Method for preparing rubusoside

Abstract

The application provides a method for preparing rubusoside comprising steps of raw material crushing, flash extraction, double centrifugations, macroporous resin purification, desorption, enzymatic hydrolysis, organic film decoloring, concentration, crystallization and drying. The application optimizes the quality control of key process such as extraction, separation, purification and decolorization, and can obtain the rubusoside product with purity more than 99%, and the product is white, no bitterness, less solvent residue, no pesticide residue, and stable quality.

Claims

1. A method for preparing rubusoside, comprising the steps: (1) providing a dry sample of Rubus suatrssimus S. Lee leaves, crushing and passing them through 40-60 mesh sieve, putting the obtained powder material into a flash extractor, in which water at the amount of 18-20 times by weight of the powder is added, extracting for 3 times, 1 minute for each time, and combining all extract solutions to get the total extract solution; (2) concentrating the total extract solution to a concentrate solution 5 times by weight of the raw leaves, centrifuging the concentrate solution through a disc centrifuge and a tube centrifuge to obtain a centrifugate, and adjusting the centrifugate to pH 5.0-6.0 using an acid solution; (3) passing the adjusted centrifugate through a macroporous resin column, adjusting the effluent to pH 7.0 using 0.2% sodium hydroxide solution, detecting the content of rubusoside and ending sample loading when the content is >1%; when the loading is completed, washing the column firstly using pure water until the effluent is colorless and clear, secondly using an alkaline solution until the effluent is PH 11 to 12, thirdly using pure water until the effluent is PH 7.0-7.5, fourthly using an acid solution until the effluent is PH 2.0-2.5, and finally using water until the effluent is PH 7.0; (4) desorbing with 45%-50% ethanol solution, collecting the effluent from the time rubusoside is detected as >1% to the time rubusoside is detected as <1%, and concentrating the effluent under reduced pressure to a concentrate solution of 5 Baume Degrees (°Bé); (5) adding a biological enzyme to the concentrate solution obtained in step (4), and digesting at a temperature of 45-60° C. for 1-3 hours to obtain an enzymatic hydrolysate; (6) passing the enzymatic hydrolysate through an organic membrane of 8000 molecular weight under the inlet pressure of 0.45-0.5 MPa, then through an organic membrane of 800 molecular weight under the inlet pressure of 1.8-2.0 MPa to obtain a decolored filtrate; (7) concentrating the filtrate under reduced pressure to obtain a thick paste of 15 Baume Degrees (°Bé), adding methanol or ethanol as a solvent at an amount 3 times that of the thick paste, crystallizing at 5-11° C. for 8-14 hours, drying the obtained crystal to obtain rubusoside.

2. The method according to claim 1, wherein the centrifugation time of the disc centrifuge in step (2) is 30-50 minutes, and the centrifugation time of the tube centrifuge is 40-60 minutes.

3. The method according to claim 1, wherein the alkaline solution in step (3) is selected from a 0.2% aqueous solution of sodium hydroxide or a 0.2% aqueous solution of potassium hydroxide; the acid solution is selected from 0.4% aqueous solution of hydrochloric acid or 0.45% aqueous solution of sulfuric acid.

4. The method according to claim 1, wherein the biological enzyme in step (5) is one or a combination of two or more enzymes selected from the group consisting of bromelain, pectinase and neutral protease.

5. The method according to claim 4, wherein the amount of the added biological enzyme is 0.5‰-1.2‰ of the total weight of the leaves.

6. The method according to claim 5, wherein the amount of the added biological enzyme is 0.6‰-0.8‰ of the total weight of the leaves.

7. The method according to claim 4, wherein the biological enzyme is a combination of pectinase and neutral protease at the ratio of 5:1.

8. The method according to claim 4, wherein the biological enzyme is a combination of pectinase and bromelain at the ratio of 1:1.

9. The method according to claim 4, wherein the biological enzyme is a combination of a neutral protease and bromelain at the ratio of 4:1.

Description

DRAWINGS

(1) FIGS. 1-8 are HPLC profiles of the rubusoside products prepared in Examples 1-8, respectively.

(2) FIGS. 9-12 are HPLC profiles of the rubusoside products prepared in Comparative Examples 1-4, respectively.

EMBODIMENTS

Example 1

(3) (1) 1.5 kg dried leaves of Sweet tea was taken, and the content of rubusoside was 5.3% according to HPLC. The dried leaves were crushed through 40 mesh sieve. The obtained material was put into a flash extractor. Pure water was used as the extraction solvent. Into the material, water at the amount of 18 times by weight of the material was added. The rotation rate was 5000 R/min. The extraction was carried out at room temperature for 3 minutes, one minute for each time and three times in total, a 2 minute-break between two extractions. The filtrates from three extractions were separated and combined to obtain a total filtrate.

(4) (2) The total filtrate was concentrated to a concentrate solution 5 times by weight of the raw leaves. The concentrate solution was cooled to 25° C., and centrifuged in a disc centrifuge for 45 minutes and then in a tube centrifuge for 60 minutes. The centrifugal deposit was discarded and the centrifugate was adjusted to pH=5.2 with acid solution.

(5) (3) The adjusted centrifugate in step (2) was passed through a macroporous resin column (T-28 resin). The effluent was adjusted to pH=7.0 using 0.2% sodium hydroxide solution. The loading into the column was ended when the content of rubusoside was detected as >1%. After the loading, the column was firstly washed using pure water until the effluent was colorless and clear, secondly using alkaline solution until the effluent was PH 11, thirdly using pure water until the effluent was PH 7.1, fourthly using an acid solution until the effluent was PH 2.0, and finally using water until the effluent was PH 7.0.

(6) (4) Desorption was carried out with 48% ethanol. The effluent was collected from the time the rubusoside was detected as >1% to the time rubusoside was detected as <1%. Ethanol was removed from the collected effluent by concentrating under reduced pressure, and a concentrate solution of 5 Baume Degrees (°Bé) was obtained.

(7) (5) The concentrate solution obtained in step (4) was added to 0.96 g bromelain, and was digested at 48° C. for 3 hours, and the obtained solution was allowed to cool to a normal temperature of 25° C. to obtain an enzymatic hydrolyzate.

(8) (6) The enzymatic hydrolyzate was passed through an organic membrane of 8000 molecular weight under the inlet pressure of 0.45 MPa, then through an organic membrane of 800 molecular weight under the inlet pressure of 1.8 MPa, to obtain the decolored filtrate.

(9) (7) The filtrate was concentrated under reduced pressure to obtain a thick paste of 15 Baume Degrees (°Bé). A solvent at an amount 3 times that of the thick paste was added, stirred thoroughly and crystallized at 6° C. for 12 hours. The obtained crystal was dried to obtain 77.1 g rubusoside. According to HPLC, the content of rubusoside was 99.3%.

Examples 2-8

(10) On the basis of the method described in Example 1, the combination of various enzymes, the temperature and time for the enzymatic hydrolysis, the first organic film (inlet pressure), the second organic film (inlet pressure), the temperature and time for crystallization and the like were adjusted. The quality and the content of the prepared rubusoside were determined by HPLC. The results are shown in Table 1.

(11) In order to verify that the present application has outstanding technical effects, the inventors conducted four comparative experiments as follows.

Comparative Example 1

(12) The rubusoside was extracted using the method of example 1 except that the extraction device used in step (1) was replaced by an extraction tank.

(13) Specifically, the method for extracting rubusoside using the extraction tank was as follows. 1.5 kg dried leaves of Sweet tea was taken, and the content of rubusoside was 5.4% according to HPLC. The dried leaves were crushed through 40 mesh sieve. The obtained material was put into an extraction tank. Distilled water at the amount of 8 times by weight of the material was added, and the extraction was carried out for 3 times, one hour for each time. The filtrates from three extractions were separated and combined to obtain a total filtrate.

Comparative Example 2

(14) The rubusoside was extracted using the method of example 1 except that the centrifugation in step (2) was carried out once using a table-top high speed centrifuge.

(15) Specifically, the method for extracting rubusoside using a table-top high speed centrifugator was as follows. The total filtrate was concentrated to the amount five times that of the raw leaves. The obtained concentrate solution was cooled to 25° C., and centrifuged in a table-top high speed centrifuge for 60 minutes. The centrifugal deposit was discarded and the centrifugate was adjusted to pH 5.2 using an acid solution.

Comparative Example 3

(16) The rubusoside was extracted using the method of example 1 except that the macroporous resin column purification in step (3) and the desorption in step (4) were carried out using national routine processes.

(17) Specifically, the macroporous resin column purification and the desorption were as follows. The filtrate with the adjusted pH value was loaded into the macroporous resin column until the effluent was no sweet. The column was washed using distilled water until the effluent was colorless and clear. Desorption was carried out with 60% ethanol from the time there was a sweet taste to the time the sweet taste was gone. A concentrate solution of 5 Baume Degrees (°Bé) was obtained.

Comparative Example 4

(18) The rubusoside was extracted using the method of example 1 except that steps (5) and (6) were omitted so that the concentrate solution was directly subjected to crystallization.

(19) The rubusoside prepared in Example 1 was compared to those obtained in Comparative Examples 1-4 in terms of quality, recovery rate, content and color. The results are shown in Table 2.

(20) According to the data in Examples 1-8 and Comparative Examples 1-4 in Table 1 and Table 2, the following can be concluded.

(21) 1. All the parameters involved in the present application may be adjusted within the feasible ranges and the obtained rubusoside all has a content of more than 99% with slight fluctuation, indicating that the method of the present application is reliable and can better control the product quality.

(22) 2. Extraction process, separation process, macroporous resin column purification, desorption process, crystallization temperature and crystallization time have great influence on the yield of rubusoside. The flash extraction process, the two-stage extraction process and the optimization of the macroporous resin purification have an outstanding contribution to the recovery rate of rubusoside.

(23) 3. The type or combination of enzymes, enzymatic hydrolysis parameters, and process parameters of organic membranes have a great influence on the color of rubusoside. The “enzymatic hydrolysis+organic film” decolorization process adopted by the application has an outstanding contribution to the improvement of whiteness of the product.

(24) TABLE-US-00001 TABLE 1 Process technical parameters and product test results of Examples 2-8 examples Example Example Example Example Example Example Example Example parameters 1 2 3 4 5 6 7 8 Raw material 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 (dried leaves)/kg Rubuscoside content 5.3 5.5 5.4 5.3 5.2 5.3 5.5 5.4 in the raw material (HPLC)/% Enzymes Bromelain Pectinase Neutral Bromelain Neutral Pectinase:Neutral Pectinase:Bromelain = Neutral protese protese protese = 5:1 1:1 protese:Bromelain = 4:1 Enzyme amount/g 0.96 1.0 0.98 0.96 0.95 1.0 g + 0.2 g 0.5 g + 0.5 g 1.0 g + 0.25 g Temperature for 45 55 48 60 48 48 48 48 enzymatic hydrolysis/° C. Time for enzymatic 3 3 1 2 3 3 3 3 hydrolysis/h Inlet pressure for 0.45 0.45 0.45 0.5 0.45 0.45 0.5 0.45 the first organic membrane/Mpa Inlet pressure 2.0 1.8 2.0 1.8 1.8 1.8 1.8 2.0 for the second organic membrane/ Mpa Crystallization 5 6 6 8 6 6 6 7 temperature/° C. Crystallization 12 8 8 8 12 12 10 12 time/h Rubuscoside product/g 77.5 80.58 78.6 77.9 76.1 77.1 79.8 78.8 Rubuscoside recovery 96.61 96.89 96.36 97.20 96.98 96.30 95.86 96.51 rate/% Rubuscoside content 99.1% 99.2% 99.3% 99.2% 99.4% 99.3% 99.1% 99.2% (HPLC)/% (FIG. 1) (FIG. 2) (FIG. 3) (FIG. 4) (FIG. 5) (FIG. 6) (FIG. 7) (FIG. 8) Visual whiteness Example 8 > Example 5 > Example 6 > Example 3 > Example 7 > Example 1 > Example 2 > Example 4 comparison

(25) TABLE-US-00002 TABLE 2 Product Comparison of Example 1 and Comparative Examples 1-4 examples Comparative Comparative Comparative Comparative parameters Example 1 Example 1 Example 2 Example 3 Example 4 Raw material (dried 1.5 1.5 1.5 1.5 1.5 leaves)/kg Betanin content in the 5.3 5.4 5.2 5.5 5.3 raw material (HPLC)/% Rubusoside product/g 77.5 65.83 63.71 66.23 71.22 Rubusoside recovery rate/% 96.61 51.08 61.52 68.52 81.88 Rubusoside content (HPLC)/% 99.1% 60.85% 75.32% 85.35% 91.4% (FIG. 1) (FIG. 9) (FIG. 10) (FIG. 11) (FIG. 12) Product color Snow white Pale yellow Brownish yellow Pale yellow Pale yellow

INDUSTRIAL APPLICABILITY

(26) The application provides a method for preparing rubusoside. The application obtains rubusoside by raw material crushing, flash extraction, double centrifugations, macroporous resin purification, desorption, enzymatic hydrolysis, organic film decoloring, concentration, crystallization and drying. The application optimizes the quality control of key process such as extraction, separation, purification and decolorization, and can obtain the rubusoside product with purity more than 99%, and the product is white, no bitterness, less solvent residue, no pesticide residue, and stable quality. The product has better economic value and application prospects.