Unravelling the contribution of lactic acid bacteria and acetic acid bacteria to cocoa fermentation using inoculated organisms.

Cocoa beans (Theobroma cacao L.) are the raw material for chocolate production. Fermentation of the bean pulp by microorganisms is essential for developing the precursors of chocolate flavour. Currently, the cocoa fermentation is still conducted by an uncontrolled traditional process via a consortium of indigenous species of yeasts, lactic acid bacteria and acetic acid bacteria. Although the essential contribution of yeasts to the production of good quality beans and, typical chocolate character is generally agreed, the roles of lactic acid bacteria and acetic acid bacteria are less certain. The objective of this study was to investigate the contribution of LAB and AAB in cocoa bean fermentation by conducting small scale laboratory fermentations under aseptic conditions, inoculated with different groups of microorganisms previously isolated from spontaneous cocoa fermentations. The inoculation protocols were: (1) four yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii, Kluyveromyces marxianus and Saccharomyces cerevisiae; (2) four yeasts plus the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum; (3) four yeasts plus the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateuri and (4) four yeasts plus two lactic acid bacteria and two acetic acid bacteria. Only the inoculated species were detected in the microbiota of their respective fermentations. Beans from the inoculated fermentations showed no significant differences in colour, shell weights and concentrations of residual sugars, alcohols and esters (p>0.05), but they were slightly different in contents of lactic acid and acetic acid (p<0.05). All beans were fully brown and free of mould. Residual sugar levels were less than 2.6 mg/g while the shell contents and ethanol were in the range of 11-13.4% and 4.8-7 mg/g, respectively. Beans fermented in the presence of LAB contained higher levels of lactic acid (0.6-1.2 mg/g) whereas higher concentrations of acetic acid (1.8-2.2 mg/g) were detected in beans inoculated with AAB. Triangle and hedonic sensory evaluations of chocolates prepared from beans taken from the three fermentations showed no significant differences (p > 0.05). It was concluded that the growth of lactic acid bacteria and acetic acid bacteria may not be essential for the fermentation of cocoa beans.

Autochthonous yeasts associated with mature pineapple fruits, freshly crushed juice and their ferments; and the chemical changes during natural fermentation.

This study investigated autochthonous yeasts and their functions in the spontaneous fermentation of freshly crushed pineapple juice samples collected from two different areas of both Thailand and Australia. Hanseniaspora uvarum and Pichia guilliermondii were the main yeast species observed on the fruit skins of Thai samples, and also in the fresh juice and ferments of all samples from both countries. P. guilliermondii was consistently present as the dominant species during the early stage of the fermentation, whereas H. uvarum became more prevalent towards the end of the six-day fermentation period, with populations increasing from an initial level of approximately 5 log CFU/mL to approximately 8 log CFU/mL at the end of fermentation. The ethanol levels in samples from both regions of Thailand were maximal at 2 days of fermentation, reaching approximately 1 to 2% (v/v) but then declined thereafter. In contrast, in the Australian samples ethanol levels continued to increase over the entire six-day fermentation period and reached approximately 3 to 4% (v/v). A significant decrease in citric acid and increase in lactic acid levels were observed throughout the fermentation period in the samples from Thailand, but not in those from Australia where the different acid contents (and pH) were relatively stable. The other wine yeasts and, in particular, Saccharomyces yeasts, were not found in any of sampled fermentation systems that is apparently different from the other fruit juices. These findings suggested that the freshly crushed pineapple juice may possibly have some effects on the other autochthonous yeasts having important role in alcoholic fermentation.

Wine yeasts for the future.

International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.

Yeasts in foods and beverages: impact on product quality and safety.

The role of yeasts in food and beverage production extends beyond the well-known bread, beer and wine fermentations. Molecular analytical technologies have led to a major revision of yeast taxonomy, and have facilitated the ecological study of yeasts in many other products. The mechanisms by which yeasts grow in these ecosystems and impact on product quality can now be studied at the level of gene expression. Their growth and metabolic activities are moderated by a network of strain and species interactions, including interactions with bacteria and other fungi. Some yeasts have been developed as agents for the biocontrol of food spoilage fungi, and others are being considered as novel probiotic organisms. The association of yeasts with opportunistic infections and other adverse responses in humans raises new issues in the field of food safety.

Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation.

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.

Genetic determinants of volatile-thiol release by Saccharomyces cerevisiae during wine fermentation.

Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation, Saccharomyces cerevisiae mediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.

Degradation of RNA during the autolysis of Saccharomyces cerevisiae produces predominantly ribonucleotides.

Autolytic degradation of yeast RNA occurs in many foods and beverages and can impact on the sensory quality of the product, but the resulting complex mixture of nucleotides, nucleosides and nucleobases has not been properly characterised. In this study, yeast autolysis was induced by incubating cell suspensions of Saccharomyces cerevisiae at 30-60 degrees C (pH 7.0), and at pH 4.0-7.0 (40 degrees C) for 10-14 days, and the RNA degradation products formed during the process were determined by reversed-phase HPLC. Up to 95% of cell RNA was degraded, with consequent leakage into the extracellular environment of mainly 3'-, 5'- and 2'-ribonucleotides, and lesser amounts of polynucleotides, ribonucleosides and nucleobases. The rate of RNA degradation and the composition of the breakdown products varied with temperature and pH. RNA degradation was fastest at 50 degrees C (pH 7.0). Autolysis at lower temperatures (30 degrees C and 40 degrees C) and at pH 5.0 and 6.0 favoured the formation of 3'-nucleotides, whereas autolysis at 40 degrees C and 50 degrees C (pH 7.0) favoured 5'- and 2'-nucleotides. The best conditions for the formation of the two flavour-enhancing nucleotides, 5'-AMP and 5'-GMP, were 50 degrees C (pH 7.0) and pH 4.0 (40 degrees C), respectively.

Pesticides as a source of microbial contamination of salad vegetables.

Ten commercially available pesticides (insecticides, herbicides and fungicides), used during the production of vegetable produce, were examined as potential sources of microbial contaminants. As purchased, none of the pesticides showed the presence of viable microorganisms (< 5 CFU/ml). Using an agar plate diffusion assay, they did not inhibit a range of bacteria of spoilage and public health significance on vegetable produce. After reconstitution in sterile water to their recommended concentration, two of the pesticides supported the survival and growth of inoculated species of Pseudomonas, Salmonella and Escherichia coli. Listeria monocytogenes did not survive after inoculation into any of the pesticides. Pesticides were reconstituted in different sources of agricultural water (bore, dam and river) and examined for survival and growth of microorganisms naturally present in these waters. On storage at 30 degrees C for 48 h, nine of the pesticides supported the growth of bacterial species present in these waters. Predominant species in the pesticide solutions, before and after storage, varied according to the source, but species of Pseudomonas, Acinetobacter and Aeromonas and various coliforms exhibited significant growth. Unless managed properly (reconstituted in potable water, and used without lengthy storage), pesticides could contribute to the microbial load of vegetable produce, thereby affecting their shelf-life and public health safety.

Variation in 4-mercapto-4-methyl-pentan-2-one release by Saccharomyces cerevisiae commercial wine strains.

The volatile thiol 4-mercapto-4-methylpentan-2-one (4MMP) is a potent contributor to wine aroma. In grape juice, 4MMP is bound to cysteine as a non-volatile compound and requires the action of yeast during fermentation to release the aroma active thiol. A method was developed to measure 4MMP release from the precursor by headspace solid-phase microextraction and separation by gas chromatography with atomic emission detection to screen the ability of wine yeast to release 4MMP. Yeast commonly used in white wine making were grown with the precursor at two different temperatures, and the amount of 4MMP released was measured. The results demonstrate that yeast strain selection and fermentation temperature can provide an important tool to enhance or modulate the grape-derived aromas formed during wine fermentation.

Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes.

The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA). After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 10(2) cfuml(-1), although this value was affected by culture age and the relative populations of the species in mixed culture. In mixed yeast populations, PCR-DGGE detected species present at 10-100-fold less than other species but not when the ratio exceeded 100-fold. Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes. It was not detected by PCR-DGGE when present at populations less than 10(3) cfug(-1). When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (10(5)-10(7) cfug(-1)), all detectable using PCR-DGGE. However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 10(4) cfug(-1). PCR -DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 10(4) cfug(-1). However, this method detected a greater diversity of species than agar plating.

Occurrence and significance of Bacillus thuringiensis on wine grapes.

Wine grapes harvested at different stages during cultivation from several vineyards in New South Wales, Australia, harboured Bacillus thuringiensis at viable populations of 10(2)-10(6) cfu/g. Commercial preparations of B. thuringiensis had been sprayed onto the grapes as a biological insecticide. B. thuringiensis (10(1)-10(3) cfu/ml) was isolated from grape juice and fermenting grape juice in a commercial winery. Although B. thuringiensis remained viable when inoculated at 10(3)-10(4) cfu/ml into grape juice and wine (pH 3.0-6.0), it did not grow. Using in vitro agar culture assays, B. thuringiensis inhibited several grape-associated yeasts and bacteria as well as various species of fungi associated with grape spoilage and ochratoxin A production. B. thuringiensis did not inhibit Saccharomyces cerevisiae in agar culture or during alcoholic fermentation of grape juice. B. thuringiensis inhibited the malolactic bacterium, Oenococcus oeni, in agar culture but not during mixed cultures in a liquid medium.

Yeast interactions and wine flavour.

Wine is the product of complex interactions between fungi, yeasts and bacteria that commence in the vineyard and continue throughout the fermentation process until packaging. Although grape cultivar and cultivation provide the foundations of wine flavour, microorganisms, especially yeasts, impact on the subtlety and individuality of the flavour response. Consequently, it is important to identify and understand the ecological interactions that occur between the different microbial groups, species and strains. These interactions encompass yeast-yeast, yeast-filamentous fungi and yeast-bacteria responses. The surface of healthy grapes has a predominance of Aureobasidium pullulans, Metschnikowia, Hanseniaspora (Kloeckera), Cryptococcus and Rhodotorula species depending on stage of maturity. This microflora moderates the growth of spoilage and mycotoxigenic fungi on grapes, the species and strains of yeasts that contribute to alcoholic fermentation, and the bacteria that contribute to malolactic fermentation. Damaged grapes have increased populations of lactic and acetic acid bacteria that impact on yeasts during alcoholic fermentation. Alcoholic fermentation is characterised by the successional growth of various yeast species and strains, where yeast-yeast interactions determine the ecology. Through yeast-bacterial interactions, this ecology can determine progression of the malolactic fermentation, and potential growth of spoilage bacteria in the final product. The mechanisms by which one species/strain impacts on another in grape-wine ecosystems include: production of lytic enzymes, ethanol, sulphur dioxide and killer toxin/bacteriocin like peptides; nutrient depletion including removal of oxygen, and production of carbon dioxide; and release of cell autolytic components. Cell-cell communication through quorum sensing molecules needs investigation.

The microbial ecology of cocoa bean fermentations in Indonesia.

Cocoa beans are the principal raw material of chocolate manufacture. The beans are subject to a microbial fermentation as the first stage in chocolate production. The microbial ecology of bean fermentation (Forastero and Trinitario cultivars) was investigated at three commercial fermentaries in East Java, Indonesia by determining the populations of individual species at 12-h intervals throughout the process. The first 2-3 days of fermentation were characterised by the successional growth of various species of filamentous fungi, yeasts, lactic acid bacteria and acetic acid bacteria. The principal species found were Penicillium citrinum, an unidentified basidiomycete, Kloeckera apis, Saccharomyces cerevisiae, Candida tropicalis, Lactobacillus cellobiosus, Lactobacillus plantarum and Acetobacter pasteurianus. The later stages of fermentation were dominated by the presence of Bacillus species, mostly, Bacillus pumilus and Bacillus licheniformis. Glucose, fructose, sucrose and citric acid of the bean pulp were utilised during fermentation, with the production of ethanol, acetic acid and lactic acid that diffused into the beans. The filamentous fungi were notable for their production of polygalacturonase activity and probably contributed to the degradation of bean pulp.

Degradation of DNA during the autolysis of Saccharomyces cerevisiae.

The autolysis of yeast cells has practical implications in the production of fermented foods and beverages and flavourants for food processing. Protein and RNA degradation during yeast autolysis are well described but the fate of DNA is unclear. Yeast cells ( Saccharomyces cerevisiae) were autolysed by incubating suspensions at 30-60 degrees C (pH 7.0), and at pH 4.0-7.0 (40 degrees C) for 10-14 days. Up to 55% of total DNA was degraded, with consequent leakage into the extracellular environment of mainly 3'- and 5'-deoxyribonucleotides, and lesser amounts of polynucleotides. The rate and extent of DNA degradation, composition of the DNA degradation products and DNase activity were affected by temperature and pH. The highest amount of DNA degradation occurred at 40 degrees C and pH 7.0, where the highest DNase activity was recorded. DNase activity was lowest at 60 degrees C and pH 4.0, where the proportion of polynucleotides in the degradation products was higher.

Rapid Detection of Salmonellae in Foods Using Immunoassay Systems.

A standard cultural method, radioimmunometric (RIMA) and enzyme immunometric (EIMA) assays were compared for detection of salmonellae in 235 food samples. The immunoassays used titanous hydroxide as the solid-phase, commercial Spicer-Edwards salmonella polyvalent H antisera (SEA) or pooled antisera produced against 10 salmonella flagellins (PFA). Nineteen food samples were positive for Salmonella by the standard cultural method. These as well as one additional sample were also positive for Salmonella by RIMA and EIMA. No false-negative results were obtained from the immunoassays using PFA, whereas two false-negative results were observed when SEA was used. The incidence of false-positive results when SEA and PFA were used were, respectively, 3.0 and 0.9% with RIMA and 2.6 and 0.9% with EIMA. The immunoassays were also able to detect 77 Salmonella serotypes when grown alone or in association with other species of Enterobacteriaceae , in mannitol selenite cystine broth. Both immunoassays performed reliably on enrichment cultures stored under refrigeration for up to 9 d. Also, of 6 non-motile salmonellae, 5 were detectable by the immunoassays. The immunoassays were simple, rapid and cost-efficient.