Caffeinated Beverages - ACS Publications - American Chemical Society

Chapter 31

The Chemistry of Guaraná: Guaraná, Brazil's Super-Fruit for the Caffeinated Beverages Industry 1

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Terry H. Walker , J. M. Chaar , C. B. Mehr , and J. L. Collins 1

Department of Biological and Agricultural Engineering, Louisiana State University Agricultural Experiment Station, Baton Rouge, L A 70803 Rua Ramos Ferreira #1442, 69000 Manaus, A M , Brazil McCormick Company, Hunt Valley, M D 21031 Department of Food Science and Technology, University of Tennessee, Knoxville, T N 37909

Downloaded by UNIV OF MONTANA on February 18, 2016 | http://pubs.acs.org Publication Date: January 15, 2000 | doi: 10.1021/bk-2000-0754.ch031

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A naturally occurring source of caffeine that has received limited attention is the seed of g u a r a n á fruit. Guaraná, a low growing bush-typeplant, is cultivated primarily in the Amazon rain-forest area of Brazil and several other Latin American countries. Guaraná seed contains 3.5-7% caffeine on a dry weight basis and is likely the richest known vegetable source of caffeine. Features of the guaraná plant are presented with an emphasis on uses for the caffeinated beverages industry. Specifics include horticulture of guaraná, treatment of unprocessed product, commercial processing and chemical changes during processing.

Approved for publication by the Director for the Louisiana Agricultural Experiment Station. Trade names are used solely to provide specific information. Mention of a trade name does not constitute a warranty by the Louisiana Agricultural Experiment Station of the L S U Agricultural Center of the product nor an endorsement to the exclusion of other products not mentioned. © 2000 American Chemical Society

In Caffeinated Beverages; Parliment, Thomas H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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Historical In the year of 1669, a missionary priest named Betendorf reported his findings o f the consumption of beverages made from the seeds of a fruit common among tribal members of the Andirâs Indians of the Amazon River Basin in Brazil (/). The explorers, Humboldt and Bonpland, collected the plant on an expedition through the upper Orinoco basin in Venezuela in 1810 (2,3). The plant was later classified in 1821 as Paullinia cupana (2). The genus gets its name from C. F. Paullini, a German medical botanist who died 1712 (2). In the 1920's the first chemical analyses of P. cupana were conducted by Vareg in Cassicourt, England and Theodore von Martius. Martius isolated a chemical component that he called 'guaranine' (4). O f the 180 species of Paullinia found strictly in the neotropics (with the exception of P. pinnata), nearly 40 have been used for centuries by the indigenous people primarily as medicines and stimulating beverages (5). Traditional cultivation and processing of guaranâ (P. cupana) is still practiced by the Saterê-Maué Indians of the Central Amazon basin. The processing steps include picking the ripened, eye-shaped seeds as the fruit shells begin to open, removing the white aril and roasting the seeds to facilitate removal of the hard seed coats (baumann). The seeds are finely ground and made into a paste with the addition of water. The dough-like paste is then shaped and dried slowly by fire for later use. The dough is converted back to powder using a tongue of the Pirarucu fish or "hioide" by a sanding action (6).

Horticultural Aspects The wild-type, caffeine-rich variety first discovered by Humboldt and Bonpland was classified as P. cupana Ducke. The cultivated plant, P. cupana H . B . K . var. sorbilis (Mart.) Ducke, is called guaranâ. Guaranâ belongs to the plant division Angiosperms, class Dicotiledonea, order Spindales, and family Sapindaceae. Until 30 years ago, guaranâ was primarily cultivated in the central Amazon basin of Brazil located in the state of Amazonas (6). Presently, only 60% (or 1500 tons/year) of guaranâ in Brazil is cultivated in the state of Amazonas. The remaining 40% is now cultivated in the adjacent states such as Mato Grosso, Bahia, Acre, Para and as far south as Mato Grosso do Sul (7). Guaranâ are also cultivated to a less extent in Venezuala and Uraguay. The guaranâ plant is low growing, which makes field conditions somewhat challenging. Fruit production occurs after the third year, but is greatest after the sixth year (8). Vegetative or asexual propagation has proven to be the most effective method where advantages include greater productivity after the third year (nearly 1kg dry seeds per plant) and greater disease resistance (9). The guaranâ fruit is elongated (2 cm) with a pointed distal extremity. The immature fruit is enclosed in a dark green shell. The mature fruit results in a striking appearance of deep yellow to red-orange pericarp, a white, scentless aril and glossy black seed coat (3).



307 Plants are typically grown in a nursery for the first nine months in rustic facilities covered with a layer of plastic and palm leaves. The palm leaves are gradually* removed after five months to increase sunlight. Cuttings are dipped in indolebutyric acid, set in soil medium and irrigated with approximately 600 mL of water per day. Soils are typically well-drained, heavy clay to sandy clay (6). Fertilizers containing urea, potassium chloride and dipotassium magnesium sulfide are applied every three months during the nursery stage. Approximately 650 plants are required to start one hectare (8). After the initial nine months in the nursery stage, the healthy plants (typically 6070%) are adapted to the field where the long growing process is initiated. Methods have been developed to add herbicides periodically (glyphosate or Round Up™ and paraquat) and urea to the soil to eliminate competition (8). Pruning of leaves from the extremities of the terminal branches is recommended after the first harvest of fruit (6). Harvesting of the fruit occur primarily between October and January when nearly 50% of the fruit cluster shows a rupture in the distal extremity (see Figure 1) of the shell. The plant has a peculiar inverted bowl shape making mechanical harvesting of the fruit a challenging task (6). A n excellent world-wide-web site for viewing the general cultivation process and ripened fruit is available at the "The Guaranâ Homepage" (10). Caffeine content in the leaves average 0.38% compared to 4.4% of that found in the seeds (8).

Treatment of Unprocessed Product Treatment of the unprocessed guaranâ fruit includes fermentation, pulping and drying. Fermentation accompanied by drying is carried out naturally for a period of three days by spreading the fruit on cloth to a depth of 25 cm, covering for protection

Figure J. Guarana fruit with exposed seeds at harvest.



308 and allowing adequate ventilation (6). The shells and pulp are removed either mechanically or manually. The manual pulping process is carried out by traditional means where individuals walk on the fruit with bare feet to separate the fermented fruit from the shells while removing the pulp. The seeds are placed on a screen (5 mm mesh) and washed with water. The seeds are then sun dried for approximately 12 hours before the final drying stage. Dehydration is carried out by one of three methods: traditional, natural, or industrial. The traditional method used mainly by small farmers include placing the seed on iron plates and roasting the seeds for five hours in wood-fired clay ovens, which are also used in the processing of cassava flour (6). Natural drying occurs in solar driers for up to four days until the seeds obtain a moisture content of 10 to 12% (wet basis). Industrial methods typically use coffee dehydrators that utilize forced heated air for only 15 minutes. Although the forced-convection dehydrators add to the expense, the product is dried more uniformly over short periods. After drying, the seed shells are removed and the seed pounded into a fine powder. The powder is either sold at this point, converted to a syrup, concentrated for the beverage industry, or made into a dried "stick" by traditional methods that require addition of water for conversion to a formed dough that is subsequently dried.

Commercial Processing Procedures Commercial processing of guaranâ converts the dried seed material to powder, syrups and concentrates for a variety of uses. The syrups must be prepared with a minimum of 1% dried seed. Nearly 80% of guaranâ is converted to concentrate for beverages. Guaranâ holds about 14% of the total market for soft drinks, which places it behind "cola" beverages (6). The concentrate is prepared with a slow ethanol-water (60:40, v/v) extraction procedure. Concentration ranges for soft drinks must not exceed 0.2% extract with the minimum concentration of 0.02% (6). Because caffeine is of primary interest in guaranâ beverages, decaffeination procedures and caffeine purification are of interest to industry. Several methods exist for caffeine extraction including liquid/liquid extraction, water decaffeination, and supercritical carbon dioxide extraction. Other solvents include ethyl acetate and natural oils. The seeds are first ground (1 mm), steamed and brought to a moisture content of 40% (wb). Further processing involves refining the caffeine to remove waxes, oils, and water-soluble pigments (//). Residual solvents are removed by steam distillation. Activated carbon is added and later filtered to remove pigments. Caffeine crystals are recovered with acetic acid (later removed by steam distillation) followed by centrifugation. A final vacuum-drying procedure produces caffeine at about 80 to 90% purity (//). Figure 2 shows the relative extraction times for methylene chloride (6,12), ethyl acetate, ethanol-water (60:40 v/v) and supercritical carbon dioxide (13) to remove caffeine from ground guaranâ seed.



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Time (hr)

Figure 2. Caffeine extraction from guarana using various solvents for comparison. MC: methylene chloride; Ε A: ethyl acetate; 95EW: ethanol: water (95:5 v/v); SFC02: supercritical C0 (55°C, 200 aim); MC(1:8): methylene chloride (1:8 seed.solvent ratio). Note: all solvents other than C0 were at room temperature. 2

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Major Chemical Changes during Processing

The chemical changes of guaranâ during processing are not well documented. A proximate analysis was conducted on guaranâ seeds processed by solar drying and traditional drying or roasting for 5 hours (6). Table 1 shows the results of this analysis. Data is also shown (drying process not known) (13,14). Differences were seen within all constituents at the 0.05 level for the two types of drying procedures (6).

Table 2 shows the minerals identified for the solar and traditionally-dried seeds by atomic absorption-emission spectrometry (Instrumentation Laboratories, Model 551 A A / A E ) . Greater amounts of iron seen in seed dried by the traditional method may be explained by the contact of the seeds with the iron heating vessels in the clay oven. Other differences could depend on many factors including cultivation conditions such as soil types, fertilizers used, etc. on different plantations.


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Table 1. Proximate analysis of solar and traditionally-dried guaranâ seed.


Constituent

Moisture Protein Fat Ash Fiber Carbohydrate Starch Caffeine

% Composition Solar Drying 6.50 14.12 1.95 1.61 34.30 48.02 NA 4.07 1

% Composition Traditional Drying 8.7 16.1 2.2 1.83 41.2 38.7 NA 4.40

% Compositio η (13) NA 9.86 3.00 1.42 NA NA 5.0-6.0 2.5-7.6

% Composition (14) 10.47 13.23 2.69 1.44 10.23 61.94 NA 3.28

1 NA: data not available

Table 2. Mineral Composition of Solar and Traditionally-Dried Guaranâ Seed. Mg% Composition Mg% Composition Mg% Composition Solar-dried Traditionally-Dried (U) Ca 29 14.0* 11.9* 337 Κ 555.2* 524.0* 83 Mg 124.3 111.5 6 Na 6.9 5.9 0.9 Cu 4.0* 2.9* 2.6 Fe 14.6* 6.1* 344 17.5 16.9 P0 2.8 Mn 1.7 2.1 1.7 Zn 2.8 3.1 * means within a factor are significantly different at 0.05 level Minerals

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Phytochemicals in G u a r a n â


Some phytochemicals associated with guaranâ include adenine, tannic acid, catechutannic-acid, choline, guanine, mucilage, saponin and timbonine (16), The methylxanthines present in guaranâ include caffeine (3,7-dihydro-l,3,7-trimethyl-lHpurine-2,6-dione, or more commonly 1,3,7- trimethylxanthine), theobromine (3,7dimethylxanthine), theophylline (1,3-dimethylxanthine), and the polyphenols including (n-)-catechin and (-)-epicatechin (16). Caffeine is also known as guaranine (from guaranâ), coffeine (from coffee), theine (from tea extract) and methyltheobromine. Theophylline is known for its stimulating effects similar to caffeine, but to a lesser extent. This compound is a pharmaceutical compound used as a bronchodilator (18). Theobromine is the primary compound in cocoa that simulates the effects of caffeine, but also to a lesser extent. Theobromine is widely known as the principle compound toxic to dogs (19). Figure 3 shows the chemical structures of the principle alkaloids present in guaranâ. The primary difference is the number and location of the methyl groups. Table 3 shows the alkaloid content including the caffeine content of various raw materials and beverage products containing the raw materials. Guaranâ is the richest known natural source of caffeine. The main components of the essential oils of guaranâ (greenish fixed oil) were identified as (2) methylbenzenes, (1) cyclic monoterpene and (2) cyclic sesquiterpene hydrocarbons, (2) methoxyphenylpropenes and (2) alkylphenol derivatives (20). Tannins that are common to guaranâ and kola nuts are associated as a carcinogen and inhibitor to protein function (21).

Table 3. Alkaloid content of different products. Product

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Guaranâ Coffee Tea Cocoa Kola N A : data

Caffeine (% dry basis) 3,5-7.0 1.0-2.0 2.5-4.0 0.07-0.39 2.5-3.5 not available

Beverage Product (per serving) Guaranâ Coffee Tea Cocoa Soft drink

(fng)

Theobromine (mg)

Theophyllin e(mg)

30-40 90-150 30-70 NA 30-55

0.3 0 NA 250 0

0.5 NA 3-4 NA 0

Caffeine

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Ο

Ο

Caffeine

Theobromine

ο Theophylline


Figure 3. Alkaloids in guarana.

Toxicity of Guarana The genotoxic and mutagenic effects of guaranâ aqueous extracts have been reported (7). Genotoxic effects were found for guaranâ at high concentrations by lysogenic induction in Escherichia coli. Related to this activity was the formation of a caffeine-flavonoid complex (identified by N M R ) in the presence of potassium (7). Mutagenesis was also found in Salmonella typhimurium as identified by the Ames test, again at high concentrations (7). The toxicological effects of guaranâ and ginseng extracts were tested in mice with results showing no toxicity as demonstrated by histopathological examination (22). The study did show that antioxidant effects were evident. The results showed inhibition of lipid peroxidation even at concentrations as low as 1.2 μg/mL (22). The ability of guaranâ to induce in vitro cytotoxic effects were also studied in Chinese hamster ovaries (23) and mice (24). The results suggested that cytotoxic effects may occur at high doses, but no toxicity was evident at low levels (similar to that found in guaranâ drinks). The primary benefit of guaranâ as advertised by the commercial beverage industry is the high natural caffeine content. The drinks are promoted to consumers as "high energy" and "natural" beverages and have recently become a commercial success. Guaranâ, once considered a drug in the United States, is now classified by the Food and Drug Administration as a diet aid and food additive (17). Mair (25) indicated concern over the most recent introduction of guaranâ (and ginseng extract) drinks by Coca Cola Industries in New Zealand, Lift Plus®, which contains nearly three times the caffeine of the average soft drink. The concern was that the diuretic effect of caffeine results in dehydration during sports activities, while the "energy" beverages are perceived to increase performance. The second major use of guaranâ is the dried extract in form of capsules for energy boost, diet aids, and headache remedies. The claim by commercial vendors is that the caffeine is absorbed into the body at slower rates causing the effect of less "shock" or stress to metabolic systems


313 in the body. Presently scientific evidence is generally lacking to validate this claim, but there may be some merit for further investigation.


Flavor Changes during Processing As with chemical changes during processing, literature is sparse concerning flavoring volatile changes. Therefore, a qualitative study was conducted to identify the compounds from ground, dried, guaranâ seeds (6). The seeds (150 g) were ground with a Viking hammer mill to pass through a 1.5 mm seive, homogenized for 5 minutes with 700 mL water (HPLC grade) at 60°C. Volatiles were steam distilled and the aqueous residue extracted for two hours with methylene chloride. The extract was concentrated to 1 mL (26). The volatiles were analyzed qualitatively with a Shimadzu Model 9 A M G C - M S . The flavor volatiles are shown with matching probabilities in Table 4. Guaranâ seed extract is used in soft drinks at low level amounts varying from 0.02 to 0.2%. Therefore, the flavoring capacity is limited. In other products, however, the volatiles that contain guaranâ seed extract in greater amounts such as syrups and concentrates may have a significant flavor contribution.

Table 4. Flavor Volatiles in Guaranâ Extracted with Methylene Chloride and Analyzed by GC-MS. Retention Time (minutes) 10.1 21.2 24.2 27.1 30.2 33.1 36.1 38.3 41.8 42.0 42.7 50.9 51.6 59.0

Volatile Component l-octen-3-ol 2,5, dymethylpyrazine Carophyllene Pyrrole Nerolidol Allyl benzoate Nerol Hexadecanal Acetophenone Dimethyl heptane Isoamylbenzyl ether Decadienal Musk xylol Ethyl caproate

Matching Probability (%) 52 52 45 39 47 32 39 58 63 22 22 78 22 25


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References 1. Schimidt, F. Campa 1930,1,74-79. 2. Nazaré, RFR; Figueiredo, P.J.C. Contribuicão ao estudo do guaraná; EMBRAPA-CPATU: Belém, PA, Brazil, 1982; 40 pp. 3. Baumann, T.W.; Schulthess, B.H.; Hanni, K. Phytochemistry, 1995, 39, 10631070. 4. Lira, M.B. Boletim de Pesquisa da CEDEAM. 1987, 6, 105-124. 5. Beck, H.T. Adv. Econ. Botany. 1990, 8, 41. 6. Chaar, J.M. Ph.D. Thesis, The University of Tennessee, Knoxville, TN, 1990. 7. Fonseca C.A.S.; Leal, J.; Costa, S.S.; Leitão, A.C. Mutation Res. 1994, 321, 165173. 8. EMBRAPA. Emprêsa Brasileira de Pesquisa Agropecuária: Manaus, AM, Brazil, 1988; 149 pp. 9. Goncalves, J. Cultura da Amazônia. 1971, 2, 1. 10. Garcia, M.V.B and J.M.P. Müller, http://www.symmetrix.ch/Public/guaraná/index.html, 1999. 11. Clifford, M.N. In CoffeeChemistry;Clark, R.J. and Macrae, R., Eds.; Elsevier Applied Science;, New York, 1985; Vol. 1, pp. 153-202. 12. Hulbert, G.J.; Biswal, R.N.; Mehr, C.B.; Walker, T.H.; Collins, J.L. Food Sci. Technol. Int. 1998, 4, 53-58. 13. Mehr, C.B.; Biswal, R.N.; Collins, J.L.; Cochran, H.D. J. Supercritical Fluids. 1996, 9, 185-191. 14. http://metalab.unc.edu/london/orgfarm/gardening/gardening-faqs/culinary-herbs/SWSBM/Constituents/Paullinia_cupana.txt 15. Angelucci, E.; Tocchini, R.P.; Lazarini, V.B.; Prado, M.A.F. Boletim do Instituto de Tecnologia de Alimentos, 1978, 56, 183. 16. http://www.rain-tree.com/guarana.htm 17. Carlson, M.; Thompson R.D. J AOAC Int. 1998, 81, 691-701. 18. http://ntp-server.niehs.nih.gov/htdocs/LT-Studies/TR473.html August, 1998. 19. http://www.vetinfo.com/dtoxin.html#Chocolate toxicity 20. Benoni H.; Dallakian P.; Taraz Κ.Ζ.Lebensm.UntersForsch.1996, 203, 95-8. 21. Morton J.F. Basic Life Sci 1992, 59, 739-65. 22. Mattei R.; Dias R.F.; Espinola E.B.; Carlini E.A.; Barros S.B. J. Ethnopharmacol. 1998, 60, 111-6. 23. Santa Maria Α.; Lopez Α.; Diaz M.M.; Munoz-Mingarro D.; Pozuelo J.M. Ecotoxicol Environ.Saf.1998, 39, 164-7. 24. Espinola E.B.; Dias R.F.; Mattei R.; Carlini E.A. J. Ethnopharmacol. 1997, 55, 223-9. 25. Mair, S. The Press. (http://www.press.co.nz:80/11/990318f5.htm) March 18, 1999. 26. Au-Yeung, D.; MacLeod, A.J. J. Agric. Food Chem. 1981, 29, 502.


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