Isothiocyanate-containing brassicaceae product and method of preparation thereof

Abstract

To provide methods for producing isothiocyanate-containing products from Brassicaceae material and lactic acid bacteria for use in such methods, and provide isothiocyanate-containing products from Brassicaceae material produced by such methods.SOLUTION: A method comprises: the step i) for pre-treating a Brassicaceae material to improve the access of myrosinase to a glucosinolate; and the step ii) for fermenting the material obtained by the step i) with lactic acid bacteria, forming an isothiocyanate-containing product.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing an isothiocyanate-containing product from Brassicaceae material and to lactic acid bacteria for use in such a method. The present invention also relates to an isothiocyanate-containing product from Brassicaceae material produced by such a method. [Background technology]

[0002] Members of the Brassicaceae family are rich in glucosinolates, which can be converted to isothiocyanates by the enzyme myrosinase, and isothiocyanates have been shown to have beneficial effects against several types of cancer (Moktari et al., 2017, Capuano et al., 2017, Kim and Park, 2016). For example, sulforaphane has been found to reduce hepatic glucose production and improve glucose control in obese patients with type 2 diabetes (Axelsson et al., 2017). However, many members of the Brassicaceae family are very perishable after harvest, and if the product is not stored properly, the quality and quantity of nutrients deteriorate rapidly.

[0003] Brassicaceae are often processed to extend their shelf life, which can lead to nutrient loss. The main methods for achieving longer shelf lives include heat treatment, freezing, modification, storage in controlled atmospheres, and the addition of chemical preservatives, which can also result in undesirable changes in chemical composition.

[0004] These processes can lead to the loss of glucosinolates or reduce the ability of myrosinase, the enzyme that converts glucosinolates to isothiocyanates. For example, conventional broccoli processing / preservation includes blanching before freezing to inactivate quality-degrading enzymes such as lipoxygenase. Inactivation of peroxidase is commonly used as an indicator of the validity of blanching. Conditions for peroxidase inactivation lead to myrosinase inactivation, and therefore the resulting product will lack isothiocyanates (Dosz and Jeffery, 2013).

[0005] Therefore, improved methods for producing Brassicaceae products containing plant nutrients such as isothiocyanates are still needed. [Overview of the project]

[0006] The inventors have developed a method for preparing isothiocyanate-containing products from Brassicaceae materials.

[0007] In one embodiment, the present invention provides a method for preparing an isothiocyanate-containing product from Brassicaceae materials, comprising: i) Pretreatment of Brassicaceae material to improve the access of myrosinase to glucosinolate, ii) Ferment the material obtained in step i) with lactic acid bacteria to form an isothiocyanate-containing product.

[0008] In one embodiment, the pretreatment is as follows: i) heating; ii) immersion; iii) Microwave processing, iv) Exposure to high-frequency sound waves (ultrasound), or v) Pulse electric field processing Includes one or more of the following: The temperature of the Brassicaceae material should not exceed approximately 75°C during pretreatment.

[0009] In one embodiment, the pretreatment reduces the activity of epithiospecifier protein (ESP) while maintaining the endogenous myrosinase activity.

[0010] In one embodiment, the pretreatment includes heating and immersing a Brassicaceae material, and the temperature of the Brassicaceae material does not exceed about 75 °C during the pretreatment. In one embodiment, the heating is performed before immersion or the heating and immersion are performed simultaneously. In one embodiment, the pretreatment includes heating the Brassicaceae material to a temperature of about 50 °C to about 70 °C and then immersing it. In one embodiment, the Brassicaceae material is immersed such that at least about 80% of the Brassicaceae material has a size of about 2 mm or less. In one embodiment, the Brassicaceae material is heated in a sealed package.

[0011] In one embodiment, the isothiocyanate-containing product contains at least about 10 times more isothiocyanates than the immersed Brassicaceae material.

[0012] In one embodiment, the isothiocyanate-containing product contains at least about 12 times more isothiocyanates than the immersed Brassicaceae material.

[0013] In one embodiment, the isothiocyanate-containing product contains at least about 14 times more isothiocyanates than the immersed Brassicaceae material.

[0014] In one embodiment, the isothiocyanate-containing product contains at least about 16 times more isothiocyanates than the immersed Brassicaceae material.

[0015] In one embodiment, the isothiocyanate-containing product contains at least about 2 times the expected maximum yield of isothiocyanates based on the extractable glucosinolate content.

[0016] In one embodiment, the lactic acid bacteria are isolated from broccoli and / or the lactic acid bacteria lack myrosinase activity.

[0017] In one embodiment, the present invention provides a method for preparing an isothiocyanate-containing product from Brassicaceae materials, comprising: i) Pretreatment of Brassicaceae material to improve the approach of myrosinase to glucosinolate, and ii) Acidify the material obtained in step i) to form an isothiocyanate-containing product.

[0018] In one embodiment, the present invention provides a method for preparing an isothiocyanate-containing product from broccoli material, comprising fermenting the material with lactic acid bacteria Leuconostoc mesenteroides and / or Lactobacillus plantarum to form an isothiocyanate-containing product, the method optionally comprising pre-treating the broccoli material to improve the access of myrosinase to glucosinolates.

[0019] In one embodiment, the present invention provides a method for preparing an isothiocyanate-containing product from Brassicaceae material, comprising fermenting the material with lactic acid bacteria Leuconostoc mesenteroides and / or Lactobacillus plantarum isolated from broccoli to form an isothiocyanate-containing product, the method optionally comprising pre-treating the Brassicaceae material to improve the access of myrosinase to glucosinolates.

[0020] In one embodiment, the present invention provides isolated strains of lactic acid bacteria selected from the following: i) BF1, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021729, and ii) BF2 deposited with the National Measurement Institute Australia on September 25, 2017, under the code V17 / 021730.

[0021] In one embodiment, the present invention provides isolated strains of lactic acid bacteria selected from the following: i) On September 25, 2017, BF1 was deposited with the National Measurement Institute Australia under the code V17 / 021729. ii) On September 25, 2017, BF2 was deposited with the National Measurement Institute Australia under V17 / 021730. iii) On September 25, 2017, B1 was deposited with the National Measurement Institute Australia under V17 / 021731. iv) On September 25, 2017, B2 was deposited with the National Measurement Institute Australia under V17 / 021732. v) On September 25, 2017, B3 was deposited with the National Measurement Institute Australia under V17 / 021733. vi) B4, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021734, and vii) B5 deposited with the National Measurement Institute Australia on September 25, 2017, under the name V17 / 021735.

[0022] In one embodiment, the present invention provides a starter culture for producing an isothiocyanate-containing product or probiotic, comprising lactic acid bacteria selected from one or more or all of the following: i) On September 25, 2017, BF1 was deposited with the National Measurement Institute Australia under the code V17 / 021729. ii) On September 25, 2017, BF2 was deposited with the National Measurement Institute Australia under V17 / 021730. iii) On September 25, 2017, B1 was deposited with the National Measurement Institute Australia under V17 / 021731. iv) On September 25, 2017, B2 was deposited with the National Measurement Institute Australia under V17 / 021732. v) On September 25, 2017, B3 was deposited with the National Measurement Institute Australia under V17 / 021733. vi) B4, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021734, and vii) B5 deposited with the National Measurement Institute Australia on September 25, 2017, under the name V17 / 021735.

[0023] In one embodiment, the starter culture is at least about 10 8 Contains lactic acid bacteria at a concentration of cfu / mL.

[0024] In one embodiment, the present invention provides a probiotic composition comprising lactic acid bacteria selected from one or more or all of the following: i) On September 25, 2017, BF1 was deposited with the National Measurement Institute Australia under the code V17 / 021729. ii) On September 25, 2017, BF2 was deposited with the National Measurement Institute Australia under V17 / 021730. iii) On September 25, 2017, B1 was deposited with the National Measurement Institute Australia under V17 / 021731. iv) On September 25, 2017, B2 was deposited with the National Measurement Institute Australia under V17 / 021732. v) On September 25, 2017, B3 was deposited with the National Measurement Institute Australia under V17 / 021733. vi) B4, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021734, and vii) B5 deposited with the National Measurement Institute Australia on September 25, 2017, under the name V17 / 021735.

[0025] In one embodiment, the present invention provides an isothiocyanate-containing product obtained by a method as described herein.

[0026] In one embodiment, the present invention provides an isothiocyanate-containing product that can be obtained by a method as described herein.

[0027] In one embodiment, the present invention provides an isothiocyanate-containing Brassicaceae product that contains at least about 10 times more isothiocyanates than the immersed Brassicaceae material.

[0028] In one embodiment, the present invention provides an isothiocyanate-containing Brassicaceae product that contains about 10 to 16 times more isothiocyanates than the immersed Brassicaceae material.

[0029] In one embodiment, the present invention provides an isothiocyanate-containing Brassicaceae product containing at least about twice the expected maximum yield of isothiocyanates based on the extractable glucosinolate content.

[0030] In one embodiment, the present invention provides an isothiocyanate-containing Brassicaceae product containing approximately 2 to 4 times the expected maximum yield of isothiocyanates, based on the extractable glucosinolate content.

[0031] In one embodiment, the present invention provides an isothiocyanate-containing Brassicaceae product containing at least 150 mg / kg dw of isothiocyanate.

[0032] In one embodiment, the present invention provides an isothiocyanate-containing product containing 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 of sulforaphane.

[0033] In one embodiment, the isothiocyanate-containing product includes Leuconostoc mesenteroides and / or Lactobacillus plantarum.

[0034] In one embodiment, the isothiocyanate-containing product has one or more or all of the following characteristics: i) When stored at approximately 4°C to 25°C, it remains stable for at least 4 weeks, or at least 8 weeks, or at least 12 weeks. ii) When stored at approximately 4°C to approximately 25°C, it will be resistant to the growth of yeast, mold and / or E. coli for at least 4 weeks, or at least 8 weeks, or at least 12 weeks, and iii) at least 10 7 Contains CFU / g of Leuconostoc mesenteroides and / or Lactobacillus plantarum.

[0035] Any embodiment described herein shall be deemed to apply mutatis mutandis to any other embodiment unless otherwise specifically stated. For example, the example of lactic acid bacteria outlined in relation to the method of the present invention, as will be understood by those skilled in the art, is equally applicable to the product of the present invention.

[0036] The present invention is not limited in scope by the specific embodiments described herein, which are intended solely for illustrative purposes. Functionally equivalent products, compositions, and methods clearly fall within the scope of the present invention as described herein.

[0037] Throughout this specification, unless otherwise specifically provided or requested by the context, references to a single step, composition, group of steps, or group of compositions shall be understood to encompass one and more (i.e., one or more) such steps, compositions, group of steps, or group of compositions.

[0038] The present invention will be described hereafter using the following non-limiting embodiments and with reference to the accompanying figures. [Brief explanation of the drawing]

[0039] [Figure 1]A shows the hydrolysis pathway from glucoraphanin to sulforaphane and sulforaphane nitrile. B shows the effect of immersion and fermentation on the sulforaphane content (mg / kg, DW) in broccoli puree. C shows the effect of fermentation on the lactic acid bacteria content (log CFU / gm) of broccoli puree during storage. [Figure 2] A shows the effect of fermentation on the stability of sulforaphane in broccoli puree stored at 4°C and 25°C (RT). B shows the effect of heat treatment conditions on the conversion of glucoraphanin to sulforaphane in the broccoli matrix. [Figure 3] A shows the total phenol content (mg GAE / 100g DW) of raw broccoli, as well as the changes in total phenol content during fermentation and storage at 25°C and 4°C. B shows the ORAC (oxygen radical absorption capacity) antioxidant capacity (μmol TE / g DW) of raw broccoli, as well as the changes in ORAC during fermentation and storage at 25°C and 4°C. [Figure 4] This shows the fermentation time required for different combinations of lactic acid bacteria strains to reach a pH of 4.4 or below. [Figure 5] A shows the sulforaphane yield (μmol / kg DW) of broccoli under different heat treatment conditions using sealed bags. B shows the sulforaphane yield (μmol / kg DW) of broccoli directly immersed in water under different heat treatment conditions. [Figure 6] This study compares the combined effects of immersion, preheating, and fermentation on sulforaphane yield (μmol / kg DW) immediately after treatment and during storage at 4°C and 25°C, with the effects of immersion and preheating alone, and immersion, preheating, and chemical acidification. Samples were pre-treated in sealed packs at 65°C for 3 minutes. [Figure 7] This study shows the effects of fermentation and storage on glucoraphanin content. Glucoraphanin content decreases in fermented samples stored at 25°C and 4°C compared to raw samples. [Figure 8]PLS-DA score plot showing the difference in polyphenol metabolite profiles between raw and fermented broccoli puree. [Figure 9] Key features that distinguish fermented and non-fermented samples identified by PLS-DA. The box on the right shows the relative concentration of each metabolite in each group. [Figure 10] This study demonstrates the effect of lactic acid fermentation on the metabolite profile of broccoli puree based on non-targeted LC-MS analysis. It shows that fermentation releases bound phytochemicals such as polyphenol glycosides and glucosinolates, improving their bioaccessibility. [Figure 11] Based on non-targeted LC-MS analysis, a volcano plot is shown indicating metabolites that exhibited a significant (p<0.05) multiplier change after fermentation. Table 8 lists the top 50 metabolites with significant multiplier changes and their individual multiplier changes. [Figure 12] The effects of lactic acid fermentation on broccoli polyphenols are shown based on targeted LC-MS analysis. A 6.6-fold change was observed in chlorogenic acid (2.4-15.8 μg / mg), a 23.8-fold increase in sinapic acid (3.6-86.6 μg / mg), a 10.5-fold increase in kaempferol (12.7-134.6 μg / mg), and a 0.48-fold decrease in p-coumaric acid. [Figure 13] This shows the SmaI and NotI restriction enzyme digestion of genomic DNA from BF1 and BF2 obtained by pulsed-field gel electrophoresis. [Modes for carrying out the invention]

[0040] General Techniques and Definitions Unless otherwise specified, all technical and scientific terms used herein shall be deemed to have the same meaning as those generally understood by those skilled in the art (e.g., enzyme, fermentation, inoculation).

[0041] The term "and / or," for example, "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be deemed to provide explicit support for both meanings or either meaning.

[0042] Throughout this specification, it will be understood that variations of the word "comprise" (including), "comprises" (including), or "comprising" (including) encompass a defined element, integer, or step, or group of elements, integers, or steps, but do not exclude any other element, integer, or step, or group of elements, integers, or steps.

[0043] As used herein, the term “about” means, unless otherwise stated, + / - 10%, more preferably + / - 5%, and even more preferably + / - 1% of the specified value.

[0044] An "allele" refers to a specific form of a gene sequence (such as a gene) within a cell, an individual plant, or a population, which differs from other forms of the same gene in at least one, and often two or more, variant sites within the gene sequence. These variant site sequences that differ between different alleles are called "dispersion," "polymorphism," or "variation."

[0045] Brassicaceae Those skilled in the art will understand that the methods described herein are suitable for producing isothiocyanate-containing products from any Brassicaceae material containing glucosinolates. As used herein, “Brassicaceae” refers to members of the Brassicaceae family, commonly known as mustard, cruciferous plants, or cabbage. Those skilled in the art will understand that the material may be derived from two or more Brassicaceae species.

[0046] In one embodiment, Brassicaceae is selected from the genera Brassica or Cardamine. In one embodiment, 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 tournefortii.

[0047] In one embodiment, Brassica is Brassica oleracea.

[0048] In one embodiment, Brassica refers to Brassica oleracea variety oleracea (wild cabbage), Brassica oleracea variety capitate (cabbage), Brassica rapa subspecies chinensis (bok choy), Brassica rapa subspecies pekinensis (Chinese cabbage), Brassica napobrassica (rutabaga), Brassica rapa variety rapa (turnip), Brassica oleracea variety alboglabra (kailan), Brassica oleracea variety viridis (collared greens), Brassica oleracea variety longata (jersey cabbage), Brassica oleracea variety acephala (ornamental cabbage), Brassica oleracea variety sabellica (kale), Brassica oleracea variety palmifolia (cavolo nero), and Brassica oleracea variety ramose (perpetual kale). The following varieties are selected: kale, Brassica oleracea variety medullosa (marrow cabbage), Brassica oleracea variety costata (tronchuda kale), Brassica oleracea variety gemmifera (Brussels sprouts), Brassica oleracea variety gongylodes (kohlrabi), Brassica oleracea variety italica (broccoli), Brassica oleracea variety botrytis (cauliflower, romanesco, broccoli di torbole), Brassica oleracea variety botrytis × italica (broccoli flower), and Brassica oleracea variety italica × alboglabra (stick senorita).

[0049] In one embodiment, Brassica refers to Brassica oleracea variety italica (broccoli).

[0050] In one embodiment, Brassicaceae includes Cardamine hirsuta, Iberis sempervirens, Sinapis arvensis, Armoracia rusticana, Pringlea antiscorbutica, Thlaspi arvense, Raphanus raphanistrum subspecies sativus, Eruca sativa, Anastatica hierochuntica, Crambe maritima, Cakile maritima, Capsella bursa-pastoris, sweet alyssum, Arabidopsis thaliana, Nasturtium officinale, and Sinapis The following are selected: alba (white mustard), Erophila verna (whitlow grass), Raphanus raphanistrum (wild radish), Isatis tinctoria (narrow-leaved woad), and Nasturtium microphyllum (yellow cress).

[0051] In one embodiment, Brassicaceae has high levels of one or more glucosinolates. In one embodiment, Brassicaceae is selectively crossbred to have high levels of one or more glucosinolates. In one embodiment, "high levels" of glucosinolates may include levels higher than those shown in Table 2 of Verkerk et al. (2009) in the corresponding Brassicaceae. In one embodiment, high levels of glucosinolates are levels higher than 3400 μmol / kg dry weight. In one embodiment, high levels of glucosinolates are levels higher than 4000 μmol / kg dry weight. In one embodiment, high levels of glucosinolates are levels higher than 5000 μmol / kg dry weight. In one embodiment, high levels of glucosinolates are levels higher than 8000 μmol / kg dry weight. In one embodiment, a high level of glucosinolate is a level of glucosinolate higher than 10,000 μmol / kg dry weight. In one embodiment, a high level of glucosinolate is a level of glucosinolate higher than 12,000 μmol / kg dry weight. In one embodiment, a high level of glucosinolate is a level of glucosinolate higher than 15,000 μmol / kg dry weight. In one embodiment, a high level of glucosinolate is a level of glucosinolate higher than 18,000 μmol / kg dry weight. In one embodiment, a high level of glucosinolate is a level of glucosinolate higher than 20,000 μmol / kg dry weight. In one embodiment, a high level of glucosinolate is a level of glucosinolate higher than 25,000 μmol / kg dry weight. In one embodiment, a high level of glucosinolate is a level of glucosinolate higher than 30,000 μmol / kg dry weight. In one embodiment, Brassicaceae are genetically modified to have high levels of one or more glucosinolates, or are subjected to biological or abiotic stress.A person skilled in the art will understand that Brassicaceae can be modified by any method known to a person skilled in the art.

[0052] In one embodiment, the glucosinolate is glucoraphanin (4-methylsulfinylbutyl). In another embodiment, the glucosinolate is glucobrassin (3-indolylmethyl).

[0053] As used herein, “Brassicaceae material” refers to any part of Brassicaceae containing glucosinolates, including, but not limited to, leaves, stems, flowers, clusters, seeds, and roots or mixtures thereof.

[0054] Those skilled in the art will understand that the methods described herein are suitable for use with different volumes of 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.

[0055] In one embodiment, the Brassicaceae material is washed. As used herein, “washed” means the removal of visible soil and contaminants. In one embodiment, the Brassicaceae material is sterilized. As used herein, “sterilized” means a reduction in pathogens on the Brassicaceae material.

[0056] In one embodiment, Brassicaceae is mixed with other plant material. In one embodiment, the other plant material is vegetable or fruit material. In one embodiment, the vegetable is carrot or beetroot.

[0057] Glucosinolates As used herein, “glucosinolate” refers to secondary metabolites found in at least the Brassicaceae family, which share a chemical structure consisting of a (Z)-N-hydroxyiminosulfate ester via a sulfur atom and a β-D-glucopyranose residue linked to a variety of R groups derived from amino acids, as described by Halkier et al. (2006). Examples of glucosinolates are provided in Halkier et al. (2006) and Agerbirk et al. (2012). Hydrolysis of glucosinolates can produce isothiocyanates, nitriles, epithionitriles, thiocyanates, and oxazolidine-2-thione (Figure 1A). Many glucosinolates play a role in plant defense mechanisms against pests and diseases.

[0058] Glucosinolates are stored in storage sites within Brassicaceae. As used herein, “storage site” refers to a site within Brassicaceae where glucosinolates are present but myrosinase is not.

[0059] As used herein, “myrosinase” is also referred to as “thioglucosidase,” “siniglase,” or “sinigrinase,” and refers to a family of enzymes (EC 3.2.1.147) involved in plant defense mechanisms that can cleave thio-bonded glucose. Myrosinase catalyzes the hydrolysis of glucosinolates, resulting in the production of isothiocyanates. Myrosinase may be conserved as myrosin granules in the vacuoles of certain heteromorphic cells called myrosin cells, but it has also been reported as a cytoplasmic enzyme that tends to bind to protein granules or vacuoles and membranes. Thus, in one embodiment, myrosinase is conserved in myrosin cells in Brassicaceae.

[0060] In one embodiment, the pretreatment described herein improves the access of myrosinase to the glucosinolate. As used herein, “improves access” or “access is improved” means increasing the availability of the glucosinolate to the myrosinase enzyme, which enables the production of isothiocyanates. In one embodiment, access is improved by the release of the glucosinolate from a glucosinolate storage site. In one embodiment, the glucosinolate storage site is mechanically ruptured (i.e., by immersion) or enzymatically degraded. In one embodiment, the glucosinolate is released from the glucosinolate storage site by the activity of one or more polysaccharide-degrading enzymes, e.g., cellulase, hemicellulase, pectinase and / or glycosidase. In one embodiment, access is improved by enabling the entry of myrosinase into the glucosinolate storage site. In one embodiment, access is improved by the release of myrosinase from myrosin cells. In one embodiment, approximately 10% to 90% of glucosinolates are released from the glucosinolate storage site. In one embodiment, approximately 20% to 80% of glucosinolates are released from the glucosinolate storage site. In one embodiment, approximately 30% to 70% of glucosinolates are released from the glucosinolate storage site. In one embodiment, approximately 40% to 60% of glucosinolates are released from the glucosinolate storage site. In one embodiment, approximately 45% to 55% of glucosinolates are released from the glucosinolate storage site. In one embodiment, approximately 10% of glucosinolates are released from the glucosinolate storage site. In one embodiment, approximately 20% of glucosinolates are released from the glucosinolate storage site. In one embodiment, approximately 30% of glucosinolates are released from the glucosinolate storage site. In one embodiment, approximately 40% of the glucosinolate is released from the glucosinolate storage site. In one embodiment, approximately 50% of the glucosinolate is released from the glucosinolate storage site. In one embodiment, approximately 60% of the glucosinolate is released from the glucosinolate storage site. In one embodiment, approximately 70% of the glucosinolate is released from the glucosinolate storage site.In one embodiment, approximately 80% of the glucosinolates are released from the glucosinolate storage site. In another embodiment, approximately 90% of the glucosinolates are released from the glucosinolate storage site.

[0061] In one embodiment, the Brassicaceae material comprises one or more glucosinolates selected from aliphatic, indole, or aromatic glucosinolates.

[0062] In one embodiment, the aliphatic glucosinolate is glucoraphanin (4-methylsulfinylbutyl or glucoraphanin), sinigrin (2-propenyl), gluconapine (3-butenyl), glucobrascanapine (4-pentenyl), progoitrin (2(R)-2-hydroxy-3-butenyl), epiprogoitrin (2(S)-2-hydroxy-3-butenyl), gluconaporeiferin (2-hydroxy-4-pentenyl), g One or more of the following are selected: lucoivervirin (3-methylthiopropyl), glucoerucin (4-methylthiobutyl), dehydroerusin (4-methylthio-3-butenyl), glucoiderin (3-methylsulfinylpropyl), glucoraphenin (4-methylsulfinyl-3-butenyl), glucolisin (5-methylsulfinylpentenyl), and glucoerythorin (3-methylsulfonylbutyl, 4-mercaptobutyl).

[0063] In one embodiment, the indole glucosinolate is selected from one or more of the following: glucobrassin(3-indolylmethyl), 4-hydroxyglucobrassin(4-hydroxy-3-indolylmethyl), 4-methoxyglucobrassin(4-methoxy-3-indolylmethyl), and neoglucobrassin(1-methoxy-3-indolylmethyl).

[0064] In one embodiment, the indole glucosinolate is selected from one or more of glucotropaeolin (benzyl) and gluconas tultiin (2-phenylethyl).

[0065] In one embodiment, the Brassicaceae material comprises one or more glucosinolates selected from benzyl glucosinolate, allyl glucosinolate, and 4-methylsulfinylbutyl. In one embodiment, the glucosinolate is glucoraphanin (4-methylsulfinylbutyl). In one embodiment, the glucosinolate is glucobrassin (3-indolylmethyl).

[0066] In one embodiment, the pretreatment described herein increases the extractable glucosinolate content compared to the extractable glucosinolate content of the Brassicaceae material before pretreatment.

[0067] As used herein, “extractable glucosinolate content” refers to the level of glucosinolate accessible in Brassicaceae material for conversion to isothiocyanates. Excluding conversion to 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 be converted to a maximum of 1 mole of isothiocyanate, 1 mole of glucose, and 1 mole of sulfate ions). Thus, for example, if the extractable glucoraphanin content of a commercially available broccoli variety is 3400 μmol glucoraphanin / kg dw, the expected maximum yield of sulforaphane from the commercially available broccoli variety is 3400 μmol sulforaphane / kg dw.

[0068] Isothiocyanates As used herein, "isothiocyanate" refers to sulfur-containing plant chemicals having a general structure RN=C=S, which are products of myrosinase activity against glucosinolates and their bioactive derivatives. In one embodiment, the isothiocyanate is sulforaphane (1-isothiocyanate-4-methylsulfinylbutane). In one embodiment, the isothiocyanate is allyl isothiocyanate (3-isothiocyanate-1-propene). In one embodiment, the isothiocyanate is benzyl isothiocyanate. In one embodiment, the isothiocyanate is phenethyl isothiocyanate. In one embodiment, the isothiocyanate is 3-butenyl isothiocyanate. In one embodiment, the isothiocyanate is 5-vinyl-1,3-oxazolidine-2-thion. In one embodiment, the isothiocyanate is 3-(methylthio)propyl isothiocyanate. In one embodiment, the isothiocyanate is 3-(methylsulfinyl)-propyl isothiocyanate. In one embodiment, the isothiocyanate is 4-(methylthio)-butyl isothiocyanate. In one embodiment, the isothiocyanate is 1-methoxyindole-3-carbinol isothiocyanate. In one embodiment, the isothiocyanate is 2-phenylethyl isothiocyanate. In one embodiment, the isothiocyanate is iverine.

[0069] In one embodiment, the isothiocyanate-containing product further comprises one or more isothiocyanate bioactive derivatives or their oligomers. In one embodiment, the isothiocyanate bioactive derivative is a derivative of any of the isothiocyanates described herein. In one embodiment, the isothiocyanate bioactive derivative is a derivative of sulforaphane. In one embodiment, the isothiocyanate bioactive derivative is iverine. In one embodiment, the isothiocyanate bioactive derivative is allyl isothiocyanate. In one embodiment, the isothiocyanate bioactive derivative is indole-3-carbinol. In one embodiment, the isothiocyanate bioactive derivative is methoxy-indole-3-carbinol. In one embodiment, the isothiocyanate bioactive derivative is ascorbigen. In one embodiment, the isothiocyanate bioactive derivative is neoascorbigen.

[0070] Pre-treatment As used herein, “pretreatment” or “pretreatment” means releasing or assisting the release of glucosinolates from glucosinolate storage sites and / or allowing myrosinase to enter glucosinolate storage sites in Brassicaceae material. In one embodiment, pretreatment increases the exposure of glucosinolates to myrosinase, allowing myrosinase to convert glucosinolates to isothiocyanates.

[0071] In one embodiment, pretreatment reduces epithio-specific protein (ESP) while maintaining endogenous myrosinase activity. As used herein, “epitio-specific protein” or “ESP” refers to a protein that induces myrosinase activity toward nitrile formation and away from isothiocyanate formation. Reducing or inhibiting the production (mRNA or protein) or activity of ESP may increase isothiocyanate formation.

[0072] As used herein, “reducing epithiospecific proteins” means reducing the protein production or activity of ESPs. In one embodiment, reducing ESPs includes inactivating (e.g., denaturing) ESPs at high temperatures. In one embodiment, ESPs are denatured at temperatures of about 50°C to about 80°C.

[0073] As used herein, “maintaining endogenous myrosinase activity” means not significantly reducing myrosinase activity compared to an untreated control. In one embodiment, endogenous myrosinase activity does not decrease by more than about 5%. In one embodiment, endogenous myrosinase activity does not decrease by more than about 10%. In one embodiment, endogenous myrosinase activity does not decrease by more than about 15%. In one embodiment, endogenous myrosinase activity does not decrease by more than about 20%. In one embodiment, endogenous myrosinase activity does not decrease by more than about 30%. In one embodiment, endogenous myrosinase activity does not decrease by more than about 40%. In one embodiment, endogenous myrosinase activity does not decrease by more than about 50%.

[0074] In one embodiment, the pretreatment includes one or more of the following: i) heating, ii) immersion, iii) microwave treatment, iv) exposure to high-frequency sound waves (ultrasound), or v) pulsed electric field treatment, wherein the temperature of the Brassicaceae material does not exceed approximately 75°C during the pretreatment.

[0075] In one embodiment, the Brassicaceae material is heated in a fuel-based heating system, an electric heating system (i.e., an oven or ohm heating), high-frequency heating, high-pressure heat treatment, or a steam-based heating system (indirect or direct application of steam). In one embodiment, the Brassicaceae material is heated in a sealed package (e.g., in a retort pouch). In one embodiment, the Brassicaceae material is heated in an oven, water bath, bioreactor, stove, water launcher, or steam launcher. In one embodiment, the Brassicaceae material is heated via high-pressure thermal heating. In one embodiment, the Brassicaceae material is heated via ohm heating. In one embodiment, the Brassicaceae material is heated via high-frequency heating. In one embodiment, the Brassicaceae material is blanched in water. In one embodiment, the Brassicaceae material is heated via high-pressure heat treatment. In one embodiment, the Brassicaceae material is placed in a sealed package for high-pressure heat treatment.

[0076] In one embodiment, the pretreatment includes heating the Brassicaceae material to about 50°C to about 70°C. In one embodiment, the pretreatment includes heating the Brassicaceae material to about 50°C to about 65°C. In one embodiment, the pretreatment includes heating the Brassicaceae material to about 50°C to about 60°C. In one embodiment, the heating includes heating the Brassicaceae material to about 55°C to about 70°C. In one embodiment, the heating includes heating the Brassicaceae material to about 60°C to about 70°C. In one embodiment, the heating includes heating the Brassicaceae material to about 65°C to about 70°C. In one embodiment, the heating includes heating the Brassicaceae material to about 30 seconds. In one embodiment, the Brassicaceae material is heated for about 1 minute. In one embodiment, the Brassicaceae material is heated for about 2 minutes. In one embodiment, the Brassicaceae material is heated for about 3 minutes. In one embodiment, the Brassicaceae material is heated for about 4 minutes. In another embodiment, the Brassicaceae material is heated for about 5 minutes.

[0077] In one embodiment, the Brassicaceae material is heated in a sealed package at approximately 60°C for approximately 1 minute. In one embodiment, the Brassicaceae material is heated in a sealed package at approximately 60°C for approximately 2 minutes. In one embodiment, the Brassicaceae material is heated in a sealed package at approximately 60°C for approximately 3 minutes. In one embodiment, the Brassicaceae material is heated in a sealed package at approximately 65°C for approximately 4 minutes. In one embodiment, the Brassicaceae material is heated in a sealed package at approximately 65°C for approximately 1 minute. In one embodiment, the Brassicaceae material is heated in a sealed package at approximately 65°C for approximately 2 minutes. In one embodiment, the Brassicaceae material is heated in a sealed package at approximately 65°C for approximately 3 minutes. In one embodiment, the Brassicaceae material is heated in a sealed package at approximately 65°C for approximately 4 minutes.

[0078] In one embodiment, the Brassicaceae material is heated in water at approximately 60°C for approximately 1 minute. In another embodiment, the Brassicaceae material is heated in water at approximately 60°C for approximately 2 minutes.

[0079] In one embodiment, heating includes steaming the Brassicaceae material. In one embodiment, pretreatment includes steaming the Brassicaceae material. In one embodiment, the Brassicaceae material is steamed to a temperature of about 50°C to about 70°C. In one embodiment, the Brassicaceae material is steamed to a temperature of about 60°C to about 70°C. In one embodiment, the Brassicaceae material is steamed for at least about 30 seconds. In one embodiment, the Brassicaceae material is steamed for at least about 1 minute. In one embodiment, the Brassicaceae material is steamed for at least about 2 minutes. In one embodiment, the Brassicaceae material is steamed for at least about 3 minutes. In one embodiment, the Brassicaceae material is steamed for at least about 4 minutes. In one embodiment, the Brassicaceae material is steamed for at least about 5 minutes.

[0080] In one embodiment, the pretreatment includes immersion of the Brassicaceae material. As used herein, “immersion,” “immersed,” or “soaking” means breaking down the Brassicaceae material into smaller fragments. In one embodiment, immersion includes decompartmenting at least about 30% to about 90% of the cells of the Brassicaceae material so that myrosinase can access its substrate, the glucosinolate. In one embodiment, immersion includes decompartmenting at least about 40% to about 90% of the cells of the Brassicaceae material. In one embodiment, immersion includes decompartmenting at least about 50% to about 90% of the cells of the Brassicaceae material. In one embodiment, immersion includes decompartmenting at least about 60% to about 90% of the cells of the Brassicaceae material. In one embodiment, immersion includes decompartmenting at least about 70% to about 90% of the cells of the Brassicaceae material. Those skilled in the art will understand that decompartmentalization of a cell involves breaking down and opening the cell wall, thereby disrupting the compartmentalization of organelles within the cell.

[0081] In one embodiment, the Brassicaceae material is immersed in a blender, grinder, or pulverizer. In one embodiment, the Brassicaceae material is immersed so that at least about 80% of the Brassicaceae material is about 2 mm or less in size. In one embodiment, the Brassicaceae material is immersed so that at least about 80% of the Brassicaceae material is about 1 mm or less in size. In one embodiment, the Brassicaceae material is immersed so that at least about 80% of the Brassicaceae material is about 0.5 mm or less in size. In one embodiment, the Brassicaceae material is immersed so that at least about 80% of the Brassicaceae material is about 0.25 mm or less in size. In one embodiment, the Brassicaceae material is immersed so that at least about 80% of the Brassicaceae material is about 0.1 mm or less in size. In one embodiment, the Brassicaceae material is immersed so that at least about 80% of the Brassicaceae material is about 0.05 mm or less in size. In one embodiment, the Brassicaceae material is immersed so that at least about 80% of the Brassicaceae material is sized to about 0.025 mm or less. In one embodiment, the Brassicaceae material is immersed so that at least about 80% of the Brassicaceae material is sized to about 0.01 mm or less. In one embodiment, the Brassicaceae material is immersed so that about 50% to about 90% of the Brassicaceae material is sized to about 2 mm or less. In one embodiment, the Brassicaceae material is immersed so that about 60% to about 80% of the Brassicaceae material is sized to about 2 mm or less. In one embodiment, the Brassicaceae material is immersed so that about 50% to about 90% of the Brassicaceae material is sized to about 1 mm or less. In one embodiment, the Brassicaceae material is immersed so that about 60% to about 80% of the Brassicaceae material is sized to about 1 mm or less. In one embodiment, the Brassicaceae material is heated to a temperature of approximately 50°C to 70°C during immersion.In one embodiment, the Brassicaceae material is heated to a temperature of approximately 55°C to approximately 70°C during immersion. In another embodiment, the Brassicaceae material is heated to a temperature of approximately 60°C to approximately 70°C during immersion. In yet another embodiment, the Brassicaceae material is heated to a temperature of approximately 65°C to approximately 70°C during immersion.

[0082] In one embodiment, the pretreatment includes heating and immersing the Brassicaceae material. In one embodiment, the pretreatment prepares a puree. As used herein, “puree” refers to Brassicaceae material mixed to a creamy paste or liquid consistency.

[0083] Those skilled in the art will understand that “microwave treatment” or “microwave processing” means heating a material, such as a brassicaceae material, by passing microwave irradiation through it. In one embodiment, pretreatment includes microwave treatment of a brassicaceae material. In one embodiment, the brassicaceae material is pretreated with a household microwave or an industrial microwave. In one embodiment, the industrial microwave is a continuous microwave system, such as, for example, the MIP 11 Industrial Microwave Continuous Cooking Oven (Ferrite Microwave Technologies). In one embodiment, pretreatment includes microwave treatment of a brassicaceae material. In one embodiment, the brassicaceae material is microwaved at about 0.9 to about 2.45 GHz. In one embodiment, the brassicaceae material is microwaved for at least about 30 seconds, or at least about 1 minute, or at least about 2 minutes, or at least about 3 minutes.

[0084] In one embodiment, the pretreatment includes exposing the Brassicaceae material to low-to-medium frequency ultrasound. In one embodiment, the pretreatment includes exposing the Brassicaceae material using thermal ultrasonic treatment (low-to-medium frequency ultrasound with heat of about 30°C to about 60°C). In one embodiment, the ultrasound is generated using an industrial-scale ultrasonic device. In one embodiment, the ultrasonic device is a continuous or batch ultrasonic device. In one embodiment, the ultrasonic device is, for example, a UIP500hd or UIP4000 (Hielscher, Ultrasound Technology). In one embodiment, the ultrasound has a frequency of about 20 kHz to about 600 kHz. In one embodiment, 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.

[0085] In one embodiment, pretreatment includes exposing the Brassicaceae material to pulsed electric field treatment. Pulsed electric field treatment is a non-thermal treatment technique that involves applying high-voltage short pulses. The pulses induce electroporation of cells in the Brassicaceae material and enhance the approach of myrosinase to glucosinolates. In one embodiment, pulsed electric field treatment heats the Brassicaceae material to a temperature of about 40 to about 70°C. In one embodiment, pulsed electric field treatment heats the Brassicaceae material to a temperature of about 50 to about 70°C. In one embodiment, pulsed electric field treatment heats the Brassicaceae material to a temperature of about 60 to about 70°C. In one embodiment, pulsed electric field treatment includes treating the Brassicaceae material with voltage pulses of about 20 to about 80 kV. In one embodiment, the pretreatment converts about 10% to about 90% of the glucosinolates to isothiocyanates. In one embodiment, approximately 20% to 80% of the glucosinolate is converted to isothiocyanate by pretreatment. In another embodiment, approximately 30% to 70% of the glucosinolate is converted to isothiocyanate by pretreatment. In another embodiment, approximately 40% to 60% of the glucosinolate is converted to isothiocyanate by pretreatment. In another embodiment, approximately 10% of the glucosinolate is converted to isothiocyanate by pretreatment. In another embodiment, approximately 20% of the glucosinolate is converted to isothiocyanate by pretreatment. In another embodiment, approximately 30% of the glucosinolate is converted to isothiocyanate by pretreatment. In another embodiment, approximately 40% of the glucosinolate is converted to isothiocyanate by pretreatment. In another embodiment, approximately 50% of the glucosinolate is converted to isothiocyanate by pretreatment. In one embodiment, approximately 60% of the glucosinolate is converted to isothiocyanate by pretreatment. In another embodiment, approximately 70% of the glucosinolate is converted to isothiocyanate by pretreatment. In yet another embodiment, approximately 80% of the glucosinolate is converted to isothiocyanate by pretreatment. In yet another embodiment, approximately 90% of the glucosinolate is converted to isothiocyanate by pretreatment.

[0086] fermentation Those skilled in the art will understand that fermentation methods as described herein may include the use of any lactic acid bacteria. As used herein, “fermentation” refers to the biochemical decomposition of Brassicaceae material by lactic acid bacteria. In one embodiment, fermentation with lactic acid bacteria is carried out by adding foreign lactic acid bacteria. As used herein, “lactic bacteria” or “lactic acid bacteria” are bacteria that produce lactic acid as the final product of carbohydrate fermentation, and these may include, but are not limited to, bacteria from the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus and Weissella. In one embodiment, the lactic acid bacteria include myrosinase activity. In one embodiment, the lactic acid bacteria are derived from the genus Leuconostoc. In another embodiment, the lactic acid bacteria are derived from the genus Lactobacillus.

[0087] In one embodiment, the lactic acid bacteria are selected from one or more of the following: Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus rhamnosus, Lactobacillus pentosus, Lactobacillus brevis, Lactococcus lactis, Pediococcus pentosaceus, and Pediococcus acidilacti.

[0088] In one embodiment, the lactic acid bacteria were isolated from Brassicaceae. In one embodiment, the lactic acid bacteria were isolated from Brassica oleracea. In one embodiment, the lactic acid bacteria were isolated from broccoli. In one embodiment, the lactic acid bacteria were isolated from broccoli leaves. In one embodiment, the lactic acid bacteria were isolated from broccoli stems. In one embodiment, the lactic acid bacteria were isolated from broccoli puree. In one embodiment, the lactic acid bacteria were isolated from Australian broccoli.

[0089] In one embodiment, the lactic acid bacteria lack myrosinase activity.

[0090] In one embodiment, the lactic acid bacteria is Lactobacillus.

[0091] In one embodiment, the lactic acid bacteria are selected from i) Leuconostoc mesenteroides, ii) Lactobacillus plantarum, iii) Lactobacillus pentosus, iv) Lactobacillus rhamnosus, v) a combination of i) and ii), vi) a combination of i), ii) and iii), and vii) a combination of i), ii) and iv).

[0092] In one embodiment, the lactic acid bacterium is Leuconostoc mesenteroides. In one embodiment, Leuconostoc mesenteroides is ATCC8293. In one embodiment, Leuconostoc mesenteroides is BF1 and / or BF2. In one embodiment, Leuconostoc mesenteroides lacks myrosinase activity.

[0093] In one embodiment, the lactic acid bacterium is Lactobacillus plantarum. In one embodiment, Lactobacillus plantarum lacks myrosinase activity.

[0094] In one embodiment, approximately 50% of the lactic acid bacteria are Leuconostoc mesenteroides, and approximately 50% are Lactobacillus species.

[0095] In one embodiment, approximately 50% of the lactic acid bacteria are Leuconostoc mesenteroides, and approximately 50% are Lactobacillus plantarum.

[0096] In one embodiment, Lactobacillus plantarum is selected from one or more or all of B1, B2, B3, B4, and B5. In one embodiment, Lactobacillus plantarum is B1. In one embodiment, Lactobacillus plantarum is B2. In one embodiment, Lactobacillus plantarum is B3. In one embodiment, Lactobacillus plantarum is B4. In one embodiment, Lactobacillus plantarum is B5.

[0097] In one embodiment, fermentation is carried out in the presence of at least two strains, at least three, at least four, at least five, or at least six strains of lactic acid bacteria selected from BF1, BF2, B1, B2, B3, B4, and B5.

[0098] In one embodiment, the lactic acid bacteria are recombinant bacteria modified to produce higher levels of myrosinase activity compared to an unmodified control. Those skilled in the art will understand that recombinant lactic acid bacteria can be produced by any technique known to those skilled in the art.

[0099] In one embodiment, lactic acid bacteria are subjected to stress from, for example, but not limited to, heat stress, cold stress, sublethal ultrasound, e.g., about 20 to about 2000 MHz, high pressure, dynamic high pressure, or pulsed electric field, to increase myrosinase activity and polysaccharide-degrading enzyme activity compared to unstressed control lactic acid bacteria. In one embodiment, heat stress includes heating the bacteria to over 40°C to about 75°C. In one embodiment, heat stress includes heating the bacteria to over 45°C to about 65°C. In one embodiment, heat stress includes heating the bacteria to over 45°C to about 55°C. In one embodiment, cold stress includes cooling the bacteria to a temperature of about 0°C to about 8°C. In one embodiment, cold stress includes cooling the bacteria to a temperature of about 2°C to about 6°C. In one embodiment, cold stress includes cooling the bacteria to a temperature of about 4°C.

[0100] In one embodiment, the Brassicaceae material is at least about 10 as described herein. 5 The lactic acid bacteria are inoculated at a concentration of CFU / g. In one embodiment, the Brassicaceae material is at least about 10 as described herein. 6 The lactic acid bacteria are inoculated at a concentration of CFU / g. In one embodiment, the Brassicaceae material is at least about 10 as described herein. 7 The lactic acid bacteria are inoculated at a concentration of CFU / g. In one embodiment, the Brassicaceae material is at least about 10 as described herein. 8 Lactobacillus is inoculated at a CFU / g level. In one embodiment, the Brassicaceae material is pre-treated.

[0101] In one embodiment, fermentation occurs at approximately 20°C to 34°C. In one embodiment, fermentation occurs at approximately 22°C to 34°C. In one embodiment, fermentation occurs at approximately 24°C to 34°C. In one embodiment, fermentation occurs at approximately 24°C to 30°C. In one embodiment, fermentation occurs at approximately 34°C to 34°C. In one embodiment, fermentation occurs at approximately 25°C. In one embodiment, fermentation occurs at approximately 30°C. In one embodiment, fermentation occurs at approximately 34°C.

[0102] In one embodiment, fermentation is approximately 8 hours to 17 days. In one embodiment, fermentation is approximately 8 hours to 14 days. In one embodiment, fermentation is approximately 8 hours to 7 days. In one embodiment, fermentation is approximately 8 hours to 5 days. In one embodiment, fermentation is approximately 8 hours to 4 days. In one embodiment, fermentation is approximately 8 hours to 3 days. In one embodiment, fermentation is approximately 8 hours to 30 hours. In one embodiment, fermentation is approximately 8 to 24 hours. In one embodiment, fermentation is approximately 10 hours to 24 hours. In one embodiment, fermentation is approximately 10 days. In one embodiment, fermentation is approximately 9 days. In one embodiment, fermentation is approximately 8 days. In one embodiment, fermentation is approximately 7 days. In one embodiment, fermentation is approximately 4 days. In one embodiment, fermentation is approximately 6 days. In one embodiment, fermentation is approximately 5 days. In one embodiment, fermentation is approximately 72 hours. In one embodiment, fermentation is approximately 60 hours. In one embodiment, fermentation is approximately 45 hours. In one embodiment, fermentation is approximately 30 hours. In one embodiment, fermentation takes about 24 hours. In one embodiment, fermentation takes about 20 hours. In one embodiment, fermentation takes about 18 hours. In one embodiment, fermentation takes about 15 hours. In one embodiment, fermentation takes about 16 hours. In one embodiment, fermentation takes about 14 hours. In one embodiment, fermentation takes about 12 hours. In one embodiment, fermentation takes about 10 hours. In one embodiment, fermentation takes about 8 hours. In one embodiment, the fermentation culture is stirred. In one embodiment, stirring is intermittent. In one embodiment, stirring is continuous. In a particularly preferred embodiment, fermentation takes 15 hours with intermittent stirring. In a particularly preferred embodiment, fermentation takes 24 hours with intermittent stirring.

[0103] In one embodiment, the fermentation reaction is completed when the composition reaches a pH of about 4.5 to about 3.8. In one embodiment, the fermentation reaction is completed when the composition reaches a pH of about 4.5 to about 3.6. In one embodiment, the fermentation reaction is completed when the composition reaches a pH of about 4.5 to about 4.04. In one embodiment, the fermentation reaction is completed when the composition reaches a pH of about 4.3 to about 4.04. In one embodiment, the fermentation reaction is completed when the composition reaches a pH of 4.5 or less, or 4.4 or less, or 4.3 or less, or 4.04 or less, or 3.8 or less. In one embodiment, the fermentation reaction is completed when the composition reaches a pH of 4.5 or less. In one embodiment, the fermentation reaction is completed when the composition reaches a pH of 4.4 or less.

[0104] In one embodiment, if present, fermentation reduces the number of one or more or all of E. coli, Salmonella, and Listeria. In one embodiment, if present, fermentation reduces the CFU / g of one or more or all of E. coli, Salmonella, and Listeria.

[0105] In one embodiment, no salt is added to the fermentation culture.

[0106] In one embodiment, fermentation increases the extractable glucosinolate content compared to the extractable glucosinolate content in the pre-treated Brassicaceae material. In one embodiment, fermentation increases the extractable glucosinolate content compared to the extractable glucosinolate content in the Brassicaceae material. In one embodiment, fermentation increases the extractable glucosinolate content by about 100% to about 500% compared to the extractable glucosinolate content in the Brassicaceae material. In one embodiment, fermentation increases the extractable glucosinolate content by about 200% to about 450% compared to the extractable glucosinolate content in the Brassicaceae material. In one embodiment, fermentation increases the extractable glucosinolate content by about 250% to about 450% compared to the extractable glucosinolate content in the Brassicaceae material. In one embodiment, fermentation increases the extractable glucosinolate content by approximately 300% to approximately 400% compared to the extractable glucosinolate content in Brassicaceae material. In one embodiment, fermentation increases the extractable glucosinolate content by approximately 300% compared to the extractable glucosinolate content in Brassicaceae material. In one embodiment, fermentation increases the extractable glucosinolate content by approximately 400% compared to the extractable glucosinolate content in Brassicaceae material. In one embodiment, the glucosinolate is glucoraphanin.

[0107] acidification The pre-treated material may be acidified to improve the microbial safety and stability (susceptibility to microbial degradation) of the product and to enhance the stability of isothiocyanates in the product. Acidification can be achieved by adding organic acids, such as, but not limited to, lactic acid, acetic acid, ascorbic acid, and citric acid. In one embodiment, acidification can be achieved by adding glucono delta-lactone. In one embodiment, acidification includes lowering the pH to about 4.4 to about 3.4. In one embodiment, acidification includes lowering the pH to 4.5, or 4.4, or 4.2, or 4, or 3.8, or 3.6, or 3.4 or lower. In one embodiment, acidification includes lowering the pH to 4.4 or lower.

[0108] Products containing isothiocyanates derived from Brassicaceae Products containing isothiocyanates derived from Brassicaceae, as described herein, may be prepared by the methods described herein. It will be understood by those skilled in the art that products containing isothiocyanates prepared using the methods described herein contain higher levels of isothiocyanates, such as sulforaphane, than Brassicaceae material or Brassicaceae material subjected only to fermentation (without the pretreatment described herein). For example, broccoli soaked from commercially available broccoli varieties has a sulforaphane concentration of approximately 800 μmol / Kg dw (approximately 149.8 mg / Kg dw), fermented broccoli has a sulforaphane concentration of approximately 1600 μmol / Kg dw (approximately 278.8 mg / Kg dw), and pre-treated fermented broccoli prepared using a method as described herein has a sulforaphane concentration of approximately 13100 μmol / Kg dw (approximately 2318.7 mg / Kg dw).

[0109] In one embodiment, the isothiocyanate-containing product contains at least about four times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about six times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about eight times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about ten times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about twelve times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about fourteen times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about sixteen times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 17 times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains about 4 to about 17 times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains about 4 to about 16 times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains about 8 to about 16 times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains about 10 to about 16 times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains about 12 to about 16 times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains approximately 14 to 16 times more isothiocyanate than the immersed Brassicaceae material. In one embodiment, the isothiocyanate is sulforaphane.

[0110] In one embodiment, the level of isothiocyanate present in the isothiocyanate-containing product is higher than that expected from the extractable glucosinolate content of the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 1 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In one embodiment, the isothiocyanate-containing product contains at least about 2 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In one embodiment, the isothiocyanate-containing product contains at least about 3 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In one embodiment, the isothiocyanate-containing product contains at least about 3.8 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In one embodiment, the isothiocyanate-containing product contains at least about 4 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In one embodiment, the isothiocyanate-containing product contains approximately 1 to 4 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In one embodiment, the isothiocyanate-containing product contains approximately 1 to 3.8 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In one embodiment, the isothiocyanate-containing product contains approximately 2 to 3.8 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In one embodiment, the isothiocyanate-containing product contains approximately 2 to 3 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.

[0111] In one embodiment, the level of sulforaphane present in the isothiocyanate-containing product is higher than that expected from the extractable glucoraphanin content of the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 1 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In one embodiment, the isothiocyanate-containing product contains at least about 2 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In one embodiment, the isothiocyanate-containing product contains at least about 3 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In one embodiment, the isothiocyanate-containing product contains at least about 3.8 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In one embodiment, the isothiocyanate-containing product contains at least about 4 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In one embodiment, the isothiocyanate-containing product contains approximately 1 to 4 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In one embodiment, the isothiocyanate-containing product contains approximately 1 to 3.8 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In one embodiment, the isothiocyanate-containing product contains approximately 1 to 3 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In one embodiment, the isothiocyanate-containing product contains approximately 2 to 3 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content.

[0112] In one embodiment, the isothiocyanate-containing product contains approximately 100 mg / kg dw to approximately 7000 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains approximately 500 mg / kg dw to approximately 7000 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains approximately 1000 mg / kg dw to approximately 7000 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains approximately 1600 mg / kg dw to approximately 4000 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains approximately 1600 mg / kg dw to approximately 3000 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains approximately 2000 mg / kg dw to approximately 4000 mg / kg dw of isothiocyanate. In another embodiment, the isothiocyanate-containing product contains approximately 2000 mg / kg dw to approximately 7000 mg / kg dw of isothiocyanate. In yet another embodiment, the isothiocyanate-containing product contains approximately 3000 mg / kg dw to approximately 7000 mg / kg of isothiocyanate. In yet another embodiment, the isothiocyanate-containing product contains approximately 2300 mg / kg dw of isothiocyanate.

[0113] In one embodiment, the isothiocyanate-containing product contains at least about 100 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 200 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 250 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 300 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 350 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 400 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 450 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 500 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 550 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 600 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 650 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 700 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 1000 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 2000 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 3000 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 4000 mg / kg dw of isothiocyanate. In one embodiment, the isothiocyanate-containing product contains at least about 5000 mg / kg dw of isothiocyanate. In another embodiment, the isothiocyanate-containing product contains at least about 6000 mg / kg dw of isothiocyanate.In one embodiment, the isothiocyanate-containing product contains at least about 7000 mg / kg dw of isothiocyanate.

[0114] In one embodiment, the isothiocyanate-containing product contains at least about 100 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 150 mg / kg of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 200 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 250 mg / kg of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 300 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 350 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 400 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 450 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 500 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 550 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 600 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 650 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 700 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 1000 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 2000 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 3000 mg / kg dw of sulforaphane. In one embodiment, the isothiocyanate-containing product contains at least about 4000 mg / kg dw of sulforaphane. In another embodiment, the isothiocyanate-containing product contains at least about 5000 mg / kg dw of sulforaphane.In one embodiment, the isothiocyanate-containing product contains at least about 6000 mg / kg dw of sulforaphane. In another embodiment, the isothiocyanate-containing product contains at least about 7000 mg / kg dw of sulforaphane.

[0115] In one embodiment, the isothiocyanate-containing product contains at least about 5% more total fiber than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 10% more total fiber than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 15% more total fiber than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 20% more total fiber than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 4% more protein than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 6% more protein than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 8% more protein than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 10% more protein than the Brassicaceae material.

[0116] In one embodiment, the isothiocyanate-containing product contains at least about 10% less carbohydrates than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 20% less carbohydrates than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 30% less carbohydrates than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 40% less carbohydrates than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 45% less carbohydrates than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains at least about 48% less carbohydrates than the Brassicaceae material. In one embodiment, the isothiocyanate-containing product contains about 10% to about 48% less carbohydrates than the Brassicaceae material.

[0117] In one embodiment, the isothiocyanate-containing product includes an increased level of polyphenol glycosides compared to Brassicaceae materials. In one embodiment, the polyphenol glycoside is an anthocyanin glycoside. In one embodiment, the polyphenol glycoside is a phenolic acid glycoside. In one embodiment, the polyphenol glycoside is a phenolic acid.

[0118] In one embodiment, the isothiocyanate-containing product includes an increased level of glucosinolate compared to the Brassicaceae material. In one embodiment, the glucosinolate is glucoraphanin. In one embodiment, glucoraphanin is increased by at least about 25 times. In one embodiment, the glucosinolate is glucobrassin. In one embodiment, glucobrassin is increased by 26 times. In one embodiment, the isothiocyanate-containing product includes indole-3-carbinol. In one embodiment, indole-3-carbinol is increased by at least about 2 times in the isothiocyanate-containing product compared to the immersed Brassicaceae material. In one embodiment, indole-3-carbinol is increased by at least about 3 times in the isothiocyanate-containing product compared to the immersed Brassicaceae material. In one embodiment, the isothiocyanate-containing product includes ascorbigen. In one embodiment, ascorbigen is increased by at least about 2 times in the isothiocyanate-containing product compared to the immersed Brassicaceae material. In one embodiment, ascorbigen increases by at least about three times in the isothiocyanate-containing product compared to the immersed Brassicaceae material.

[0119] In one embodiment, the isothiocyanate-containing product includes an increase in the level of one or more of the following compared to the Brassicaceae material: ferulic acid, syringic acid, phenyl lactic acid, chlorogenic acid, rutin, sinapic acid, methyl syringate, hesperetin, quercetin, and kaempferol. In one embodiment, the isothiocyanate-containing product includes an increase in the level of chlorogenic acid compared to the Brassicaceae material. In one embodiment, chlorogenic acid increases by about 6.6 times. In one embodiment, the isothiocyanate-containing product includes an increase in the level of sinapic acid compared to the Brassicaceae material. In one embodiment, sinapic acid increases by about 23.8 times. In one embodiment, the isothiocyanate-containing product includes an increase in the level of kaempferol compared to the Brassicaceae material. In one embodiment, kaempferol increases by about 10.5 times.

[0120] In one embodiment, the isothiocyanate-containing product contains a reduction in the level of one or more of the following compared to the Brassicaceae material: protocatechuic acid, gallic acid, 4,hydroxybenzoic acid, vanillic acid, 2,3-dihydroxybenzoic acid, p-coumaric acid, cinnamic acid, catechin, rosmarinic acid, and caffeic acid.

[0121] In one embodiment, approximately 40% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In another embodiment, approximately 50% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In yet another embodiment, approximately 60% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In yet another embodiment, approximately 70% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In yet another embodiment, approximately 80% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In yet another embodiment, approximately 90% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In one embodiment, approximately 95% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In another embodiment, approximately 97% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In yet another embodiment, approximately 98% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In yet another embodiment, approximately 99% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In yet another embodiment, approximately 100% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In yet another embodiment, approximately 40% to 100% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing product. In one embodiment, approximately 40% to 80% of the glucosinolates present in the Brassicaceae material are converted to isothiocyanates in the isothiocyanate-containing Brassicaceae product.

[0122] In one embodiment, the isothiocyanate in the isothiocyanate-containing product is stable for at least one week, or at least two weeks, or at least three weeks, or at least four weeks, or at least six weeks, or at least eight weeks, or at least ten weeks, or at least twelve weeks, or at least fourteen weeks, when stored at about 4°C to about 25°C. In one embodiment, the isothiocyanate in the isothiocyanate-containing product is stable for at least four weeks, when stored at about 4°C to about 25°C. In one embodiment, the isothiocyanate in the isothiocyanate-containing product is stable for at least eight weeks, when stored at about 4°C to about 25°C. In one embodiment, the isothiocyanate in the isothiocyanate-containing product is stable for at least twelve weeks, when stored at about 4°C to about 25°C.

[0123] As used herein, “stable” means that the isothiocyanate concentration does not decrease at all or only slightly when stored at 4°C for 6 weeks. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 1% to about 30%. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 5% or less. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 10% or less. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 15% or less. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 20% or less. In one embodiment, a slight decrease refers to a decrease in isothiocyanate concentration of about 30% or less. Isothiocyanate analysis may be carried out by any method known to those skilled in the art, for example, by the method shown in Example 1 for sulforaphane.

[0124] In one embodiment, the isothiocyanate is sulforaphane.

[0125] In one embodiment, when stored at approximately 4°C to approximately 25°C, the isothiocyanate-containing product is resistant to the growth of yeast, mold and / or E. coli for at least one week, or at least two weeks, or at least three weeks, or at least four weeks, or at least six weeks, or at least eight weeks, or at least ten weeks, or at least twelve weeks, or at least fourteen weeks.

[0126] In one embodiment, an isothiocyanate-containing product is resistant to the growth of yeast, mold, and / or E. coli for at least four weeks when stored at approximately 4°C to approximately 25°C. In another embodiment, an isothiocyanate-containing product is resistant to the growth of yeast, mold, and / or E. coli for at least eight weeks when stored at approximately 4°C to approximately 25°C. In yet another embodiment, an isothiocyanate-containing product is resistant to the growth of yeast, mold, and / or E. coli for at least twelve weeks when stored at approximately 4°C to approximately 25°C.

[0127] As used herein, “resistant” to the growth of yeast, mold and / or E. coli means that <1 Log CFU / g of yeast, mold and / or E. coli is detectable in the sample after the periods listed above using the method described in Example 1. In one embodiment, the isothiocyanate-containing product contains about 20 g / 100 gdw to about 32 g / 100 gdw of total fiber. In one embodiment, the isothiocyanate-containing product contains about 20 g / 100 gdw of total fiber. In one embodiment, the isothiocyanate-containing product contains about 25 g / 100 gdw of total fiber. In one embodiment, the isothiocyanate-containing product contains about 28 g / 100 gdw of total fiber. In one embodiment, the isothiocyanate-containing product contains about 29 g / 100 gdw of total fiber. In one embodiment, the isothiocyanate-containing product contains about 30 g / 100 gdw of total fiber. In one embodiment, the isothiocyanate-containing product contains approximately 32 g / 100 gdw of total fiber.

[0128] In one embodiment, the isothiocyanate-containing product contains an ORAC antioxidant capacity of approximately 14,000 μmol TE / 100gdw to approximately 19,000 μmol TE / 100gdw. In one embodiment, the isothiocyanate-containing product contains an ORAC antioxidant capacity of approximately 14,000 μmol TE / 100gdw. In one embodiment, the isothiocyanate-containing product contains an ORAC antioxidant capacity of approximately 15,000 μmol TE / 100gdw. In one embodiment, the isothiocyanate-containing product contains an ORAC antioxidant capacity of approximately 16,000 μmol TE / 100gdw. In one embodiment, the isothiocyanate-containing product contains an ORAC antioxidant capacity of approximately 17,000 μmol TE / 100gdw. In one embodiment, the isothiocyanate-containing product contains an ORAC antioxidant capacity of approximately 18,000 μmol TE / 100gdw. In one embodiment, the isothiocyanate-containing product contains an ORAC antioxidant capacity of approximately 18,695 μmol TE / 100 gdw. In another embodiment, the isothiocyanate-containing product contains an ORAC antioxidant capacity of approximately 19,000 μmol TE / 100 gdw.

[0129] In one embodiment, the isothiocyanate-containing product contains a total polyphenol content of approximately 1750 mg GAE / 100 gdw to approximately 2600 mg GAE / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total polyphenol content of approximately 1750 mg GAE / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total polyphenol content of approximately 2000 mg GAE / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total polyphenol content of approximately 2100 mg GAE / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total polyphenol content of approximately 2200 mg GAE / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total polyphenol content of approximately 2300 mg GAE / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total polyphenol content of approximately 2360 mg GAE / 100 gdw.

[0130] In one embodiment, the isothiocyanate-containing product contains a total titratable acidity of about 0.9% to about 1.1% lactic acid equivalent.

[0131] In one embodiment, the isothiocyanate-containing product contains a total protein content of approximately 23 g / 100 gdw to approximately 39 g / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total protein content of approximately 23 g / 100 gdw to approximately 30 g / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total protein content of approximately 25 g / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total protein content of approximately 27 g / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total protein content of approximately 28 g / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total protein content of approximately 29 g / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total protein content of approximately 30 g / 100 gdw. In one embodiment, the isothiocyanate-containing product contains a total protein content of approximately 32 g / 100 gdw.

[0132] In one embodiment, the isothiocyanate-containing product comprises at least about 100 mg / kg dw of isothiocyanate and one or more or all of the following:

[0133] i) Total fiber, approximately 29-36g / 100gdw ii) ORAC antioxidant capacity of approximately 15,000 to 18,695 μmol TE / 100gdw, iii) Total polyphenol content of approximately 2310 to 2600 mg GAE / 100gdw iv) Total titration acidity of approximately 0.9 to 1.1% lactate equivalents, v) Total protein content of approximately 27-39 g / 100 gdw, and vi) Leuconostoc mesenteroides and / or Lactobacillus plantarum.

[0134] In one embodiment, the isothiocyanate-containing product is made from broccoli.

[0135] Brassicaceae products as described herein may contain live lactic acid bacteria that can assist in the conversion of glucosinolates present in the isothiocyanate-containing product to isothiocyanates during digestion of the glucosinolate-containing product in a subject (i.e., the bacteria function as a probiotic). In one embodiment, the lactic acid bacteria is Leuconostoc mesenteroides. In one embodiment, the lactic acid bacteria is of the Lactobacillus species. In one embodiment, the lactic acid bacteria is Lactobacillus plantarum.

[0136] In one embodiment, the isothiocyanate-containing product contains lactic acid bacteria at a concentration of at least about 10 2 CFU / g. In one embodiment, the isothiocyanate-containing product contains lactic acid bacteria at a concentration of at least about 10 2 CFU / g. In one embodiment, the isothiocyanate-containing product contains lactic acid bacteria at a concentration of at least about 10 5 CFU / g. In one embodiment, the isothiocyanate-containing product contains lactic acid bacteria at a concentration of at least about 10 6 CFU / g. In one embodiment, the isothiocyanate-containing product contains lactic acid bacteria at a concentration of at least about 10 7 CFU / g. In one embodiment, the isothiocyanate-containing product contains lactic acid bacteria at a concentration of at least about 10 8 CFU / g. In one embodiment, the isothiocyanate-containing product contains lactic acid bacteria at a concentration of at least about 10 9 CFU / g.

[0137] In one embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 10 days. In one embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 20 days. In one embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 30 days. In one embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 40 days. In one embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 50 days. In one embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 60 days. In one embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 70 days. In one embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 80 days. In another embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 85 days. In yet another embodiment, when stored at approximately 4°C to approximately 25°C, the raw lactic acid bacteria are present in the isothiocyanate-containing product for at least 90 days.

[0138] In one embodiment, the lactic acid bacteria are of the Lactobacillus species. In one embodiment, the lactic acid bacteria are Lactobacillus plantarum. In one embodiment, the lactic acid bacteria are Leuconostoc mesenteroides. In one embodiment, the bacteria are at least about 10 7 It exists at a concentration of CFU / g.

[0139] In one embodiment, the isothiocyanate-containing product contains one or more bacteriocins produced by lactic acid bacteria. In one embodiment, the bacteriocin is a class I bacteriocin. In one embodiment, the bacteriocin is a class II bacteriocin. In one embodiment, the bacteriocin is a class III bacteriocin. Examples of bacteriocins produced by lactic acid bacteria can be found in Alvarez-Sieiro et al. (2016).

[0140] In one embodiment, the isothiocyanate-containing product is a food. In one embodiment, the isothiocyanate-containing product is a nutritional supplement. In one embodiment, the isothiocyanate-containing product is a supplement. In one embodiment, the isothiocyanate-containing product is a food ingredient. In one embodiment, the isothiocyanate-containing product is a probiotic. In one embodiment, the isothiocyanate-containing product is animal feed. The animals may be aquatic animals such as fish or livestock. In one embodiment, the isothiocyanate-containing product is an insecticide. In one embodiment, the isothiocyanate-containing product is a medicated cosmetic. In one embodiment, the isothiocyanate-containing product is formulated topically.

[0141] In one embodiment, the isothiocyanate-containing product is a solid, liquid, puree, or powder. In one embodiment, the isothiocyanate-containing product is dried after fermentation to become a powder. In one embodiment, the isothiocyanate-containing product is freeze-dried after fermentation. In one embodiment, the isothiocyanate-containing product is microencapsulated after fermentation as described in WO2005030229. In one embodiment, the isothiocyanate-containing product is formulated as pills.

[0142] Post-processing In one embodiment, after fermentation or acidification, the isothiocyanate-containing product may be post-treated, for example, to inactivate microorganisms that contribute to the degradation of the product or pathogens in the event of ingestion.

[0143] As used herein, “post-treatment” or “to post-treatment” refers to the treatment of an isothiocyanate-containing product as described herein after fermentation for the purpose of inactivating microorganisms. As used herein, “microorganisms” refers to the activity of bacteria, viruses, fungi or eukaryotes that may cause the decomposition or spoilage of an isothiocyanate-containing product. As used herein, “inactivating” or “inactivating” microorganisms refers to reducing the number of viable microorganisms by about 1 to about 7 log. In one embodiment, the number of viable microorganisms is reduced by about 1 to 6 log. In one embodiment, the number of viable microorganisms is reduced by about 2 to 6 log. In one embodiment, the number of viable microorganisms is reduced by about 3 to 6 log.

[0144] Those skilled in the art will understand that the post-treatment may include any method for inactivating microorganisms, such as heat treatment, UV treatment, ultrasonic treatment, pulsed electric field treatment, or high-pressure treatment. In one embodiment, the isothiocyanate-containing product is post-treated by heat treatment. In one embodiment, the isothiocyanate-containing product is post-treated by high-pressure treatment. In one embodiment, the isothiocyanate-containing product is in a sealed package during post-treatment. In one embodiment, the isothiocyanate-containing product is in a sealed package during high-pressure treatment. In one embodiment, the isothiocyanate-containing product is in a sealed package during heat treatment. In one embodiment, the high-pressure treatment includes treating the isothiocyanate-containing product at a hydrostatic pressure of about 300 to about 600 MPa. In one embodiment, the high-pressure treatment includes treating the isothiocyanate-containing product at a hydrostatic pressure of about 350 to about 550 MPa. In one embodiment, the high-pressure treatment includes treating the isothiocyanate-containing product at a hydrostatic pressure of about 300 to about 400 MPa. In one embodiment, the heat treatment includes heating the sample to a temperature of about 60°C to about 121°C. In one embodiment, the heat treatment includes heating the sample to a temperature of about 65°C to about 100°C. In one embodiment, the heat treatment includes heating the sample to a temperature of about 65°C to about 80°C. In one embodiment, the heat treatment includes heating the sample to a temperature of about 65°C to about 75°C.

[0145] Isolated strains and starter cultures In one embodiment, the present invention provides an isolated strain of lactic acid bacteria suitable for use in methods and products as described herein.

[0146] In one embodiment, the present invention provides an isolated strain of lactic acid bacteria selected from the following: i) On September 25, 2017, BF1 was deposited with the National Measurement Institute Australia under the code V17 / 021729. ii) On September 25, 2017, BF2 was deposited with the National Measurement Institute Australia under V17 / 021730. iii) On September 25, 2017, B1 was deposited with the National Measurement Institute Australia under V17 / 021731. iv) On September 25, 2017, B2 was deposited with the National Measurement Institute Australia under V17 / 021732. v) On September 25, 2017, B3 was deposited with the National Measurement Institute Australia under V17 / 021733. vi) B4, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021734, and vii) B5 deposited with the National Measurement Institute Australia on September 25, 2017, under the name V17 / 021735.

[0147] In one embodiment, the present invention provides an isolated strain of Leuconostoc mesenteroides containing genomic DNA that, when cleaved at SmaI and / or NotI, produces the same SmaI and / or NotI fingerprint as BF1 or BF2. The SmaI and NotI fingerprints of BF1 and BF2 are shown in Figure 13.

[0148] In one embodiment, the present invention provides an isolated strain of Lactobacillus plantarum containing genomic DNA that, when cleaved by SmaI and / or NotI, produces the same SmaI and / or NotI fingerprint as B1, B2, B3, B4, or B5.

[0149] In one embodiment, the present invention provides an isolate of Leuconostoc mesenteroides containing one or more or all of the polymorphisms listed in Table 18 or 19 that are different from ATCC8293. In one embodiment, the isolate of Leuconostoc mesenteroides contains five or more of the polymorphisms listed in Table 18 or 19 that are different from ATCC8293. In one embodiment, the isolate of Leuconostoc mesenteroides contains ten or more of the polymorphisms listed in Table 18 or 19 that are different from ATCC8293. In one embodiment, the isolate of Leuconostoc mesenteroides contains fifteen or more of the polymorphisms listed in Table 18 or 19 that are different from ATCC8293. In one embodiment, the isolate of Leuconostoc mesenteroides contains nineteen or more of the polymorphisms listed in Table 18 or 19 that are different from ATCC8293. In one embodiment, the Leuconostoc mesenteroides isolate contains 20 or more of the polymorphisms listed in Table 19 that are different from ATCC8293. In one embodiment, the Leuconostoc mesenteroides isolate contains 30 or more of the polymorphisms listed in Table 19 that are different from ATCC8293. In one embodiment, the Leuconostoc mesenteroides isolate contains 50 or more of the polymorphisms listed in Table 19 that are different from ATCC8293. In one embodiment, the Leuconostoc mesenteroides isolate contains 80 or more of the polymorphisms listed in Table 19 that are different from ATCC8293. In one embodiment, the Leuconostoc mesenteroides isolate contains 100 or more of the polymorphisms listed in Table 19 that are different from ATCC8293. In one embodiment, the isolated strain of Leuconostoc mesenteroides contains 150 or more polymorphisms listed in Table 19 that are different from ATCC8293. In another embodiment, the isolated strain of Leuconostoc mesenteroides contains 200 or more polymorphisms listed in Table 19 that are different from ATCC8293.In one embodiment, the isolated strain of Leuconostoc mesenteroides contains more than 300 polymorphisms listed in Table 19 that are different from ATCC8293. In another embodiment, the isolated strain of Leuconostoc mesenteroides contains more than 400 polymorphisms listed in Table 19 that are different from ATCC8293.

[0150] In one embodiment, the present invention provides an isolate of Lactobacillus plantarum that contains one or more or all of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16, or Table 17, which is different from ATCC8014. In one embodiment, the present invention provides an isolate of Lactobacillus plantarum that contains five or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16, or Table 17, which is different from ATCC8014. In one embodiment, the present invention provides an isolate of Lactobacillus plantarum that contains ten or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16, or Table 17, which is different from ATCC8014. In one embodiment, the present invention provides an isolate of Lactobacillus plantarum that contains fifteen or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16, or Table 17, which is different from ATCC8014. In one embodiment, the present invention provides an isolated strain of Lactobacillus plantarum containing 20 or more polymorphisms listed in Table 13, Table 14, Table 15, Table 16, or Table 17, which are different from ATCC8014. In one embodiment, the present invention provides an isolated strain of Lactobacillus plantarum containing 25 or more polymorphisms listed in Table 13, Table 14, Table 15, Table 16, or Table 17, which are different from ATCC8014. In one embodiment, the present invention provides an isolated strain of Lactobacillus plantarum containing 30 or more polymorphisms listed in Table 13, Table 14, Table 15, Table 16, or Table 17, which are different from ATCC8014. In one embodiment, the present invention provides an isolated strain of Lactobacillus plantarum containing 35 or more polymorphisms listed in Table 13, Table 14, Table 15, Table 16, or Table 17, which are different from ATCC8014. In one embodiment, the present invention provides an isolated strain of Lactobacillus plantarum that is different from ATCC8014 and contains 40 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16, or Table 17.

[0151] In one embodiment, the present invention provides a starter culture for producing an isothiocyanate-containing product or probiotic, comprising a lactic acid bacterium containing one or more isolated strains as described herein. As used herein, “starter culture” is a culture of live microorganisms intended for fermentation. In one embodiment, the present invention provides a starter culture for producing an isothiocyanate-containing product or probiotic, comprising a lactic acid bacterium selected from one or more or all of the following: i) On September 25, 2017, BF1 was deposited with the National Measurement Institute Australia under the code V17 / 021729. ii) On September 25, 2017, BF2 was deposited with the National Measurement Institute Australia under V17 / 021730. iii) On September 25, 2017, B1 was deposited with the National Measurement Institute Australia under V17 / 021731. iv) On September 25, 2017, B2 was deposited with the National Measurement Institute Australia under V17 / 021732. v) On September 25, 2017, B3 was deposited with the National Measurement Institute Australia under V17 / 021733. vi) B4, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021734, and vii) B5 deposited with the National Measurement Institute Australia on September 25, 2017, under the name V17 / 021735.

[0152] In one embodiment, the Brassicaceae material is at least about 10 as described herein. 5A starter culture of CFU / g is inoculated. In one embodiment, the Brassicaceae material is at least about 10 as described herein. 6 A starter culture of CFU / g is inoculated. In one embodiment, the Brassicaceae material is at least about 10 as described herein. 7 A starter culture of CFU / g is inoculated. In one embodiment, the Brassicaceae material is at least about 10 as described herein. 8 A starter culture of CFU / g is inoculated. In one embodiment, the Brassicaceae material is at least about 10 as described herein. 10 A starter culture of CFU / g is inoculated. In one embodiment, the Brassicaceae material is about 10 as described herein. 5 CFU / g ~ approximately 10 10 The starter cultures with a CFU / g concentration are inoculated.

[0153] Probiotics In one embodiment, the present invention provides a probiotic comprising one or more lactic acid bacteria isolated from Brassicaceae. As used herein, “probiotic” refers to a live microorganism that provides health benefits to a host when administered in appropriate amounts. In one embodiment, the lactic acid bacteria were isolated from Brassica oleracea. In one embodiment, the lactic acid bacteria were isolated from broccoli. In one embodiment, the lactic acid bacteria were isolated from Australian broccoli. In one embodiment, the lactic acid bacteria are selected from i) Leuconostoc mesenteroides, ii) Lactobacillus plantarum, iii) Lactobacillus pentosus, iv) Lactobacillus rhamnosus, v) a combination of i) and ii), vi) a combination of i), ii) and iii), and vii) a combination of i), ii) and iv). In one embodiment, the lactic acid bacteria are selected from one or more or all of BF1, BF2, B1, B2, B3, B4, and B5. In one embodiment, the lactic acid bacteria are B1. In one embodiment, the lactic acid bacteria are B2. In one embodiment, the lactic acid bacteria are B3. In one embodiment, the lactic acid bacteria are B4. In one embodiment, the lactic acid bacteria are B5. In one embodiment, the probiotic is a capsule, tablet, powder, or liquid. In one embodiment, the probiotic is microencapsulated as described in WO2005030229. [Examples]

[0154] Example 1 - Method Chemical substances and reagents HPLC-grade methanol, sodium dihydrogen phosphate, sodium hydroxide (NaOH), and hydrochloric acid (HCl) were purchased from Merck (Damstadt, Germany). Forin-Chiocalt reagent, sodium carbonate (Na2CO3), gallic acid, sodium fluorescein salt, and dipotassium hydrogen phosphate were purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium dihydrogen phosphate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and 2,20-azobis(2-methylpropionamidine) dihydrochloride (AAPH) were purchased from Sapphire Bioscience (Redfern, NSW, Australia).

[0155] lactic acid bacteria The lactic acid bacteria used during fermentation were selected from one or more of the following: LP:Lactobacillus plantarum ATCC8014, LGG: Lactobacillus rhamnosus ATCC53103, B1: Lactobacillus plantarum, isolated from broccoli, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021731. B2: Lactobacillus plantarum, isolated from broccoli, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021732. B3:Lactobacillus plantarum isolated from broccoli, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021733. B4:Lactobacillus plantarum isolated from broccoli, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021734. B5: Lactobacillus plantarum, isolated from broccoli, deposited with the National Measurement Institute Australia on September 25, 2017, under V17 / 021735. BF1: Leuconostoc mesenteroides isolated from broccoli puree, deposited on September 25, 2017, under V17 / 021729, at the National Measurement Institute Australia. BF2: Deposited on September 25, 2017, at the National Measurement Institute Australia under V17 / 021730, BF2, Leuconostoc mesenteroides isolated from broccoli puree. BP: Pooled BF1, BF2, and LAB: Pooled B1, B2, B3, B4, and B5.

[0156] BF1 and BF2 were identified as Leuconostoc mesenteroides based on their 16s-RNA sequences (Australian Genome Research Facility, data not shown). B1-B5 were identified as Lactobacillus plantarum based on their 16S-RNA sequences. Identification information for all isolates was confirmed by whole-genome sequencing analysis.

[0157] Isolation of lactic acid bacteria from broccoli and broccoli puree Lactobacillus plantarum strains B1, B2, B3, B4, and B5 were isolated from broccoli leaves and stems. The leaves and stems were washed with water and homogenized with peptone-added saline using a stomacher. The immersion solutions were serially diluted and spread onto De Man, Rogosa, and Sharpe (MRS) agar. These plates were incubated at 37°C for 48–72 hours under anaerobic conditions to isolate putative mesophilic lactic acid bacteria. Based on the different colony morphologies on the MRS plates, colonies were isolated, cultured in MRS broth, screened using staining and biochemical characterization techniques, and cryopreserved at -80°C using glycerol. The isolates were identified at the species level using 16s RNA sequencing with AGRF.

[0158] For the isolation of Leuconostoc mesenteroides BF1 and BF2, instead of using the suspension described above for isolation from broccoli leaves, a puree of broccoli florets was used after serial dilution.

[0159] Preparation of starter cultures Lactobacillus strains Leuconostoc mesenteroides and Lactobacillus plantarum were isolated from broccoli and identified by Australian Genome Research Facility Ltd. To obtain primary cultures, lactic acid bacteria cultures stored at -80°C were inoculated into 10 mL of MRS broth (Oxoid, Victoria, Australia) and incubated at 30°C for 24 hours to obtain an initial biomass of 8 log colony-forming units / milliliter (CFU / mL). 2 mL of each primary inoculum was inoculated into 200 mL of MRS broth and incubated at 30°C for 24 hours. The cultures were collected by centrifugation at 2000 g for 15 minutes at 4°C, washed twice with sterile phosphate-buffered saline (PBS), and all Lactobacillus plantarum cultures were mixed together, followed by all Leuconostoc mesenteroides cultures. The two culture suspensions were diluted to 10 log CFU / ml and mixed in the same volume ratio. The mixture was then stored at -80°C with glycerol until ready to be used as a mixed starter culture for broccoli fermentation.

[0160] Fermentation method 30 kg of broccoli florets (Brassica oleracea L. Italic) were cut approximately 2 cm from the canopy and finely chopped into smaller pieces. These were then immersed for 1 minute in Milli-Q water at a 3:2 ratio using a magic bullet blender. The broccoli slurry was thoroughly mixed and placed in sterile plastic bottles (200 mL) with screw caps. Each bottle (200 mL) of broccoli puree was inoculated with a starter culture prepared at an initial concentration of 8 log CFU / g. Fermentation experiments were carried out in parallel at 30°C with 48 bottles until the pH reached approximately 4.0 (day 4). After the fermentation stage was complete, three samples were taken out as storage samples for day 0, and the remaining samples were divided into two lots for storage experiments: one lot was stored in a refrigerator (4°C), and the other lot was stored in a constant temperature room at 25°C. Samples were periodically collected over 12 weeks for microbiological, physicochemical, and phytochemical analysis. Fermented broccoli puree was compared to raw broccoli puree stored at -20°C after homogenization. The puree samples were incubated for the same period as the fermented samples without inoculation in the laboratory.

[0161] Sample collection In the time-course experiment, samples were collected on days 10, 20, 30, 40, 50, 60, 70, 80, and 90, as well as on days 14, 28, 42, 56, 70, and 84, for samples stored at 25°C and 4°C, respectively. Sample collection was repeated three times using color measured on the surface and pH measured immediately after opening the fermentation bottle. Subsequently, samples were collected for microbiological analysis and titration acidity analysis. The remaining material was divided into two parts: the first part was frozen, freeze-dried, and ground into a fine powder, then stored in a desiccator for further analysis; and the second part was frozen and stored at -20°C until glucoraphanin and sulforaphane analysis.

[0162] Microbiological analysis In the microbiological analysis, CFU / g of broccoli puree was measured for different microorganisms using three different media: plate counts of total lactic acid bacteria on DeMan-Rogosa-Sharp (MRS) agar, plate counts of total enterobacteria on violet-red bile-glucose agar (VRBGA), and plate counts of yeasts and molds on potato dextrose agar (PDA). For each sample, the broccoli suspension was serially diluted in sterile peptone saline diluent, and 0.1 mL of the dilution was seeded twice on an agar plate. CFU was counted after aerobic incubation at 25°C for 72 hours (PDA), after aerobic incubation at 37°C for 24 hours (VRBGA), and after anaerobic incubation at 30°C for 72 hours (MRS).

[0163] Determination of pH and titration acidity The pH value was directly determined using a pH meter (PHM240, MeterLab) in a fermentation bottle containing broccoli puree. The titratable acidity (TA) of the broccoli sample was measured using an automated titrator (Titralab 854 titration manager, Radiometric Analytical, France). In short, diluted broccoli puree (10 mL) was titrated with 0.1 M NaOH to an endpoint pH of 8.1, and the results were expressed as the gram equivalent of lactic acid per liter of sample according to the following formula:

number

[0164] Total protein and color analysis The total protein content of the broccoli sample was determined by multiplying the total nitrogen content by 6.25. The total nitrogen content of the broccoli was analyzed using the Dumas combustion method with a LECO TruMac instrument (LECO Corporation, Michigan, USA). The color index (L, a, b) of the fermented broccoli sample was determined using a CR-200 tristimulus colorimeter (Minolta, Osaka, Japan). The obtained color values ​​were converted to lightness / darkness (L). * (as), redness / greenness (a * ) and yellowness / blueness (b * It was expressed as ). The sum of the color differences (ΔE) was calculated according to the following formula:

number

[0165] Determination of total polyphenol content Total phenol content (TPC) was measured by spectrophotometric analysis using a modified version of the Forin-Ciocalt colorimetric method (Singleton and Rossi, 1965). Briefly, 50 mg of broccoli powder was suspended in 10 mL of acidified (1% HCl) methanol / water (70:30, v / v) solution and extracted in an ultrasonic bath (IDK technology Pty Ltd, VIC, Australia) for 8 minutes. The extract was stored at 4°C for 16 hours, filtered through a 0.2 μM filter, and stored at 4°C until analysis. 1 mL of 0.2 N Forin-Ciocalt reagent, 800 μL of aqueous sodium carbonate solution (7.5% p / v), and 180 μL of Milli-Q grade water were added to the extract (20 μL). After incubation at 37°C for 1 hour in the dark, absorbance was measured three times at 765 nm using a spectrophotometer (UV-1700 Pharma Spec, SHIMADZU). Gallic acid was used as a standard substance, and TPC was expressed in mg (mg GAE / 100g FW) as gallic acid equivalents (GAE) per 100g of fresh body weight, based on a calibration curve created using gallic acid of known concentrations.

[0166] Oxygen radical absorption capacity assay Lyophilized broccoli powder (10 mg) was suspended in 10 mL of methanol / water (80:20, v / v) as the extraction solvent. This slurry was extracted on a Heidolph Multi-Reax (John Morris Scientific, NSW, Australia) at 650 rpm for 1 hour at room temperature. The slurry was then centrifuged at 25,000 g for 15 minutes at 4°C, and the supernatant was collected. After dilution by 100 × with 75 mM potassium phosphate buffer (pH 7.4), the supernatant was prepared for analysis. ORAC analysis was performed according to the procedure reported by Huang et al. (2002), with minor modifications. The assay was performed in an opaque 96-well plate (black optical bottom, Waltham, MA, USA). The assay reaction mixture contained 81.6 nM fluorescein, 153 mM AAPH, Trolox standards at different concentrations (100, 50, 25, 12.5, and 6.25 μM), and 75 mM phosphate buffer as a blank. The reaction mixture was added in the following order: 25 μL of diluted sample, 25 μL of 75 mM phosphate buffer, 25 μL of Trolox standard, and 150 μL of fluorescein. After adding fluorescein, the plate was incubated at 37°C for 10 minutes, and then AAPH (25 μL) was added. Immediately after adding AAPH, the plate was placed in a fluorescence plate reader (BMG Labtech ClarioStar, Germany), and fluorescence was measured every 3 minutes until it decreased to less than 5% of the original fluorescence. ORAC values ​​were calculated as the area under the curve (AUC) and expressed as micromoles of trolox equivalents (TE) per gram of dry weight of broccoli (μmol TE / g DW). Each sample was assayed three times.

[0167] Sulforaphane analysis Sulforaphane extraction from broccoli matrix was performed according to the method of Li et al. (2012), with some modifications. Briefly, 2 g of frozen broccoli was mixed with 2 mL of Milli-Q water and vortexed for 1 minute. Then, 20 mL of ethyl acetate was added to the slurry, followed by sonication for 5 minutes and shaking at 4°C for 20 minutes. The slurry was then centrifuged at 15,000 g for 10 minutes, and the supernatant was collected. A further 15 mL of ethyl acetate was added to the precipitate for secondary extraction. Pooled extracts from each sample were evaporated to dryness at room temperature using a vacuum centrifuge (SC250EXP, Thermo Fisher Scientific, CA, USA) and stored at -20°C until analysis. The sulforaphane concentration was determined using an Acquity® Ultra Performance LC system (Waters Corporation, Milford, MA, USA), which is equipped with a binary solvent delivery manager and a sample manager. Chromatographic separation was performed on a 2.1 × 50 mm Acquity BEH C18 chromatography column. Mobile phases A and B were 0.1% formic acid milliq aqueous solution and 0.1% formic acid acetonitrile solution, respectively. The gradient elution system consisted of mobile phases A (0.1% formic acid milliq aqueous solution) and B (0.1% formic acid acetonitrile solution), 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 maintained at a constant 30°C. The flow rate was 0.350 mL / min, and the injection volume was 5 μL.

[0168] Prior to analysis, all samples were dissolved in 1 mL of 30% acetonitrile and filtered through a 0.22 μm membrane filter (Merk Millipore, Billerica, MA, USA). Identification of each peak was based on the retention time of known standards and chromatography. The concentration of each compound was calculated according to a calibration curve, and the results were expressed as micromoles (μmol / kg DW) per kilogram of broccoli DW.

[0169] Glucoraphanin analysis Extraction of glucoraphanin from raw or fermented broccoli was carried out according to the method of Cai and Wang (2016), with some modifications. Specifically, 2 g of frozen broccoli puree was added to 10 mL of boiling Milli-Q water, and the mixture was incubated in a boiling water bath for 5 minutes. It was then cooled and centrifuged at 15000 × g for 15 minutes to collect the supernatant. The precipitate was extracted again with 8 mL of boiling water. Pooled extracts from each sample were evaporated to dryness at 3°C ​​using a vacuum centrifuge (Speedvac SC250EXP, Thermo Fisher Scientific, CA, USA) and stored at -20°C until analysis. Glucoraphanin concentration was quantified using an Alliance HPLC instrument (Waters Corporation, Milford, MA, USA) equipped with a 2998 photodiode array detector. An HPLC column—Luna® 3 μM hydrophilic interaction liquid chromatography (HILIC) 200 Å (100 × 4.6 mm, Phenomenex, Torrance, CA, USA)—was used for analysis at a column temperature of 25°C. The mobile phase consisted of 30 mM ammonium formate (Solution A) and acetonitrile (Solution B) in acetonitrile / water (85:15, v / v), using the following isocratic flow program: 70% Solution A, 30% Solution B. Other chromatographic conditions included a constant flow rate of 2.0 mL / min, an injection volume of 100 μL, a run time of 8 minutes, and a detection wavelength of 235 nm. Prior to analysis, the entire sample was dissolved in 1 mL of solvent A and filtered through a 0.22 μm membrane filter (Merk Millipore, Billerica, MA, USA). Identification of each peak was based on the retention time of known glucoraphanin standards and chromatography. The concentration of glucoraphanin was calculated using a calibration curve, and the results were expressed as micromoles of glucoraphanin per kilogram of dry weight (DW) of broccoli (μmol / kg DW).

[0170] statistical analysis All experiments were repeated three times, and the results were expressed as mean values. One-way analysis of variance (ANOVA) was applied to evaluate the significance of differences between means at a significance level of 0.05 (p<0.05). Statistical analysis was performed using statistical software, SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA).

[0171] Example 2 - Microbial analysis of lactic acid fermented broccoli florets The broccoli puree was fermented as described in the fermentation section of Example 1. As shown in Table 1, the total number of lactic acid bacteria was lower in raw broccoli compared to inoculated broccoli. After 4 days of fermentation, the pH of the sample reached 4.04, so fermentation was stopped, and the fermented sample before the storage experiment was considered the day 0 sample. As is clear from Table 1 and Figure 1C, the total number of lactic acid bacteria in the day 0 sample was significantly increased compared to raw broccoli (8 log CFU / g). During the first two weeks of storage, the viable number of total lactic acid bacteria increased to a maximum of 9 log CFU / g for samples stored at both 25°C and 4°C (Tables 1 and 2). During storage at 25°C, the total number of lactic acid bacteria increased to 9 log CFU / g on day 10, slowly decreased during storage to 5 log CFU / g by day 50, and further decreased to a nearly undetectable level after day 70. In contrast, the LAB count in samples stored at 4°C remained high (6 log CFU / g) even after 84 days of storage.

[0172] [Table 1]

[0173] [Table 2]

[0174] The total number of yeasts and molds in the raw broccoli sample was 2 log CFU / g. The number of Enterobacteriaceae in the raw broccoli was 3 log CFU / g. No fungi, molds, or enterobacteria were detected in the fermented sample after fermentation or after storage under both temperature conditions. No pathogenic or spoilage bacteria were detected after fermentation or during storage. The results indicate that the fermentation process produced a safe and stable product in which potentially pathogenic Enterobacteriaceae and spoilage yeasts and molds were undetectable, and that high levels of total lactic acid bacteria were maintained when stored at 4°C. After approximately 3 months at 4°C, approximately 10 6 Lactobacillus is present in CFU / g amounts.

[0175] Example 3 - Evaluation of pH and titratable acidity of lactic acid fermented broccoli florets after storage. The pH and titratable acidity (TA) of raw broccoli, fermented broccoli, and fermented broccoli stored at 25°C and 4°C were 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 lactic acid bacteria lower the pH of the sample. As shown in Table 1, the TA rose to 10.7 g / L in the day 0 sample. When stored at 25°C, the pH decreased to 3.87 during storage after 10 days, and in addition, the TA value reached 14.4 g / L, a significant increase (p<0.05, see Table 1). The results indicate that during the first few days of storage, substrates for further acid production were still present and consumed by the lactic acid bacteria. Neither the pH nor the TA value changed significantly during the remainder of the storage period (Table 1).

[0176] When the temperature was lowered to 4°C, the activity of lactic acid bacteria decreased at low temperatures, resulting in a reduction in the rate of decrease in pH and TA in the stored samples (see Table 2). After storage at 4°C for approximately 3 months, the pH was 3.85 and the TA value was 13.7 g / L.

[0177] Example 4 - Evaluation of broccoli immersion and fermentation for the conversion of glucoraphanin to sulforaphane. Broccoli florets were cut into small pieces and mixed with water in a 3:2 broccoli:water ratio. The mixture was then pureed using a blender. 200 g of the puree sample was divided into sterile plastic bottles. A pooled culture of lactic acid bacteria (Leuconostoc mesenteroides and Lactobacillus plantarum) isolated from Australian broccoli was added to the sample. 8 The samples were inoculated with CFU / gm. The samples were incubated in a water bath maintained at 30°C until the pH dropped to approximately 4.0, which was achieved 4 days after fermentation. The control uninoculated samples were frozen immediately after immersion. Sodium benzoate was added to a second set of uninoculated control samples to inhibit microbial growth, and these samples were incubated with the inoculated samples at 30°C for 4 days until fermentation of the inoculated samples was complete. The experiment was repeated three times. All samples were frozen and stored until sulforaphane and glucoraphanin analysis. As shown in Figure 1B and Table 3, fermentation following immersion increased sulforaphane yield compared to immersion and incubation alone.

[0178] [Table 3]

[0179] Example 5 - Evaluation of total protein content and color of lactic acid fermented broccoli florets after storage. The total protein content and color of lactic acid-fermented broccoli florets after fermentation were evaluated as described above in the Methods section. Compared to raw broccoli (26.9 ± 0.03 mg), the total protein content of fermented broccoli was significantly increased (29.6 ± 0.8 mg / g, p < 0.05). This may be due to the large number of lactic acid bacteria inoculated into the sample, which led to the proliferation of lactic acid bacteria during fermentation and subsequent protein synthesis by these bacteria. The total protein content remained stable during storage at both 25°C and 4°C (Tables 1 and 2), and there were no significant differences between samples.

[0180] The color values ​​(L, a, b) and total color difference (ΔE) of the broccoli samples are summarized in Tables 1 and 2. As shown in Tables 1 and 2, significant differences in color parameters and total color difference values ​​(ΔE) were recorded between raw and fermented samples. * While the value (brightness) did not change significantly, a * Value (greenness) and b * The value (yellowness) decreased after fermentation of the broccoli puree. * Value and b * The decrease in values ​​may be due to the decomposition of pigment compounds such as chlorophyll, which is converted to pheophytin under low pH conditions. The high ΔE value (12.5) of the day 0 sample indicated a significant change in the color of the broccoli puree after fermentation, which was visually confirmed. During storage (Tables 1 and 2), there were no significant changes in ΔE values ​​for any of the samples at 25°C or 4°C.

[0181] Broccoli fermented using LAB+BP (B1, B2, B3, B4, B5 of Lactobacillus plantarum isolated from broccoli, and BF1, BF2 of Leuconostoc mesenteroides) exhibited a brighter, deeper green color compared to broccoli fermented using LAB alone (Lactobacillus plantarum (B1, B2, B3, B4, B5) isolated from broccoli), and was closer in color to raw soaked broccoli.

[0182] Example 6 - Changes in total phenol content and antioxidant activity of lactic acid bacteria in fermented broccoli florets The total phenol content (TPC) and antioxidant activity of lactic acid fermented broccoli florets were evaluated as described above in the Methods section. The TPC of raw broccoli was 127.6 ± 12.4 mg GAE / 100g fresh body weight (Figure 3A). The TPC value on day 0 was significantly increased to 236.9 ± 23.4 mg GAE / 100g (p<0.05) compared to raw broccoli. There was no significant difference in TPC after storage between samples stored at 25°C and 4°C (Figure 3A). When stored at 25°C, the TPC value of fermented broccoli was 246.2 ± 19.3 mg GAE / 100g on day 10 and 248.1 ± 25.0 mg GAE / 100g on day 90. When stored at 4°C, the TPC values ​​were 274.1±20.2 and 267.2±3.3 mg GAE / 100g for 14 days and 84 days, respectively.

[0183] Figure 3B shows the antioxidant activity of the sample, expressed as an ORAC value. The ORAC value of the raw sample was 110.1 ± 0.05 μmol TE / g. After fermentation, the ORAC value increased significantly by approximately 70% to 186.9 ± 3.3 μmol TE / g compared to raw broccoli. This result suggests that antioxidant compounds may have increased during fermentation and is consistent with the change in TPC after fermentation.

[0184] During storage, the antioxidant activity of fermented broccoli did not change significantly. As shown in Figure 3B, when stored at 25°C, the ORAC values ​​at 10 and 90 days were 173.0±14.4 and 150±5.5 μmol TE / g, respectively. Similar results were obtained for samples stored at 4°C. The ORAC value was 172.0±15.5 μmol TE / g at the start of storage and increased to a maximum value (188.7±12.9 μmol TE / g) after storage.

[0185] Example 7 - Evaluation of fermentation time for different combinations of lactic acid bacteria Soaked broccoli was prepared as described above in the Method section, with a broccoli-to-water ratio of 3:2 and a soaking time of 1 minute. 7 CFU / g or 10 8One of the following was inoculated at a CFU / g level: LGG, LAB (Lactobacillus plantarum (B1, B2, B3, B4, B5) isolated from Australian broccoli), LAB+LP (Lactobacillus plantarum and Lactobacillus species ATCC8014 isolated from broccoli), BP (Leuconostoc mesenteroides isolated from broccoli), or LAB+BP (a mixture of the two groups as described in the Methods section). Fermentation was carried out at 25°C, 30°C, or 34°C to reach the target pH of 4.4. As shown in Figure 4, the addition of lactic acid bacteria isolated from broccoli and / or broccoli puree resulted in a pH of 4.4 after approximately 4 days of fermentation, while the LAB+BP combination significantly reduced the fermentation time. An example composition of the fermented broccoli product is shown in Table 4.

[0186] [Table 4]

[0187] Example 8: Effect of storage on the sulforaphane content of fermented broccoli Figure 2A shows the effects of storage at 4°C and 25°C on the sulforaphane content of fermented broccoli puree. As can be seen in Figure 2A, the sulforaphane content of the sample stored at 25°C dramatically decreased to 770.7 ± 34.9 μmol / kg after 20 days of storage (a 52% loss), and then gradually decreased for the remainder of the storage period, reaching a total loss of 69.5%. Interestingly, no statistically significant change in sulforaphane content was observed in the fermented broccoli sample during the first two weeks of storage at 4°C. A significant decrease of approximately 23.7% occurred in the following two weeks, and then gradually decreased for the remainder of the storage period. At the end of storage (day 84), the sulforaphane content in the sample stored at 4°C was 1012.9 ± 57.6 μmol / kg, representing a total sulforaphane loss of approximately 37.4% compared to the sample on day 0. During the first two weeks of storage, the sulforaphane content was likely maintained in samples stored at 4°C, as a slight decrease in glucoraphanin content was observed over the same period, suggesting that sulforaphane was being produced and broken down simultaneously.

[0188] Example 9: Effects of fermentation and storage on glucoraphanin content Figure 7 shows the effects of immersion 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 μmol / kg (Figure 7), and after fermentation, the glucoraphanin content rapidly decreased to 712.4 ± 64.2 μmol / kg (day 0 sample). Since glucoraphanin is relatively stable in intact tissue, this degradation may be due to myrosinase-catalyzed hydrolysis caused by increased enzyme-substrate interactions in immersed tissue during fermentation. The period of rapid decrease in glucoraphanin coincided with the fermentation period.

[0189] No significant changes in glucoraphanin content were observed in fermentation samples stored at 25°C or 4°C. However, a slightly higher glucoraphanin content was observed in the sample stored at 25°C. This may be related to the rapid decrease in pH of the sample stored at 25°C (pH 3.87 at the second measurement point) compared to the sample stored at 4°C (pH 4.04 at the second measurement point). The optimal pH range for myrosinase-catalyzed hydrolysis of glucoraphanin is 5 to 6, decreasing to a minimum value of pH 3.0 (Dosz & Jeffery, 2013). The relatively higher pH of the sample stored at 4°C may have contributed to slightly higher glucoraphanin degradation during storage at 4°C compared to 25°C.

[0190] Example 10 - Evaluation of heat treatment conditions to maximize the conversion of glucoraphanin to sulforaphane in broccoli matrix Broccoli florets packed in retort pouches were heat-treated at temperatures ranging from 60°C to 80°C for processing times of 0 to 5 minutes. The treatment involved preheating to the experimental temperature in a water bath maintained 5°C above the experimental temperature, followed by incubation in a second water bath maintained at the experimental temperature. After heat treatment, the samples were cooled in ice water and immersed in water with a water-to-broccoli ratio of 2:3 as described above. The immersed samples were incubated at 30°C for 1 hour and then frozen for sulforaphane analysis. The results are shown in Figure 2B and Table 5. As shown in Table 5, preheating the samples to 60°C, 65°C, or 80°C, followed by immersion, increased the sulforaphane yield compared to raw broccoli florets immersed without preheating.

[0191] [Table 5]

[0192] Example 11 - Evaluation of preheating before lactic acid fermentation of broccoli in relation to sulforaphane content This study evaluated the effect of slight preheating of broccoli florets, aimed at inactivating epithio-specific proteins (ESPs), on the sulforaphane content of broccoli puree when combined with lactic acid bacteria.

[0193] material Broccoli (variety "Viper") was purchased from a local supermarket (Coles, Werribee South, VIC, Australia). DeMan-Rogosa-Sharp (MRS) broth (1823477, CM0359, Oxoid) was purchased from Thermo Fisher Scientific (Australia). DL-sulforaphane was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). All other chemical and biochemical reagents were analytical grade or higher and purchased from local chemical suppliers.

[0194] Experiment to optimize some preheating conditions for maximizing sulforaphane yield. Broccoli florets were cut to approximately 2 cm from the top, and 30 g of randomly mixed florets were used for the preheating experiment. Two types of preheating experiments were performed: in-pack processing and direct water blanching. In the in-pack experiment, the broccoli florets were filled into retort pouches (Caspak Australia, Melbourne), sealed, and preheated at various points in constant-temperature water batches maintained at 60°C, 65°C, and 80°C. The temperature of the broccoli sample at the latest heating point was measured using a thermometer. Time 0 was defined as the time it took for the core temperature to reach the specified experimental temperature. Processing times were 0 minutes, 1 minute, 3 minutes, and 5 minutes at 60°C and 65°C, and 0 minutes, 1 minute, 2 minutes, and 3 minutes at 80°C. In the direct water blanching experiment, the broccoli florets were immersed in Milli-Q water in a glass beaker heated in a constant-temperature water bath. Direct water blanching experiments were performed at 60°C and 65°C. The temperature of the broccoli samples was continuously measured using a thermometer, and timing was started when the temperature of the slowest heating point reached the specified experimental temperature as described above. All heat treatment experiments were repeated three times. Unheated broccoli florets were used as a control. Immediately after heat treatment, the samples were cooled in ice water and homogenized for 1 minute using Milli-Q water in a 3:2 ratio of broccoli to water using a kitchen-scale magic bullet blender (Nutribullet pro 900 series, LLC, USA). The homogenized samples were incubated in the dark at 25°C for 4 hours to allow for enzymatic hydrolysis of glucoraphanin. After incubation, all samples were frozen at -20°C until sulforaphane analysis.

[0195] Preparation of starter cultures Pooled cultures of Leuconostoc mesenteroides (BF1, BF2) and Lactobacillus plantarum (B1, B2, B3, B4, B5) isolated from broccoli, as described in the method of Example 1, were used in the fermentation experiment. Lactic acid bacteria cultures stored at -80°C were inoculated into 10 mL of MRS broth (Oxoid, Victoria, Australia) and activated by incubation at 30°C for 24 hours to obtain primary inoculum. 2 mL of primary culture was inoculated into 200 mL of MRS broth to obtain secondary cultures. After 24 hours of incubation, the six secondary cultures were centrifuged, washed twice with sterile phosphate-buffered saline (PBS), and each culture was resuspended in Milli-Q water at a concentration of 10 log colony-forming units / milliliter (CFU / mL) to obtain an initial biomass of 8 log CFU / mL in 100 g / m³ of broccoli puree sample. L. plantarum culture was mixed with L. mesenteroides culture in a 1:1 ratio and then inoculated into a broccoli puree sample.

[0196] Sample preparation Broccoli florets were cut approximately 2 cm from the canopy and divided into two lots: heat-treated and untreated. After heat treatment under optimal conditions selected based on the experimental results described above, the samples were cooled in ice water, shredded, and homogenized for 1 minute with Milli-Q water in a 3:2 ratio using a kitchen-scale magic bullet blender (Nutribullet pro 900 series, LLC, USA). Untreated broccoli was homogenized in the same manner. After thorough mixing, the broccoli puree was portioned into sterile plastic containers with screw caps (100 mL) (Technoplast Australia) for further experiments.

[0197] fermentation In this example, LAB cultures prepared as described above were inoculated into broccoli puree samples (preheated and untreated). Preheating of broccoli florets was performed in a pack at 65°C for 3 minutes based on experimental results, and the preheating conditions were optimized. To evaluate the effect of acidification without fermentation on the conversion of glucoraphanin to sulforaphane, an acidification experiment was performed using glucono delta-lactone (GDL) on preheated, untreated broccoli puree to obtain the pH of fermented broccoli puree. Furthermore, preheated broccoli puree without further treatment and untreated broccoli puree were used as controls.

[0198] In the fermentation experiment, each broccoli puree sample was inoculated with a starter culture prepared at an initial level of 8 log CFU / g. The fermentation experiment was carried out at 30°C until the pH reached approximately 4.0 after 15 hours of incubation. Once fermentation was complete, three samples (day 0 samples) were taken from each fermentation group and stored at -20°C until analysis. The remaining fermented material was randomly divided into two lots for storage testing: one lot was stored under refrigerated conditions (4°C) and the second lot was stored at 25°C to evaluate the sulforaphane stability of the samples after 14 days of storage. Similarly, untreated broccoli puree, preheated broccoli puree, and preheated GDL-treated broccoli puree were also sampled at day zero and stored at 25°C and 4°C for 14 days of storage testing. After 14 days of storage, all samples were frozen and stored at -20°C until sulforaphane analysis.

[0199] Sulforaphane analysis and statistical analysis The procedure was carried out as described in Example 1.

[0200] Optimization of heat treatment conditions to improve sulforaphane yield Figure 5A shows the effect of heat treatment on sulforaphane formation in packaged broccoli florets at three different temperatures (60°C, 65°C, and 80°C) and various treatment times (0, 1, 3, and 5 minutes at 60°C or 65°C, and 0, 1, 2, and 3 minutes at 80°C). The results showed an increased sulforaphane yield in all heat-treated samples compared to raw broccoli. Time 0 indicates samples heated until their centers reached the experimental temperature.

[0201] As shown in Figure 5A, when packed broccoli samples were heated at 60°C for 0 minutes, 1 minute, 3 minutes, and 5 minutes, the sulforaphane yield increased. The sulforaphane concentrations in these samples were 2343.5±124.1, 2661.5±10.9, 2780.9±270.8, and 3147.7±148.0 μmol / kg DW, respectively. On the other hand, when broccoli was treated at 65°C, the sulforaphane yield initially increased with treatment time, from 3585.9±119.2 (0 minutes) to a maximum of 3983.4±30.5 μmol / kg DW (3 minutes). Further increases in treatment time resulted in a decrease in yield, with a minimum value of 3620.1±240.7 μmol / kg observed after 5 minutes of treatment. In contrast to treatments at 60°C and 65°C, samples treated at 80°C showed a consistent decrease in sulforaphane yield with increasing treatment time, with sulforaphane yields of 1451.5±43.5, 1446.8±17.5, 1043.1±94.2, and 981.2±35.1 μmol / kg DW after 0, 1, 2, and 3 minutes of treatment, respectively. Generally, for in-pack treatment of broccoli, the highest sulforaphane yield (3983.4±30.5 μmol / kg) was obtained from samples preheated at 65°C for 3 minutes, which is approximately five times higher than that of raw broccoli (817.5±9.3 μmol / kg DW). In contrast, when broccoli was heated directly in water, the sulforaphane yield was generally lower compared to in-pack treatment, as shown in Figure 5B. In direct water blanching at 60°C, the sulforaphane yield increased with treatment time from 1698.00±121.9 μmol / kg DW (0 min) to 2833.3±118.6 μmol / kg DW (1 min), and then gradually decreased to a minimum value of 2345.8±57.7 μmol / kg DW after 5 minutes of treatment at 60°C. When the sample was blanched at 65°C, a sharp decrease in sulforaphane yield was observed compared to 60°C. The sulforaphane yield of broccoli after heat treatment at 65°C for 5 minutes was 503.7±23.8 μmol / kg DW, which was even lower than the value obtained from raw broccoli. This may be because glucoraphanin leached into the blanching water, reducing the sulforaphane yield.In direct water blanching, the optimal treatment temperature for maximizing sulforaphane yield was 60°C, compared to 65°C in in-pack treatment.

[0202] In this study, the highest sulforaphane yield was obtained from broccoli florets treated at 65°C for 3 minutes in a pack. This condition indicated that it promotes greater inactivation of ESP while maintaining sufficient myrosinase activity, resulting in optimal conversion to sulforaphane. Under these conditions, the glucoraphanin content of the broccoli sample was determined to be 3423.7 ± 39.7 μmol / kg DW. Assuming a 1:1 conversion, it is likely that a large portion of the extractable glucoraphanin is converted to sulforaphane.

[0203] The finding that fermenting heat-treated broccoli yielded higher levels of sulforaphane than expected based on the extractable glucoraphanin levels in raw broccoli suggests that heat treatment may have increased the accessibility of glucoraphanin to myrosinase, resulting in a higher sulforaphane yield than expected based on the quantifiable amount of glucoraphanin present in untreated broccoli.

[0204] Broccoli florets blanched directly in water yielded a low sulforaphane yield, likely due to the water-soluble nature of glucoraphanin and its subsequent leaching into the blanching water. It is also noteworthy that when broccoli florets were directly heated in water, the maximum amount of sulforaphane was obtained by heating them at 60°C for 1 minute, compared to heat-treating them in a pack at 65°C for 3 minutes. This may be because the leaching rate into the blanching water was higher at 65°C, offsetting the effect of inactivating ESP at a high level.

[0205] Effects of LAB fermentation and chemical acidification on sulforaphane yield Broccoli florets were preheated in a pack under the optimal treatment conditions selected above (65°C, 3 minutes). The samples were then subjected to either fermentation with lactic acid bacteria or acidification using an acidifying agent (GDL). Consistent with the pretreatment experiment, the sulforaphane levels of the broccoli significantly increased after heat treatment (p<0.05), with sulforaphane yields of 806.2±7.0 μmol / kg DW and 3536.0±136.9 μmol / kg DW for raw and preheated broccoli, respectively. The value of 3536 μmol / kg DW obtained from this batch of preheated broccoli before fermentation was comparable to that obtained from different batches of broccoli, with a yield of 3983 μmol / kg DW, indicating minimal inter-batch variability.

[0206] As shown in Table 6, the sulforaphane content of broccoli samples after fermentation varied depending on the treatment of the broccoli before fermentation. The sulforaphane content of raw broccoli puree after fermentation (1617.4 ± 10.2 μmol / kg DW) was approximately twice that of raw broccoli puree. Preheating the broccoli before pureeing resulted in a significant increase in sulforaphane content after fermentation. The sulforaphane content of preheated fermented broccoli (13121.3 ± 440.8 μmol / kg DW) was approximately eight times that of raw fermented broccoli puree. The sulforaphane yield observed after combined preheating and fermentation was much higher than expected based on the quantifiable amount of glucoraphanin in raw broccoli samples (3423.7 ± 39.7 μmol / kg). The combined preheating and fermentation process appears to enhance the release and accessibility of glucoraphanin for conversion, in addition to the inactivation of ESP by the preheating process. The preheating process, interacting with microbial cell wall-degrading enzymes, may have improved the disruption of cell compartments and the release of bound glucosinolates in the matrix that were unextractable or inaccessible in raw broccoli. Some lactic acid strains produce polysaccharide-degrading enzymes such as cellulases and pectinases that can degrade cell wall structures, enhancing the release of wall-bound components.

[0207] In contrast, chemical acidification of preheated broccoli puree with GDL resulted in significantly lower sulforaphane content compared to preheated and preheated fermented samples (p<0.05) (Table 6). The sulforaphane content of the GDL-acidified sample was 2169.4±176.0 μmol / kg DW, which is 40% lower than that of the preheated broccoli sample (3536.0±136.9 μmol / kg DW) (P<0.05). The rapid decrease in pH to 4.04 during acidification may have reduced the conversion of glucoraphanin to sulforaphane in the GDL sample. It is well known that the conversion of glucosinolates is highly pH-dependent, and acidic pH is favorable for conversion to nitriles (Latte et al., 2011).

[0208] In the case of preheated fermentation samples, acidification occurs gradually over >15 hours, and after preheating at 65°C for 3 minutes, the activity of ESP is expected to decrease significantly, which mainly enables the conversion of glucoraphanin to sulforaphane.

[0209] Changes in sulforaphane content during storage The sulforaphane concentration in all samples decreased after 14 days of storage at 25°C (see Table 6 and Figure 6). Interestingly, an increase in sulforaphane content was observed in all samples except for the fermented sample stored at 4°C for 14 days. The sulforaphane content of the raw puree nearly doubled during storage at 4°C. Similarly, the sulforaphane content of the preheated sample increased by approximately 2.6 times, while that of the preheated GDL sample increased by approximately 2.3 times. This suggests that glucoraphanin is continuously released from the matrix during storage, enabling further conversion to sulforaphane and an increase in concentration, offsetting the results of sulforaphane degradation during storage. For the preheated fermented sample, a decrease in sulforaphane content was observed during storage at both temperatures. Since all accessible glucoraphanin may have been converted in large quantities to sulforaphane during fermentation, no further conversion occurred during storage; rather, degradation occurred, to varying degrees depending on the temperature. Therefore, while only a slight decrease (approximately 6%) was observed during storage at 4°C, a decrease of approximately 70% was observed during storage at 25°C.

[0210] This study demonstrated that preheating in conjunction with lactic acid fermentation significantly improves the sulforaphane content of broccoli-based products. Preheating broccoli florets in a pack at 65°C for 3 minutes, followed by immersion and fermentation, resulted in a sulforaphane yield approximately 16 times higher than that of raw broccoli puree. Under these conditions, preheating increased the sulforaphane yield of broccoli puree from 806 μmol / KgDW (dry weight) in untreated broccoli to 3536 μmol / KgDW, indicating that the treatment substantially inhibits ESP while maintaining sufficient myrosinase activity for the conversion of glucoraphanin to sulforaphane. The optimal preheating condition during direct water blanching was 60°C for 1 minute, resulting in a sulforaphane yield of 2833 μmol / KgDW. The low yield during direct blanching may be due to the leaching of water-soluble glucoraphanin into the blanching medium. Combining in-pack preheating of broccoli florets (65°C / 3 min) with lactic acid fermentation further increased the sulforaphane content to 13121 μmol / KgDW, which is approximately a 16-fold increase compared to raw broccoli. Chemical acidification of in-pack preheating (65°C, 3 min) combined with acidification of broccoli puree with glucono delta-lactone resulted in a sulforaphane yield of 2169 μmol / KgDW, which is lower than preheating alone. The sulforaphane content of the preheated and fermented puree remained stable during storage at 4°C for two weeks (approximately 94% retention).

[0211] [Table 6]

[0212] Example 12 - Effect of lactic acid fermentation on the polyphenol profile of broccoli To determine the effect of fermentation on polyphenol metabolites in broccoli samples, metabolome analysis based on target solution c chromatography-mass spectrometry (LC-MS) was performed on raw and fermented broccoli puree samples. The obtained multivariate data were analyzed using Metaboanalyst software (Metaboanalyst 3.0, Xia and Wishart, 2016). Fermentation resulted in significant changes in the metabolite profiles of the broccoli samples. Partial least squares discriminant analysis (PLS-DA) of the data showed a clear distinction in the polyphenol profiles between fermented and unfermented samples (Figure 8).

[0213] The top 15 metabolites identified as contributing to the difference between the two groups are shown in Figure 9. These are phenolic acids and phenol aglycones, which exhibit higher bioactivity and bioavailability compared to their phenolic acid esters and phenolic glycoside precursors. The concentrations of many of these metabolites showed a significant increase after fermentation, indicating a beneficial effect of fermentation on the polyphenol profile of broccoli puree. Table 7 shows the multiplier changes of some metabolites.

[0214] After fermentation, significant increases of 24-fold and 16-fold were observed in sinapic acid and kaempferol, respectively. Similarly, fermentation induced an 8-fold increase in chlorogenic acid and phenyllactic acid. The concentrations of hesperetin, quercetin, methyl syringate, and syringic acid also increased significantly after fermentation. The increase in the concentrations of aglycones, such as kaempferol, hesperetin, and quercetin, may be due to the conversion of their glycoside precursors by the activity of microbial glycosidases. The increase in the concentrations of phenolic acids such as sinapic acid may be due to the conversion of phenolic acid esters in broccoli by the activity of microbial esterases. After fermentation, slight decreases were observed in caffeic acid and gallic acid. The conversion of caffeic acid to its corresponding vinylcatechol and gallic acid to pyrogallol by the activity of microbial decarboxylases may be involved in the decrease in their concentrations (Filanino et al., 2015, Guzman-Lopez et al., 2009).

[0215] [Table 7]

[0216] Example 13 - Identification of metabolites produced by lactic acid fermentation of broccoli by targeted and non-targeted LC_MS analysis of samples. Fermented and unfermented broccoli puree samples were frozen and lyophilized. Each sample (100 mg of lyophilized powder) was extracted using 1 ml of ice-cold methanol and Milli-Q water (50:50, v:v) containing 100 mg / ml of caffeine as an internal standard. The samples were then vortexed for 2 minutes and sonicated for 30 minutes (40 Hz). The samples were then centrifuged at 20,000 rpm at 4°C for 30 minutes, and the supernatant was transferred to a clean silane-treated LC-MS vial. The samples were analyzed by injecting 1.4 μl into an Agilent 6410 LC-QQQ HPLC (Agilent Technologies, Santa Clara, California, USA). The analysis was performed using a reversed-phase Agilent Zorbax Eclipse Plus C18, Rapid Resolution HD, 2.1 × 50 mm, 1.8 μm column (Agilent Technologies, Santa Clara, California, USA) at a column temperature of 30°C and a flow rate of 0.3 ml / min. The mobile phase was operated isocratically at 95:5 (A:B) for 1 minute, then switched to 1:99 (A:B) for a further 12 minutes, and then returned to 95:5 (A:B) for a further 2 minutes, for a total run time of 15 minutes. Mobile phase "A" consisted of 100% H2O and 0.1% formic acid, and mobile phase "B" contained 75% acetonitrile, 25% isopropanol, and 0.1% formic acid. MS data was collected in the mass range of 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. Generally, instrument conditions were similar in both positive electrospray (+ESI) and negative electrospray (-ESI) modes. The scan time was 500, the source temperature was maintained at 350°C, the gas flow rate was 12 L / min, and the nebulizer pressure was 35 psi.

[0217] For the identification of compounds in non-target analysis, the criterion was set at a coincidence rate of >90%. When the coincidence rate decreased to between 70% and 89%, the compounds were identified with parentheses (for example, if the compounds were between 70% and 89%, they were annotated as "<name>"). Any coincidence less than 70% was removed. Overall, there were about 1000 - 1500 features to be identified, many of which had insufficient coincidence (removed) or a S / N ratio less than 10 times from the baseline. Therefore, the compounds / peaks used were actual peaks and the IDs were quite certain (i.e., >70%).

[0218] The non-target LC-MS metabolomics study showed a 2 - 360-fold increase in certain polyphenol glycosides including anthocyanin glycosides, phenolic acid glycosides, and phenolic acids, a 27-fold increase in glucoraphanin, a 5 - 60-fold increase in some glucosinolates, and an approximately 3 - 4-fold increase in indole-3-carbinol and ascorbigen. The results were summarized in Table 8 and shown in the volcano plots of Figures 10 and 11. The top 50 metabolites that increased after fermentation included some polyphenol glycosides and glucosinolates, indicating that the process improved their extractability and bioaccessibility.

[0219]

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

[0220] To determine the effect of fermentation on polyphenol metabolites in broccoli samples, metabolome analysis based on targeted liquid chromatography-mass spectrometry (LC-MS) was performed on raw and fermented broccoli puree samples. Statistical analysis was performed without pretreatment. Fermentation resulted in significant changes in the metabolite profiles of the broccoli samples.

[0221] In targeted LC-MS analysis, polyphenol standards were used for the identification and quantification of metabolites. Increases in chlorogenic acid, ferulic acid, syringic acid, phenyllactic acid, rutin, sinapic acid, methyl syringate, hesperetin, quercetin, and kaempferol were confirmed in fermented broccoli (Figure 12). Decreases in protocatechuic acid, gallic acid, 4,hydroxybenzoic acid, vanillic acid, 2,3-dihydroxybenzoic acid, p-coumaric acid, cinnamic acid, catechin, rosmarinic acid, and caffeic acid were confirmed in fermented broccoli (Figure 12). Of particular note are the 6.6-fold change in chlorogenic acid (2.4-15.8 μg / mg), the 23.8-fold increase in sinapic acid (3.6-86.6 μg / mg), the 10.5-fold increase in kaempferol (12.7-134.6 μg / mg), and the 0.48-fold decrease in p-coumaric acid observed in the fermented samples (Figure 12).

[0222] Example 14 - Evaluation of broccoli fermentation culture to inhibit the growth of intentionally introduced microorganisms Load tests were conducted to evaluate the ability of broccoli fermentation cultures to inhibit the growth of intentionally introduced microorganisms, which are often observed in food preparations of interest.

[0223] Lab cultures / starter cultures 10 samples in the fermented product 8 10 ml of 10 10 Inoculum with cfu / mL.

[0224] pathogen culture E. coli isolates FSAW 1310, FSAW 1311, FSAW 1312, FSAW 1313, and FSAW 1314 were incubated in NB (nutrient broth) at a rate of 1-4 × 10⁻¹⁴ 8 The cultures were grown separately at 37°C overnight until the concentration reached cfu / mL. The cultures were combined (1 mL each), and the combined cultures were diluted 10 times: the first two dilutions were diluted with MRD (Maximum Recovery Diluent), and the last two dilutions were diluted with water. 4 It was diluted to this extent.

[0225] Salmonella strains S. Infantis 1023, S. Singapore 1234, S. Typhimurium 1657 (PT135), S. Typhimurium 1013 (PT9), and S. Virchow 1563 were collected in NB at a rate of 1-4 × 10⁻⁶. 8 The cultures were grown separately at 37°C overnight until the concentration reached cfu / mL. The cultures were combined (1 mL each), and the combined cultures were diluted using MRD for the first two dilutions and with water for the last two dilutions (10 ml each). 4 It was diluted to this extent.

[0226] Listeria isolates Lm2987(7497), Lm2965(7475), Lm2939(7449), Lm2994(7537), and Lm2619(7514) were grown separately in 10 mL of BHI (Brain Heart Infusion Broth) overnight at 37°C with agitation. All cultures were then combined (1 mL each), and this cocktail was diluted using MRD for the first two (1 / 10) dilutions and with sterile deionized water for the last two dilutions.

[0227] B. cerus spores were prepared from isolates B3078, B2603, 2601, 7571, and 7626.

[0228] method Broccoli puree was prepared before preparing the inoculum, using a broccoli:sterilized tap water ratio of 3:2 (900g broccoli:600g water). The broccoli heads were rinsed with tap water, and the stems were cut off using a sterile knife on a cutting board sterilized with 80% ethanol. The broccoli florets (900g) were cut into small pieces. 450g of broccoli pieces were placed in a Thermomix bowl with all 600g of water. A translucent Thermomix cup / lid, sterilized with 80% ethanol, was placed over the hole in the lid. The broccoli was shredded at speed 4 for 1 minute. A second 450g of broccoli pieces was added to the Thermomix bowl and shredded at speed 4 for 1 minute. The contents were shredded for a further 5 minutes at speed 10 (maximum). After confirming that the puree was sufficiently smooth, the Thermomix bowl was placed in a cooling chamber and the contents were cooled for 30 minutes. After that, the bowl was placed in an incubator and equilibrated to 30°C. Meanwhile, starter cultures and pathogen cultures (E. coli, B. cereus, Salmonella, Listeria monocytogenes) were prepared. 10 mL of LAB culture and 7.5 mL of 10-4 diluted loaded microbial cocktail (10 4 A culture in water at cfu / mL was added to broccoli puree (10 5 (B. cereus). Before mixing the culture, foil was placed over the large hole in the Thermomix lid and held down. The culture was mixed into the puree at maximum speed for 1 minute. The Thermomix heat setting was switched off, and the Thermomix was placed in a 30°C incubator, and fermentation was started at 10:45 AM. pH and temperature were measured hourly from 7 hours after mixing the puree at a speed of 4.5 for 1 minute (end of work time). The pH meter was calibrated and sterilized with 80% ethanol. The temperature probe was also sterilized with 80% ethanol before measurement.

[0229] The growth of the inoculated microorganisms was evaluated by counting their numbers on the selective media MRS, DRBX, and NA+S containing raw broccoli at pre-fermentation (T0), 4 h (T4), and 22 h (T22) after the start of fermentation.

[0230] Results Yeast and mold significantly decreased within 4 h and were not detected at the end of fermentation (T22). E. coli and Salmonella were not detected at all at the end of fermentation (T22). A small number of Listeria were detected at the end of fermentation, just exceeding the inoculum of 10 3 cfu / mL at the start. B. cereus spores were not affected by fermentation overall and did not germinate. The results of the inoculation test indicate that the lactic acid bacterial strains isolated by the inventors from broccoli can completely inactivate Salmonella and E. coli and inhibit the growth of the most acid-tolerant Listeria strains. They can also inhibit the sporulation of B. cereus spores. Table 9. Example of a microbial load test using E. coli. E. coli (a mixture of five E. coli strains EC1605, EC1606, EC1607, EC1608) was inoculated into an immersion broccoli (3:2 broccoli-water ratio) fermentate (2.2×102 CFU / gm), and it was evaluated whether the fermentation starters (a mixture of B1, B2, B3, B4, B5, BF1, BF2) could inhibit the growth of E. coli. The experiment was repeated three times. Fermentation was carried out at 30 °C for 22 h until the pH was less than 4.0.

[0231] [Table 9]

[0232] Table 10. Example of a microbial load test using Salmonella. A mixture of five Salmonella strains (S. Infantis 1023, S. Singapore 1234, S. Typhimurium 1657 (PT135), S. Typhimurium 1013 (PT9), and S. Virchow 1623) was inoculated (1.1 × 10³) into a fermented product of soaked broccoli (broccoli-water ratio of 3:2), and it was evaluated whether the fermentation starter (a mixture of B1, B2, B3, B4, B5, BF1, and BF2) inhibited the growth of Salmonella. The experiment was repeated three times. Fermentation was carried out at 30°C for 22 hours until the pH fell below 4.0.

[0233] [Table 10]

[0234] Table 11. Example of a microbial load test using Listeria monocytogenes. A mixture of five strains of Listeria monocytogenes (Lm2987 (7497), Lm2965 (7475), Lm2939 (7449), Lm2994 (7537), and Lm2919 (7514)) was inoculated into a fermented product of soaked broccoli (broccoli-water ratio of 3:2) (1.9 × 10³), and it was evaluated whether the fermentation starter (a mixture of B1, B2, B3, B4, B5, BF1, and BF2) inhibited the growth of acid-tolerant Listeria. The experiment was repeated three times, and the final Listeria count at the end of fermentation was <10 (undetectable) to 1.1 × 10³. 2 The CFU / gm range was used. Fermentation was carried out at 30°C for 22 hours, until the pH fell below 4.0.

[0235] [Table 11]

[0236] Table 12. Example of a microbial load test using Bacillus cereus. Bacillus cereus (a mixture of five strains B3078, B2603, B2601, B7571, and B7626) was inoculated into a fermented product of soaked broccoli (broccoli-water ratio of 3:2) (1.9 × 10³), and it was evaluated whether the fermentation starter (a mixture of B1, B2, B3, B4, B5, BF1, and BF2) inhibited the growth of acid-tolerant Listeria. The experiment was repeated three times. Fermentation was carried out at 30°C for 22 hours until the pH was less than 4.0.

[0237] [Table 12]

[0238] Example 15 - Pulsed-field gel electrophoresis of Leuconostoc mesenteroloides isolate Leuconostoc mesenteroides derived from vegetables were evaluated by SmaI and NotI restriction enzyme digestion using pulsed-field gel electrophoresis, modified from the method described by Chat and Dalmasso (2015).

[0239] method: Day 1 The evaluated isolates were inoculated into 10 mL of MRS broth and incubated overnight (16 hours) in a 30°C incubator.

[0240] Day 2 The isolate was centrifuged at 3500g for 10 minutes, and the supernatant was discarded. The pellet was mixed, washed with 5 mL of deionized water, centrifuged at 3500g for 10 minutes, and the supernatant was discarded. The pellet was mixed with 5 mL of TES (1 mM EDTA, 10 mM Tris-HCl, 0.5 M saccharose) and vortexed. Next, the sample was centrifuged at 3500g for 15 minutes, and the supernatant was discarded. 700 μL of lysis solution (TE buffer containing 10 mg / mL of lysozyme (1 mM EDTA, 10 mM Tris-HCl, pH 8.0, normally sterilized)) was added to the pellet, mixed, and incubated at 56°C for 2 hours to lyse the bacteria. Next, 700 μL of agarose (1% SeaChem Gold agarose containing 50 μL of EDTA / 100 mL) was added to the cell mixture, mixed, and dispensed into plug molds. 2 mL of deproteinization solution (660 μL of proteinase K buffer, 11 μL of proteinase K) was added to each plug for one sample, and the plugs were placed in tubes and incubated overnight at 55°C.

[0241] Day 3 Next, the plug was heated at 55°C in 100 mL of sterile deionized water to remove the deproteinizing solution, and the plug was transferred to a 15 mL centrifuge tube. It was then washed with 4 mL of sterile deionized water and heated at 55°C at room temperature for 10 minutes, followed by four washes at room temperature for 10 minutes each with 4 mL of TE buffer.

[0242] Consumption of restrictions A 2mm cut plug was placed in an Eppendorf tube containing 100 μL of 1× restriction buffer, incubated at room temperature for 20 minutes, the restriction buffer was removed and replaced with 40-100 μL of SmaI (20U) or NotI, and incubated at optimal temperature (25°C) for 4 hours.

[0243] Day 4 Separation of restrictive fragments 1 mL of 0.5× TBE buffer was added to each tube, and the reaction was stopped by standing for at least 15 minutes. Bacteriophage λDNA ladders (New England Biolab) were incubated in TBE buffer. The buffer was removed, and the slices were placed on a comb, with the ladder used every 5 lanes. 1.0% ultra-high purity DNA-grade agarose (pulsed-field certified agarose) was prepared in 0.5× TBE electrophoresis buffer.

[0244] Electrophoresis conditions The buffer solution was maintained at 14°C (model 1000 Mini-chiller, BioRad). In BioRad's "Chef Mapper™," the Two State Program (not Auto Algorithm) was selected. The pulse time was linearly sloped from 2 to 25 seconds (press Enter when "a" is displayed). The gradient was 6V / cm (voltage), the crossing angle was 120°, and the electrophoresis time was 24 hours.

[0245] Day 5 The gel was stained with GelRed for approximately 30 minutes, then destained and visualized.

[0246] result The restriction fingerprint of BF1 was different but similar to that of Leuconostoc mesenteroides isolated from carrots (Figure 13). The restriction fingerprint of BF2 was different from all of the Leuconostoc mesenteroides strains evaluated (Figure 13).

[0247] Example 16 - Variant analysis of Leuconostoc mesenteroides and Lactobacillus plantarum isolates For SNP analysis of Lactobacillus plantarum isolates (B1-B5), B1 Prokka gbk was used as a reference for Snippy SNP analysis - standard method. Single comparisons were performed using the read data for each isolate. B1 reads were used as the control.

[0248] The command example was as follows: snippy --cpus 24 --outdir B5 --ref B1_S1mod.gbk --pe1 B5_S17_L001_R1_001.fastq.gz --pe2 B5_S17_L001_R2_001.fastq.gz

[0249] Individual comparisons and cores were calculated using B1 GBK as a reference. snippy-core --prefix core B1 B2 B3 B4 B5

[0250] The comparison was also performed between B1 and the lead data of a reference strain downloaded from the SRA of Lactobacillus plantarum ATCC8014 (SRR1552613). The download was performed using the standard method, with prefetching and conversion to fastq using -sratoolkit.2.9.2-win64. A similar approach was used to compare Leuconostoc mesenteroides isolates BF1 and BF2 with Leuconostoc mesenteroides ATCC 8293 as a reference.

[0251] result Variants (41) were observed between B1 and ATCC8014 (Table 13). Variants (1-4) were observed between B1 and other B isolates B2, B3, B4, and B5 (Tables 14-17). BF1 and BF2 are significantly different from each other. Variant (19) was observed between BF1 and ATCC8293 (Table 18). Variants (approximately 7000) were observed between BF2 and ATCC8293. 459 composite variants were identified between BF2 and ATCC8293 and are summarized in Table 19.

[0252] [Table 13-1] [Table 13-2] Table 13-3 Table 13-4 Table 13-5 Table 13-6

[0253] Table 14

[0254] Table 15

[0255] Table 16

[0256] Table 17

[0257] Table 18-1 Table 18-2

[0258] Table 19-1 Table 19-2 Table 19-3 Table 19-4 Table 19-5 Table 19-6 Table 19-7 Table 19-8 Table 19-9 Table 19-10 Table 19-11 Table 19-12 Table 19-13 Table 19-14 Table 19-15 Table 19-16 Table 19-17 Table 19-18 Table 19-19 Table 19-20 Table 19-21 Table 19-22 Table 19-23 Table 19-24 Table 19-25 Table 19-26 Table 19-27 Table 19-28 Table 19-29 Table 19-30 Table 19-31 Table 19-32 Table 19-33 Table 19-34 Table 19-35 Table 19-36 Table 19-37 Table 19-38 Table 19-39 Table 19-40 Table 19-41 Table 19-42 Table 19-43 Table 19-44 Table 19-45 Table 19-46 Table 19-47 Table 19-48 Table 19-49 Table 19-50 Table 19-51 Table 19-52 Table 19-53 [Table 19-54] [Table 19-55] [Table 19-56] [Table 19-57] [Table 19-58] [Table 19-59] [Table 19-60] [Table 19-61]

[0259] Those skilled in the art will understand that many variations and / or modifications may be made to the invention as shown in particular embodiments, without departing from the spirit and scope of the invention as extensively described. Therefore, these embodiments are descriptive in all respects and should not be considered limiting.

[0260] This application claims priority to Australian Provisional Application No. 2017903944, filed on 28 September 2017, entitled "Isothiocyanate containing Brassicaceae products and method of preparation thereof," the entirety of which is incorporated herein by reference.

[0261] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0262] Any consideration of documents, actions, materials, devices, articles, etc., included herein is solely for the purpose of providing context to the present invention. Because these matters existed before the priority date of each claim of this application, none of them, in whole or in part, should be deemed to constitute part of the foundation of the prior art or to be common general knowledge in the relevant field of the present invention.

[0263] References Agerbirk et al. (2012) Phytochemistry 77:16-45. Alvarez-Sieiro et al. (2016) Applied Microbiology and Biotechnology 7:2939-2951. Axelsson et al. (2017) Sci Transl Med 9(394). Cai and Wang(2016)Food Chem 1;210:451-6. Capuano et al. (2017) Curr Pharm Des 19:2697-2721. Chuat and Dalmasso (2015) p. 241-251. In Jordan and Dalmasso (ed.), Pulse Field Gel Electrophoresis: Methods and Protocols, vol. 1301. Springer, New York, NY. Dosz and Jeffery (2013) Journal of Functional Foods 5:987-990. Filannino et al. (2015).Food microbiology 46:272-279. Guzman-Lopez et al. (2009).J Ind Microbiol Biotechnol 36:11-20. Halkier et al. (2006) Annual Reviews in Plant Biology 57:303-33. Huang et al.(2002)Journal of agricultural and food chemistry 50(16),4437-4444. Jeffery and Araya(2009)Phytochemistry Reviews 8:283-298. Kim and Park(2016)Excli J 15:571-577. Latte et al.(2011)Food&Chemical Toxicology,49(12),3287-3309. Li et al.(2012)Journal of Medicinal Plants Research 6:4796-4803. Moktari et al.(2017)J Cell Commun Signal Jul 23. Singleton and Rossi(1965)American Journal of Enology and Viticulture 16:144-158. Verkerk et al.(2009)Molecular Nutrition and Food Research 53:S219-S265. Xia and Wishart(2016)Current Protocols in Bioinformatics 55:14.10.1-14.10.91.

Claims

1. i) BF1, deposited with the National Measurement Institute Australia on September 25, 2017 as V17 / 021729; ii) BF2, deposited with the National Measurement Institute Australia on September 25, 2017 as V17 / 021730; iii) B1, deposited on September 25, 2017 at the National Measurement Institute Australia under accession number V17 / 021731; iv) B2, deposited on 25 September 2017 at the National Measurement Institute Australia as V17 / 021732; v) B3, deposited on September 25, 2017 at the National Measurement Institute Australia under V17 / 021733; vi) B4, deposited on September 25, 2017 at the National Measurement Institute Australia under V17 / 021734; and vii) B5, deposited with the National Measurement Institute Australia on September 25, 2017 as V17 / 021735 A probiotic composition comprising lactic acid bacteria selected from one or more of the following:

2. i) BF1, deposited with the National Measurement Institute Australia on September 25, 2017 as V17 / 021729; ii) BF2, deposited with the National Measurement Institute Australia on September 25, 2017 as V17 / 021730; iii) B1, deposited on September 25, 2017 at the National Measurement Institute Australia under accession number V17 / 021731; iv) B2, deposited on 25 September 2017 at the National Measurement Institute Australia as V17 / 021732; v) B3, deposited on September 25, 2017 at the National Measurement Institute Australia under V17 / 021733; vi) B4, deposited on September 25, 2017 at the National Measurement Institute Australia under V17 / 021734; and vii) B5, deposited with the National Measurement Institute Australia on September 25, 2017 as V17 / 021735 A starter culture comprising lactic acid bacteria selected from one or more of the following:

3. The starter culture is at least about 10 8 3. The starter culture of claim 2, comprising lactic acid bacteria at a concentration of 1000 cfu / mL.

4. i) BF1, deposited with the National Measurement Institute Australia on September 25, 2017 as V17 / 021729; ii) BF2, deposited with the National Measurement Institute Australia on September 25, 2017 as V17 / 021730; iii) B1, deposited on September 25, 2017 at the National Measurement Institute Australia under accession number V17 / 021731; iv) B2, deposited on 25 September 2017 at the National Measurement Institute Australia as V17 / 021732; v) B3, deposited on September 25, 2017 at the National Measurement Institute Australia under V17 / 021733; vi) B4, deposited on September 25, 2017 at the National Measurement Institute Australia under V17 / 021734; and vii) B5, deposited with the National Measurement Institute Australia on September 25, 2017 as V17 / 021735 An isolated strain of lactic acid bacteria selected from

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