WO2016097270A1

WO2016097270A1 - Process for bleaching a food product

Info

Publication number: WO2016097270A1
Application number: PCT/EP2015/080449
Authority: WO
Inventors: Pieter Cornelis Langeveld; DE René Marcel JONG; Rémon BOER
Original assignee: Dsm Ip Assets B.V.
Priority date: 2014-12-19
Filing date: 2015-12-18
Publication date: 2016-06-23

Abstract

The present invention relates to a process for bleaching a food product, comprising incubating an intermediate form of the food product with a laccase which comprises a conserved prosite pattern according to amino acid sequence W-Y-H-X-H- X(3)-Q-Y-C-D-G-X-R-G, wherein X is an amino acid residue A, R, N, D, C, Q, E, G, H, I, L K, M, F, P, S, T, W, Y, or V, and wherein the intermediate form of the food product comprises a colored substance, and producing the food product, wherein the food product is bleached.

Description

PROCESS FOR BLEACHING A FOOD PRODUCT

The present invention relates to a process for bleaching a food product, and a food product obtainable by the process.

Background

In several food products the presence of a colored substance is seen as undesirable, such as in dairy products for example cheese, whey, butter, and milk powder and flour based products for example bread and noodles.

The raw materials or intermediary forms however of such food products may contain colored substances, for instance beta-carotenes or annatto.

Annatto is a coloring agent derived from the achiote trees (Bixa orellana) and is used in the coloring of cheese. The annatto that remains after the production of cheese may end up in the whey, which is undesirable.

Several enzymatic methods have been disclosed to reduce the amount of a colored substance in a food product, also called bleaching of food product. For instance, EP0282663 discloses a method for decolorizing annatto-containing whey by oxidation. A disadvantage of this method is that hydrogen peroxide is required for bleaching of annatto.

Another method is disclosed in WO2005004616 wherein a method for whitening or bleaching a dairy product using a lipoxygenase is disclosed.

In EP1703808 an enzymatic process for the reduction of a pigment is disclosed, wherein an enzyme composition derived from Marasmius scorodonius is used to directly act on a pigment. The enzyme composition from Marasmius scorodonius comprises a peroxidase.

Laccases can be used to cross-link whey proteins like alpha-lactalbumin (Faeregmand et. al. 1998, J. Agric. Food. Chem. 46, 1326-1333). Laccases may also be used in bread making where they can improve bread volume (W094/28728)

WO2005/073381 teaches a laccase from Pycnoporus cinnabarinus and its use in food.

However, there is no teaching that laccases can be used to bleach a food product.

The aim of the present invention is a method for bleaching of at least part of a food product in a process for the production of a food product. Summary

In one aspect the present invention relates to a process for bleaching a food product, comprising incubating an intermediate form of the food product with a laccase which comprises a conserved prosite pattern according to amino acid sequence W-Y- H-X-H-X(3)-Q-Y-C-D-G-X-R-G, wherein X is an amino acid residue A, R, N, D, C, Q, E, G, H, I ,L K, M, F, P, S, T, W, Y, or V, and wherein the intermediate form of the food product comprises a colored substance, and producing the food product, wherein the food product is bleached Surprisingly, it was found that a laccase comprising a prosite pattern as identified above effectively converted a colored substance into an uncolored substance in a food product.

In another aspect the present invention relates to the use of a laccase which comprises a conserved prosite pattern according to amino acid sequence W-Y-H-X-H- X(3)-Q-Y-C-D-G-X-R-G, wherein X is an amino acid residue A, R, N, D, C, Q, E, G, H, I ,L K, M, F, P, S, T, W, Y, or V to bleach a food product.

Detailed description

In one aspect the present invention relates to a process for bleaching a food product, comprising incubating an intermediate form of the food product with a laccase which comprises a conserved prosite pattern according to amino acid sequence W-Y- H-X-H-X(3)-Q-Y-C-D-G-X-R-G, wherein X is an amino acid residue A, R, N, D, C, Q, E, G, H, I ,L K, M, F, P, S, T, W, Y, or V, and wherein the intermediate form of the food product comprises a colored substance, and producing the food product, wherein the food product is bleached.

It was surprisingly found that a laccase comprising a conserved prosite pattern as identified above effectively converted a colored substance into an uncolored substance. Accordingly, a process for bleaching a food product as disclosed herein comprises converting a colored substance into an uncolored substance.

Any suitable food product can be produced in a process for bleaching a food product as disclosed herein. The production of a food product is generally known. A food product may be a dairy product, for instance milk, milk powder, cheese, butter oil, cream or whey. A dairy product as defined herein contains at least 10 wt%, such as at least 30 wt%, or at least 50 wt% or 70 wt% or at least 80 wt% on dry solid basis of components originating from milk, such as cow's milk. Components originating from milk are fats, proteins, for example whey, cheese curd, casein etc. Milk, such as cow's milk, may contain coloring substances such as carotenoids, for example beta-carotene. A food product may also be a flour based product, for instance a baked product from wheat flour and/or other cereal flours. Usually a baked product is prepared from a dough or a batter.

An intermediate form of a food product may be any form of a food product during the production of the food product, for instance an intermediate form of a food product may be a food product that is not intended for consumption, even though still edible. For instance, whey comprising a colored substance, for instance annatto, may be an intermediate form of whey; milk or cheese curd may be an intermediate form of a food product soft cheese ; a dough or a batter may be an intermediate form of a baked food product.

Any suitable colored substance can be present in a food product or an intermediate form thereof, for instance beta-carotene or annatto, or lutein, zeaxanthin astaxanthin lycopene, gossypol, chlorophyll, caramels, or carminic acid.

A dairy product product may for instance comprise annatto. Annatto is typically used as a coloring agent for cheese. Annatto as used herein is an orange-red condiment and food coloring derived from seeds of the achiote tree (Bixa orellana).

Laccase (EC 1 .10.3.2) in a process for the production of a dairy product as disclosed herein is a copper containing oxidase enzyme, which requires oxygen as a second substrate for the enzymatic action. Laccase is also known as a polyphenol oxidase. A laccase which is incubated with an intermediate form of a food product comprises a conserved prosite pattern according to amino acid sequence W-Y-H-X-H- X(3)-Q-Y-C-D-G-X-R-G, wherein X is an amino acid residue A, R, N, D, C, Q, E, G, H, I ,L K, M, F, P, S, T, W, Y, or V. The numerical value (3) after X in the prosite sequence means that there are three amino acid residues, or in other words X(3) corresponds to X-X-X. The amino acid residues are indicated with their lUPAC single letter code, wherein A = Alanine, R = Arginine, N= Asparagine, D = Aspartic acid, , C= cysteine, Q= glutamine, E = Glutamic acid, G= glycine, H= Histidine, I = Isoleucine, L = Leucine, K = Lysine, M = Methionine, F = Phenylalanine, P = Proline, S= Serine, T = Threonine, W = Tryptophan, Y = Tyrosine, V = Valine.

Protein sequences can be classified into subclasses by conserved patterns determined from a sequence alignment. One such systematic pattern description is a PROSITE pattern. The syntax of prosite patterns follows pattern syntax rules which can be found on the website of hpa-bioinfotools and is as follows:

The standard lUPAC one-letter codes for the amino acids are used.

The symbol ^"x' is used for a position where any amino acid is accepted. Ambiguities are indicated by listing the acceptable amino acids for a given position, between square brackets ^"[ ]'. For example: [ALT] stands for Ala or Leu or Thr.

Ambiguities are also indicated by listing between a pair of curly brackets ^"{ }' the amino acids that are not accepted at a given position. For example: {AM} stands for any amino acid except Ala and Met.

Each element in a pattern is separated from its neighbor by a

Repetition of an element of the pattern can be indicated by following that element with a numerical value or, if it is a gap ('χ'), by a numerical range between parentheses. Examples: x(3) corresponds to x-x-x; x(2,4) corresponds to x-x or x-x-x or x-x-x-x A(3) corresponds to A-A-A; Note: You can only use a range with 'x', i.e. A(2,4) is not a valid pattern element.

When a pattern is restricted to either the N- or C-terminal of a sequence, that pattern either starts with a symbol or respectively ends with a ' symbol. In some rare cases (e.g. PS00267 or PS00539), '>' can also occur inside square brackets for the C- terminal element. 'F-[GSTV]-P-R-L-[G>]' means that either 'F-[GSTV]-P-R-L-G' or 'F- [GSTV]-P-R-L>' are considered.

A laccase may for instance comprise a prosite pattern according to amino acid sequences that can be found by searching the UNIREF protein sequence database with the UNIREF codes in Table 1 below. The pattern in the sequence is underlined.

Table 1. Examples of laccases having a prosite sequence W-Y-H-X-H-X(3)-Q-Y- C-D-G-X-R-G

UNIREF Species Prosite sequence in laccase

code

Q02497 Trametes hirsuta 128-WYHSHLSTQYCDGLRG-143

Q716A1 Trametes sp. I-62 128-WYHSHLSTQYCDGLRG-143

G4XU43 Lenzites gibbosa 128-WYHSHLSTQYCDGLRG-143

059896 Pycnoporus cinnabarinus 128-WYHSHLSTQYCDGLRG-143

I3NL60 Lentinus sp. WR2 128-WYHSHLSTQYCDGLRG-143

013422 Trametes sp. I-62 132-WYHSHLSTQYCDGLRG-147

G9M4T7 Trametes versicolor 132-WYHSHLSTQYCDGLRG-147

A3F8Z8 Polyporus brumalis 128-WYHSHLSTQYCDGLRG-143

Q01679 Phlebia radiata 128-WYHSHLSTQYCDGLRG-143

M2RE78 Ceriporiopsis subvermispora 128-WYHSHLATQYCDGLRG-143

(strain B) Other laccase sequences that contain a prosite pattern as defined herein can be found in the uniref database using, but not limiting to, the following codes: Q12718; Q9HG17; D0VWU3; Q1W6B1 ; A3F8Z8; UPI000; Q716A3; B5G551 ; D9J214; R7SP52; 061263; E1 U754; G4XU44; Q50JG6; C1 KDZ5; C1 KDZ6; V9PQZ5; Q308Q9; UPI000; Q5EBY5; V5IVB7; Q6RYA5; C1 KDZ8; Q5I7J0; B5G555; U3PH31 ; A0A060; Q5MBH4; Q9HDS8; R7T0U4; C1 KDZ7; Q5MBH3; Q12719; V9PR04; Q6RYA2; R7SVI6; E0W6S1 ; Q01679; Q9UVU8; Q12717; Q8NID5; R7T2I7; I 1VE65; E7BLQ8; E7AIS4; UPI000; B5B8U2; E7BLQ9; I1W1V7; Q6UNT7; Q2HWK1 ; Q5MBH2; A0A060; Q9HDS7; V9PQQ3; D2D3A8; I 1VE66; D2CSE5; M2QYC3; M2QK34; M2QK38; R4JRR8; Q5MBH0; D2CSG0; E7BLR0; V9PR01 ; M2RE78; V5IWI6; Q1 EPM3; Q5MBH7; D2CSF2; V9PQU4; UPIOOO; M2QJC6; Q8WZG3; I 1VE67; E1 CGD5; E1 CGD6; Q6UPQ6; W4K128; UPIOOO; I 1 SB14; UPIOOO; J3S8K6; UPIOOO; UPIOOO; A0A067; C5HL40; UPIOOO; A5ACB1 ; R7SPI3; Q08AC5; Q0KHD1 ; V2Y238; W8SB57; A8W7J6; H9BT71 ; UPIOOO; W4K0R6; UPIOOO; A0A067; W4K2W6; B0CT29; V9XS33; Q7Z8S6; G8A545; E2LYT9; M2QJC0; V2X2N8; H9C4A3; W4JR24; Q12739; V2WNA2; B0DC12; V2X7U4; A0A067; A0A067; V9XS35; A0A067; B5G550; B5MAF5; V9PQQ1 ; K5XN09; B0DD15; Q08AC4; B8YQ97; UPIOOO; V2WSP2; V2XHV1 ; UPIOOO; D8QME3; B5MAF4; B0DD17; A0A067; UPIOOO; V2XJ10; V2X1 13; I6V2C5; A0A067; D2KZ05; B6V331 ; A6N8N5; V9PQV5; Q08AB3; Q6H9H7; S7QCZ9; B5G547; V9XTV1 ; Q08AB5; Q08AB4; V9PQV7; V2WLL0; A0A060; UPIOOO; R7SWM9; D2KZ02; A0A067; H9BT70; Q69FX0; V9PQZ0; B0DD16; V9QM82; Q2AAD1 ; B0E060; V2X2H5; R7SPG3; B5G549; Q08AC6; W4KFM8; D2KZ06; W4KFP2; A5ACB4; UPIOOO; Q99049; Q5VKI5; B8PJH3; UPIOOO; Q08AB9; W4KI25; D2KZ01 ; G8A529; A8N4I7; M2R2P1 ; UPIOOO; V2X3Y4; B0DS50; A0A067; V2WYW9.

It was found that a laccase having a prosite pattern according to amino acid sequence W-Y-H-X-H-X(3)-Q-Y-C-D-G-X-R-G has a more open active site structure compared to laccases lacking this prosite pattern. Examples of laccases with a prosite pattern and this open structure can be found in the PDB structural database using, but not limited to, the following PDB identifiers: 1 KYA, 3V9C, 3FPX, and 2XYB. In contrast, laccases with a more closed active site structure, which are not active, can be found using for instance the PDB identifiers: 3FU7, 3PPS and 3SQR.

Surprisingly, it was found that a laccase having a prosite pattern according to amino acid sequence W-Y-H-X-H-X(3)-Q-Y-C-D-G-X-R-G efficiently converts at least part of a colored substance in a food product into an uncolored substance. Accordingly, a laccase having a prosite pattern as defined herein efficiently bleaches a food product comprising a colored substance. Advantageously, a laccase having a prosite sequence as defined herein efficiently bleaches a dairy product, for instance whey, comprising annatto.

A laccase used in a process as disclosed herein may be obtained from any suitable origin, for instance from fungi such as from the genus Trametes, Phlebia, Ceriporiopsis, Polyporus. Advantageously, a laccase is derived from a fungal species Trametes versicolor, Trametes hirsuta, Dichomitus squalens, Ganoderma lucidum, Lenzites gibbosa, Lentinus tigrinus, Trametes sanguinea, Ganoderma fornicatum, Ganoderma weberianum, Rigidoporus microporus, Agaricomycetes incertae sedis, Pycnoporus cinnabarinus, Steccherinum murashkinskyi, Meripilus giganteus, Phlebia tremellosa, Phlebia radiata Ceriporiopsis rivulosa, Ceriporiopsis subvermispora Heterobasidion irregulare, Polyporus brumalis, Postia placenta, Flammulina velutipes, Coprinopsis cinerea, Cerrena unicolor, Moniliophthora roreri, or Schizophyllum commune. Advantageously a laccase is derived from Trametes hirsuta.

A laccase in a process as disclosed herein may comprise a polypeptide which has at least 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to amino acids 22 to 520 of amino acid sequence according to SEQ ID NO: 1 1 , and which has a prosite pattern according to amino acid sequence W-Y-H-X- H-X(3)-Q-Y-C-D-G-X-R-G as defined herein above. Accordingly, a laccase in a process as disclosed herein may comprise a polypeptide which comprises the mature polypeptide sequence of SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16. For instance a laccase in a process as disclosed herein may comprise amino acids 22 to 520 of SEQ ID NO: 1 1 , or amino acids 22 to 520 of SEQ ID NO: 12, or amino acids 22 to 520 of SEQ ID NO: 14, or amino acids 22 to 522 of SEQ ID NO: 16.

For the purpose of this invention sequence identity is defined here that in order to determine the percentage of sequence identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between two sequences, gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region. The percent sequence identity between two amino acid sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice,P. LongdenJ. and BleasbyA Trends in Genetics 16, (6) pp. 276— 277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as "longest-identity".

A laccase may be produced in any suitable host cell, by heterologous or homologous expression of the laccase in the host cell, fermenting the host cell in a fermentation medium under conditions that allow expression and secretion of the laccase, known to a skilled person in the art. Usually the production of laccase comprises recovery of the laccase from the fermentation medium. A suitable host cell may for instance be Aspergillus, for instance Aspergillus niger or Aspergillus oryzae or Trichoderma, for example Trichoderma reesei (Hypocrea jacorina).

Any suitable amount of laccase may be incubated with an intermediate form of a food product, such as between 100 and 10.000 mg/l, or between 200 and 5.000 mg/l or between 500 and 1 .000 mg / 1.

The present invention also relates to the use of a laccase which comprises a conserved prosite pattern according to amino acid sequence W-Y-H-X-H-X(3)-Q-Y-C- D-G-X-R-G, wherein X is an amino acid residue A, R, N, D, C, Q, E, G, H, I ,L K, M, F, P, S, T, W, Y, or V to bleach a dairy product.

A laccase in a use as disclosed herein is a laccase as defined herein above. The present invention also relates to a food product, such as a dairy obtainable by a process as defined herein.

FIGURES

Figure 1. Schematic representation of the vector pGBFINOXL-1

Figure 2. Schematic representation of the vector pGBFINOXI-1

Figure 3. Schematic representation of the vector pGBTOP-16

EXAMPLES

Example 1. Cloning and expression of two laccase genes oxl and oxi from Aspergillus niger

Example 1.1. Cloning and expression oxl and oxi

The genome of Aspergillus, niger strain CBS513.88 was sequenced (Pel H.J et all, 2007 "Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88" Nat. Biotechnol 25(2):221-231 ). The open reading frames, An08g08450 and An01 g13660, were named oxl and oxi , respectively. The oxl and oxi genes encode two putative laccases OXL and OXI. The protein sequences of OXL and OXI of Aspergillus niger CBS513.88 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The OXL sequence consists of a signal sequence of 17 amino acids for efficient secretion in A. niger, and a deduced mature protein sequence of 577 amino acids. The OXI sequence consists of a signal sequence of 21 amino acids for efficient secretion in A. niger, and a deduced mature protein sequence of 575 amino acids.

The genomic DNA of A. niger strain CBS513.88 (deposited at the CBS Institute, Utrecht, the Netherlands under the deposit number CBS 513.88) was isolated to allow the amplification of the oxl and oxi genes from the chromosomal DNA. A. niger strain CBS513.88 was grown for 3 days at 30 degrees Celsius in PDB (Potato dextrose broth, Difco) and chromosomal DNA was isolated from the mycelium using the Q-Biogene kit (catalog nr. 6540-600; Omnilabo International BV, Breda, the Netherlands), using the instructions of the supplier.

Four PCR primers were designed based on the genomic DNA of the oxl and oxi loci. The sequence of the oxl gene including 103 nucleotides downstream of the oxl coding sequence is shown SEQ ID NO: 3. The sequence of the oxi gene including 80 nucleotides downstream of the oxi coding sequence is shown in SEQ ID NO: 5. Primer (Oxl-dir) contains 22 nucleotides OXL coding sequence starting at the ATG start codon, preceded by a 23 nucleotides sequence including a Pad restriction site (SEQ ID NO:7). The reverse primer (Oxl-rev) contains nucleotides complementary to the reverse strand of the region downstream of the OXL coding sequence preceded by an Asd restriction site (SEQ ID NO:8). Using these primers a 2.0 kb sized fragment was amplified with chromosomal DNA from A. niger CBS513.88 as template. The obtained 2.0 kb sized fragment was isolated, digested with Pad and Asd and purified. The Pad / Asd fragment comprising the OXL coding sequences was exchanged with the Pad I Asd phyk fragment from pGBFIN-5 (WO 99/32617). Resulting plasmid is the OXL expression vector named pGBFINOXL-1 (see Figure 1 ). The cloned insert in pGBFINOXL-1 contains the full oxl gene with one intron interrupting the coding sequence. The cDNA sequence encoding the OXL protein is shown in SEQ ID NO: 4.

Primer (Oxi-dir) contains 24 nucleotides OXI coding sequence starting at the ATG start codon, preceded by a 23 nucleotides sequence including a Pad restriction site (SEQ ID NO:9). The reverse primer (Oxi-rev) contains nucleotides complementary to the reverse strand of the region downstream of the OXI coding sequence preceded by an Asd restriction site (SEQ ID NO:10). Using these primers a 2.0 kb sized fragment was amplified with chromosomal DNA from A. niger CBS513.88 as template. The obtained 2.0 kb sized fragment was isolated, digested with Pad and Asd and purified. The Pad / Asd fragment comprising the OXL coding sequences was exchanged with the Pad I Asd phyk fragment from pGBFIN-5 (WO 99/32617). Resulting plasmid is the OXI expression vector named pGBFINOXI-1 (see Figure 2). The cloned insert in pGBFINOXI-1 contains the full oxi gene with two introns interrupting the coding sequence. The cDNA sequence encoding the OXI protein is shown in SEQ ID NO: 6.

The expression vectors pGBFINOXL-1 and pGBFINOXI-1 were linearized by digestion with A/oil, which removes all E. coli derived sequences from the expression vectors. The digested DNA was purified using phenol: chloroform: isoamylalcohol (24:23:1 ) extraction and precipitation with ethanol. Subsequently, A. niger GBA 306 was transformed with the linearized pGBFINOXL-1 and pGBFINOXI-1 expression vectors with strain and methods as described in WO 201 1/009700 and references therein, and selected on acetamide containing media and colony purified according to standard procedures. Transformation and selection was performed as described in WO 98/46772 and WO 99/32617. Preferably, A. niger transformants containing multiple copies of the expression cassette were selected for further generation of sample material. For the pGBFINOXL-1 and pGBFINOXI-1 expression vector 10 A. niger transformants were purified; first by plating individual transformants on selective medium plates followed by plating a single colony on PDA (potato dextrose agar: PDB + 1 .5% agar) plates. The spores were collected after growth for 1 week at 30 degrees Celsius. Spores were stored refrigerated and were used for the inoculation of liquid media. Single transformants containing the pGBFINOXL-1 or pGBFINOXI-1 vectors were selected and named strain OXL1 and OXI 1 , respectively.

Example 1.2. Fermentation of A niger OXL1 and OXI1 strains

A. niger strains OXL1 and OXI 1 were used for generating sample material by cultivation of the strains in 24 deep well plates (Axygen, Union City, USA) containing 3 ml cultivation medium (70 g glucose, 10 g yeast extract, 10 g (NH₄)₂S0₄, 2 g K₂S0₄, 2 g KH₂P0₄, 6.75 mg ZnS0₄7H₂0, 1 .5 mg MnCI₂4H₂0, 0.45 mg CoCI₂ 6H₂0, 15.45 mg CuS0₄5H₂0, 0.6 mg Na₂Mo0₄2H₂0, 6.75 mg CaCI₂2H₂0_, 4.5 mg FeS0₄7H₂0, 1.5 mg H₃BO₃ 0.5 g MgS0₄7H₂0, 0.25 g Tween-80, 10 g Citric acid, 30 g MES, EDTA (triplex) 22.5 mg, pH5.5 per liter medium). The 24 deep well plates were covered with a Breathseal (Greiner bio-one, Frickenhausen, Germany) and a lid. After 6 days of growth at 34^°C, 550 rpm and 80% humidity in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland) 1.5 ml samples were taken, the mycelium was separated from the supernatant by centrifugation for 30 min at 4000g and the supernatants were stored at -20°C until further analyses.

Example 2. Cloning, Expression of other motif-containing laccases and Recovery Example 2.1. Cloning and Expression

Further motif-containing laccases derived from Polyporus brumalis (SEQ ID NO: 12), Phlebia radiata (SEQ ID NO 14), and Ceriporiopsis subvermispora (SEQ ID NO: 16) were analyzed using SignalP 4.1 to determine the location of a signal sequence for efficient secretion in Aspergillus niger. The P. brumalis, P. radiata and C. subvemispora laccases comprise a signal sequence of 21 amino acids and a deduced mature protein sequence of 499, 499 and 501 amino acids, respectively. Codon-adapted DNA sequences for expression of the protein in Aspergillus niger were designed containing additional restriction sites for subcloning in an Aspergillus expression vector. Codon adaptation was performed as described in WO2008/000632. The codon optimized DNA sequences for expression of the genes encoding the Polyporus brumalis,, Phlebia radiata, and Ceriporiopsis subvermispora laccases in A. niger is shown in SEQ ID NO: 13, 15 and 17, respectively. The translational initiation sequence of the glucoamylase glaA promoter was modified into 5 -CACCGTCAAA ATG-3' (SEQ ID NO:3) and an optimal translational termination sequence 5'-TAAA-3' was used in the generation of the expression constructs (as also detailed in WO2006/077258). A DNA fragment (SEQ ID NO: 4), containing a.o. part of the glucoamylase promoter and the laccase encoding genes, was synthesized completely, purified and digested with EcoRI and Pad.

Figure 3 shows a physical map of the pGBTOP-16 vector used for cloning of the laccase genes. The pGBTOP-16 vector is derived from the pGBTOP-12 vector described in WO201 1/009700. In addition to pGBTOP-12, it contains the ccdB gene from E.coli for positive selection for presence of an insert between the EcoRI and Pad cloning sites. The Pad restriction site replaces the SnaBI restriction site present in pGBTOP-12. The pGBTOP-16 vector was linearized by EcoRI/PacI digestion and the linearized vector fragment was subsequently purified by gel-extraction. The DNA fragments were cloned into the pGBTOP-16 vector and the resulting vector was named pGBTOP-NBL0496, pGBTOP-NBLO503 and pGBTOP-NBLO505. Subsequently, A. niger GBA 306 was transformed with pGBTOP-NBLO0496, pGBTOP-NBLO503 and pGBTOP-NBLO505 in a co-transformation protocol with pGBAAS-4, with strain and methods as described in WO 201 1/009700 and references therein, and selected on acetamide containing media and colony purified according to standard procedures. Transformation and selection was performed as described in WO 98/46772 and WO 99/32617. Strains containing the laccase genes were selected via PCR with primers amplifying the introduced laccase gene to verify presence of the vector. The strains were named NBL0496, NBLO503 and NBLO505 expression cassette. A single transformant expressing each laccase was selected, and further replica-plated to obtain a single strain inoculum.

Example 2.2. Fermentation of A. niger NBL0496, NBLO503 and NBLO505 strains

Fresh A. niger spores of A. niger NBL0496, NBLO503 and NBLO505 strains were prepared and used for generating sample material by cultivation of the strain in 24 deep well plates (Axygen, Union City, USA) containing 3 ml cultivation medium (70 g glucose, 10 g yeast extract, 10 g (NH₄)₂S0₄, 2 g K₂S0₄, 2 g KH₂P0₄, 6.75 mg ZnS0₄7H₂0, 1.5 mg MnCI₂4H₂0, 0.45 mg CoCI₂ 6H₂0, 15.45 mg CuS0₄5H₂0, 0.6 mg Na₂Mo0₄2H₂0, 6.75 mg CaCI₂2H₂0_, 4.5 mg FeS0₄7H₂0, 1 .5 mg H₃B0₃ 0.5 g MgS0₄7H₂0, 0.25 g Tween-80, 10 g Citric acid, 30 g MES, EDTA (triplex) 22.5 mg, pH5.5 per liter medium). The 24 deep well plates were covered with a Breathseal (Greiner bio-one, Frickenhausen, Germany) and a lid. After 6 days of growth at 34^°C, 550 rpm and 80% humidity in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland) 1.5 ml_ samples were taken, the mycelium was separated from the supernatant by centrifugation for 30 min at 4000g and the supernatants were stored at -20°C until further analyses.

Example 3. Bleaching of whey containing annatto with different laccases

Bleaching experiments were performed in whey from a cheddar cheese production on a pilot scale at DSM. Cheddar cheese was manufactured following a generic English recipe. Following renneting and cutting, the cooking step was started. The temperature was increased in 30 minutes from 31 to 38 degrees Celsius Once the pH had reached 6.2, the curd and whey were separated. At this time point, a whey sample was taken.

Bleaching experiments were performed in a 200 microliter scale microtiterplate. A volume of 180 microliter of whey colored with 300 ppm Annatto having a pH of 6.6, was incubated at temperature of 21 °C.

The reaction was started with 20 microliter laccase from Trametes hirsuta (Amano M120), and laccase OXL and laccase OXI from Aspergillus niger prepared as disclosed in Example 1 and 2. The blank is whey plus 300 ppm annatto plus 20 microliter of water.

The color of the whey was followed in time by means of a reflection spectrophotometer where 3 components were determined: L=black/white, a=green/red, b=yellow/blue. It appeared that component b is most sensitive for yellow color of whey colored with annatto and therefore was used to determine bleaching efficiency.

The results in Table 1 show that Amano M120 laccase reduced the color in whey significantly after 2 and 4 hours. Amano M120 laccase, which is a Trametes hirsuta laccase, contains a prosite sequence WYHSHLSTQYCDGLRG (see also Table 1 ). The laccases OXL and OXI from Aspergillus niger which lack a prosite sequence did not reduce the color in whey.

Table 2. Color of whey (b-value) after incubation of whey containing annatto with different laccases after incubation at 21 °C for 2 and 4 hrs.

Example 4. Bleaching of whey containing Annatto with laccase having a prosite sequence WYHSHLSTQYCDGLRG at different concentrations

Bleaching experiments were performed in whey from a cheddar cheese production on a pilot scale at DSM as described in Example 3.

300 ppm Annatto was added to the whey (pH 6.6) and the bleaching experiment was performed on a 5 ml scale at a temperature of 21 °C. The reaction was started by adding laccase (0.25-1 -4 and 10 g/l) Amano M120. The blank is whey plus 300 ppm annatto. The color of the whey was followed as described in Example 3.

The results in Table 3 show that a laccase (Amano M120) having a prosite sequence WYHSHLSTQYCDGLRG effectively reduces color already at low concentration.

Table 3. Color of whey (b-value) after incubation of whey with different amounts of Amano M120 laccase after incubation at 21 °C for 4 hrs.

NA: Not applicable

Example 5. Sequence alignment of laccases having a prosite pattern of W-Y-H-X- H-X(3)-Q-Y-C-D-G-X-R-G

Sequence alignment of the mature polypeptide sequences of SEQ ID NO: 1 1 , 12 , 14 and 16 laccases from T. hirsuta, P. brumalis, P. radiata and C. subvemispora was performed according to the longest identity with the Needle computer program using the settings as disclosed in the description.

The results in Table 4 show that the amino acid sequences of the laccases have more than 60% identity.

Table 4. Percentage of identity of the mature sequences of laccases according to SEQ ID NO: 1 1 , 12, 14 and 16

Example 6. Bleaching of whey containing annatto with laccases from Polyporus brumalis, Phlebia radiata, and Ceriporiopsis subvermispora

Bleaching experiments were performed in 50mM acetate buffer pH5 to which 300 ppm annatto was added.

Laccases from Polyporus brumalis, Phlebia radiata, and Ceriporiopsis subvermispora as prepared according to Example 2 were used.

The bleaching experiment was performed on a 200μΙ scale and experimental conditions were:

-Temperature: 21 °C

- pH 5

- Volume: MTP with 180ul buffer + annatto per well

- The reaction was started with 20 μΙ enzyme sample

The color of the buffer was followed in time and measured as disclosed in Example 3. The results in Table 5 show that the laccases from Polyporus brumalis, Phlebia radiata and Ceriporiopsis subvermispora which comprise a prosite sequence according to W-Y- H-X-H-X(3)-Q-Y-C-D-G-X-R-G reduce the color resulting from annatto in buffer as compared to the blank which was not treated with laccase. Table 5. Color (b value) of buffer comprising annatto that has been incubated with laccases from Polyporus brumalis, Phlebia radiata, and Ceriporiopsis subvermispora for 4 and 8 hours

b-value

Laccase of mature Organism

sequence of: 4 hours 8 hours

SEQ ID NO: 12 Polyporus brumalis 5.36 4.43

SEQ ID NO: 14 Phlebia radiata 6.29 5.39

SEQ ID NO: 16 Ceriporiopsis subvermispora 4.92 4.09

Blank 9.46 8.97

Claims

1 . Process for bleaching a food product, comprising incubating an intermediate form of the food product with a laccase which comprises a conserved prosite pattern according to amino acid sequence W-Y-H-X-H-X(3)-Q-Y-C-D-G-X-R-G, wherein X is an amino acid residue A, R, N, D, C, Q, E, G, H, I ,L K, M, F, P, S, T, W, Y, or V, and wherein the intermediate form of the food product comprises a colored substance, and producing the food product, wherein the food product is bleached.

2. Process according to claim 1 , wherein the food product is a dairy product.

3. Process according to claim 2, wherein the dairy product is milk, cheese, butter oil, cream or whey.

4. Process according to any one of claims 1 to 3, wherein the colored substance comprises annatto.

5. Process according to any one of the claims 1 to 4, wherein the laccase comprises a polypeptide which has at least 60% identity to amino acids 22 to 520 of the amino acid sequence according to SEQ ID NO: 1 1.

6. Process according to any one of the claims 1 to 5 wherein said incubating comprises converting at least part of the colored substance into a non-colored substance.

7. Use of a laccase comprising a conserved prosite pattern according to amino acid sequence W-Y-H-X-H-X(3)-Q-Y-C-D-G-X-R-G, wherein X is an amino acid residue A, R, N, D, C, Q, E, G, H, I ,L K, M, F, P, S, T, W, Y, or V to bleach a food product, for instance a dairy product.

8. A food product obtainable by a process according to any one of the claims 1 to 6.