24 hours. In one embodiment, the fermentation is conducted for about 10 hours to about 24 hours. In one embodiment, the fermentation is conducted for about 10 days. In one embodiment, the fermentation is conducted for about 9 days. In one embodiment, the fermentation is conducted for about 8 days. In one embodiment, the fermentation is conducted for about 7 days. In one embodiment, the fermentation is conducted for about 4 days. In one embodiment, the fermentation is conducted for about 6 days. In one embodiment, the fermentation is conducted for about 5 days. In one embodiment, the fermentation is conducted for about 72 hours. In one embodiment, the fermentation is conducted for about 60 hours. In one embodiment, the fermentation is conducted for about 45 hours. In one embodiment, the fermentation is conducted for about 30 hours. In one embodiment, the fermentation is conducted for about 24 hours. In one embodiment, the fermentation is conducted for about 20 hours. In one embodiment, the fermentation is conducted for about 18 hours. In one embodiment, the fermentation is conducted for about 15 hours. In one embodiment, the fermentation is conducted for about 16 hours. In one embodiment, the fermentation is conducted for about 14 hours. In one embodiment, the fermentation is conducted for about 12 hours. In one embodiment, the fermentation is conducted for about 10 hours. In one embodiment, the fermentation is conducted for about 8 hours. In one embodiment, the fermentation culture is stirred. In one embodiment, the agitation is intermittent. In one embodiment, the agitation is continuous. In a particularly preferred embodiment, the fermentation is carried out for 15 hours with intermittent stirring. In a particularly preferred embodiment, the fermentation is carried out for 24 hours with intermittent stirring.

In one embodiment, the fermentation reaction is complete when the composition reaches a pH of about 4.5 to about 3.8. In one embodiment, the fermentation reaction is complete when the composition reaches a pH of about 4.5 to about 3.6. In one embodiment, the fermentation reaction is complete when the composition reaches a pH of about 4.5 to about 4.04. In one embodiment, the fermentation reaction is complete when the composition reaches a pH of about 4.3 to about 4.04. In one embodiment, the fermentation reaction is complete when the composition reaches a pH of 4.5 or less, or 4.4 or less, or 4.3 or less, or 4.04 or less, or 3.8 or less. In one embodiment, the fermentation reaction is complete when the composition reaches a pH of 4.5 or less. In one embodiment, the fermentation reaction is complete when the composition reaches a pH of 4.4 or less.

In one embodiment, if present, fermentation reduces the number of one or more or all of the following: coli (e.coli), salmonella, and listeria. In one embodiment, if present, the fermentation reduces the CFU/g of one or more or all of: coli (e.coli), salmonella, and listeria.

In one embodiment, no salt is added to the fermentation culture.

In one embodiment, fermentation increases the extractable glucosinolate content as compared to the extractable glucosinolate content in the pretreated cruciferous material. In one embodiment, fermentation increases the extractable glucosinolate content as compared to the extractable glucosinolate content of cruciferous material. In one embodiment, the fermentation increases the extractable glucosinolate content by about 100% to about 500% as compared to the extractable glucosinolate content in cruciferous material. In one embodiment, fermentation increases the extractable glucosinolate content by about 200% to about 450% as compared to the extractable glucosinolate content in cruciferous material. In one embodiment, fermentation increases the extractable glucosinolate content by about 250% to about 450% as compared to the extractable glucosinolate content in cruciferous material. In one embodiment, fermentation increases the extractable glucosinolate content by about 300% to about 400% as compared to the extractable glucosinolate content in cruciferous material. In one embodiment, fermentation increases the extractable glucosinolate content by about 300% as compared to the extractable glucosinolate content in cruciferous material. In one embodiment, fermentation increases the extractable glucosinolate content by about 400% as compared to the extractable glucosinolate content in cruciferous material. In one embodiment, the glucosinolate is glucoraphanin.

Acidification

The pretreated material can be acidified, so that the microbial safety and stability (susceptibility to microbial degradation) of the product are improved, and the stability of isothiocyanate in the product is improved. Acidification may be achieved by the addition of organic acids such as, but not limited to, lactic acid, acetic acid, ascorbic acid and citric acid. In embodiments, acidification may be achieved by the addition of glucono-lactone. In one embodiment, acidifying comprises lowering the pH to a pH of about 4.4 to about 3.4. In one embodiment, acidification comprises lowering the pH to a pH of 4.5, or 4.4, or 4.2, or 4, or 3.8, or 3.6, or 3.4 or below. In one embodiment, acidifying comprises lowering the pH to a pH of 4.4 or below.

Isothiocyanate-containing products from Brassicaceae

Isothiocyanate-containing products from the cruciferae family, as described herein, may be produced by methods as described herein. Those skilled in the art will appreciate that the isothiocyanate containing product produced using the methods described herein contains higher levels of isothiocyanate, e.g., sulforaphane, than cruciferous material or cruciferous material that has been subjected to fermentation alone (without pretreatment as described herein). For example, macerated broccoli from a commercial broccoli cultivar has a sulforaphane concentration of 800 μmol/Kg dw (-149.8 mg/Kg dw), fermented macerated broccoli has a sulforaphane concentration of 1600 μmol/Kg dw (-278.8 mg/Kg dw), and pretreated and fermented broccoli produced using the methods described herein has a sulforaphane concentration of 13100 μmol/Kg dw (-2318.7 mg/Kg d).

In one embodiment, the isothiocyanate containing product comprises at least about 4 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 6 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 8 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 10 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 12 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 14 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 16 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 17 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises from about 4 times to about 17 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises from about 4 to about 16 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises from about 8 times to about 16 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises from about 10 times to about 16 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises from about 12 times to about 16 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises from about 14 to about 16 times more isothiocyanate than impregnated cruciferous material. In one embodiment, the isothiocyanate is sulforaphane.

In one embodiment, the level of isothiocyanate present in the isothiocyanate containing product is greater than would be expected from the extractable glucosinolate content of cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 1-fold the expected maximum yield of isothiocyanate, based on extractable glucosinolate content. In one embodiment, the isothiocyanate containing product comprises at least about 2 times the expected maximum yield of isothiocyanate, based on extractable glucosinolate content. In one embodiment, the isothiocyanate containing product comprises at least about 3 times the expected maximum yield of isothiocyanate, based on extractable glucosinolate content. In one embodiment, the isothiocyanate containing product comprises at least about 3.8 times the expected maximum yield of isothiocyanate, based on extractable glucosinolate content. In one embodiment, the isothiocyanate containing product comprises at least about 4 times the expected maximum yield of isothiocyanate, based on extractable glucosinolate content. In one embodiment, the isothiocyanate containing product comprises from about 1-fold to about 4-fold the expected maximum yield of isothiocyanate, based on extractable glucosinolate content. In one embodiment, the isothiocyanate containing product comprises from about 1-fold to about 3.8-fold the expected maximum yield of isothiocyanate, based on extractable glucosinolate content. In one embodiment, the isothiocyanate containing product comprises from about 2-fold to about 3.8-fold the expected maximum yield of isothiocyanate, based on extractable glucosinolate content. In one embodiment, the isothiocyanate containing product comprises from about 2-fold to about 3-fold the expected maximum yield of isothiocyanate, based on extractable glucosinolate content.

In one embodiment, the level of sulforaphane present in the isothiocyanate containing product is greater than would be expected from the extractable glucoraphane content of cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 1-fold the expected maximum yield of sulforaphane, based on the extractable sulforaphane content. In one embodiment, the isothiocyanate containing product comprises at least about 2 times the expected maximum yield of sulforaphane, based on the extractable sulforaphane content. In one embodiment, the isothiocyanate containing product comprises at least about 3 times the expected maximum yield of sulforaphane, based on the extractable sulforaphane content. In one embodiment, the isothiocyanate containing product comprises at least about 3.8 times the expected maximum yield of sulforaphane, based on the extractable sulforaphane content. In one embodiment, the isothiocyanate containing product comprises at least about 4 times the expected maximum yield of sulforaphane, based on the extractable sulforaphane content. In one embodiment, the isothiocyanate containing product comprises from about 1-fold to about 4-fold the expected maximum yield of sulforaphane, based on the extractable sulforaphane content. In one embodiment, the isothiocyanate containing product comprises from about 1-fold to about 3.8-fold the expected maximum yield of sulforaphane, based on the extractable sulforaphane content. In one embodiment, the isothiocyanate containing product comprises from about 1-fold to about 3-fold the expected maximum yield of sulforaphane, based on the extractable sulforaphane content. In one embodiment, the isothiocyanate containing product comprises from about 2-fold to about 3-fold the expected maximum yield of sulforaphane, based on the extractable sulforaphane content.

In one embodiment, the isothiocyanate containing product comprises from about 100mg/kg dw to about 7000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises from about 500mg/kg dw to about 7000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises from about 1000mg/kg dw to about 7000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises from about 1600mg/kgdw to about 4000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises from about 1600mg/kgdw to about 3000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises from about 2000mg/kg dw to about 4000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises from about 2000mg/kg dw to about 7000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises from about 3000mg/kg dw isothiocyanate to about 7000mg/kg dw isothiocyanate. In one embodiment, the isothiocyanate containing product comprises about 2300mg/kg dw of isothiocyanate.

In one embodiment, the isothiocyanate containing product comprises at least about 100mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 200mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 250mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 300mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 350mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 400mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 450mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 500mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 550mg/kgdw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 600mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 650mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 700mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 1000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 2000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 3000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 4000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 5000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 6000mg/kg dw of isothiocyanate. In one embodiment, the isothiocyanate containing product comprises at least about 7000mg/kg dw of isothiocyanate.

In one embodiment, the isothiocyanate containing product comprises at least about 100mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 150mg/kg of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 200mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 250mg/kg of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 300mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 350mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 400mg/kgdw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 450mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 500mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 550mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 600mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 650mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 700mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 1000mg/kg of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 2000mg/kgdw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 3000mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 4000mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 5000mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 6000mg/kg dw of sulforaphane. In one embodiment, the isothiocyanate containing product comprises at least about 7000mg/kg dw of sulforaphane.

In one embodiment, the isothiocyanate containing product comprises at least about 5% more total fibers than cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 10% more total fibers than cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 15% more total fibers than cruciferous material. In one embodiment, the isothiocyanate containing product comprises at least about 20% more total fibers than cruciferous material. In one embodiment, the isothiocyanate containing product includes at least about 4% more protein than cruciferous material. In one embodiment, the isothiocyanate containing product includes at least about 6% more protein than cruciferous material. In one embodiment, the isothiocyanate containing product includes at least about 8% more protein than cruciferous material. In one embodiment, the isothiocyanate containing product includes at least about 10% more protein than cruciferous material.

In one embodiment, the isothiocyanate containing product includes at least about 10% less carbohydrate than the cruciferous material. In one embodiment, the isothiocyanate containing product includes at least about 20% less carbohydrate than the cruciferous material. In one embodiment, the isothiocyanate containing product includes at least about 30% less carbohydrate than the cruciferous material. In one embodiment, the isothiocyanate containing product includes at least about 40% less carbohydrate than the cruciferous material. In one embodiment, the isothiocyanate containing product includes at least about 45% less carbohydrate than the cruciferous material. In one embodiment, the isothiocyanate containing product includes at least about 48% less carbohydrate than the cruciferous material. In one embodiment, the isothiocyanate containing product includes from about 10% to about 48% less carbohydrate than cruciferous material.

In one embodiment, the isothiocyanate containing product includes increased levels of polyphenol glycosides as compared to cruciferous material. In one embodiment, the polyphenol glycoside is an anthocyanin glycoside. In one embodiment, the polyphenol glycoside is a phenolic acid glycoside. In one embodiment, the polyphenol glycoside is a phenolic acid.

In one embodiment, the isothiocyanate containing product includes increased levels of glucosinolates as compared to cruciferous material. In one embodiment, the glucosinolate is glucoraphanin. In one embodiment, glucoraphanin is increased by at least about 25-fold. In one embodiment, the glucosinolate is canola glucosinolate. In one embodiment, the canola glucosinolates are increased 26-fold. In one embodiment, the isothiocyanate containing product comprises indole-3-methanol. In one embodiment, indole-3-carbinol is increased by at least about 2-fold in the isothiocyanate containing product as compared to the impregnated cruciferous material. In one embodiment, indole-3-carbinol is increased in the isothiocyanate containing product by at least about 3-fold as compared to the impregnated cruciferous material. In one embodiment, the isothiocyanate containing product comprises procarbazine. In one embodiment, the increase in pro-ascorbate in the isothiocyanate containing product is at least about 2-fold as compared to the impregnated cruciferous material. In one embodiment, the increase in pro-ascorbate in the isothiocyanate containing product is at least about 3-fold as compared to the impregnated cruciferous material.

In one embodiment, the isothiocyanate containing product includes an increased level of one or more of the following, as compared to cruciferous material: ferulic acid, syringic acid, phenyllactic acid, chlorogenic acid rutin, sinapic acid, methyl syringate, hesperetin, quercetin and kaempferol. In one embodiment, the isothiocyanate containing product includes increased levels of chlorogenic acid as compared to cruciferous material. In one embodiment, chlorogenic acid is increased by about 6.6 fold. In one embodiment, the isothiocyanate containing product includes increased levels of sinapic acid as compared to cruciferous material. In one embodiment, sinapinic acid is increased by about 23.8 fold. In one embodiment, the isothiocyanate containing product includes increased levels of kaempferol as compared to cruciferous material. In one embodiment, the kaempferol is increased by a factor of about 10.5.

In one embodiment, the isothiocyanate containing product includes reduced levels, as compared to cruciferous material, of one or more of the following: protocatechuic acid, gallic acid, 4, hydroxybenzoic acid, vanillic acid, 2, 3-dihydroxybenzoic acid, p-coumaric acid, cinnamic acid, catechin, rosmarinic acid, and caffeic acid.

In one embodiment, about 40% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 50% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 60% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 70% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 80% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 90% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 95% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 97% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 98% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 99% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 100% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, from about 40% to about 100% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing product. In one embodiment, about 40% to about 80% of the glucosinolates present in the cruciferous material are converted to isothiocyanates in the isothiocyanate containing cruciferous product.

In one embodiment, the isothiocyanate in the isothiocyanate containing product is stable when stored at about 4 ℃ to about 25 ℃ for at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 6 weeks, or at least 8 weeks, or at least 10 weeks, or at least 12 weeks, or at least 14 weeks. In one embodiment, the isothiocyanate in the isothiocyanate containing product is stable for at least 4 weeks when stored at about 4 ℃ to about 25 ℃. In one embodiment, the isothiocyanate in the isothiocyanate containing product is stable for at least 8 weeks when stored at about 4 ℃ to about 25 ℃. In one embodiment, the isothiocyanate in the isothiocyanate containing product is stable for at least 12 weeks when stored at about 4 ℃ to about 25 ℃.

As used herein, "stable" means that the isothiocyanate concentration does not decrease or only slightly decreases when stored at 4 ℃ for six weeks. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 1% to about 30%. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 5% or less. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 10% or less. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 15% or less. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 20% or less. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 30% or less. Isothiocyanate analysis may be performed by any method known to those skilled in the art, such as sulforaphane as shown in example 1.

In one embodiment, the isothiocyanate is sulforaphane.

In one embodiment, the isothiocyanate containing product is resistant to yeast, mold, and/or coliform growth for at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 6 weeks, or at least 8 weeks, or at least 10 weeks, or at least 12 weeks, or at least 14 weeks when stored at about 4 ℃ to about 25 ℃.

In one embodiment, the isothiocyanate containing product is resistant to yeast, mold, and/or coliform growth for at least 4 weeks when stored at about 4 ℃ to about 25 ℃. In one embodiment, the isothiocyanate containing product is resistant to yeast, mold, and/or coliform growth for at least 8 weeks when stored at about 4 ℃ to about 25 ℃. In one embodiment, the isothiocyanate containing product is resistant to yeast, mold, and/or coliform growth for at least 12 weeks when stored at about 4 ℃ to about 25 ℃.

As used herein, "tolerant" of yeast, mold, and/or coliform growth means that < 1 LogCFU/g of yeast, mold, and/or coliform can be detected in a sample after the above-listed time periods using the methods described in example 1. In one embodiment, the isothiocyanate containing product comprises from about 20g/100gdw to about 32g/100gdw total fiber. In one embodiment, the isothiocyanate containing product comprises about 20g/100gdw total fiber. In one embodiment, the isothiocyanate containing product comprises about 25g/100gdw total fiber. In one embodiment, the isothiocyanate containing product comprises about 28g/100gdw total fiber. In one embodiment, the isothiocyanate containing product comprises about 29g/100gdw total fiber. In one embodiment, the isothiocyanate containing product comprises about 30g/100gdw total fiber. In one embodiment, the isothiocyanate containing product comprises about 32g/100gdw total fiber.

In one embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 14000. mu. mol TE/100gdw to about 19000. mu. mol TE/100 gdw. In one embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 14000. mu. mol TE/100 gdw. In one embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 15000. mu. mol TE/100 gdw. In one embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 16000. mu. mol TE/100 gdw. In one embodiment, the isothiocyanate containing product includes an ORAC antioxidant capacity of about 17000. mu. mol TE/100 gdw. In one embodiment, the isothiocyanate containing product includes an ORAC antioxidant capacity of about 18000. mu. mol TE/100 gdw. In one embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 18695. mu. mol TE/100 gdw. In one embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 19000. mu. mol TE/100 gdw.

In one embodiment, the isothiocyanate containing product comprises a total polyphenol content of from about 1750mg GAE/100gdw to about 2600mg GAE/100 gdw. In one embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 1750mg GAE per 100 gdw. In one embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2000mg GAE per 100 gdw. In one embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2100mg GAE per 100 gdw. In one embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2200mg GAE per 100 gdw. In one embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2300mg GAE per 100 gdw. In one embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2360mg GAE per 100 gdw.

In one embodiment, the isothiocyanate containing product comprises total titratable acidity in the range of about 0.9% to about 1.1% lactic acid equivalents. In one embodiment, the isothiocyanate containing product includes total titratable acidity of about 1.1% lactic acid equivalents.

In one embodiment, the isothiocyanate containing product comprises a total protein content of about 23g/100gdw to about 39g/100 gdw. In one embodiment, the isothiocyanate containing product comprises a total protein content of about 23g/100gdw to about 30g/100 gdw. In one embodiment, the isothiocyanate containing product comprises a total protein content of about 25g/100 gdw. In one embodiment, the isothiocyanate containing product comprises a total protein content of about 27g/100 gdw. In one embodiment, the isothiocyanate containing product comprises a total protein content of about 28g/100 gdw. In one embodiment, the isothiocyanate containing product comprises a total protein content of about 29g/100 gdw. In one embodiment, the isothiocyanate containing product comprises a total protein content of about 30g/100 gdw. In one embodiment, the isothiocyanate containing product comprises a total protein content of about 32g/100 gdw.

In one embodiment, the isothiocyanate containing product comprises at least about 100mg/kg dw of isothiocyanate and one or more or all of the following.

i) About 29 to about 36g/100gdw total fiber;

ii) an ORAC antioxidant capacity of about 15000 to about 18695. mu. mol TE/100 gdw;

iii) a total polyphenol content of about 2310 to about 2600mg GAE/100 gdw;

iv) total titratable acidity of about 0.9 to about 1.1% lactic acid equivalents;

v) a total protein content of about 27 to about 39g/100 gdw; and

vi) Leuconostoc mesenteroides and/or Lactobacillus plantarum.

In one embodiment, the isothiocyanate containing product is produced from broccoli.

The cruciferous products described herein may include active lactic acid bacteria that can aid in the conversion of glucosinolates present in the isothiocyanate containing product to isothiocyanates (i.e., they act as probiotics) during digestion of the glucosinolate containing product in a subject. In one embodiment, the lactic acid bacterium is leuconostoc mesenteroides. In one embodiment, the lactic acid bacterium is lactobacillus. In one embodiment, the lactic acid bacterium is lactobacillus plantarum.

In one embodiment, the isothiocyanate containing product comprises a concentration of at least about 10²CFU/g lactic acid bacteria. In one embodiment, the isothiocyanate containing product comprises a concentration of at least about 10²CFU/g lactic acid bacteria. In one embodiment, the isothiocyanate containing product comprises a concentration of at least about 10⁵CFU/g lactic acid bacteria. In one embodiment, the isothiocyanate containing product comprises a concentration of at least about 10⁶CFU/g lactic acid bacteria. In one embodiment, the isothiocyanate containing product comprises a concentration of at least about 10⁷CFU/g lactic acid bacteria. In one embodiment, the isothiocyanate containing product comprises a concentration of at least about 10⁸CFU/g lactic acid bacteria. In one embodiment, the isothiocyanate containing product comprises a concentration of at least about 10⁹CFU/g lactic acid bacteria.

In one embodiment, the isothiocyanate containing product is present in the active lactic acid bacteria for at least 10 days when stored at about 4 ℃ to about 25 ℃. In one embodiment, the active lactic acid bacteria are present in the isothiocyanate containing product for at least 20 days when stored at about 4 ℃ to about 25 ℃. In one embodiment, the active lactic acid bacteria are present in the isothiocyanate containing product for at least 30 days when stored at about 4 ℃ to about 25 ℃. In one embodiment, the active lactic acid bacteria are present in the isothiocyanate containing product for at least 40 days when stored at about 4 ℃ to about 25 ℃. In one embodiment, the active lactic acid bacteria are present in the isothiocyanate containing product for at least 50 days when stored at about 4 ℃ to about 25 ℃. In one embodiment, the active lactic acid bacteria are present in the isothiocyanate containing product for at least 60 days when stored at about 4 ℃ to about 25 ℃. In one embodiment, the active lactic acid bacteria are present in the isothiocyanate containing product for at least 70 days when stored at about 4 ℃ to about 25 ℃. In one embodiment, the active lactic acid bacteria are present in the isothiocyanate containing product for at least 80 days when stored at about 4 ℃ to about 25 ℃. In one embodiment, the active lactic acid bacteria are present in the isothiocyanate containing product for at least 85 days when stored at about 4 ℃ to about 25 ℃. In one embodiment, the active lactic acid bacteria are present in the isothiocyanate containing product for at least 90 days when stored at about 4 ℃ to about 25 ℃.

In one embodiment, the lactic acid bacterium is lactobacillus. In one embodiment, the lactic acid bacterium is lactobacillus plantarum. In one embodiment, the lactic acid bacterium is leuconostoc mesenteroides. In one embodiment, the bacteria are present in an amount of at least about 10⁷The concentration of CFU/g is present.

In one embodiment, the isothiocyanate containing product comprises one or more bacteriocins produced by the lactic acid bacteria. In one embodiment, the bacteriocin is a class I bacteriocin. In one embodiment, the bacteriocin is a class II bacteriocin. In one embodiment, the bacteriocin is a class III bacteriocin. Examples of bacteriocins produced by lactic acid bacteria can be found in Alvarez-Sieiro et al (2016).

In one embodiment, the isothiocyanate containing product is a food product. In one embodiment, the isothiocyanate containing product is a nutraceutical. In one embodiment, the isothiocyanate containing product is a supplement. In one embodiment, the isothiocyanate containing product is a food ingredient. In one embodiment, the isothiocyanate containing product is a probiotic. In one embodiment, the isothiocyanate containing product is an animal feed. The animal may be an aquatic animal, such as fish or livestock. In one embodiment, the isothiocyanate containing product is a pesticide. In one embodiment, the isothiocyanate containing product is a cosmeceutical. In one embodiment, the isothiocyanate containing product is formulated topically.

In one embodiment, the isothiocyanate containing product is a solid, liquid, paste, or powder. In one embodiment, the isothiocyanate containing product is dried to a powder after fermentation. In one embodiment, the isothiocyanate containing product is freeze-dried after fermentation. In one embodiment, the isothiocyanate containing product is microencapsulated after fermentation as described in WO 2005030229. In one embodiment, the isothiocyanate containing product is formulated as a pill.

Post-treatment

In one embodiment, after fermentation or acidification, the isothiocyanate-containing product may be post-treated to inactivate microorganisms that, for example, contribute to product degradation or are pathogenic if consumed.

As used herein, "post-treatment" or "post-treating" refers to treating the isothiocyanate containing product described herein after fermentation to inactivate microorganisms. As used herein, "microorganism" refers to a bacterial, viral, fungal, or eukaryotic activity that can lead to degradation or spoilage of isothiocyanate-containing products. As used herein, "inactivation" or "inactivation" of a microorganism means a reduction of about 1-7 logs of viable microorganisms. In one embodiment, viable microorganisms are reduced by about 1-6 log. In one embodiment, viable microorganisms are reduced by about 2-6 log. In one embodiment, viable microorganisms are reduced by about 3-6 log.

One skilled in the art will appreciate that the post-treatment may be any method of inactivating microorganisms including, for example, heat treatment, UV treatment, sonication, pulsed electric field treatment, or high pressure treatment. In one embodiment, the isothiocyanate containing product is post-treated with heat treatment. In one embodiment, the isothiocyanate containing product is post-treated with high pressure treatment. In one embodiment, the isothiocyanate containing product is in a sealed package during post-processing. In one embodiment, the isothiocyanate containing product is in a sealed package during the high pressure treatment. In one embodiment, the isothiocyanate containing product is in a sealed package during the heat treating. In one embodiment, the high pressure treatment comprises treating the isothiocyanate containing product with isostatic pressure at about 300 to about 600 MPa. In one embodiment, the high pressure treatment comprises treating the isothiocyanate containing product with isostatic pressure at about 350 to about 550 MPa. In one embodiment, the high pressure treatment comprises treating the isothiocyanate containing product with isostatic pressure at about 300 to about 400 MPa. In one embodiment, the heat treatment comprises heating the sample to a temperature of about 60 ℃ to about 121 ℃. In one embodiment, the heat treatment comprises heating the sample to a temperature of about 65 ℃ to about 100 ℃. In one embodiment, the heat treatment comprises heating the sample to a temperature of about 65 ℃ to about 80 ℃. In one embodiment, the heat treatment comprises heating the sample to a temperature of about 65 ℃ to about 75 ℃.

Isolated strains and starter cultures

In one embodiment, the present invention provides isolated strains of lactic acid bacteria suitable for use in the methods and products described herein.

In one embodiment, the present invention provides an isolated lactic acid bacterial strain selected from the group consisting of:

i) BF1, deposited as V17/021729 at 9/25.2017 at the national institute of Mediterranean research, Australia;

ii) BF2 deposited as V17/021730 at 9, 25.2017 at the national Mediterranean Kangh, Australia;

iii) B1, deposited at the national institute for metrological hyperthyroidism in australia as V17/021731, 25/9/2017;

iv) B2 deposited as V17/021732 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017;

v) B3 deposited at the national institute for metrological and hyperthyroidism in australia as V17/021733 on 25/9/2017;

vi) B4, deposited at the national institute for metrological hyperthyroidism in australia as V17/021734 on 25/9/2017; and

vii)2017, 9, 25, B5 at the national institute for metrological hyperthyroidism in australia as V17/021735.

In one embodiment, the invention provides an isolated leuconostoc mesenteroides strain comprising genomic DNA which when cleaved by SmaI and/or NotI produces the same SmaI and/or NotI fingerprint as BF1 or BF 2. SmaI and NotI fingerprints for BF1 and BF2 are shown in fig. 13.

In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising genomic DNA that, when cleaved by SmaI and/or NotI, produces the same SmaI and/or NotI fingerprint as B1, B2, B3, B4 or B5.

In one embodiment, the invention provides an isolated leuconostoc mesenteroides strain comprising one or more or all of the polymorphisms listed in tables 18 or 19 other than ATCC 8293. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 5 or more polymorphisms other than ATCC8293 listed in table 18 or 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 10 or more polymorphisms other than ATCC8293 listed in table 18 or 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 15 or more polymorphisms other than ATCC8293 listed in table 18 or 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 19 or more polymorphisms other than ATCC8293 listed in table 18 or 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 20 or more polymorphisms other than ATCC8293 listed in table 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 30 or more polymorphisms other than ATCC8293 listed in table 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 50 or more polymorphisms other than ATCC8293 listed in table 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 80 or more polymorphisms other than ATCC8293 listed in table 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 100 or more polymorphisms listed in table 19 other than ATCC 8293. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 150 or more polymorphisms other than ATCC8293 listed in table 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 200 or more polymorphisms other than ATCC8293 listed in table 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 300 or more polymorphisms other than ATCC8293 listed in table 19. In one embodiment, the isolated leuconostoc mesenteroides strain comprises 400 or more polymorphisms other than ATCC8293 listed in table 19.

In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising one or more or all of the polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014. In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising 5 or more polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014. In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising 10 or more polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014. In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising 15 or more polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014. In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising 20 or more polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014. In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising 25 or more polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014. In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising 30 or more polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014. In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising 35 or more polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014. In one embodiment, the invention provides an isolated lactobacillus plantarum strain comprising 40 or more polymorphisms listed in table 13, table 14, table 15, table 16 or table 17, other than ATCC 8014.

In one embodiment, the invention provides a starter culture for the production of an isothiocyanate containing product or a probiotic comprising a lactic acid bacterium comprising one or more of the isolated strains described herein. As used herein, a "starter culture" is a culture of a living microorganism used for fermentation. In one embodiment, the invention provides a starter culture for the production of an isothiocyanate containing product or a probiotic comprising a lactic acid bacterium selected from one or more or all of the following:

i) BF1, deposited as V17/021729 at 9/25.2017 at the national institute of Mediterranean research, Australia;

ii) BF2 deposited as V17/021730 at 9, 25.2017 at the national Mediterranean Kangh, Australia;

iii) B1, deposited at the national institute for metrological hyperthyroidism in australia as V17/021731, 25/9/2017;

iv) B2 deposited as V17/021732 at the national institute of metrological and hyperthyroidism in australia on 25/9/2017;

v) B3 deposited at the national institute for metrological and hyperthyroidism in australia as V17/021733 on 25/9/2017;

vi) B4, deposited at the national institute for metrological hyperthyroidism in australia as V17/021734 on 25/9/2017; and

vii)2017, 9, 25, B5 at the national institute for metrological hyperthyroidism in australia as V17/021735.

In one embodiment, at least about 10 is used⁵CFU/g of the starter culture described herein is inoculated with cruciferous material. In one embodiment, at least about 10 is used⁶CFU/g of the starter culture described herein is inoculated with cruciferous material. In one embodiment, at least about 10 is used⁷CFU/g of the starter culture described herein is inoculated with cruciferous material. At one endIn one embodiment, at least about 10 is used⁸CFU/g of the starter culture described herein is inoculated with cruciferous material. In one embodiment, at least about 10 is used¹⁰CFU/g of the starter culture described herein is inoculated with cruciferous material. In one embodiment, about 10 is used⁵CFU/g to about 10¹⁰CFU/g of the starter culture described herein is inoculated with cruciferous material.

Probiotics

In one embodiment, the present invention provides probiotics comprising one or more lactic acid bacteria isolated from the brassicaceae family. As used herein, "probiotic" refers to live microorganisms that, when administered in sufficient amounts, confer a health benefit to the host. In one embodiment, the lactic acid bacteria are isolated from cabbage. In one embodiment, the lactic acid bacteria are isolated from broccoli. In one embodiment, the lactic acid bacteria are isolated from broccoli australia. In one embodiment, the lactic acid bacteria are selected from: i) leuconostoc mesenteroides; ii) Lactobacillus plantarum; iii) Lactobacillus pentosus; iv) lactobacillus rhamnosus; v) a combination of i) and ii); vi) a combination of i), ii), and iii); and vii) combinations of i), ii) and iv). In one embodiment, the lactic acid bacteria are selected from one or more or all of BF1, BF2, B1, B2, B3, B4 and B5. In one embodiment, the lactic acid bacterium is B1. In one embodiment, the lactic acid bacterium is B2. In one embodiment, the lactic acid bacterium is B3. In one embodiment, the lactic acid bacterium is B4. In one embodiment, the lactic acid bacterium is B5. In one embodiment, the probiotic is a capsule, tablet, powder or liquid. In one embodiment, the probiotic bacteria are microencapsulated as described in WO 2005030229.