Böle Biehl’s research while affiliated with Technische Universität Braunschweig and other places

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Publications (49)


Subunit structure of the vicilin-like globular storage protein of cocoa seeds and the origin of cocoa- and chocolate-specific aroma precursors
  • Article

April 2009

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334 Reads

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61 Citations

Food Chemistry

Ulrich Kratzer

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Essential cocoa-specific aroma precursors are generated during fermentation of cocoa seeds by proteolysis of the vicilin-like globulin. This particular storage protein consists of three subunits with apparent molecular masses of 47 kDa, 31 kDa, and 15 kDa, respectively, which are derived from a common 66-kDa precursor. Since all these subunits resist N-terminal sequencing by Edman degradation, we have analysed the localisation of these vicilin subunits on their common precursor sequence by MALDI-TOF-MS analyses of their tryptic fragments. The results were corroborated by epitope mapping of polyclonal antibodies using 185 overlapping pentadeca-peptides covering the whole sequence of the precursor. The results show that the cocoa vicilin is encoded by a single gene and that heterogeneity of the vicilin subunits must be attributed to statistical post-translational modifications. Localisation of the precursor regions, from which the various subunits are derived, will be helpful for the future identification of aroma-relevant peptides generated during the fermentation process.





Analysis of vicilin (7S)‐class globulin in cocoa cotyledons from various genetic origins

May 2002

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163 Reads

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23 Citations

Cocoa cotyledons contain vicilin (7S)‐class globulin (VCG), a major storage protein. It is the native source of oligopeptides and free amino acids which have been identified as precursors of cocoa‐specific aroma and are formed through proteolysis during fermentation. High‐resolution electrophoresis of native proteins isolated from ripe, unfermented cocoa cotyledons harvested from different cultivars was used to determine genetic differences of the genotypes. Flavour differences have been reported to exist after standard fermentation in cocoa beans harvested from various genotypes. In this paper, SDS‐PAGE and 2D IEF/SDS‐PAGE polypeptide separation patterns are shown which were separately isolated from cotyledons of various genetic origins. Cotyledons from three cultivars belonging to genetically distant varieties and a hybrid, Criollo, Forastero and Trinitario, did not reveal any analytical identity differences of VCG subunit polypeptide bands. Additionally, proteins of cotyledons harvested from three of those clones which were reported to produce genotype‐specific flavour differences in raw cocoa after standard fermentation, SCA 12, UIT1 and PBC 140, when analysed in the same way, did not indicate differences. Thus the cotyledon storage proteins from various genetically different cocoa trees are, within methodological limits, the same. We conclude that aroma differences in raw cocoa harvested from various genotypes are the result of other genetic, physiological or curing‐related factors, but are not due to genetic differences of aroma precursors derived from storage proteins. © 2002 Society of Chemical Industry


Oligopeptide patterns produced from Theobroma cacao L of various genetic origins

May 2002

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73 Reads

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17 Citations

Acetone dry powder (AcDP) was prepared for six cocoa genotypes, namely Forastero (Amelonado type), Criollo, Trinitario, SCA 12, UIT1 and PBC 140. Hydrophobic oligopeptides were produced when autolysis of AcDP was carried out at pH 3.5. Comparative HPLC analysis showed that autolysis of AcDP from various genotypes revealed a similar pattern of oligopeptides. Most of the hydrophobic oligopeptides were not generated during autolysis of AcDP in the presence of protease inhibitor (Pepstatin A), indicating that the generation of these oligopeptides was due to the action of cocoa cotyledon aspartic endoprotease. This finding implies that the splitting action of aspartic endoprotease on vicilin (7S)‐class globulin (VCG) from various genotypes was the same. The information from the study provides additional evidence that there are no obvious differences in VCG composition between various genotypes. © 2002 Society of Chemical Industry


Aspartic proteinase levels in seeds of different angiosperms

February 1997

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82 Reads

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37 Citations

Phytochemistry

Aspartic proteinases have been detected in seeds of numerous angiosperms and of gymnosperms but their biological function is still unknown. We have analysed the specific activities of these endoproteinases in ungerminated seeds of different monocot and dicot plants. Rather similar aspartic endoproteinase levels were found in the seeds of most of the investigated plants but considerably elevated specific activities were measured in the recalcitrant seeds of Hevea brasiliensis and especially in the case of Theobroma cacao. In all the different plants, the levels of cysteine proteinases in the ungerminated seeds were lower than the specific activities of aspartic proteinases. Only small variations of aspartic proteinase activities were observed during germination.


Fig. 8: The illuminated chloroplast is a cyanide trap. In non-illuminated chloroplasts the pH of the chloroplast stroma is below pH 7.4. At environmental conditions HCN would dissociate at pH values higher than 7.4. In its undissociated, uncharged state HCN diffuses freely through membranes into the cellular compartments. During illumination the pH of the chloroplast stroma rises above 7.4, HCN starts to dissociate and produyes cyanide ions, which are captured within the membrane-surrounded chloroplast. Cyanide is accumulated as long as free diffusible HCN is present outside the chloroplast in the symplastic space.
Cyanogenesis inhibits active pathogen defence in plants: Inhibition by gaseous HCN of photosynthesis CO2 fixation and respiration in intact leaves
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  • Full-text available

December 1996

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244 Reads

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29 Citations

Angewandte Botanik

The action of cyanide, a compound liberated from injured or infected cyanogenic plants, on plant respiration and photosynthesis of intact tissues has been studied. A method for quantified application of HCN to plant tissue is presented. HCN diffuses rapidly through the leaf blades and it inhibits photosynthetic CO2 fixation (PCF) and respiration in intact cells. Inhibition of PCF consists of (1) a reversible acute inhibition and (2) an irreversible, destructive photoinhibition of the photosynthetic dark reaction with subsequent photooxidation of the photosynthetic apparatus. As long as free HCN is present in leaf tissue, the PCF is blocked. It is suggested that this block in carbon flow is the main reason for the impairment of active, synthesis-dependent defence reactions.

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Characterization of two cyano glucosyltransferases from cassava leaves

August 1996

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8 Reads

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11 Citations

Phytochemistry

The terminal step in the biosynthesis of cyanogenic glucosides is the glucosylation of labile hydroxynitriles by UDP-glucose:hydroxynitrile-β-d-glucosyltransferases. These enzymes have not been characterized previously because this reaction is strongly affected by the instability of the hydroxynitriles, which dissociate to carbonyls and cyanide. Dissociation constants of cyanohydrins in aqueous solutions have been determined. Using these constants we are able to calculate the equilibrium concentrations of undissociated hydroxynitriles at a specific pH and thereby to determine the Km for several cyanohydrins of glucosyltransferases that synthesize cyanogenic glucosides. Two soluble glucosyltransferases occurring in cassava leaves were separated by chromatographic methods and their kinetic properties determined. The two glucosyltransferases could be distinguished by different Km-values, supporting the assumption that they represent distinct isoenzymes.


Effect of storage of cocoa seed AcDP on carboxy- peptidase activity
Specificity and stability of the carboxypeptidase activity in ripe, ungerminated seeds of Theobroma cacao L

December 1995

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175 Reads

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33 Citations

Food Chemistry

As previously reported the carboxypeptidase activity present in ripe, ungerminated Theobr oma cacao seeds is involved in the proteolytic formation of the cocoa-specific aroma precursors. Therefore, we have investigated the specificity of this particular enzyme activity using crude homogenates of acetone dry powder prepared from unfermented, ripe cocoa seeds. Both oligopeptide mixtures derived from cocoa seed proteins and synthetic peptides have been found to be suitable substrates for this enzyme activity. However, peptides with carboxyterminal arginine, lysine or proline residues are resistant against degradation by the cocoa seed carboxypeptidase. The enzyme preferentially liberates hydrophobic amino acids, whereas acidic amino acids are released very slowly. The rate of hydrolysis is not only determined by the carboxyterminal, but also affected by the neighbouring amino acid residue. Furthermore, the specificity of the enzyme is influenced by the pH-value. The specificity of the cocoa seed carboxypeptidase activity is remarkably similar to the specificity of carboxypeptidase A from porcine pancreas which (in addition to the cocoa aspartic endoprotease) has recently been successfully used for the in vitro formation of the cocoa-specific aroma precursors. These results are discussed in the light of the pH-dependent generation of the cocoa-specific aroma precursors during fermentation of the cocoa seeds.


Citations (48)


... Although the importance of endogenous enzymes in modulating the above mentioned reactions is well-documented, the roles played by other factors, particularly pH, in determining the extent to which these reactions occur within the nib, during fermentation, have also been highlighted [22][23][24]. Recent research indicates pH levels of 3.5-5.5, ...

Reference:

Effect of Partial pulp removal and fermentation duration on drying behavior, nib acidification, fermentation quality, and flavor attributes of Ghanaian cocoa beans.
pH-dependent enzymatic formation of oligopeptides and amino acids, the aroma precursors in raw cocoa beans
  • Citing Article
  • January 1993

... The acidic condition associated with the low pH in the fermentation process allows proteins to be hydrolysed by aspartic endoprotease and carboxypeptidase generating FAAs and peptides (Biehl et al., 1993;Voigt et al., 1994). There is a diversity of FAAs found in DCB (Araujo et al., 2021;Brunetto et al., 2020;Deus et al., 2020;Hinneh et al., 2018;Marseglia et al., 2014;Pätzold and Brückner, 2006;Tchouatcheu et al., 2019) and these can be classified biochemically according to their specific side chain (R group): 1) aliphatic -glycine (Gly), alaline (Ala), valine (Val), leucine (Leu) , isoleucine (Ile), cyclic aliphatic-proline (Pro), and, aliphatic and sulfur-containingmethionine (Met); 2) aromatic-phenylalanine (Phe), tyrosine (Tyr), and, tryptophan (Trp); 3) polar neutral -serine (Ser) and threonine (Thr); 4) amide-asparagine (Asn) and glutamine (Gln); 5) cationic -histidine (His), lysine (Lys), and arginine (Arg); 6) anionic -aspartic acid (Asp) and glutamic acid (Glu). ...

The proteases of ungerminated cocoa seeds and their role in the fermentation process
  • Citing Article
  • January 1993

... Significantly, free cyanide has not thetic steps are localized on the tonoplast and that the been found in processed cassava foods due to its volatility soluble UDP-glucosyltransferase is presumably vacuolar Rosling et al., 1993). The health (Gruhnert et al., 1994;Mederacke et al., 1995). A vacu disorders associated with subsistence on a high cyanogen-olar site of linamarin synthesis is supported also by the containing diet include (1) hyperthyroidism, resulting observation that vacuoles contain more than sufficient from thiocyanate interference in iodine metabolism, (2) quantities of linamarin to account for that present in tropical ataxic neuropathy, a neurological disorder, and intact cells (White et al., 1994). ...

Glucosyltransferases in Cassava Manihot esculenta
  • Citing Article
  • January 1995

... Furthermore, excess moisture is removed during conching, which is crucial for achieving suitable flow properties of the final product for further processing and may support the aroma development throughout the process (Beckett, 2003;Afoakwa et al., 2008b). The conching process can be divided into three phases that describe the texture changes during the process: the dry conching, the plastic conching, and the liquid conching (Biehl and Ziegleder, 2003). The dry conching describes the beginning of the process as the refined material turns into a firmer, plastic mass and the plastic conching phase starts. ...

COCOA | Production, Products, and Use
  • Citing Chapter
  • December 2003

... Sucrose (around 90% of total sugars), fructose and glucose (both 6% of total sugars) are the polysaccharides mostly contained in cocoa beans, followed by low quantities (less than 0.5 mg/g) of sugar alcohols, such as mannitol and inositol [70,71]. The enzyme invertase breaks down sucrose into reducing sugars, which play a role in the Maillard and Strecker reactions during roasting [7], leading to the formation of flavor compounds like pyrazines, aldehydes, and esters responsible for fruity, floral, and nutty notes [72,73]. ...

Cocoa: chemistry of processing
  • Citing Article
  • December 2003

... The cocoa pulp surrounding the raw cocoa beans has been shown to play an important role during fermentation [25][26][27]. The bioconversion of the pulp, first by yeast, and then by lactic and acetic acid bacteria, leads to the formation of the respective organic acids. ...

Analysis of Cocoa Flavour Components and Flavour Precursors
  • Citing Chapter
  • January 1988

... For quantification of low-concentration protein extracts (prepared from culture medium), the NanoOrange protein quantitation kit (Invitrogen) was used according to the manufacturer's instructions and fluorescence was determined with a luminescence spectrometer (LS 55, Perkin Elmer). Total in vitro PPO activity was determined polarographically according to the method of Lieberei and Biehl (1976) as described in Richter et al. (2005). ...

Freisetzung und Aktivierung von Polyphenoloxydasen aus Thylakiodmembranen der Spinat-Chloroplasten
  • Citing Article
  • October 1976

Plant Biology

... Unfermented cocoa beans contain low levels of free amino acids [23]. During fermentation, the overall amino acid content increased by roughly 150%-200% [24,25], but the final concentration was highly dependent on the pH. The dried bean fermentation approach yielded more acidic amino acids (aspartic acid and glutamic acid), possibly because of the use of additional acid at pH 4, resulting in over-acidification, as indicated in Table 3. ...

Kinetics of the formation of free amino acids in cocoa seeds during fermentation
  • Citing Article
  • December 1989

Food Chemistry

... Cyanogenesis is a broadly distributed chemical defense that is reported in over 2,500 plant species (Poulton, 1990). Cyanogenic glucosides are the most common cyanogenic compounds that are synthesized as inactive precursors and require modification by b-glucosidases and a-hydroxynitrile lyases for hydrogen cyanide (HCN) release (Conn, 1981;Frehner and Conn, 1987;Selmar et al., 1987Selmar et al., , 1989Poulton, 1990;Møller and Seigler, 1999;Zagrobelny et al., 2004). Cyanide is a potent inhibitor of mitochondrial oxidative phosphorylation (Isom and Way, 1984) and is an effective deterrent against herbivory (Tattersall et al., 2001;Hay-Roe et al., 2011;Wybouw et al., 2012Wybouw et al., , 2014. ...

Hevea Linamarase--A Nonspecific Glycosidase
  • Citing Article
  • March 1987

Plant Physiology