Rosane Freitas Schwan

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°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.

Microbial diversity during maturation and natural processing of coffee cherries of Coffea arabica in Brazil.

The magnitude and diversity of the microbial population associated with dry (natural) processing of coffee (Coffea arabica) has been assessed during a 2-year period on 15 different farms in the Sul de Minas region of Brazil. Peptone water-washed samples were taken of maturing cherries on trees (cherries, raisins and dried cherries) and from ground fermentations. The microbial load varied from 3 x 10(4) to 2.2 x 10(9) cfu/cherry with a median value of 1.6 x 10(7) cfu/cherry. The microbial load increased after heavy rainfall on cherries that were drying on the ground. At all stages, bacteria were usually the most abundant group, followed by filamentous fungi and finally yeasts. Counts of bacteria, yeasts and fungi varied considerably between farms and at different stages of maturation and processing and no consistent pattern could be seen. Yeasts showed an increase during the fermentation process. Median counts were not significantly different for fungi, yeasts and bacteria between the 2 years although Gram-negative bacteria dominated in the wet year and Gram-positive bacteria dominated in the dry year. Of a total of 754 isolates, 626 were identified to at least genus level comprising 44 genera and 64 different species. The 164 isolates of Gram-negative bacteria included 17 genera and 26 species, the most common of which were members of the genera Aeromonas, Pseudomonas, Enterobacter and Serratia. Of 191 isolates of Gram-positive bacteria, 23 were spore-forming and included six Bacillus species, and 118 were non-spore-formers of which over half were Cellulomonas with lesser numbers of Arthrobacter, Microbacterium, Brochothrix, Dermabacter and Lactobacillus. Of the 107 yeast isolates, 90 were identified into 12 genera and 24 different species and almost all were fermentative. The most common genera, in decreasing frequency, were Pichia, Candida, Arxula and Saccharomycopsis. There were many rarely described yeasts including Pichia lynferdii and Arxula adeninivorans. Almost all 292 fungal isolates were identified to genus level and 52 were identified to species level. Cladosporium, Fusarium and Penicillium each comprised about one third of the isolates and were found on all farms. Only 3% of the isolates were Aspergillus. Beauvaria, Monilia, Rhizoctonia and Arthrobotrys species were also occasionally found. The microbial flora is much more varied and complex than found in wet fermentations. The genera and species identified include members known to have all types of pectinase and cellulase activities.

Toxigenic fungi associated with processed (green) coffee beans (Coffea arabica L.)

Processed (green) coffee beans from Coffea arabica in Brazil were assessed for the presence of Aspergillus and Penicillium species both before and after surface sterilisation, the aflatoxigenic and ochratoxigenic potential of the isolates and ochratoxin A levels. Contamination by Aspergillus and Penicillium species was found on 96% and 42%, respectively, of 45 samples from 11 localities. After disinfection with 1% sodium hypochlorite, the levels fell to 47% and 24%, respectively. One hundred and eighty isolates were identified to species level and comprised Aspergillus sections Circumdati (10 species), Flavi (3), Nigri (3), Versicolores (4), while two were teleomorphic species. Eight species of Penicillium were isolated. Within section Circumdati, 75% of the isolates produced ochratoxin A and all except Aspergillus elegans and Aspergillus insulicola have previously been reported to produce ochratoxin A. One-third of the 18 isolates of Aspergillus flavus produced aflatoxin B1 and B2. None of the isolates belonging to Aspergillus section Nigri or Penicillium produced ochratoxin A. Of the 40 bean samples analysed, 58% were infected with potentially ochratoxigenic fungi but only 22% of these were contaminated with ochratoxin A at levels that varied from 0.47 to 4.82 ng/g, with an average contamination level of 2.45 ng/g.

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.

A Multiphasic Approach for the Identification of Endophytic Bacterial in Strawberry Fruit and their Potential for Plant Growth Promotion

This study used a multiphasic approach, characterized by the simultaneous use of culture-dependent and culture-independent methods, to investigate endophytic bacterial communities in strawberry (Fragaria ananassa) fruit. A total of 92 bacterial endophytes were isolated and initially grouped by their repetitive extragenic palindromic (rep)-PCR banding pattern and biochemical features. Phylogenetic analysis of the 16S rRNA gene sequences of 45 representatives showed that the isolates belonged to the species Bacillus subtilis (eight isolates), Bacillus sp. (seven isolates), Enterobacter sp. (seven isolates), Enterobacter ludwigii (six isolates), Lactobacillus plantarum (six isolates), Pseudomonas sp. (five isolates), Pantoea punctata (three isolates), and Curtobacterium citreum (three isolates). Nucleic acids were extracted from the strawberry fruit and subjected to 16S rRNA gene directed polymerase chain reaction denaturing gradient gel electrophoresis (16S rRNA PCR-DGGE). The species B. subtilis, Enterobacter sp., and Pseudomonas sp. were detected both by isolation and DGGE. The DGGE fingerprints of total bacterial DNA did not exhibit bands corresponding to several of the representative species isolated in the extinction dilution (L. plantarum, C. citreum, and P. punctata). In contrast, bands in the DGGE profile that were identified as relatives of Arthrobacter sp. and one uncultivable Erythrobacter sp. were not recovered by cultivation techniques. After isolation, the nitrogen fixation ability and the in vitro production of indole-3-acetic acid (IAA) equivalents and siderophores were evaluated. A high percentage of isolates were found to possess the ability to produce siderophores and IAA equivalents; however, only a few isolates belonging to the genera Pseudomonas and Enterobacter showed the ability to fix nitrogen. Plant growth promotion was evaluated under greenhouse conditions and revealed the ability of the Bacillus strains to enhance the number of leaves, shoot length, root dry weight, and shoot dry weight. The activity of the bacterial isolate identified as B. subtilis NA-108 exerted the greatest influence on strawberry growth and showed a 42.8% increase in number of leaves, 15.26% for high shoot, 43.5% increase in root dry weight, and a 77% increase in shoot dry weight when compared with untreated controls.

Microbiology and physiology of Cachaca (Aguardente) fermentations

Cachaça (aguardente) is a rum-style spirit made from sugar cane juice by artisanal methods in Brazil. A study was made of the production, biochemistry and microbiology of the process in fifteen distilleries in Sul de Minas. Identification of 443 yeasts showed Saccharomyces cerevisiae to be the predominant yeast but Rhodotorula glutinis and Candida maltosa were predominant in three cases. Bacterial infection is a potential problem, particularly in older wooden vats, when the ratio of yeasts:bacteria can be 10:1 or less. A study of daily batch fermentations in one distillery over one season in which 739 yeasts were identified revealed that S. cerevisiae was the predominant yeast. Six other yeast species showed a daily succession: Kluyveromyces marxianus, Pichia heimii and Hanseniaspora uvarum were present only at the beginning, Pichia subpelliculosa and Debaryomyces hansenii were detected from mid to the end of fermentation, and Pichia methanolica appeared briefly after the cessation of fermentation. Despite a steady influx of yeasts from nature, the species population in the fermenter was stable for at least four months suggesting strong physiological and ecological pressure for its maintenance. Cell densities during the fermentation were: yeasts – 4 × 108/ml; lactic acid bacteria – 4 × 105/ml; and bacilli – 5 × 104/ml. Some acetic acid bacteria and enterobacteriaceae appeared at the end. Sucrose was immediately hydrolysed to fructose and glucose. The main fermentation was complete after 12 hours but not all fructose was utilised when harvesting after 24 hours.

Microbiological and Physicochemical Characterization of Small-Scale Cocoa Fermentations and Screening of Yeast and Bacterial Strains To Develop a Defined Starter Culture

ABSTRACT Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.

Determination of dynamic characteristics of microbiota in a fermented beverage produced by Brazilian Amerindians using culture-dependent and culture-independent methods.

Cauim is a fermented beverages prepared by Tapirapé Amerindians in Brazil from substrates such as cassava, rice, peanuts, pumpkin, cotton seed and maize. Here we study the microorganisms associated with peanut and rice fermentation using a combination of culture-dependent and -independent methods. The bacterial population varied from 7.4 to 8.4 log CFU/ml. The yeast population varied from 4.0 to 6.6 log CFU/ml. A total of 297 bacteria and yeasts strains were isolated during fermentation, with 198 bacteria and 99 yeast. The Lactobacillus genus was dominant throughout fermentation. Bacteria and yeast community dynamics during the fermentation process were monitored by PCR-DGGE analysis. Both culture-dependent and -independent methods indicated that the bacterial species L. plantarum, L. fermentum, L. paracasei and L. brevis as well as the yeast species P. guilliermondii, K. lactis, Candida sp, R. toruloides and Saccharomyces cerevisiae, were dominant during fermentation. Multivariate analysis of microorganisms during beverage fermentation showed that the microbial community changed during the fermentation process.

Production of fermented cheese whey-based beverage using kefir grains as starter culture : evaluation of morphological and microbial variations

Whey valorization concerns have led to recent interest on the production of whey beverage simulating kefir. In this study, the structure and microbiota of Brazilian kefir grains and beverages obtained from milk and whole/deproteinised whey was characterized using microscopy and molecular techniques. The aim was to evaluate its stability and possible shift of probiotic bacteria to the beverages. Fluorescence staining in combination with Confocal Laser Scanning Microscopy showed distribution of yeasts in macro-clusters among the grains matrix essentially composed of polysaccharides (kefiran) and bacteria. Denaturing gradient gel electrophoresis displayed communities included yeast affiliated to Kluyveromyces marxianus, Saccharomyces cerevisiae, Kazachatania unispora, bacteria affiliated to Lactobacillus kefiranofaciens subsp. Kefirgranum, Lactobacillus kefiranofaciens subsp. Kefiranofaciens and an uncultured bacterium also related to the genus Lactobacillus. A steady structure and dominant microbiota, including probiotic bacteria, was detected in the analyzed kefir beverages and grains. This robustness is determinant for future implementation of whey-based kefir beverages.

Cultivo do cogumelo Pleurotus sajor-caju em diferentes resíduos agrícolas

Diferentes residuos agricolas disponiveis na regiao sul de Minas Gerais foram testados para o cultivo do cogumelo Pleurotus sajor-caju. Foram avaliados os seguintes substratos: palha de feijao pura (PFP), palha de milho pura (PMP), casca de cafe pura (CCP), palha de feijao enriquecida com 2% de calcario, 2% de gesso e 10% de farelo de trigo (PFE), palha de milho enriquecida (PME) e casca de cafe enriquecida (CCE). Todos os substratos receberam 2% de inoculante e foram incubados a 24°C. Apos a colonizacao, os sacos foram mantidos abertos em ambiente a 24°C e umidade a 80%. PFP, PFE e PME apresentaram os melhores resultados na producao de cogumelos, com uma eficiencia biologica de 85,7; 81,4 e 83,4%, respectivamente. A palha de feijao foi considerada o melhor residuo para a producao do cogumelo P. sajor-caju, porque apresentou a melhor eficiencia biologica sem necessidade de enriquecimento.