Method of enhancing colour in a tea based foodstuff

Abstract: A method of inducing the formation of colour determining compounds in a tea-based food product, characterised in that the tea leaves are treated with an exogenous laccase, polyphenol oxidase or peroxidase, in combination with a pretreatment with a tannase. Also, a treatment with a fungal laccase form a Pleurotus species can be performed to enhance the colour of a tea-based product.

Description: The present invention relates a method of inducing the formation of coloured compounds in a tea-based product. More specifically, the invention involves using certain fungal laccases to brighten or intensify the colour of tea. Colour can be one of the most important factors affecting a consumer's decision to choose one product over another. Apart from the visual impact it can influence one's perception of the product's taste and quality. This can be especially true for tea-based beverages. Whether freshly brewed, taken with or without milk, instant or ready-to-drink, tea can be enjoyed or dismissed by consumers before the first sip. Black tea is traditionally produced by oxidising and drying freshly plucked green tea leaves. The harvested leaves are usually the so-called flush or new growth comprising the apical bud and several new leaves below it (typically two leaves and a bud). The oxidation step involves converting the colourless catechins in the leaves to a complex mixture of yellow and orange to dark-brown substances and producing a large number of aromatic volatile compounds. The colourful oxidation products include theaflavins and thearubigins. Theaflavins comprise six well-defined catechin condensation products that are characterised by their benztropolone ring. Thearubigins are a group of undefined molecules with a large variance in molecular weight. They have a variety of colours ranging from yellow to dark red and brown. It is well known to use exogenous enzymes such as tannases and cellulases to increase the yield of tea liquor from tea leaves. It is also known to use polyphenol oxidases (PPO's) in producing tea-like colours. JP-A-49-4394 (CA 1974: 81, 103275v) discloses fermentation of tea leaves with PPO's from Alternaria tenuis or Cladosporium species, resulting in the production of black tea. Similarly, the use of Alternaria tenuis PPO was reported to shorten the fermentation time and to increase the colour of black tea (Kato et al, CA 1987: 107, 11452g) and to darken cocoa, coffee and tea extracts (Motoda, Nippon Shokuhin Kogyo Gakkaishi 1982, 1, 11-15). JP-A-57-206391 (=JP-B-5393) discloses the production of flavonoid pigments by treating plant tissue, such as green tea, with a tyrosine or Alternaria tenuis PPO. Li and Guo (Linchan Huaxue Yu Gongye, 1988, 8, 33-38) describe the catalytic oxidation of green tea tannin by immobilised laccases and native laccases from plant origin (Rhus vernicifera). The use of enzymes in the manufacture of black tea and instant tea was reviewed by Sanderson and Coggon (ACS Symposium Series 1977, 81, 12-26). The enzymes include catechol oxidase for black tea production. No laccases are mentioned. GB-A-1318035 (Tenco Brooke Bond) discloses a process for enzymatically converting green tea into a liquor which is similar to that obtained from black tea leaf. US-A-3,812,266 and US-A-4,051,264 (Sanderson) disclose methods of treating tea leaves with tannase with consequent colour changes. The present inventors have found that treating black tea leaves with certain fungal laccases, especially from a Pleurotus species, leads to an unexpectedly enhanced colour. Accordingly the present invention relates to a method of enhancing the colour of black tea-based products, characterised in that black tea leaves or black tea extracts are treated with a fungal laccase that has a redox potential higher than 550 mV in the presence of oxygen. The present inventors have found using such a laccase in this way can optimise the intensity of the colour of a tea extract and/or increase the theaflavin content. This can result in a more desirable product. Laccases (EC 1.10.3.2) are sometimes referred to as p-diphenol oxidases. However, this name is too restricted since their substrate specificity is much broader. While they are sometimes grouped under the polyphenol oxidases (PPO's), laccases can be discriminated from tyrosinases and catechol oxidases by the fact that they contain at least three copper ions per molecule/subunit (amino acid chain). Tyrosinase and catechol oxidase contain 1 or 2 copper ions. The enzyme urishol oxidase is also considered to be a laccase. Their natural function is not well understood but it has been suggested that they play a role in the mineralisation of lignin. The laccases also require oxygen as an electron acceptor. Laccases can also be distinguished from other PPO's on the basis of their activity range. Laccase can oxidise hydroquinones, whereas other PPO's cannot. The preferred laccases show good activity towards syringaldazine (more than 100 U/mg protein; 1 unit (U) id defined as 1nmole/s-lnkatal) and catechin (more than 1,500 AU/mg protein, wherein 1 AU is defined as 0.001 ΔA 400 ), while the other PPO's are less active with these substrates. The syringaldazine assay is performed as follows: 50 µl of enzyme solution is added to a mixture of 1200 µl 25 mM sodium acetate, pH 5.5, 250 µl 1 g/l plyethylene glycol (PEG. 8000) in Milli Q water and 500 µl 800. µM syringaldazine solution in methanol; ΔA 530 /min between t = 60s and t = 120 s is measured at 30 °C; e = 104 mM -1 . cm -1 . pathlength in cuvette = 1 cm. The catechin assay is performed as follows: 50µl of enzyme solution is mixed with 0.2 M catechin dissolved in oxygen-free ethanol and 900 µl of air sturated McIlvain buffer pH = 6. ΔA 400 is measured. Also, the preferred laccases are not seriously inhibited by 1mM 4-hexylresorcinol or salicyhydroxamic acid, whereas catechol oxidases are inhibited by these agents. The laccases suitable for use in the method of the present invention have a redox potential of more than 550mV. Laccases can be derived from plant sources such as Rhus, Daucus, Iris, Rosmarinus, Solanum, or Vinca species or aleurone layer of grains. However for the purposes of the present invention, laccases from microbial, specifically fungal sources such as Agaricus, Armillaria, Aspergillus, Basidiomycete PM1, Botrytis, Cerrena, Coriolus (= Polyporus = Trametes), Cryptococcus, Curvularia, Daedalea, Flammulina, Ganoderma, Lentinus, Monocillium, Neurospora, Phlebia, Pleurotus, Pycnoporus, Rhizoctonia, Schizophyllum, and Trichoderma are used. Preference is given in particular to laccases derived from basidiomycetes (mushrooms with a stem) and/or white-rot fungi. Most preferred laccase is derived from food-grade mushrooms, such as Pleurotus species, in particular P. ostreatus, an edible basidiomycete having white-rot fungal properties. For each of the laccases described above, preference is given to those enzymes derived from organisms which are acceptable for food purposes, i.e. which are food-grade. The above-mentioned laccases can also be obtained from genetically modified organisms. This would be the case when an enzyme-encoding gene of one of the organisms mentioned is transferred and expressed in another organism. Known examples include the production of Phlebia radiata laccase in Trichoderma reesei (M. Saloheimo et al in Bio/Technology 9, 987 (1991)) and Polyporus sintinus laccase in Aspergillus oryzae (D.S. Yaver et al, Genetics and cellular biology of basidiomycetes III, Congress abstracts, London, C.F. Thurston and D.A. Wood organizers, 16-19 June 1995) and Myceliophthora thermophila or Scytalidium thermophilium laccase in A. Oryzae (WO 95/33836, WO 95/33837). Preferred host organisms are food-grade microorganisms, in particular yeast species, such as Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha, and fungal species such as Aspergillus sp., e.g. A. oryzae or A. awamori. For the purposes of the present invention suitable enzyme concentrations range between about 0.0001 and about 2.5% on the basis of soluble tea solids, preferably between 0.001 and 0.3% dry enzyme weight). Soluble tea solids are determined by filtering, drying and weighing. The laccase treatment can be performed using black tea leaves, preferably in the presence of water. The treatment can also be performed using black tea extracts, either direct extracts, or reconstituted extracts, i.e. redissolved dry extracted tea components. Furthermore, the laccase can be added to a conventional black tea "fermentation", i.e. during the primary oxidation of the green tea. The preferred pH during the enzymatically catalyzed oxidation should be between pH 3 and pH 8, especially between pH 4 and pH 7. Suitable oxidation temperatures range from 10 to 70°C, the best temperatures being 20 and 65°C. suitable oxidation times depend on the temperature and the enzyme strength, and can be e.g. from 1 minute to 6 hours, in particular from 5 minutes to 3 hours. The enzyme may be removed afterwards e.g. by means of filtration, but in general removal of the enzyme is not necessary. According to a particular embodiment of the invention, the enzymes are not added in soluble form, but are immobilised on a solid support. The solid support can be a conventional resin e.g. packed in a column. The solid support can also be formed by beads, filters, and the like, made from polyacrylamide, polysaccharides, polyamides or other insoluble organic or inorganic polymers. The method of the invention can be used to produce tea-like beverages as such or to produce dry tea material such as soluble powders ("instant tea" and the like), which can be reconstituted to form tea-like beverages, or also to produce other edible products wherein a tea-like colour is desirable. The method of the present invention will now be described with reference to the following examples. EXAMPLES In the Examples 1 to 3 colour was measured using the CIE (Commission Internationale d'Eclairage) scale on a Hunter Lab DP 1000 or a standard spectrophotometer with appropriate software. The colour was measured using a sample path length of 1 cm. Measurements were performed at a concentration of 0.15% soluble tea solids and at pH 4, unless stated otherwise. The colour was analysed by means of parameters L* (0 = black, 100 = colourless), a* (negative = green, positive = red) and b* (negative = blue, positive = yellow). Control values refer to the colour before treatment. Example 1 Fifty ml of 0.1 McIlvain Buffer (pH=6) containing 3 mg/l of laccase obtained from Pleurotus ostreatus and 30 g/l of instant black tea (Lipton, USA) were stirred at room temperature with air sparging. After 90 min. the colour was measured as shown in Table 1 below. L* a* b* Control before treatment 98 -3 12 + air sparging 97 -1 1 + air sparging + laccase 84 7 87 These results demonstrate that laccase darkens an extract of black tea. Example 2 A black tea extract (3% soluble solids) was treated with 0.5% laccase (weight % based on tea solids) from Rigidoporous zonalis (Takara, Japan) for 30 min at 50 °C under air sparging in 50 ml of McIlvain Buffer (pH=6). After 30 min. the colour was measured as shown in Table 2. L* a* b* Control before treatment 89 0 42 + air sparging 89 -3 41 + air sparging + laccase 71 15 76 Example 3 Tea leaves (Broken Mixed Fannings, BMF, from Sri Lanka, 50 g/l) were boiled in 50 ml of water for 10 min. The mixture was allowed to cool to 50 °C and was treated with laccase from Pleurotus ostreatus at a final concentration of 37 mg/l, for 60 min under air sparging. Solid material was removed. Colour was measured at 0.2 % soluble solids at pH = 4 and the results are shown in table 3 L a b Control before treatment 77 13 88 + air sparging 74 16 89 + air sparging + laccase 38 40 63

Claims: A method of enhancing the colour determining of black tea-based products, characterised in that black tea leaves or black tea extracts are treated with a fungal laccase that has a redox potential higher than 550 mV in the presence of oxygen. A method according to claim 1, wherein the laccase is derived from a Pleurotus species. A method according to claim 1 or 2, wherein between 0.0001 and 2.5% wt. of said laccase, on the basis of soluble tea solids, is used. A method according to claim 3, wherein between 0.001 and 0.3% wt. of said laccase, on the basis of soluble tea solids, is used. A method according to any one of the preceding claims, wherein the leaves or extracts are treated at a temperature between 20 and 65 °C. A method according to any one of the preceding claims, wherein the leaves or extracts are treated at a pH between 3 and 8. A method according to claim 6, wherein the pH is between 4 and 7. A method according to any one of the preceding claims, wherein the laccase is derived from Pleurotus ostreatus.

Inventors: BOUWENS ELISABETH CORNELIA MAR, TRIVEDI KETAN, VAN VLIET CORNELIS, WINKEL CORNELIS, BOUWENS, ELISABETH CORNELIA MARIA, TRIVEDI, KETAN, VAN VLIET, CORNELIS, WINKEL, CORNELIS
Applicants: UNILEVER NV, UNILEVER PLC, UNILEVER N.V.
Application year: 1996
Main classification: A23F3
IPC codes: A23F3