Spoilage yeasts: What are the sources of contamination of foods and beverages?

Foods and beverages are nutrient-rich ecosystems in which most microorganisms are able to grow. Moreover, several factors, such as physicochemical characteristics, storage temperature, culinary practices, and application of technologies for storage, also define the microbial population of foods and beverages. The yeast population has been well-characterised in fresh and processed fruit and vegetables, dairy products, dry-cured meat products, and beverages, among others. Some species are agents of alteration in different foods and beverages. Since the most comprehensive studies of spoilage yeasts have been performed in the winemaking process, hence, these studies form the thread of the discussion in this review. The natural yeast populations in raw ingredients and environmental contamination in the manufacturing facilities are the main modes by which food contamination occurs. After contamination, yeasts play a significant role in food and beverage spoilage, particularly in the alteration of fermented foods. Several mechanisms contribute to spoilage by yeasts, such as the production of lytic enzymes (lipases, proteases, and cellulases) and gas, utilisation of organic acids, discolouration, and off-flavours. This review addresses the role of yeasts in foods and beverages degradation by considering the modes of contamination and colonisation by yeasts, the yeast population diversity, mechanisms involved, and the analytical techniques for their identification, primarily molecular methods.

Combined effect of antagonistic yeast and modified atmosphere to control Penicillium expansum infection in sweet cherries cv. Ambrunés.

Fruit decay caused by pathogenic moulds is a major concern in the postharvest quality and shelf life of fruit. Blue mould decay is caused by Penicillium expansum (P. expansum) and is one of the most important postharvest diseases in cherries (Prunus avium L.). Synthetic fungicides are the main medium used to control pathogenic moulds. However, alternative approaches are available for developing safer technologies to control postharvest disease. An integrated approach that combines biological control, using antagonistic yeasts and modified atmosphere packaging (MAP) with cold storage is a promising alternative to synthetic fungicide treatment. In this work, two microperforated films (M10 and M50) and two antagonistic yeast strains (Hanseniaspora opuntiae L479 and Metschnikowia pulcherrima L672) were evaluated for their effectiveness to control the development of P. expansum in wounded cherries stored at 1°C. Results showed that the microperforated films had fungistatic effects, particularly M50, due to the level of CO2 achieved (mean CO2 of 11.2kPa at 35days), and the decrease in disease severity. Antagonistic yeasts, particularly Metschnikowia pulcherrima L672, delayed the development of P. expansum and decreased disease incidence and severity. The combination of MAP and antagonistic yeasts was the most effective approach to control P. expansum, during cold storage.

Yeasts isolated from figs (Ficus carica L.) as biocontrol agents of postharvest fruit diseases.

Fresh fruit is highly perishable during postharvest life, mainly due to fungal growth. Thus, fungal control is an important goal for the fruit industry. In this work, a selection of antagonistic yeasts isolated from fig and breba crops were screened in vitro. The isolated yeasts were challenged with three moulds isolated from decayed figs and breba crops, identified as Penicillium expansum M639 and Cladosporium cladosporioides M310 and M624, and pathogenic moulds Botrytis cinerea CECT20518 and Monilia laxa CA1 from culture collections. Two yeast isolates, Hanseniaspora opuntiae L479 and Metschnikowia pulcherrima L672, were selected for their ability to inhibit the growth of aforementioned moulds. These yeasts reduced the radial growth of moulds on PDA by between 45.23% and 66.09%. Antagonistic activity was associated with the interaction of live yeast cells with moulds. M. pulcherrima L672 apparently parasitised C. cladosporioides isolates. In addition, challenges were assayed using wounded apples and nectarines, with significant reductions in percent infection and lesion size for all moulds tested. To our knowledge, this is the first report identifying H. opuntiae as an antagonist against different pathogenic moulds.

Application of ISSR-PCR for rapid strain typing of Debaryomyces hansenii isolated from dry-cured Iberian ham.

Yeast populations of dry-cured Iberian ham isolated from seven industries in the province of Badajoz were characterized by ISSR-PCR using the (CAG)4 primer and PCR-RFLP of the ITS1-5.8S rRNA-ITS2 fragment, and identified by DNA sequencing. A total of 242 isolates were analyzed, indicating the primary species present was Debaryomyces hansenii at 80.9% of the isolates followed by Candida zeylanoides at 10.3% of the isolates. The remainders of isolates were identified as Yamadazyma triangularis, Sporobolomyces roseus, Meyerozyma guilliermondii, Rhodotorula slooffiae, and Cryptococcus victoriae. The ISSR-PCR method was a fast and reliable method which was able to discriminate species at a level comparable to restriction analyses of the ITS1-5.8S rRNA-ITS2 region. This method allowed for strain typing of D. hansenii, yielding 29 different PCR patterns within 196 isolates. Moreover, ISSR-PCR using the (CAG)4 primer indicated that this technique could be a promising tool for rapid discrimination of yeast starter cultures and spoilage species in dry-cured Iberian ham.

Characterization and selection of autochthonous lactic acid bacteria isolated from traditional Iberian dry-fermented salchichón and chorizo sausages.

A total of 192 lactic acid bacteria were isolated from 2 types of naturally fermented dry sausages at 3 different stages of the ripening process in order to select the most suitable strains as starter cultures in dry-cured sausage manufacture according to their technological characteristics such as glucose fermentation, lactic and acetic acid production, and proteolytic, lipolytic, and antimicrobial activities. Identification of the isolates revealed that 31.2% were Pediococcus pentosaceus, 26.9% Lactococcus lactis, 18.6% Pediococcus acidilactici, 17% Lactobacillus brevis, and sporadic isolates of Leuconostoc mesenteroides, Lactobacillus plantarum, and Lactobacillus curvatus. Most of the strains did not produce gas from glucose and showed the capacity to produce lactic acid rapidly. Some 25% of the strains were able to degrade tributyrin (esterase activity), but none showed lipolytic activity against olive oil and pork fat. Only 3 strains of P. acidilactici showed weak proteolytic activity against myofibrillar or sarcoplasmic proteins. Also, the same strains showed antimicrobial activity against Listeria monocytogenes. Nine strains with the best properties were preselected and tested for biogenic amine production. The results showed that two of the strains, identified as P. acidilactici by polymerase chain reaction, had the potential to be further tested as starter cultures in pilot processing of Iberian sausages.

Differentiation of Clostridium perfringens and Clostridium botulinum from non-toxigenic clostridia, isolated from prepared and frozen foods by PCR-DAN based methods.

During the elaboration process of prepared and frozen foods, Clostridium sp. have been reported. From those microorganisms, C. perfringens and C. botulinum may pose a high risk for the consumers. To avoid these pathogenic organisms an HACCP program should be implemented, but in addition sensitive and moderately time consuming microbiological methods for monitoring C. perfringens and C. boulinum should be established. In this work, an RFLP analysis of the 16S rDNA will be developed to differentiate C. perfringens from other Clostridium sp. In addition, a PCR protocol, will be assayed to detect C. botulinum. Both two methods will be compared with biochemical characterization by API system. The restriction analysis of the 16S rDNA with Taq I and Rsa I showed at least the same sensitivity to differentiate C. perfringens from clostridial isolates as biochemical identification. However, the former method takes only 8-10 h of analysis as compared with 24-48 h required for biochemical characterization. With the specific PCR protocol to detect C. botulinum a band of 1.1 kbp was obtained derived from the specific amplification of BoNT genes, taking 6-8 h for analysis. Both two molecular DNA based methods should be considered as verification techniques of pathogenic clostridia in the HACCP program.

Rapid differentiation of Staphylococcus aureus from staphylococcal species by arbitrarily primed-polymerase chain reaction.

An arbitrarily primed-polymerase chain reaction (AP-PCR) method was optimized to differentiate Staphylococcus aureus from other staphylococcal species, using DNA from crude cell extract. From the different assays carried out, the best resolution of the band patterns was obtained when the reaction mixture contained 200 micromol l(-1) dNTPs, 200 ng primer, 1 U Taq DNA polymerase and 3 mmol l(-1) MgCl2 and the amplification conditions were: initial denaturation of 94 degrees C for 1 min, primer annealing of 30 degrees C for 1.5 min, DNA extension at 55 degrees C for 5 min and final extension at 55 degrees C for 5 min. The results of the characterization of the staphylococcal isolates by AP-PCR are in accordance with those of the biochemical identification by the API Staph System, time of analysis of the AP-PCR being only 6-7 h. Thus, this technique could be a useful method for microbial quality assurance.

Evaluation of microbial hazards during processing of Spanish prepared Flamenquín.

Flamenquín is a traditional, prepared, frozen meat product from the south of Spain made with minced pork, chicken, and cooked ham. Since it is a prepared raw meat product some microbial hazards could be associated with the process of making it. Microbiological analyses have been performed throughout the various steps of processing over a 1-year period to evaluate microbial hazards in the commercial process. High levels of microorganisms were observed all through the processing of this product, the mincing and mixing steps being where major microbial contamination was observed. Pathogenic bacteria such as Staphylococcus aureus, Clostridium perfringens, Escherichia coli and Pseudomonas aeruginosa were detected during processing. Raw materials and food handlers were the principal sources of microbial contamination. A modification of processing to include a heating step after mincing and mixing and an improvement in hygiene practices could eliminate the microbial hazards. Both modifications should be noted for the implementation of a hazard analysis of critical control points (HACCP) program in commercial flamenquín processing.

Aflatoxin-Producing Strains of Aspergillus flavus Isolated from Cheese.

Contamination by fungi of the genus Aspergillus with special reference to the possible detection of aflatoxin-producing strains of Aspergillus flavus was studied in 52 samples of commercial cheeses made with different types of milk (8 of cow's milk, 12 of ewe's milk, 13 of goat's milk, and 19 of milk mixtures of various species: cow, ewe, and goat) produced in Southern Spain. The frequency of appearance of various species of Aspergillus , A. glaucus , A. niger , A. nidulans , A. sulphureus , A. Terreus , and A. flavus , in the different types of cheese was determined. In 4 (2 of goat's milk cheese and 2 of cheeses made with milk from various species) out of 52 samples (7.69%), aflatoxin-producing strains of A. flavus were detected.