WO2019060963A1 - ISOTHIOCYANATE-CONTAINING BRASSICACEAE PRODUCTS AND PROCESS FOR PREPARING THE SAME - Google Patents

ISOTHIOCYANATE-CONTAINING BRASSICACEAE PRODUCTS AND PROCESS FOR PREPARING THE SAME Download PDF

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Publication number
WO2019060963A1
WO2019060963A1 PCT/AU2018/051063 AU2018051063W WO2019060963A1 WO 2019060963 A1 WO2019060963 A1 WO 2019060963A1 AU 2018051063 W AU2018051063 W AU 2018051063W WO 2019060963 A1 WO2019060963 A1 WO 2019060963A1
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WO
WIPO (PCT)
Prior art keywords
isothiocyanate
brassicaceae
containing product
september
deposited under
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/AU2018/051063
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English (en)
French (fr)
Inventor
Mary Ann Augustin
Netsanet SHIFERAW TEREFE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commonwealth Scientific and Industrial Research Organization CSIRO
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Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from AU2017903944A external-priority patent/AU2017903944A0/en
Priority to GB2005625.5A priority Critical patent/GB2583586B/en
Priority to JP2020517996A priority patent/JP2020534850A/ja
Priority to KR1020257011503A priority patent/KR20250051163A/ko
Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Priority to CA3077235A priority patent/CA3077235A1/en
Priority to US16/651,974 priority patent/US12227780B2/en
Priority to KR1020207011661A priority patent/KR20200074953A/ko
Priority to AU2018340868A priority patent/AU2018340868B2/en
Priority to SG11202002850SA priority patent/SG11202002850SA/en
Priority to IL273661A priority patent/IL273661B2/en
Priority to MYPI2020001627A priority patent/MY200000A/en
Priority to EP18861205.5A priority patent/EP3688179A4/en
Priority to CN201880074464.3A priority patent/CN111601896A/zh
Publication of WO2019060963A1 publication Critical patent/WO2019060963A1/en
Priority to PH12020550169A priority patent/PH12020550169A1/en
Anticipated expiration legal-status Critical
Priority to JP2023127796A priority patent/JP2023155265A/ja
Priority to US18/990,423 priority patent/US20250197898A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
    • A23B7/10—Preserving with acids; Acid fermentation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
    • A23B7/10—Preserving with acids; Acid fermentation
    • A23B7/105—Leaf vegetables, e.g. sauerkraut
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01147—Thioglucosidase (3.2.1.147), i.e. myrosinase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/01—Instant products; Powders; Flakes; Granules
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/31—Leuconostoc
    • A23V2400/321—Mesenteroides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • the present invention relates to methods for producing isothiocyanate containing products from Brassicaceae material and lactic acid bacteria for use in such methods.
  • the present invention also relates to isothiocyanate containing products from Brassicaceae material produced by such methods.
  • Brassicaceae are often processed to increase the shelf life which can result in the loss of nutrients.
  • the main methods to obtain a longer shelf life include thermal processing, freezing, modified and controlled atmosphere storage and the addition of chemical preservatives which also would bring undesirable changes in chemical composition.
  • the present inventors have developed methods for preparing isothiocyanate containing products from Brassicaceae material.
  • the present invention provides a method of preparing an isothiocyanate containing product from Brassicaceae material comprising:
  • step ii) fermenting the material obtained by step i) with lactic acid bacteria to form the isothiocyanate containing product.
  • pre-treating comprises one or more of the following:
  • pre-treating reduces epithiospecifier protein (ESP) activity while maintaining endogenous myrosinase activity.
  • ESP epithiospecifier protein
  • pre-treating comprises heating and macerating the Brassicaceae material and wherein the temperature of the Brassicaceae material does not exceed about 75 °C during pre-treating. In an embodiment, heating occurs before macerating or wherein heating and macerating occur at the same time. In an embodiment, pre-treating comprises heating the Brassicaceae material to a temperature of about 50°C to about 70°C followed by maceration. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 2 mm or less. In an embodiment, the Brassicaceae material is heated in a sealed package.
  • the isothiocyanate containing product comprises at least about 10 times more isothiocyanate than macerated Brassicaceae material.
  • the isothiocyanate containing product comprises at least about 12 times more isothiocyanate than macerated Brassicaceae material.
  • the isothiocyanate containing product comprises at least about 14 times more isothiocyanate than macerated Brassicaceae material.
  • the isothiocyanate containing product comprises at least about 16 times more isothiocyanate than macerated Brassicaceae material.
  • the isothiocyanate containing product comprises at least about 2 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.
  • lactic acid bacteria was isolated from a broccoli and/or the lactic acid bacteria lacks myrosinase activity.
  • the present invention provides a method of preparing a isothiocyanate containing product from Brassicaceae material comprising:
  • step ii) acidifying the material obtained by step i) forming the isothiocyanate containing product.
  • the present invention provides a method of preparing an isothiocyanate containing product from broccoli material comprising fermenting the material with lactic acid bacteria Leuconostoc mesenteroides and/or Lactobacillus plantarum to form the isothiocyanate containing product, wherein the method optionally comprises pre-treating the broccoli material to improve the access of myrosinase to a glucosinolate.
  • the present invention provides a method of preparing an isothiocyanate containing product from a Brassicaceae material comprising fermenting the material with lactic acid bacteria Leuconostoc mesenteroides and/or Lactobacillus plantarum isolated from broccoli to form the isothiocyanate containing product, wherein the method optionally comprises pre-treating the Brassicaceae material to improve the access of myrosinase to a glucosinolate.
  • the present invention provides an isolated strain of lactic acid bacteria selected from:
  • the present invention provides a starter culture for producing an isothiocyanate containing product or a probiotic comprising lactic acid bacteria selected from one or more or all of:
  • the present invention provides a probiotic composition
  • a probiotic composition comprising lactic acid bacteria selected from one or more or all of:
  • the present invention provides an isothiocyanate containing product obtained by the method as described herein.
  • the present invention provides an isothiocyanate containing product obtainable by the method as described herein.
  • the present invention provides an isothiocyanate containing Brassicaceae product comprising at least about 10 times more isothiocyanate than the macerated Brassicaceae material.
  • the present invention provides an isothiocyanate containing Brassicaceae product comprising about 10 times to about 16 times more isothiocyanate than the macerated Brassicaceae material.
  • the present invention provides an isothiocyanate containing Brassicaceae product comprising at least about 2 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.
  • the present invention provides an isothiocyanate containing
  • Brassicaceae product comprising about 2 times to about 4 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.
  • the present invention provides an isothiocyanate containing Brassicaceae product comprising at least 150 mg/kg dw of isothiocyanate.
  • the present invention provides an isothiocyanate containing product comprises at least 150 mg/kg dw, at least 200 mg/kg dw, at least 300 mg/kg dw, at least 400 mg/kg dw, or at least 450 mg/kg dw, or at least 500 mg/kg dw, or at least 550 mg/kg dw, or at least 600 mg/kg dw, or at least 650 mg/kg dw, or at least 700 mg/kg dw, or at least 1000 mg/kg dw, or at least 2000 mg/kg dw, or at least 3000 mg/kg dw, or at least 4000 mg/kg dw, or at least 5000 mg/kg dw, or at least 6000 mg/kg dw, or at least 7000 mg/kg dw sulforaphane.
  • the isothiocyanate containing product comprises Leuconostoc mesenteroides and/or Lactobacillus plantarum.
  • the isothiocyanate containing product has one or more or all of the following features: i) is stable for at least 4 weeks, or for at least 8 weeks, or for at least 12 weeks when stored at about 4°C to about 25°C;
  • ii) is resistant to yeast, mould and/or coliform growth for at least 4 weeks, or for at least 8 weeks, or for at least 12 weeks when stored at about 4°C to about 25°C; and iii) comprises at least 10 7 CFU/g Leuconostoc mes enter oides and/or
  • Lactobacillus plantarum Lactobacillus plantarum.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • Figure 1 Shows the pathways of hydrolysis of glucoraphanin to sulforaphane and sulforaphane nitrile. B) Shows the effects of maceration and fermentation on sulforaphane content (mg/kg, DW) in broccoli puree. C) Shows the effect of fermentation on lactic acid bacteria count (log CFU/gm) of broccoli puree during storage. Figure 2. A) Shows the effects of fermentation on the stability of sulforaphane in broccoli puree stored at 4°C and 25°C (RT). B) Effects of heat treatment condition on the conversion of glucoraphanin into sulforaphane in broccoli matrix.
  • Figure 3 Shows the total phenolic content (mg GAE/100 g DW) of raw broccoli and its changes during fermentation and storage at 25°C and 4°C, respectively.
  • Figure 4 Shows the fermentation time taken to reach a pH of 4.4 or lower for different combinations of lactic acid bacteria strains.
  • Figure 5 Shows sulforaphane yield ( ⁇ /kg DW) under different heat treatment conditions of broccoli with a sealed bag.
  • Figure 7 Shows the effect of fermentation and storage on glucoraphanin content. Glucoraphanin content is reduced in fermented samples stored at 25°C and 4°C compared to raw samples.
  • Figure 8 PLS-DA score plot showing the difference in polyphenolic metabolite profile of raw and fermented broccoli puree.
  • Figure 10 Shows the effect of lactic acid fermentation on metabolite profile of broccoli puree- based on untargeted LC-MS analysis. It demonstrates that fermentation releases bound phytochemicals such as polyphenolic glycosides and glucosinolates and enhances their bioaccessibility.
  • Figure 11 Shows a volcano plot indicating metabolites with significant (p ⁇ 0.05) fold changes after fermentation based on untargeted LC-MS analysis. The top 50 metabolites with significant fold changes and their individual fold changes are recited in Table 8.
  • Figure 12. Shows the effect of lactic acid fermentation on broccoli polyphenols based on targeted LC-MS analysis. A 6.6 fold change is observed in chlorogenic acid (2.4 to 15.8 ⁇ g/mg), a 23.8 fold increase is observed in sinapic acid (3.6 to 86.6 ⁇ ig/mg), a 10.5 increase in kaempferol (12.7 to 134.6 ⁇ g mg) and a 0.48 fold decrease is observed in p- coumaric acid.
  • an “allele” refers to one specific form of a genetic sequence (such as a gene) within a cell, an individual plant or within a population, the specific form differing from other forms of the same gene in the sequence of at least one, and frequently more than one, variant sites within the sequence of the gene.
  • the sequences at these variant sites that differ between different alleles are termed "variances", “polymorphisms”, or “mutations”.
  • Brassicaceae refers to members of the Family Brassicaceae commonly referred to as mustards, cruicifers or the cabbage family.
  • material can be from more than one Brassicaceae.
  • the Brassicaceae is selected from the genus Brassica or Cardamine.
  • the Brassica is selected from Brassica balearica, Brassica carinata, Brassica elongate, Brassica fruticulosa, Brassica hilarionis, Brassica juncea, Brassica napus, Brassica narinosa, Brassica nigra, Brassica oleracea, Brassica perviridis, Brassica rapa, Brassica rupestris, Brassica septiceps, and Brassica tournamentfortii.
  • the Brassica is Brassica oleracea.
  • the Brassica is selected from Brassica oleracea variety oleracea (wild cabbage), Brassica oleracea variety capitate (cabbage), Brassica rapa subsp. chinensis (bok choy), Brassica rapa subsp. pekinensis (napa cabbage), Brassica napobrassica (rutabaga), Brassica rapa var.
  • rapa (turnip), Brassica oleracea variety alboglabra (kai-lan), Brassica oleracea variety viridis (collard greens), Brassica oleracea variety longata Oersey cabbage), Brassica oleracea variety acephala (ornamental kale), Brassica oleracea variety sabellica (kale), Brassica oleracea variety palmifolia (lacinato kale), Brassica oleracea variety ramose (perpetual kale), Brassica oleracea variety medullosa (marrow cabbage), Brassica oleracea variety costata (tronchuda kale), Brassica oleracea variety gemmifera (brussels sprout), Brassica oleracea variety gongylodes (kohlrabi), Brassica oleracea variety italica (broccoli), Brassica oleracea variety botrytis (cauliflower, Romanesco broccoli,
  • the Brassica is Brassica oleracea, variety italica (broccoli). In an embodiment, the Brassicaceae is selected from Cardamine hirsuta
  • the Brassicaceae has a high level of one or more glucosinolate/s. In an embodiment, the Brassicaceae has been selectively bred to have a high level of one or more glucosinolate/s. In an embodiment, "high level" of a glucosinolate can comprise a higher level of a glucosinolate than shown in Table 2 of Verkerk et al. (2009) in the corresponding Brassicaceae. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 3400 ⁇ /kg dry weight.
  • a high level of glucosinolate is a level of glucosinolate higher than 4000 ⁇ /kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 5000 ⁇ /kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 8000 ⁇ /kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 10,000 ⁇ /kg dry weight.
  • a high level of glucosinolate is a level of glucosinolate higher than 12,000 ⁇ /kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 15,000 ⁇ /kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 18,000 ⁇ /kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 20,000 ⁇ /kg dry weight.
  • a high level of glucosinolate is a level of glucosinolate higher than 25,000 ⁇ /kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 30,000 ⁇ /kg dry weight.
  • the Brassicaceae has been genetically modified or subjected to biotic or abiotic stress to have a high level of one or more glucosinolate/s. A person skilled in the art will appreciate that the Brassicaceae can be modified by any method known to a person skilled in the art.
  • the glucosinolate is glucoraphanin (4-Methylsulphinylbutyl). In an embodiment, the glucosinolate is glucobrassicin (3-Indolylmethyl).
  • Brassicaceae material refers to any part of the Brassicaceae which comprises a glucosinolate, including, but not limited to, the leaves, stems, flowers, florets, seeds, and roots or mixtures thereof.
  • Brassicaceae material for example, but not limited to, at least 30 kg, or at least 50 kg, or at least 80 kg, or at least 100 kg, or at least 1,000 kg, or at least 2,000 kg, or at least 5,000 kg, or at least 8,000 kg, or at least 10,000 kg, or at least 15,000 kg, or at least 20,000 kg.
  • the Brassicaceae material has been washed. As used herein “washing” removes visible soil and contamination. In an embodiment, the Brassicaceae material has been sanitized. As used herein “sanitized” refers to a reduction of pathogens on the Brassicaceae material. In an embodiment, the Brassicaceae is mixed with other plant material. In an embodiment, the other plant material is vegetable or fruit material. In an embodiment, the vegetable is a carrot or beetroot. Glucosinolates
  • glucosinolate refers to a secondary metabolite found at least in the Brassicaceae family that share a chemical structure consisting of a ⁇ -D- glucopyranose residue linked via a sulfur atom to a (Z)-N-hydroximinosulfate ester, plus a variable R group derived from an amino acid as described in Halkier et al. (2006). Examples of glucosinolates are provided in Halkier et al. (2006) and Agerbirk et al. (2012).
  • glucosinolate The hydrolysis of glucosinolate can produce isothiocyanates, nitriles, epithionitrile, thiocyanate and oxazolidine-2-thione ( Figure 1A). Many glucosinolates play a role in plant defence mechanisms against pests and disease.
  • Glucosinolates are stored in Brassicaceae in storage sites.
  • a "storage site” is a site within the Brassicaceae where glucosinolates are present and myrosinase is not present.
  • myrosinase also referred to as “thioglucosidase”, “sinigrase”, or “sinigrinase” refers to a family of enzymes (EC 3.2.1.147) involved in plant defence mechanisms that can cleave thio-linked glucose.
  • Myrosinases catalyze the hydrolysis of glucosinolates resulting in the production of isothiocyanates.
  • Myrosinase is stored sometimes as myrosin grains in the vacuoles of particular idioblasts called myrosin cells, but have also been reported in protein bodies or vacuoles, and as cytosolic enzymes that tend to bind to membranes.
  • myrosinase is stored in a myrosin cell in Brassicaceae.
  • pre-treating as described herein improves the access of myrosinase to a glucosinolate.
  • improved the access or “access is improved” refers to increasing the availability of glucosinolate to the myrosinase enzyme allowing for the production of an isothiocyanate.
  • access is improved by the release of a glucosinolate from a glucosinolate storage site.
  • the glucosinolate storage site is mechanically ruptured (i.e. by maceration) or enzymatically degraded.
  • glucosinolate is released from a glucosinolate storage site by the activity of one or more polysaccharide degrading enzymes e.g. a cellulase, hemicellulase, pectinase and/or glycosidase.
  • access is improved by allowing the entry of myrosinase into a glucosinolate storage site.
  • access is improved by the release of myrosinase from myrosin cells.
  • about 10% to about 90% of a glucosinolate is released from a glucosinolate storage site.
  • a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 30% to about 70% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 40% to about 60% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 45% to about 55% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 10% of a glucosinolate is released from a glucosinolate storage site.
  • about 20% of a glucosinolate is released from a glucosinolate storage site.
  • about 30% of a glucosinolate is released from a glucosinolate storage site.
  • about 40% of a glucosinolate is released from a glucosinolate storage site.
  • about 50% of a glucosinolate is released from a glucosinolate storage site.
  • about 60% of a glucosinolate is released from a glucosinolate storage site.
  • about 70% of a glucosinolate is released from a glucosinolate storage site.
  • a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 90% of a glucosinolate is released from a glucosinolate storage site.
  • the Brassicaceae material comprises one or more glucosinolate/s selected from an aliphatic, indole or aromatic glucosinolate.
  • the aliphatic glucosinolate is selected from one or more of glucoraphanin (4-Methylsulphinylbutyl or glucorafanin), sinigrin (2-Propenyl), gluconapin (3-Butenyl), glucobrassicanapin (4-Pentenyl), progoitrin (2(R)-2-Hydroxy- 3-butenyl, epiprogoitrin (2(S)-2-Hydroxy-3-butenyl), gluconapoleiferin (2-Hydroxy-4- pentenyl), glucoibervirin (3-Methylthiopropyl, glucoerucin (4-Methylthiobutyl), dehydroerucin (4-Methylthio-3-butenyl, glucoiberin (3-Methylsulphinylpropyl), glucoraphenin (4-Methylsulphinyl)
  • Methylsulphinylpentenyl Methylsulphinylpentenyl
  • glucoerysolin 3-Methylsulphonylbutyl, 4- Mercaptobutyl
  • the indole glucosinolate is selected from one or more of glucobrassicin (3-Indolylmethyl), 4-hydroxyglucobrassicin (4-Hydroxy-3- indolylmethyl), 4-methoxyglucobrassicin (4-Methoxy-3-indolylmethyl), and neoglucobrassicin (1 -Methoxy-3-indolylmethyl).
  • the indole glucosinolate is selected from one or more of Glucotropaeolin (Benzyl) and Gluconasturtiin (2-Phenylethyl).
  • the Brassicaceae material comprises one or more glucosinolate/s selected from benzylglucosinolate, allylglucosinolate and 4- methylsulfmylbutyl.
  • the glucosinolate is glucoraphanin (4- Methylsulphinylbutyl).
  • the glucosinolate is glucobrassicin (3- Indolylmethyl).
  • pre-treating as described herein increases the extractable glucosinolate content compared to the extractable glucosinolate content of the Brassicaceae material before pre-treatment.
  • extractable glucosinolate content refers to the level of glucosinolate accessible in the Brassicaceae material for conversion to isothiocyanate. Excluding conversion into nitriles and other compounds the expected maximum yield of isothiocyanate from 1 mole of glucosinolate is 1 mole of isothiocyanate (1 mole of glucosinolate can maximally be converted to 1 mole of isothiocyanate, 1 mole of glucose and 1 mole of sulphate ion).
  • the extractable glucoraphanin content of a commercial broccoli cultivar is 3400 ⁇ glucoraphanin/kg dw and the expected maximum yield of sulforaphane from the commercial broccoli cultivar is 3400 ⁇ sulforaphane /kg dw.
  • the isothiocyanate is sulforaphane (l-isothiocyanato-4-methylsulfinylbutane).
  • the isothiocyanate is allyl isothiocyanate (3-isothiocyanato-l-propene).
  • the isothiocyanate is benzyl isothiocyanate.
  • the isothiocyanate is phenethyl isothiocyanate.
  • the isothiocyanate is 3-Butenyl isothiocyanate.
  • the isothiocyanate is 5-vinyl-l,3-oxazolidine-2- thione.
  • the isothiocyanate is 3-(methylthio)propyl isothiocyanate.
  • the isothiocyanate is 3-(methylsulfinyl)-propyl isothiocyanate.
  • the isothiocyanate is 4-(methylthio)-butyl isothiocyanate.
  • the isothiocyanate is l-methoxyindol-3-carbinol isothiocyanate.
  • the isothiocyanate is 2-phenylethyl isothiocyanate.
  • the isothiocyanate is iberin.
  • the isothiocyanate containing product further comprises one or more isothiocyanate bioactive derivative/s or oligomers thereof.
  • the isothiocyanate bioactive derivative is a derivative of any of the isothiocyanates as described herein.
  • the isothiocyanate bioactive derivative is a derivative of sulforaphane.
  • the isothiocyanate bioactive derivative is iberin.
  • the isothiocyanate bioactive derivative is allyl isothiocyanate.
  • the isothiocyanate bioactive derivative is indole-3- caribinol.
  • the isothiocyanate bioactive derivative is methoxy-indole- 3-carbinol. In an embodiment, the isothiocyanate bioactive derivative is ascorbigen. In an embodiment, the isothiocyanate bioactive derivative is neoascorbigen.
  • pre-treatment releases or aids in the release of a glucosinolate from glucosinolate storage site and/or allows myrosinase to enter a glucosinolate storage site in the Brassicaceae material.
  • pre-treating increases the exposure of a glucosinolate to myrosinase allowing myrosinase to convert a glucosinolate to an isothiocyanate.
  • pre-treating reduces epithio specifier protein (ESP) while maintaining endogenous myrosinase activity.
  • epithiospecifier protein or "ESP” refers to a protein that directs myrosinase activity towards the production of nitriles and away from isothiocyanate production. Reducing or inhibiting ESP production (mRNA or protein) or activity can increase production of isothiocyanates.
  • reducing ESP refers to decreasing the protein production or activity of ESP.
  • reducing ESP comprises inactivating (e.g. denaturing) ESP at high temperature.
  • ESP is denatured at temperatures of about 50°C to about 80°C.
  • maintaining endogenous myrosinase activity means not significantly reducing myrosinase activity compared to an untreated control.
  • endogenous myrosinase activity is not reduced by about 5% or more.
  • endogenous myrosinase activity is not reduced by about 10% or more.
  • endogenous myrosinase activity is not reduced by about 15% or more.
  • endogenous myrosinase activity is not reduced by about 20% or more.
  • endogenous myrosinase activity is not reduced by about 30%) or more.
  • endogenous myrosinase activity is not reduced by about 40%) or more.
  • endogenous myrosinase activity is not reduced by about 50% or more.
  • pre-treating comprises one or more of the following: i) heating; ii) macerating; iii) microwaving; iv) exposure to high frequency sound waves (ultrasound), or v) pulse electric field processing, wherein the temperature of the Brassicaceae material does not exceed about 75 °C during pre-treating.
  • the Brassicaceae material is heated in a fuel based heating system, an electricity based heating system (i.e. an oven or ohmic heating), radio frequency heating, high pressure thermal processing or a steam based heating system (indirect or direct application of steam).
  • the Brassicaceae material is heated in a sealed package (e.g. in a retort pouch).
  • the Brassicaceae material is heated in an oven, water bath, bioreactor, stove, water blancher, or steam blancher.
  • the Brassicaceae material is heated via high pressure thermal heating.
  • the Brassicaceae material is via ohmic heating.
  • the Brassicaceae material is via radio frequency heating.
  • the Brassicaceae material is blanched in water.
  • the Brassicaceae material is heated via high pressure thermal processing.
  • the Brassicaceae material is placed in a sealed package for high pressure thermal processing.
  • pre-treating comprises heating the Brassicaceae material to about 50°C to about 70°C. In an embodiment, pre-treating comprises heating the Brassicaceae material to about 50°C to about 65°C. In an embodiment, pre-treating comprises heating the Brassicaceae material to about 50°C to about 60°C. In an embodiment, heating comprises heating the Brassicaceae material to about 55°C to about 70°C. In an embodiment, heating comprises heating the Brassicaceae material to about 60°C to about 70°C. In an embodiment, heating comprises heating the Brassicaceae material to about 65°C to about 70°C. In an embodiment, the Brassicaceae material is heated for about 30 seconds. In an embodiment, the Brassicaceae material is heated for about 1 minute.
  • the Brassicaceae material is heated for about 2 minutes. In an embodiment, the Brassicaceae material is heated for about 3 minutes. In an embodiment, the Brassicaceae material is heated for about 4 minutes. In an embodiment, the Brassicaceae material is heated for about 5 minutes.
  • the Brassicaceae material is heated in a sealed package for about 1 min at about 60°C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 2 mins at about 60°C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 3 mins at about 60°C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 4 mins at about 65°C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 1 min at about 65°C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 2 mins at about 65°C.
  • the Brassicaceae material is heated in a sealed package for about 3 mins at about 65°C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 4 mins at about 65°C. In an embodiment, the Bmssicaceae material is heated in water for about 1 min at about 60°C. In an embodiment, the Bmssicaceae material is heated in water for about 2 mins at about 60°C.
  • heating comprises steaming the Bmssicaceae material.
  • pre-treating comprises steaming the Bmssicaceae material.
  • the Bmssicaceae material is steamed to a temperature of about 50°C to about 70°C.
  • the Bmssicaceae material is steamed to a temperature of about 60°C to about 70°C.
  • the Bmssicaceae material is steamed for at least about 30 seconds.
  • the Bmssicaceae material is steamed for at least about 1 minute.
  • the Bmssicaceae material is steamed for at least about 2 minutes.
  • the Bmssicaceae material is steamed for at least about 3 minutes. In an embodiment, the Bmssicaceae material is steamed for at least about 4 minutes. In an embodiment, the Bmssicaceae material is steamed for at least about 5 minutes.
  • pre-treating comprises macerating the Bmssicaceae material.
  • macerating refers to breaking the Bmssicaceae material into smaller pieces.
  • macerating comprising decompartmentalizing at least about 30% to about 90% of the cells of the Bmssicaceae material to allow myrosinase access to its substrate glucosinolates.
  • macerating comprising decompartmentalizing at least about 40% to about 90% of the cells of the Bmssicaceae material.
  • macerating comprising decompartmentalizing at least about 50% to about 90% of the cells of the Bmssicaceae material.
  • macerating comprising decompartmentalizing at least about 60% to about 90% of the cells of the Bmssicaceae material. In an embodiment, macerating comprising decompartmentalizing at least about 70% to about 90% of the cells of the Bmssicaceae material.
  • the Bmssicaceae material is macerated with a blender, grinder or pulveriser. In an embodiment, the Bmssicaceae material is macerated so that at least about 80% of the Bmssicaceae material is of a size of about 2 mm or less. In an embodiment, the Bmssicaceae material is macerated so that at least about 80% of the Bmssicaceae material is of a size of about 1 mm or less. In an embodiment, the Bmssicaceae material is macerated so that at least about 80% of the Bmssicaceae material is of a size of about 0.5 mm or less.
  • the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 0.01 mm or less. In an embodiment, the Brassicaceae material is macerated so that about 50% to about 90% of the Brassicaceae material is of a size of about 2 mm or less. In an embodiment, the Brassicaceae material is macerated so that about 60% to about 80% of the Brassicaceae material is of a size of about 2 mm or less. In an embodiment, the Brassicaceae material is macerated so that about 50% to about 90% of the Brassicaceae material is of a size of about 1 mm or less.
  • the Brassicaceae material is macerated so that about 60% to about 80% of the Brassicaceae material is of a size of about 1 mm or less.
  • the Brassicaceae material is heated to a temperature of about 50°C to about 70°C during maceration.
  • the Brassicaceae material is heated to a temperature of about 55°C to about 70°C during maceration.
  • the Brassicaceae material is heated to a temperature of about 60°C to about 70°C during maceration.
  • the Brassicaceae material is heated to a temperature of about 65°C to about 70°C during maceration.
  • pre-treating comprises heating and macerating the Brassicaceae material. In an embodiment, pre-treating produces a puree.
  • a "puree” refers to Brassicaceae material blended to the consistency of a creamy paste or liquid.
  • the ultrasounds waves are at a frequency of about 20 kHz to about 600 kHz.
  • the Brassicaceae 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.
  • pre-treating comprises exposing the Brassicaceae material to pulse electric field processing.
  • Pulse electric field processing is a non-thermal processing technique comprising the application of short, high voltage pulses. The pulses induce electroporation of the cells of the Brassicaceae material enhancing the access of myrosinase to glucosinolates.
  • pulse electric field processing heats the Brassicaceae material to a temperature of about 40 to about 70°C.
  • pulse electric field processing heats the Brassicaceae material to a temperature of about 50°C to about 70°C.
  • pulse electric field processing heats the Brassicaceae material to a temperature of about 60°C to about 70°C.
  • pulse electric field processing comprises treating the Brassicaceae material with voltage pulses of about 20 to about 80 kV.
  • pre-treating converts about 10% to about 90% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 20% to about 80% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 30% to about 70%) of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 40% to about 60%> of a glucosinolate to an isothiocyanate.
  • pre-treating converts about 10% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 20% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 30% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 40% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 50% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 60% of a glucosinolate to an isothiocyanate.
  • pre-treating converts about 70%o of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 80% of a glucosinolate to an isothiocyanate. In an embodiment, pre- treating converts about 90% of a glucosinolate to an isothiocyanate.
  • fermentation method as described herein can comprise the use of any lactic acid bacteria.
  • “fermentation” refers to the biochemical breakdown of the Brassicaceae material by lactic acid bacteria.
  • fermentation with lactic acid bacteria is performed using the addition of exogenous lactic acid bacteria.
  • the lactic acid bacteria is selected from one or more of Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus rhamnosus, Lactobacillus pentosus, Lactobacillus brevis, Lactococus lactis, Pediococcus pentosaceus and Pedicoccus acidilacti.
  • the lactic acid bacteria was isolated from a Brassicaceae. In an embodiment, the lactic acid bacteria was isolated from a Brassica oleracea. In an embodiment, the lactic acid bacteria was isolated from broccoli. In an embodiment, the lactic acid bacteria was isolated from broccoli leaves. In an embodiment, the lactic acid bacteria was isolated from broccoli stem. In an embodiment, the lactic acid bacteria was isolated from broccoli puree. In an embodiment, the lactic acid bacteria was isolated from Australian broccoli.
  • the lactic acid bacteria lacks myrosinase activity.
  • the lactic acid bacteria is a Lactobacillus.
  • the lactic acid bacteria is Leuconostoc mesenteroides.
  • the Leuconostoc mesenteroides is ATCC8293.
  • the Leuconostoc mesenteroides is BFl and/or BF2.
  • the Leuconostoc mesenteroides lacks myrosinase activity.
  • the lactic acid bacteria is Lactobacillus plantarum. In an embodiment, the Lactobacillus plantarum lacks myrosinase activity.
  • about 50% of the lactic acid bacteria is Leuconostoc mesenteroides and about 50% of the lactic acid bacteria is Lactobacillus plantarum.
  • the Lactobacillus plantarum is selected from one or more or all of Bl, B2, B3, B4 and B5.
  • the Lactobacillus plantarum is Bl .
  • the Lactobacillus plantarum is B2.
  • the Lactobacillus plantarum is B3.
  • the Lactobacillus plantarum is B4.
  • the Lactobacillus plantarum is B5.
  • 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 strains of lactic acid bacteria selected from BFl, BF2, Bl, B2, B3, B4 and B5.
  • the lactic acid bacteria is a recombinant bacteria modified to produce a high level of myrosinase activity compared to a control bacteria lacking the modification.
  • a person skilled in the art will appreciate that the recombinant lactic acid bacteria is produced by any technique known to a person skilled in the art.
  • the lactic acid bacteria is stressed, for example but not limited to, heat stress, cold stress, sub-lethal ultrasonic waves e.g. about 20 to about 2000 MHz, high pressure, dynamic high pressure or pulsed-electric field, to increase myrosinase activity and the activity of polysaccharide degrading enzymes compared to a control lactic acid bacteria that has not been stressed.
  • heat stress comprises heating the bacteria to greater than about 40°C to about 75°C.
  • heat stress comprises heating the bacteria to greater than about 45 ° C to about 65°C.
  • heat stress comprises heating the bacteria to greater than about 45 °C to about 55 °C.
  • cold stress comprises lower the bacteria to temperature of about 0°C to about 8°C. In an embodiment, cold stress comprises lower the bacteria to temperature of about 2°C to about 6 ° C. In an embodiment, cold stress comprises lower the bacteria to temperature of about 4°C.
  • fermentation is at about 20°C to about 34°C. In an embodiment, fermentation is at about 22°C to about 34°C. In an embodiment, fermentation is at about 24°C to about 34°C. In an embodiment, fermentation is at about 24°C to about 30°C. In an embodiment, fermentation is at about 34°C to about 34°C. In an embodiment, fermentation is at about 25 °C. In an embodiment, fermentation is at about 30°C. In an embodiment, fermentation is at about 34°C.
  • fermentation is for about 8 hours to about 17 days. In an embodiment, fermentation is for about 8 hours to about 14 days. In an embodiment, fermentation is for about 8 hours to about 7 days. In an embodiment, fermentation is for about 8 hours to about 5 days. In an embodiment, fermentation is for about 8 hours to about 4 days. In an embodiment, fermentation is for about 8 hours to about 3 days. In an embodiment, fermentation is for about 8 hours to about 30 hours. In an embodiment, fermentation is for about 8 to about 24 hours. In an embodiment, fermentation is for about 10 hours to about 24 hours. In an embodiment, fermentation is for about 10 days. In an embodiment, fermentation is for about 9 days. In an embodiment, fermentation is for about 8 days. In an embodiment, fermentation is for about 7 days. In an embodiment, fermentation is for about 4 days.
  • fermentation is for about 6 days. In an embodiment, fennentation is for about 5 days. In an embodiment, fermentation is for about 72 hours. In an embodiment, fermentation is for about 60 hours. In an embodiment, fermentation is for about 45 hours. In an embodiment, fermentation is for about 30 hours. In an embodiment, fermentation is for about 24 hours. In an embodiment, fermentation is for about 20 hours. In an embodiment, fermentation is for about 18 hours. In an embodiment, fermentation is for about 15 hours. In an embodiment, fermentation is for about 16 hours. In an embodiment, fermentation is for about 14 hours. In an embodiment, fermentation is for about 12 hours. In an embodiment, fermentation is for about 10 hours. In an embodiment, fermentation is for about 8 hours. In an embodiment, the fermentation culture is stirred. In an embodiment, stirring is intermittent.
  • stirring is continuous.
  • fermentation is for 15 hours with intermittent stirring.
  • fennentation is for 24 hours with intermittent stirring.
  • the fermentation reaction is complete when the composition reaches a pH of about 4.5 to about 3.8.
  • the fermentation reaction is complete when the composition reaches a pH of about 4.5 to about 3.6.
  • the fermentation reaction is complete when the composition reaches a pH of about 4.5 to about 4.04.
  • the fermentation reaction is complete when the composition reaches a pH of about 4.3 to about 4.04.
  • 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 an embodiment, the fermentation reaction is complete when the composition reaches a pH of 4.5 or less. In an embodiment, the fermentation reaction is complete when the composition reaches a pH of 4.4 or less.
  • if present fermentation reduces the number of one or more or all of: E. coli, Salmonella and Listeria. In an embodiment, if present fermentation reduces the CFU/g of one or more or all of: E. coli, Salmonella and Listeria.
  • fermentation increases the extractable glucosinolate content compared to the extractable glucosinolate content in the pre-treated Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content is increased by about 100% to about 500% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content by about 200% to about 450% compared to the extractable glucosinolate content in the Brassicaceae material.
  • fermentation increases the extractable glucosinolate content by about 250% to about 450% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content by about 300% to about 400% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content by about 300% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content by about 400% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, the glucosinolate is glucoraphanin. Acidification
  • the pre-treated material can by acidified to improve the microbial safety and stability (susceptibility to microbial degradation) of the product and increase the stability of isothiocyanate in the product.
  • Acidification can be achieved by the addition of organic acids, such as, but not limited to lactic, acetic, ascorbic, and citric acid.
  • acidification can be achieved with the addition of glucono-delta-lactone.
  • acidification comprises lowering the pH to a pH of about 4.4 to about 3.4.
  • 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 less.
  • acidification comprises lowering the pH to a pH of 4.4 of less.
  • An isothiocyanate containing product from Brassicaceae as described herein can be produced by the methods as described herein. It will be appreciated be a person skilled in the art that an isothiocyanate containing product produced using the methods as described herein contains higher levels of isothiocyanates, for example sulforaphane, than the Brassicaceae material or Brassicaceae material subjected to fermentation alone (without pre-treatment as described herein).
  • macerated broccoli from a commercial broccoli cultivar has a sulforaphane concentration of ⁇ 800 ⁇ 1/3 ⁇ 4 dw (-149.8 mg/Kg dw)
  • fermented macerated broccoli has a sulforaphane concentration of ⁇ 1600 ⁇ 1/13 ⁇ 4 dw (-278.8 mg/Kg dw)
  • pre-treated and fermented broccoli produced using the methods as described herein has a sulforaphane concentration of ⁇ 13100 ⁇ 1/3 ⁇ 4 dw (-2318.7 mg/Kg dw).
  • the isothiocyanate containing product comprises at least about 4 times more isothiocyanate than macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 6 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 8 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 10 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 12 times more isothiocyanate than the macerated Brassicaceae material.
  • the isothiocyanate containing product comprises at least about 14 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 17 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 4 times to about 17 times more isothiocyanate than the macerated Brassicaceae material.
  • the isothiocyanate containing product comprises about 4 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 8 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 10 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 12 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 14 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate is sulforaphane.
  • the level of isothiocyanate present in the isothiocyanate containing product is higher than what would be expected from the extractable glucosinolate content of the Brassicaceae material.
  • the isothiocyanate containing product comprises at least about 1 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.
  • the isothiocyanate containing product comprises at least about 2 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.
  • the isothiocyanate containing product comprises at least about 3 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.
  • the isothiocyanate containing product comprises at least about 3.8 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises at least about 4 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises about 1 times to about 4 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises about 1 times to about 3.8 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.
  • the isothiocyanate containing product comprises about 2 times to about 3.8 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises about 2 times to about 3 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.
  • the level of sulforaphane present in the isothiocyanate containing product is higher than what would be expected from the extractable glucoraphanin content of the Brassicaceae material.
  • the isothiocyanate containing product comprises at least about 1 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content.
  • the isothiocyanate containing product comprises at least about 2 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content.
  • the isothiocyanate containing product comprises at least about 3 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content.
  • the isothiocyanate containing product comprises at least about 3.8 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises at least about 4 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises about 1 times to about 4 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises about 1 times to about 3.8 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content.
  • the isothiocyanate containing product comprises about 1 times to about 3 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises about 2 times to about 3 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content.
  • the isothiocyanate containing product comprises about 100 mg/kg dw to about 7000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 500 mg/kg dw to about 7000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 1000 mg/kg dw to about 7000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 1600 mg/kg dw to about 4000 mg/kg dw of isothiocyanate.
  • the isothiocyanate containing product comprises about 1600 mg/kg dw to about 3000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 2000 mg/kg dw to about 4000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 2000 mg/kg dw of to about 7000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 3000 mg/kg dw isothiocyanate to about 7000 mg/kg of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 2300 mg/kg dw of the isothiocyanate.
  • the isothiocyanate containing product comprises at least about 100 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 200 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 250 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 300 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 350 mg/kg dw of the isothiocyanate.
  • the isothiocyanate containing product comprises at least about 400 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 450 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 500 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 550 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 600 mg/kg dw of the isothiocyanate.
  • the isothiocyanate containing product comprises at least about 650 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 700 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 1000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 2000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 3000 mg/kg dw of the isothiocyanate.
  • the isothiocyanate containing product comprises at least about 4000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 5000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 6000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 7000 mg/kg dw of the isothiocyanate.
  • the isothiocyanate containing product comprises at least about 100 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 150 mg/kg of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 200 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 250 mg/kg of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 300 mg/kg dw of sulforaphane.
  • the isothiocyanate containing product comprises at least about 350 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 400 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 450 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 500 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 550 mg/kg dw of sulforaphane.
  • the isothiocyanate containing product comprises at least about 600 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 650 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 700 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 1000 mg/kg of sulforaphane dw. In an embodiment, the isothiocyanate containing product comprises at least about 2000 mg/kg dw of sulforaphane.
  • the isothiocyanate containing product comprises at least about 3000 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 4000 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 5000 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 6000 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 7000 mg/kg dw of sulforaphane.
  • the isothiocyanate containing product comprises at least about 10% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 20% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 30% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 40% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 45% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 48% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 10% to about 48% less carbohydrate than the Brassicaceae material.
  • the isothiocyanate containing product comprises an increased level of polyphenolic glycosides compared to the Brassicaceae material.
  • the polyphenolic glycosides are anthocyanin glycosides.
  • the polyphenolic glycosides are phenolic acid glycosides.
  • the polyphenolic glycosides are phenolic acids.
  • the isothiocyanate containing product comprises an increased level of glucosinolates compared to the Brassicaceae material.
  • the glucosinolate is glucoraphanin.
  • glucoraphanin is increased at least about 25 fold.
  • the glucosinolate is glucobrassicin.
  • the glucobrassicin is increased by 26 times.
  • the isothiocyanate containing product comprises indole-3 -carbinol.
  • indol-3 carbinol is increased at least about 2 fold in the isothiocyanate containing product compared to the macerated Brassicaceae material.
  • indol-3 - carbinol is increased at least about 3 fold in the isothiocyanate containing product compared to the macerated Brassicaceae material.
  • the isothiocyanate containing product comprises ascorbigen.
  • ascorbigen is increased at least about 2 fold in the isothiocyanate containing product compared to the macerated Brassicaceae material.
  • ascorbigen is increased at least about 3 fold in the isothiocyanate containing product compared to the macerated Brassicaceae material.
  • the isothiocyanate containing product comprises an decreased level of one or more of protocatechuic acid, gallic acid, 4,hydroxybenzoic acid, vanillic acid, 2,3dihydroxybenzoic acid, p-cuomaric acid, cinnamic acid, catechin, rosmarinic acid, caffeic acid compared to the Brassicaceae material.
  • about 40% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product.
  • about 50% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product.
  • about 60% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product.
  • about 70% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product.
  • a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product.
  • about 90% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product.
  • about 95% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product.
  • about 97% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product.
  • stable refers to no decrease or only a minor decrease in isothiocyanate concentration when stored at 4°C for six weeks.
  • a minor decrease refers to a decrease in isothiocyanate concentration of about 1% to about 30%.
  • a minor decrease refers to a decrease in isothiocyanate concentration of about 5% or less.
  • a minor decrease refers to a decrease in isothiocyanate concentration of about 10% or less.
  • a minor decrease refers to a decrease in isothiocyanate concentration of about 15% or less.
  • a minor decrease refers to a decrease in isothiocyanate concentration of about 20% or less.
  • a minor decrease refers to a decrease in isothiocyanate concentration of about 30% or less.
  • Isothiocyanate analysis can be performed by any method know to a person skilled in the art and for example as shown in Example 1 for sulforaphane.
  • the isothiocyanate containing product is resistant to yeast, mould and/or coliform growth for at least a week, or for at least two weeks, or for at least 3 weeks, or for at least 4 weeks, or for at least 6 weeks, or for at least 8 weeks, or for at least 10 weeks, or for at least 12 weeks, or for at least 14 weeks when stored at about 4°C to about 25°C.
  • the isothiocyanate containing product is resistant to yeast, mould and/or coliform growth for at least 4 weeks when stored at about 4°C to about 25°C. In an embodiment, the isothiocyanate containing product is resistant to yeast, mould and/or coliform growth for at least 8 weeks when stored at about 4°C to about 25°C. In an embodiment, the isothiocyanate containing product is resistant to yeast, mould and/or coliform growth for at least 12 weeks when stored at about 4°C to about 25°C.
  • resistant to yeast, mould and/or coliform growth means that ⁇ 1 Log CFU/g of yeast, mould and/or coliform is detectable in the sample after the above listed time periods using the methods described in Example 1.
  • the isothiocyanate containing product comprises about 20 g/lOOgdw to about 32 g/lOOgdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 20 g/lOOgdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 25 g/lOOgdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 28 g/lOOgdw total fibre.
  • the isothiocyanate containing product comprises about 29 g/lOOgdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 30 g/lOOgdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 32 g/lOOgdw total fibre.
  • the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 14000 ⁇ TE/100 gdw to about 19000 ⁇ TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 14000 ⁇ TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 15000 ⁇ TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 16000 ⁇ TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 17000 ⁇ TE/100 gdw.
  • the isothiocyanate containing product comprises a total polyphenol content of about 1750 mg GAE/lOOgdw to about 2600 mg GAE/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 1750 mg GAE/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2000 mg GAE/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2100 mg GAE/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2200 mg GAE/lOOgdw.
  • the isothiocyanate containing product comprises a total polyphenol content of about 2300 mg GAE/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2360 mg GAE/lOOgdw.
  • the isothiocyanate containing product comprises a total titratable acidity of about 0.9% to about 1.1% lactic acid equivalent. In an embodiment, the isothiocyanate containing product comprises a total titratable acidity of about 1.1% lactic acid equivalent.
  • the isothiocyanate containing product comprises a total protein content of about 23 g/lOOgdw to about 39 g/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 23 g/lOOgdw to about 30 g/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 25 g/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 27 g/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 28 g/lOOgdw.
  • the isothiocyanate containing product comprises a total protein content of about 29 g/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 30 g/lOOgdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 32 g/lOOgdw.
  • the isothiocyanate containing product comprises at least about 100 mg/kg dw of an isothiocyanate and one or more or all of the following.
  • the isothiocyanate containing product is produced from broccoli.
  • the Brassicaceae products as described herein can comprise live lactic acid bacteria which can aid the conversion of glucosinolate present in the isothiocyanate containing product to an isothiocyanates during digestion of a glucosinolate containing product in a subject (i.e. they act as a probiotic).
  • the lactic acid bacteria is a Leuconostoc mes enter vide.
  • the lactic acid bacteria is Lactobacillus sp.
  • the lactic acid bacteria is Lactobacillus plantarum.
  • the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 10 2 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 10 2 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 10 5 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 10 6 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 10 7 CFU/g.
  • live lactic acid bacteria are present in the isothiocyanate containing product for at least 10 days when stored at about 4°C to about 25°C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 20 days when stored at about 4°C to about 25°C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 30 days when stored at about 4°C to about 25°C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 40 days when stored at about 4°C to about 25°C.
  • live lactic acid bacteria are present in the isothiocyanate containing product at least 50 days when stored at about 4°C to about 25°C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 60 days when stored at about 4°C to about 2 °C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 70 days when stored at about 4°C to about 25 °C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 80 days when stored at about 4°C to about 25 °C.
  • live lactic acid bacteria are present in the isothiocyanate containing product at least 85 days when stored at about 4°C to about 25 °C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 90 days when stored at about 4°C to about 25°C. In an embodiment, the lactic acid bacteria is a Lactobacillus sp.. In an embodiment, the lactic acid bacteria is Lactobacillus plantarum. In an embodiment, the lactic acid bacteria is Leuconostoc mes enter oides. In an embodiment, the bacteria are present at a concentration of at least about 10 7 CFU/g.
  • the isothiocyanate containing product comprises one or more bacteriocin/s produced by lactic acid bacteria.
  • the bacteriocin is a Class I bacteriocin.
  • the bacteriocin is a Class II bacteriocin.
  • the bacteriocin is a Class III bacteriocin. Examples of bacteriocins produced by lactic acid bacteria can be found in Alvarez-Sieiro et al. (2016).
  • the isothiocyanate containing product is a solid, liquid, puree or a powder. In an embodiment, the isothiocyanate containing product is dried to a powder after fermentation. In an embodiment, the isothiocyanate containing product is freeze dried after fermentation. In an embodiment, the isothiocyanate containing product is microencapsulated as described in WO2005030229 after fermentation. In an embodiment, the isothiocyanate containing product is formulated as a pill.
  • the isothiocyanate containing product can be post-treated to inactivate microbes that for example contribute to degradation of the product or a pathogenic if consumed.
  • post-treatment or “post-treating” refers to treatment of the isothiocyanate containing product as described herein after fermentation to inactivate microbes.
  • microbes refers to bacterial, viral, fungal or eukaryotic activity that can result in degradation or spoilage of the isothiocyanate containing product.
  • inactivate or “inactivation” of microbes refers to reducing the viable microbes by about 1 to about 7 logs. In an embodiment, the viable microbes are reduced by about 1 to 6 logs. In an embodiment, the viable microbes are reduced by about 2 to 6 logs. In an embodiment, the viable microbes are reduced by about 3 to 6 logs.
  • the post treatment can be any method that inactivates microbes, including for example, heat treatment, UV treatment, ultrasonic processing, pulsed electric field processing or high pressure processing.
  • the isothiocyanate containing product is post-treated with heat processing.
  • the isothiocyanate containing product is post-treated with high pressure processing.
  • the isothiocyanate containing product is in a sealed package during post-treatment.
  • the isothiocyanate containing product is in a sealed package during high pressure processing.
  • the isothiocyanate containing product is in a sealed package during heat treatment.
  • high pressure processing comprises treating the isothiocyanate containing product with isostatic pressure at about 300 to about 600 MP a. In an embodiment, high pressure processing comprises treating the isothiocyanate containing product with isostatic pressure at about 350 to about 550 MPa. In an embodiment, high pressure processing comprises treating the isothiocyanate containing product with isostatic pressure at about 300 to about 400 MPa.
  • heat treatment comprises heating the sample to a temperature of about 60°C to about 121°C. In an embodiment, heat treatment comprises heating the sample to a temperature of about 65 °C to about 100°C. In an embodiment, heat treatment comprises heating the sample to a temperature of about 65 °C to about 80°C. In an embodiment, heat treatment comprises heating the sample to a temperature of about 65°C to about 75°C.
  • the present invention provides isolated strains of lactic acid bacteria suitable for use in the methods and products as described herein.
  • the present invention provides an isolated strain of lactic acid bacteria selected from:
  • the present invention provides an isolated strain of Lactobacillus plantarum comprising genomic DNA which when cleaved with Smal and/or Notl produces a Smal and/or Notl fingerprint identical to B 1 , B2, B3, B4 or B5.
  • the present invention provides an isolated strain of Leuconostoc mesenteroides comprising one or more or all of the polymorphisms listed in Table 18 or 19 that differs from ATCC8293.
  • the isolated strain of Leuconostoc mesenteroides comprises 5 or more of the polymorphisms listed in Table 18 or 19 that differs from ATCC8293.
  • the isolated strain of Leuconostoc mesenteroides comprises 10 or more of the polymorphisms listed in Table 18 or 19 that differs from ATCC8293.
  • the isolated strain of Leuconostoc mesenteroides comprises 15 or more of the polymorphisms listed in Table 18 or 19 that differs from ATCC8293.
  • the isolated strain of Leuconostoc mesenteroides comprises 19 or more of the polymorphisms listed in Table
  • the isolated strain of Leuconostoc mesenteroides comprises 20 or more of the polymorphisms listed in Table
  • the isolated strain of Leuconostoc mesenteroides comprises 30 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 50 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 80 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 100 or more of the polymorphisms listed in Table 19 that differs from ATCC8293.
  • the isolated strain of Leuconostoc mesenteroides comprises 150 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mes enter oides comprises 200 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mes enter oides comprises 300 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 400 or more of the polymorphisms listed in Table 19 that differs from ATCC8293.
  • the present invention provides an isolated strain of Lactobacillus plantarum comprising one or more or all the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 5 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 10 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014.
  • the present invention provides an isolated strain of Lactobacillus plantarum comprising 15 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 20 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 25 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014.
  • the present invention provides an isolated strain of Lactobacillus plantarum comprising 30 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 35 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 40 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014.
  • the present invention provides a starter culture for producing an isothiocyanate containing product or a probiotic comprising lactic acid bacteria comprising one or more of the isolated strains as described herein.
  • a starter culture is a culture of live microorganisms for fermentation.
  • the present invention provides a starter culture for producing an isothiocyanate containing product or a probiotic comprising lactic acid bacteria selected from one or more or all of:
  • the Brassicaceae material is inoculated with at least about 10 5 CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with at least 10 6 about CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with at least about 10 7 CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with at least about 10 8 CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with at least about 10 10 CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with about 10 5 CFU/g to about 10 10 CFU/g of a starter culture as described herein.
  • the present invention provides for a probiotic comprising one or more of the lactic acid bacteria isolated from a Brassicaceae.
  • a probiotic refers to a live microorganism which when administered in an adequate amount confers a health benefit to the host.
  • the lactic acid bacteria was isolated from a Brassica oleracea.
  • the lactic acid bacteria was isolated from broccoli.
  • the lactic acid bacteria was isolated from Australian broccoli.
  • the lactic acid bacteria is selected from: i) a Leuconostoc mesenteroides; ii) a Lactobacillus plantarum; iii) a Lactobacillus pentosus; iv) a Lactobacillus rhamnosus; v) a combination of i) and ii); vi) a combination of i), ii) and iii); and vii) a combination of i), ii) and iv).
  • the lactic acid bacteria is selected from one or more or all of BF1, BF2, Bl, B2, B3, B4 and B5.
  • the lactic acid bacteria is Bl .
  • the lactic acid bacteria is B2. In an embodiment, the lactic acid bacteria is B3. In an embodiment, the lactic acid bacteria is B4. In an embodiment, the lactic acid bacteria is B5. In an embodiment, the probiotic is a capsule, tablet, powder or liquid. In an embodiment, the probiotic is microencapsulated as described in WO 2005030229.
  • Lactic acid bacteria used during fermentation were selected from one or more of:
  • LGG Lactobacillus rhamnosus ATCC53103;
  • BP pooled BF1. BF2;
  • BF1 and BF2 were identified as Leuconostoc mes enter oides via a 16s-RNA sequence (Australian Genome Research Facility; data not shown).
  • Bl to B5 were identified as Lactobacillus plantarum based on 16S-RNA sequence. The identity of all the isolates were confirmed by whole genome sequence analysis.
  • Lactobacillus plantarum Bl, B2, B3, B4 and B5 were isolated from broccoli leaves and stem.
  • the leaves and stem were washed with water and homogenised with added peptone saline using a stomacher.
  • the soaking solution was serially diluted and spread plated on De Man, Rogosa and Sharpe (MRS) agar.
  • the plates were incubated under anaerobic condition for 48 to 72 hrs at 37°C for isolating presumptive mesophilic lactic acid bacteria. Based on different colonial morphology on MRS plates, colonies were isolated, cultivated in MRS broth, screened using staining and biochemical characterisation techniques, and kept frozen with glycerol at -80°C.
  • the isolates were identified at species level using 16s RNA sequencing at AGRF.
  • lactic acid bacteria cultures which were stored at -80°C were inoculated into 10 mL of MRS broth (Oxoid, Victoria, Australia) and incubated at 30°C for 24 h to obtain an initial biomass of 8 log colony-forming units per milliliter (CFU/mL).
  • CFU/mL log colony-forming units per milliliter
  • the cultures were collected by centrifugation at 2000g for 15min at 4°C, washed twice with sterile phosphate buffer saline (PBS), and all the Lactobacillus plantarum cultures were mixed together and all the Leuconostoc mesenteroides cultures were mixed together.
  • the two culture suspensions were diluted to 10 log CFU/ml and were mixed at the same volumetric proportion and stored with glycerol at -80°C until use as a mixed starter culture for broccoli fermentation.
  • Broccoli (Brassica oleracea L. ssp. Italic; 30 kg) florets were cut approximately 2 cm from the crown, shredded to smaller pieces and, were macerated with Milli-Q water in ratio of 3:2 for 1 min using magic bullet blender.
  • the broccoli slurry was mixed well and placed into sterile plastic bottles (200 mL) with screw lids. Each bottle of broccoli puree (200 mL) was inoculated with the prepared starter culture at an initial concentration of 8 log CFU/g.
  • the fermentation experiment was carried out in 48 bottles in parallel at 30°C, until a pH value of about 4.0 was reached (Day 4).
  • Microbiological analysis For microbial analysis, three different media were used to measure CFU per g broccoli puree of the different microorganisms; the plate counts for total lactic acid bacteria on DeMan-Rogosa-Sharp (MRS) agar, for total enterobacteria on violet red bile glucose agar (VRBGA), and the yeasts and mould on potato dextrose agar (PDA). For each sample, serial dilution of the broccoli suspension in sterilized peptone saline diluent were made and 0.1 mL of the dilutions were plated onto agar plates in duplicates.
  • MRS DeMan-Rogosa-Sharp
  • VRBGA violet red bile glucose agar
  • PDA potato dextrose agar
  • v is titer volume of NaOH.
  • the acid factor for lactic acid is 0.009.
  • the total protein content of broccoli samples was determined as total nitrogen content multiplied by 6.25.
  • Total nitrogen content of broccoli was analyzed using a Dumas combustion method with LECO TruMac apparatus (LECO Coiporation, Michigan, USA).
  • the color indexes (L, a, b) of fermented broccoli sample were determined using a Chroma meter CR-200 tristimulus colorimeter (Minolta, Osaka, Japan).
  • the color values obtained were expressed as lightness/darkness (as L * ), redness/greenness (a * ) and yellow/blueness (b * ).
  • the total color difference ( ⁇ ) was calculated according to the following equation:
  • TPC total phenolic content
  • 50 mg of broccoli powder was suspended in 10 mL of acidified (1 % HC1) methanol/water (70:30, v/v) solution and extracted in ultrasonic bath (IDK technology Pty Ltd, VIC, Australia) for 8 min.
  • the extracts were kept for 16 h at 4°C and filtered with 0.2 ⁇ filter and stored at 4°C until analysis.
  • Freeze-dried broccoli powder (10 mg) was suspended in 10 mL of methanol/water (80:20, v/v), the extraction solvent. The slurry was extracted at 650rpm on a Heidolph Multi-Reax (John Moms Scientific, NSW, Australia) at room temperature for an hour. Then it was centrifuged at 25,000g for 15 min in 4°C, the supernatant was collected, and was ready for analysis after lOOx dilution with 75 mM potassium phosphate buffer (pH 7.4). ORAC analysis was conducted according to the procedure reported by Huang et al. (2002) with minor modifications. The assay was carried out in opaque 96-well plates (dark optical bottom, Waltham, MA, USA).
  • the assay reactants included 81.6 nM of fluorescein, 153 mM of AAPH, Trolox standard of different concentration (100, 50, 25, 12.5, and 6.25 ⁇ ), and 75 mM phosphate buffer as the blank.
  • the reactants were added in the following order: 25 of diluted sample; either 25 ⁇ ⁇ of 75 mM phosphate buffer, 25 ⁇ Trolox standard and 150 ⁇ , fluorescein. After adding the fluorescein, the plate was incubated at 37°C for 10 min and then the AAPH (25 ⁇ ) was added.
  • the plate was placed in the fluorescence plate reader (BMG Labtech ClarioStar, Germany) and the fluorescence was measured every 3 min until it decreased to less than 5% of original fluorescence.
  • the ORAC values were calculated as the area under the curve (AUC) and expressed as micromoles of trolox equivalent (TE) per gram dry weight of broccoli ( ⁇ TE/g DW). Each sample was assayed triplicate.
  • the gradient elution system consisted of mobile phase A (0.1% formic acid in millique water) and B (0.1% formic acid in acetonitrile) and separation was achieved using the following gradient: 0-2 min, 10% B; 2-5 min, 20% B; 5-10 min, 10% B.
  • the column temperature was kept constant at 30°C.
  • the flow-rate was 0.350 mL/min and the injection volume was 5 ⁇ .
  • glucoraphanin from raw or fermented broccoli was carried out according to the method of Cai and Wang (2016) with some modifcation. Accordingly, to 2 g of frozen broccoli puree, 10 mL of boiling Milli-Q water was added, and the mixture was incubated for 5 min in a boiling water bath. It was then cooled and centrifuged at 15000xg for 15 min, and the supernatant was collected. The precipitate was extracted once more with 8 mL of boiling water. Pooled extracts from each sample were evaporated to dryness with a vacuum spin dryer (Speedvac SC250EXP, Thermo Fisher Scientific, CA, USA) at 3°C, and stored at -20°C until analysis.
  • a vacuum spin dryer Speedvac SC250EXP, Thermo Fisher Scientific, CA, USA
  • the concentration of glucoraphanin was quantified using an Alliance HPLC instrument (Waters Corporation, Milford, MA, USA) equipped with Photo Diode Array Detector 2998.
  • the mobile phase consisted of an acetonitrile/water (85: 15, v/v) with 30mM Ammonium formate (solution A) and acetonitrile (solution B) with the following isocratic flow program: solution A 70%; solution B 30%.
  • chromatographic conditions included a constant flow rate of 2.0 mL/ min, an injection volume of 100 ⁇ L, a run time of 8 min, and detection wavelength of 235 nm. Prior to analysis, all samples were dissolved in 1 mL solvent A, and filtered through a 0.22 ⁇ membrane filter (Merk Millipore, Billerica, MA, USA). The identification of each peak was based on the retention time and the chromatography of an authentic glucoraphanin standard. The concentrations of glucoraphanin were calculated using a standard curve, and the results were expressed as micromoles glucoraphanin per kilogram DW ( ⁇ /kg DW) of broccoli.
  • Example 2 Microbial analysis of lactic acid bacteria fermented broccoli florets
  • MRS de Man-Rogosa-Sharpe agar for LAB
  • PDA potato dextrose agar for total yeasts and moulds
  • VRBGA violet red bile glucose agar for Enterobacteriaceae
  • TA titratable acidity
  • TP total protein
  • ⁇ total color difference.
  • MRS de Man-Rogosa-Sharpe agar for LAB
  • PDA potato dextrose agar for total yeasts and moulds
  • VRBGA violet red bile glucose agar
  • TA titratable acidity
  • TP total protein
  • ⁇ total color difference
  • the total counts of yeast and moulds in the raw broccoli sample was 2 log CFU/g.
  • No fungi, moulds and enterobacteria were detected after fermentation or on the fermented samples after storage at both temperature conditions.
  • No pathogenic and spoilage organisms were detected following fermentation and during storage.
  • the results indicate that the fermentation process resulted in a safe and stable product with undetectable level of potentially pathogenic eneterobacteriaceae and spoilage yeast and mould, which maintained high levels of total lactic acid bacteria when stored at 4°C. There are ⁇ 10 6 CFU/g lactic acid bacteria after ⁇ 3 months at 4°C.
  • the pH and titratable acidity (TA) of raw broccoli, fermented broccoli and fermented broccoli after storage at 25°C and 4°C was analyzed as described in Example 1.
  • the determination of TA was used to estimate the amount of lactic acid and acetic acid, the main acids produced by lactic acid bacteria, during fermentation. During fermentation, the acids produced by the lactic acid bacteria decrease the pH of the sample. As shown in Table 1, the TA was increased to 10.7 g/L in Day 0 samples. When stored in 25°C, the pH was decreased to 3.87 during storage after 10 days, along with the significantly increased values of TA which reached 14.4 g/L (p ⁇ 0.05; see Table 1). The results indicate that there were still substrates present for lactic acid bacteria to consume and further produce acid during the early days of storage. Neither the pH nor TA value were significantly changed during the remaining storage period (Table 1).
  • Broccoli florets were cut into small pieces, mixed with water at 3:2 broccoli: water ratio and the mixture was macerated into a puree using a blender. Puree samples (200 gm) were aliquoted into sterile plastic bottles. The samples were inoculated at 10 8 CFU/gm with pooled culture of lactic acid bacteria Leuconostoc mesenteroides and Lactobacillus plantarum) isolated from Australian broccoli. Samples were incubated in a water bath maintained at 30°C until the pH dropped to -4.0, which was attained after four days of fermentation. Control non-inoculated samples were immediately frozen after maceration.
  • the total protein content and color of lactic acid fermented broccoli florets after fermentation was assessed as described above in the methods section. Compared to raw broccoli (26.9 ⁇ 0.03), the total protein content of fermented broccoli was significantly increased (29.6 ⁇ 0.8 mg/g; p ⁇ 0.05). This could be due to the high number of lactic acid bacteria inoculated into the sample and the growth during fermentation and protein synthesis by the lactic acid bacteria. The total protein content stayed stable during storage both at 25°C and 4°C (Table 1 and Table 2), with no significant difference between samples.
  • the color values (L, a, b) and the total color difference ( ⁇ ) of broccoli samples are summarized in Table 1 and Table 2. As presented in Table 1 and Table 2, significant differences in the color parameters and the total color difference value ( ⁇ ) were recorded between raw and fermented samples. The L * value (lightness) did not change significantly, whereas a * (greenness) and b * (yellowness) values decreased after the fermentation of broccoli puree. The decrease in a * and b * values may be attributed to the degradation in the color pigmented compounds, such as chlorophyll which would convert to pheophytins under the low pH. The high ⁇ value (12.5) of Day 0 sample indicate that the color of broccoli puree was significantly changed after fermentation, which was visually noticeable. During storage (Table 1 and Table 2) there was no significant change in the ⁇ value in neither 25 °C nor 4°C samples.
  • Example 6 Changes of total phenolic content and antioxidant activity of lactic acid bacteria in fermented broccoli florets
  • the total phenolic content (TPC) and antioxidant activity of lactic acid fermented broccoli florets after fermentation was assessed as described above in the methods section.
  • the TPC of raw broccoli was 127.6 ⁇ 12.4 mg GAE/100 g ( Figure 3A) of fresh weight.
  • the values of TPC on Day 0 significantly increased to 236.9 ⁇ 23.4 mg GAE/100 g (p ⁇ 0.05) compared to raw broccoli.
  • the value of TPC in fermented broccoli was 246.2 ⁇ 19.3 mg GAE/100 g on Days 10, and 248.1 ⁇ 25.0 mg GAE/100 g on Days 90.
  • the values of TPC was 274.1 ⁇ 20.2 and 267.2 ⁇ 3.3 mg GAE/100 g for Days 14 and Days 84, respectively.
  • the antioxidant activities of sample expressed as ORAC values are shown in Figure 3B.
  • the ORAC value of the raw sample was 1 10.1 ⁇ 0.05 ⁇ TE/g. Fermentation significantly increased the ORAC value by -70% to 186.9 ⁇ 3.3 ⁇ TE/g when compared to raw broccoli. This result suggested that antioxidant compounds may have increased during fermentation and was consistent with the change in TPC after fermentation.
  • Example 7 Assessment of fermentation time for different combinations of lactic acid bacteria
  • Lactic acid bacteria count ⁇ 10 8 CFU/gm
  • Example 8 Effect of storage on sulforaphane content of fermented broccoli
  • Figure 2 A shows the effects of storage at 4 and 25 °C on sulforaphane content of fermented broccoli puree.
  • the sulforaphane content of samples stored at 25°C dramatically decreased to 770.7 ⁇ 34.9 pmol/kg (a 52% loss) after 20 days storage, followed by a slower decline during the rest of the storage period, reaching a total loss of 69.5%.
  • no statistically significant change in sulforaphane content was observed during the first 2 weeks of storage of fermented broccoli samples at 4°C.
  • a significant decrease of -23.7 % occurred during the subsequent two weeks followed by a slow degradation during the rest of the storage period.
  • the sulforaphane content was 1012.9 ⁇ 57.6 pmol/kg in samples stored at 4°C, making the total loss of sulforaphane -37.4 % compared to the Day 0 samples.
  • the sulforaphane content during the first two weeks of storage was maintained perhaps due to simultaneous production and degradation of sulforaphane since some decrease in glucoraphanin content was observed in the 4°C stored samples over the same period.
  • Example 9 Effect of fermentation and storage on glucoraphanin content
  • Figure 7 shows the effect of maceration and fermentation on glucoraphanin content and its stability during storage at 4°C and 25 °C.
  • the glucoraphanin content of raw broccoli was 3423.7 ⁇ 39.7 ⁇ /kg ( Figure 7), After fermentation, the glucoraphanin content sharply decreased to 712.4 ⁇ 64.2 ⁇ /kg (Day 0 sample).
  • Glucoraphanin is relatively stable in intact tissue and the degradation in this case can be attributed to myrosinase catalyzed hydrolysis due to increased enzyme- substrate interaction in the macerated tissue during fermentation. The period of sharp decrease in glucoraphanin coincided with the fermentation period.
  • Example 10 Assessment of heat treatment conditions to maximise conversion of glucoraphanin into sulforaphane in broccoli matrix
  • Broccoli florets packed in retort pouches were subjected to thermal processing at temperatures ranging from 60°C to 80°C and treatment times of 0 to 5 minutes.
  • the treatment involved pre-heating to the experimental temperature in a water bath maintained at 5°C higher than the experimental temperature followed by incubation in a second water bath maintained at the experimental temperature.
  • samples were cooled in ice-water and were macerated with water added at 2:3 water to broccoli ratio as described above.
  • the macerated samples were incubated for 1 hr at 30°C and kept frozen until sulforaphane analysis. Results are shown in Figure 2B and Table 5.
  • pre-heating the sample at 60°C, 65°C or 80°C followed by maceration increased the sulforaphane yield relative to raw broccoli floret which was macerated without pre-heating.
  • Table 5 Effects of heat treatment on sulforaphane production in broccoli matrix.
  • Broccoli (cv. 'Viper') was purchased from a local supermarket (Coles, Werribee South, VIC, Australia).
  • DL- Sulforaphane was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). All the other chemical and biochemical reagents were analytical grade or higher and were purchased from local chemical vendors.
  • the treatment time were 0, 1 , 3, and 5 min for 60°C and 65°C and 0, 1, 2, 3 min for 80°C.
  • the direct water blanching experiments were conducted at 60°C and 65°C.
  • the temperature of the broccoli samples was continuously measured using a thermometer and timing started once the temperature at the slowest heating point attained the designated experimental temperature as described above. All thermal treatment experiments were carried out in triplicate. Unheated broccoli florets were used as controls.
  • the samples were cooled in ice water and were homogenized with Milli-Q water in ratio of 3 parts broccoli to 2 parts of water for 1 min using a kitchen scale magic bullet blender (Nutribullet pro 900 series, LLC, USA).
  • the homogenized samples were incubated in the dark for 4 h at 25°C to allow the enzymatic hydrolysis of glucoraphanin. After incubation, all the samples were frozen in -20°C until sulforaphane analysis.
  • the 6 secondary cultures were centrifuged, washed twice with sterile phosphate buffer saline (PBS) and each of the culture was resuspended in Milli-Q water at a concentration of 10 log colony- forming units per millilitre (CFU/mL) to obtain an initial biomass of 8 log CFU/mL in 100 gm broccoli puree samples.
  • PBS sterile phosphate buffer saline
  • the L. plantarum cultures were mixed with the L. mesenteroides cultures at 1 :1 proportion prior to inoculation into the broccoli puree samples.
  • Broccoli florets were cut at approximately 2 cm below the crown and were separated into two lots; heat treated and non-treated. After heat treatment at the optimal condition selected based on the results of the experiments as described above, the samples were cooled in ice-water, shredded and homogenized with Milli-Q water in ratio of 3:2 for 1 min using a kitchen scale magic bullet blender (Nutribullet pro 900 series, LLC, USA). The non-treated broccoli were also homogenized in a similar way. The broccoli puree, after mixing well, was aliquoted into sterile plastic containers (100 mL) with screw lids (Technoplast Australia) for further experiments.
  • each broccoli puree sample was inoculated with the prepared starter culture at an initial level of 8 log CFU/g.
  • the fermentation experiment was carried out at 30°C until the pH reached ⁇ 4.0 after 15 hrs of incubation.
  • 3 samples (day 0 samples) of each fermented group were taken and stored at -20°C until analysis.
  • the rest of the ferments were randomly separated into two lots for the storage trials: one lot was stored under refrigerated condition (4°C) and the second lot was stored at 25°C for the assessment of the sulforaphane stability of the samples after 14 days storage.
  • the untreated broccoli puree, preheated broccoli puree and the preheated-GDL treated broccoli puree were also sampled at time zero and stored at 25 and 4°C for the 14 days storage trials. After 14 days storage, all the samples were frozen and kept at -20°C until sulforaphane analyses.
  • the concentration of sulforaphane in these samples were 2343.5 ⁇ 124.1, 2661.5 ⁇ 10.9, 2780.9 ⁇ 270.8, and 3147.7 ⁇ 148.0 ⁇ /kg DW, respectively.
  • the sulforaphane yield initially increased with processing time from 3585.9 ⁇ 119.2 (0 min) to the highest value of 3983.4 ⁇ 30.5 ⁇ /kg DW (3 min). Further increase in treatment time resulted in lower yield with the lowest value of 3620.1 ⁇ 240 ⁇ mol/kg observed after 5 min treatment time.
  • the sulforaphane yield was 503.7 ⁇ 23.8 ⁇ /kg DW of broccoli after 5 min thermal treatment at 65°C, which was even lower than the value obtained for raw broccoli.
  • the reason could be the leaching of glucoraphanin into the blanching water resulting in low yield of sulforaphane.
  • the optimum treatment temperature for maximizing sulforaphane yield was 60°C compared to 65°C for the in-pack processing.
  • Broccoli florets were pre-heated in-pack at the best treatment condition selected above (65°C, 3 min). Samples were then either fermentation by lactic acid bacteria or acidified using the acidulant (GDL). Consistent with the pre-treatment experiments, the sulforaphane value of broccoli significantly increased (p ⁇ 0.05) after the heat treatment; with 806.2 ⁇ 7.0 ⁇ /kg DW and 3536.0 ⁇ 136.9 ⁇ /kg DW of sulforaphane yield for raw and pre-heated broccoli, respectively. The value of 3536 ⁇ /kg DW obtained with this separate batch of broccoli preheated prior to fermentation is of the same order obtained when a different batch of broccoli was used, where 3983 ⁇ /kg DW was obtained indicating slight batch to batch variation.
  • the sulforaphane content of broccoli samples varied depending on the treatment of the broccoli prior to fermentation.
  • the sulforaphane content of raw broccoli puree after fermentation (1617.4 ⁇ 10.2 ⁇ 1/ ⁇ 3 ⁇ 4 DW) was approximately twice the sulforaphane content of raw broccoli puree.
  • Pre-heating of broccoli prior to pureeing resulted in much higher increase in sulforaphane content after fermentation.
  • the sulforaphane content of preheated-fermented broccoli 13121.3 ⁇ 440.8 ⁇ /kg DW was about 8 times of the raw-fermented broccoli puree.
  • the observed sulforaphane yield after the combined preheating- fermentation treatment is much higher than what would be expected based on the quantifiable amount of glucoraphanin (3423.7 ⁇ 39.7 ⁇ /kg) in the raw broccoli sample. It seems that the combined preheating and fermentation process enhances the release and accessibility of glucoraphanin for conversion over and above the inactivation of ESP by the pre-heating process.
  • the pre-heating process coupled with microbial cell wall degrading enzymes may have enhanced the disruption of the cell compartment and release of bound glucosinolates in the matrix, that were not extractable or accessible in the raw broccoli.
  • Some lactic acid strains produce polysaccharide degrading enzymes such as cellulases and pectinases capable of degrading the cell wall structure and enhance the release of wall bound components.
  • the acidification occurs gradually over a period of > 15 hi- enabling the conversion of glucoraphanin mainly to sulforaphane since the activity of ESP is expected to be significantly reduced after preheating at 65°C for 3 min.
  • the best preheating condition during direct water blanching was 1 min at 60°C and resulted in sulforaphane yield of 2833 ⁇ /KgDW.
  • the lower yield during direct blanching can be attributed to leaching of the water- soluble glucoraphanin into the blanching media.
  • Preheating of broccoli florets in-pack (65°C/3min) combined with lactic acid bacteria fermentation further enhanced the sulforaphane content to 13121 ⁇ /KgDW, which is ⁇ 16 times increase compared to raw broccoli.
  • DW dry weight
  • GDL acidified using glucono-delta-lactone. Preheating was conducted at 6 °C in pack for 3 minutes.
  • LC-MS liquid chromatography-mass spectrometry
  • the top 15 metabolites that were identified to be responsible for the differences between the two groups are shown in Figure 9. They are phenolic acids and phenolic aglycones, with higher bioactivity and bioavailability compared to their phenolic acid ester and phenolic glycoside precursors. The concentrations of most of these metabolites showed substantial increase following fermentation indicating the beneficial effect of ferendingation on the polyphenol profile of broccoli puree. The fold changes for some of the metabolites are shown in Table 7.
  • the concentrations of hesperetin, quercetin, methyl syringate and syringic acid also increased substantially after fermentation.
  • the increase in the concentration of aglycones such as kaempferol, hesperetin and quercetin can be attributed to conversion of their glycoside precursors by the activity of microbial glycosidases.
  • the increase in the concentration of phenolic acids such as sinapic acid could be due to the conversion of phenolic acid esters in broccoli by the activity of microbial esterases.
  • caffeic acid and gallic Some decrease in caffeic acid and gallic was observed following fermentation.
  • the activity of microbial decarboxylases convert caffeic acid into the corresponding vinyl catechol and gallic acid into pyrgallol, which may be responsible for the decrease in their concentration (Filanino et al., 2015; Guzman-Lopez et al., 2009).
  • Example 13 Identification of metabolites produced by lactic acid bacteria fermentation of broccoli by targeted and untargeted LC MS analyses of samples
  • the fermented and non-fermented broccoli puree samples were frozen and freeze dried.
  • the samples (100 mg freeze dried powder each) were extracted using 1 ml of ice-cold methanol and Milli-Q water (50:50, v:v), which comprised 100 mg/ml of caffeine as an internal standard.
  • the samples were then vortexed for 2 minutes prior to being sonicated (40 Hz) for 30 minutes.
  • Samples were then centrifuged at 20,000 rpm at 4°C for 30 minutes, and the supernatant transferred to clean silanised LC-MS vials.
  • Samples were analyzed by injecting 1.4 ⁇ into an Agilent 6410 LC-QQQ HPLC (Agilent Technologies, Santa Clara, California, USA).
  • the analyses were performed using a reversed-phase Agilent Zorbax Eclipse Plus CI 8, Rapid Resolution HD, 2.1 x 50 mm, 1.8 um (Agilent Technologies, Santa Clara, California, USA), with a column temperature of 30°C and a flow rate of 0.3 ml/min.
  • the mobile phase was operated isocratically for 1 min 95:5 (A:B) then switched to 1 :99 (A:B) for a further 12 min before returning back to 95:5 (A:B) for an additional 2 min; providing a total run time of 15 min.
  • Mobile phase 'A' consisted of 100% H 2 0 and 0.1% formic acid, and mobile phase 'B' contained 75% acetonitrile, 25% isopropanol and 0.1% formic acid.
  • the MS was collecting data in the mass range 50-1000 m/z.
  • Qualitative identification of the compounds was performed according to the Metabolomics Standard Initiative (MSI) Chemical Analysis Workgroup using several online LC-MS metabolite databases, including Massbank and METLIN. Overall, the instrumental conditions were similar for both positive electrospray (+ESI) and negative electrospray (-ESI) modes.
  • Scan time was 500, the source temperature was maintained at 350°C, the gas flow was 12 L/min and the nebuliser pressure was 35 psi.
  • the criteria was set at >90% match rate. Where the match rate dropped to between 70-89%, the compounds are identified with brackets (for example, if a compound was between 70- 89% they are annotated as " ⁇ name>"). Any matches below 70% were removed. In total, there was ca. 1000-1500 fatures to identify; many were poorly matched (and removed) or were less than 10 x S/N ratio from the baseline. As such, the compounds/peaks used were actual peaks and the IDs are fairly strong (i.e. >70%).
  • CDP-choline 617.84 9.2711 0.073728 1.1324 adenine 2.0623 1.0442 0.074004 1.1307 raphanusamate 5.5593 2.4749 0.074387 1.1285
  • LC-MS liquid chromatography-mass spectrometry
  • E. coli isolates FSAW 1310, FSAW 1311, FSAW 1312, FSAW 1313 and FSAW 1314 were grown separately to 1-4 xlO 8 cfu/mL in NB (nutrient broth) overnight at 37°C, static. The cultures were combined (1 mL of each) and the combined culture diluted to 10 4 with MRD (maximum recovery diluent) for first two dilutions and water for last two dilutions.
  • MRD maximum recovery diluent
  • Salmonella strains S. Infantis 1023, S. Singapore 1234, S. Typhimurium 1657 (PT135), S. Typhimurium 1013 (PT9) and S. Virchow 1563 were grown separately to 1- 4 xlO 8 cfu/mL in NB overnight at 37°C, static. The cultures were combined (1 mL of each) and combined culture diluted to 10 4 with MRD for first two dilutions and water for last two dilutions.
  • Listeria isolates Lm2987 (7497), Lm2965 (7475), Lm2939 (7449), Lm2994 (7537) and Lm2619 (7514) were grown separately in 10 mL BHI (brain heart infusion broth) overnight at 37°C under agitation. All cultures were then combined (1 mL of each) and this cocktail was diluted using MRD for first two (1/10) dilutions and sterile deionised water for last two dilutions.
  • Broccoli puree was prepared prior to preparing the inoculums, Broccoli: Sterile Tap Water 3:2 (900 g broccoli: 600 g water). Broccoli heads were rinsed in tap water, the stalks were cut off the broccoli with a sterile knife on a cutting board sanitised with 80% ethanol. Broccoli florets (900 g) were cut into small pieces. 450 g of broccoli pieces were placed into Thermomix bowl with all 600 g of the water. The translucent Thermomix cup/lid was sanitised with 80 % ethanol and placed over the lid hole. The broccoli was chopped at speed 4 for 1 min.
  • the second 450 g of broccoli pieces were added to the Thermomix bowl and chopped at speed 4 for 1 min. The contents were chopped for a further 5 min at speed 10 (max). After making sure the puree was indeed smooth enough, the Thermomix bowl was placed in the cool room to cool down the contents for 30 min. Following this, the bowl was put in the incubator and equilibrated to 30°C. Meanwhile the starter culture and pathogen culture (E. coli, B. cereus, Salmonella, Listeria monocytogenes) were prepared. 10 mL of LAB culture and 7.5 mL of the 10-4-diluted challenge microorganism cocktail (10 4 cfu/mL culture in water) were added into the broccoli puree (10 5 of B. cereus).
  • starter culture and pathogen culture E. coli, B. cereus, Salmonella, Listeria monocytogenes
  • Foil was held down over the large hole in the Thermomix lid prior to mixing culture.
  • the cultures were mixed into the puree for 1 min on maximum speed.
  • the heat setting for the Thermomix was switched off and the Thermomix was placed inside the 30°C incubator and the fermentation started at 10:45 am. pH and temperature measurements were taken every hour up until 7 h (end of work time) after mixing the puree for 1 min speed 4.5.
  • the pH meter was calibrated and sanitised using 80% ethanol.
  • the temperature probe was also sanitised prior to measurements with 80% ethanol.
  • the growth of the challenge microorganisms was assessed by counts on growth on the selective media MRS, DRBX and NA +S of raw broccoli, before fermentation (TO) and after fermentation commenced at 4 hours (T4) and 22 hours (T22).
  • the yeast and mould were significantly reduced by 4 hours, and were not detected at the end of fermentation (T22). E. coli and Salmonella were never detected at the end of fennentation (T22). Listeria was detected in low numbers at the end of fermentation, with a starting inoculum just over 10 3 cfu/mL. B. cereus spores were generally not affected by the fermentation, but did not germinate.
  • the result of the challenge study indicates that the lactic acid bacteria strains that we isolated from broccoli are able to completely inactivate Salmonella and E. coli and inhibit the growth of the most acid resistant strains of Listeria. They are also able to inhibit the sporulation of B. cerus spores. Table 9. Example of microbial challenge study with E. coli. E.
  • coli (mix of 5 E. coli strains EC1605, EC1606, EC1607, EC1608 inoculated (2.2 xl02 CFU/gm) into the macerated broccoli (3:2 broccoli-water ratio) ferment to evaluate if the fermentation starter (a consortia of Bl, B2, B3, B4, B5, BF1, BF2) inhibits the growth of E.coli. Experiments were repeated three times. Fermentation was conducted at 30°C for 22 hrs to pH below 4.0.
  • Salmonella A mix of 5 strains S. Infantis 1023, S. Singapore 1234, S. Typhimurium 1657 (PT135), S. Typhimurium 1013 (PT9), S. Virchow 1623) inoculated (1.1 x 103) into macerated broccoli (3:2 broccoli-water ratio) ferment to evaluate if the fermentation starter (a consortia of Bl, B2, B3, B4, B5, BF1, BF2) inhibits the growth of Salmonella. Experiments were repeated three times. Fermentation was conducted at 30°C for 22 hrs to pH below 4.0.
  • Listeria monocytogenes (A mix of 5 strains Lm2987 (7497), Lm2965 (7475), Lm2939 (7449), Lm2994 (7537), Lm2919 (7514)) inoculated (1.9 xl03) into macerated broccoli (3:2 broccoli-water ratio) ferment to evaluate if the fermentation starter (a consortia of Bl, B2, B3, B4, B5, BF1, BF2) inhibits the growth of acid resistant Listeria. Experiments were repeated three times and the final Listeria count at the end of fermentation ranged from ⁇ 10 (undetected) to 1.1 x 10 2 CFU/gm. Fermentation was conducted at 30°C for 22 hrs to pH below 4.0. Time (hrs) Lactic acid Yeast and mould Listeria (CFU/gm) bacteria (CFU/gm) (CFU/gm) (CFU/gm)
  • Isolates were centrifuge at 3500 g for 10 min and the supernatant discarded.
  • the pellet was mixed and washed with 5 mL deionised water and centrifuged at 3500 g for 10 min and the supernatant discarded.
  • the pellet was mixed with 5 mL TES (1 mM EDTA, 10 mM Tris-HCl, 0.5 M saccharose) and vortexcd. Next the samples were centrifuged at 3500 g for 15 min and the supernatant discarded.
  • the plugs were heated in 100 mL of sterile deionised water at 55 °C, the deproteinisation solution was removed and the plugs transferred to 15 mL centrifuge tubes, washed with 4 mL of sterile deionised water and heated to 55 °C for 10 min at room temperature followed by washing four times with 4 mL TE buffer for 10 min at room temperature.
  • TBE buffer The buffer was removed and the slices loaded onto comb, with the ladder in every five lanes.
  • 1.0 % ultra-pure DNA grade agarose (pulsed field certified agarose) was prepared in 0.5X TBE running buffer.
  • Example 16 Variant analysis of Leuconostoc mesenteroides and Lactobacillus plantarum isolates

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WO2025078970A1 (en) 2023-10-09 2025-04-17 Universidade Católica Portuguesa - Ucp Green method for obtaining isothiocyanates from cruciferous vegetables
CN117481281B (zh) * 2023-11-15 2024-11-05 江南大学 一种发酵贝塔菜汁及其制备方法
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CN111269850B (zh) * 2020-01-20 2022-07-22 吉林农业大学 一株具有高黏附能力及降血脂功效的戊糖片球菌pp04
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