Fermentation of cocoa beans: influence of microbial activities and polyphenol concentrations on the flavour of chocolate

BACKGROUND: Spontaneous cocoa bean fermentation is characterised by a succession of microbial activities. Cocoa flavour precursors are developed during fermentation and drying of cocoa beans. Polyphenols and alkaloids contribute to astringency and bitterness of cocoa and chocolate. RESULTS: Population dynamics, metabolite target analyses, and chocolate production were performed for seven independent spontaneous cocoa bean heap fermentations in Ghana. Although the same micro‐organisms were involved in these heaps, carried out at different farms or in different seasons, heap temperatures and microbial metabolite concentrations were different. This could be due to heterogeneity and size of the heaps, but was mainly ascribed to microbial variability. Indeed, differences in microbial activity could be linked with the flavour of chocolates made from the corresponding dried, fermented cocoa beans. Whereas the polyphenol and alkaloid contents of cocoa beans were crop‐ and heap‐dependent, epicatechin and theobromine levels decreased during fermentation due to diffusion out of the bean cotyledons and polyphenol oxidation and condensation. Residual levels were responsible for the degree of bitterness of the final chocolates. CONCLUSION: Differences in microbial activities between different heap fermentations can result in dried fermented cocoa beans and chocolates with different flavour characteristics. Hence, fermentation control may direct the flavour of chocolate. Copyright © 2008 Society of Chemical Industry     

IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARP

Myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55–60C as shown by SDS‐PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as α‐actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril‐bound serine proteinase.     

PURIFICATION AND CHARACTERIZATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF SILVER CARP

Recently, a myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was identified. MBSP could be dissociated from myofibrils by treatment at pH 4.0. Following ultrafiltration concentration and chromatography on Sephacryl S‐200, High Q ion‐exchange and affinity column of Arginine Sepharose‐4B, MBSP was partially purified. The enzyme with an estimated molecular weight of 28 kDa cleaves synthetic fluorogenic substrates specifically at the carboxyl sites of arginine and lysine residues. MBSP activity is suppressed by serine proteinase inhibitors such as Pefabloc SC, lima bean trypsin inhibitor and benzamidine; it is insensitive to Pepstatin, l ‐3‐carboxy‐trans‐2, 3‐epoxypropionyl‐l ‐leucine‐4‐guanidinobutylamide and ethylenediaminetetraacetic acid, suggesting MBSP is a trypsin‐like serine proteinase. Optimal profiles of pH and temperature of the enzyme are 8.5 and 55C, respectively. Hydrolysis of myofibrillar proteins such as myosin heavy chain, actin and tropomyosin by purified MBSP occurred especially at around 55C, consistent with our proposal that MBSP plays a significant role in the Modori phenomenon.     

Extraction,identification and semi-quantification of oligopeptides in cocoa beans

Peptides and amino acids generated during cocoa bean fermentation are known to be the most important precursors for the development of cocoa aroma. Although cocoa fermentation and aroma development have been extensively studied, the cocoa oligopeptide fraction is under-investigated. In particular, the identification of specific peptide sequences and the quantification of cocoa peptides are scarce in the literature. The aim of this study was to investigate the presence of oligopeptides in fermented cocoa beans. Peptides were analysed by reversed phase LC/ESI-MS and LC/ESI-MS/MS, and the molecular masses of 44 different peptides were obtained by analysing the mass spectra associated with the most intense chromatographic peaks. Peptides were identified based on the exact molecular masses, mass fragmentation patterns and by comparison with vicilin and 21 kDa cocoa seed protein sequences. Semi-quantitative data on peptide presence in fermented cocoa samples of different geographic origin, different fermentation levels and on roasted products were also provided.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

Factors influencing quality variation in cocoa (Theobroma cacao) bean flavour profile — A review

This review examined the factors that influence flavour volatiles of cocoa beans and the volume of work that needs to be done on these factors and their impact on the flavour volatiles of commercial cocoa beans. Cocoa bean flavour is one of the most important quality attributes as flavour is central to acceptability of cocoa beans and cocoa products such as chocolate. The complex composition of cocoa bean flavour depends on bean genotype, postharvest treatments such as pulp pre-conditioning, fermentation and drying, industrial processes such as roasting as well as the type of soil and age of cocoa tree. The bean genotype determines the chemical composition of the bean, specifically the contents of bean storage proteins, polysaccharides, and polyphenols. This determines the quantities and type of precursors formed during fermentation and drying processes leading to flavour formation, hence, influencing both flavour type and intensity. Cocoa bean fermentation and drying result in the breakdown of the storage proteins by endogenous proteases into amino acids and short chain oligopeptides while the polysaccharides are also degraded by invertase to glucose and fructose. The amino acids, oligopeptides, glucose and fructose react with each other during the roasting process to produce the typical cocoa flavour volatiles. Polyphenols are also oxidized by polyphenol oxidase during fermentation and drying which reduce the astringency and bitterness of the beans, thus, enhancing the flavour of cocoa beans. However, the extent to which other factors such as age of the cocoa tree and soil chemical compositions influence the formation of flavour precursors and their relationships with final flavour quality remains unclear. With increasing demand for sustainable production of high quality cocoa beans, greater understanding of factors contributing to the variations in flavour character would have significant commercial implications.     

Pod storage time and spontaneous fermentation treatments and their impact on the generation of cocoa flavour precursor compounds

Most of volatile flavour compounds occurred during the cocoa postharvest operations including pod storage and fermentation whom effects on these metabolites were aimed by the present study. Three different spontaneous fermentation treatments were performed on the farm, and the main isolated yeasts were identified using PCR-DGGE technique. Fermented cocoa beans were solar-dried, and their flavour compounds were identified using solid phase microextraction technique–gas chromatography–mass spectrometry. The main results showed that Pichia kudriavzevii was common yeast involved in the cocoa fermentation. Thirty flavour precursor compounds grouped into six chemical families including alcohols and pyrazines were identified in raw cocoa samples. ACP showed that short durations of pod storage and fermentation promote the formation of flavour precursors of cocoa. No spontaneous cocoa fermentation treatment influenced the occurrence of aroma compounds. The generation of flavour compounds depends only on the pod storage and the fermentative microorganisms of cocoa.     

Preparation and characterisation of glyceollin‐enriched soya bean protein using solid‐state fermentation

Glyceollins, stress‐induced phytoalexins from parent soya bean isoflavones, were elicited with Aspergillus oryzae. This solid‐state fermentation facilitated the conversion of isoflavone glycosides into aglycones and glyceollins, which could mainly enrich in soya bean protein isolate (SPI) during protein preparation due to protein–polyphenol interactions. Glyceollin‐enriched SPI exhibited less flavour volatiles, higher solubility, lower whiteness and higher antioxidant activity than unfermented SPI. Fermented SPI was more easily to be digested during in vitro pepsin–trypsin digestion. This may be attributed to partial degradation of protein, especially α′ and α subunits of β‐conglycinin and acidic subunit of glycinin. The antioxidant activity of digestive products derived from fermented SPI was obviously enhanced with increasing digestion time due to simultaneous release of antioxidant peptides and glyceollins involved in the interior of protein molecule. These results suggest an effective technique for producing a nutrient‐enhanced SPI as novel functional ingredients applied in food industry.     

Variations of peroxidase activity in cocoa (Theobroma cacao L.) beans during their ripening,fermentation and drying

An increase of peroxidase activity in the seeds of cocoa (Theobroma cacao L.) during their ripening has been determined. An additional increase of the peroxidase activity (about 10 times) is observed during the fermentation and drying of the beans. The residual activity determined in the cocoa beans after sun-drying was higher than that in unfermented ripe seeds. The major cocoa isoperoxidase was shown to be an acidic enzyme with pI 4.7. Using isoelectrofocusing, the appearance of two basic isoenzymes of the peroxidase with pI 8.6 and 9.0 during the process of the fermentation has been detected. ©     

Flavor Chemistry of Cocoa and Cocoa Products—An Overview

Cocoa originates from beans of the cocoa tree (Theobroma cacao L.) and it is an important commodity in the world and the main ingredient in chocolate manufacture. Its value and quality are related to unique and complex flavors. Bulk cocoas (Forastero type) exhibit strong basic cocoa notes, whereas fine varieties (Criollo, Nacional) show aromatic, floral, or smoother flavor characteristics. About 600 various compounds (alcohols, carboxylic acids, aldehydes, ketones, esters, and pyrazines) have been identified as odor‐active components. The specific cocoa aroma arises from complex biochemical and chemical reactions during the postharvest processing of raw beans, and from many influences of the cocoa genotype, chemical make‐up of raw seeds, environmental conditions, farming practices, processing, and manufacturing stages. There has been much research on cocoa flavor components. However, the relationships between all chemical components that are likely to play a role in cocoa flavor, their sensory properties, and the sources and mechanisms of flavor formation are not fully understood. This paper provides an overview on cocoa flavor from a compositional and a sensory perspective. The nonvolatile and volatile chemical components of cocoa and chocolate flavor, and their sensory properties correlated to the main influences involved in flavor formation, are reviewed.     

Potential of lactic acid bacteria to improve the fermentation and quality of coffee during on‐farm processing

This study describes, for the first time, the potential use of selected lactic acid bacteria (LAB) to conduct improved coffee bean fermentation during on‐farm wet processing. Among different strains tested, Lactobacillus plantarum LPBR01 showed a suitable production of organic acids and flavour‐active esters in a coffee‐pulp simulation medium and was used as starter culture under field conditions. The results indicated that L. plantarum LPBR01 was able to establish an accelerated coffee‐pulp acidification process and potentially reduced the fermentation time from 24 to 12 h. The inoculation of LPBR01 strain also increased significantly the formation of volatile aroma compounds during fermentation process (such as ethyl acetate, ethyl isobutyrate and acetaldehyde) and enabled the production of beverage with distinct sensory notes and a remarkable increase in quality compared to the conventional process. Our results suggest that the use of LAB in coffee processing is an ideal alternative way to conduct faster and improved coffee bean fermentation.     

Improving Malaysian cocoa quality through the use of dehumidified air under mild drying conditions

BACKGROUND: Various studies have been conducted in the past to improve the quality of Malaysian cocoa beans. However, the processing methods still remain crude and lack technological advancement. In terms of drying, no previous study has attempted to apply advanced drying technology to improve bean quality. This paper presents the first attempt to improve the quality of cocoa beans through heat pump drying using constant air (28.6 and 40.4 °C) and stepwise (step‐up 30.7–43.6–56.9 °C and step‐down 54.9–43.9 °C) drying profiles. Comparison was made against hot air drying at 55.9 °C. RESULTS: Product quality assessment showed significant improvement in the quality of Malaysian cocoa beans. Quality was found to be better in terms of lower acidity (higher pH) and higher degree of browning (cut test) for cocoa beans dried using the step‐up profile. All heat pump‐dried samples showed flavour quality comparable to that of Ghanaian and better than that of Malaysian and Indonesian commercial samples. Step‐up‐dried samples showed the best flavour profile with high level of cocoa flavour, low in sourness and not excessive in bitterness and astringency. CONCLUSION: Dried cocoa samples from the step‐up drying profile showed the best overall quality as compared with commercial samples from Malaysia, Indonesia and Ghana. The improvement of Malaysian cocoa bean quality is thus achievable through heat pump drying. Copyright © 2010 Society of Chemical Industry     

Fatty acid and triacylglycerol composition and thermal behaviour of fats from seeds of Brazilian Amazonian Theobroma species

Raw materials for cocoa butter substitutes, replacements or equivalents depend mostly on the unsteady supply from wild stands of plants, while there is no current supply of Neotropical origin. Seed fats from Theobroma species (T cacao, T bicolor, T grandiflorum, T obovatum, T subincanum, T speciosum, T sylvestre and T microcarpum, plus the closely related species Herrania mariae) were analysed for fatty acid and triacylglycerol composition by gas and liquid chromatography respectively, for iodine value, for melting point by open capillary tube and for solid fat content (SFC) by nuclear magnetic resonance. All Theobroma species had significantly lower palmitate levels than T cacao, except for T sylvestre and T speciosum, T microcarpum presented highly unsaturated fat (C18:2), while H mariae had high levels of arachidate. Fats from T sylvestre and T speciosum had a similar iodine value to T cacao and a higher melting point. No fat from the other species presented a similar melting profile to cocoa butter. T sylvestre and T bicolor were the most similar to T cacao but had a higher SFC at human body temperature. T sylvestre and T speciosum seed fats had more POP than cocoa butter. Fats from seeds of T speciosum, T sylvestre and T bicolor could be recommended as cocoa butter substitutes, while fats from species of the section Glossopetalum could be employed in products requiring fats with a lower melting point. © 2002 Society of Chemical Industry     

Biochemical changes in fermenting African locust bean (Parkia biglobosa) during 'iru' fermentation

The enzymic activities and biochemical changes in the principal food constituents of African locust bean were investigated. The reducing sugar level increased from 63 mg/g to 134 mg/g during the first 24 hr but subsequently decreased. Amylase activity was not detected. The lipase activities were detectable with the peak at 48 hr after the start of fermentation. The most significant activity during the fermentation is the rapid and steady increase in the quantity of free amino acids throughout the fermentation. This is due to a consistently active proteinase activity by the fermentative microorganisms. The number and quantities of each amino acids analysed also increased in the fermented beans. Glutamic acid, valine, aspartic acid and alanine were rapidly liberated during the fermentation. The changes observed are compared with the fermentation of other protein rich seeds.     

黑豆多肽的制备及其对乙醇脱氢酶活性的 影响研究

目的制备黑豆多肽,研究其对乙醇脱氢酶(alcohol dehydrogenase,ADH)活性的影响。方法采用碱性蛋白酶与胰蛋白酶分步水解法制备黑豆多肽。用截留分子量分别为5000、3500、2000、1000 Da的透析袋将黑豆多肽分离成4个相对分子量的肽段,对不同相对分子量的黑豆多肽进行抗氧化能力以及体外醒酒活性实验。结果黑豆多肽在碱性蛋白酶与胰蛋白酶分步水解后的水解度达35.64%。不同分子量的黑豆多肽均具有对乙醇脱氢酶的激活作用和对羟自由基(·OH)的清除能力,且都随着黑豆多肽相对分子质量的减小而增强。相对分子质量在1000 Da以下的黑豆多肽对乙醇脱氢酶的激活率和羟自由基(·OH)的清除能力最强,分别达22.43%和24.39%。结论黑豆多肽具有较强的乙醇脱氢酶激活率,并且与黑豆蛋白质和其他来源的多肽相比,相对分子质量在500~2000 Da的黑豆多肽中亮氨酸和丙氨酸的含量较多,因此推测黑豆多肽具有较好的醒酒效果。     

Effect of the solid state fermentation of cocoa shell on the secondary metabolites,antioxidant activity,and fatty acids

During cocoa (Theobroma cacao L.) processing, the accumulated cocoa shell can be used for bioconversion to obtain valuable compounds. Here, we evaluate the effect of solid-state fermentation of cacao flour with Penicillium roqueforti on secondary metabolite composition, phenol, carotenoid, anthocyanin, flavonol, and fatty acids contents, and antioxidant activity. We found that the total concentrations of anthocyanins and flavonols did not change significantly after fermentation and the phenolic compound and total carotenoid concentrations were higher. The fermentation process produced an increase in saponin concentration and antioxidant activity, as well as significant changes in the levels of oleic, linoleic, gamma-linolenic, and saturated fatty acids. Based on our findings, we propose that the reuse of food residues through solid state fermentation is viable and useful.     

Angiotensin I-converting enzyme inhibitory activity of enzymatic hydrolysates of goat milk protein fractions

Casein and whey protein fractions from goat milk were hydrolysed by subtilisin and trypsin, individually and in combination, to release angiotensin converting enzyme (ACE)-inhibitory peptides. Selected hydrolysates were fractionated by size exclusion chromatography (SEC) and further characterised. The highest ACE-inhibitory activity was obtained from the casein fraction hydrolysed by the combination of enzymes. SEC presented 4 fractions with fraction F2 (<2.3 kDa) containing the highest concentration of peptides and the highest activity. F2 contained a number of peptides not previously identified from caprine caseins but with structural similarity to other ACE-inhibitory peptides. The most active fraction in relation to protein content was F4 with IC₅₀ between 9.3 and 5.1 μg mL⁻¹. This fraction contained a compound tentatively identified as WY, an active dipeptide not previously reported from caseins. The high inhibitory capacity of these fractions points towards the advantage of implementing a membrane process to concentrate the most active peptides.     

Response of intestinal proteinase activities to the feeding of isolated winged bean (Psophocarpus tetragonolobus) and soya bean (Glycine max) trypsin inhibitors in rats

The antinutritional activities of trypsin inhibitors (TIs) were compared between winged beans (Psophocarpus tetragonolobus) and soya beans (Glycine max). The inhibitors of the two beans were isolated by trypsin‐bound Sepharose 4B, and 50 mg of lyophilised powders were intubated intragastrically into 24 h fasted rats. The activities of trypsin and chymotrypsin were compared after 30, 60 and 180 min in the washings of the upper, middle and lower parts of the small intestine. The elution profiles of TI and non‐TI compounds in the affinity chromatography were similar in the two beans, and the antitryptic activities were concentrated 5.5 and 6.2 times (based on specific activity) for winged beans and soya beans respectively. Regardless of the TI fed to rats, trypsin activity in the upper intestine was suppressed to almost undetectable levels at 30 and 60 min after intubation. The activities in the middle and lower intestines were also substantially lowered when rats were fed winged bean TI, and significant differences were detected at 30 and 60 min after intubation when compared with rats fed soya bean TI. However, at 180 min after feeding, no differences were found in the trypsin activity in any gut segments. Similar inhibitory properties of isolated TIs were observed in chymotrypsin activities in the small intestine. The results suggest that winged bean TI may have greater inhibitory activity on the intestinal proteinase compared with soya bean TI. © 2001 Society of Chemical Industry     

Individual variation in the amino acid and chemical composition of defatted cocoa bean meal of three varieties of cocoa (Theobroma cacao) from south-eastern Nigeria

Cocoa in the world market is the ripened bean of the cocoa tree (Theobroma cacao L) that has been properly fermented and dried. A number of varieties are found such as Amelonado and F₃Amazon which are considered ‘bulk’ cocoas by the FAO. Chocolate produced by these cocoas does not, possess the additional flavours associated with the ‘finey’ types such as Trinitario and Criollo. Literature exists on the composition of cocoa bean meal but the work reported has been on bulk samples received by chocolate manufacturers or found in warehouses. These are beans harvested from several trees and of varied and unknown species growing in different localities. Therefore differences due to environmental and varietal factors will be masked. This work, which was carried out on cocoas from south-eastern Nigeria, is aimed at highlighting the variation in composition (if any)of the cocoa bean between (a)individual species, (b) individual trees of each species planted at the same time and in the same geographical area, (c) individual trees of the same species, planted in the same area but having an age difference of some ten years.     

Potentiality of Bacillus amyloliquefaciens KFCC11574P isolated from Korean traditional doenjang as a starter in the production of functional soya bean paste

Proteolytic activity, fibrinolytic activity and angiotensin converting enzyme (ACE)‐inhibitory activity, volatile compound profile, off‐flavour compound and sensory characteristics in soya bean paste made with Bacillus amyloliquefaciens KFCC11574P (KHG19) strain were compared with those in three different commercial soya bean paste samples. The KHG19 strain was isolated from doenjang, a traditional Korean soya bean paste, and the maximum dominance in soya bean paste prepared in this study was 68.8% after 2 months of ageing. Proteolytic and fibrinolytic activities in soya bean paste made with KHG19 strain (KHG19 soya bean paste) were higher than those in commercial soya bean pastes. The maximum proteolytic activity (2.47 cm²) was observed after 3 months of ageing, and the maximum relative fibrinolytic activity (236.98%) was appeared after 2 months of ageing. ACE‐inhibitory activity in KHG19 soya bean paste was similar to those in commercial samples. The volatile compound profile of KHG19 soya bean paste was closest to that of cheonggukjang according to the result from principal component analysis. The sensory score for off‐flavour character was significantly low in KHG19 soya bean paste (2.9) compare with the score (6.8) for Korean traditional doenjang. KHG19 strain can be used for the production of functional soya bean paste that would be affordable to global consumer.