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Cacao endoproteinases and production of cocoa flavor from same

a technology of cacao endoproteinase and cocoa flavor, which is applied in the direction of hydrolases, biochemistry apparatus and processes, food preparations, etc., can solve the problem of peptides exhibiting differing sizes

Inactive Publication Date: 2008-02-28
NESTEC SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides two novel aspartic endoproteinases (TcAP1 and TcAP2) derived from Th. cacao that can improve the formation of cocoa flavor during processing and manufacturing. These enzymes are capable of cleaving the vicilin-class globulins isolated from cacao beans, resulting in a mixture of peptides and amino acids that yields cocoa flavor upon a reaction with reducing sugars. The DNA sequences coding for these enzymes can be incorporated into a suitable expression vector and used for recombinant production in various host cells. The endoproteinases can also be used for the manufacture of cocoa flavor by treating a suitable starting material with the endoproteinases and carboxypeptidase. The invention provides a method for generating cocoa flavor without the need for fermenting and roasting cacao beans.

Problems solved by technology

Second, fermentation also results in a release of peptides exhibiting differing sizes and a generation of a high level of hydrophobic free amino acids.

Method used

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  • Cacao endoproteinases and production of cocoa flavor from same
  • Cacao endoproteinases and production of cocoa flavor from same
  • Cacao endoproteinases and production of cocoa flavor from same

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of mRNA

[0087] Two beans were grounded in liquid nitrogen to a fine powder and extraction was directly performed with a lysis buffer containing 100 mM Tris HCl pH8, 1% SDS and 0.1M β-mercaptoethanol. RNA was extracted with one volume phenol / chloroform / isoamylalcohol (25 / 24 / 1) and centrifuged at 8000 rpm for 10 min at 4° C. The aqueous phase was washed three times with chloroform / isoamylalcohol (24 / 1). RNA was precipitated with 0.3M sodium acetate pH 5.2 in two volumes of ethanol. The RNA pellet obtained after centrifugation was resuspended in 100 mM Tris HCl pH 8 and a second precipitation with 2M lithium chloride was performed. The RNA pellet was washed with 70% ethanol and resuspended in DEPC treated water.

example 2

Cloning of Aspartic Proteinase cDNAs

[0088] A search for aspartic proteinase sequences in the GenBank database led to the identification of several plant sequences. A multiple alignment of these sequences revealed the presence of conserved regions, which have been used to design two degenerate oligonucleotides:

[0089] A sense primer, pAP0 (SEQ ID NO:7) (5′-GAYACNGGNAGYTCYAAYYTVTGG) has been synthesized according to the sequence Asp-Thr-Gly-Ser-Ser-Asn-Leu-Trp (SEQ ID NO:17), which contains an active site (Asp-Thr-Gly) of the protein.

[0090] An antisense primer pAP4r (5′-CCATMAANACRTCNCCMARRATCC) (SEQ ID NO:8) has been synthesized according to the sequence Trp-Ile-Leu-Gly-Asp-Val-Phe (SEQ ID NO:18), located in the C-terminal part of the protein.

[0091] Total RNA as prepared in example 1 was used to synthesize first strand cDNA with the SMART PCR cDNA Synthesis Kit (Clontech, USA). Synthesis has been performed exactly as described in the kit instructions using 1 μg of total RNA and th...

example 3

Sequencing and Analysis of DNA Sequences

[0104] cDNA sequencing has been performed according to standard techniques (Maniatis, A Laboratory Manual, Cold Spring Harbor, 1992). Sequence analysis and comparison were done using DNAStar programme. The sequences are shown under SEQ ID Nos. 1 and 2.

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Abstract

The present invention pertains to novel aspartic endoproteinases from Th. cacao which are involved in the production of cocoa flavor and DNA sequences coding for them. These enzymes are advantageously used in the manufacture of cocoa flavor.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 339,351, filed Jan. 10, 2003, which is a continuation of International application PCT / EP01 / 07255 filed Jun. 26, 2001, the entire content of both of which is expressly incorporated herein by reference thereto.TECHNICAL FIELD [0002] A recombinant aspartic endoproteinase and a method of preparing cocoa flavor using it. BACKGROUND ART [0003] The present invention pertains to novel endoproteinases involved in the production of cocoa flavor and the DNA coding for them. In particular, the present invention relates to the use of said enzymes for the manufacture of cocoa flavor. [0004] It is known that in processing cacao beans the generation of the typical cocoa flavor requires two steps—the fermentation step, which includes air-drying of the fermented material and the roasting step. Though roasting seems to be the key stage of obtaining cocoa flavor subjecting non fermented be...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A23G1/02A23G1/30A01H5/00C12N9/50C12N15/57C12P21/06
CPCA23G1/02C12N9/50A23L1/234A23L27/28C12N9/63
Inventor BUCHELI, PETERLALOI, MARYSEMCCARTHY, JAMES
Owner NESTEC SA
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