Method for producing roasted coffee beans

Abstract

The present invention relates to a method for producing roasted coffee beans comprising treating raw coffee beans with steam followed by contacting with asparaginase.

Description

[0001] This application is a divisional application of an invention patent application with an application date of March 20, 2014, application number 201480016836.9, and invention name “Method for producing roasted coffee beans”.

[0002] Reference to a sequence listing

[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference. Field of the Invention

[0004] The present invention relates to a method for producing roasted coffee beans, the method comprising contacting raw coffee beans with asparaginase. Background of the Invention

[0006] It is well known that the formation of acrylamide in heated food products can be reduced by treatments which reduce the amount of asparagine in the food material, for example by subjecting the food material to the action of asparaginase (see for example, WO 2004 / 026042).

[0007] In uncooked roasted coffee grounds, acrylamide has been measured at concentrations between 45 and 374 ng / g (Andrzejewski D. et al., "Ahalysis of Coffee for the Presence of Acrylamide by LC-MS / MS", Journal of Agricultural and Food Chemistry, Vol. 52, No. 7, 2004, pp. 1996-2002).

[0008] Asparaginase treatment of coffee beans has been disclosed to reduce the acrylamide content in roasted coffee (WO 2004 / 037007; WO 2008 / 151807; WO 2013 / 005145).

[0009] It has been disclosed to improve the quality of coffee beans by treating raw coffee beans with steam at a temperature of 135°C-140°C (US 5,019,413). SUMMARY OF THE INVENTION

[0011] The inventors have found that the formation of acrylamide in asparaginase treated roasted coffee beans, in particular Robusta coffee beans, can be further reduced by subjecting the coffee beans to a steam treatment prior to the asparaginase treatment.

[0012] Accordingly, the present invention provides a method for producing roasted coffee beans, the method comprising:

[0013] (a) treating raw Robusta coffee beans with steam;

[0014] (b) contacting the steam-treated raw coffee beans with asparaginase; and

[0015] (c) roasting the raw coffee beans treated with asparaginase.

[0016] Specifically, treating raw coffee beans with steam at a temperature of about 100°C-115°C (e.g., about 100°C or 110°C), followed by asparaginase treatment and roasting, results in lower levels of acrylamide in the roasted coffee beans compared to coffee beans not so treated.

[0017] In a preferred embodiment, raw Robusta coffee beans are treated with steam at a temperature of about 100°C-120°C, preferably about 105°C-115°C.

[0018] The present invention also provides a method for producing roasted coffee beans, the method comprising:

[0019] (a) treating raw coffee beans with steam at a temperature of about 105° C. to 115° C.;

[0020] (b) contacting the steam-treated raw coffee beans with asparaginase; and

[0021] (c) roasting the raw coffee beans treated with asparaginase.

[0022] In a preferred embodiment, the coffee beans are Robusta coffee beans.

[0023] Detailed description of the invention

[0024] There are generally considered to be two main commercial varieties of coffee: Arabica beans (from the plant Coffea arabica) and Robusta beans (from the plant Coffea canephora).

[0025] The present invention provides in a first aspect a method for producing roasted coffee beans, the method comprising:

[0026] (a) treating raw Robusta coffee beans with steam;

[0027] (b) contacting the steam-treated raw coffee beans with asparaginase; and

[0028] (c) roasting the raw coffee beans treated with asparaginase.

[0029] Preferably, in the context of the present invention, Robusta coffee beans are coffee beans from the plant Coffea canephora.

[0030] In a preferred embodiment of the first aspect, the raw Robusta coffee beans are treated with steam at a temperature of about 100°C-120°C, preferably about 105°C-115°C.

[0031] In a second aspect, the present invention provides a method for producing roasted coffee beans, the method comprising:

[0032] (a) treating raw coffee beans with steam at a temperature of about 105° C. to 115° C.;

[0033] (b) contacting the steam-treated raw coffee beans with asparaginase; and

[0034] (c) roasting the raw coffee beans treated with asparaginase.

[0035] In a preferred embodiment of the second aspect, the coffee beans are Robusta coffee beans.

[0036] With respect to the two aspects of the present invention, the method will be described in further detail below.

[0037] In a preferred embodiment, the method is for producing roasted coffee beans having reduced acrylamide levels compared to roasted coffee beans that have not been steam treated and subsequently contacted with asparaginase.

[0038] Preferably, the acrylamide content of these roasted coffee beans is reduced by at least 25%, more preferably at least 40%, even more preferably at least 50% compared to the acrylamide content of roasted coffee beans that have not been steam treated subsequently with asparaginase.

[0039] In another preferred embodiment, the method is for producing roasted coffee beans having reduced acrylamide levels compared to roasted coffee beans obtained in the same manner except that the roasted coffee beans were not contacted with asparaginase.

[0040] Preferably, the acrylamide content of the roasted coffee beans is reduced by at least 10%, more preferably by at least 25%, such as at least 30%, at least 35%, at least 40%, at least 45% or at least 50% compared to the acrylamide content of roasted coffee beans obtained in the same way (except that the roasted coffee beans were not contacted with asparaginase).

[0041] In another preferred embodiment, the method is for producing roasted coffee beans having reduced acrylamide levels compared to roasted coffee beans obtained in the same way except that the roasted coffee beans were not subjected to steam treatment.

[0042] Preferably, the acrylamide content of these roasted coffee beans is reduced by at least 10%, more preferably by at least 25%, for example at least 30%, at least 35%, at least 40%, at least 45% or at least 50% compared to the acrylamide content of roasted coffee beans obtained in the same way (except that the roasted coffee beans were not subjected to steam treatment).

[0043] The coffee beans to be used in the method of the present invention are raw coffee beans. Raw coffee beans can also be called green coffee beans (greencoffee beans) or unroasted coffee beans.

[0044] In the method of the present invention, raw coffee beans are subjected to steam treatment. Preferably, raw coffee beans are treated with saturated steam.

[0045] In a preferred embodiment, prior to or during step (a), the moisture content of the coffee beans is increased to a level ranging from about 30% w / w to about 45% w / w, such as about 35% w / w.

[0046] Preferably, the moisture content of the coffee beans is increased by steam treatment, ie during step (a).

[0047] Alternatively, the moisture content of the coffee beans may be increased by pre-soaking the coffee beans in water, for example at a temperature of about 30°C-80°C, for example about 60°C.

[0048] In a preferred embodiment, the steam treatment is carried out for about 5-60 minutes, preferably for about 10-30 minutes, for example for about 15 minutes. Preferably, the steam treatment is carried out for about 5-60 minutes after the desired temperature has been obtained, more preferably for about 10-30 minutes, for example for about 15 minutes after the desired temperature has been obtained.

[0049] The steam-treated raw coffee beans are contacted with the asparaginase for an appropriate time interval to allow the asparaginase to exert its effect.

[0050] The time interval for contacting with asparaginase depends on various factors. The amount of time required for enzyme and asparagine reaction will depend on factors including, but not limited to, the desired level of asparagine (and therefore acrylamide) reduction, the characteristics of specific coffee beans (e.g., the type of beans, composition, particle size), the level of residue or remaining pulp, production technology, asparagine concentration, and the specific asparaginase added. The technician will be able to easily determine the contact time. Preferably, the amount of time allowed for enzyme reaction is sufficient to produce coffee beans, wherein the level of asparagine is reduced by at least about 10%, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40%, and even more preferably at least about 50%. Generally speaking, the longer the time allowed for enzyme reaction, the higher the level of asparagine reduction in the roasted coffee beans, and therefore the higher the level of acrylamide reduction.

[0051] In a preferred embodiment, the contacting in step (b) lasts for about 10-240 minutes, preferably for about 30-180 minutes, and more preferably for about 45-180 minutes.

[0052] The step of allowing sufficient time for the enzyme to react may be performed in any suitable manner; for example, asparaginase may be added at one temperature followed by gradually increasing or decreasing the temperature.

[0053] Contacting with asparaginase constitutes asparaginase treatment.

[0054] As known in the art, pH and temperature are factors that affect enzyme activity. Those skilled in the art should be able to easily determine the optimum conditions for these and other parameters (e.g., water content). In addition, optimum pH and temperature conditions for specific enzymes typically can be obtained in the literature and / or from enzyme suppliers.

[0055] In case the asparaginase used is active, the contacting with the asparaginase is carried out at a temperature. The skilled person will be able to easily determine the temperature for contacting with the asparaginase.

[0056] In a preferred embodiment, the contacting in step (b) is carried out at a temperature of about 25°C-75°C, preferably about 50°C-70°C, for example about 60°C.

[0057] In another embodiment, a thermostable asparaginase is used and the contacting in step (b) is performed at a higher temperature, for example, at a temperature of about 75°C-100°C.

[0058] A thermostable enzyme in the context of the present invention may be defined as an asparaginase having a residual activity of at least 75% after incubation at 70°C for 10 minutes.

[0059] The coffee beans may be contacted with the asparaginase by any method known in the art, such as by spraying, soaking, showering, or a main bath.

[0060] In one embodiment, coffee beans are contacted with asparaginase by spraying a solution of asparaginase onto the beans while gently agitating the beans to provide even application to all surfaces of the beans.

[0061] In another embodiment, the coffee beans are contacted with asparaginase by soaking the beans in a solution of asparaginase. Slight agitation may be applied during contacting.

[0062] The skilled person will be able to determine the concentration of asparaginase to add to the coffee beans. The coffee beans may be contacted with asparaginase at a concentration of about 100-30,000 ASNU / kg coffee beans, preferably about 500-20,000 ASNU / kg coffee beans, for example about 3,000 ASNU / kg coffee beans.

[0063] After contacting, the asparaginase may be inactivated by any method known in the art. The enzyme may be inactivated by heating, so the optional inactivation step and roasting of the coffee beans may be performed simultaneously. Heat treatment may denature and inactivate the enzyme, such that the roasted coffee beans are not subjected to continued enzyme activity.

[0064] After step (b) and before step (c), the raw coffee beans may be dried. The raw coffee beans may be dried to a moisture content of about 5%-15% w / w, preferably 7%-12% w / w. Suitable methods of drying may include freeze drying, belt drying, vacuum drying, oven drying, fluidized bed drying, and combinations thereof.

[0065] After asparaginase treatment and optional drying, the coffee beans are roasted to form roasted coffee beans. Any suitable process including roasting can be used. As used herein, the term "roasting" includes any suitable heat treatment of coffee beans to produce a coffee-indicative flavor. Suitable roasting techniques may include, but are not limited to, oven roasting, extrusion roasting, steam roasting (e.g., without post-roasting), infrared roasting, microwave roasting, dielectric / induction heating roasting, and combinations thereof.

[0066] Coffee beans can be roasted to any desired roast color. This roast color can be monitored by different equipment, HunterLab colorimeter or color testing

[0067] The coffee beans may optionally be ground. Grinding may be performed at any stage, for example before steaming or before or after roasting. Preferably, milling is performed after roasting.

[0068] The roasted coffee beans can be used as is or can be used to make a variety of roasted coffee products, such as roasted and ground coffee, liquid concentrates, instant or powdered coffee, coffee beverages (e.g., hot and cold ready-to-drink coffee, vending coffee, commercial and home brewed coffee, Kahlua™, latte, cappuccino), blended coffee (e.g., latte blended coffee), confectionery (e.g., candy), desserts (e.g., cakes, ice cream, mousse, custard), pastries (e.g., Danish, donuts), sauces, and soups (e.g., chili). In one embodiment, the coffee beans are dried, roasted, and then ground to form roasted and ground coffee.

[0069] Coffee beans produced according to the method of the invention may be blended with other coffee beans. For example, Robusta coffee beans produced according to the invention may be blended with Arabica coffee beans, which may or may not be treated with asparaginase. Such blending may be performed at any stage, for example, before or after optional drying, before or after roasting, and / or before or after optional grinding.

[0070] In another aspect, the present invention provides roasted coffee beans obtainable by the method of the present invention as described above.

[0071] Asparaginase

[0072] Asparaginase in the context of the present invention means an enzyme with asparaginase activity, ie an enzyme which catalyzes the hydrolysis of asparagine to aspartic acid (EC 3.5.1.1).

[0073] For example, the asparaginase activity can be determined according to the asparaginase activity (ASNU) assay described in the examples. In one embodiment, the asparaginase to be used in the method of the invention has at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the asparaginase activity of the mature polypeptide of SEQ ID NO: 1 when measured at pH 7 and 37°C. The asparaginase activity per microgram of asparaginase can be determined.

[0074] Asparaginase can be obtained from any source, for example, from microorganisms, from plants or from animals.

[0075] Asparaginase may be obtained from microorganisms of any genus, for example from bacteria, archaea or fungi. For the purposes of the present invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the asparaginase encoded by the polynucleotide is produced by the source or by a strain into which the polynucleotide from the source has been inserted.

[0076] It can be a wild-type asparaginase, i.e. an asparaginase found in nature; or it can be an asparaginase variant, i.e. an asparaginase comprising an alteration, i.e. a substitution, insertion, and / or deletion at one or more (e.g., several) positions compared to a parent asparaginase from which it is derived. Substitution means that the amino acid occupying a position is replaced by a different amino acid; deletion means the removal of an amino acid occupying a position; and insertion means the addition of an amino acid adjacent to and immediately following the amino acid occupying a position.

[0077] The asparaginase or its parent (preferably the asparaginase) can be a bacterial asparaginase. For example, the asparaginase can be a Gram-positive bacterial asparaginase, such as Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces. or a Gram-negative bacterial asparaginase, such as Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma.

[0078] In one embodiment, the asparaginase is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis asparaginase.

[0079] In another embodiment, the asparaginase is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus asparaginase.

[0080] In another embodiment, the asparaginase is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans asparaginase.

[0081] In a preferred embodiment, the asparaginase or its parent (preferably the asparaginase) is a fungal asparaginase. For example, the asparaginase can be a yeast asparaginase, such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia asparaginase;or asparaginases from filamentous fungi, such as Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, C. optotermes), Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, M. agnaporthe), Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudoplectania udotrichonympha), Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella or Xylaria. ;

[0082] In one embodiment, the asparaginase is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis asparaginase.

[0083] In another embodiment, the asparaginase is Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium stoloniferum ... merdarium), Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum), Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusariumtrichothecioides, Fusariumvenenatum), Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspore microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0084] It will be understood that for the above-mentioned species, the present invention encompasses both perfect and imperfect states, as well as other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. One of ordinary skill in the art will readily recognize the identity of appropriate equivalents.

[0085] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), the Netherlands Type Culture Collection (Centraalbureau Voor Schimmelcultures, CBS), and the Northern Regional Research Center (NRRL) of the Agricultural Research Service Type Collection (ARS).

[0086] Can be from other sources, comprise from nature (for example, soil, compost, water etc.) separate microorganism or directly from natural material (for example, soil, compost, water etc.) obtain DNA sample identification and obtain this asparaginase.Techniques for directly separating microorganism and DNA from natural living environment are well known in the art.Then can be by similarly screening the genomic DNA of another microorganism or cDNA library or mixed DNA sample to obtain the polynucleotide encoding this asparaginase.Once the polynucleotide encoding asparaginase has been detected, can be by utilizing the technology known to those of ordinary skill in the art to separate or clone this polynucleotide (for example, see, Sambrook et al., 1989, Molecular Cloning, Laboratory Manual (A Laboratory Manual), 2nd edition, Cold Spring Harbor (Cold Spring Harbor), New York).

[0087] In a preferred embodiment, the asparaginase or a parent thereof (preferably the asparaginase) is obtainable from the genus Aspergillus, for example, from Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae or Aspergillus terreus.

[0088] In another preferred embodiment, the asparaginase or its parent (preferably the asparaginase) can be obtained from Aspergillus oryzae, for example, the asparaginase or its mature polypeptide of SEQ ID NO: 1. In another preferred embodiment, the asparaginase or its parent (preferably the asparaginase) can be obtained from Aspergillus niger, for example, the asparaginase or its mature polypeptide of SEQ ID NO: 2.

[0089] In another preferred embodiment, the asparaginase has at least 50% sequence identity to the mature polypeptide of any one of SEQ ID NO: 1 or 2, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the mature polypeptide of any one of SEQ ID NO: 1 or 2.

[0090] SEQ ID NO: 1 is the amino acid sequence of asparaginase from Aspergillus oryzae.

[0091] In the context of the present invention, the term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0092] Based on N-terminal sequencing and mass spectrometry (MS) analysis, it appears that the N-terminal processing of the asparaginase of SEQ ID NO: 1 is quite diverse. In one embodiment, based on SignalP (Nielsen et al., 1997, Protein Engineering 10: 1-6), which predicts that amino acids 1 to 19 of SEQ ID NO: 1 are a signal peptide, the mature polypeptide is amino acids 20 to 378 of SEQ ID NO: 1.

[0093] SEQ ID NO: 2 is the amino acid sequence of asparaginase from Aspergillus niger. In one embodiment, the mature polypeptide of SEQ ID NO: 2 is amino acids 18 to 378 of SEQ ID NO: 2 based on the predicted signal peptide being amino acids 1-17.

[0094] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the "longest identity" labeled by Needle (obtained using the -non-simplified option) is used as the percentage identity and is calculated as follows:

[0095] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0096] In another preferred embodiment, the asparaginase is a variant of a parent asparaginase, which has at least 50% sequence identity to the mature polypeptide of any one of SEQ ID NO: 1 or 2, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the mature polypeptide of any one of SEQ ID NO: 1 or 2.

[0097] In a more preferred embodiment, the asparaginase is a variant of a parent asparaginase, which has at least 50% sequence identity to the mature polypeptide of SEQ ID NO: 1, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 1.

[0098] In another preferred embodiment, the asparaginase comprises at most 100, preferably at most 80 or at most 50, more preferably at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid differences compared to the mature polypeptide of any of SEQ ID NO: 1 or 2.

[0099] In another embodiment, the asparaginase is a thermostable asparaginase, for example, the asparaginase from Pyrococcus furiosus disclosed in WO2008 / 151807.

[0100] Examples

[0101] Materials and Methods

[0102] Asparaginase activity (ASNU) assay

[0103] The activity of asparaginase can be measured as ASNU. One asparaginase unit (ASNU) is defined as the amount of enzyme required to produce 1.0 micromole of ammonia in 0.1 M MOPS buffer with 9.2 mg / ml asparagine in 1 minute at 37°C and pH 7.0.

[0104] Asparaginase hydrolyzes asparagine to aspartic acid and ammonium. The ammonium produced combines with α-ketoglutarate to form glutamate, whereby NADH is oxidized to NAD+. The reaction is catalyzed by the remaining glutamate dehydrogenase. The consumption of NADH is measured by photometry at 340nm. NADH has an absorbance at 340nm, whereas NAD+ does not. Therefore, the decline in color is measured and can be correlated with asparaginase activity.

[0105] Activity is determined relative to an asparaginase standard of known activity. A commercial product with declared activity (such as ) was used as a standard.

[0106] Quantification of Asparagine and Aspartic Acid Using HPLC

[0107] The asparagine and aspartic acid content of the samples can be analyzed on a ThermoFisher WPS3000 high pressure liquid chromatography system, which includes a quaternary pump, an autosampler with temperature control, a column oven, and an adjustable fluorescence detector.

[0108] After automatic pre-column derivatization, analyze the sample. 30 μL of milli-Q water, 10 μL of 0.4M borate buffer pH 10.2, 2 μL of sample, and 2 μL of o-phthalaldehyde 10 g / L in 0.4M borate buffer (pH 10.2) were collected and mixed by pipetting up and down in a mixing bottle; 100 μL of milli-Q water was added, and finally 2 μL was injected on an Agilent zorbax eclipse AAA column (4.6 mm by 150 mm, 3.5 μm particle size) with a corresponding guard column for chromatographic analysis.

[0109] The pump is set to a constant flow rate of 2 ml / minute, initially with a 20 mM phosphate buffer pH 7.5 balance column, and asparagine is eluted with a linear gradient from 0% to 100% of a mixture of 45% methanol, 45% acetonitrile, 10% water from 4 minutes to 12 minutes. The fluorescence of the asparagine derivative excites under the light of 340 nm, and the emission value is quantified at 450 nm. By comparing the analysis of the sample with the standard aspartic acid and asparagine in the concentration range from 0.05 g / l to 0.75 g / L.

[0110] Example 1

[0111] Asparaginase treatment of Robusta coffee beans with and without steaming Targeting Vietnam Robs Asparaginase test setup for Vietnam Robusta bean

[0112] Steaming process

[0113] In the autoclave used, only a final moisture content in the beans of 20% w / w can be obtained, since the water / steam inlet cannot be adjusted above this level. Therefore, a pre-soaking step is introduced before the steaming procedure in the autoclave. However, more flexible steaming equipment is commercially available for the given purpose, in which case pre-soaking may be unnecessary, since the moisture content of the coffee beans can be increased to the desired level by the steam treatment.

[0114] Pre-soak: Gently mix 5.0kg Vietnamese Robusta beans + up to 2.25L of tap water. Mix until all the water is absorbed by the beans, about 20-30 minutes.

[0115] Steaming: Steaming was performed by Systec XV 65 autoclave, program No. 3, steaming took place at 110-120°C for 15 min.

[0116] Enzyme treatment and drying

[0117] To 5.0 kg beans (initial amount) was added 4.5 L of tap water and 0 or 3000 ASNU / kg CB, L beans, with gentle mixing at 60°C for 60-120 minutes. Slowly add water and enzymes.

[0118] After enzyme treatment, the green coffee beans were dried to about 10% w / w water in a fluidized bed with an inlet temperature of 60°C and a flow rate of 300 m / s. 3 The air flow rate is 50 °C and 250 m / h for the first 1.5 h. 3 The air flow rate of / h is continued for another 2.5h.

[0119] Baking and acrylamide analysis

[0120] 125 g of dried coffee beans from the enzyme impact test were roasted to an LRU color value of 80. The beans were then analyzed for acrylamide using LC-MS / MS.

[0121] result

[0122] Initially, 5 kg of raw Vietnamese Robusta beans were steamed at 110°C at a total moisture content of 35% w / w (obtained during pre-soaking and steaming as described above). 0 or 3000 ASNU / kg were added CB, L coffee beans and allowed to react for 120 min with gentle mixing. After drying and roasting, while the control produced a 3% reduction in acrylamide, a 38% reduction in acrylamide was measured for the enzyme treated. In both cases, the reduction was compared to the corresponding process control without enzyme.

[0123] The time of enzyme treatment was then reduced from 120 min to 60 min; the other parameters were kept constant. The latter produced a 33% reduction in acrylamide content. Subsequently, an increase in the steaming temperature from 110°C to 120°C was tested. Surprisingly, the acrylamide reduction obtained so far was only 4%.

[0124]

[0125] Nos. 3 and 4 show that asparaginase treatment without a preceding steaming step results in almost no acrylamide reduction. Comparing Nos. 5 and 6 with Nos. 7 and 8 shows that the steaming temperature is important for acrylamide reduction. Comparing the acrylamide levels for the raw (unroasted) beans of Nos. 5 and 6, which have a moisture content of 35%, with No. 10 (where steaming was performed on green beans with an as-is moisture content of 8%) shows that moisture content is important. When green beans with low moisture content were steamed, 140 ppb of acrylamide was measured before roasting. Acrylamide gained during steaming will not be reduced by subsequent asparaginase treatment.

[0126] Example 2

[0127] Asparaginase treatment of Arabica coffee beans with and without steaming

[0128] Arabica beans treated with asparaginase were tested on a 5 g scale with or without steaming prior to enzyme treatment. In this case, only asparagine reduction was monitored; however, it is well known that the main precursor for acrylamide reduction is asparagine.

[0129] Unwashed green Arabica coffee beans were obtained from CR3 (CR3-Kaffeeveredelung M. Hermsen GmbH, Bremen, Germany) in 2010. CB, L and they were tested with and without steaming.

[0130] A portion of 5.0 g of unwashed green Arabica coffee beans with a moisture content of 6.5% was steamed at 100° C. for 45 minutes in a small wire basket embedded in a steam boiler containing 1 L of boiling deionized water. After steaming, the amount of absorbed water was quantitatively determined. The beans were then added to 4.2 ml of heated deionized water (60° C.) containing 0 ASNU. CB, L / kg or 3000ASNU / kg CB, L. Only the other 5 g portion of the beans was added to 4.2 ml of heated deionized water (60°C) containing 0ASNU CB, L / kg or 3000ASNU / kg CB, L. All doses were tested in quadruplicate. The samples were incubated at 60°C for 45 minutes with a stirring speed of 20 rpm. After 45-60 minutes, the water was completely absorbed by the beans.

[0131] An amount corresponding to 5 g of initially dry green beans was ground; hydrochloric acid was added to inactivate the enzymes and used to extract asparagine. After extraction, the beans were filtered and kept at -18°C until further analysis.

[0132] Asparagine content was analyzed using a high pressure liquid chromatography system, amino acids were analyzed by pre-column derivatization, and fluorescence was quantified.

[0133]

[0134]

[0135] When Arabica coffee beans were used, a significant enzyme-induced reduction of asparagine was observed without a previous steaming step.

Claims

1. A method for producing roasted coffee beans, the method comprising: (a) treating raw Robusta coffee beans with steam; (b) contacting the steam-treated raw coffee beans with asparaginase; and (c) roasting the raw coffee beans treated with asparaginase.

2. The method of claim 1, wherein the raw Robusta coffee beans are treated with steam at a temperature of about 100°C-120°C, preferably about 105°C-115°C.

3. A method for producing roasted coffee beans, the method comprising: (a) treating raw coffee beans with steam at a temperature of about 105° C. to 115° C.; (b) contacting the steam-treated raw coffee beans with asparaginase; and (c) roasting the raw coffee beans treated with asparaginase.

4. The method of claim 3, wherein the coffee beans are Robusta coffee beans.

5. The method of any one of the preceding claims, wherein before or during step (a), the moisture content of the coffee beans is increased to a level ranging from about 30% w / w to about 45% w / w, such as about 35% w / w.

6. The method of claim 5, wherein the moisture content of the coffee beans is increased by steam treatment.

7. A method as claimed in any one of the preceding claims, wherein step (a) is performed for about 5-60 minutes, preferably about 10-30 minutes, for example about 15 minutes.

8. The method of any one of the preceding claims, wherein the contacting in step (b) is for about 10-240 minutes, preferably for about 30-180 minutes, more preferably for about 45-180 minutes.

9. The method of any one of the preceding claims, wherein step (b) is performed by contacting with asparaginase at a concentration of about 100-30,000 ASNU / kg coffee beans, preferably about 500-20,000 ASNU / kg coffee beans, such as about 3,000 ASNU / kg coffee beans.

10. The method of any one of the preceding claims, wherein after step (b) and before step (c), the raw coffee beans are dried to a moisture content of about 5%-15% w / w, preferably 7%-12% w / w.

11. The method of any one of the preceding claims, wherein after step (c), the coffee beans are ground.

12. The method according to any one of the preceding claims, wherein the asparaginase is obtained from Aspergillus, preferably Aspergillus oryzae or Aspergillus niger, or is a variant of a parent asparaginase obtained from Aspergillus, preferably Aspergillus oryzae or Aspergillus niger.

13. The method of any one of the preceding claims, wherein the asparaginase comprises at most 100, preferably at most 80 or at most 50, more preferably at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid differences compared to the mature polypeptide of any one of SEQ ID NO: 1 or 2.

14. The method of any of the preceding claims, wherein the asparaginase has at least 50% sequence identity to the mature polypeptide of any of SEQ ID NO: 1 or 2, such as at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the mature polypeptide of any of SEQ ID NO: 1 or 2.

15. The method of any one of the preceding claims, wherein the asparaginase is a thermostable asparaginase.

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