Biochemical fate of vicilin storage protein during fermentation and drying of cocoa beans

Key cocoa-specific aroma precursors are generated during the fermentation of cocoa beans via the proteolysis of the vicilin-like globulin. Previous studies had shown that degradation of this particular 566 amino acid-long storage protein leads to three distinct subunits with different molecular masses. Although oligopeptides generated from the proteolysis of vicilin-like globulin have been studied previously, changes occurring to vicilin at different stages of fermentation have not yet been explored in detail. The aim of this study was to investigate the fate of vicilin protein from the non-fermented stage up to the dried cocoa beans. Our results showed a remarkable shift in the electrophoretic mobility of vicilin towards higher pI during the onset of fermentation. The pI-shifted subunit was found susceptible to further degradation into a lower-molecular-weight vicilin subunit. The observed pI shift correlated with, but did not depend on protein phosphorylation. Glycosylation of some but not all vicilin subunits occurred at different stages of the fermentation process. Peptides generated from vicilin throughout fermentation were analyzed by UHPLC-ESI-MS/MS revealing an initial increase and subsequent decrease in the diversity of peptides with an increasing degree of fermentation. We furthermore describe the rate of degradation of different vicilin subunits. The detected diversity and dynamics of vicilin peptides will help to define biochemical markers of distinct steps of the fermentation process.     

Antidiabetic,angiotensin-converting enzyme inhibitory and anti-inflammatory activities of fermented camel milk and characterisation of novel bioactive peptides from lactic-fermented camel milk with molecular interaction study

This study was to separate and purify antidiabetic and angiotensin-converting enzyme (ACE) inhibitory peptides from Lactiplantibacillus plantarum KGL3A fermented camel milk. After 48 h of fermentation at 37°C, ɑ-amylase inhibition, ɑ-glucosidase inhibition, lipase inhibition and ACE inhibitory activities were 80.94%, 64.45%, 63.93%, and 77.53%, respectively in fermented camel milk. Optimisation of growth condition for the evaluation of maximum peptide production was evaluated by measuring proteolytic activity (O-phthalaldehyde, OPA method) with different inoculation rates and incubation times and highest proteolytic activity (9.21 mg/mL) was observed after 48 h of fermentation at 2.5% rate of inoculation. The antidiabetic and ACE inhibitory activity of 3 kDa permeate fraction were higher as compared with other fractions. Purification of antidiabetic and ACE inhibitory peptides from fermented camel milks was performed through sodium dodecyl-sulphate polyacrylamide gel electrophoresis, and 2-dimensional polyacrylamide gel electrophoresis and maximum number of protein bands were present in between 25 and 10 kDa. The generated peptide sequences were matched with antihypertensive peptide database (AHTPDB) and BIOPEP databases for confirming the antidiabetic and ACE inhibitory activity. Peptides, that is. TDVMPQWW and MMSLVSLLLVGILFPTIQAK were having highest peptide ranker score among the all sequences. Furthermore, anti-inflammatory activity (TNF-α, IL-6 and IL-1β) of fermented camel milk was evaluated in macrophage cell line RAW 264.7 (Ralph and William's cell line). Furthermore, peptides were predicted to have improved binding affinity against Human Angiotensin converting enzyme (hACE) through molecular docking.     

Proposing sequences for peptides derived from whey fermentation with potential bioactive sites

In fed-batch fermentation by Kluyveromyces marxianus var. marxianus, whey-soluble proteins were converted into oligopeptides. To assess whether bioactive peptides could be produced during whey fermentation, K. marxianus was cultured in batch in deproteinized media containing 5 or 15% (wt/vol) dehydrated whey for 20 h and then was in fed-batch mode for 50 h. After harvesting the biomass (25,000 x g, 15 min), at 6-h intervals, the wort was analyzed to determine protein consumption and oligopeptide production by HPLC. The proteins in the wort showed an oscillatory degradation with a constant increase in the production of oligopeptides. Four major peaks were collected and were analyzed by API mass spectroscopy. Sequences of fermented peptides were compared with sequences of known bioactive peptides. On the basis of their molecular weights, two amino acid sequences were proposed. The presence of sites containing the peptide sequence of beta-lactorphin (YLLF) suggests that these oligopeptides may have antihypertensive properties.     

Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate

The sensory quality of chocolate is widely determined by the qualitative and quantitative composition of volatile compounds resulting from microbial metabolism during fermentation, and Maillard reactions taking place during drying, roasting and conching. The influence of applying mixed starter cultures on the formation of flavour precursors, composition of volatile aroma compounds and sensory profile was investigated in cocoa inoculated with cultures encompassing a highly aromatic strain of Pichia kluyveri or a pectinolytic strain of Kluyveromyces marxianus, and compared to commercially fermented heap and tray cocoa. Although only minor differences in the concentration of free amino acids and reducing sugars was measured, identification and quantification by dynamic headspace gas chromatography–mass spectrometry (HS/GC–MS) revealed pronounced differences in the composition of volatiles in roasted cocoa liquors and finished chocolates. 19 of the 56 volatile compounds identified in the chocolates were found in significantly higher amounts in the tray fermented sample, whilst significantly higher amounts of 2-methoxyphenol was measured in the two inoculated chocolates. The P. kluyveri inoculated chocolate was characterized by a significantly higher concentration of phenylacetaldehyde and the K. marxianus inoculated chocolate by significantly higher amounts of benzyl alcohol, phenethyl alcohol, benzyl acetate and phenethyl acetate compared to a spontaneously fermented control. Sensory profiling described the heap and tray fermented chocolates as sweet with cocoa and caramel flavours, whilst the inoculated chocolates were characterized as fruity, acid and bitter with berry, yoghurt and balsamic flavours. The choice of fermentation technique had the greatest overall impact on the volatile aroma and sensory profile, but whilst the application of starter cultures did affect the volatile aroma profile, differences were too small to significantly change consumer perception of the chocolates as compared to a spontaneously fermented control.     

Aroma precursors and methylpyrazines in underfermented cocoa beans induced by endogenous carboxypeptidase

Cocoa-specific aroma precursors and methylpyrazines in underfermented cocoa beans obtained from fermentation induced by indigenous carboxypeptidase have been investigated. Fermentation conditions and cocoa bean components were analyzed during 0 to 3 d of fermentation. Underfermented cocoa beans were characterized as having hydrophilic peptides and free hydrophobic amino acids much higher than unfermented ones. These 2 key components of cocoa aroma precursors may be produced from the breakdown of proteins and polypeptides by endogenous carboxypeptidase during the fermentation process. The enzyme was activated during fermentation. Polypeptides of 47, 31, and 19 kDa were observed in the samples throughout the 3-d fermentation period; however, only the first 2 polypeptides were remarkably reduced during fermentation. Since the 1st day of fermentation, underfermented cocoa beans contained methylpyrazines, a dominant group of cocoa-specific aroma. This might be due to microbial activities during fermentation, observed through a decrease of pH value and an increase of temperature of cocoa beans. The concentration of tetramethylpyrazines was significantly increased during the 3 d of fermentation. This may increase the cocoa-specific flavor to the beans.     

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

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.     

Bioactive peptides and lactic fermentations

Milk proteins contain regions within their primary structures that encrypt for many latent biological activities. The beneficial health effects associated with some fermented dairy products may, in part, be attributed to the release of bioactive peptide sequences during the fermentation process. Peptides displaying opioid, mineral binding, cytomodulatory and hypotensive activities, for example, have been identified in cheese and yogurt. Much effort has to date concentrated on the release of angiotensin-converting enzyme (ACE) inhibitory peptides due to their potential to act as hypotensive agents. Peptide fractions obtained by hydrophobic interaction chromatography of different cheese varieties (Blue, Camembert, Edam, Emmental, Gouda and Havarti) were reported to give systolic blood pressure (SBP) decreases in spontaneously hypertensive rats (SHR) ranging from 7.1 to 29.3 mm mercury (Hg). Skim milks fermented with various strains of Lactobacillus helveticus , and in one case also with Saccharomyces cerevisiae , have been reported to display SBP decreases in mildly hypertensive human volunteers ranging from 4.6 to 14.1 mmHg. These human hypotensive effects have, in part, been attributed to the release of potent casein-derived tripeptide inhibitors of ACE during fermentation. In general, the likelihood of any bioactive peptide released during fermentation mediating a physiological response is dependent on the ability of that peptide to reach an appropriate target site. Therefore, peptides may need to be resistant to further degradation by gastrointestinal and serum proteinases/peptidases following oral ingestion in order to display a functional food effect.     

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.     

锡盟蒙古族传统发酵马乳中功能性特征肽段的研究

乳基中的小分子活性肽的相关研究,近几年被重点关注,它可以提高机体免疫能力,是调节生理代谢的功能成分。发酵马乳已被证实具有较高的营养价值和医疗保健作用。该研究针对锡盟蒙古族传统发酵马乳,用高效凝胶过滤色谱与超高效液相色谱-电喷雾串联三重四极杆质谱分析发酵马乳样品中蛋白水解物的分子质量分布,并对主要的功能二肽进行定性定量检测。研究结果表明,发酵后马乳的分子质量在1000 Da以下的蛋白水解物比例有显著提升,检测出24条具有潜在血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制功能的二肽,标记含量较高且有功能活性的肽段VP、AF为该发酵马乳样品的特征肽段,并对以上功能肽段的构效关系进行探讨。该研究为进一步开发来源于蒙古族传统发酵食品中的功能乳肽提供参考。     

Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase

Peptides from hydrolysates of fish proteins and from cheeses were analysed for inhibition of prolyl endopeptidase (PE) isolated from porcine muscle. Muscles of cod, salmon, and trout were homogenised and incubated at pH 4.0 with pepsin and then at pH 7.5 with trypsin to obtain fish protein hydrolysates. Homogenates were incubated without exogenous enzymes at pH 4.0 and 7.5 to obtain fish protein autolysates. Water-soluble extracts from "rakfisk" (a Norwegian fermented/autolysed trout muscle dish) and water-soluble extracts from Cheddar, Norvegia, Jarlsberg, and Blue cheese were also prepared. Peptides in the supernatants obtained after heat-treatment of fish hydrolysates, autolysates and water-soluble extracts of rakfisk and cheeses at 95 degrees C for 15 min were analysed for inhibition of PE. Inhibition was also measured in peptide fractions separated by reversed-phase high-performance chromatography and by gel permeation chromatography. The peptide fractions from fish hydrolysates, fish autolysates, and water-soluble extracts of cheeses inhibited PE in hydrolysing Z-Gly-Pro-amidomethylcoumarin. Inhibition by peptides from rakfisk was negligible. Pepsin + trypsin hydrolysates from the three fish species contained PE inhibitory peptides with a broad range of apparent hydrophobicity and apparent molecular mass. Autolysates from muscles of the 3 fish species contained narrow peptide peaks of different molecular mass and different apparent hydrophobicity with strong PE inhibitory activity. The content of hydrophilic inhibitory peptides was lower in cheeses than in pepsin + trypsin hydrolysates of fish muscle.     

Ghanaian Cocoa Bean Fermentation Characterized by Spectroscopic and Chromatographic Methods and Chemometrics

Abstract: Export of cocoa beans is of great economic importance in Ghana and several other tropical countries. Raw cocoa has an astringent, unpleasant taste, and flavor, and has to be fermented, dried, and roasted to obtain the characteristic cocoa flavor and taste. In an attempt to obtain a deeper understanding of the changes in the cocoa beans during fermentation and investigate the possibility of future development of objective methods for assessing the degree of fermentation, a novel combination of methods including cut test, colorimetry, fluorescence spectroscopy, NIR spectroscopy, and GC-MS evaluated by chemometric methods was used to examine cocoa beans sampled at different durations of fermentation and samples representing fully fermented and dried beans from all cocoa growing regions of Ghana. Using colorimetry it was found that samples moved towards higher a* and b* values as fermentation progressed. Furthermore, the degree of fermentation could, in general, be well described by the spectroscopic methods used. In addition, it was possible to link analysis of volatile compounds with predictions of fermentation time. Fermented and dried cocoa beans from the Volta and the Western regions clustered separately in the score plots based on colorimetric, fluorescence, NIR, and GC-MS indicating regional differences in the composition of Ghanaian cocoa beans. The study demonstrates the potential of colorimetry and spectroscopic methods as valuable tools for determining the fermentation degree of cocoa beans. Using GC-MS it was possible to demonstrate the formation of several important aroma compounds such 2-phenylethyl acetate, propionic acid, and acetoin and the breakdown of others like diacetyl during fermentation. Practical Application: The present study demonstrates the potential of using colorimetry and spectroscopic methods as objective methods for determining cocoa bean quality along the processing chain. Development of objective methods for determining cocoa bean quality will be of great importance for quality insurance within the fields of cocoa processing and raw material control in chocolate producing companies.     

Activation of remaining key enzymes in dried under-fermented cocoa beans and its effect on aroma precursor formation

Incubation-activation of remaining key enzymes in dried under-fermented cocoa beans and its effect on aroma precursor formation has been studied using defatted unfermented and partly fermented cocoa bean powders. Results of the study showed that aspartic endoprotease, carboxypeptidase and invertase were significantly inactivated during fermentation and drying, and the effect of fermentation was significantly lower than that of drying. The enzyme activities remaining in these beans were still sufficient to carry out enzymatic reaction during incubation. Peptide patterns, resulting from incubation of unfermented and partly fermented beans powders, were quite similar to the well-fermented patterns. Meanwhile, free amino acid concentrations of the unfermented beans were significantly increased during the first 4 h of incubation and then remained constant; however, with partly fermented beans, the formation continued and the hydrophobic and total free amino acid concentrations reached the value of well-fermented beans after 24 h of incubation. Reducing sugar concentrations of both unfermented and partly fermented cocoa beans could reach the level of well-fermented beans by incubation.     

Prediction of peptides in the fermented cocoa storage protein,with reference to the modelled 3D structure

The amino acid sequence and a model for the three-dimensional (3D) structure of the cocoa storage protein have been combined with available data from protease digestions to aid understanding of factors that contribute to the characteristic cocoa aroma. Data reported for the free amino acids liberated during extensive digestions, modelling the fermentation process, are compared to computer predictions. Since good agreement is obtained between experiment and theory for the free amino acids, the modelling can be used to study properties not yet reported experimentally. In particular, the peptides that remain after digestion are predicted, highlighting those sequences which may play important roles in the production of cocoa aroma. This approach could prove equally useful in understanding other such systems. © 1998 SCI.     

The Precursors of Chocolate Aroma: Production of Free Amino Acids during Fermentation of Cocoa Beans

SUMMARY— Amino acids are produced during the fermentation of cocoa beans at a rate which is in agreement with the already established rapid rate of flavour and aroma development. The role of these compounds as aroma precursors receives further confirmation as a result of these studies, and an objective method of assessing the "degree of fermentation" of cocoa beans has been described and tested on a number of different commercial varieties of cocoa.     

不同加工工艺对蒲公英叶茶品质及抗氧化活性的影响

以蒲公英叶为研究对象,分别利用传统工艺、渥堆发酵以及添加β-葡萄糖苷酶发酵工艺加工成蒲公英绿茶和红茶,并通过汤色色差评价、功能性成分定量分析、香气组分分析、抗氧化能力分析以及感官评价,以期确定蒲公英叶茶加工的最佳工艺。经过β-葡萄糖苷酶发酵的蒲公英叶茶能获得较佳的汤色品质,未经过发酵工艺的蒲公英绿茶能保留较高的黄酮、多糖以及蛋白质成分,分别为1.42%、7.23%以及1.81%。未添加β-葡萄糖苷酶渥堆发酵的蒲公英红茶含有最多嗅感化合物,包括糠醇、5-甲基呋喃醛、大茴香醛、乙酸甲酯、苯乙酸甲酯、2-乙基丁酸、2-甲基四氢呋喃-3-酮、2,3-辛二酮等18种特有香气物质,主要表现为甜香、山楂、可可、肉类、爆米花等令人愉快的香气,为其获得较高感官评价香气评分奠定了物质基础,且其总抗氧化能力、DPPH清除率以及羟基自由基清除率分别为3.87 mmol/L FeSO₄·7H₂O、73.99%和34.21%,具有显著性的抗氧化能力。不同加工工艺制作的蒲公英叶茶品质及抗氧化活性存在差异性,其中渥堆发酵工艺较为适合蒲公英叶茶的制作。     

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.     

Investigations of peptides and proteases in green coffee beans

Cof. canephora var. robusta. The coffee types were not significantly different in peptide content, but were significantly different in peptide composition. Peptides with weakly acid pI values were mainly found in the Cof. robusta samples. In comparison, peptides from Cof. arabica samples ranged evenly from the weakly acidic up to the weakly basic pH range. The apparent molecular masses of the peptides in the two groups of samples lie between 4 kDa and 10 kDa. The cysteine content of the peptides was relatively high. Model roastings permitted the assumption that peptides make a contribution to coffee flavour. Peptides are already available in freshly harvested coffee. Model tests have suggested that processing after the coffee harvest has an influence on peptide composition but not on the amounts of peptide. All coffee samples contained some extractable proteases. The electrophoretically obtained enzyme patterns of Cof. arabica and Cof. robusta were different. Received: 6 September 1999     

ACE inhibitory and hydrolytic enzyme activities in textured vegetable protein in relation to the solid state fermentation period using Bacillus subtilis HA

Textured vegetable protein (TVP) was fermented by solid-state fermentation using Bacillus subtilis HA and production of biologically active compounds were examined with fermentation time. The TVP fermented resulted in the highest protease activity at 48 hr, where as α-amylase activity was the highest at 24 hr. During the fermentation the peptide and tyrosine content increases drastically and then it decrease gradually after 48 hr. The fast protein transition to low molecular peptides in early fermentation period was confirmed with sodium dedecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) analysis. Fibrinolytic enzyme activity proportionately increased with the fermentation time and the highest enzyme activity was 25.2 unit/g after fermentation for 120 hr. In the evaluation of ACE inhibitory effect, IC₅₀ value of fermented TVP and its crude peptides was similar with 2.19 and 1.21 mg/mL, respectively. Peptide fraction below 3,000 Da showed 0.5 mg/mL of IC₅₀ value. The peptide fraction (III) with molecular weight of 500–999 Da separated from SEC was consisted of 7 amino acids, indicated 0.094 mg/mL of IC₅₀ value which is 23 times higher than the fermented TVP.     

Profiles,antioxidative and ACE inhibitory activity of peptides released from fermented buttermilk before and after simulated gastrointestinal digestion

Bioactive peptides, released from buttermilk by fermentation and/or gastrointestinal proteases, may have health promoting effects. Thus, a comprehensive analysis of the peptide fraction of fermented buttermilk, before and after different phases of simulated gastrointestinal digestion, was performed using ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS). Results showed that digestion simulation substantially changed the peptide profile of fermented buttermilk. A total of 81, 120 and 46 peptides were identified in fermented buttermilk, its gastric and intestinal digests, respectively. These peptides released mostly from β-casein followed by αs1-casein, κ-casein and β-lactoglobulin. In addition, 14 peptides released from milk fat globule membrane proteins (lactadherin, butyrophilin and GlyCAM-1). Bioactivity, mainly angiotensin converting enzyme (ACE) inhibitory activity, has been reported before for only 54 of the detected peptides. Radical scavenging, ferric reducing and ACE inhibitory activities of fermented buttermilk peptides increased significantly after digestion, indicating promotion in fermented buttermilk-peptide bioactivity by gastrointestinal digestion.     

Angiotensin-converting enzyme inhibitory peptides from Lactobacillus delbrueckii QS306 fermented milk

Angiotensin-converting enzyme inhibitory peptides were isolated and identified from milk fermented using Lactobacillus delbrueckii QS306. The peptide with the highest angiotensin-converting enzyme inhibitory activity (C5) was purified using ultrafiltration with 10 and 3 kDa molecular mass cut-off membranes, Sephadex G-15 (Sigma-Aldrich, St. Louis, MO) gel filtration chromatography, reversed-phase HPLC, and Orbitrap Elite (Thermo Fisher Scientific Inc., Waltham, MA) liquid chromatography-tandem mass spectrometry. We obtained peptide LPYPY by microbial fermentation, which was derived from κ-casein f (AA 77–81). We synthesized LPYPY using an Fmoc solid-phase synthesis method and explored the secondary structure of the pentapeptide. The half maximal inhibitory concentration for the angiotensin-converting enzyme inhibitory activity of LPYPY was 12.87 μg/mL. The results provide additional information for ongoing research and the development of functional foods having antihypertensive effects.