Enzyme agent, processed coffee and application thereof

Abstract

The present invention addresses the problem of providing a coffee beverage in which aggregation of coffee oil is suppressed. The present invention relates to an enzyme agent for suppressing aggregation of coffee oil, the enzyme agent containing a lipase derived from the genus Candida. The present invention also relates to a processed coffee and a method for producing a processed coffee, comprising allowing an enzyme agent to act on coffee.

Description

Enzymes, processed coffee and their applications

[0001] The present invention relates to an enzyme preparation, processed coffee, a method for producing processed coffee, and a method for inhibiting coffee oil aggregation.

[0002] Consumption of coffee drinks is increasing year by year, and in particular, the Chinese market is showing explosive growth due to rising national income and changes in consumer awareness. In the coffee drink manufacturing process, extracted coffee is subjected to filtration and sterilization processes.

[0003] In the extraction and filtration processes of coffee beverage production, the addition of various enzymes has been investigated to increase the amount of components in the extract and improve filterability. For example, mannanase is known to act on galactomannan contained in coffee to improve the extraction rate of coffee components and prevent precipitation. Also, cellulase is known to act on cellulose, the main component of coffee bean cell walls, and increase the solubility of coffee beans, thereby improving the extraction rate and preventing precipitation.

[0004] Furthermore, in the coffee beverage production process, the addition of enzymes to enhance the umami and flavor of coffee has been considered. For example, Patent Document 1 discloses a method for producing a coffee flavor by treating coffee oil with lipase. In this method, it is considered to produce a coffee flavor product that has a smooth texture, a smooth throat feel, and an espresso flavor by treating oil derived from coffee beans with lipase.

[0005] Japanese Patent Application Laid-Open No. 2007-61046

[0006] Coffee beans contain fats and oils, and during the storage process of coffee extracted from the beans, these fats continue to rise to the surface of the coffee and agglomerate (phase separation), which can result in the formation of an oil ring.

[0007] Therefore, an object of the present invention is to provide a coffee beverage in which aggregation of coffee oil is suppressed.

[0008] Examples of specific embodiments of the present invention are given below.

[0009] [1] An enzyme preparation for inhibiting aggregation of coffee oil, comprising a lipase derived from the genus Candida. [2] Processed coffee obtained by allowing the enzyme preparation according to [1] to act on coffee. [3] Processed coffee according to [2], wherein the coffee has a moisture content of 80% by mass or more. [4] A method for producing processed coffee, comprising allowing a lipase derived from the genus Candida to act on coffee. [5] A method for producing processed coffee according to [4], wherein the coffee has a moisture content of 80% by mass or more. [6] A method for inhibiting aggregation of coffee oil, comprising allowing a lipase derived from the genus Candida to act on coffee.

[0010] According to the present invention, a coffee beverage in which aggregation of coffee oil is suppressed can be provided. Furthermore, according to the present invention, a coffee beverage in which aggregation of coffee oil is suppressed can be provided which has excellent storage stability.

[0011] The present invention will be described in detail below. The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0012] (Enzyme Preparation) The present invention relates to an enzyme preparation containing a lipase for inhibiting the aggregation of coffee oil. In particular, a preferred embodiment of the present invention relates to an enzyme preparation containing a lipase derived from the genus Candida for inhibiting the aggregation of coffee oil. In this embodiment, by allowing the lipase derived from the genus Candida to act on coffee, the aggregation of coffee oil can be inhibited, thereby suppressing the occurrence of oil rings. In this specification, the term "oil ring" refers to a ring-shaped oil layer that forms on the surface of the liquid in a container as a result of coffee oil contained in coffee being separated due to hydrophobic interactions during storage.

[0013] The enzyme preparation may be any preparation containing a lipase derived from the genus Candida, or may consist of a lipase derived from the genus Candida. The enzyme preparation may be in any form, such as powder, solid, gel, or liquid.

[0014] The enzyme preparation may also contain a carrier, in which case the enzyme in the enzyme preparation may be immobilized on a carrier such as a porous material. In the immobilized enzyme, the enzyme and the carrier may be covalently bonded, may be attracted to each other by electrostatic interaction, or may be encapsulated in protein in the porous material. The carrier used for the immobilized enzyme is not particularly limited, but is preferably insoluble. The insoluble carrier may be inorganic or organic.

[0015] Lipase Lipase is an enzyme that acts on triacylglycerol (triglyceride) to liberate fatty acids depending on the specificity of the enzyme, catalyzing the reaction to produce diacylglycerol (DAG) or monoacylglycerol (MAG), or the reverse reaction.

[0016] Examples of lipases include lipases derived from the genus Penicillium, Aspergillus, Rhizopus, Candida, Mucor, Rhizomucor, and Penicillium. In this embodiment, the lipase is preferably a lipase derived from the genus Candida, and particularly preferably a lipase derived from Candida cylindracea. In this embodiment, the use of a lipase derived from the genus Candida can more effectively suppress the occurrence of oil rings. Furthermore, in this embodiment, the use of a lipase derived from the genus Candida can also suppress the occurrence of off-flavors in coffee (e.g., stale coffee odors).

[0017] Lipase can be prepared from the culture medium of the microorganism from which the lipase is derived. Specific preparation methods include recovering lipase from the culture medium or cells of the microorganism. For example, when a lipase-secreting microorganism is used, the cells are recovered from the culture medium in advance by filtration, centrifugation, or the like, as necessary, and the enzyme is then separated and / or purified. When a lipase-nonsecreting microorganism is used, the cells are recovered from the culture medium in advance by pressure treatment, ultrasonic treatment, or the like, as necessary, to expose the enzyme, and the enzyme is then separated and / or purified. The enzyme separation and / or purification method is not particularly limited, and known protein separation and / or purification methods can be used, including, for example, centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins. The separated and / or purified enzyme can be powdered by drying methods such as freeze-drying and vacuum drying, or by using appropriate excipients and / or drying aids in the drying methods. The isolated and / or purified enzyme can also be liquefied by adding an appropriate additive and sterilizing by filtration.

[0018] Commercially available lipases can also be used. Preferred examples of commercially available lipases include those derived from Candida cylindracea manufactured by Amano Enzyme Inc.

[0019] When the enzyme preparation is allowed to act on coffee, the amount of lipase added is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and even more preferably 0.05% by mass or more, relative to the total mass of coffee, and is preferably 20% by mass or less, more preferably 10% by mass or less, relative to the total mass of coffee.

[0020] There are no particular limitations on the amount of lipase added, but it is preferable that the amount added is such that the enzymatic activity per gram of coffee is at least 0.1 U, more preferably at least 0.5 U, even more preferably at least 1 U, still more preferably at least 10 U, even more preferably at least 50 U, and even more preferably at least 100 U. The amount of lipase added is preferably such that the enzymatic activity per gram of coffee is at most 50,000 U, more preferably at most 30,000 U, even more preferably at most 10,000 U, still more preferably at most 5,000 U, even more preferably at most 2,000 U, and even more preferably at most 1,000 U.

[0021] The enzymatic activity of lipase is measured as follows. First, 18 g of polyvinyl alcohol I and 2 g of polyvinyl alcohol II are weighed out, 800 mL of water is added, and the mixture is dissolved by heating at 75-80°C for approximately 1 hour while stirring. The solution is made up to 1000 mL with water, and 150 mL of the emulsion and 50 mL of olive oil (Japanese Pharmacopoeia) are emulsified using a homogenizer at 10°C or below to obtain an olive oil emulsion. After preparation, the olive oil emulsion is stored refrigerated and allowed to stand for at least 1 hour before use. Next, 4 mL of McIlvain buffer (pH 7.0) and 5 mL of the olive oil emulsion are mixed, and the mixture is left to stand at 37°C for 10-15 minutes. 1 mL of the enzyme solution diluted to the desired concentration is added to the mixture, mixed, and left to stand at 37°C for 30 minutes. Add 10 mL of ethanol / acetone mixture (1:1) and mix. Add 10 mL of 0.05 mol / L sodium hydroxide and 10 mL of ethanol / acetone mixture (1:1), and then add phenolphthalein reagent as an indicator. While stirring with a stirrer under nitrogen substitution, titrate with 0.05 mol / L hydrochloric acid until the pH reaches 10.00. ３０ As a blank, add 10 mL of ethanol / acetone mixture (1:1) to 4 mL of buffer solution and 5 mL of olive oil emulsion, mix, and then add 1 mL of water. ０Lipase activity is calculated using the following formula, with 1 unit (1 U) being the amount of enzyme that increases 1 micromole of fatty acid per minute when treated with olive oil (Japanese Pharmacopoeia) as a substrate at 37°C.

[0022]

[0023] <Coffee> Coffee is liquid or semi-liquid coffee. For example, coffee may be an extract obtained by extracting coffee ingredients (green coffee beans, roasted coffee beans, or ground coffee beans) with water (including hot water or steam), or a concentrate obtained by concentrating the extract. The concentrate may also be a semi-liquid substance such as a paste obtained by concentrating coffee. Coffee may also be instant coffee obtained by dissolving a dried product (instant coffee powder, etc.) obtained by drying the above-mentioned extract or concentrate in water (including hot water or steam). Coffee also includes slurries in which coffee ingredients are dispersed and solutions in which the coffee ingredients have settled.

[0024] In particular, the coffee in this embodiment is preferably green coffee beans, roasted coffee beans, or an extract of ground coffee beans. The type of coffee beans used for the coffee is not particularly limited, and examples of cultivated tree species include Arabica, Robusta, and Liberica, and examples of coffee varieties include Mocha, Brazil, Colombia, Guatemala, Blue Mountain, Kona, Mandheling, and Kilimanjaro. One type of coffee bean may be used, or multiple types may be blended. In this embodiment, coffee beans are preferably roasted to obtain roasted coffee beans, and the coffee is preferably an extract of roasted coffee beans.

[0025] The coffee of this embodiment is preferably an extract obtained by extracting roasted coffee beans or crushed coffee beans with water (including hot water or steam). The coffee is preferably a water (including hot water or steam) extract, and the water content of the coffee is preferably 80% by mass or more, more preferably 85% by mass or more, even more preferably 90% by mass or more, even more preferably 95% by mass or more, and particularly preferably 99% by mass or more.

[0026] The pH of coffee is, for example, 3.0 to 7.0, preferably 3.5 to 6.5, more preferably 4.0 to 6.0, and even more preferably 4.2 to 5.0.

[0027] The coffee in this embodiment is preferably coffee beans or an extract of ground coffee beans, and the fat and oil content of the coffee beans is preferably 1.0% by mass or more, more preferably 1.2% by mass or more, even more preferably 1.4% by mass or more, and particularly preferably 1.5% by mass or more.

[0028] (Processed Coffee) This embodiment relates to processed coffee obtained by treating coffee with the enzyme agent described above. The processed coffee contains enzyme-treated products of some components of coffee, and in this embodiment, fatty acids are preferably included.

[0029] In this embodiment, the coffee to be treated with the enzyme agent is preferably a water (including hot water and steam) extract, and therefore the moisture content of the resulting processed coffee is preferably 80% by mass or more, more preferably 85% by mass or more, even more preferably 90% by mass or more, even more preferably 95% by mass or more, and particularly preferably 99% by mass or more.

[0030] The acid value of the processed coffee is preferably 1.10 mg KOH or more. The acid value of the processed coffee is preferably 10.0 mg KOH or less, more preferably 8.00 mg KOH or less, and even more preferably 5.00 mg KOH or less. The acid value of the processed coffee can be calculated by neutralization titration using potassium hydroxide and a pH indicator.

[0031] In this specification, processed coffee also includes a slurry in which coffee ingredients are dispersed, a solution in which coffee ingredients have settled, and a solid form of dried processed coffee.

[0032] (Processed Coffee-Containing Beverage) This embodiment may relate to a processed coffee-containing beverage containing the processed coffee described above. The processed coffee-containing beverage of this embodiment may contain optional ingredients as appropriate. For example, optional ingredients may include milk (animal milk, vegetable milk), flavorings, sweeteners, acidulants, bittering agents, colorants, antioxidants, pH adjusters, vitamins, amino acids, minerals, antifoaming agents, emulsifiers (glycerin fatty acid esters, sucrose fatty acid esters, lecithin, saponin, etc.), thickening polysaccharides (pectin, carboxymethylcellulose, etc.), salts (table salt, calcium salts, phosphates, etc.), etc.

[0033] A pH adjuster can be used in processed coffee-containing beverages to further improve the effect of suppressing the occurrence of oil rings. The pH adjuster is not limited as long as it exhibits alkaline properties when dissolved in water, and specific examples include baking soda (sodium bicarbonate), sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, trisodium phosphate, and tripotassium phosphate. These pH adjusters may be used alone or in combination. Among these pH adjusters, baking soda (sodium bicarbonate) is preferred from the viewpoint of further improving the effect.

[0034] When a pH adjuster is added to a processed coffee-containing beverage, there are no particular restrictions on the amount of pH adjuster used, but for example, the amount of pH adjuster per 100 parts by mass of coffee is preferably 1 to 1,000 parts by mass, more preferably 1 to 500 parts by mass, even more preferably 1 to 100 parts by mass, and particularly preferably 1 to 50 parts by mass.

[0035] (Method for Producing Processed Coffee) This embodiment relates to a method for producing processed coffee, which comprises allowing a lipase derived from the genus Candida to act on coffee.

[0036] The reaction time, temperature, and pH of the reaction solution for allowing the enzyme agent to act on the coffee are not particularly limited. The reaction temperature is, for example, 5 to 75°C, preferably 10 to 75°C, more preferably 15 to 75°C, even more preferably 20 to 60°C, even more preferably 25 to 55°C, even more preferably 30 to 55°C, and particularly preferably 40 to 55°C. The pH of the reaction solution is, for example, 3.0 to 10.0, preferably 3.0 to 7.0, and more preferably 4.0 to 7.0. The reaction time is, for example, 1 minute to 24 hours, preferably 2 minutes to 12 hours, and more preferably 10 minutes to 6 hours. The above reaction conditions can more effectively suppress the occurrence of oil rings and can also more effectively suppress the occurrence of coffee off-flavors (e.g., stale coffee odors). Furthermore, in this embodiment, even when the enzyme treatment is performed in a relatively low-temperature environment of 15°C or below, the occurrence of oil rings can be more effectively suppressed and the occurrence of coffee off-flavors (e.g., stale coffee odors) can also be more effectively suppressed. These reaction conditions are appropriately selected depending on the type of coffee used, etc. The optimal reaction conditions can be determined through preliminary experiments.

[0037] One aspect of the method for producing processed coffee according to the present embodiment includes the following steps (1) and (2). Note that an enzyme deactivation step may be added after step (2): (1) preparing coffee; and (2) treating the prepared coffee with an enzyme preparation containing lipase derived from the genus Candida. By using the above production method, the occurrence of oil rings is suppressed, and processed coffee can be produced in which off-flavors of coffee (e.g., stale coffee odors) are suppressed.

[0038] The coffee prepared in step (1) above is preferably an extract obtained by extracting roasted coffee beans or their crushed material with water (including hot water or steam). The water content of the coffee prepared in step (1) is preferably 80% by mass or more, more preferably 85% by mass or more, even more preferably 90% by mass or more, even more preferably 95% by mass or more, and particularly preferably 99% by mass or more.

[0039] (Aggregation Inhibition Method) This embodiment relates to a method for inhibiting aggregation of coffee oil, which comprises allowing a lipase derived from the genus Candida to act on coffee. In the aggregation inhibition method of this embodiment, by allowing the enzyme agent described above to act on coffee, it is possible to inhibit aggregation of coffee oil, thereby suppressing the occurrence of oil rings.

[0040] The features of the present invention will be explained in more detail below with reference to examples and comparative examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0041] (Materials used)

[0042] (Method for Measuring Enzyme Activity) 18 g of polyvinyl alcohol I and 2 g of polyvinyl alcohol II were weighed, 800 mL of water was added, and the mixture was dissolved by heating at 75-80°C for approximately 1 hour while stirring. The solution was made up to 1000 mL with water to prepare a dissolution solution. 150 mL of the emulsion and 50 mL of olive oil (Japanese Pharmacopoeia) were emulsified using a homogenizer at 10°C or below to prepare an olive oil emulsion. After preparation, the olive oil emulsion was stored refrigerated and allowed to stand for at least 1 hour before use. 4 mL of McIlvain buffer (pH 7.0) and 5 mL of the olive oil emulsion were mixed and then allowed to stand at 37°C for 10-15 minutes. 1 mL of enzyme solution diluted to the desired concentration was added, mixed, and allowed to stand at 37°C for 30 minutes. 10 mL of a 1:1 ethanol / acetone mixture was added and mixed. To this solution, 10 mL of 0.05 mol / L sodium hydroxide and 10 mL of an ethanol / acetone mixture (1:1) were added, and phenolphthalein reagent was added as an indicator. While the solution was purged with nitrogen and stirred with a stirrer, 0.05 mol / L hydrochloric acid was titrated until the pH reached 10.00, and the titer was recorded as T ３０ As a blank, 10 mL of an ethanol / acetone mixture (1:1) was added to 4 mL of buffer solution and 5 mL of olive oil emulsion, and after mixing, 1 mL of water was added. This was used, and 0.05 mol / L hydrochloric acid was titrated until the pH reached 10.00, and the titer was recorded as T ０ Lipase activity was calculated using the following formula, with 1 unit (1 U) being the amount of enzyme that increases 1 micromole of fatty acid per minute when treated with olive oil (Japanese Pharmacopoeia) as a substrate at 37°C.

[0043]

[0044] In the above formula, f is a factor of 0.05 mol / L hydrochloric acid (for quantitative determination), and n is a dilution factor per ml of sample.

[0045] [Example 1] (Coffee Manufacturing Method) The coffee machine was connected to a power source and preheated by pressing the heating button. 9.0 g of weighed ground coffee beans were placed in a powder bowl, flattened, and set in the coffee maker. When the temperature of the preheated water reached 100-104°C, the extraction button was pressed to perform extraction. The timer was started when the first drop of coffee (extract) flowed out, and the extraction button was pressed 11 seconds later to stop the water injection. Coffee continued to flow even after extraction was complete, and coffee liquid collection was stopped after 1 minute. 48.6 g of coffee was recovered. 26.1 g of hot water was added to adjust the Brix to 1.0%. In this way, 74.7 g of coffee was obtained. The above procedure was repeated to obtain the desired amount of coffee.

[0046] 95 mL of coffee was dispensed into a reaction vessel. Candida cylindracea-derived lipase was added to the coffee at 0.1% W / V (503 U lipase activity per 1 g of coffee). The reaction was carried out in a 30°C water bath for 50 minutes. After the reaction was completed, the enzyme was inactivated by treating in a boiling water bath for 10 minutes, and a sample was taken for acid value measurement. After the reaction, the processed coffee was transferred to a 100 mL glass vial and stored refrigerated at 5°C, and the presence or absence of an oil ring (agglomeration of coffee oil) was observed.

[0047] Comparative Example 1 Coffee was produced in the same manner as in Example 1, and coffee (control) was obtained in the same manner as in Example 1, except that the lipase derived from Candida cylindracea was not added.

[0048] Comparative Examples 2 and 3 Processed coffee was obtained in the same manner as in Example 1, except that the lipase derived from Candida cylindracea was replaced with lipase derived from Rhizopus delemer (Comparative Example 2) or lipase derived from Rhizopus oryzae (1) (Comparative Example 3).

[0049] [Evaluation 1] Oil Ring Formation The coffee or processed coffee obtained in the Examples and Comparative Examples was stored at 5°C, and the presence or absence of oil rings was observed on days 1, 7, 14, and 21. The results were evaluated as follows: "+++: Thick oil rings formed, ++: Oil rings formed, +: A small amount of oil rings formed, -: No oil rings formed."

[0050]

[0051] "Evaluation 2" Calculation of Relative Acid Value and Sensory Test The acid values ​​of the coffee or processed coffee obtained in the Examples and Comparative Examples were calculated by neutralization titration using potassium hydroxide and a pH indicator. The acid value was calculated based on the weight (mg) of potassium hydroxide required to neutralize the free fatty acids contained in 1 g of sample. The acid value of the untreated sample was set at 100%, and the relative acid value of the processed coffee liquid was calculated and shown in Table 4. The processed coffee was also stored at 5°C for 14 days, and then evaluated for coffee off-flavor by four panelists. Scoring was performed based on the following criteria: 3 points = "Strongly detectable off-flavor of stale coffee (stale coffee smell)," 2 points = "Detectable off-flavor of stale coffee," 1 point = "Slightly detectable off-flavor of stale coffee," and 0 point = "No detectable off-flavor of stale coffee." The average scores are shown in Table 4 below.

[0052]

[0053] In the Examples, no off-flavor was detected despite an increase in relative acid value (fatty acids were produced) compared to Comparative Examples 2 and 3. In Comparative Example 3, an off-flavor was detected despite the relative acid value not increasing significantly compared to Comparative Example 2. This is thought to be because the fatty acids produced change depending on the substrate specificity of the enzyme, affecting how the off-flavor is perceived.

[0054] Example 2: 95 mL of coffee was dispensed into a reaction vessel. Candida cylindracea-derived lipase was added to the coffee at 0.1% W / V (503 U lipase activity per 1 g of coffee). The reaction was carried out in a 30°C water bath for 50 minutes. After the reaction was completed, the enzyme was inactivated by treating in a boiling water bath for 10 minutes, and a sample was taken for acid value measurement. The processed coffee after the reaction was transferred to a 100 mL glass vial and stored refrigerated at 5°C. The presence or absence of an oil ring (agglomerate of coffee oil) was observed. After one week of storage, the processed coffee was shaken and then continued to be stored at 5°C, and the presence or absence of an oil ring was observed.

[0055] Comparative Example 4 Coffee was produced in the same manner as in Example 1, and the coffee (control) was observed in the same manner as in Example 2, except that the Candida cylindracea-derived lipase was not added.

[0056] [Evaluation 1] Oil Ring Formation The coffee or processed coffee obtained in Example 2 and Comparative Example 4 was stored at 5°C and observed for the presence or absence of oil rings on days 0, 7, 14, 21, 28, 35, 42, and 72. The results were evaluated as follows: "+++: Thick oil ring formed, ++: Oil ring formed, +: Slight oil ring formed, -: No oil ring formed."

[0057]

[0058] As shown in the table above, by inserting an industrially feasible process (shaking during transportation), the period during which no oil rings are formed can be extended from 21 days of storage to 28 days or more of storage. It was also shown that long-term storage (72 days) is possible.

[0059] Example 3 The coffee liquid shown in Table 1 was dispensed into a 95 mL reaction vessel. Candida cylindracea-derived lipase was added to a concentration of 0.05% W / V (252 U lipase activity per gram of coffee). The reaction was carried out for exactly 1 hour in a constant temperature bath at 10°C. After the reaction was completed, the enzyme was inactivated by treatment in a boiling water bath for 10 minutes. The processed coffee after the reaction was transferred to a 100 mL glass vial and stored refrigerated at 5°C, and the presence or absence of an oil ring (agglomeration of coffee oil) was observed.

[0060] Comparative Example 5 The coffee liquid shown in Table 1 was used, and the coffee (control) was observed in the same manner as in Example 3, except that the Candida cylindracea-derived lipase was not added.

[0061] [Evaluation 1] Oil Ring Formation The coffee or processed coffee obtained in Example 3 and Comparative Example 5 was stored at 5°C, and the presence or absence of oil rings was observed on the 28th day. The results were evaluated as follows: "+++: Thick oil rings formed, ++: Oil rings formed, +: A small amount of oil rings formed, -: No oil rings formed."

[0062]

[0063] It was found that even when lipase treatment was carried out at 10°C, there was a certain effect of reducing oil ring formation.

Claims

1. An enzyme preparation for inhibiting the aggregation of coffee oil, comprising a lipase derived from the genus Candida.

2. Processed coffee obtained by allowing the enzyme preparation according to claim 1 to act on coffee.

3. The processed coffee according to claim 2, wherein the moisture content of the coffee is 80% by mass or more.

4. A method for producing processed coffee, which comprises allowing a lipase derived from the genus Candida to act on coffee.

5. The method for producing processed coffee according to claim 4, wherein the water content of the coffee is 80% by mass or more.

6. A method for inhibiting coffee oil aggregation, which comprises subjecting coffee to the action of lipase derived from the genus Candida.

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