Method for preparing broccoli with increased sulforaphane content and use of broccoli prepared thereby

Abstract

Disclosed is a method for preparing broccoli with increased sulforaphane content using high-voltage pulsed electric field treatment. According to the method, broccoli with increased sulforaphane content can be eaten raw or can be chopped to appropriate sizes before eating without the need to crush the broccoli. In addition, sulforaphane can be extracted from the broccoli with increased sulforaphane content. The broccoli with increased sulforaphane content and the sulforaphane extract produced therefrom can be used as an active ingredient of a food, feed or cosmetic composition to exhibit the activities of sulforaphane, including antioxidative and anti-inflammatory activities.

Claims

1. A method for preparing broccoli with increased sulforaphane content, comprising: introducing broccoli into a batch or continuous type vessel containing a liquid medium; and treating the liquid medium 1 to 100 times with a high-voltage pulsed electric field of 0.1 to 200 kV/cm at a frequency of 1 to 100 Hz for 0.1 to 300 seconds.

2. The method according to claim 1, wherein the high-voltage pulsed electric field treatment is performed at 0.2 to 20 kV/cm for 1 to 50 seconds.

3. The method according to claim 1, wherein the broccoli being introduced into the vessel is either intact or is chopped into pieces, the broccoli being not subject to pulverization or trituration.

4. The method according to claim 1, wherein the liquid medium is water, ethyl alcohol or a mixture thereof.

5. The method according to claim 1, wherein the broccoli having undergone the high-voltage pulsed electric field treatment is subjected to drying, pulverization, pulverization after drying, or drying after pulverization.

6. Broccoli treated by a high-voltage pulsed electric field method of claim 1, wherein the treated broccoli has increased sulforaphane content, wherein the sulforaphane content after the high-voltage pulsed electric field treatment is higher by at least 100% than its original sulforaphane content before the treatment.

7. A food composition comprising the high-voltage pulsed electric field treated broccoli with increased sulforaphane content according to claim 6.

8. A feed composition comprising the high-voltage pulsed electric field treated broccoli with increased sulforaphane content according to claim 6.

9. A cosmetic composition comprising the high-voltage pulsed electric field treated broccoli with increased sulforaphane content according to claim 6.

10. The high-voltage pulsed electric field treated broccoli according to claim 6, wherein the sulforaphane content after the high-voltage pulsed electric field treatment is higher by at least 200% than its original sulforaphane content before the treatment.

11. The high-voltage pulsed electric field treated broccoli according to claim 6, wherein the sulforaphane content after the high-voltage pulsed electric field treatment is higher by at least 300% than its original sulforaphane content before the treatment.

12. A method for producing a sulforaphane extract from the high-voltage pulsed electric field treated broccoli with increased sulforaphane content according to claim 6, comprising extracting sulforaphane from the high-voltage pulsed electric field treated broccoli with water, an organic solvent, or a mixture thereof.

13. A food composition comprising a sulforaphane extract produced by the method according to claim 12.

14. A feed composition comprising a sulforaphane extract produced by the method according to claim 12.

15. A cosmetic composition comprising a sulforaphane extract produced by the method according to claim 12.

16. A method of increasing sulforaphane content and sterilizing broccoli, comprising: introducing broccoli into a batch or continuous type vessel containing a liquid medium; and treating the liquid medium 1 to 100 times with a high-voltage pulsed electric field of 0.1 to 200 kV/cm at a frequency of 1 to 100 Hz for 0.1 to 300 seconds.

17. A method of extracting sulforaphane from fresh broccoli, comprising: introducing broccoli into a batch or continuous type vessel containing a liquid medium; and treating the liquid medium 1 to 100 times with a high-voltage pulsed electric field of 0.1 to 200 kV/cm at a frequency of 1 to 100 Hz for 0.1 to 300 seconds; adding an extraction solvent to the vessel; and extract with stirring at room temperature for 1 hour.

18. The method of extracting sulforaphane from fresh broccoli according to claim 17, wherein the extraction solvent is selected from the group consisting of water, methanol, ethanol, and a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1a shows an image of broccoli of Comparative Example 1 (left) and an image of broccoli of Example 1 (right) immediately after high-voltage pulsed electric field treatment;

(2) FIG. 1b shows an image of broccoli of Comparative Example 1 (left) after storage at room temperature for 1 day and an image of broccoli of Example 1 (right) after storage at room temperature for 1 day following high-voltage pulsed electric field treatment;

(3) FIG. 1c shows an image of broccoli of Comparative Example 1 (left) after storage at room temperature for 3 days and an image of broccoli of Example 1 (right) after storage at room temperature for 3 days following high-voltage pulsed electric field treatment;

(4) FIG. 1d shows an image of broccoli of Comparative Example 1 (left) after storage at room temperature for 4 days and an image of broccoli of Example 1 (right) after storage at room temperature for 4 days following high-voltage pulsed electric field treatment; and

(5) FIG. 1e shows an image of broccoli of Comparative Example 1 (left) after storage in a cold place for 6 days and an image of broccoli of Example 1 (right) after storage in a cold place for 6 days following high-voltage pulsed electric field treatment.

BEST MODE

(6) The present invention provides a method for preparing broccoli with increased sulforaphane content, including: introducing broccoli into a batch or continuous type vessel containing a liquid medium; and treating the liquid medium 1 to 50 times with a high-voltage pulsed electric field of 0.1 to 200 kV/cm at a frequency of 1 to 100 Hz for 0.1 to 300 seconds.

Mode for Invention

(7) The present invention will be explained in more detail with reference to the following examples. However, these examples are not intended to limit the scope of the invention.

(8) Fresh broccoli harvested from Jeju-Do, Korea and fresh turnips harvested from Gangwha Island, Korea were purchased and used as samples. An ELCRACK HVP5 apparatus (German Institute of Food Technology, Germany) was used for high-voltage pulsed electric field treatment.

Experimental Example 1: Analysis of Sulforaphane Content of Vegetables Belonging to the Family Brassicaceae

(9) The contents of sulforaphane in raw radish, turnip, cabbage, red cabbage, Brussels sprout, broccoli, and cauliflower as vegetables of the family Brassicaceae were analyzed by the following procedure.

(10) A standard solution was prepared using a sulforaphane standard product (90%, Sigma) and was analyzed using UPLC-MS/MS (Waters) to construct a standard calibration curve. 1 g of each sample (on a dry weight basis) was ground in 10 ml of water using a mixer and kept immersed for 2 h such that glucoraphanin was converted to sulforaphane by myrosinase. After a 2-h reaction, the reaction mixture was extracted with 90 ml of methanol with stirring. 1 ml was taken from the extract (100 ml), diluted 10-fold, and analyzed for sulforaphane content by UPLC-MS/MS. The UPLC system was an Acquity UPLC system (Waters corporation, milford, MA) comprised of a binary solvent manager and a sample manager. The MS system was a Waters Quattro Premier XE Tandem MS system (Micromass UK limited). The systems were controlled using Masslynx V4.1 software.

(11) TABLE-US-00001 TABLE 1 Red Kind Radish Turnip Cabbage cabbage Broccoli Content (mg/100 g 0.7 1.5 1.9 18.6 37.4 dry weight)

(12) From the results in Table 1, it could be confirmed that red cabbage and broccoli were most suitable for use as sulforaphane sources among the vegetables of the family Brassicaceae.

Experimental Example 2: Analysis of Contents of Sulforaphane Precursor in Broccoli and Red Cabbage

(13) To determine the contents of glucoraphanin, a sulforaphane precursor, in the red cabbage and broccoli whose sulforaphane contents were found to be high in Experimental Example 1, the following procedure for simultaneous analysis of sulforaphane and glucoraphanin was established.

(14) A standard solution was prepared using a glucoraphanin standard product (90%, PhytoLab) and was analyzed using UPLC-MS/MS (Waters) to construct a standard calibration curve. 1 g of the sample (on a dry weight basis) was ground in 10 ml of water using a mixer and kept immersed for 2 h such that glucoraphanin was converted to sulforaphane by myrosinase. After a 2-h reaction, 90 ml of ethyl alcohol was added to simultaneously extract glucoraphanin and sulforaphane with stirring, instead of methanol. 1 ml was taken from the extract (100 ml), diluted 10-fold, and analyzed for glucoraphanin and sulforaphane contents by UPLC-MS/MS. The UPLC system was the same as that used in Experimental Example 1. The MS/MS method was carried out by scanning in positive ion mode (MS 178 m/z.fwdarw.Daughter 113.9 m/z) for sulforaphane and in negative ion mode (MS 436 m/z.fwdarw.Daughter 372, 96.9 m/z) for glucoraphanin.

(15) TABLE-US-00002 TABLE 2 Kind Red cabbage Broccoli Glucoraphanin content (mg/100 g dry weight) 22.2 235.2 Sulforaphane content (mg/100 g dry weight) 18.6 37.4

(16) The results in Table 2 show that the sulforaphane content of the red cabbage was about half that of the broccoli but the content of glucoraphanin, a sulforaphane precursor, in the red cabbage was less than one tenth of that in the broccoli. In addition, it was predicted that the conversion of glucoraphanin to sulforaphane in the broccoli would be resulted in a significant increase in sulforaphane content.

Example 1

(17) Inedible parts of fresh broccoli were discarded and the broccoli florets were cut out (50 g at a time). The cut florets were introduced into a water-filled batch type vessel of a high-voltage pulsed electric field treatment system and were once treated at an electric field of 2 kV/cm and a frequency of 10 Hz for 10 sec.

Example 2

(18) The procedure of Example 1 was repeated except that broccoli was once treated at an electric field of 2 kV/cm and a frequency of 20 Hz for 10 sec.

Comparative Example 1

(19) For comparison with the broccoli florets having undergone the high-voltage pulsed electric field treatment, fresh broccoli was used as a control. Inedible parts of the broccoli were discarded and the broccoli florets were cut out (50 g at a time).

Comparative Example 2

(20) Inedible parts of fresh broccoli were discarded and the broccoli florets were cut out (50 g at a time). The cut florets were blanched in boiling water at 100 C. for 1 min.

Comparative Example 3

(21) Inedible parts of fresh broccoli were discarded and the broccoli florets were cut out (50 g at a time). The cut florets were ground using a mixer for 2 min.

Experimental Example 3: Analysis of Sulforaphane Contents

(22) A standard solution was prepared using a sulforaphane standard product (90%, Sigma) and analyzed using UPLC-MS/MS (Waters) to construct a standard calibration curve. Based on the standard calibration curve, each broccoli of Examples 1-2 and Comparative Examples 1-3 was analyzed for sulforaphane content. The analysis was conducted in the same manner as in Experimental Example 1.

(23) TABLE-US-00003 TABLE 3 Comparative Comparative Comparative Kind Example 1 Example 2 Example 3 Example 1 Example 2 Content (mg/100 g dry weight) 37.4 1.75 65.6 118.6 138.8 Relative proportion (%) 100.0 4.7 175.4 317.1 371.1

Experimental Example 4

(24) The number of bacteria in each broccoli of Examples 1-2 and Comparative Examples 1-2 was measured in accordance with the testing method for counting the number of bacteria described in the Korean Food Standards Codex. The results are shown in Table 4. The samples were collected from the broccoli florets where microorganisms are most likely to be viable after washing.

(25) TABLE-US-00004 TABLE 4 Comparative Comparative kind Example 1 Example 2 Example 1 Example 2 CFU/g 2.7 10.sup.3 0.97 10.sup.1 2.05 10.sup.1 1.05 10.sup.1

(26) When the broccoli of Comparative Example 2 was blanched in boiling water, the number of bacteria in the broccoli was lowered by 99% than that in the fresh broccoli of Comparative Example 2. The effect of the high-voltage pulsed electric field treatment on the sterilization of each broccoli of Examples 1-2 was substantially equal to the sterilization effect in Comparative Example 2.

Experimental Example 5

(27) To predict the storage conditions and shelf life of each broccoli having undergone the high-voltage pulsed electric field treatment, changes in the color of the broccoli of Example 1 and the broccoli of Comparative Example 1 were observed during storage at room temperature (25 C.) and in a refrigerator (5 C.). The images are shown in FIGS. 1a to 1e.

(28) In both the fresh broccoli (Comparative Example 1) and the broccoli having undergone the high-voltage pulsed electric field treatment (Example 1), no browning was observed at room temperature for 3 d but browning occurred on the fourth day. Particularly, the florets of the broccoli of Comparative Example 1 were broken into pieces from the fourth day.

(29) In contrast, both the fresh broccoli (Comparative Example 1) and the broccoli having undergone the high-voltage pulsed electric field treatment (Example 1) did not turn brown under refrigeration conditions even on the sixth day.

(30) From these results, it can be predicted that the shelf life of the broccoli having undergone the high-voltage pulsed electric field treatment will be at least equal to or longer than that of the fresh broccoli.

Example 3

(31) 50 g of the broccoli of Example 1 was dried with hot air at 40 C. and powdered.

Example 4

(32) 250 ml of an aqueous solution of 70 wt % ethanol was added to 50 g of the broccoli of Example 1. The mixture was pulverized with a homogenizer for 3 min, extracted with stirring at 20 C. for 12 h, concentrated under reduced pressure, and freeze-dried to prepare a sulforaphane extract powder.

Example 5

(33) 250 ml of ethyl acetate was added to 50 g of the broccoli florets of Example 1. The mixture was pulverized with a homogenizer for 3 min and extracted with stirring at 20 C. for 1 h. The ethyl acetate layer was concentrated under reduced pressure and freeze-dried to prepare a sulforaphane extract powder.

Experimental Example 6: Analysis of Sulforaphane Contents

(34) The contents of sulforaphane in the broccoli powder of Example 3 and sulforaphane extract powders of Examples 4-5 were analyzed. The analysis was conducted in the same manner as in Experimental Example 1. The results are shown in Table 5.

(35) TABLE-US-00005 TABLE 5 kind Example 1 Example 3 Example 4 Example 5 Content (mg/100 g 1,210 445 1,830 3,580 solid content)

Experimental Example 7

(36) Fresh broccoli florets each was placed in a water-filled batch type treatment vessel of a high-voltage pulsed electric field treatment system. The florets were treated with varying electric fields, frequencies, treatment times, and numbers of treatments. The treated florets were analyzed for sulforaphane contents and were organoleptically evaluated.

(37) 20 panelists were requested for the organoleptic evaluation. The evaluation was based on a 9-point scale, where the fresh broccoli of Comparative Example 1 was defined as the highest score (9) and the others were expressed as relative scores. The treatment conditions and the sulforaphane contents are shown in Table 6.

(38) TABLE-US-00006 TABLE 6 Fre- Treat- Num- Organoleptic Electric quen- ment ber of evaluation field cy time treat- Sulforaphane (9-point kind (kV/cm) (Hz) (sec) ments (mg/100 g) scale) Compar- 37.4 9 ative Example 1 1-1 0.1 20 10 1 77.1 9 1-2 1.0 20 10 1 82.4 9 1-3 2.0 20 10 1 138.8 9 1-4 5.0 20 10 1 132.1 8 1-5 10.0 20 10 1 145.1 6 1-6 100.0 20 10 1 107.0 3 2-1 2.0 0.5 10 1 90.5 9 2-2 2.0 5 10 1 119.2 9 2-3 2.0 50 10 1 151.4 6 2-4 2.0 100 10 1 155.0 4 3-1 2.0 5 0.5 1 66.3 9 3-2 2.0 5 5 1 58.2 9 3-3 2.0 5 50 1 121.1 7 3-4 2.0 5 300 1 103.5 4 3-5 2.0 5 600 1 99.8 2 4-1 2.0 5 5 2 49.4 9 4-2 2.0 5 5 10 88.2 8 4-3 2.0 5 5 30 82.1 6 4-4 2.0 5 5 60 56.2 4

Experimental Example 8

(39) Fresh broccoli florets each was once treated in a liquid medium in a batch type treatment vessel of a high-voltage pulsed electric field treatment system at an electric field of 2 kV/cm and a frequency of 5 Hz for 5 sec. The liquid medium was an aqueous solution of 50 vol % of ethanol, an aqueous solution of 20 vol % of ethanol or 0.5 wt % brine. The sulforaphane contents were analyzed and are shown in Table 7.

(40) TABLE-US-00007 TABLE 7 Preparative 50% 20% 0.5% kind Example 3-2 ethanol ethanol brine Sulforaphane content 58.2 32.2 42.1 65.5. (mg/100 g)

(41) Food compositions containing the broccoli with increased sulforaphane content or the sulforaphane extract produced therefrom according to the present invention were prepared into the following formulations. It should be understood that the proportions of the ingredients in the formulations may be arbitrarily changed depending on the consumers' regional and national preferences, such as classes, nationalities, and purposes of use.

Formulation Example 1: Production of Tablets

(42) TABLE-US-00008 Broccoli powder 10 mg (Example 3) Corn starch 100 mg Lactose 100 mg Magnesium stearate 2 mg

(43) The ingredients were mixed together and the mixture was compressed to produce tablets in accordance with a general method known in the art.

Formulation Example 2: Production of Capsules

(44) TABLE-US-00009 Sulforaphane extract powder 10 mg (Experimental Example 4) Crystalline cellulose 3 mg Lactose 14.8 mg Magnesium stearate 0.2 mg

(45) The ingredients were mixed together and the mixture was filled in gelatin capsules to produce capsules in accordance with a general method known in the art.

Formulation Example 3: Production of Powdered Health Functional Food

(46) TABLE-US-00010 Broccoli powder 1,000 mg (Example 3) Vitamin mixture q.s. Vitamin A acetate 70 g Vitamin E 1.0 mg Vitamin B1 0.13 mg Vitamin B2 0.15 mg Vitamin B6 0.5 mg Vitamin B12 0.2 g Vitamin C 10 mg Biotin 10 g Nicotinamide 1.7 mg Folic acid 50 g Calcium pantothenate 0.5 mg Mineral mixture q.s. Ferrous sulfate 1.75 mg Zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Potassium dihydrogen phosphate 15 mg Calcium monohydrogen phosphate 55 mg Potassium citrate 90 mg Calcium carbonate 100 mg Magnesium chloride 24.8 mg

(47) The vitamin and mineral mixtures were prepared using the ingredients suitable for health functional foods. However, it should be understood that the compositions of the mixtures are provided as preferred embodiments and may be arbitrarily changed. The ingredients were mixed together and the mixture was granulated. In accordance with a general method known in the art, the granules were used to produce a health functional food.

Formulation Example 4: Production of Beverages

(48) TABLE-US-00011 Sulforaphane extract powder 1,000 mg (Example 4) Citric acid 1,000 mg Oligosaccharides 100 g Plum concentrate 2 g Taurine 1 g Purified water To 900 mL

(49) In accordance with a general method known in the art, the ingredients were mixed together, heated with stirring at 85 C. for about 1 h, filtered, sealed and sterilized in a sterile 2 L vessel, and kept refrigerated. The resulting solution was used to produce functional beverage compositions.

(50) Cosmetic compositions containing the broccoli with increased sulforaphane content or the sulforaphane extract produced therefrom according to the present invention were prepared into the following formulations. It should be understood that the proportions of the ingredients in the formulations may be arbitrarily changed depending on the consumers' regional and national preferences, such as classes, nationalities, and purposes of use.

Formulation Example 5: Production of Soaps

(51) TABLE-US-00012 Sulforaphane extract powder 5 wt % (Example 4) Oils and fats 75 wt % Sodium hydroxide 5 wt % Perfume 10 wt % Purified water Balance

(52) In accordance with a general method known in the art, the ingredients were mixed together and the mixture was formulated into soaps.

Formulation Example 6: Production of Lotions

(53) TABLE-US-00013 Sulforaphane extract powder 3 wt % (Example 4) L-ascorbic acid-2-phosphate magnesium salt 1 wt % Water soluble collagen (1% aqueous solution) 1 wt % Sodium citrate 0.1 wt % Citric acid 0.05 wt % Licorice extract 0.2 wt % 1,3-Butylene glycol 3 wt % Purified water Balance

(54) In accordance with a general method known in the art, the ingredients were mixed together and the mixture was formulated into lotions.

Formulation Example 7: Production of Creams

(55) TABLE-US-00014 Sulforaphane extract powder 3 wt % (Example 4) Polyethylene glycol monostearate 2 wt % Self-emulsified monostearic acid glycerin 5 wt % Cetyl alcohol 4 wt % Squalene 6 wt % Glyceryl tri(2-ethylhexanoate) 6 wt % Sphingoglycolipid 1 wt % 1.3-Buthylene glycol 7 wt % Purified water Balance

(56) In accordance with a general method known in the art, the ingredients were mixed together and the mixture was formulated into creams.

Formulation Example 8: Production of Packs

(57) TABLE-US-00015 Broccoli powder 2 wt % (Example 3) Polyvinyl alcohol 13 wt % L-ascorbic acid-2-phosphate magnesium salt 1 wt % Lauroyl hydroxyproline 1 wt % Water soluble collagen (1% aqueous solution) 2 wt % 1,3-Butylene glycol 3 wt % Ethanol 5 wt % Purified water Balance

(58) In accordance with a general method known in the art, the ingredients were mixed together and the mixture was formulated into packs.

(59) As is apparent from the foregoing, the method for preparing broccoli with increased sulforaphane content according to the present invention uses high-voltage pulsed electric field treatment. According to the method of the present invention, broccoli with increased sulforaphane content can be eaten raw or can be chopped to appropriate sizes before eating without the need to crush the broccoli. In addition, sulforaphane can be extracted from the broccoli with increased sulforaphane content. The broccoli with increased sulforaphane content and the sulforaphane extract produced therefrom can be used as an active ingredient of a food, feed or cosmetic composition to exhibit the activities of sulforaphane, including antioxidative and anti-inflammatory activities.