TEPZZ_555885B_T
(19)
(11)
EP 1 555 885 B1
EUROPEAN PATENT SPECIFICATION
(12)
(51) Int Cl.:
A23F 5/00 (2006.01)
(45) Date of publication and mention
of the grant of the patent:
14.08.2013 Bulletin 2013/33
(86) International application number:
PCT/US2003/034154
(21) Application number: 03779358.5
(87) International publication number:
WO 2004/037007 (06.05.2004 Gazette 2004/19)
(22) Date of filing: 23.10.2003
(54) METHOD FOR REDUCTION OF ACRYLAMIDE IN ROASTED COFFEE BEANS, ROASTED COFFEE
BEANS HAVING REDUCED LEVELS OF ACRYLAMIDE, AND ARTICLE OF COMMERCE
VERFAHREN ZUR SENKUNG DES ACRYLAMIDGEHALTS VON RÖSTKAFFEEBOHNEN,
RÖSTKAFFEEBOHNEN MIT NIEDRIGEM ACRYLAMIDGEHALT, UND HANDELSPRODUKT
PROCEDE DE REDUCTION DE LA TENEUR EN ACRYLAMIDE DANS DES GRAINS DE CAFE
TORREFIES, GRAINS DE CAFE TORREFIES PRESENTANT UNE TENEUR EN ACRYLAMIDE
REDUITE, ET ARTICLE DE COMMERCE
(84) Designated Contracting States:
AT BE BG CH CY CZ DE DK EE ES FI FR GB GR
HU IE IT LI LU MC NL PT RO SE SI SK TR
(74) Representative: Cabinet Plasseraud
52, rue de la Victoire
75440 Paris Cedex 09 (FR)
(30) Priority: 25.10.2002 US 421344 P
05.12.2002 US 431150 P
(56) References cited:
• ANONYMOUS: "Mechanism(s) of Formation of
Acrylamide in Food: Background" INTERNET
ARTICLE, October 2002 (2002-10), XP002237515
Retrieved from the Internet: <URL:http:
//www.jitsan.umd.edu/Acrylamide/
WG1/WG1_Mech_BG_Paper.pdf> [retrieved on
2003-04-03]
• MOTTRAM D.S., WEDZICHA B.L., DODSON A.T.:
"ACRYLAMIDE IS FORMED IN THE MAILLARD
REACTION" NATURE, MACMILLAN JOURNALS
LTD. LONDON, GB, vol. 419, 3 October 2002
(2002-10-03), pages 448-449, XP002235161 ISSN:
0028-0836
• STADLER R H ET AL: "Acrylamide from Maillard
reaction products" NATURE, MACMILLAN
JOURNALS LTD. LONDON, GB, vol. 419, 3
October 2002 (2002-10-03), pages 449-450,
XP002237518 ISSN: 0028-0836
• YAYLAYAN V A ET AL: "Why asparagine needs
carbohydrates to generate acrylamide"
JOURNAL OF AGRICULTURAL AND FOOD
CHEMISTRY, AMERICAN CHEMICAL SOCIETY.
WASHINGTON, US, vol. 51, 11 February 2003
(2003-02-11), pages 1753-1757, XP002237519
ISSN: 0021-8561
(43) Date of publication of application:
27.07.2005 Bulletin 2005/30
EP 1 555 885 B1
(73) Proprietor: Pringles S.a.r.l.
2220 Luxembourg (LU)
(72) Inventors:
• DRIA, Glenn, James
Okeana, OH 45053 (US)
• ZYZAK, David, Vincent
Mason, OH 45040 (US)
• GUTWEIN, Roger, William
Cincinnati, OH 45239 (US)
• VILLAGRAN, Francisco, Valentino
Mason, OH 45040 (US)
• YOUNG, Herbert, Thomas
Cincinnati, OH 45231 (US)
• BUNKE, Ralph, Paul
Cincinnati, OH 45248 (US)
• LIN, Peter, Yau Tak
Liberty Township, OH 45044 (US)
• HOWIE, John, Keeney
Oregonia, OH 45054 (US)
• SCHAFERMEYER, Richard, Gerald
Cincinnati, OH 45231 (US)
Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent
Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the
Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been
paid. (Art. 99(1) European Patent Convention).
Printed by Jouve, 75001 PARIS (FR)
(Cont. next page)
EP 1 555 885 B1
• MOTTRAM D., WEDZICHA B.: "Suggested
mechanism for the formation of acrylamide in
foods" INTERNET ARTICLE, [Online] 2002,
XP002276622 Retrieved from the Internet: <URL:
http://www.jifsan.umd.edu/presentatio ns/
acrylamide2002/WG1_Mottram_D.pdf>
[retrieved on 2004-04-08]
• FALBE J ET AL: "Römpp Lexikon
Lebensmittelchemie, ASPARAGIN" , ROEMPP
LEXIKON LEBENSMITTELCHEMIE, XX, XX,
PAGE(S) 76 XP002268124
• CLARKE R.J.; MCRAE R.: "Coffee. Vol. 1:
Chemistry", 1985, ELSEVIER APPLIED SCIENCE
PUBLISHERS * page 94 *
• ANONYMUS: ’Health Implications of Acrylamide
in Food’, [Online] 27 June 2002, ISBN:
92-4-156218-8 WORLD HEALTH ORGANIZATION
Retrieved from the Internet: <URL:http:
//www.who.int/foodsafety/publicat ions/
chem/en/acrylamide_full.pdf>
• JACKSON L: "Formation of Acrylamide in Food"
FOOD ADVISORY COMMITTEE.
CONTAMINANTS AND NATURAL TOXICANTS.
SUBCOMMITTEE MEETING, XX, XX, 4 December
2002 (2002-12-04), XP002237522
• ZYZAK D.V. ET AL: "Acrylamide formation
mechanism in heated foods" JOURNAL OF
AGRICULTURAL AND FOOD CHEMISTRY, vol.
51, no. 16, 28 June 2003 (2003-06-28), pages
4782-4787, XP002276619 ISSN: 0021-8561
• FRIEDMAN M.: "Chemistry, biochemistry, and
safety of acrylamide. A review" JOURNAL OF
AGRICULTURAL AND FOOD CHEMISTRY, vol.
51, no. 16, 3 July 2003 (2003-07-03), pages
4504-4526, XP002276620 ISSN: 0021-8561
• ELMORE J.S., MOTTRAM D.S.: "Compilation of
free amino acids data for various food raw
materials, showing the relative contributions of
asparagine, glutamine, aspartic acid and
glutamic acid to the free amino acid composition"
INTERNET ARTICLE, [Online] October 2002
(2002-10), XP002276621 Retrieved from the
Internet: <URL:http://www.jifsan.umd.edu/
presentatio ns/acrylamide2002/wg1_aspargine_
in_foods.p df> [retrieved on 2004-04-08]
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EP 1 555 885 B1
Description
FIELD OF INVENTION
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[0001] The present invention relates to the reduction of acrylamide in roasted coffee beans, the reduction of asparagine
in coffee beans, roasted coffee beans having reduced levels of acrylamide, and coffee beans having reduced levels of
asparagine. The invention further relates to an article of commerce.
BACKGROUND OF THE INVENTION
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[0002] With over 400 billion cups consumed every year, coffee is the world’s most popular beverage. Although coffee
has been enjoyed for thousands of years, researchers have only recently discovered that coffee contains acrylamide.
In April 2002, the Swedish National Food Administration and researchers from Stockholm University announced their
findings that acrylamide, a potentially cancer-causing chemical, is formed in many types of foods and beverages that
undergo heat processing. Acrylamide has a carcinogenic potency in rats that is similar to that of other carcinogens in
food, but for humans, the relative potency in food and beverages is not known. Only limited human population data are
available for acrylamide and these provide no evidence of cancer risk from occupational exposure. (FAO/WHO Consultation on the Health Implication of Acrylamide in Food: Summary Report; Geneva, Switzerland, 25-27 June 2002.)
[0003] The publication "Mechanism(s) of formation of acrylamaide in food: background" retrieved from the internet
(www.jitsan.umd.edu/Acrylamide/WG1/WG1 Mech BG Paper. pdf) is a summary of the discussions which were held in
Chicago on October 28-30, 2002, i.e. after the date of filing of the invention, at the general meeting which dealt with the
significance of food-borne acrylamide in foods. This document discloses that before the meeting of October 28-30, 2002,
at lot of theories concerning the synthesis of acrylamide in food products coexisted.
[0004] Mottram D.S. et al., Nature, Vol. 419, pages 448-449, 3 October 2002, and Stadler R.H. et al., Nature, Vol.
419, pages 449-450, 3 October 2002, disclose one of the numerous proposed syntheses of Acrylamide in fried and
oven-cooked foods, i.e. the Maillard synthesis pathway.
[0005] Elmore J.S. and Mottram D.S. « Compilation of free amino acids data for various food raw materials, showing
the relative contributions of asparagine, glutamine, aspartic acid and glutamic acid to the free amino acid composition,
published in October 2002, discloses that coffee contains asparagine, aspartic acid and glutamic acid.
[0006] Although further research is needed to assess what health effects, if any, may result from human consumption
of acrylamide at the levels commonly found in roasted coffee products, many consumers have voiced concern. Accordingly, it is an object of the present invention to provide a method for reducing the level of acrylamide in roasted coffee
beans. It is also an object of the present invention to provide roasted coffee beans having reduced levels of acrylamide.
Further, it is an object of the present invention to provide an article of commerce that communicates to the consumer
that a roasted coffee product has reduced or low levels of acrylamide.
SUMMARY OF THE INVENTION
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[0007] In one aspect, the present invention provides a method for reducing the level of acrylamide in roasted coffee
beans as in claim 1. In one embodiment, the method comprises adding an asparagine-reducing enzyme to coffee beans.
[0008] In another aspect, the present invention provides a method for reducing the level of asparagine in coffee beans.
In one embodiment, the method comprises adding an asparagine-reducing enzyme to coffee beans.
[0009] In another aspect, the present invention provides roasted coffee beans having reduced levels of acrylamide.
[0010] In another aspect, the present invention provides coffee beans having reduced levels of asparagine.
[0011] In yet another aspect, the present invention provides an article of commerce that communicates to the consumer
that a product comprising roasted coffee beans has reduced or low levels of acrylamide. ,
[0012] In still another aspect, the present invention provides an article of commerce that communicates to the consumer
that a product comprising coffee beans has reduced or low levels of asparagine.
[0013] All documents cited herein are, in relevant part, incorporated herein by reference; the citation of any document
is not to be construed as an admission that it is prior art with respect to the present invention.
[0014] As used herein, all percentages (%) are by weight unless otherwise indicated.
BRIEF DESCRIPTION OF THE DRAWINGS
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[0015]
Figure 1. Figure 1 sets forth the proposed reaction mechanism by which acrylamide forms from asparagine and a
carbonyl source (such as glucose). R1 and R2 can = H, CH3, CH2OH, CH2(CH2)nCH3, or any other component
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making up a reducing sugar; n can be any integer less than 10.
Figure 2. Figure 2 sets forth the proposed reaction mechanism by which asparaginase reacts with asparagine to
prevent the formation of acrylamide.
Figure 3. Figure 3 sets forth a sample chromatogram for LC analysis of asparagine and aspartic acid. The x-axis
represents retention time and the y-axis represents response.
DETAILED DESCRIPTION OF THE INVENTION
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[0016] Applicants have discovered that asparagine, a naturally occurring amino acid found in virtually all living systems,
can form acrylamide when heated. Thus, materials richer in asparagine, when heated, tend to contain higher levels of
acrylamide; this is especially the case when asparagine-containing materials are heated in the presence of reducing
sugars.
[0017] While not being limited by theory, it is believed that acrylamide forms via the reaction mechanism set forth in
Figure 1. It is believed that the alpha-amine group of free asparagine reacts with a carbonyl source, forming a Schiff
base. Under heat, the Schiff base adduct decarboxylates, forming a product that can either: (1) hydrolyze to form betaalanine amide (which can, under heat, further degrade to form acrylamide) or (2) decompose to form acrylamide and
the corresponding imine. (Applicants have discovered that the circled precursor atoms comprise the carbons and nitrogens in acrylamide.)
[0018] Accordingly, Applicants have further discovered that acrylamide formation in roasted coffee beans can be
reduced by removing the asparagine or converting the asparagine in the coffee beans to another substance before final
roasting of the beans. When such beans containing reduced levels of asparagine undergo final roasting, the amount of
acrylamide formed is reduced.
[0019] Applicants have found that adding an enzyme that hydrolyzes the amide group on the side chain of asparagine
prior to final roasting of the coffee beans reduces the level of acrylamide present in the roasted coffee beans. While not
being limited by theory, it is believed that the addition of such an enzyme degrades the side chain of asparagine, thus
preventing the asparagine from forming acrylamide. In doing so, the amide bond is hydrolyzed and asparagine is converted to aspartic acid. This reaction mechanism is set forth in Figure 2.
[0020] Preferred enzymes for use in the method herein include, but are not limited to, asparaginase. However, any
enzyme capable of hydrolyzing the amide group of free asparagine to prevent the formation of acrylamide is within the
scope of the present invention.
[0021] The advantages of using enzymes are numerous. These advantages include: (a) they are natural, nontoxic
substances; (b) they generally catalyze a given reaction without causing unwanted side reactions; (c) they are active
under very mild conditions of temperature and pH; (d) they are active at low concentrations; (e) the rate of reaction can
be controlled by adjusting temperature, pH, and the amount of enzyme employed; and (f) they can be inactivated after
the reaction has proceeded to the desired extent. (Food Chemistry, 4th Ed., Owen R. Fennema, Ed., Marcel Dekker,
Inc., New York, 1985, pp. 427, 433.)
A. Method for Reduction of Acrylamide in Roasted Coffee Beans
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[0022] In one aspect, the present invention provides a method for the reduction of acrylamide in roasted coffee beans.
In one embodiment, the method comprises reducing the level of asparagine in coffee beans. In another aspect, the
method comprises adding an asparagine-reducing enzyme to coffee beans. The preferred enzyme is asparaginase.
[0023] In a preferred embodiment, the present invention provides a method for reducing the level of acrylamide in
roasted coffee beans, comprising:
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(1) providing coffee beans containing asparagine;
(2) optionally pre-treating the coffee beans;
(3) adding an asparagine-reducing enzyme to the coffee beans;
(4) allowing a sufficient time for the enzyme to react with the asparagine;
(5) optionally deactivating or optionally removing the enzyme; and
(6) roasting the coffee beans to form roasted coffee beans.
[0024] In another aspect, the present invention provides a method for the reduction of asparagine in coffee beans. In
one embodiment, the method comprises adding an asparagine-reducing enzyme to coffee beans. The preferred enzyme
is asparaginase.
[0025] In a preferred embodiment, the present invention provides a method for reducing the level of asparagine in
coffee beans, comprising:
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(1) providing coffee beans containing asparagine;
(2) optionally pre-treating the coffee beans;
(3) adding an asparagine-reducing enzyme to the coffee beans;
(4) allowing a sufficient time for the enzyme to react with the asparagine; and
(5) optionally deactivating or optionally removing the enzyme.
1. Providing coffee beans containing asparagine
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[0026] Coffee beans are the seeds of cherries which grow from coffee trees in a narrow subtropical belt around the
world. There are many coffee species, however, it is generally recognized that there are two primary commercial coffee
species: Coffea arabica and Coffea canephora var. robusta. Coffees from the species arabica are described as "Brazils,"
which come from Brazil, or "Other Milds" which are grown in other premium coffee producing countries. Premium arabica
countries are generally recognized as including Colombia, Guatemala, Sumatra, Indonesia, Costa Rica, Mexico, United
States (Hawaii), El Salvador, Peru, Kenya, Ethiopia and Jamaica. Coffees from the species canephora var. robusta are
typically used as a low cost extender or as a source of additional caffeine for arabica coffees. These robusta coffees
are typically grown in the lower regions of West and Central Africa, India, South East Asia, Indonesia, and Brazil. After
the coffee cherries are harvested, the fruit is typically removed from the seed.
[0027] Any suitable coffee beans, including mixtures of various types of beans, can be used in accordance with the
present invention. The preferred coffee beans are arabica, robusta, or a mixture thereof.
[0028] As used herein, the term "coffee beans" or "beans" includes coffee beans in any suitable form. Non-limiting
examples include coffee beans in bean form or in the form of a green coffee bean extract (e.g., dry or wet green coffee
bean extracts). The coffee beans can be whole or can be reduced in particle size. The size of the coffee beans can be
reduced by cracking, chopping, dicing, macerating, grinding, flaking, or any other suitable method. The size of the coffee
beans may be reduced at any suitable stage of the method, including any time before, during, or after the addition of
the asparagine-reducing enzyme, but before the end of the time period in which the enzyme is allowed to react with the
coffee beans.
[0029] Furthermore, the coffee beans for use herein optionally, but preferably, have their fruits removed. In one embodiment, coffee beans that have undergone decaffeination are used. In another embodiment, coffee beans that have
not undergone decaffeination are used. In still another embodiment, a mixture of decaffeinated and un-decaffeinated
beans is used. Preferably green coffee beans are used, but any suitable beans that have not been subjected to final
roasting can be utilized in accordance with the method herein.
2. Optionally, pre-treating the coffee beans
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[0030] The coffee beans may optionally be pre-treated before or during the addition of enzyme. Suitable pre-treatment
includes drying, hydrating, rinsing without or with mechanical action (e.g., wet-brushing), pressurizing, steaming, blanching, heating, reduced pressure processing (e.g., vacuum), particle size reduction, or combinations thereof. Suitable pretreatment methods can also include the exposure of the coffee beans to one or more cellulose-degrading enzymes; this
method can be used alone or in combination with one or more other pre-treatment methods. Preferred cellulose-degrading
enzymes include cellulase, hemicellulase, pectinase, and mixtures thereof, although any suitable cellulose-degrading
enzyme can be used.
[0031] Pre-treatment can facilitate removal and/or extraction of the asparagine from inside the beans, allowing the
asparagine to be brought into more intimate contact with the asparagine-reducing enzyme outside of the beans. Pretreatment can also facilitate the migration of asparagine-reducing enzyme into the beans, allowing for more intimate
contact with the asparagine-reducing enzyme inside of the beans, as well as more uniform distribution of asparaginereducing enzyme within the bean.
[0032] The beans can be dried to open up their pores. In one embodiment, the beans are dried in preparation for
soaking in an asparagine-reducing enzyme solution. Drying creates a driving force for the enzyme solution to penetrate
the beans, and thus for the enzyme to reach the beans’ interior. Any suitable means of drying the beans may be used,
as long as the drying method employed does not reach a temperature where asparagine could start reacting to form
significant levels of acrylamide. Preferably, drying methods employing temperature below those typically used for roasting
are used; for example, in one embodiment coffee beans are dried at a temperature of below about 49°C (120°F). Suitable
methods of drying can include freeze drying, belt drying, vacuum drying, oven drying, fluid bed drying, and combinations
thereof. Preferably, the dried beans have a moisture content of less than about 10% in order to create a driving force
for moisture absorption.
[0033] Suitable means of hydration can include treating with low pressure or atmospheric steam, spraying the coffee
with the desired amount of water and allowing it to be absorbed, soaking the beans in an aqueous solution of the desired
amount of water in order to create moistened beans without excess water remaining, or soaking the beans in an aqueous
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solution of the desired amount of water in order to create a mixture of hydrated beans and excess water (i.e., water that
is not completely absorbed by the beans). Soaking the beans in excess solution is less preferred, because coffee solids
may be extracted into the remaining solution, resulting in decreased flavor and lower quality beans.
[0034] In one embodiment, the beans are steamed to open up their pores in preparation for soaking in an asparaginereducing enzyme solution. In another embodiment, the coffee beans are soaked in water and allowed to hydrate to from
about 15% to about 75%, preferably from about 20% to about 55%, and more preferably from about 25% to about 40%
moisture before the addition of asparagine-reducing enzyme to the solution. Hydrating the beans with aqueous solution
or steam to a moisture content of greater than about 15% can cause the beans to swell and can facilitate the formation
of pathways for the extraction of asparagine out of the beans, and/or for the transfer of an asparagine-reducing enzyme
solution into the beans.
[0035] Subjecting a mixture of beans and excess water or asparagine-reducing enzyme solution to vacuum and/or
pressure can result in more water or asparagine-reducing enzyme solution penetrating the beans and entering the beans’
interior. Pressure can force the water or asparagine-reducing enzyme solution into the structure of the beans, while
vacuum can pull residual air from within the beans and allow the water or asparagine-reducing enzyme solution to more
readily penetrate.
[0036] Particle size reduction can create larger surface areas and can allow solution uptake and/or extraction to occur
more completely, more uniformly, and more rapidly. The size of the coffee beans can be reduced by cracking, chopping,
dicing, macerating, grinding, flaking, or any other suitable method. For example, cracking (such as when the bean is
broken into quarter sections or smaller), or grinding (such as that performed when processing roast and ground coffee)
can be used to facilitate solution uptake and/or the extraction of asparagine.
3. Adding an asparagine-reducing enzyme to the coffee beans
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[0037] As used herein, "asparagine-reducing enzyme" includes any enzyme capable of reducing the level of asparagine
in coffee beans. In one embodiment, the asparagine-reducing enzyme is an enzyme capable of hydrolyzing the amide
group of free asparagine. A preferred enzyme for use herein is asparaginase. A preferred source of asparaginase is
Sigma-Aldrich, catalog #A2925.
[0038] As used herein, the terms "asparagine-reducing enzyme" and "enzyme" include one or more enzymes; for
example, a mixture of two or more enzymes is encompassed by the terms. For example, deamidases that have asparagine-reducing functionality are included in the terms.
[0039] The enzyme may be added to the coffee beans in any suitable form. For instance, the enzyme may be added
as a powder or in the form of a solution. Furthermore, the enzyme may be added to the coffee beans in any suitable
manner, such as directly (for example, sprinkled, poured, or sprayed on the coffee beans, or the coffee beans can be
soaked in an enzyme solution) or indirectly. As used herein, "adding" the enzyme to the coffee beans includes, but is
not limited to, any means of bringing the asparagine and the enzyme together.
[0040] The enzyme may be added at any suitable stage of the method before completion of final roasting (as set forth
at step 6 of the method herein) to form the roasted coffee beans. For example, the enzyme may be added to the coffee
beans during or after the optional pre-treating step. Furthermore, enzyme can be added during more than one stage of
the method. In one embodiment, enzyme is added to the beans post-harvest before their fruit is removed, then again
after the fruit has been removed and the beans have been dried.
[0041] Enzymes are marketed by units of activity, rather than by weight or volume. Thus, the effective amount of
enzyme required to achieve the desired level of acrylamide reduction will depend upon the activity of the particular
enzyme product used.
[0042] The amount of enzyme to add can depend upon the level of asparagine reduction, and accordingly the level
of acrylamide reduction, that is desired. The amount of enzyme to add can also depend upon the amount of asparagine
present in the coffee beans; coffee beans higher in asparagine will generally require increased levels of enzyme or
increased reaction time to achieve the same percentage of acrylamide reduction. The amount of enzyme to add can
also depend upon the particular enzyme used (for example, the particular enzyme’s enzymatic activity) and the particular
type of coffee beans treated. One skilled in the art will be able to determine the effective amount of enzyme based upon
the specific type of coffee, the specific enzyme, the enzyme’s specific activity, and the desired result.
[0043] Preferred methods of adding the enzyme to the coffee beans include spraying, soaking, sprinkling, and dominant
bath. In one embodiment, enzyme solution is applied by spraying the solution onto the beans along with gentle agitation
of the beans in order to create a uniform application to all the bean surfaces.
[0044] In another embodiment, coffee beans are soaked in an enzyme solution to hydrate the beans. The amount of
solution used depends upon the desired end moisture content of the beans. Enzyme solution can be used in such an
amount that all the liquid is absorbed by the beans, or in such an amount that excess solution remains after solution
absorption by the coffee beans. In yet another embodiment, the coffee beans are hydrated in a solution then an enzyme
powder is sprinkled on the hydrated coffee beans. The beans can be removed from solution by any suitable means of
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separating particulates from a solution, such as by screening.
[0045] In still another embodiment, enzyme is added to the beans by means of a dominant bath. In succession, several
batches of beans are soaked in an enzyme containing solution until the soluble materials that extract from the beans
are in or near equilibrium with the solution. In one embodiment, the enzyme in the dominant bath converts asparagine
to aspartic acid, thus creating a driving force for additional asparagine extraction on subsequent additions of batches of
beans. Extractable materials can equilibrate with the beans such that additional soluble coffee components do not extract
out, with the exception of asparagine, which continues to react and be converted by the enzyme. The aspartic acid that
is formed from the asparagine soaks back into the beans and equilibrates. Additional water and/or enzyme-containing
solution is added back after every batch of beans to make up for the solution going into the previous batch of beans;
this maintains a constant volume of the dominant bath.
[0046] In one embodiment, at least a portion of the asparagine is extracted from the coffee beans, the resulting extract
is treated with the enzyme, then at least a portion of the extract is added back into at least a portion of the coffee beans;
for example, the enzyme may be added to the extract, or the extract may be pumped through a bed or column of
immobilized enzyme (enzyme either adsorbed or chemically bonded to a substrate, preferably an inert substrate, e.g.,
pieces of plastic or beads in a column).
4. Allowing a sufficient time for the enzyme to react with the asparagine
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[0047] The amount of time needed for the enzyme to react with the asparagine will depend upon factors including,
but not limited to, the desired level of asparagine (and thus acrylamide) reduction, the characteristics of the particular
coffee beans (e.g., chemical composition, amount of asparagine present, particle size), and the particular enzyme added.
Preferably, the enzyme is allowed to react for a sufficient amount of time to result in coffee beans wherein the level of
asparagine is reduced by at least about 10%, preferably at least about 30%, more preferably at least about 50%, still
more preferably at least about 70%, and even more preferably at least about 90%. In general, the longer the enzyme is
allowed to react, the greater the level of asparagine reduction and thus the greater the level of acrylamide reduction in
the roasted coffee beans. The step of allowing a sufficient time for the enzyme to react can be carried out in any suitable
manner; for example, it can be carried out simultaneously with adding the enzyme to the coffee beans, mixing the enzyme
with the coffee beans, the absorption of the enzymatic solution by the coffee beans, or combinations thereof.
[0048] As known in the art, pH and temperature are factors that affect enzymatic activity. One skilled in the art should
readily be able to determine optimal conditions of these and other parameters (e.g., water content). In addition, optimal
pH and temperature conditions for specific enzymes are typically available in the literature and/or from enzyme suppliers.
[0049] Coffee beans prepared according to the method herein can have a reduction in the asparagine level of at least
about 10%, preferably at least about 30%, more preferably at least about 50%, still more preferably at least about 70%,
and even more preferably at least about 90%.
[0050] In one embodiment, the coffee beans comprise less than about 500 ppm asparagine, preferably less than about
300 ppm, more preferably less than about 200 ppm, and still more preferably less than about 100 ppm
5. Optionally deactivating or optionally removing the enzyme
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[0051] After the enzyme has reacted to the desired extent, it can optionally be inactivated or removed from the coffee
beans. When an enzyme that is safe for consumption (e.g., naturally occurring and found in common foods) is used,
one may choose not to deactivate or remove the enzyme. Alternatively, the enzyme can be deactivated by any suitable
means that inactivates the enzyme. For example, the enzyme can be deactivated through the use of heat, pH adjustment,
treatment with a protease, or combinations thereof. Furthermore, the enzyme can be removed from the coffee beans
by any suitable means including, but not limited to, extraction. The enzyme can be deactivated, removed, or subjected
to a combination of deactivation and removal.
6. Roasting the coffee beans to form roasted coffee beans
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[0052] The coffee beans are then roasted to form roasted coffee beans. Any suitable process comprising roasting can
be used. As used herein, the term "roasting" includes any suitable thermal treatment of coffee beans to create flavors
that are indicative of coffee. Suitable roasting techniques can include, but are not limited to, oven roasting, extrusion
roasting, steam roasting (e.g., with no post roasting), infrared roasting, microwave roasting, di-electric/induction heating
roasting, and combinations thereof. Typical roasting equipment and methods for roasting coffee beans are described,
for example, in Sivetz & Foote, Coffee Processing Technology, Avi Publishing Co., Westport, Conn., Vol. 1 (1963), pp.
203-226. The roasted coffee beans can be in any suitable form, such as decaffeinated versions, caffeinated versions,
or mixtures thereof.
[0053] The coffee beans can be roasted to any desired roast color. Preferably, the beans are roasted to a Hunter color
7
EP 1 555 885 B1
5
10
15
20
25
level of from about 10L (very dark) to about 25L (very light). As used herein, Hunter color is measured on a Hunter
colorimeter from the Hunter CIE scale. See pages 985-95 of R. S. Hunter, "Photoelectric Color Difference Meter," J. of
the Optimal Soc. of Amer., Volume 48 (1958).
[0054] Roasted coffee beans prepared according to the method herein can have a reduction in the acrylamide level
of at least about 10%, preferably at least about 30%, more preferably at least about 50%, still more preferably at least
about 70%, and even more preferably at least about 90%.
[0055] In one embodiment, the roasted coffee beans comprise less than about 160 ppb acrylamide, preferably less
than about 150 ppb acrylamide. In another embodiment, the roasted coffee beans comprise less than about 135 ppb
acrylamide, preferably less than about 120 ppb, more preferably less than about 100 ppb, still more preferably less than
about 50 ppb, even more preferably less than about 20 ppb, and most preferably less than about 10 ppb.
[0056] The roasted coffee beans can be used as is or can be used to make a variety of roasted coffee products, such
as roast and ground coffees, liquid concentrates, instant or powdered coffees, coffee beverages (e.g., hot and cold ready
to serve coffees, vended coffees, commercial and at-home brewed coffees, Kahlua™, lattes, cappuccinos), mixes (e.g.,
café latte mixes), confectionaries (e.g., candy), desserts (e.g., cakes, ice creams, mousses, custards), pastries (e.g.,
danish, donuts), sauces, and soups (e.g., chili). In one embodiment, the coffee beans are dried, roasted, then ground
to form roast and ground coffee. Typical grinding equipment is described, for example in Sivetz & Foote, supra, pp.
239-250.
[0057] Roasted coffee products comprising the roasted coffee beans of the present invention can have a reduction in
the acrylamide level of at least about 10%, preferably at least about 30%, more preferably at least about 50%, still more
preferably at least about 70%, and even more preferably at least about 90%. In one embodiment, roast and ground
coffee comprises less than about 160 ppb acrylamide, preferably less than about 150 ppb. In another embodiment, roast
and ground coffee comprises less than about 135 ppb acrylamide, preferably less than about 120 ppb, more preferably
less than about 100 ppb, still more preferably less than about 50 ppb, even more preferably less than about 20 ppb, and
most preferably less than about 10 ppb. In another embodiment, a roast and ground coffee brew comprises less than
about 7 ppb acrylamide, preferably less than about 5 ppb acrylamide.
[0058] Deactivating the enzyme may occur through heating, thus the optional deactivation step and roasting the coffee
beans may be carried out simultaneously. Heat processing can denature and inactivate the enzyme such that the roasted
coffee beans are not subjected to continuing enzymatic activity. Furthermore, at least a portion of the time allowed for
enzymatic reaction may be carried out during the roasting step.
30
B. Means of Practicing the Method
[0059] The present invention can be practiced by any suitable means. For example, the method herein can be practiced
in batch, semi-batch, or continuous mode.
35
C. Preferred Embodiments
40
45
50
55
[0060] Although the method herein will generally be described in terms of preferred embodiments set forth below, it
should be understood by one skilled in the art that the method herein can be practiced in any suitable manner.
[0061] In one embodiment coffee beans are dried to a moisture content of less than about 15%, preferably less than
about 10%, more preferably less than about 5%, and most preferably from about 1.5% to about 4%. For example, the
beans can be dried at 49°C (120°F) overnight. Drying increases the potential for the beans to absorb more enzymatic
solution. Optionally, the beans can be cracked or ground. The beans are then put into a solution comprising enzyme
and allowed to soak. The enzyme is allowed to react for from about 45 minutes to about 1 hour at a temperature of about
38°C (100°F); then, optionally the enzyme is deactivated by microwaving. The beans are then dried to a moisture content
of from about 7% to about 11% at 74°C (165°F). The beans are then roasted to a roast color of from about 16L to about
24L on a Probat Duett™ roaster, then ground to form roast and ground coffee.
[0062] In yet another embodiment, a dominant bath is employed. Coffee beans are optionally pre-wet with steam to
a moisture content of from about 15% to about 30%. They are then soaked in excess water to create a dominant bath
(i.e., not all the water is taken in by the beans). The beans are then separated from the water, such as by screening
them out of the solution. This procedure is repeated several times to form a dominant bath. In the dominant bath, an
equilibrium can be established between the water soluble components that are in the beans and in the bath. Enzyme is
then added to the bath. The enzyme selectively converts the asparagine to aspartic acid. New beans are then added to
the enzyme-containing dominant bath. Asparagine extracts out of the beans into the dominant bath and the enzyme
converts the asparagine to aspartic acid. The excess aspartic acid in the bath establishes equilibrium with the beans.
Thus, the net effect is the conversion from asparagine to aspartic acid. The beans are removed from the dominant bath.
Additional batches of beans can be processed in a continuous or semi-continuous fashion. The beans can then be
processed in the typical manner known in the art, such as by drying, roasting, then grinding to form roast and ground coffee.
8
EP 1 555 885 B1
D. Article of Commerce
[0063] In another aspect, the present invention provides an article of commerce. In one embodiment, the article of
commerce comprises:
5
(a) a product comprising roasted coffee beans, wherein said roasted coffee beans have a reduced level of acrylamide;
(b) a container for containing the product; and
(c) a message associated with the container.
10
15
20
[0064] The message associated with the container informs the consumer that the roasted coffee beans, the product
comprising the roasted coffee beans, and/or the article of commerce has a reduced level of acrylamide. In one embodiment, the message informs the consumer that the roasted coffee beans, the product comprising roasted coffee beans,
and/or the article of commerce is made with coffee beans having reduced or low levels of asparagine. The message
can be printed material attached directly or indirectly to the container, attached directly or indirectly near the container,
or alternatively can be a printed, electronic, or broadcast message associated with the container. Suitable messages
include, but are not limited to, messages that communicate "reduced" or "low" levels of acrylamide, messages that
communicate that less than a specified amount of acrylamide is present, and messages that communicate that the
roasted coffee beans, product comprising roasted coffee beans, and/or article of commerce meet or exceed a suggested
or mandatory level (e.g., regulatory threshold or signal level).
[0065] In another embodiment, the article of commerce comprises:
(a) a product comprising coffee beans, wherein said coffee beans have a reduced level of asparagine;
(b) a container for containing the coffee beans; and
(c) a message associated with the container.
25
30
35
[0066] The message associated with the container informs the consumer that the coffee beans, the product comprising
the coffee beans, and/or the article of commerce has a reduced level of asparagine. The message can be printed material
attached directly or indirectly to the container, attached directly or indirectly near the container, or alternatively can be
a printed, electronic, or broadcast message associated with the container. Suitable messages include, but are not limited
to, messages that communicate "reduced" or "low" levels of asparagine, messages that communicate that less than a
specified amount of asparagine is present, and messages that communicate that the coffee beans, product comprising
coffee beans, and/or article of commerce meet or exceed a suggested or mandatory level (e.g., regulatory threshold or
signal level).
[0067] Any container from which the product comprising the roasted coffee beans or coffee beans can be dispensed,
presented, displayed, or stored is suitable. Suitable containers include, but are not limited to, bags, canisters, boxes,
bowls, plates, tubs, and cans.
ANALYTICAL METHODS
40
[0068] Parameters used to characterize elements of the present invention are quantified by particular analytical methods. These methods are described in detail as follows.
1. Acrylamide
45
Method for Measuring Acrylamide (AA) in Food Products
Summary
50
[0069] Roast and ground coffee is spiked with 13C-AA and extracted with hot water. The aqueous supernatant is
extracted twice with ethyl acetate, and the ethyl acetate extracts are concentrated and analyzed by LC/MS with selected
ion monitoring for specific detection of AA and 13C-AA.
Extraction of Ground Coffee Beans (Both Green and Roasted)
55
[0070]
1. Weigh 6.00 6 0.01 g of sample into a 125-mL Erlenmeyer flask.
2. Add 120 mL of 100 ng/mL 13C-AA in de-ionized distilled water (ISTD 2), with an adjustable 1000-mL pipette
9
EP 1 555 885 B1
5
10
15
(calibrated), directly onto the sample.
3. Using a dispenser, add 40 mL of de-ionized distilled water to the flask and cover with foil.
4. Place into a 65°C water bath for 30 min.
5. With a dispenser, add 10 mL of ethylene dichloride to the flask, and homogenize with a Tekmar Tissumizer™
(SDT-1810) or an Ultra Turrax® (T18 Basic) for 30 seconds. Rinse the dispersing element into the flask with deionized
distilled water.
6. Place 25 g of the homogenate into an 8-dram vial
7. Tightly cap the vial and centrifuge for 30 minutes at 2500-5200 RPM.
8. Condition a Sep-Pak® Plus C18 (Waters #WAT020515) with 3 mL acetonitrile followed by 6 mL of deionized
distilled water.
9. Pass 6 mL of the supernatant through the cartridge and elute into an 8-dram vial.
10. Add 10 mL of ethyl acetate to the vial with a dispenser, cap, and vortex for 10 seconds.
11. Allow any emulsion to break up; help by swirling or shaking once or twice and then allowing layers to split.
12. Transfer as much of the top layer as possible to a scintillation vial, without transferring any liquid from the
interface. Extract once more with a 10-mL portion of ethyl acetate and add to the same scintillation vial. Then, add
approximately 2 g of anhydrous sodium sulfate.
13. Concentrate the extract with a gentle stream of nitrogen in a 60-65°C water bath to about 1 mL. Transfer the
extract to a Pierce REACTI-VIAL™ (or equivalent conical shaped glass vial) and further concentrate the extract to
a final volume of approximately 100-200 mL. Place this extract into an autosampler vial with a conical sleeve and cap.
20
Preparation of Standards
Stock Solutions and Internal Standards
25
[0071]
Solution
Weight
Volumetric Flask (mL)
Solvent
Concentration (ppm)
Stock 1
0.1000 g Acrylamide
(AA)
100
Ethyl Acetate
1000
ISTD 1
0.0100g 13CAcrylamide
100
Ethyl Acetate
100
Stock 2
0.1000 g Acrylamide
(AA)
100
Deionized Distilled
Water
1000
ISTD 2
0.0100g 13CAcrylamide
100
Deionized Distilled
Water
100
30
35
40
Intermediate Standards
[0072]
Solution
Volume Stock 1 AA (mL)
Volumetric Flask (mL)
Solvent
Concentration (ppm)
INT 1
100
10
Ethyl Acetate
10
INT 2
1000
10
Ethyl Acetate
100
45
50
Calibration Standards
[0073]
Standard
Volume
INT 1 (mL)
Volume
INT 2 (mL)
Volume
ISTD 1 (mL)
Volumetric
Flask (mL)
Solvent
Conc. AA
(ppm)
0
0
0
450
10
Ethyl
Acetate
0
55
10
Conc. ISTD
1 (ppm)
4.50
EP 1 555 885 B1
(continued)
5
10
15
Standard
Volume
INT 1 (mL)
Volume
INT 2 (mL)
Volume
ISTD 1 (mL)
Volumetric
Flask (mL)
Solvent
Conc. AA
(ppm)
Conc. ISTD
1 (ppm)
0.25
250
0
450
10
Ethyl
Acetate
0.250
4.50
0.75
750
0
450
10
Ethyl
Acetate
0.750
4.50
1.5
0
150
450
10
Ethyl
Acetate
1.50
4.50
3.0
0
300
450
10
Ethyl
Acetate
3.00
4.50
5.0
0
500
450
10
Ethyl
Acetate
5.00
4.50
Homogenizer Cleaning Procedure
20
25
30
[0074]
Use this cleaning procedure between every sample.
1. Fill a 1-L Erlenmeyer flask with hot tap water (≈80% full) and add a drop of Dawn™ dishwashing liquid (available
from the Procter & Gamble Co.) or equivalent.
2. Insert the homogenizer dispersing element into the water as far as possible.
3. Homogenize the solution for about 10-15 seconds.
4. Empty the cleaning solution from the Erlenmeyer; rinse and refill the flask with hot tap water.
5. Homogenize again for about 10-15 seconds.
6. Empty the flask and refill with hot tap water; homogenize again for about 10-15 seconds.
7. If the water is not clear and free of particulates, continue homogenizing clean hot tap water as many times as
necessary to achieve this condition.
8. When the hot tap water is clear and free of particulates, rinse the dispersing element with deionized distilled water.
Analysis by LC/MS
35
40
45
[0075]
Samples are analyzed using a Waters 2690 LC interfaced to a Micromass LCZ mass spectrometer.
Mobile Phase
100% H2O, 10 mM NH4Ac, adjusted to pH 4.6 w/ formic acid
Column
2.0 mm x 150 mm, YMC C18 AQ-3mm, 120 Å´ (available from Waters Corp.)
Flow rate
0.2 mL/min
Interface
Direct (no split)
Injection volume
5 mL
MS ionization mode
Electrospray, positive ion mode
MS detection mode
Selected ion monitoring: m/z 72 (AA), m/z 73 (13C-AA); dwell times: 0.5 s
Data Analysis
50
55
[0076] Response ratios (area of AA peak/area of 13C-AA peak) are plotted against the corresponding concentration
ratios for a series of six standards in ethyl acetate. All standards contain 4.50 mg/mL 13C-AA, and AA concentrations
ranging from 0 to 5.0 ppm. Linear regression results in a calibration curve from which concentration ratios in extracts
are determined from measured response ratios. When this concentration ratio is multiplied by the accurately known 13CAA level (nominally 2 ppm) added to sample in step three of the extraction procedure, the level of AA in ppm results.
[0077] Sample Calculation for LC/MS:
The calibration curve is generated by plotting the response ratio (area mz/72 / area m/z 73) on the y axis vs. the
11
EP 1 555 885 B1
concentration ratio ([acrylamide] / [13C-acrylamide]) on the x axis. For this example, the equation of that line is y =
0.899x + 0.0123.
Measured area of AA peak (m/z 72) at 4.0 min: 100,000
5
Measured area of 13C-AA peak (m/z 73) at 4.0 min: 500,000
10
Response ratio Rr = 0.200. From the slope and intercept of the calibration curve, the concentration ratio Rc is
calculated: Rc = (0.200 - 0.0123) / 0.899 = 0.209
Given the spike level of 13C-AA in the sample (2 ppm), the measured level of AA is 0.209 x 2 ppm = 0.418 ppm
Quality Assurance/Quality Control (QA/QC)
15
20
25
[0078]
1. All balances used in the preparation of standards and/or samples, must have their calibrations checked weekly
with a set of qualified weights. The balances should be checked with at least three weights covering the range of
sample/standard weights to be measured.
2. A six-point calibration curve should be performed daily.
3. A working reference material (WRM) should be analyzed with each set of samples. The concentration of this
material should be within 2 σ of the running mean. If it is not, the instrument should be recalibrated and the WRM
recalculated.
2. Asparagine
Determination of Asparagine and Aspartic Acid in Food and Beverage Products
PRINCIPLE
30
35
[0079] A weighed amount of sample is mixed with 5% HCl and heated for 30 minutes, then homogenized. A portion
of the homogenate is centrifuged and then a portion of the supernatant is diluted and treated with FMOC reagent (9fluorenylmethyl chloroformate), which reacts with asparagine and aspartic acid to form a highly fluorescent derivative.
Reverse-phase HPLC is then used to resolve FMOC-asparagine from other sample matrix components. Detection is by
fluorescence emission at 313 nanometers (nm) upon excitation at 260 nm. Analysis of standards of known concentration
permits quantification.
LINEARITY
40
[0080] Working calibration curve of four standards (50 - 600ppm) give a correlation of 0.998 or better. A curve taken
out to 2000ppm also gives a correlation of 0.998.
ACCURACY
45
Coffee samples:
[0081] A roast and ground coffee sample is spiked at four levels of both asparagine and aspartic acid (40, 200, 400,
and 600 ppm). Asparagine is recovered at 86% (Relative standard deviation of less than 4%) and aspartic acid is
recovered at 92% (Relative standard deviation of less than 4%).
50
REFERENCES
[0082]
55
1. Herbert, P.; Santos, L; Alves, A. Journal of Food Science (2001), 66(9), 1319-1325.
2. Heems, Dany; Luck, Geneviewe; Fraudeau, Chrisophe; Verette, Eric. Journal of Chromatography, A (1998), 798
(1 + 2), 9-17.
12
EP 1 555 885 B1
[0083] BELOW ARE SUGGESTED CHEMICALS AND EQUIPMENT; HOWEVER, SUBSTITUTIONS OF EQUIVALENT MATERIALS ARE ACCEPTABLE.
CHEMICALS
5
[0084]
Water, HPLC or Milli-Q™ Grade (Millipore)
Acetonitrile, HPLC Grade
Methanol, HPLC Grade
Ethyl Acetate
Pentane
Asparagine monohydrate
Aspartic acid
aminoisobutyric acid
9-Fluorenyl Chloroformate (FMOC)
Sodium Borate
Boric Acid
Sodium Bicarbonate
Tetramethyl Ammonium Chloride
Sodium Citrate anhydrous
Citric Acid
Acetone
Hydrochloric Acid, 0.1N
Calcium Chloride Dihydrate
10
15
20
25
30
35
40
Burdick & Jackson #AH015-4
Fisher #A452-4
Baker #9280-3
Burdick & Jackson #GC312-4
EM Science
Sigma #A-8949
Sigma #A-8379
ICN #150200
EM Science #SX 0355-1
Fisher #A-73
ICN #194847
Fisher #04640-500
MCB #SX445
Baker #0122-01
Burdick & Jackson #010-4
Fisher #SA48-500
Aldrich #22,350-6
EQUIPMENT
[0085] Transfer Pipettes, polyethylene (Samco #222)
[0086] Volumetric Flasks (25, 100, 250, 1000 ml)
[0087] Volumetric Pipet (10 ml)
[0088] Graduated Cylinders (100-1000ml)
[0089] HPLC reservoirs (500 ml, 1 or 2 liter)
[0090] Beakers
[0091] Magnetic stirrers/stir bars
[0092] Analytical (4-place) balance
[0093] Scintillation Vials
[0094] Centrifuge tubes, screw cap (100x16 mm) with caps
[0095] Autosampler vials (8x30 mm, 1 ml), with crimp caps
[0096] Safety: This method requires the use of a fume hood, and involves exposure to chemicals. Please review Safe
Practices for Fume Hood Use and Chemical Spills.
45
INSTRUMENT
MODEL
MANUFACTURER
50
Robot
Pump/HPLC injector
Detector
Data System
Microlab® SPE
HP 1100
RF10AXL
Chemstation
Hamilton
Agilent
Shimadzu
Agilent
Column
55
[0097]
Phenomenex Luna 100 x 4.6 mm C-18(2) 3 micron # 00D-4251-EO
13
EP 1 555 885 B1
PREPARATION OF REAGENTS
[0098]
5
10
1. Weigh 3.0 grams of Sodium Borate, 3.0 grams of Boric Acid, and 8.0 grams of Sodium Bicarbonate into a
dry tared beaker.
2. Place an empty 800 ml beaker on a magnetic stirrer. Add about 500 ml of Milli-Q™ water and a stir bar. Stir the
water vigorously without splashing.
3. Quantitatively transfer the reagents from step 1 to the water; stir until they are completely dissolved.
4. Quantitatively transfer the solution from step 3 to a 1-liter volumetric flask and dilute to volume with Milli-Q™
water; mix well. Stable for up to six (6) months.
[0099]
15
Diluent (pH 8.3-8.5; 1000ml).
Calcium Chloride Solution (100 grams).
1. Weigh 40 grams of Calcium Chloride Dihydrate into a tared 250 ml beaker.
2. Add 60 grams of Milli-Q™ water. Mix well. Store at ambient conditions in a capped glass bottle. Stable for up to
1 year.
Extraction Solvent (Pentane: Ethyl Acetate 80: 20; 500 ml)
20
[0100]
Safety: pentane and ethyl acetate are volatile and flammable. Perform the following operations in a Fume Hood.
1. Transfer 400 ml of pentane to a 500 ml HPLC reservoir bottle.
2. Add 100 ml ethyl acetate. Mix well. Store capped in/under the Fume Hood.
25
[0101]
30
35
Mobile Phase (Buffer:Methanol:Acetonitrile 60:5:35,pH 3.2, 2 L)
1. Weigh 1.35 grams of Tetramethyl Ammonium Chloride, 3.65 grams of Citric Acid, and 1.60 grams of Sodium
Citrate into a dry tared beaker.
2. Place an empty 800 ml beaker on a magnetic stirrer. Add about 500 ml of Milli-Q™ water and a stir bar. Stir the
water vigorously without splashing.
3. Quantitatively transfer the reagents from step 1 to the water; stir until they are completely dissolved.
4. Quantitatively transfer the solution from step 3 to a 1 liter graduated cylinder and dilute to 1000 ml with Milli-Q™
water; mix well.
5. Transfer to a 2-liter HPLC mobile phase reservoir.
6. Add 200 ml Milli-Q™ water, 100 ml methanol and 700 ml acetonitrile. Add the latter two solvents slowly with
vigorous stirring. Perform this operation in a hood, and wear personal protective equipment. Refer to the relevant
Material Safety Data Sheets (MSDS) for specific details.
7. Degas the mobile phase by vacuum aspiration while stirring.
40
FMOC Reagent Solution (in acetone)
[0102]
45
50
1. Weigh 0.10 grams of FMOC reagent into a tared 100 ml volumetric flask.
2. Add acetone to dissolve and dilute to volume with same. Mix well. Perform this operation in a hood. Wear PPE
specified in the MSDS for the chemicals.
3. Store refrigerated for no more than six (6) months.
Acid solution for sample extraction (5% HCl)
[0103]
55
1. Add 100ml of Milli-Q™ water into a 200ml volumetric.
2. Add 4ml of 1N HCl to volumetric.
[0104]
Bring to volume with Milli-Q ™ water.
14
EP 1 555 885 B1
PREPARATION OF INTERNAL STANDARD (AMINOISOBUTYRIC ACID)
ISTD A - Internal Standard StockA
5
[0105]
1. Weigh 0.5 grams of aminoisobutyric acid into a tared 250 ml volumetric
2. Add 25 ml of 1.0N HCl and about 100 ml Milli-Q™ water. Mix by swirling until dissolved. Dilute to volume with
Milli-Q™water and mix well. Store refrigerated for no more than six (6) months.
10
[0106]
ISTD B - Working Internal Standard Solution B (this solution is added to calibration
standards)
15
[0107]
1. Pipet 1 ml of Internal Standard Stock A into a 100 ml volumetric flask.
2. Dilute to volume with Milli-Q ™ water. Stable for one month.
20
25
PREPARATION OF CALIBRATION STANDARD(S)
[0108] Stock Calibration Solution.
[0109] Into a tared 50 ml volumetric, weigh 0.100 g (+/- 0.001 g) asparagine and 0.100 g (+/- 0.001 g) aspartic acid.
Add 25 mL Milli-Q™ water and 1 mL 1 N HCl. Place in sonic bath until dissolved, then bring to volume with Milli-Q™
H2O. Solution is good for 6 months refrigerated.
[0110] Working Standards.
[0111] Prepare the following working calibration standards:
30
35
[0112]
40
Std #
mL stock
final volume (mL)
ppm
1
5
200
50
2
5
100
100
3
1
10
200
4
3
10
600
Solutions are good for one month refrigerated.
PREPARATION OF SAMPLES
[0113]
45
50
1. Weigh 6 grams of ground coffee* into 125 ml Erlenmeyer flask.
2. Add 48.0ml of 5% HCl solution to each sample.
3. Add 2 ml ISTD A to each sample.
4. Cover each flask with aluminum foil and place in 60C water bath for 30 minutes.
5. Add 10 mL dicloroethane to each sample.
6. Homogenize sample for 60 seconds.
7. Pour portion of sample into 30ml centrifuge tube.
8. Centrifuge at 10000 rpm for 32 minutes at 5C. The supernatant is used in "Samples - Diluting" step 1.
* If coffee samples are not finely ground, grind in small food processor before weighing. If samples are high in moisture,
add several pieces of dry ice while grinding.
55
Preparation of Standards and Samples
[0114]
Three Microlab methods are run in order to dilute the samples/standards, add the internal standard, and form
15
EP 1 555 885 B1
the FMOC derivative. These are summarized below.
Operation
Dilution
Addition of Internal Standard
Formation of FMOC derivative
5
Microlab® method used
TRANSDIL
ADDISTD
ADDFMOC
PREPARATION OF SAMPLES AND STANDARDS USING MICROLAB® ROBOT
10
Step 1: Standards - Adding ISTD and Dilution Step
[0115]
15
20
25
1. Prepare two sets of tubes for each standard. Place approximately 2 mL of standard in one set of tubes, place
these filled tubes on the left most position of the Microlab®.
2. Place the rack with empty tubes in the rightmost rack position of the Microlab®.
3. Fill a 20 ml glass (scintillation) vial with Working Internal Standard Solution B and place on the Microlab®
workspace.
4. Select method ADDISTD. (Mixes 200 ul ISTD B, 50 ul standard solution, to 4000ul total volume with Milli-Q ™ water).
5. Execute the method.
6. Remove the tube set from the left position and set aside for discard.
7. Remove the Working Internal Standard Solution from the Microlab® work space and refrigerate.
[0116]
Set aside right side tubes for step 3.
Step 2: Samples - Dilution Step (ISTD was already added during sample preparation)
[0117]
30
35
1. Prepare two sets of tubes for each sample. Place approx. 2 mL of sample in one set of tubes, place these filled
tubes on the left most position of the Microlab®.
2. Place the rack with the empty tubes in the rightmost rack position of the Microlab®.
3. Select method TRANSDIL. (Set # of samples, 50ul for amount of sample, and 4000ul for final dilution amount
with Milli-Q™ water.)
4. Execute the method.
5. Remove the tube set from the left position and set aside for discard.
[0118]
Set aside right side tubes for step 3.
40
Step 3: Addition of FMOC Reagent - Making Fluorescent derivative
[0119]
45
50
55
1. Prepare a rack of 100x16 mm screw-cap tubes.
2. Place the rack in the rightmost rack position of the Microlab®.
3. Place standard and sample tubes from above dilution steps in leftmost rack position of Microlab®.
4. Transfer an aliquot (22 mL) of FMOC reagent solution to a glass scintillation vial. Add approximately 100 mL of
40% Calcium Chloride solution; mix well. (Calcium chloride is added to make the FMOC reagent "charged" - necessary
for detection by Microlab®).
5. Place the vial on the Microlab® workspace.
6. Select method ADDFMOC.
7. Switch syringes 1 & 2 from water to Diluent (pH 8.3-8.5).
8. Perform a wash of at least five (5) cycles for syringes 1 & 2 using Diluent (pH 8.3-8.5)
9. Execute method ADDFMOC. (mixes 450ul of FMOC solution, 250 ul sample from ADDISTD abo to final volume
of 1300 ul with diluent solution).
10. Remove the tube set from the SAMPLE rack position and set aside.
11. Remove the FMOC reagent solution from the Microlab® workspace and refrigerate.
16
EP 1 555 885 B1
5
10
13. Remove the tube set from the rightmost position and place in fume hood. Let stand for at least 10 minutes or
until solution is clarified (but no longer than 20 minutes).
14. Add 2 ml of Extraction Solvent to each tube. Cap and vortex at high speed for two (2) minutes to extract
unreacted FMOC reagent.
15. Prepare another tube set of 55x16 mm tubes. Add 1 ml of mobile phase solution to each tube.
16. Transfer the 1.0 mL of aqueous (lower) layer from the centrifuge tubes to the 55x16 mm tubes.
17. Discard the upper (organic) layer.
18. Transfer samples to autosampler vials and seal.
CHROMATOGRAPHY
Operating Conditions
HP 1100 with Chem Station software
15
20
[0120]
[0121]
[0122]
[0123]
Em 313
[0124]
[0125]
[0126]
[0127]
Detector: Waters 474 Scanning Fluorescence detector
Mode: Norm
Signal: 0.0000
Wavelength: Ex 260
Gain: 10
Atten: 1
Response: FST
Column: Phenomex Luna C18 (2) 100 x 4.6 mm 3 u
25
LC METHOD
30
[0128]
[0129]
[0130]
[0131]
Flow: 1.000 ml/min
Isocratic run (see preparation of reagents - Mobile Phase)
Injection volume: 10.0 ul
Temperature settings: not controlled
CALCULATIONS
35
[0132]
Sample solutions are calculated against a standard curve of known amounts using area counts:
[0133]
[0134]
ppm asparagine = (area aspargine/area ISTD - intercept)/slope
[ppm = ug/mL]
40
45
50
Example:
55
[0135]
17
EP 1 555 885 B1
[0136]
Correction for dilution/homogeniization in sample preparation step.
5
10
[ppm = ug/mL]
[0137]
Example:
15
20
RUN ACCEPTABILITY CRITERIA:
[0138]
25
•
•
the Check Sample of Working Reference Material accuracy must be within 10% of known result for asparagine.
the linearity of the calibration curve (r2) must be 0.995 or greater.
SAMPLE CHROMATOGRAM OF LC ANALYSIS
30
[0139]
Figure 3 sets forth a sample chromatogram of LC analysis.
RT
35
40
Compound
4.5 min
Asparagine
6.6 min
aspartic acid
11.5 min
FMOC reagent
20.7 min
ISTD
3. % Reduction of Acrylamide
[0140]
45
50
[0141] The control sample is prepared in the conventional manner as known in the art. Both the control and the enzymetreated sample are roasted in the same manner and to about the same Hunter L color.
4. % Reduction of Asparagine
[0142]
55
18
EP 1 555 885 B1
[0143] The control sample is prepared in the conventional manner as known in the art. If roasted, both the control and
the enzyme-treated sample are roasted in the same manner and to about the same Hunter L color.
EXAMPLES
5
[0144]
The following examples are illustrative of the present invention but are not meant to be limiting thereof.
Example 1- Enzyme Solution Absorption
10
15
20
25
[0145] An enzyme solution is formulated consisting of 1,000 units of Asparaginase and 0.1% (of coffee weight) of a
cellulase dissolved in 600 grams of distilled water. Twelve hundred grams of washed arabica green coffee beans from
Guatemala are placed in a 3 liter round bottom flask, which is then placed on a rotovap with a water bath set at 35°C.
A 25 inch vacuum is pulled on the rotating flask for 5 minutes, then enough enzyme solution is added to the flask to wet
the surface of the coffee beans. The vacuum is then released. The vacuum is applied to the system for about 10 seconds
and then released 2 more times. The flask of coffee beans is then left to rotate at atmospheric pressure until all of the
enzyme solution on the surface of the beans is absorbed. This process is repeated until 600 grams of enzyme solution
is added to the coffee.
[0146] The coffee is then transferred to a pressure vessel and 80 psi pressure is applied using nitrogen gas. The wet
coffee is kept under pressure for 2 hours, with samples being removed at 1 and 2 hours. The enzyme is deactivated by
microwave heating. The samples are then dried at 50-60°C and roasted to a roast color of about 17L. Optionally, the
samples are then ground to form roast and ground coffee.
Enzyme-Treated Samples
Asparagine (green)*
Acrylamide (roasted)
1 hour of pressure
384 ppm
346 ppb
2 hours of pressure
393 ppm
280 ppb
*Asparagine values are reported on a 0% moisture basis.
30
35
[0147] For calculation of the percent reduction of asparagine and acrylamide, the asparagine and acrylamide values
for coffee beans that have not been treated with asparaginase solution are measured. The asparagine value is 661 ppm
(on a 0% moisture basis), and the acrylamide value is 397 ppb. The percent reduction of both asparagine and acrylamide
are set forth in the table below.
Enzyme-Treated Samples
% Asparagine Reduction
% Acrylamide Reduction
1 hour of pressure
42
13
2 hours of pressure
40
29
40
Example 2 - Dominant Bath
45
50
55
[0148] Washed arabica green coffee beans (Sample #1, Green) are moistened to about 25% moisture using atmospheric pressure steam. The coffee beans are then soaked in water in a ratio of steamed coffee beans to water of about
1:4 with gentle agitation for about 30 minutes. The extracted coffee beans are removed and the extract is then used to
soak another batch of pre-moistened coffee beans. This process is repeated, with a coffee beans to extract ratio of
between about 1:4 and about 1:1, until the solids contained in the extract achieve a constant level.
[0149] This extract is used to soak a fresh batch of pre-moistened green coffee beans in a ratio of 270 g pre-moistened
coffee to 504 g of extract for 20 minutes with gentle agitation. The coffee beans are separated from the extract (this
extract is Sample #2) , dried (these dried beans are Sample #3 Green), and roasted (these dried and roasted beans are
Sample #3 Roasted). The roasted coffee beans are then optionally ground to form roast and ground coffee.
[0150] 300 units of asparaginase is added to 350 grams of extract and is allowed to sit for 1 hour to allow the asparaginase to convert the asparagine to aspartic acid (the enzyme-treated extract is Sample #4). The extract is then used
to soak another fresh batch of pre-moistened arabica green coffee beans for 1 hour in a ratio of coffee beans to extract
of about 0.8:1. The coffee beans are separated from the extract, the enzyme is de-activated, and the beans are dried
(these dried beans are Sample #5 Green) and roasted (these dried and roasted beans are Sample #5 Roasted). The
beans are roasted to a roast color of about 17L. The roasted coffee beans are then optionally ground to form roast and
ground coffee. An aliquot of Sample #1, Green, is roasted to form Sample #1, Roasted.
19
EP 1 555 885 B1
[0151] Analytical measurements of coffee beans that are soaked in the enzyme-treated extract show the following
levels of asparagine and acrylamide:
5
10
Sample
Asparagine (Green)*
Acrylamide (Roasted)
Sample #1, Control Coffee Beans
661 ppm
397 ppb
Sample #2, Extract Before Enzymatic Treatment
385 ppm
N/A**
Sample #3, Beans Soaked in Sample #2
436 ppm
399 ppb
Sample #4, Extract After Enzymatic Treatment
8 ppm
N/A**
Sample #5, Beans Soaked in Sample #4
240 ppm
291 ppb
*Asparagine values are reported on a 0% moisture basis.
** Not applicable
15
[0152] The percent reduction of both asparagine and acrylamide for coffee beans and extracts are set forth in the
table below.
20
Sample
% Asparagine Reduction
% Acrylamide Reduction
Sample #5 (compared versus Sample #1)
64%
27%
Sample #4 (compared versus Sample #2)
98%
N/A*
* Not applicable
25
Example 3 - Dominant Bath with Deactivated Enzyme
30
35
40
45
[0153] Washed arabica green coffee beans (Sample #1, Green) are moistened to about 25% moisture using atmospheric pressure steam. The coffee beans are then soaked in water in a ratio of steamed coffee beans to water of about
1:4 with gentle agitation for about 30 minutes. The extracted coffee beans are removed and the extract is then used to
soak another batch of pre-moistened coffee beans. This process is repeated, with a coffee beans to extract ratio of
between about 1:4 and about 1:1, until the solids contained in the extract achieve a constant level.
[0154] This extract is used to soak a fresh batch of pre-moistened green coffee beans in a ratio of 270 g pre-moistened
coffee to 504 g of extract for 20 minutes with gentle agitation. The coffee beans are separated from the extract (this
extract is Sample #2), dried (these dried beans are Sample #3 Green), and roasted. The roasted coffee beans are then
optionally ground to form roast and ground coffee.
[0155] 300 units of asparaginase is added to 350 grams of extract and is allowed to sit for 1 hour to allow the asparaginase to convert the asparagine to aspartic acid. The enzyme treated extract is then deactivated (the enzyme-treated,
deactivated extract is Sample #4). The extract is then used to soak more pre-moistened arabica green coffee beans for
1 hour in a ratio of coffee beans to extract of about 0.8:1. The coffee beans are separated from the extract. The beans
are dried (these dried beans are Sample #5 Green) and roasted. The roasted coffee beans are then optionally ground
to form roast and ground coffee.
[0156] Analytical measurements of coffee beans that are soaked in the enzyme-treated extract show the following
levels of asparagine:
50
Sample
Asparagine (Green)
Sample #1, Control Coffee Beans
661 ppm
Sample #2, Extract Before Enzymatic Treatment
385 ppm
Sample #3, Beans Soaked in Sample #2
436 ppm
Sample #4, Extract After Enzymatic Treatment (deactivated)
7 ppm
Sample #5, Beans Soaked in Sample #4
320 ppm
55
[0157]
The percent reduction of asparagine for coffee beans and extracts are set forth in the table below.
20
EP 1 555 885 B1
5
Sample
% Asparagine Reduction
Sample #5 (compared versus Sample #1)
52
Sample #4 (compared versus Sample #2)
98
[0158] The level of acrylamide in the enzyme-treated roasted coffee beans, the roast and ground coffee, and coffee
brews prepared therefrom is reduced by at least about 10% compared to conventionally processed products.
10
15
20
25
30
35
Example 4 - Dominant Bath with Immobilized Enzyme
[0159] Washed arabica green coffee beans (Sample #1, Green) are moistened to about 25% moisture using atmospheric pressure steam. The coffee beans are then soaked in water in a ratio of steamed coffee beans to water of about
1:4 with gentle agitation for about 30 minutes. The extracted coffee beans are removed and the extract is then used to
soak another batch of pre-moistened coffee beans. This process is repeated, with a coffee beans to extract ratio of
between about 1:4 and about 1:1, until the solids contained in the extract achieve a constant level.
[0160] This extract is used to soak a fresh batch of pre-moistened green coffee beans in a ratio of 270 g pre-moistened
coffee to 504 g of extract for 20 minutes with gentle agitation. The coffee beans are separated from the extract (this
extract is Sample #2) , dried (these dried beans are Sample #3 Green), and roasted (these dried and roasted beans are
Sample #3 Roasted). The roasted coffee beans are then optionally ground to form roast and ground coffee.
[0161] The extract is contacted with immobilized asparaginase (this enzyme-treated extract is Sample #4). The extract
is then used to soak more pre-moistened arabica green coffee beans for 1 hour in a ratio of coffee beans to extract of
about 0.8:1. The coffee beans are separated from the extract, then the beans are dried (these dried beans are Sample
#5 Green) and roasted (these dried and roasted beans are Sample #5 Roasted). The roasted coffee beans are then
optionally ground to form roast and ground coffee. An aliquot of Sample #1, Green, is roasted to form Sample #1,
Roasted. The level of acrylamide in the enzyme-treated roasted coffee beans, the roast and ground coffee, and coffee
brews prepared therefrom is reduced by at least about 10% compared to conventionally processed products.
Example 5 - Decaffeinated Roast and Ground Coffee
[0162] The dried enzyme-treated (2 hour pressure) green arabica coffee beans from Example 1 are taken prior to
roasting and subjected to a decaffeination process. The decaffeination process as outlined in U.S. Patent No. 4,474,821
to Morrison, Jr. et al. is followed to decaffeinate the beans to a point where 97% of the original caffeine level in the beans
has been extracted. The beans are then roasted and ground to produce a decaffeinated roast and ground coffee having
at least about 10% acrylamide reduction compared to conventionally processed products.
Example 6 - Decaffeinated Roast and Ground Coffee
40
45
50
[0163] Green arabica coffee beans of Example 2, Sample #5, are taken prior to roasting and subjected to a decaffeination process. The decaffeination process as outlined in U.S. Patent No. 4,474,821 to Morrison, Jr. et al. is followed
to decaffeinate the beans to a point where 97% of the original caffeine level in the beans has been extracted. The beans
are then roasted and ground to produce a decaffeinated roast and ground coffee having at least about 10% acrylamide
reduction compared to conventionally processed products.
Example 7 - Decaffeinated Roast and Ground Coffee
[0164] Green arabica coffee beans are decaffeinated according to the decaffeination process of U.S. Patent No.
4,474,821 to Morrison, Jr. et al., to a point where 97% of the original caffeine level in the beans has been extracted. The
decaffeinated beans are then subjected to the process outlined in Example 1 above. The resulting product is a decaffeinated roast and ground coffee having at least about 10% acrylamide reduction compared to conventionally processed
products.
Example 8 - Decaffeinated Roast and Ground Coffee
55
[0165] Green arabica coffee beans are decaffeinated according to the decaffeination process of U.S. Patent No.
4,474,821 to Morrison, Jr. et al., to a point where 97% of the original caffeine level in the beans has been extracted. The
decaffeinated beans are then subjected to the process outlined in Example 2 above. The resulting product is a decaf-
21
EP 1 555 885 B1
feinated roast and ground coffee having at least about 10% acrylamide reduction compared to conventionally processed
products.
Example 9 - Decaffeinated Roast and Ground Coffee
5
10
[0166] Coffee beans are decaffeinated by a process wherein caffeine-containing green coffee beans are concurrently
extracted with a water solution of coffee solubles as is known in the art (such as that set forth in U.S. Patent No. 3,989,850
to Erb, et al.), except that the water solution also comprises asparaginase in solution as in Example 2 above. The
decaffeinated coffee removed from the extraction zone is treated to remove surface solids contained thereon and is then
dried then roasted to form a decaffeinated roast and ground coffee. The level of acrylamide in the enzyme-treated roasted
coffee beans, the roast and ground coffee, and coffee brews prepared therefrom is reduced by at least about 10%
compared to conventionally processed products.
Example 10 - Instant Coffee Product
15
20
[0167] The acrylamide-reduced roast and ground coffee produced from any of Examples 1-9 above is subjected to
countercurrent extraction with heated water in a series of extraction columns at temperatures of 177°C (350°F) and
pressures of 8 bar (120 psi) to produce a concentrated coffee extract of 20% solids. The extract is stripped of its aromatic
components, and then condensed to a concentration of 60%. The aromatics are condensed and then recombined with
the concentrated extract and the entire stream fed to a spray dryer where the liquid is sprayed into a countercurrent flow
of heated air at 121°C (250°F). The final dried product is a soluble or instant coffee form having at least about 10% less
acrylamide than conventionally processed products.
Example 11- Ready to Serve Sweetened Cappuccino Product
25
[0168] The acrylamide-reduced roast and ground coffee produced from any of Examples 1-9 above is subjected to
an aqueous extraction to produce a coffee concentrate containing 7.5% solids. This concentrate is then used in the
following formulation (on a weight basis) to make a ready-to-drink coffee product.
30
35
Water
2% low-fat milk
Fructose
Vanilla powder
Coffee concentrate of this invention
50%
35%
7%
2%
6%
[0169] These ingredients are mixed uniformly and subjected to ultra-high temperature (UHT) process conditions to
sterilize the product, and then aseptically filled into individual package containers. This ready-to-drink coffee beverage
has at least about 10% less acrylamide compared to conventionally processed products.
40
Example 12 - Instant Coffee Beverage
[0170] The instant coffee produced in Example 10 is used in the following formulation (on a weight basis) to make an
instant creamy coffee powder used to make a café latte type beverage.
45
Instant Coffee of Example 10
Foaming Creamer*
Sucrose
Flavors
50
55
16%
50%
33.5%
0.5%
(*The foaming creamer is 68% skim milk,
30% coconut oil, 1% silicon dioxide flow
agent and 1% sodium dihydrogen
orthophosphate for stabilization of the
protein structure during dissolution.)
[0171] The composition is prepared by weighing each of the particulate dry ingredients into a mixer and dry mixing in
a paddle mixer until uniform. The resulting product is a dry instant mix that can be used to prepare a café latte upon
22
EP 1 555 885 B1
addition of hot water. The dry instant mix and beverages prepared therefrom have at least about 10% less acrylamide
than conventionally processed products.
Example 13 - Liquid Coffee Concentrate
5
10
[0172] The acrylamide-reduced roast and ground coffee produced from any of Examples 1-9 above is subjected to
an aqueous extraction with heated water in a batch extraction vessel at a temperature of 82°C (180°F) and a pressure
of 6 bar (90 psi) to produce a concentrated coffee extract of 4.5% solids. This extract is then frozen and reconstituted
with the addition of hot water to make a finished coffee beverage with 0.7% solids. The extract and the finished coffee
beverage prepared therefrom have at least about 10% less acrylamide than conventionally processed products.
Example 14 - Article of Commerce
15
[0173] The acrylamide-reduced roast and ground coffees produced from any of Examples 1-9 above are packaged
in cans for sale to consumers. Printed on the cans is a message stating, "Acrylamide-free product!"
Example 15 - Article of Commerce
20
[0174] Green coffee beans treated with enzyme according to any of Examples 1-9 above are packaged in drums. The
drums are labeled, "Low in Asparagine."
Example 16 - Article of Commerce
25
[0175] The ready to serve sweetened cappuccino product of Example 11 is packaged in a bottle for sale to consumers.
A label on the bottle states, "Acrylamide reduced by over 90%!" A television commercial for the product communicates
the message, "Acrylamide-reduced product."
Example 17 - Article of Commerce
30
[0176] Brewed coffee made from roast and ground coffee of any of Examples 1-9 above is sold to consumers served
in a cup. A sign posted inside the retail establishment where the brewed coffee is sold communicates the message, "We
serve only acrylamide-free coffee."
35
Claims
1.
A method for reducing the level of acrylamide in roasted coffee beans, said method comprising the steps of:
(1) forming a dominant bath, this step comprising:
40
a) soaking coffee beans in excess water to create a dominant bath,
b) separating the coffee beans from the water,
c) repeating several times steps a) and b) to form a dominant bath wherein an equilibrium is established
between the water soluble components that are in the coffee beans and in the bath;
45
(2) adding an asparagine-reducing enzyme to the dominant bath, said enzyme converting selectively the asparagine to aspartic acid;
(3) adding coffee beans containing asparagine to the dominant bath of step (2), said asparagine extracting out
of the beans into the dominant bath and said enzyme converting the asparagine to aspartic acid;
(4) removing the coffee beans from the dominant bath;
(5) optionally processing additional batches of coffee beans containing asparagine in a continuous or semicontinuous fashion by repeating steps (3) and (4);
(6) optionally deactivating or optionally removing the enzyme; and
(7) roasting the coffee beans to form roasted coffee beans.
50
55
2.
The method of claim 1, wherein said asparagine-reducing enzyme is asparaginase.
3.
The method of claim 1 or 2, wherein said asparagine-reducing enzyme is an enzyme capable of hydrolyzing the
23
EP 1 555 885 B1
amide group of free asparagine.
4.
5
The method of any of claims 1-3, wherein the level of asparagine in said coffee beans is reduced from 10% to 100%,
preferably from 30% to 100%, more preferably from 50% to 100%, even more preferably from 70% to 100%, and
most preferably from 90% to 100%.
Patentansprüche
10
1.
Ein Verfahren zur Reduzierung des Acrylamidgehalts in gerösteten Kaffeebohnen, wobei das Verfahren die Schritte
umfasst:
(1) Bilden eines dominierenden Bades, wobei dieser Schritt umfasst:
a) Einweichen von Kaffeebohnen in einem Überschuss an Wasser, um ein dominierendes Bad herzustellen,
b) Abtrennen der Kaffeebohnen vom Wasser,
c) mehrmaliges Wiederholen der Schritte a) und b), um ein dominierendes Bad zu bilden, wobei zwischen
den wasserlöslichen Komponenten, die sich in den Kaffeebohnen und im Bad befinden, ein Gleichgewicht
hergestellt wird;
15
20
(2) Zugeben eines Asparagin-reduzierenden Enzyms zum dominierenden Bad, wobei das Enzym das Asparagin
selektiv in Asparaginsäure umwandelt;
(3) Zugeben von Asparagin enthaltenden Kaffeebohnen zum dominierenden Bad von Schritt (2), wobei das
Asparagin aus den Bohnen in das dominierende Bad extrahiert wird und wobei das Enzym das Asparagin in
Asparaginsäure umwandelt;
(4) Entfernen der Kaffeebohnen aus dem dominierenden Bad;
(5) gegebenenfalls Verarbeiten weiterer Chargen an Asparagin enthaltenden Kaffeebohnen in einer kontinuierlichen oder halbkontinuierlichen Weise durch Wiederholen der Schritte (3) und (4);
(6) gegebenenfalls Deaktivieren oder gegebenenfalls Entfernen des Enzyms; und
(7) Rösten der Kaffeebohnen, um geröstete Kaffeebohnen zu bilden.
25
30
2.
Das Verfahren nach Anspruch 1, wobei das Asparagin-reduzierende Enzym Asparaginase ist.
3.
Das Verfahren nach Anspruch 1 oder 2, wobei das Asparagin-reduzierende Enzym ein Enzym ist, das in der Lage
ist, den Amidrest von freiem Asparagin zu hydrolysieren.
4.
Das Verfahren nach einem der Ansprüche 1 - 3, wobei der Asparagingehalt in den Kaffeebohnen um 10% bis 100%,
vorzugsweise um 30% bis 100%, stärker bevorzugt um 50% bis 100%, noch stärker bevorzugt um 70% bis 100%
und am meisten bevorzugt um 90% bis 100% reduziert wird.
35
40
Revendications
1.
45
Procédé pour réduire le taux d’acrylamide dans des grains de café torréfiés, ledit procédé comprenant les étapes
consistant à :
(1) former un bain dominant, cette étape comprenant :
50
55
a) la trempe de grains de café dans de l’eau en excès pour créer un bain dominant,
b) la séparation des grains de café de l’eau,
c) la répétition plusieurs fois des étapes a) et b) pour former un bain dominant dans lequel un équilibre est
établi entre les composants solubles dans l’eau qui sont dans les grains de café et dans le bain ;
(2) ajouter une enzyme réduisant l’asparagine dans le bain dominant, ladite enzyme transformant sélectivement
l’asparagine en acide aspartique ;
(3) ajouter des grains de café contenant de l’asparagine au bain dominant de l’étape (2), ladite asparagine étant
extraite des grains dans le bain dominant et ladite enzyme transformant l’asparagine en acide aspartique ;
(4) retirer les grains de café du bain dominant ;
24
EP 1 555 885 B1
(5) éventuellement traiter des lots supplémentaires de grains de café contenant de l’asparagine d’une manière
continue ou semi-continue en répétant les étapes (3) et (4) ;
(6) éventuellement désactiver ou éventuellement retirer l’ enzyme ; et
(7) torréfier les grains de café pour obtenir des grains de café torréfiés.
5
2.
Procédé selon la revendication 1, dans lequel ladite enzyme réduisant l’asparagine est l’asparaginase.
3.
Procédé selon la revendication 1 ou 2, dans lequel ladite enzyme réduisant l’asparagine est une enzyme capable
d’hydrolyser le groupe amide de l’asparagine libre.
4.
Procédé selon l’une quelconque des revendications 1 à 3, dans lequel le taux d’asparagine dans lesdits grains de
café est réduit de 10 % à 100 %, de manière préférée de 30 à 100 %, de manière davantage préférée de 50 % à
100 %, de manière encore davantage préférée de 70 % à 100 %, et de manière préférée entre toutes de 90 % à 100 %.
10
15
20
25
30
35
40
45
50
55
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REFERENCES CITED IN THE DESCRIPTION
This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European
patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be
excluded and the EPO disclaims all liability in this regard.
Patent documents cited in the description
•
US 4474821 A, Morrison, Jr. [0162] [0163] [0164]
[0165]
•
US 3989850 A, Erb [0166]
•
R. S. HUNTER. Photoelectric Color Difference Meter.
J. of the Optimal Soc. of Amer., 1958, vol. 48, 985-95
[0053]
HERBERT, P. ; SANTOS, L; ; ALVES, A. Journal of
Food Science, 2001, vol. 66 (9), 1319-1325 [0082]
HEEMS, DANY ; LUCK, GENEVIEWE ; FRAUDEAU, CHRISOPHE ; VERETTE, ERIC. Journal of
Chromatography, A, 1998, vol. 798 (1 + 2), 9-17
[0082]
Non-patent literature cited in the description
•
•
•
•
•
Mechanism(s) of formation of acrylamaide in food:
background,
www.jitsan.umd.edu/Acrylamide/WG1/WG1 Mech BG Paper. pdf [0003]
MOTTRAM D.S. et al. Nature, 03 October 2002, vol.
419, 448-449 [0004]
STADLER R.H. et al. Nature, 03 October 2002, vol.
419, 449-450 [0004]
Food Chemistry. Marcel Dekker, Inc, 1985, 427, 433
[0021]
SIVETZ ; FOOTE. Coffee Processing Technology.
Avi Publishing Co, 1963, vol. 1, 203-226 [0052]
•
•
29