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.     

IDENTIFICATION AND QUANTITATION OF SEVERAL NONVOLATILE ORGANIC ACIDS OF COCOA BEANS

SUMMARY— Organic acids precipitated as lead salts from water extracts of cocoa beans were converted to trimethylsilyl ethers. TMS ethers were then separated and identified using a combined gas chromatograph-mass spectrometer. Citric, phosphoric, lactic, oxalic, malic, tartaric, succinic and gluconic acids were present in every sample examined, irrespective of geographic origin. Five of the acids were quantitated using a GLC procedure which involved the preparation of methyl esters from freeze-dehydrated, water extracts. Concentration ranges found, g/100g beans, were: lactic, 0.11–0.71; oxalic, 0.24–0.43; succinic, 0.02–0.07; malic, 0.02–0.10; citric, 0.56–1.32. Concentrations of lactic acid were lowest and citric acid levels tended to be highest in commercial samples from countries where cocoa beans usually are not subjected to a planned fermentation. These trends were confirmed in an experiment involving beans from Trinidad which had undergone a carefully controlled fermentation. Only minor differences were found in organic acid concentrations between roasted and unroasted cocoa beans.     

Acidic Characteristics of Fermented and Dried Cocoa Beans from Different Countries of Origin

Thirty-nine fermented and dried cocoa bean samples from 13 countries were evaluated for pH, titratable acidity and concentrations of volatile and nonvolatile acids. The correlation coefficient between pH and log₁₀ titratable acidity was -0.94. Cocoa beans from Brazil and Far Eastern countries were highly acidic while those from Central American and South American countries were low in acidity. Samples from West African countries were intermediate with titratable acidity values from 0.12 to 0.15 meq NaOH/g sample and pH values from 5.20 to 5.49. Highly acidic beans were characterized by high concentrations of acetic and lactic acids. The high correlation between acetic acid and both pH (r=0.86) and titratable acidity (r = 0.91) indicated that this acid could be primarily responsible for high acidity in cocoa beans.     

Non-conventional fermentation at laboratory scale of cocoa beans: Using probiotic microorganisms and substitution of mucilage by fruit pulps

In a non-conventional lab-scale fermentation of cocoa beans using probiotic microorganisms and substituting the cocoa pulp for fruit pulp, physicochemical, microbiological and quality parameters were investigated. Two hundred grams of beans were fermented in a controlled environmental chamber (temperature ramp rate of 25°C for 48 h; 35°C for 48 h and 45°C for 48 h; and 65% HR). pH, titratable acid, citric, lactic and acetic acids, as well as sugars and ethanol were measured. A cut test was also performed on the cocoa beans fermented 5 and 6 days. As the fermentation time progressed, citric acid concentration decreased until 0.53 g kg⁻¹, whereas both lactic and acetic acids increased until 0.44 and 16.58 g kg⁻¹, respectively. Sucrose content decreased from 12.26 g kg⁻¹ (in fresh) to 6.54 g kg⁻¹ on the 6th day. Fructose and glucose contents increased in the cotyledons from day five, reaching a maximum concentration of 1.14 and 1.01 g kg⁻¹, respectively, on day six. Yeasts were the main microorganisms during the first 24–48 h (8.4 log CFU g⁻¹), while bacterial counts reached its highest number (7.8 log CFU g⁻¹) on day four. Beans fermented 5 and 6 days resulted in more fermented beans (>81%) and less violet ones (<18.4%) than the control.     

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.     

Spontaneous organic cocoa bean box fermentations in Brazil are characterized by a restricted species diversity of lactic acid bacteria and acetic acid bacteria

Spontaneous organic cocoa bean box fermentations were carried out on two different farms in Brazil. Physical parameters, microbial growth, bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the fermented dry cocoa beans. The main end-products of the catabolism of the pulp substrates (glucose, fructose, and citric acid) by yeasts, LAB, and AAB were ethanol, lactic acid, mannitol, and/or acetic acid. Lactobacillus fermentum and Acetobacter pasteurianus were the predominating bacterial species of the fermentations as revealed through (GTG)₅-PCR fingerprinting of isolates and PCR-DGGE of 16S rRNA gene PCR amplicons of DNA directly extracted from fermentation samples. Fructobacillus pseudoficulneus, Lactobacillus plantarum, and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Also, three novel LAB species were found. This study emphasized the possible participation of Enterobacteriaceae in the cocoa bean fermentation process. Tatumella ptyseos and Tatumella citrea were the prevailing enterobacterial species in the beginning of the fermentations as revealed by 16S rRNA gene-PCR-DGGE. Finally, it turned out that control over a restricted bacterial species diversity during fermentation through an ideal post-harvest handling of the cocoa beans will allow the production of high-quality cocoa and chocolates produced thereof, independent of the fermentation method or farm.     

Aspergillus spp., distribution, population composition and ochratoxin A production in wine producing vineyards in Greece

Vineyard surveys of 11 wine producing grape cultivars, were carried out in sixteen vineyards, in five winemaking regions in Greece, during 2002 and 2003. The occurrence of various Aspergillus spp. in bunches of berries at setting, veraison and ripening at harvest time was investigated. Aspergillus niger aggregate and A. carbonarius were predominantly isolated from sampled berries. Although the prevailing Aspergillus spp. isolates belonged to A. niger aggregate, isolates of A. carbonarius were the most efficient ochratoxin A (OTA) producers. Of 50 tested isolates of A. carbonarius 42% produced amounts of OTA, exceeding 25 ppb, while none of the 85 isolates of A. niger aggregate tested produced above 16 ppb.     

Hydrogen production by fermentation using acetic acid and lactic acid

Microbial hydrogen production from sho-chu post-distillation slurry solution (slurry solution) containing large amounts of organic acids was investigated. The highest hydrogen producer, Clostridium diolis JPCC H-3, was isolated from natural environment and produced hydrogen at 6.03+/-0.15 ml from 5 ml slurry solution in 30 h. Interestingly, the concentration of acetic acid and lactic acid in the slurry solution decreased during hydrogen production. The substrates for hydrogen production by C. diolis JPCC H-3, in particular organic acids, were investigated in an artificial medium. No hydrogen was produced from acetic acid, propionic acid, succinic acid, or citric acid on their own. Hydrogen and butyric acid were produced from a mixture of acetic acid and lactic acid, showing that C. diolis. JPCC H-3 could produce hydrogen from acetic acid and lactic acid. Furthermore, calculation of the Gibbs free energy strongly suggests that this reaction would proceed. In this paper, we describe for the first time microbial hydrogen production from acetic acid and lactic acid by fermentation.     

发酵前后甜石榴汁中有机酸的变化研究

本试验利用反相液相色谱（RP-HPLC）法分离测定发酵前后甜石榴汁中有机酸种类和含量。在甜石榴汁中检测到的有机酸包括草酸、苹果酸、α-酮戊二酸、乳酸、乙酸、柠檬酸与富马酸,其中主要有机酸为乳酸（占总有机酸的51.2%）、草酸（占总有机酸的26.8%）和柠檬酸（占总有机酸的16.8%）。发酵后甜石榴汁中的草酸与乳酸含量下降,乙酸含量增加,新生成酒石酸、丙酮酸与琥珀酸;苹果酸与柠檬酸含量增加。发酵后甜石榴汁的有机酸总量下降了7.8%.     

葡萄酒酿造过程中有机酸变化规律研究

采用离子色谱法分析了葡萄酒中9种有机酸含量。通过对赤霞珠葡萄酒发酵过程中主要有机酸变化分析,研究其从葡萄汁到原酒的酿造过程中有机酸含量变化规律和量变的幅度。结果表明,葡萄酒中有机酸主要有酒石酸、苹果酸、乳酸、柠檬酸和琥珀酸,葡萄汁经发酵产生了乳酸、乙酸和琥珀酸。酿造过程中酒石酸、苹果酸、抗坏血酸和柠檬酸含量一直呈下降趋势;草酸和琥珀酸含量先增高后减少,乳酸含量一直呈上升趋势。乙酸是葡萄酒酒精发酵的主要副产物,含量为0.2～0.3 g/L。     

Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations,underlining the importance of these microbial species for a successful cocoa bean fermentation process

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it.     

Ochratoxin A-producing fungi from grapes intended for liqueur wine production

The ochratoxigenic mycobiota of grapes intended for liqueur wines from four Spanish vineyards were studied. The specific wine-making technology of these wines requires overripening of the grapes on the vine or extended post-harvest exposure of the grapes in the sun. In every vineyard, samples were taken at three different developmental stages: veraison, harvesting time and after over-ripening. With the maturation of the berries there was a clear increase of Aspergillus spp. In the last sampling time studied, they were isolated from the 90.3% of the plated berries. Black aspergilli (mainly A. niger aggregate and A. carbonarius) were predominant among the different Aspergillus spp. isolated and constituted 98.5% of the total Aspergillus strains isolated. At harvesting time and after over-ripening, the percentage of colonized berries with A. carbonarius exceeded that of Aspergillus niger aggregate. Due to their low frequency of isolation, Penicillium spp. and Aspergillus spp. outside black aspergilli are not an important source of ochratoxin A in grapes for liqueur wine production. On the contrary, 98.5% of the A. carbonarius isolates screened were able to produce ochratoxin A. Although the possible participation of different ochratoxin A-producing species may occur, our results confirm that A. carbonarius is the most important source of ochratoxin A in liqueur wines, increasing its occurrence along the ripening of grapes.     

Dynamics of volatile and non-volatile compounds in cocoa (Theobroma cacao L.) during fermentation and drying processes using principal components analysis

Different volatile and non-volatile compounds produced during the fermentation-drying process are considered as indicatives of cocoa beans quality. We found thirty-nine different compounds identified by SPME-HS/GC-MS and related to the desirable notes and off-flavor that have been reported. Volatile and non-volatile compounds were associated with acidity and changes of pH, such as acetic and lactic acid. Using the principal component analysis (PCA), relations between compounds and fermentation and drying day were associated with dynamics of these compounds. The identification of principal compound produced during the fermentation and drying processes can be helpful in searching for off-flavor indicator and as a fermentation index, such as isobutyric, isovaleric and propionic acids. Oxidation of 3-methyl-1-butanol-to-3-methyl-1-butanol acetate can be of use in evaluating the degree of fermentation. At drying, the compounds with the highest levels were acetic and isobutyric acid, ethyl and 3-methyl-1-butanol acetate, pentanal and 2,3-pentanedione, and 1,3-butanediol and 2,3-butanediol. Therefore, acetic acid and isobutyric acid, due to their high levels and their low threshold value could play an important role in the aromatic quality of cacao drying.     

葡萄酵素发酵过程中代谢产物的动态变化

为探究葡萄酵素接种发酵过程中物质变化规律,以葡萄为原料,分析了接种酵母菌、醋酸菌以及乳酸菌发酵过程中总酸、有机酸、乙醇体积分数、总酚、主要功效酶（超氧化物歧化酶及β-葡萄糖苷酶）以及香气成分等代谢产物的变化。结果表明：发酵过程中总酸质量浓度呈先上升后下降的趋势,于第34天达到最大值19.37 g/L;在接种酵母菌（0～6 d）发酵过程中有机酸主要以柠檬酸、苹果酸、酒石酸为主,且乙醇主要产生于该阶段;接种醋酸菌后（6～27 d）主要产生乙酸;而后接种乳酸菌后（27～41 d）,乳酸、柠檬酸质量浓度逐渐增加至最大值102.09 mg/L和67.70 mg/L,而苹果酸减少,此时乙醇体积分数降低至最小值0.47%并趋于稳定;总酚含量与β-葡萄糖苷酶活力的变化趋势一致,主要于接种酵母菌（0～6 d）发酵以及接种乳酸菌发酵后期（34～41 d）产生,超氧化物歧化酶活力总体呈波动上升趋势;葡萄酵素发酵过程中产生的挥发性物质主要以醇类、酸类和酯类化合物为主。以上研究结果可为葡萄酵素的精准制备以及品质优化提供理论依据。     

Toxigenic fungi isolated from dried vine fruits in Argentina

To evaluate the potential for mycotoxin production by fungi in dried vine fruits, the mycobiota was determined both before and after surface disinfection. Predominant genera were Aspergillus (50.2%), Eurotium (21.4%) and Penicillium (13.5%). Aspergillus section Nigri ("black aspergilli") were isolated with relatively high frequency. Aspergillus niger was the most common species but only 3 of 293 isolates screened were ochratoxin A (OTA) producers. Aspergillus carbonarius was less common but 96% of 48 strains screened were ochratoxigenic. OTA was not produced by A. japonicus. Other toxigenic fungi detected were A. ochraceus (3 strains produced OTA), Aspergillus flavus (5 strains produced cyclopiazonic acid but not aflatoxins), P. citrinum (19 strains were strong citrinin producers) and Alternaria alternata (15 strains were producers of tenuazonic acid, alternariol and alternariol methyl ether). In spite of the high incidence of A. carbonarius capable of producing OTA, low levels of this toxin were detected in the samples analysed.     

Filamentous fungi producing ochratoxin a during cocoa processing in Cameroon

Ochratoxin A (OTA) is the main mycotoxin occurring in cocoa. A study was conducted in Cameroon to assess how filamentous fungi and toxigenesis were affected by the type of cocoa post-harvest treatment (boxes or heaps). The filamentous fungi isolated were almost identical when fermentation was carried out in boxes or heaps, with the presence of abundant black Aspergillus filamentous fungi: A. niger and A. carbonarius. Filamentous fungi were more abundant at the end of the harvesting season. Factors affecting bean integrity (poor handling, deferred processing) resulted in a qualitative and quantitative increase in contamination, when the total number of filamentous fungi could reach a maximum value of 5.5+/-1.4x10(7) CFU g(-1) and black Aspergilli a maximum value of 1.42+/-2.2x10(7) CFU g(-1). A toxigenesis study showed that Aspergillus carbonarius was the main OTA-producing strain isolated. Its maximum production could reach 2.77 microg g(-1) on rice medium. Aspergillus niger strains did not always produce OTA and their toxigenesis was much lower. Fermented dried cocoa from poor quality pods was the most contaminated by OTA: up to 48 ng g(-1).     

乳酸菌协同小麦胚芽油发酵对黑曲霉生长活性的影响

本文通过初筛和复筛考察不同乳酸菌的抗真菌活性,并将小麦胚芽油添加入乳酸菌培养液中进行发酵,旨在明确乳酸菌协同小麦胚芽油发酵对抗黑曲霉活性的影响。研究表明,小麦胚芽油不具有抑制黑曲霉生长的能力,而乳杆菌LF5发酵液抑菌率随着小麦胚芽油浓度的升高明显提升,且该现象具有菌株特异性。通过加热、中和pH、添加蛋白酶及过氧化氢酶发现,乳酸菌协同发酵上清液中的抗真菌活性物质主要由有机酸以及部分具有良好热稳定性的非蛋白类组分组成。此外,采用乳杆菌LF5协同小麦胚芽油发酵制备面包,在第5 d发现肉眼可见霉菌,与空白组(第2~3 d)相较具有显著抑制黑曲霉生长活性的作用,为乳酸菌在发酵米面食品中的应用提供了新的方向。     

Occurrence of ochratoxin A in wine and ochratoxigenic mycoflora in grapes and dried vine fruits in South America

Grape and wine production in South America represents about 6.6% and 10% respectively of the world grape and wine production. The available information on the ochratoxigenic mycoflora and ochratoxin A (OTA) presence in wine grapes, wines, grape juices and dried vine fruits is limited. Surveys have been carried out in Argentina and Brazil which showed that Aspergillus niger aggregate are predominant in the Argentinean varieties while from the Brazilian varieties the species A. niger, Aspergillus ochraceus and Aspergillus carbonarius were isolated. A mycobiota survey from wine grapes in Argentina showed that while Alternaria alternata was predominant, Aspergillus section Nigri species were isolated from 60% of samples. About 41% of black Aspergilli isolates produced OTA with levels ranging from 2 to 24.5 ng mL(-1). In another study, about 83% of A. carbonarius isolates from dried vine fruits produced OTA, with levels ranging from 2 to 5200 ng mL(-1). A survey of grape juices and wines of Brazilian, Argentinean and Chilean origin were found to contain very low levels of OTA. Studies are in progress in Latin America on the ecophysiology of ochratoxigenic fungi and OTA occurrence to reduce the impact of this toxin in the food chain.     

藏灵菇发酵奶发酵特性的研究

本文研究了藏灵菇发酵奶发酵过程中酸度、有机酸、挥发性风味物质及微生物数量的变化,在发酵0、4、8、12、16、20h时采样,用高效液相色谱(HPLC)跟踪发酵过程中乳酸、乙酸和柠檬酸的变化;用气相色谱(GC)跟踪了发酵过程中乙醇、丁二酮、乙醛和丙酮的变化。实验结果表明,乳酸和乙酸在发酵过程中都呈上升趋势,发酵4h时,乳酸的生成量约是乙酸的20倍,发酵结束乳酸的量为8.56g/L(TK1),7.73g/L(TK2);乙酸的量为0.71g/L(TK1),0.81g/L(TK2),乳酸的量约是乙酸的9～12倍。柠檬酸从发酵开始就呈下降趋势。挥发性风味物质中乙醇的生成量最大,发酵结束分别达到3134mg/L(TK1),4994mg/L(TK2);丁二酮和乙醛的量较普通酸奶高,分别达到62.4mg/L(TK1)和37.8mg/L(TK1)。本研究也对发酵过程中微生物进行了分析,发酵结束,发酵奶中乳杆菌达到2.3×109CFU/ml(TK1),1.3×108CFU/ml(TK2);乳球菌达到1.3×109CFU/ml(TK1),1.1×108CFU/ml(TK2);酵母菌1.6×106CFU/ml(TK1),6.6×106CFU/ml(TK2)。TK1和TK2之间存在较大差异,TK1的风味好于TK2。     

原汁苹果醋中的有机酸

以苹果汁为原料 ,采用液态酒精发酵和醋酸发酵法制作苹果醋 ,采用反相高效液相色谱法分析苹果醋中有机酸 ,研究了苹果品种、醋酸菌种、醋酸发酵方法及过程对有机酸的影响。认为苹果酸是苹果醋中的主要有机果酸 ,其与液态粮食醋有机酸组成的差别主要体现在苹果酸、酒石酸和乳酸的含量上 ,苹果品种是影响原汁苹果醋中有机酸的种类和含量主要因素 ,醋酸菌在醋酸发酵过程中有代谢各种有机酸的作用 ,发酵方法会影响醋酸菌对有机酸代谢作用的程度 ,试验中所采用的 5种醋酸菌在醋酸发酵过程中通过消耗苹果酸、乳酸、琥珀酸 ,而产生和积累柠檬酸。