Diversity and probiotic characterisation of yeast isolates in the bovine gastrointestinal tract.

The use of yeasts as a feed supplement for cattle can promote animal development and performance. However, for the positive results to be consistent, strains with probiotic properties must be selected. The objective of this study was to isolate and identify yeasts present in the bovine feces and evaluate their probiotic potential together with strains previously isolated from the rumen (preliminary study). A total of 193 isolates were studied, including 139 isolates (19 species) from fecal samples from 11 different animals (Bos taurus and Bos indicus) and 54 strains previously isolated from rumen fluid (Bos taurus). The yeast population in the feces ranged from 3.51 to 4.99 log CFU/g, with Candida pararugosa being the most abundant (isolated from the feces of six samples analysed). Isolates were selected that had negative results in the safety tests (hemolytic activity, DNAse, and gelatinase) and had percentages greater than 35 and 70% for hydrophobicity and auto-aggregation, respectively. In addition, selected isolates had percentages greater than 77.7 and 74.7% for coaggregation with pathogenic strains of Escherichia coli and Clostridium perfringens, respectively. The isolates with percentage growth at 39 °C greater than 64.6% and viability greater than 96.7% were selected for survival testing under bovine gastrointestinal conditions. After the tests, the seven best isolates were selected, belonging to the species Candida pararugosa (L60, CCMA 928 and CCMA 930) and Pichia kudriavzevii (L97, L100, CCMA904, CCMA 907). The selected isolates were exopolysaccharide producers. Based on the results of the evaluated properties, the seven selected isolates were classified as potential probiotics for cattle.

Probiotic properties of yeasts isolated from Brazilian fermented table olives.

AIMS: To investigate the probiotic potential of yeasts isolated from naturally fermented Brazilian table olives. METHODS AND RESULTS: Eighteen yeast strains were tested in terms of: safety; survival of gastrointestinal and digestion conditions; antimicrobial activity; cellular hydrophobicity; autoaggregation ability and adhesion to epithelial cells; coaggregation and inhibition of pathogenic bacteria adhesion. Six yeasts showed favourable results for all probiotic attributes: Saccharomyces cerevisiae CCMA 1746, Pichia guilliermondii CCMA 1753, Candida orthopsilosis CCMA 1748, Candida tropicalis CCMA 1751, Meyerozyma caribbica CCMA 1758 and Debaryomyces hansenii CCMA 1761. These yeasts demonstrated resistance to 37°C, pH 2·0 and bile salts, and survived in vitro digestion (≥106  CFU per ml). Furthermore, the yeasts exhibited a hydrophobic cell surface (42·5-92·2%), autoaggregation capacity (41·0-91·0%) and adhesion to Caco-2 (62·0-82·8%) and HT-29 (57·6-87·3%) epithelial cell lines. Also, the strains showed antimicrobial activity against Salmonella Enteritidis as well as the ability to coaggregate and reduce the adhesion of this pathogen to intestinal cells. CONCLUSIONS: Autochthonous yeasts from naturally fermented Brazilian table olives have probiotic properties, with potential for development of new probiotic food products. SIGNIFICANCE AND IMPACT OF STUDY: These data are important and contribute to the knowledge of new potential probiotic yeasts capable of surviving gastrointestinal tract conditions and inhibiting pathogenic bacteria.

Criteria for lactic acid bacteria screening to enhance silage quality.

The main challenge of ensiling is conserving the feed through a fermentative process that results in high nutritional and microbiological quality while minimizing fermentative losses. This challenge is of growing interest to farmers, industry and research and involves the use of additives to improve the fermentation process and preserve the ensiled material. Most studies involved microbial additives; lactic acid bacteria (LAB) have been the focus of much research and have been widely used. Currently, LABs are used in modern and sustainable agriculture because of their considerable potential for enhancing human and animal health. Although the number of studies evaluating LABs in silages has increased, the potential use of these micro-organisms in association with silage has not been adequately studied. Fermentation processes using the same strain produce very different results depending on the unique characteristics of the substrate, so the choice of silage inoculant for different starting substrates is of extreme importance to maximize the nutritional quality of the final product. This review describes the current scenario of the bioprospecting and selection process for choosing the best LAB strain as an inoculant for ensiling. In addition, we analyse developments in the fermentation process and strategies and methods that will assist future studies on the selection of new strains of LAB as a starter culture or inoculant.

Identification and characterization of yeasts from bovine rumen for potential use as probiotics.

AIMS: The aim was to isolate, identify and characterize yeasts present in rumen fluid and to select strains showing potential as probiotics. METHODS AND RESULTS: Rumen fluid was sampled from 4 herds of dairy and beef cattle and 77 yeast isolates were identified. Initial screening was based on the capacity to maintain viability in a medium with different ruminal conditions. A second screening in fresh rumen fluid to assess the growth of inoculated yeasts and evaluate in vitro neutral detergent fibre digestibility (NDF-D), pH and acid accumulation was conducted. The yeast population ranged from 3·84 to 6·76 log10 CFU per ml. The main species of yeast found were Pichia kudriavzevii, Candida rugosa, C. pararugosa, C. ethanolica and Magnusiomyces capitatus. Strains CCMA 933 (C. rugosa) and CCMA 970 (C. pararugosa) showed greater ability to survive in ruminal fluid and stimulated the production of acids. Isolate CCMA 967 (C. ethanolica) survived and improved the NDF-D. CONCLUSION: Pichia kudriavzevii was the dominant yeast found in the cattle herds. Strains CCMA 933, CCMA 970 and CCMA 967 showed properties that could be useful as potential probiotics for cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to select yeasts from the rumen fluid, with the potential to be used as probiotic, based on the ruminal conditions.

Effect of the inoculation of sugarcane silage with Lactobacillus hilgardii and Lactobacillus buchneri on feeding behavior and milk yield of dairy cows.

Despite its low NDF digestibility, sugarcane is an option for feeding dairy cattle in tropical regions. We evaluated the effect of sugarcane silages inoculated with CCMA 0170 (LH; an epiphytic bacteria isolated from sugarcane) or with NCIMB 40788 (LB; a commercial strain isolated from temperate grasses) on dairy cow performance and feeding behavior. The microbial inoculums were previously grown in the laboratory to obtain 5 log cfu/g of fresh forage. Nine tons of each inoculated silage and a noninoculated control silage (CON) were harvested from the same field and stored for at least 35 d in experimental 20 × 2.1 × 0.4 m bunker silos. Fifteen Holstein cows in late lactation (336 ± 175 days in milk at the start of the experiment) received the treatments in five 3 × 3 Latin squares with 21-d periods. The diets contained 20% of DM of sugarcane silage and 41% of DM of corn silage. Milk yield was increased from 18.0 kg/d for CON to18.8 kg/d for LH, but LB did not elicit a detectable increase in milk yield (18.1 kg/d). The daily yields of fat, protein, lactose, and total solids were increased by LH. Daily DMI and total tract apparent digestibility of nutrients did not differ among treatments. Both inoculated silages reduced acetate and increased butyrate proportions in ruminal VFA, but only LH silage reduced the acetate to propionate ratio (3.0 vs 3.3). First meal duration was shorter for CON compared to LH and LB. The proportion of daily intake between 0700 and 1300 h tended to be increased, and the proportion between 1900 and 0700 h was reduced by LH. The inoculation of sugarcane silage with affected rumen fermentation profile and feeding behavior of late lactation dairy cows, increasing the yield of milk solids.

Fermentation profile and identification of lactic acid bacteria and yeasts of rehydrated corn kernel silage.

AIMS: The aim of this study was to evaluate the chemical and microbiological characteristics and to identify the lactic acid bacteria (LAB) and yeasts involved in rehydrated corn kernel silage. METHODS AND RESULTS: Four replicates for each fermentation time: 5, 15, 30, 60, 90, 150, 210 and 280 days were prepared. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and PCR-based identification were utilized to identify LAB and yeasts. Eighteen bacteria and four yeast species were identified. The bacteria population reached maximum growth after 15 days and moulds were detected up to this time. The highest dry matter (DM) loss was 7·6% after 280 days. The low concentration of water-soluble carbohydrates (20 g kg-1 of DM) was not limiting for fermentation, although the reduction in pH and acid production occurred slowly. Storage of the rehydrated corn kernel silage increased digestibility up to day 280. CONCLUSIONS: This silage was dominated by LAB but showed a slow decrease in pH values. This technique of corn storage on farms increased the DM digestibility. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to evaluate the rehydrated corn kernel silage fermentation dynamics and our findings are relevant to optimization of this silage fermentation.

Occurrence of mycotoxins and yeasts and moulds identification in corn silages in tropical climate.

AIMS: This study was aimed to identify yeasts and moulds as well as to detect mycotoxin in corn silages in southern Minas Gerais, Brazil. METHODS AND RESULTS: Corn silages from 36 farms were sampled to analyse dry matter, crude protein, ether extract, ash, neutral detergent fibre, nonfibre carbohydrates and mycotoxins contents, yeasts and moulds population, pH and temperature values. The mycotoxins found in high frequency were aflatoxin in 77·7% of analysed samples, ochratoxin (33·3%) and zearalenone (22·2%). There was no significant correlation between the mycotoxin concentration and the presence of moulds. The pH was negatively correlated with ochratoxin concentration. Aspergillus fumigatus was identified in all silages that presented growth of moulds. Ten different yeast species were identified using the culture-dependent method: Candida diversa, Candida ethanolica, Candida rugosa, Issatchenkia orientalis, Kluyveromyces marxianus, Pichia manshurica, Pichia membranifaciens, Saccharomyces cerevisiae, Trichosporon asahii and Trichosporon japonicum. Another six different yeast species were identified using the culture-independent method. CONCLUSIONS: A high mycotoxin contamination rate (91·6% of the analysed silages) was observed. The results indicated that conventional culturing and PCR-DGGE should be combined to optimally describe the microbiota associated with corn silage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information about the corn silage fermentation dynamics and our findings are relevant to optimization of this silage fermentation.

Fermentative profile and bacterial diversity of corn silages inoculated with new tropical lactic acid bacteria.

AIMS: This study aimed to evaluate the effects of inoculation of strains of lactic acid bacteria (LAB) isolated from sugarcane grown in a Brazil on the quality of corn silage. METHODS AND RESULTS: Three strains of Lactobacillus buchneri (UFLA SLM11, UFLA SLM103 and UFLA SLM108), five strains of Lactobacillus plantarum (UFLA SLM08, UFLA SLM41, UFLA SLM45, UFLA SLM46 and UFLA SLM105), and one strain of Leuconostoc mesenteroides (UFLA SLM06) were evaluated at 0, 10, 30, 60 and 90 day after inoculating corn forage. The inoculation of the LAB strains did not influence the chemical composition of the silage, but pH, acetic acid and 1,2-propanediol were affected by treatment. The silages inoculated with UFLA SLM11 and SLM108 contained the lowest yeast and filamentous fungi counts during fermentation. Bacteria belonging to the Enterobacteriaceae family, Clostridium genus were detected in the silages inoculated with Lact. buchneri UFLA SLM 11, 103 and 108, as shown by DGGE analysis. Silages inoculated with Lact. buchneri UFLA SLM 11 showed higher aerobic stability. CONCLUSIONS: The Lact. buchneri UFLA SLM11 strain was considered promising as a starter culture or inoculant for corn silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of microbial inoculants for each crop promotes improvement of silage quality. Studies on the chemical and microbiological characteristics of silage provide useful information for improving ensiling techniques.

A new alternative use for coffee pulp from semi-dry process to ß-glucosidase production by Bacillus subtilis.

UNLABELLED: Coffee is among the most preferred nonalcoholic drinks, and its consumption is distributed globally. During the coffee fruiting process, however, a large amount of waste is generated in the form of pulp, mucilage, husks, and water waste. The pulp and mucilage have the chemical composition to support the growth of micro-organisms and the production of value-added product. The aim was testify pulp coffee can be considered as carbon and inductor source for ß-glucosidase by Bacillus subtilis CCMA 0087. The response surface methodology (RSM) based on a central composite rotatable design (CCRD) was employed for this optimization. The methodology used in the optimization process was validated by testing the best conditions obtained and comparing them with the values predicted by the model. The highest ß-glucosidase production (22·59 UI ml(-1) ) was reached in 24 h of culturing at coffee pulp concentration of 36·8 g l(-1) , temperature of 36·6°C, and pH of 3·64. SIGNIFICANCE AND IMPACT OF THE STUDY: Countries whose economy is based on agricultural activities generate a great deal of liquid and solid waste. Thus, it is important to develop new alternatives for using this waste rather than disposing it in the environment. The production of enzymes, and particularly cellulase, is one such alternative. In this study, we proposed to produce ß-glucosidase production from pulp coffee extract using a Bacillus subtilis strain.

Enumeration, identification and safety proprieties of lactic acid bacteria isolated from pork sausage / Enumeração, identificação e propriedades de segurança de bactérias ácido-lácticas isoladas de linguiça suína

Lactic Acid Bacteria (LAB) are indigenous microorganisms occurring in pork sausages. The utilization of selected autochthonous LAB may improve the safety of meat products. This study aims to enumerate and identify LAB in pork sausage and to characterize their safety properties, such as antimicrobial susceptibility and antibacterial activity. A total of 189 sealed packages of pork sausages were collected in seven municipalities (27 samples in each city) of Minas Gerais, Brazil. Microbiological analyses were performed to enumerate LAB. Two pre-selection criteria were applied to 567 isolates of LAB: catalase activity and tolerance to pH 2. A total of 32 strains of UFLA SAU were selected, characterized phenotypically and identified through 16S rDNA region sequencing. The susceptibility to antimicrobial and antibacterial activities of isolates was evaluated. The LAB count ranged from 3.079 to 8.987 log10 CFU/g. Lactobacillus plantarum and Lactobacillus paracasei were identified in the samples. UFLA SAU 11, 20, 34, 86, 131 and 258 showed a profile of susceptibility to four antimicrobials: erythromycin, ampicillin, chloramphenicol and gentamycin. In the antibacterial activity test, with exception of UFLA SAU 1, all other strains showed efficiency in inhibiting Escherichia coli, Salmonella Typhiand Listeria monocytogenes. In the statistical analysis there was interaction among strains of Lactobacillus against the pathogens tested. L. monocytogenes (P=0.05) was more sensitive to Lactobacillus strains and the highest inhibitory activity against this pathogen was achieved by strains UFLA SAU 135, 226, 238 and 258. Thus, UFLA SAU 11, 20, 34, 86, 131, 135, 226, 238 and 258 possess safety characteristics for application in meat products.

Bactérias ácido-lácticas (BAL) são microrganismos indígenas em linguiças. A utilização de selecionadas BAL autóctones pode melhorar a segurança dos produtos cárneos. Este estudo objetivou enumerar e identificar BAL em linguiças suínas e caracterizar suas propriedades de segurança, como a susceptibilidade antimicrobiana e a atividade antibacteriana. Um total de 189 embalagens fechadas de linguiça suína foi adquirido em sete municípios (27 amostras em cada cidade) de Minas Gerais, Brasil. Análises microbiológicas para a enumeração de BAL foram realizadas. Dois critérios de pré-seleção foram aplicados para os 567 isolados de BAL: atividade catalase e tolerância ao pH 2. Um total de 32 estirpes UFLA SAU foi selecionado, caracterizado fenotipicamente e identificado por meio do sequenciamento da região 16S rDNA. A susceptibilidade a antimicrobianos e a atividade antimicrobiana dos isolados foram avaliadas. Nas linguiças, a contagem de BAL variou de 3,079 a 8,987log10 UFC/g. Lactobacillus plantarum e Lactobacillus paracasei foram identificados nas amostras. UFLA SAU 11, 20, 34, 86, 131 e 258 apresentaram um perfil de suscetibilidade a quatro antimicrobianos: eritromicina, ampicilina, cloranfenicol e gentamicina. No teste de atividade antibacteriana, com exceção da UFLA SAU 1, todas as outras estirpes mostraram eficiência em inibir Escherichia coli, Salmonella Typhi e Listeria monocytogenes. Na análise estatística, houve interação entre estirpes de Lactobacillus contra os patógenos testados. L. monocytogenes (P=0,05) foi mais sensível às estirpes de Lactobacillus, e a maior atividade inibitória contra este patógeno foi apresentada por estirpes UFLA SAU 135, 226, 238 e 258. Assim, estirpes UFLA SAU 11, 20, 34, 86, 131, 135, 226, 238 e 258 possuem características de segurança para aplicação em produtos cárneos.

The use of Lactobacillus species as starter cultures for enhancing the quality of sugar cane silage.

Sugar cane (Saccharum spp.) is a forage crop widely used in animal feed because of its high dry matter (DM) production (25 to 40 t/ha) and high energy concentration. The ensiling of sugar cane often incurs problems with the growth of yeasts, which leads to high losses of DM throughout the fermentative process. The selection of specific inoculants for sugar cane silage can improve the quality of the silage. The present study aimed to select strains of lactic acid bacteria (LAB) isolated from sugar cane silage and to assess their effects when used as additives on the same type of silage. The LAB strains were inoculated into sugar cane broth to evaluate their production of metabolites. The selected strains produced higher concentrations of acetic and propionic acids and resulted in better silage characteristics, such as low yeast population, lower ethanol content, and lesser DM loss. These data confirmed that facultative heterofermentative strains are not good candidates for sugar cane silage inoculation and may even worsen the quality of the silage fermentation by increasing DM losses throughout the process. Lactobacillus hilgardii strains UFLA SIL51 and UFLA SIL52 resulted in silage with the best characteristics in relation to DM loss, low ethanol content, higher LAB population, and low butyric acid content. Strains UFLA SIL51 and SIL52 are recommended as starter cultures for sugar cane silage.

Identification and assessment of kefir yeast potential for sugar/ethanol-resistance.

Biochemical and molecular analysis was used for identification of different kefir yeasts species from Brazil, Canada and the United States of America. The sugar/ethanol-resistant activity of the yeasts was evaluated. Saccharomyces cerevisiae and Kluyveromyces marxianus had the highest growth rates, suggesting biotechnological applications possible for these strains.

Selection of tropical lactic acid bacteria for enhancing the quality of maize silage.

The objective of this study was to select lactic acid bacteria (LAB) strains isolated from silage and assess their effect on the quality of maize silage. The LAB strains were inoculated into aqueous extract obtained from maize to evaluate their production of metabolites and pH reduction. The ability to inhibit the pathogenic and silage-spoilage microorganisms' growth was evaluated. Nine LAB strains that showed the best results were assessed in polyvinyl chloride experimental silos. The inoculation of the LAB strains influenced the concentration of lactic and acetic acids and the diversity of Listeria. The inoculation of silages with Lactobacillus buchneri (UFLA SLM11 and UFLA SLM103 strains) resulted in silages with greater LAB populations and improvements after aerobic exposure. The UFLA SLM11 and SLM103 strains identified as L. buchneri showed to be promising in the treatment of maize silage.

Identification and assessment of kefir yeast potential for sugar/ethanol-resistance

Biochemical and molecular analysis was used for identification of different kefir yeasts species from Brazil, Canada and the United States of America. The sugar/ethanol-resistant activity of the yeasts was evaluated. Saccharomyces cerevisiae and Kluyveromyces marxianus had the highest growth rates, suggesting biotechnological applications possible for these strains.

Identification and characterization of yeasts in sugarcane silages.

AIMS: To enumerate the micro-organisms and to identify the yeast species present during the ensilage of different sugarcane (Saccharum spp.) cultivars. METHOD: Samples of sugarcane silage were collected at 10, 20, 30 and 40 days from the start of fermentation. Population levels of lactic acid bacteria (LAB), mesophilic facultative anaerobic (MFA) bacteria, filamentous fungi and yeasts were determined. Nine species of yeasts were classified according to traditional methods and confirmed using molecular techniques. CONCLUSIONS: LAB dominated the ensiling process of sugarcane, although yeasts were present at relatively high population levels throughout the whole fermentation period. The detected species of yeasts varied according to sugarcane cultivar and time of fermentation. Torulaspora delbrueckii was the predominant yeast, followed by Pichia anomala and Saccharomyces cerevisiae. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the population of micro-organisms in general, and of yeasts in particular, present during the fermentation of sugarcane is of fundamental importance in the development of more effective ensiling processes.

Use of specific PCR primers to identify three important industrial species of Saccharomyces genus: Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces pastorianus.

AIM: To develop species-specific primers capable of distinguishing between three important yeast species in alcoholic fermentation: Saccharomyces bayanus, Saccharomyces cerevisiae and Saccharomyces pastorianus. METHODS AND RESULTS: Two sets of primers with sequences complementary to the HO genes from Saccharomyces sensu stricto species were used. The use of the ScHO primers produced a single amplificon of c. 400 or 300 bp with species S. cerevisiae and S. pastorianus, respectively. The second pair of primers (LgHO) was also constructed, within the HO gene, composed of perfectly conserved sequences common for S. bayanus species, which generate amplicon with 700 bp. No amplification product was observed in the DNA samples from non-Saccharomyces yeasts. Saccharomyces species have also been characterized via electrophoretic karyotyping using pulsed-field gel electrophoresis to demonstrate chromosomal polymorphisms and to determine the evolutionary distances between these species. CONCLUSIONS: We conclude that our novel species-specific primers could be used to rapidly and accurately identify of the Saccharomyces species most commonly involved in fermentation processes using a PCR-based assay. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be used for routine identification of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes in less than 3 h.

Features of Saccharomyces cerevisiae as a culture starter for the production of the distilled sugar cane beverage, cachaça in Brazil.

AIMS: To evaluate the dominance and persistence of strains of Saccharomyces cerevisiae during the process of sugar cane fermentation for the production of cachaça and to analyse the microbial compounds produced in each fermentative process. METHODS AND RESULTS: Three S. cerevisiae strains were evaluated during seven consecutive 24-h fermentation batches using recycled inocula. The UFLA CA 116 strain had the largest population of viable organisms, and the maximum population was achieved in the fourth batch after 96 h of fermentation. The UFLA CA 1162 and UFLA CA 1183 strains grew more slowly, and the maximum population was reached in the seventh batch. Molecular characterization of isolated yeast cells using PFGE (pulse field gel electrophoresis) revealed that more than 86% of the isolates corresponded to the initially inoculated yeast strain. The concentration of aldehydes, esters, methanol, alcohol and volatile acids in the final-aged beverages were within the legal limits. CONCLUSIONS: Cachaça produced by select yeast strains exhibits analytical differences. UFLA CA 1162 and UFLA CA 116 S. cerevisiae isolates can be considered the ideal strains for the artisanal production of cachaça in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of select yeast strains can improve the quality and productivity of cachaça production. Our findings are important for the appropriate monitoring of yeast during sugar cane fermentation. In addition, we demonstrate that UFLA CA 116 and UFLA CA 1162, the ideal yeast strains for cachaça production, are maintained at a high population density. The persistence of these yeast strains in the fermentation of sugar cane juice promotes environmental conditions that prevent or decrease bacterial contamination. Thus, the use of select yeast strains for the production of cachaça is a viable economic alternative to standardize the production of this beverage.

Microbiology and physiology of Cachaça (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 x 108/ml; lactic acid bacteria -4 x 10(5)/ml; and bacilli - 5 x 10(4)/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.

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.

Cocoa fermentations conducted with a defined microbial cocktail inoculum.

Cocoa fermentations were performed in wooden boxes under the following four experimental regimens: beans naturally fermented with wild microflora; aseptically prepared beans with no inoculum; and beans inoculated with a defined cocktail containing microorganisms at a suitable concentration either at zero time or by using phased additions at appropriate times. The cocktail used consisted of a yeast, Saccharomyces cerevisiae var. chevalieri, two lactic acid bacterial species, Lactobacillus lactis and Lactobacillus plantarum, and two acetic acid bacterial species, Acetobacter aceti and Gluconobacter oxydans subsp. suboxydans. The parameters measured were cell counts (for yeasts, filamentous fungi, lactic acid bacteria, acetic acid bacteria, and spore formers, including reisolation and identification of all residual cell types), sugar, ethanol, acetic acid, and lactic acid contents (and contents of other organic acids), pH, and temperature. A cut test for bean quality and a sensorial analysis of chocolate made from the beans were also performed. The natural fermentation mimicked exactly the conditions in 800-kg boxes on farms. The aseptic box remained largely free of microflora throughout the study, and no significant biochemical changes occurred. With the zero-time inoculum the fermentation was almost identical to the natural fermentation. The fermentation with the phased-addition inoculum was similar, but many changes in parameters were slower and less pronounced, which led to a slightly poorer end product. The data show that the nearly 50 common species of microorganisms found in natural fermentations can be replaced by a judicious selection and concentration of members of each physiological group. This is the first report of successful use of a defined, mixed starter culture in such a complex fermentation, and it should lead to chocolate of more reliable and better quality.