The microbiology of cocoa fermentation and its role in chocolate quality

The first stage of chocolate production consists of a natural, seven-day microbial fermentation of the pectinaceous pulp surrounding beans of the tree Theobroma cacao. There is a microbial succession of a wide range of yeasts, lactic-acid, and acetic-acid bacteria during which high temperatures of up to 50 degrees C and microbial products, such as ethanol, lactic acid, and acetic acid, kill the beans and cause production of flavor precursors. Over-fermentation leads to a rise in bacilli and filamentous fungi that can cause off-flavors. The physiological roles of the predominant micro-organisms are now reasonably well understood and the crucial importance of a well-ordered microbial succession in cocoa aroma has been established. It has been possible to use a synthetic microbial cocktail inoculum of just 5 species, including members of the 3 principal groups, to mimic the natural fermentation process and yield good quality chocolate. Reduction of the amount of pectin by physical or mechanical means can also lead to an improved fermentation in reduced time and the juice can be used as a high-value byproduct. To improve the quality of the processed beans, more research is needed on pectinase production by yeasts, better depulping, fermenter design, and the use of starter cultures.     

Comparison of the bacterial species diversity of spontaneous cocoa bean fermentations carried out at selected farms in Ivory Coast and Brazil

To compare the spontaneous cocoa bean fermentation process carried out in different cocoa-producing regions, heap and box (one Ivorian farm) and box (two Brazilian farms) fermentations were carried out. All fermentations were studied through a multiphasic approach. In general, the temperature inside the fermenting mass increased throughout all fermentations and reached end-values of 42-48 °C. The main end-products of pulp carbohydrate catabolism were ethanol, lactic acid, acetic acid, and/or mannitol. In the case of the fermentations on the selected Ivorian farm, the species diversity of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) was restricted. Lactobacillus fermentum and Leuconostoc pseudomesenteroides were the predominant LAB species, due to their ethanol and acid tolerance and citrate consumption. The levels of mannitol, ascribed to growth of L. fermentum, were fermentation-dependent. Also, enterobacterial species, such as Erwinia soli and Pantoea sp., were among the predominating microbiota during the early stages of both heap and box fermentations in Ivory Coast, which could be responsible for gluconic acid production. Consumption of gluconic acid at the initial phases of the Ivorian fermentations could be due to yeast growth. A wider microbial species diversity throughout the fermentation process was seen in the case of the box fermentations on the selected Brazilian farms, which differed, amongst other factors, regarding pod/bean selection on these farms as compared to fermentations on the selected Ivorian farm. This microbiota included Lactobacillus plantarum, Lactobacillus durianis, L. fermentum, Lactobacillus mali, Lactobacillus nagelii, L. pseudomesenteroides, and Pediococcus acidilactici, as well as Bacillus subtilis that was present at late fermentation, when the temperature inside the fermenting mass reached values higher than 50 °C. Moreover, AAB seemed to dominate the Brazilian box fermentations studied, explaining higher acetic acid concentrations in the pulp and the beans. To conclude, it turned out that the species diversity and community dynamics, influenced by local operational practices, in particular pod/bean selection, impact the quality of fermented cocoa beans.     

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.     

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.     

Succession of bacterial and fungal communities during natural coffee (Coffea arabica) fermentation

Bacteria, yeasts and filamentous fungi were isolated during natural coffee processing. Bacteria were isolated in greater numbers at the beginning of the fermentation, when the moisture of the coffee beans was around 68%. Gram-positive bacteria represented 85.5% of all bacteria isolated, and Bacillus was the predominant genus (51%). Gram-negative species of the genera Serratia, Enterobacter and Acinetobacter were also found. Approximately 22% of 940 randomly chosen isolates of microorganisms were yeasts. Debaryomyces (27%), Pichia (18.9%) and Candida (8.0%) were the most commonly found genera, and these three genera tended to appear more often as the fruit was fermented and dried. Aspergillus was the most abundant genus besides Penicillium, Fusarium and Cladosporium, with 42.6% of the total fungi isolates. The genera and species identified included members known to have pectinase and cellulase activities. Of the 10 organic acids analyzed and quantified in coffee beans, acetic and lactic acids may have been generated by microbial activity. Butyric acid was not detected in any sample.     

Characterisation of the microflora of attiéké, a fermented cassava product, during traditional small-scale preparation

Attiéké is a fermented cassava product consumed mainly in Cote d'Ivoire. The aim of this study was to characterise the attiéké fermentation by examining products from 15 small-scale production sites at various stages of its preparation. For the preparation of attiéké, fresh cassava is grated to a pulp and inoculated with 10% of a spontaneous traditional inoculum. The inocula contained aerobic mesophiles at mean numbers of 8.2 x 10(7) cfu/g and lactic and acetic acids at mean concentrations of 0.2% and 0.1%, respectively. The mean pH was 5.0. Lactic acid bacteria were the dominant microorganisms in cassava pulp throughout fermentation with the mean numbers being 1.2 x 10(9) cfu/g after 15 h. The identification to the species level of microorganisms from one representative attiéké production of good quality showed that, at the start of fermentation, Leuconostoc mesenteroides subsp. mesenteroides was present in the highest numbers, accounting for 20% of all lactic acid bacteria. As the fermentation proceeded, this species was replaced by homofermentative lactic acid bacteria, Lactobacillus salivarius and Lactobacillus delbrueckii subsp. delbrueckii, present at 20% and 16%, respectively, and obligate heterofermentatives, Lactobacillus fermentum and Lactobacillus confusus at 12% and 10%, respectively, of total lactic acid bacteria in the flora at the end of fermentation. High numbers of acid-sensitive microorganisms, Bacillus circulans, Bacillus lentus, Enterobacter sakazakii, Enterobacter cloacae and Klebsiella pneumoniae subsp. pneumoniae, were transferred to the pulp in the inocula, but acidification to a mean pH of 4.4 with mean lactic and acetic acid concentrations of 0.59% and 0.2%, respectively, prevented their growth and reduced their numbers to less than 10(2) cfu/g at the end of fermentation. The mean numbers of Candida tropicalis, the main yeast present, remained relatively constant at about 10(5) cfu/g throughout attiéké production. The mean numbers of aerobic mesophiles decreased to below 10(2) cfu/g as a result of the steaming process. The finished attiéké had a mean pH of 4.4 and mean lactic and acetic acid concentrations of 0.6% and 0.1%, respectively.     

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.     

酱香型白酒4轮次堆积发酵理化因子、风味物质与微生物群落相关性分析

基于传统可培养方法和高通量测序技术分析酱香型白酒4轮次堆积发酵过程微生物群落组成及动态变化,采用气相色谱法解析酒醅中风味物质结构,并分析理化指标、风味物质和微生物群落间的相关性。结果表明,4轮次堆积发酵过程优势细菌主要为Bacillusamyloliquefaciens、B.licheniformis、unculturedKroppenstedtiasp.、K. eburnea、Sphingobium yanoikuyae和Lactobacillus sp.,优势真菌主要为Pichia kudriavzevii、Kazachstania humilis、Thermomyces languginosus、Byssochlamys spectabilis、Zygosaccharomyces parabilii和Aspergillus sp.。微生物群落与理化指标、风味物质形成存在复杂的作用关系：水分含量与P. kudriavzevii等酵母菌呈正相关,与T. lanuginosus、B.spectabilis、A.costiformis等丝状真菌呈负相关;淀粉和还原糖含量与Bacillus...     

海棠酵素微生物菌相分析及其功能初步研究

采用传统微生物培养法和高通量测序技术对海棠酵素微生物菌相进行分析,并对海棠酵素改善小鼠肠道菌群进行初步研究。结果表明,海棠酵素主要由乳酸菌（4.4±0.1）×107 CFU/mL、醋酸菌（5.4±0.1）×108 CFU/mL和酵母菌（5.0±0.2）×105 CFU/mL组成;经分离鉴定共获得9株优势菌株,其中4株乳酸菌、2株醋酸菌和3株酵母菌。发酵0～4 d,细菌主要为乳杆菌属（Lactobacillus）、酒球菌属（Oenococcus）、消化链球菌属（Peptostreptococcus）、魏斯氏菌属（Weissella）,发酵13～31 d,主要为乳杆菌属、酒球菌属;发酵0～31 d,真菌主要为酵母属（Saccharomyces）和假丝酵母属（Candida）。经α多样性分析可知,海棠酵素细菌丰富度较高,真菌多样性较高。连续灌胃小鼠0.4 mL海棠酵素28 d可增加小鼠粪便中乳杆菌属、瘤胃球菌属等有益菌属,具有调节小鼠肠道菌群的能力。     

Changes in the concentration of organic acids during the soaking of soybeans for tempe production

Soybeans were soaked for 24h in tap water at 30°C in preparation for tempe fermentation. Soaking was conducted under conditions that give a microbial fermentation, and in the presence of antibiotics where microbial growth was inhibited. Valeric, propionic, formic and acetic were the main acids in the beans. Their concentrations decreased progressively during soaking as they were leached from the bean into the soak-water. At the end of soaking, lactic and malic were the main acids in the beans. They were produced by microbial fermentation in the soak-water and diffused into the beans. The concentration of acids produced in the soak-water was determined by the microbial species responsible for the fermentation.     

Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed. The early stage was characterized by the coexistence of Saccharomyces sp. and lactic acid bacteria. Almost all of the bacterial DGGE bands related to lactic acid bacteria were replaced by bands derived from Lactobacillus acetotolerance and Acetobacter pasteurianus at the stage at which acetic acid started to accumulate. The microbial succession, tested in three different pots, was found to be essentially identical. Among the bacteria isolated at the early stage, some species differed from those detected by DGGE. This is the first report to reveal the microbial community succession that occurs during a unique vinegar fermentation process, as determined by a culture-independent method.     

Antimicrobial interactions of microbial species involved in the fermentation of cassava dough into agbelima with particular reference to the inhibitory effect of lactic acid bacteria on enteric pathogens

Lactic acid bacteria, Bacillus species and yeasts are involved in the fermentation of cassava dough into agbelima. Microbial interactions within and between these groups of microorganisms were investigated in addition to the survival of five enteric pathogens inoculated into agbelima under various conditions. Nine out of 10 cultures of lactic acid bacteria isolated at the end of agbelima fermentation showed inhibitory effect against 10 cultures of lactic acid bacteria isolated at the start of fermentation. Only 3 out of 10 isolates of Bacillus subtilis were inhibited by 10 isolates of lactic acid bacteria tested. No interactions were observed between yeasts and the lactic acid bacteria, whereas three of the Bacillus isolates showed inhibitory effects against the yeasts. Twelve isolates of Lactobacillus plantarum tested inhibited the growth of an isolate each of Lactobacillus fermentum and Lactobacillus brevis but none tested positive for bacteriocin production. The antimicrobial effect of the lactic acid bacteria was attributed to acid production. In fermenting cassava dough, enteric pathogens survived to different extents depending on pH and their sensitivity to acids. Vibrio cholerae C-230, Salmonella typhimurium 9 and Salmonella enteritidis 226 were not detectable in 10 g of sample after 4 h when inoculated into the 48-h fermented product, agbelima, whereas Shigella dysenteriae 2357T and Escherichia coli D2188 were detectable up to 8 h in the product.     

保宁醋醋曲中产多糖乳酸菌的筛选及其代谢产物分析

保宁醋因其复杂微生物体系和酿造工艺而形成独特风味,其风味物质和营养成分主要由各种功能性微生物发酵生成,其中乳酸菌是非常关键的一类菌株,其对保宁醋的风味和营养物质的形成具有重要作用。本研究以稀释涂布平板法从保宁醋醋曲中分离得到12株乳酸菌,通过定性试验、产酸率、耐酸试验和胞外多糖测定等实验,得到一株产多糖量为191.98 mg/L、产酸率为1.62%的乳酸菌L7,鉴定为发酵乳酸杆菌(Lactobacillus femertum),其发酵液中3-羟基-2-丁酮和乙酸相对含量较高,分别为36.22%、45.76%,HPLC检测到乳酸、乙酸,含量分别为66.56 mg/100 mL和104.08 mg/100 mL,这表明L7将有利于提高食醋酸度以及川芎嗪含量。产多糖乳酸菌在食醋发酵过程中的具有重要作用,而此次所得乳酸杆菌对保宁醋生产和发酵工艺改良具有指导意义。     

The effect of cocoa fermentation and weak organic acids on growth and ochratoxin A production by Aspergillus species

The acidic characteristics of cocoa beans have influence on flavor development in chocolate. Cocoa cotyledons are not naturally acidic, the acidity comes from organic acids produced by the fermentative microorganisms which grow during the processing of cocoa. Different concentrations of these metabolites can be produced according to the fermentation practices adopted in the farms, which could affect the growth and ochratoxin A production by fungi. This work presents two independent experiments carried out to investigate the effect of some fermentation practices on ochratoxin A production by Aspergillus carbonarius in cocoa, and the effect of weak organic acids such as acetic, lactic and citric at different pH values on growth and ochratoxin A production by A. carbonarius and Aspergillus niger in culture media. A statistical difference (ρ<0.05) in the ochratoxin A level in the cured cocoa beans was observed in some fermentation practices adopted. The laboratorial studies demonstrate the influence of organic acids on fungal growth and ochratoxin A production, with differences according to the media pH and the organic acid present. Acetic acid was the most inhibitory acid against A. carbonarius and A. niger. From the point of view of food safety, considering the amount of ochratoxin A produced, fermentation practices should be conducted towards the enhancement of acetic acid, although lactic and citric acids also have an important role in lowering the pH to improve the toxicity of acetic acid.     

Theobroma cacao L., "The food of the Gods": quality determinants of commercial cocoa beans, with particular reference to the impact of fermentation

The quality of commercial cocoa beans, the principal raw material for chocolate production, relies on the combination of factors that include the type of planting material, the agricultural practices, and the post-harvest processing. Among these, the fermentation of the cocoa beans is still the most relevant since it is the process whereby the precursors of the cocoa flavor arise. The formation of these precursors depends on the activity of different microbial groups on the beans pulp. A comparison of fermentations in different countries showed that a well-defined microbial succession does not always take place and that the role of Bacillus spp. in this process remains unclear. Considering the overriding importance of the fermentation to achieve high quality commercial cocoa beans, we discuss the need of addressing the impact of the farming system, the ripeness state of the pods, and the role of microbial interactions on the fermentation in future research. In addition, the problem of high acidification cocoa beans, aspects dealing with the volatile fraction of the flavor, and the cocoa butter properties, all were identified as critical aspects that need further investigation. The standardization of the microbiological methods and the application of metagenomic approaches would magnify the knowledge in this domain.     

Complete Heterolactic Acid Fermentation of Green Beans by Lactobacillus cellobiosis

Only Lactobacillus cellobiosus, among eight strains of heterofermentative lactic acid bacteria, removed all fermentable sugars from green beans. Proper blanching of the beans was required to prevent growth by natural lactic acid bacteria. An inoculum of 10 CFU/ml Lactobacillus plantarum and 10⁶ CFU/ml L. cellobiosus resulted in the formation of twice as much lactic acid as inoculation with L. cellobiosus alone. A maximum of 3.74% sugar was metabolized by L. cellobiosus in bean juice containing 2.5% NaCI and 0.08% acetic acid. Fructose was nearly quantitatively reduced to mannitol with a concomitant accumulation of acetic acid. Ethanol was not observed until most of the fructose was metabolized. The L. cellobiosus-fermented beans had a more mild acid flavor than beans fermented with L plantarum.     

Study on the microflora and biochemistry of cocoa fermentation in the Dominican Republic

Cocoa fermentation was monitored at the IDIAF (Instituto Dominicano de Investigaciones Agropecuarias y Forestales) "Mata Larga" experimental station, in San Francisco de Macoris, Dominican Republic. The maximum average fermentation temperature reached 51 degrees C after 48 h and the pH reached 4.5 after 144 h of fermentation. A significant decrease in glucose, fructose and citric acid was seen in the pulp over the first 48 h. There was a delay of 24 h between maximum microbial growth and maximum concentrations of the respective metabolites, which occurred after 48 h for ethanol and after 72 h for acetic acid. A maximum concentration in lactic acid was found after around 120 h of fermentation. The aerobic mesophilic flora increased from 6.1x10(6) to a maximum of 4.2x10(7) CFU g(-1) of dry matter after 48 h of fermentation. Yeasts displayed maximum development after 24 h (6.1x10(7) CFU g(-1) of dry matter), whilst for lactic and acetic acid bacteria it occurred after 48 h (7.3x10(7) and 1.5x10(8) CFU g(-1) of dry matter respectively). The yeasts isolated belonged to the genera Hanseniaspora and Candida, the lactic acid bacteria to the genus Lactobacillus, and the acetic acid bacteria to the genus Acetobacter. The differences compared to other fermentation trials concerned the micropopulation from a qualitative point of view.     

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     

Fermentation Characteristics of Heterolactic Acid Bacteria in Green Bean Juice

Green bean juice was fermented with 10 species (14 strains) of heterofermentative and two homofermentative lactic acid bacteria to select organisms which might be used to carry out a complete fermentation. Lactobacillus cellobiosus was the only organism to remove all fermentable sugars from bean juice with or without 2.5% NaCI. Nine other cultures used from 75–95% of the sugars. Lactobacillus cellobiosus also produced the lowest final pH among the 14 strains. A complete analysis of the major fermentation substrates and products was done for each of the organisms. Fermentation balance calculations showed a range from 74–132% carbon recovery. These bacteria showed considerable variation in the ability to degrade malic acid and to form mannitol and acetic acid.     

Apparent antifungal activity of several lactic acid bacteria against Penicillium discolor is due to acetic acid in the medium

Fifty-six dairy bacteria belonging to the genera Lactococcus, Lactobacillus, Pediococcus, Propionibacterium, Streptococcus, Enterococcus, Leuconostoc, and Brevibacterium were screened for antifungal activity against four species of fungi relevant to the cheese industry (Penicillium discolor, Penicillium commune, Penicillium roqueforti, and Aspergillus vesicolor). Most of the active strains belonged to the genus Lactobacillus, whereas Penicillium discolor was found to be the most sensitive of the four fungi investigated. Further studies on P. discolor showed antifungal activity only below pH 5. This effect of pH suggests that organic acids present in the culture could be involved in the detected activity. Determination of acid composition revealed lactic acid production for active dairy strains and the presence of acetic acid in active as well as inactive strains. It was demonstrated that the undissociated acetic acid originates from the bacterial growth medium. The synergistic effect of the acetic acid present and the lactic acid produced was likely the main factor responsible for the antifungal properties of the selected bacteria. These results could explain some discrepancies in reports of the antifungal properties of lactic acid bacteria, since the role of acetic acid has not been considered in previous studies.