Method for simultaneously extracting lycopene and citrulline from watermelon

Abstract

A method for simultaneously extracting Lycopene and Citrulline from a watermelon includes: separating the rind and the pulp of the watermelon, preprocessing the rind, and using the preprocessed rind to extract the Citrulline; subjecting the pulp to biological enzymolysis and filtering, centrifuging a filtrate, using a precipitate and a filter residue obtained after the centrifuging to extract the Lycopene, and using a supernatant obtained after the centrifuging to extract the Citrulline. By using the method for synchronously extracting Lycopene and Citrulline from the watermelon of the present invention, about 0.5 kg of Lycopene (6% content) and more than 1.2 kg of Citrulline which are worthy of nearly ten thousand yuan can be extracted from each ton of imperfect watermelons, the economic benefit of each ton of watermelons can be increased by more than 5000 yuan after extraction costs are deducted, and the method is high in economic benefits.

Claims

1. A method for simultaneously extracting Lycopene and Citrulline from a watermelon, comprising: separating a rind and a pulp of the watermelon; extracting Citrulline from the rind after a pretreatment of the rind; processing the pulp with a biological enzymolysis, then filtering to obtain a filter residue and a filtrate; centrifuging the filtrate to obtain a precipitate and a supernatant, wherein the precipitate is mixed with the filter residue to be used to extract Lycopene, and the supernatant is used to extract Citrulline.

2. The method for simultaneously extracting Lycopene and Citrulline from the watermelon according to claim 1, wherein the biological enzymolysis comprises: adding a biological enzyme in an amount of 0.1-0.3% (m/m), with a temperature maintaining at 40-50 C., and then performing an enzymolysis for 1-2 hours.

3. The method for simultaneously extracting Lycopene and Citrulline from the watermelon according to claim 2, wherein the biological enzyme is at least one selected from the group consisting of pectinase, cellulase and protease.

4. The method for simultaneously extracting Lycopene and Citrulline from the watermelon according to claim 1, wherein the biological enzymolysis comprises: beating the pulp to form a slurry after seeds are removed; adding 0.3% (m/m) of biological enzymes to the slurry to perform an enzymatic hydrolysis for 1.5 h at 45 C.; filtering the slurry to obtain the filter residue and the filtrate, and centrifuging the filtrate to collect the precipitate; after being lyophilized, combining the precipitate and filter residue and grounding into powder for extraction of Lycopene; and using the supernatant after centrifugation for extraction of Citrulline.

5. The method for simultaneously extracting Lycopene and Citrulline from the watermelon according to claim 4, wherein the extraction of Lycopene comprises: (1) ultrasound-assisted organic solvent leaching: adding ethyl acetate to the powder grounded from the filter residue and the precipitate at a ratio of 1:4-1:8 (kg/L) and mixing evenly at a temperature of 40-50 C., an ultrasonic output power of 80-120 W, an extraction time of 30-90 min, and extracting twice to obtain of a crude Lycopene extract solution; (2) concentrated under reduced pressure: concentrating the crude Lycopene extract solution under reduced pressure at 40 C. to obtain a crude Lycopene; (3) purification: Anhydrous adding anhydrous ethanol to the crude Lycopene at a material-liquid ratio of 1:3 (kg/L) to embathe for twice; and then removing the anhydrous ethanol to obtain a Lycopene powder.

6. The method for simultaneously extracting Lycopene and Citrulline from the watermelon according to claim 1, wherein the pretreatment of the rind comprises: removing an outer skin of the rind, and then squeezing the rind to obtain the filter residue and the filtrate; subjecting the filter residue to an ultrasound-enzyme assisted solvent leaching to obtain an extract solution; and then mixing the extract solution with the filtrate and a pulp supernatant which is obtained by centrifuging the pulp to obtain a pretreatment material for extracting Citrulline.

7. The method for simultaneously extracting Lycopene and Citrulline from the watermelon according to claim 1, wherein the pretreatment of the rind comprises: removing an outer skin of the rind, and grounding the rind into a powder after being dried; subjecting the powder to an ultrasound-enzyme assisted solvent leaching and filtering to obtain a filter residue and a primary extraction solution; subjecting the filter residue to the ultrasound-enzyme assisted solvent leaching, and then filtering to obtain a secondary extraction solution; and mixing the secondary extraction solution with the primary extraction solution and a pulp supernatant which is obtained by centrifuging the pulp to obtain a pretreatment material for extracting Citrulline.

8. The method for simultaneously extracting Lycopene and Citrulline from the watermelon according to claim 6, wherein the ultrasonic-enzyme-assisted solvent leaching comprises: adding water to the filtered residue at a material-liquid ratio of 1:10-1:20 (kg/L), and then adding 0.1% (m/m) of pectinase and 0.1% (m/m) of cellulose to the filtered residue and water; adjusting pH to 4.0, ultrasonic power to 100-140 W, extraction temperature to 30-50 C., extraction time to 60-120 min, and extraction times to twice.

9. The method for simultaneously extracting Lycopene and Citrulline from the watermelon according to claim 6, wherein extracting Citrulline comprises: (1) microbial fermentation: adding 5% (v/v) yeast to the supernatant to ferment for 24 h to remove sugar; (2) ion-exchange resin purification: filtering the supernatant after microbial fermentation to remove yeasts, and adding to an activated wet resin to adsorb Citrulline, and then eluting with 0.5 mol/L ammonia solution at an elution rate of 2 BV/h to obtain a Citrulline eluent; (3) macroporous adsorption resin discoloration: mixing an activated wet resin with the Citrulline eluent at 1:20 (g/mL), and performing a static adsorption on condition of 100-150 r/min for 2 h at room temperature to obtain a crude Citrulline extract; (4) crystallization and recrystallization: a. concentrating the crude Citrulline extract under vacuum to a soluble solid content of 30%, adjusting the pH of the concentrated solution to 5.97, and precipitating Citrulline crystals at 4 C.; centrifuging, washing, and drying to obtain a white powdered solid of Citrulline crystals; b. dissolving the white powdered solid of step (a) in water, adjusting the pH to 5.97, adding 0.1% Citrulline crystals as seed crystals, precipitating the Citrulline crystals at 4 C., and centrifuging to separate the Citrulline crystals; c. repeating the step (b) 1-2 times to obtain a refined solid powder of Citrulline.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Terms used in the present invention have the meanings generally understood by those of ordinary skill in the art unless otherwise specified.

(2) The present invention will be described in further detail with reference to specific embodiments and with reference to data. The following examples are only for the purpose of illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

Embodiment 1

(3) A method for synchronously extracting Lycopene and Citrulline from watermelon, the steps are as follows:

(4) Isolated the rind and pulp of watermelon;

(5) Treatment of pulp:

(6) Watermelon pulp was beaten after the seeds were removed. The slurry was added with 0.1% pectinase, 0.1% cellulase, and 0.1% protease to perform enzymatic hydrolysis for 1.5 h at 45 C. Then the enzymatic slurry was filtered to obtain filter residue and filtrate; the filtrate is centrifuged (4000 r/min, room temperature, 10-20 min) to obtain precipitate and supernatant. After being lyophilized, the precipitate and filter residue which have been already combined were ground into powder for extraction of Lycopene; the supernatant after centrifugation would be used for extraction of Citrulline.

(7) The extraction method of Lycopene is:

(8) (1) Ultrasound-assisted organic solvent leaching: added ethyl acetate to the powder ground from the filter residue and the precipitate at a ratio of 1:6 (kg/L) and mixed evenly with the temperature 45 C., ultrasonic output power 100 W, extraction time 30 min, and extraction times twice to obtain liquid of crude Lycopene extract;

(9) (2) Concentrated under reduced pressure: the liquid of crude Lycopene extract was concentrated under reduced pressure at 40 C., and the solvent was recovered to obtain a crude Lycopenet;

(10) (3) Purification: Anhydrous ethanol was added to the crude Lycopene at a material-liquid ratio of 1:3 (kg/L) to embathe for twice. And then a Lycopene powder was obtained after removing the anhydrous ethanol.

(11) Treatment of watermelon rind:

(12) Remove the outer skin of watermelon rind, and then squeeze the rind to obtain the filter residue and rind juice.

(13) Water was added to the filter residue at a material-liquid ratio of 1:10 (kg/L), and then 0.1% (m/m) of pectinase and 0.1% (m/m) of cellulose were also added to the above filter residue. Adjusted pH to 4.0, ultrasonic power to 100 W, extraction temperature to 50 C., extraction time to 90 min, and extraction times to twice. And then the combined extract was obtained.

(14) The above combined extract was mixed with the supernatant which was obtained by centrifugation of the rind juice and pulp to obtain a pretreatment material for extracting Citrulline.

(15) Extraction method of Citrulline:

(16) (1) Microbial fermentation: 5% (v/v) yeast was added to the pretreated material to ferment for 24 hours to remove sugar.

(17) (2) Ion-exchange resin purification: the pretreated material after microbial fermentation was filtered to remove yeasts, and added to HD-8 activated wet resin to perform the dynamic exchange adsorption of Citrulline, and then eluted with 0.5 mol/L ammonia solution for 2 BV/h of the elution rate.

(18) The dynamic exchange adsorption is that 180 g of activated wet resin was packed in a glass chromatography column with an inner diameter of 26 mm and a height of 400 mm, and the Citrulline extract solution after fermentation by microorganisms was passed through the resin column at a speed of 2 BV/h to adsorb Citrulline.

(19) (3) Macroporous adsorption resin discoloration: mixed XAD-761 activated wet resin with Citrulline eluent at 1:20 (g/mL), and performed static adsorption on condition of 100-150 r/min for 2 h at room temperature.

(20) (4) Crystallization and recrystallization:

(21) a. The crude Citrulline extract was concentrated under vacuum to a soluble solid content of 30%, the pH of the concentrated solution was adjusted to 5.97, and the Citrulline crystals were precipitated at 4 C. The precipitate was centrifuged, washed, and dried to obtain a white powdered solid of Citrulline crystals.

(22) b. Dissolved the Citrulline powder of step (a) with water, adjusted the pH to 5.97, added 0.1% Citrulline crystals as seed crystals, precipitated the Citrulline crystals at 4 C., and centrifuged to separate the precipitates.

(23) c. Repeated step (b) 1-2 times, centrifuged the precipitate, washed, and dried to obtain a refined solid powder of Citrulline.

(24) After testing, the extraction rate of Lycopene was 91.57%; the extraction rate of Citrulline was 93.43%, and the purity of Citrulline was 99.35%.

Embodiment 2

(25) A method for synchronously extracting Lycopene and Citrulline from watermelon, the steps are as follows:

(26) Isolated the rind and pulp of watermelon;

(27) Treatment of pulp:

(28) Watermelon pulp was beaten after the seeds were removed. The slurry was added with 0.1% pectinase, 0.1% cellulase, and 0.1% protease to perform enzymatic hydrolysis for 1.5 h at 45 C. Then the enzymatic slurry was filtered to obtain filter residue and filtrate; the filtrate is centrifuged (4000 r/min, room temperature, 10-20 min) to obtain precipitate and supernatant. After being lyophilized, the precipitate and filter residue which have been already combined were ground into powder for extraction of Lycopene; the supernatant after centrifugation would be used for extraction of Citrulline.

(29) The extraction method of Lycopene is:

(30) (1) Ultrasound-assisted organic solvent leaching: added ethyl acetate to the powder ground from the filter residue and the precipitate at a ratio of 1:6 (kg/L) and mixed evenly with the temperature 45 C., ultrasonic output power 100 W, extraction time 30 min, and extraction times twice to obtain liquid of crude Lycopene extract;

(31) (2) Concentrated under reduced pressure: the liquid of crude Lycopene extract was concentrated under reduced pressure at 40 C., and the solvent was recovered to obtain a crude Lycopenet;

(32) (3) Purification: Anhydrous ethanol was added to the crude Lycopene at a material-liquid ratio of 1:3 (kg/L) to embathe for twice. And then a Lycopene powder was obtained after removing the anhydrous ethanol.

(33) Treatment of watermelon rind:

(34) The watermelon rind which the outer skin was removed was ground into powder after being dried;

(35) Water was added to the powder at a material-liquid ratio of 1:10 (kg/L), then 0.1% (m/m) of pectinase and 0.1% (m/m) of cellulose were also added to the above powder. Adjusted pH to 4.0, ultrasonic power to 100 W, extraction temperature to 50 C., and extraction time to 90 min, and a primary extraction solution was obtained after being filtered. Water was added to the filter residue at a material-liquid ratio of 1:10 (kg/L), then 0.1% (m/m) of pectinase and 0.1% (m/m) of cellulose were also added to the above filter residue. Adjusted pH to 4.0, ultrasonic power to 100 W, extraction temperature to 50 C., and extraction time to 90 min, and a secondary extraction solution was obtained after being filtered.

(36) The secondary extraction solution was mixed with the primary extraction solution and the supernatant after centrifugation of pulp to obtain a pretreatment material for extracting Citrulline.

(37) Extraction method of Citrulline:

(38) (1) Microbial fermentation: 5% (v/v) yeast was added to the pretreated material to ferment for 24 hours to remove sugar.

(39) (2) Ion-exchange resin purification: the pretreated material after microbial fermentation was filtered to remove yeasts, and added to HD-8 activated wet resin to perform the dynamic exchange adsorption of Citrulline, and then eluted with 0.5 mol/L ammonia solution for 2 BV/h of the elution rate.

(40) The dynamic exchange adsorption is that 180 g of activated wet resin was packed in a glass chromatography column with an inner diameter of 26 mm and a height of 400 mm, and the Citrulline extract solution after fermentation by microorganisms was passed through the resin column at a speed of 2 BV/h to adsorb Citrulline.

(41) (3) Macroporous adsorption resin discoloration: mixed XAD-761 activated wet resin with Citrulline eluent at 1:20 (g/mL), and performed static adsorption on condition of 100-150 r/min for 2 h at room temperature.

(42) (4) Crystallization and recrystallization:

(43) a. The crude Citrulline extract was concentrated under vacuum to a soluble solid content of 30%, the pH of the concentrated solution was adjusted to 5.97, and the Citrulline crystals were precipitated at 4 C. The precipitate was centrifuged, washed, and dried to obtain a white powdered solid of Citrulline crystals.

(44) b. Dissolved the Citrulline powder of step (a) with water, adjusted the pH to 5.97, add 0.1% Citrulline crystals as seed crystals, precipitated the Citrulline crystals at 4 C., and centrifuged to separate the precipitates.

(45) c. Repeat step (b) 1-2 times, centrifuged the precipitate, washed, and dried to obtain a refined solid powder of Citrulline.

(46) After testing, the extraction rate of Lycopene was 91.76%; the extraction rate of Citrulline was 92.98%, and the purity of Citrulline was 98.77%.

(47) 1. Analysis and test results of Lycopene and Citrulline extract of the present invention

(48) (1) Analysis of Lycopene

(49) The hygienic indicators such as heavy metals and microorganisms in the watermelon Lycopene extract extracted in Example 1 were tested and compared with the quality standards of commercially available Lycopene oleoresin products. The results are shown in Table 1.

(50) TABLE-US-00001 TABLE 1 Hygienic indicators of watermelon Lycopene extract Commercial Test item Unit Test result product standard Arsenic mg/kg 5.3 10.sup.2 5 Lead mg/kg Not detected 10 Total bacteria CFU/g <10 1000 Yeast and mold CFU/g <10 100 E.coli MPN/100 g <30 40

(51) From the test results in Table 1, it can be known that the indexes of heavy metals and microorganisms of the watermelon Lycopene extract extracted by the method of the present invention meet the relevant sanitary requirements and product quality standards.

(52) (2) Analysis of Citrulline

(53) The physicochemical and hygienic indicators of the Citrulline extract extracted in Example 1 were analyzed and compared with the quality standards of commercially available Citrulline products. The results are shown in Table 2.

(54) TABLE-US-00002 TABLE 2 Physicochemical and hygienic indicators of Citrulline extract Commercial Test item Unit Test result product standard Citrulline content % 92.48 Ammonium content % <0.02 0.02 (NH.sub.4) Chloride % <0.02 0.02 Slsfate (SO.sub.4) % <0.02 0.02 Iron mg/kg <10 10 Arsenic mg/kg 2.7 10.sup.2 1 Lead mg/kg Not detected 10

(55) From the test results in Table 2, it can be known that the heavy metal and microbe indexes of the Citrulline extract extracted by the method of the present invention meet the relevant health requirements and product quality standards.

(56) Economic Benefits

(57) The current price of Lycopene on the Chinese market is about 2,000 yuan/kg (6% content), and Citrulline extracted from plants can be sold for about $1,000/kg in the United States. By using the method of the present invention, about 0.5 kg (6% content) of Lycopene and more than 1.2 kg of Citrulline can be extracted per ton of inferior watermelon, and the value is nearly 10,000 yuan. After deducting the extraction cost, the economic benefit can still increase by more than 5000 yuan per ton of watermelon. Based on the annual output of 70 million tons of watermelon in China, if 10% of the processing capacity can be achieved, the output value can reach more than 60 billion yuan. If the watermelon with higher content of Lycopene and Citrulline is used as the raw material, the extraction yield and economic benefits will be higher.

(58) The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, the technical solutions described in the embodiments are still possibly modified, or some of the technical features are equivalently replaced by those skilled in the art by referring to the foregoing. These modifications or replacements do not depart from the spirit and scope of the technical solutions claimed in the present invention.