EXTRACTION METHOD

Abstract

The present invention relates to the methods for extraction of natural caffeine from caffeine containing biological material.

Claims

1. A method for extraction of natural caffeine from a caffeine containing biological material comprising: (i) contacting h caffeine containing biological material with an extraction solvent; (ii) separating solids and collecting the solvent extract; (iii) contacting the solvent extract from step (ii) with 1.0% to 15% (w/v) of MgO at a temperature from about 70° C. to about 90° C.; (iv) waiting until two phases (A and B) are formed in the mixture of step (iii) and recuperating the upper phase (phase A) containing the caffeine; (v) optionally concentrating the phase A from step (iv); (vi) and optionally crystalizing the caffeine.

2. The method according to claim 1, wherein the caffeine containing biological material is selected from guarana, coffee, tea, Kola, mate leaves and/or cocoa.

3. The method according to claim 1, wherein the caffeine containing biological material is milled or grounded dried guarana seeds.

4. The method according to claim 1, wherein the solvent used is water.

5. The method according to claim 1, wherein the ratio of biological material to solvent is 1:5.

6. The method according to claim 1, wherein step (i) is performed at a temperature from about 50° C. to about 100° C.

7. The method according to claim 6, wherein the temperature gradient is progressively increased from about 50° to about 100° C.

8. The method according to claim 7, wherein the temperature in step (i) is first from about 60±5° C., then increased to about 75±5° C., then increased to about 85±5° C., and finally increased to about 95±5° C.

9. The method according to claim 1, wherein steps (i) and (ii) are repeated more than one time.

10. The method according to claim 9, wherein steps (i) and (ii) are repeated 4 times, and wherein the temperature in step (i) is from about 605° C. in the first repetition, then increased to about 75±5° C. in the second repetition, then increased to about 85±5° C. in the third repetition, and finally increased to about 95±5° C. in the fourth repetition.

11. The method according to claim 1, wherein in steps (i) and/or (ii) the components are mixed applying mechanical energy.

12. The method according to claim 1, wherein the MgO concentration in step (iii) is from about 2.5% to about 3.0% w/v.

13. The method according to claim 1, wherein the temperature in step (iii) is about 75±5° C.

14. The method according to claim 1, wherein in step (v) the phase A is concentrated at 80±5° C. in a falling film evaporator.

15. The method according to claim 1, wherein in step (vi) of the crystallization of the caffeine is performed at a temperature from about 3° C. to about 25° C.

16. (canceled)

17. The method according to claim 1, wherein the extraction is dene performed without enzymatic treatment.

18. Natural caffeine obtained by the process according to claim 1.

19. The natural caffeine according to claim 18, wherein the purity of the caffeine is of more than about 95%.

20. A method of utilizing the natural caffeine according to claim 18 in a nutraceutical formulation, a dietary, food or beverage product for humans or animals, a nutritional supplement, a sports supplement, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation.

21. A nutraceutical formulation, a dietary, food or beverage product for humans or animals, a nutritional supplement, a sports supplement, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation comprising the natural caffeine according to claim 18.

22. A beverage comprising the natural caffeine according to claim 18.

Description

FIGURES

[0187] FIG. 1—Flowchart with each step of the process necessary to obtain 95-100% purified caffeine.

[0188] FIG. 2—Condensed tannin molecule.

[0189] FIG. 3—Caffeine molecules.

[0190] FIGS. 4, 5, 6 and 7—Different steps of the process.

[0191] FIG. 8—Guarana seeds chromatogram in 270 nm.

[0192] FIG. 9—Guarana seeds scanning chromatogram from 200 to 400 nm.

[0193] FIG. 10—Drainage chromatogram in 270 nm.

[0194] FIG. 11—Drainage scanning chromatogram from 200 to 400 nm.

[0195] FIG. 12—Purified caffeine chromatogram in 270 nm.

[0196] FIG. 13—Purified caffeine scanning chromatogram from 200 to 400 nm.

EXAMPLES

1—Materials and Methods

[0197] Materials

[0198] To produce Guaranine caffeine, guarana seed (Paullinia cupana Kunth) from Bahia (northern region of Brazil) is used as the main raw material. Table 1 shows guarana seeds properties used.

TABLE-US-00001 TABLE 1 Specification of guarana seeds. Item Results Insoluble ash in acid (%) 0.48-0.69 Total ash (%) 1.50-1.90 Loss on drying (%) 12.10-12.00 Methylxanthines calculated a 4.77-4.80 caffeine (%) Total tannins (%) 8.30-9.68 Caffeine from HPLC analysis 3.50-4.50 (%)

[0199] Magnesium oxide (MgO) is also used in the process to react with guarana. MgO used is provided by KYOWA Chemical Co., Ltd (Japan), sales name: Kyowamag 150. MgO is min. 98% of purity and this is really important because the reaction will not happen if the degree of purity is less than 98%. Table 2 shows properties of magnesium oxide given by the provider of the raw material.

TABLE-US-00002 TABLE 2 Magnesium oxide properties. Item Unit Results Batches — 180421 180422 180423 MgO (after ignited) % 97.6 97.8 97.9 Ignition Loss % 4.8 4.7 4.6 Drying Loss % 0.3 0.2 0.4 Fe.sub.2O.sub.3—Al.sub.2O.sub.3 % 0.04 0.04 0.05 Acid insoluble substances % 0.02 0.02 0.01 Soluble matter % 0.75 0.76 0.65 Specific surface area (BET) m.sup.2/g 146 140 141 Apparent specific gravity g/mL 0.34 0.35 0.34 Screen analysis on 150 μm % Trace Trace Trace Screen analysis through % 99.8 min 99.7 min 99.7 min 75 μm

[0200] Analytical Method

[0201] Highly-performance liquid chromatography method was used to quantify the amount of caffeine present in the samples. Intern method developed by GNutra qualified and authenticated was applied to the samples. The equipment used was a HPLC from Waters and the software used was Empower.

[0202] Sample Preparation (Raw Material)

[0203] Approximately 0.2 g of guarana seeds already milled was weighted and 50 mL of ultrapure water were added into a 250 mL Erlenmeyer and let into reflux for 1 hour at 70-80° C. After the solution cooled down to ambient temperature it was filtered in cotton to a volumetric flask of 100 mL. The empty volume of the flask was filled with ultrapure water. Then, the solution was filtered with a 0.45 μm membrane to a HPLC vial.

[0204] Sample Preparation (Steps of the Process)

[0205] Samples from other steps of the process were also prepared to identify the amount of caffeine. In those cases, the samples were weighted in different amounts and a dilution with ultrapure water was made. Then, the solution was filtered with a 0.45 μm membrane to a HPLC vial.

[0206] Standard Preparation

[0207] Approximately 1.0 mg of caffeine standard was weighted into a 10 mL volumetric flask. 7 mL of ultrapure water was added and the flask was put into ultrasonic bath for 30 minutes to dissolve the caffeine. After the solution cooled to ambient temperature, the flask was filled with ultrapure water and homogenized. Then, the solution was filtered into 0.45 μm membrane to a HPLC vial.

TABLE-US-00003 Column Lichrospher 100 RP-18 (125 × 4.0 mm × 5 μm) Mobile phase (A) Ultrapure water (B) Acetonitrile Detection 270 nm Flow 1.0 mL/min Injection volume 20 μL Analysis time 30 minutes

[0208] Table 3 shows the conditions to the analysis.

TABLE-US-00004 TABLE 4 Mobile phases were used in gradient as shown. Time (min) (%) A (%) B 0 100 0 10 90 10 15 80 20 20 100 0 30 100 0

[0209] To identify the percentage of caffeine, the equation was used.

[00001]$\left(\%\right)\mathrm{Caffeine}=\frac{A_{\mathrm{sample}}\times M_{\mathrm{standard}}\times P_{\mathrm{standard}}\times D_{\mathrm{sample}}\times 100}{A_{\mathrm{standard}}\times D_{\mathrm{standard}}\times M_{\mathrm{sample}}}$

TABLE-US-00005 Simbol Meaning A.sub.sample Sample area M.sub.standard Standard weight (g) P.sub.standard Standard purity (decimal) D.sub.sample Sample dilution (mL) A.sub.standard Standard average area D.sub.standard Standard dilution (mL) M.sub.sample Sample weight (g) 100 Coefficient to result in percentage

[0210] Caffeine standard was injected five times in the equipment and then the samples were read.

2—Experimental Procedure of Extraction of Highly Purified Caffeine

[0211] First, the experiments were conducted in small scale at the laboratory. Then, they were executed in medium scale in a pilot plant. And finally they were conducted in industrial scale. High purify caffeine was obtained in small laboratory scale, medium scale and at industrial level (the pilot plant).

[0212] Milling

[0213] The first step of the process is milling. Guarana seeds are ground in a hammer mill with a 3 mm sieve resulting in particles with 0.1-0.3 cm of diameter.

[0214] Extraction

[0215] The ground seeds are extracted using water as solvent. For an efficient extraction it is necessary to use high temperatures, but in the beginning of the extraction it is essential that temperature is low and next to 605° C. to avoid the formation of gel from starches present in the smaller particles of the seeds generated by the milling process.

[0216] In this experiment four extractions of the same seeds are necessary to make sure that the process is optimized beginning with low temperature and increasing in the following extractions until 95° C.±5. At each extraction an amount of water is added to the seeds that represents times the amount of the seeds. Table 5 contains the conditions of all four extractions.

TABLE-US-00006 TABLE 5 Extraction conditions. Extraction 1.sup.st 2.sup.nd 3.sup.rd 4.sup.th Temperature (° C.) 60 ± 5 75 ± 5 85 ± 5 95 ± 5 Time (h) 2 2 2 2 Agitation Constant Constant Constant Constant

[0217] The first extraction begins with putting the milled seeds into the reactor and then adding water five times the amount of seeds. At that time, agitation is on and the temperature will rise. After 2 hours of extraction, the liquid now containing molecules of guarana will be drained through the bottom of the reactor where there is a 3-5 mm inox sieve which retains the seeds into the reactor and let the liquid get by.

[0218] Then, another amount of water (5 times the amount of seeds) is added to the same seeds, agitation is on and the temperature is higher to this second extraction following the conditions showed in Table 5. The same happens to the third extraction and the process goes on until the last extraction: after that the seeds are discarded. All of the four drainages are pulled together.

[0219] Filtration

[0220] After extraction, total drainage still contains small particles that went through the sieve, so the next step is a filtration. This happens in a rotary filter where the drainage goes through a polyester mesh of 30 μm. The bigger particles are discarded while the liquid filtered continues the process.

[0221] Reaction

[0222] Then, the filtered goes into a reactor. And to the filtered it is added 2.5-3% of magnesium oxide based on the total amount of filtered. The mixture is heated to 75±5° C. with constant stirring until the reaction takes place after about 1 hour.

[0223] In guarana extract we have caffeine and tannin molecules in solution, their structures are shown in FIG. 2 and FIG. 3, respectively. When tannins are present, part of the caffeine probably binds to the aromatic ring hydroxyl through hydrogen bonds, thus complexing and precipitating so they are not available in the solution.

[0224] When MgO is added to the drainage, Mg+2 ions are released in aqueous solution which will probably bind with the two oxygen molecules attached to the tannin ring resulting in tannin-Mg complex. When that happens, caffeine cannot combine with the tannins because they are not available anymore, so caffeine stays available and soluble in the solution without binding with the hydrogen in the tannins hydroxyl.

[0225] Separation

[0226] After one hour of reaction the stirring is turned off and the mixture rests. A separation naturally occurs and two phases appear. The upper phase is transparent and contains water and caffeine while the lower phase looks like sludge and contains other guarana substances such as tannins and a small amount of caffeine.

[0227] To recovery as many as caffeine possible, the phases are separated and the lower phase goes through a recovery.

[0228] Recovery

[0229] To recovery the caffeine in the lower phase, water is added to it. The amount of water used is the same amount of resulting lower phase. Then, there will be two phases again, upper phase with water and solubilized caffeine and the lower phase with other guarana substances and with less caffeine than before.

[0230] After that, the lower phase has no use because the amount of caffeine left there is negligible so it is discarded, but the upper phase is the main phase which has caffeine and we want to purify so this phase will join the upper phase of the reaction.

[0231] Concentration

[0232] Both upper phase from reaction and from recovery are united and then concentrated at 80±5° C. in falling film. In the equipment, water is evaporated until 8-10% of total solids are reached in the concentrated.

[0233] Cooling

[0234] After concentration, the concentrated is cooled down in an inox cooling tank until temperature reaches 5 to 10° C. and that is when caffeine crystals are formed.

[0235] Filtration

[0236] After the crystallization, washing the crystals with cold water is necessary because the purity wanted is not reached yet. Water in 5° C. is used to wash the crystals that are retained by the filter paper in a vacuum filter. In that temperature, caffeine is not solubilized but other guarana substances are and they leave the crystals with the cold water. If there is any MgO residue it will be removed as well with the water. This water can be reincorporated before concentration in other batches.

[0237] The water with other substances is discarded while the crystals continue to the final step of the process.

[0238] Oven Drying

[0239] The crystals are oven dried at 1055° C. in a stove from 10 to 15 hours with air circulation to evaporate the water left in the crystals resulting in purified caffeine

3—Results

[0240] The quantity of each step of the process was weighted as well as total solids. After that, the amount of caffeine was determined through HPLC analysis in dry base for the dry crystal and in liquid base for other steps of the process. It was also possible to calculate process efficiency and the losses associated with caffeine based in the previous step. That is the percentage of caffeine that continues in the process from one step to another. The pH was also monitored during the experiments. All the results are shown in Table 6.

[0241] The ratio between the amount of raw material and the final product is 40-50 which means that to produce 1 kg of caffeine (95-100% purity) it is necessary 40-50 kg of guarana seeds.

[0242] To compare the peak of caffeine in different steps of the process, chromatogram of samples of the raw material (guarana seeds), total drainage and purified caffeine were analysed as shown in Table 6 and FIGS. 8 to 13.

TABLE-US-00007 TABLE 6 Results of the experiment Caffeine Sample Amount content Caffeine weight Dilution liquid Total liquid Amount content Process for for base solids base dry base dry base Caffeine efficiency Losses HPLC HPLC Step (kg) (%) (%) (kg) (%) (kg) (%) (%) (g) (mL) pH Raw material 136.00 — — 136.00 3.66 4.98 — — 0.20 100.00 5.00 Drainage 1.sup.st ext 468.00 2.75 0.48-0.58 12.87 21.18 2.73 53.95-54.76 45.24-46.05 0.20 10.00 5.70 Drainage 2.sup.nd ext 577.00 0.50 0.19-0.26 2.89 51.76 1.49 25.20-30.00 70.00-74.80 0.30 10.00 5.30 Drainage 3.sup.rd ext 675.00 0.50 0.07-0.11 3.38 22.06 0.74  8.86-14.96 85.04-91.14 0.30 10.00 6.70 Drainage 4.sup.th ext 680.00 0.60 0.01-0.06 4.08 9.50 0.39 1.12-7.79 92.21-98.88 0.50 10.00 7.10 Total drainage 2400.00 1.40 0.18-0.20 33.60 14.29 4.80 89.22-96.43  3.57-19.78 0.30 10.00 6.20 Supernatant 1400.00 0.49 0.18-0.19 6.86 38.31 2.63 38.64-52.79 45.20-49.20 0.30 10.00 9.90 from reaction Total supernatant 2260.00 0.27 0.11-0.18 6.10 68.33 4.17 74.36-83.77 14.20-18.20 0.30 10.00 9.20 (reaction + recovery) Concentrated 83.00 8.70 2.70-6.70 7.22 54.02 3.90 76.40-80.40 19.60-21.60 0.10 100.00 9.20 Washing water 120.00 0.80 0.25-0.75 0.96 58.75 0.56  9.30-13.30 86.70-90.60 0.10 100.00 9.00 Dry crystal — 100 — 3.30 98.00 3.23 63.00-66.00 33.10-37.10 0.01 100.00 6.40

4—Conclusions

[0243] The purification process removes all the dark substances that make the extract turbid and insoluble, resulting in isolated caffeine (95-100% of purity). It is an efficient alternative to obtain purified guarana caffeine from seeds with a less environmentally aggressive methodology, without using dichloromethane, with lower costs, obtaining the same degree of purity.

[0244] It may be a new commercial source of natural caffeine in addition to those traditionally used in beverage production such as coffee and green tea.

REFERENCES

[0245] ATTWOOD, T. K.; CAMMACK, R. Oxford dictionary of biochemistry and molecular biology, 2006. ISBN 0198529171. [0246] EDWARDS, H. G. M.; FARWELL, D. W.; OLIVEIRA, L. F. C.; ALIA, J. M.; LE HYARIC, M.; AMEIDA, M. V. FT-Raman spectroscopic studies of guarana and some extracts. Analytica Chimica Acta, Amsterdam, v. 532, p. 177-186, 2005. http://dx.doi.org/10.1016/j. aca.2004.10.055 [0247] GRASES, F.; RODRIGUEZ, A.; COSTA-BAUZA, A. Theobromine Inhibits Uric Acid Crystallization. A Potential Application in the Treatment of Uric Acid Nephrolithiasis. PloS one, 2014. 9. e111184. 10.1371/journal.pone.0111184. [0248] PAGLIARUSSI, R. S.; BASTOS, J. K.; FREITAS, L. A. P. Fluid Bed Drying of Guarana (Paullinia cupana HBK) Extract: Effect of Process Factors on Caffeine Content. AAPS PharmSciTech, New York, v. 7, n. 2, p. E160-E166, Article 54, 2006. http://dx.doi. org/10.1208/pt070254. [0249] SPENCER, C. M.; CAI, Y.; MARTIN, R.; GAFFNEY, S. H.; GOULDING, P. N.; MAGNOLATO, D.; LILLEY, T. H.; HASLAM, E. Polyphenol Complexation—Some thoughts and observations. Phytochemistry, Oxford, v. 27, n. 8, p. 2397-2409, 1988. http://dx.doi.org/10.1016/0031-9422(88)87004-3.