Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Changes in polyphenol ability to produce astringency during roasting of cocoa liquor

Polyphenols in cocoa products determine the astringent sensation due to their interaction with salivary proline‐rich protein. Changes in the ability of polyphenols to produce astringency during cocoa roasting have been studied through an evaluation of the polyphenol–protein interaction in cocoa cake/liquor roasted at 120 °C for 45 min, with and without enrichment with polyphenol extract. Roasting decreased the capacity of polyphenols to interact with protein, causing a decrease in astringency. However, the polyphenol–protein interaction products after roasting could still be oxidized enzymatically and most probably would still give cocoa products beneficial effects as functional food. Copyright © 2005 Society of Chemical Industry     

Oxidation of polyphenols in unfermented and partly fermented cocoa beans by cocoa polyphenol oxidase and tyrosinase

Unfermented and partly fermented dried cocoa beans have an excessively astringent and bitter flavour owing to their high polyphenol content. Studies on the remaining polyphenol oxidase activity and the oxidation of polyphenols in these beans have been conducted in relation to two factors, ie incubation time (0, 1, 2, 4, 8 and 16 h) and pH of incubation (3.5, 4.5, 5.5 and 6.5). Owing to unsuccessful polyphenol oxidation during incubation of partly fermented beans, incubation–enrichment treatments were carried out by combining incubation time (0, 8, 16, 24 and 32 h) and addition of crude cocoa polyphenol oxidase and purified tyrosinase from mushroom at 88 and 8800 units g^?1 and pH 5.5. Results showed that the remaining polyphenol oxidase activities of unfermented and partly fermented dried beans were 1 and 0.08% with specific activities of 9 and 1% respectively. The polyphenols of unfermented beans were effectively oxidised by incubation at 45 °C and pH 3.5–6.5 without enzyme enrichment; while those of partly fermented beans required enzyme enrichment. Both crude cocoa polyphenol oxidase and tyrosinase could be used for enzyme enrichment, but tyrosinase seemed to be more effective. © 2002 Society of Chemical Industry     

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)₅-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~ 88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.     

Assessment of the flavour of fresh uncooked onions by taste‐panels and analysis of flavour precursors,pyruvate and sugars

Flavour in fresh onions is dominated by volatile sulphenic and thiosulphenic acids that are liberated once alk(en)yl cysteine sulphoxide (ACSO) flavour precursors are cleaved by the enzyme alliinase after tissue disruption. The levels of pyruvate and ACSOs in over 100 samples of onions marketed in the UK were measured, and compared with assessment by taste‐panels. There was a linear relationship between the content of ACSOs and pyruvate. Measurements of pyruvate indicated that the marketing classification of some types of onion did not correspond to their pyruvate levels. A significant linear relationship was found between a sensory measure of strength and pyruvate over the range 1.2–9.3 µmol pyruvate g^?1 fresh weight. In most cases, when a flavour classification of sweet, mild or strong was applied to a sample of onions based on pyruvate content, the taste‐panels agreed with the categorization. The taste‐panels were unable to identify a sweet flavour in onions, except at low levels of pyruvate. Taste‐panels were able to define a likeability character (attractiveness of flavour) for onions, which correlated with the level of pyruvate. However, for some varieties, the flavour classification or likeability did not correspond to predictions based on pyruvate levels alone. Pyruvate measurements were seen as a suitable method for routine quality control once the characteristics of a variety of onion had been established, but initial evaluations should include well‐designed taste‐panel assessments. Copyright © 2004 Society of Chemical Industry     

Effect of fermentation time and drying temperature on volatile compounds in cocoa

The effects of fermentation time and drying temperature on the profile of volatile compounds were evaluated after 2, 4, 6, and 8 fermentation days followed by drying at 60, 70 and 80 °C. These treatments were compared with dry cocoa controls produced in a Samoa drier and by a sun-drying process. A total of 58 volatile compounds were identified by SPME-HS/GC-MS and classified as: esters (20), alcohols (12), acids (11), aldehydes and ketones (8), pyrazines (4) and other compounds (3). Six days of fermentation were enough to produce volatile compounds with flavour notes desirable in cocoa beans, as well as to avoid the production of compounds with off-flavour notes. Drying at 70 and 80 °C after six fermentation days presented a volatile profile similar to the one obtained by sun drying. However, drying at 70 °C represents a lower cost. Given the above results, in the present study the optimal conditions for fermentation and drying of cocoa beans were 6 days of fermentation, followed by drying at 70 °C.     

Water-soluble precursors of beef flavour. Part II: Effect of post-mortem conditioning

Changes in glycolytic metabolites, nucleotide degradation products, free amino acids and other amino compounds were monitored in beef muscle (M. longissimus lumborum), stored for 21 days at 4 °C, in order to evaluate how post-mortem conditioning may affect flavour formation in beef. The major effects observed in sugar-related substances were the dephosphorylation of the phosphates of glucose, fructose and mannose, to yield their free sugars, as well as the breakdown of inosine 5′-monophosphate, to give a sixfold increase in ribose. Total reducing sugars increased by only 15% during conditioning, while glycogen levels remained unchanged from 2 days post-slaughter. Free amino acids increased during conditioning, particularly between days 7 and 14. Phenylalanine, methionine, lysine, leucine and isoleucine were the amino acids showing the greatest increase with conditioning time, with methionine, in particular, showing a sevenfold increase during the conditioning period. The effects of these precursor changes on cooked beef flavour are discussed.     

On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof

Cocoa bean fermentations controlled by means of starter cultures were introduced on several farms in two different cocoa-producing regions (West Africa and Southeast Asia). Two starter culture mixtures were tested, namely one composed of Saccharomyces cerevisiae H5S5K23, Lactobacillus fermentum 222, and Acetobacter pasteurianus 386B (three heaps and one box), and another composed of L. fermentum 222 and A. pasteurianus 386B (seven heaps and one box). In all starter culture-added cocoa bean fermentation processes, the inoculated starter culture species were able to outgrow the natural contamination of the cocoa pulp-bean mass and they prevailed during cocoa bean fermentation. The application of both added starter cultures resulted in fermented dry cocoa beans that gave concomitant milk and dark chocolates with a reliable flavour, independent of cocoa-producing region or fermentation method. The addition of the lactic acid bacterium (LAB)/acetic acid bacterium (AAB) starter culture to the fermenting cocoa pulp-bean mass accelerated the cocoa bean fermentation process regarding citric acid conversion and lactic acid production through carbohydrate fermentation. For the production of a standard bulk chocolate, the addition of a yeast/LAB/AAB starter culture was necessary. This enabled an enhanced and consistent ethanol production by yeasts for a successful starter culture-added cocoa bean fermentation process. This study showed possibilities for the use of starter cultures in cocoa bean fermentation processing to achieve a reliably improved fermentation of cocoa pulp-bean mass that can consistently produce high-quality fermented dry cocoa beans and flavourful chocolates produced thereof.     

Thin-layer high-vacuum distillation to isolate volatile flavour compounds of cocoa powder

The volatile fraction of commercial cocoa powders was isolated using thin-layer high-vacuum distillation (TLHVD) of Soxhlet extracts. Calculated and experimental recovery of the internal standard n-undecanoic acid methylester did agree, and a good reproducibility was found for the procedure. Around 70 volatile compounds were identified and semi-quantified using internal standard-based gas chromatography (GC), coupled GC–mass spectrometry (GC–MS) and GC-olfactometry (GC-O). Dehydromevalonic acid lactone, (R)-(−)-pantolactone and two diastereomer solerols were identified for the first time after purification by micropreparative GC and re-analysis using GC–MS and chiral GC. Strong sensory contributions also came from acetic acid, 2/3-methyl butanoic acid, phenylacetaldehyde, furaneol, dihydroxymaltol, vanillin and phenylacetic acid.     

Acidification,proteolysis and flavour potential in fermenting cocoa beans

Cocoa fermentations in Ghana and Trinidad as well as anaerobic fermentation-like incubations of fresh cocoa beans in Germany were carried out under controlled conditions. Samples of beans were taken during the course of these treatments and determinations were made as to acidification (pH, acetic acid content), proteolysis (free α-amino nitrogen, peptide nitrogen and SDS electrophoresis of the protein peptides) and flavour potential (gas chromatography of the highly volatile compounds, in particular isopentanal and organoleptic analysis after thin layer roasting). A positive correlation between acidification, proteolysis and the development of flavour potential during anaerobic fermentation can be demonstrated in principle. However, the flavour potential is increased if the temperature rise is comparatively slow in both normal fermentation and laboratory incubation. Strong acidification and high accumulation of amino acids and peptides were not essential for a good flavour potential. The isopentanal content proved to be a useful indicator of the progress of normal fermentation in the tropics. These findings can be interpreted on the basis of earlier results about germination-like processes in the protein vacuoles, pre- and post-mortem subcellular structures and the special characteristics of acetic acid diffusion. Conclusions which are relevant to the practice of cocoa fermentation are discussed in more detail.     

咖啡主要烘焙风味物质的形成及变化规律

咖啡以其独特的风味和功能特性受到人们的喜爱。烘焙温度及烘焙时间决定了咖啡的烘焙程度,并对咖啡风味物质的产生及变化产生重要影响。咖啡主要烘焙风味物质是与咖啡风味直接相关且代表性强的香气成分,本文通过综合分析烘焙程度与咖啡主要风味物质的形成和变化规律的关系,为系统了解和掌握精品咖啡烘焙工艺提供科学参考。      

The effects of the organic acids in cocoa on the flavour of chocolate

Samples (54) of dried fermented cocoa beans from different world regions were analysed for levels of organic acids, pH and titratable acidity. The effects of the organic acids on the flavour characteristics of cocoa were examined by sensory evaluation of chocolate made from samples of cocoa beans. Concentrations (g kg^?1) of acids ranged from 1.3 to 11.8 for acetic, 1-6 to 9-9 for citric, 0.6 to 11.1 for lactic and 2.1 to 6.5 for oxalic. pH values ranged from 4.6 to 5.8, while titratable acidity ranged from 0.08 to 0.31 equivalents of sodium hydroxide per kg sample. Cocoas from South East Asia and the South Pacific tended to be more acidic than West African beans in terms of both chemical and sensory characteristics. Lactic and acetic acids were found to be in greater concentrations in cocoas from the former regions and were considered to be largely responsible for higher acid flavour scores. In contrast, citric and oxalic acids were generally lower in these beans. Flavour assessments of cocoas with and without added organic acids indicated that oxalic acid played an important role in chocolate flavour. These results suggest that a reduction in the levels of acetic and lactic acids only, may not be sufficient to produce a desirable flavour balance.     

Extraction and determination of methylpyrazines in cocoa beans using coupled steam distillation-microdistillator

The formation of methylpyrazines was determined in fermented cocoa beans (Ivory Coast), in laboratory and industrially roasted samples. The determination of these methylpyrazines was studied by coupled steam distillation-microdistillation as the extraction method and gas chromatography using capillary column and a thermionic detector. The monomethyl-; 2,3-dimethyl-; 2,5-dimethyl-; 2,6-dimethyl-; trimethyl- and the tetramethylpyrazine were detected in non-roasted cocoa beans. Their concentration increased rapidly in laboratory roasted cocoa beans and industrial samples, only the tetramethylpyrazine showed a maximum peak of concentration. The principal compound was the tetramethylpyrazine in fermented and roasted cocoa beans. The determination of the different methylpyrazines in cocoa beans would permit both the evaluation of cocoa mass quality and the control of the cocoa roasting process.     

Comparison of enzyme activities involved in flavour precursor formation in unfermented beans of different cocoa genotypes

The activities of endoprotease, aminopeptidase, carboxypeptidase and invertase (cotyledon and pulp) were studied in unfermented beans of 10 genotype samples with different flavour characteristics (high and low cocoa flavour). Analysis of variance showed that significant differences in enzyme activities exist between certain genotypes. Aminopeptidase and endoprotease activities in beans of the PA7 genotype were higher than in all others. Principal component analysis (PCA) showed that the PA7 genotype (high cocoa flavour) was very different from the UIT1 genotype (low cocoa flavour). Although significant differences exist, no simple and general relationship is established between the flavour potential of a genotype and the level of key enzyme activities in unfermented beans. Carboxypeptidase is of key importance for peptide and free amino acid formation, but differences in enzyme activity could not be correlated to flavour potential of the genotype. It is suggested that the level of enzyme activities present in unfermented beans is not a limiting factor for optimal formation of flavour precursors during the fermentation process. © 2000 Society of Chemical Industry     

Effect of sugar, cocoa particles and lecithin on cocoa butter crystallisation in seeded and non-seeded chocolate model systems

The effect of major chocolate ingredients (sugar, cocoa particles and lecithin), in combination with the two pre-crystallization techniques, seeding and non-seeding, was investigated with respect to the kinetics of cocoa butter crystallisation and the resulting microstructure. Confocal laser scanning microscopy (CLSM) was used to monitor microstructural evolution under dynamic thermal conditions. DSC measurements and image analysis were also applied in order to quantify the impacts of processing and formulation on microstructure. All ingredients and pre-crystallisation techniques considered proved to have a large impact on fat crystallisation kinetics and the resulting microstructure. Seeded samples tended to form multiple nucleation sites, inducing rapid growth of a crystal network. The non-seeded samples showed an altering structure, with some domains developing large spherical crystals while in other domains a more heterogeneous microstructure resulted. Lecithin showed a remarkable impact on crystallisation kinetics in both the seeded and non-seeded samples. For the seeded samples, the effect was most noteworthy in samples containing cocoa butter and sugar, where lecithin significantly reduced the induction time. In the absence of seeds, lecithin itself acted as the nucleation site for fat crystallisation.     

Effect of autoclaving cocoa nibs before roasting on the precursors of the Maillard reaction and pyrazines

Nibs of raw cocoa (N) were roasted (R) at 150 °C for 38 min. Some samples were autoclaved at 121 °C for 15 min (A) and roasted (AR) at 150 °C for 38 min. The samples were then analysed for moisture content, water activity (a_w), reducing sugar and amino group (α-N) levels and, absorption at 275 nm (A₂₇₅) for a trichloro acetic acid (TCA) extract and at 280 nm (A₂₈₀) for the distillate. Tri- and tetramethylpyrazines were quantified by solid phase micro extraction-gas chromatography (SPME-GC). There was a reduction in the levels of α-N, in the order N > A > R > AR. The same sequence was observed for reducing sugars. The tetramethylpyrazine had an Area_pyrazine/Area_{internal standard} of N < R < A < AR and for trimethylpyrazine the order was N < A < R < AR. After autoclaving, the A_tetra/A_tri decreased from 31.30 (N) to 11.06 (A). With roasting, this ratio was 2.18 (R) and 2.58 (AR). Autoclaving nibs before roasting significantly influenced the levels of compounds which contribute to cocoa flavour formation and increased the concentration of tri- and tetramethylpyrazines in the headspace of cocoa samples. Autoclaving prior to roasting also affected the sensory properties of the samples.     

Water-soluble precursors of beef flavour: I. Effect of diet and breed

The effects of diet and breed on the concentration of water-soluble flavour precursors, namely sugars, free amino acids, ribonucleotides, creatinine, carnosine and creatine, were studied in beef longissimus lumborum muscle. Diet had a significant effect on the concentration of free amino acids, with animals fed on grass silage having higher free amino acid levels than animals fed on a concentrate diet, whereas animals fed concentrates had a higher total reducing sugar content. Differences between a beef breed (Aberdeen Angus×Holstein-Friesian) and a dairy breed (Holstein-Friesian) were generally small.     

Effects of pH and temperature on metmyoglobin solubility in a model system

From a series of experiments heating metmyoglobin solutions at pH 5.0 through 7.0, the effects of temperature and pH on the thermal stability of metmyoglobin were investigated. The percent metmyoglobin denatured at temperatures from 25 to 80 °C was determined. pHs lower than 6.5 caused metmyoglobin denaturation at various temperatures from 25 to 80 °C, but it was particularly apparent when pH was < 5.6. Thermal stability of metmyoglobin increased as pH increased. Metmyoglobin denaturation occurred at 55 °C at pH 5, however, denaturation did not occur until 60 °C at pHs from 5.3 to 7.0. A slower heating rate (0.9 °C/min) resulted in more metmyoglobin thermal denaturation than a faster heating rate (1.3 °C/min) when the temperature was above 55 to 60 °C. The denaturation caused by low pH alone was reversible, while that caused by high temperature was not. Techniques which increase muscle pH, such as the injection of sodium bicarbonate, could effectively improve the color condition of PSE meat.