Marcela P. Sangorrín

Patagonian wines: the selection of an indigenous yeast starter.

The use of selected yeasts for winemaking has clear advantages over the traditional spontaneous fermentation. The aim of this study was to select an indigenous Saccharomyces cerevisiae yeast isolate in order to develop a regional North Patagonian red wine starter culture. A two-step selection protocol developed according to physiological, technological and ecological criteria based on killer interactions was used. Following this methodology, S. cerevisiae isolate MMf9 was selected among 32 indigenous yeasts previously characterized as belonging to different strains according to molecular patterns and killer biotype. This isolate showed interesting technological and qualitative features including high fermentative power and low volatile acidity production, low foam and low sulphide production, as well as relevant ecological characteristics such as resistance to all indigenous and commercial S. cerevisiae killer strains assayed. Red wines with differential volatile profiles and interesting enological features were obtained at laboratory scale by using this selected indigenous strain.

Killer behaviour in wild wine yeasts associated with Merlot and Malbec type musts spontaneously fermented from Northwestern Patagonia (Argentina)

The occurrence of killer wine yeasts in Comahue Region (Patagonia, Argentina) was studied. Wild wine yeasts were isolated from spontaneously fermenting Merlot and Malbec type musts. Out of 135 isolates analyzed 37% were sensitive to some well characterized killer toxins as K1–K10 and did not show killer activity (sensitive phenotype, S), 21% showed neutral phenotype (N) and 42% demonstrated killer activity (killer phenotype, K). All but two killer strains, identified as Candida pulcherrima and Kluyveromyces marxianus, were Saccharomyces cerevisiae. Additionally, all killer strains were sensitive to some killer reference strains, showing a killer‐sensitive phenotype (KS); neither Saccharomyces or non‐Saccharomyces wild yeasts were phenotype killer‐resistant (KR).

Efficacy and putative mode of action of native and commercial antagonistic yeasts against postharvest pathogens of pear.

Putative mechanisms of action associated with the biocontrol capacity of four yeast strains (Cryptoccocus albidus NPCC 1248, Pichia membranifaciens NPCC 1250, Cryptoccocus victoriae NPCC 1263 and NPCC 1259) against Penicillium expansum and Botrytis cinerea were studied by means of in vitro and in situ assays. C. albidus(YP), a commercial yeast was also evaluated for comparative purposes. The yeast strains exhibited a variety of different mechanisms including: wound colonization, germination inhibition, biofilm formation, secretion of killer toxins, competition for nutrient and secretion of hydrolytic enzymes (protease, chitinase and glucanase). The relationship between strains (and their associated antagonist mechanisms) and in situ antagonist activity was also evaluated. Results indicate that mechanisms such as production of hydrolytic enzymes, the ability for colonization of wounds, production of killer toxin and inhibition of germination are the most important for biocontrol activity. Our study indicate that multiple modes of action may explain why P. membranifaciens NPCC 1250 and C. victoriae NPCC 1263 provided excellent control of postharvest pears disease.

Yeast biocontrol of fungal spoilage of pears stored at low temperature.

To reduce the use of fungicides, biological control with yeasts has been proposed in postharvest pears. Most studies of antagonists selection have been carried out at room temperature. However, in regions like North Patagonia where fruits are stored at -1/0 °C during 5-7 months the selection of potential antagonist agents must be carried out at low temperature. In this study, 75 yeast cultures were isolated from healthy pears from two Patagonian cold-storage packinghouses. Aureobasidium pullulans, Cryptococcus albidus, Cryptococcus difluens, Pichia membranifaciens, Pichia philogaea, Rhodotorula mucilaginosa and Saccharomyces cerevisiae yeast species were identified. Additionally, 13 indigenous isolates of Penicillium expansum and 10 isolates of Botrytis cinerea were obtained from diseased pears, characterized by aggressiveness and tested for sensitivity to postharvest fungicides. The yeasts were pre-selected for their ability to grow at low temperature. In a first biocontrol assay using the most aggressive and the most sensitive isolate of each pathogen, two epiphytic isolates of A. pullulans and R. mucilaginosa were the most promising isolates to be used as biocontrol agents. They reduced the decay incidence by P. expansum to 33% and the lesion diameter in 88% after 60 days of incubation in cold. Foreign commercial yeast used as a reference in assays, only reduced 30% of lesion diameter in the same conditions. Yeasts were not able to reduce the incidence of B. cinerea decay. The control activity of the best two yeasts was compared with the control caused by the fungicides in a second bioassay, obtaining higher levels of protection against P. expansum by the yeasts. These two regional yeasts isolates could be promising tools for the future development of commercial products for biological control.

The use of killer biotyping in an ecological survey of yeast in an old patagonian winery

An ecological study of Saccharomyces cerevisiae strains in spontaneous alcoholic fermentation has been made in the same winery on two consecutive years (1993 and 1994) with Merlot type musts, and with Malbec type must on a third year (1998). Saccharomyces cerevisiae strains associated with winery surfaces were also analysed. Differential killer sensitivity patterns related to a killer reference panel of 10 killer yeasts belonging to nine species of four genera were used as a quick and simple procedure to discriminate between indigenous S. cerevisiae isolates at the strain level. Although a great diversity of wild strains was observed, two main indigenous S. cerevisiae strains, designated as S. cerevisiae 9 and S. cerevisiae 13, took over the Merlot type fermentation in both years. These strains also appeared in Malbec must fermentation during the year 1998 and they were again found on the winery surface the next year. These results show that some few and stable indigenous S. cerevisiae strains remained in the environmental winery over the considered period of time (1993–1999) and they represent an additional evidence of the taking over of musts by local strains of S. cerevisiae.

Production of volatile phenols by Pichia manshurica and Pichia membranifaciens isolated from spoiled wines and cellar environment in Patagonia.

In order to detect spoilage yeast species in wines showing off-odors, different yeast isolation protocols were evaluated. Independently of the isolation method, only Saccharomyces cerevisiae and Pichia manshurica were detected. The spoilage capacity of P. manshurica regional isolates was evaluated in red wine and the production of volatile phenols was evidenced. To evaluate the possible source of contamination, yeasts from both grapes and cellar surfaces were obtained. Hanseniaspora uvarum and Zygoascus hellenicus were detected in both sound and damaged grapes from sunny areas. The most frequent species in cellar surfaces was Candida boidinii, Pichia membranifaciens and P. manshurica were detected in filters. The intra-specific genetic characterization of the P. manshurica isolates by mtDNA-RFLP demonstrated that the same strain was detected in both wine and filter. Most P. membranifaciens isolates produced 4-EP (maximum level of 1.895 mg/L) and particularly high levels of 4-EG (maximum level of 10.260 mg/L) were produced by P. manshurica isolates in synthetic wine-like medium. In this work the capacity of P. manshurica and P. membranifaciens species to produce volatile phenols was shown for the first time.

A new improved strategy for the selection of cold-adapted antagonist yeasts to control postharvest pear diseases

Abstract Postharvest diseases cause considerable losses of harvested fruits during transportation and storage. Many yeast species have been reported as good antagonists against postharvest pear pathogens. In this work, we used a novel selection strategy that involves the isolation of yeasts from washing fluids, showing biocontrol activity against a regional Penicillium expansum strain (primary screening), originally obtained from fruit wounds after long time storage at −1/0°C. About 26 isolates representative of the 11 yeast species identified in the 27 selected washing waters were chosen to be evaluated in a secondary screening against a regional Botrytis cinerea strain on pear wounds. Among yeasts tested, 38% showed complete control of P. expansum, but only 15% reduced the decay incidence of B. cinerea to 60–80% at −1/0°C. These results reveal that some of the yeasts found can be biological alternatives to fungicides in the control of P. expansum and B. cinerea infections. Based on the data obtained, our strategy seems to be much more effective than the previously reported methods in obtaining successful biocontrol agents.

Saccharomyces uvarum is Responsible for the Traditional Fermentation of Apple CHICHA in Patagonia.

Abstract Apple chicha is a fresh low alcoholic beverage elaborated by aboriginal communities of Andean Patagonia (Argentina and Chile). In the present work, we identified the yeast microbiota associated with this fermentation, and characterized genetically those belonging to the genus Saccharomyces. Both Saccharomyces cerevisiae and S. uvarum were found in the analyzed fermentations. Phylogenetic and population structure analyses based on genes sequence analysis were carried out for both S. cerevisiae and S. uvarum strains obtained in this study and a set of additional strains from diverse origins. The results demonstrate that S. cerevisiae strains from apple chicha belong to the big group of wine/European strains of this species, while S. uvarum strains were included in the Holartic population of this species. Additionally, some S. uvarum strains from chichas evidenced as an admixture of both pure Holartic and pure South American populations. Our results suggest that Holartic strains could have been introduced in South America together with the domestication of apple trees by Mapuche communities. This Holartic population suffered admixis with the naturally present South American population of this species, originating strains bearing genetic features from the two populations, detectable in both chichas and natural habitats.

Cold-Adapted Yeasts as Biocontrol Agents: Biodiversity, Adaptation Strategies and Biocontrol Potential

After harvest, many fruits are kept in cold storage in order to prolong their availability and shelf life. Often, this requires the application of a chemical fungicide to prevent postharvest decay from decay fungi. An alternative approach for preventing postharvest fungal decay during storage could be based on the treatment of the commodity with antagonistic yeasts. In this regard, the use of cold-adapted yeasts may offer a distinct advantage. Numerous cold-adapted yeast species have been isolated from artificial cold environments, as well as cold-stored fruits. Since the method employed to isolate potential antagonists has a major impact on the type and properties of the antagonist to be identified, it is important to evaluate the consequences of the methods that are presently being utilized and to appraise whether or not they can be improved. Although the mechanism(s) by which yeast antagonists suppress postharvest diseases can be quite variable, competition for nutrients and space plays a major role in their antagonistic activity. Additionally, production of antibiotics, direct parasitism and the induction of resistance in the harvested commodity are other modes of action that have been documented and suggested to play a role in how yeasts suppress postharvest pathogens in harvested fruits. While a few yeast-based products have been on the market, this field of study is still in its infancy and it is likely that several new products will enter the market in near future. Nonetheless, it is necessary to continue to identify new potential microorganisms and to develop a better understanding of the biology of yeast biocontrol systems by involving yeasts, pathogens and host commodity, in order to increase the potential of postharvest biocontrol as a viable alternative to synthetic postharvest fungicides. While the results of this technology are encouraging, we need to continue to explore the potential use of appropriate yeasts worldwide where management practices, types of fruit and decay pathogens can vary considerably.

Patagonian Antagonist Yeasts for Food Biopreservation

Worldwide, microbial growth destroys large amounts of various products, causing yield losses in the agronomical and biotechnological industries. Traditionally, biocides have been used to manage these problems, but different disadvantages such as the establishment of resistant strains and the suppression of natural competitors have made alternatives such as biological control necessary. After harvest, many fruits are kept in cold storage to prolong their availability and shelf life. Often, this requires the application of a chemical fungicide to prevent postharvest decay from decay fungi. An alternative approach for preventing postharvest fungal decay during storage could be based on the treatment of the commodity with antagonistic yeasts. In this regard, the use of cold-adapted yeasts may offer a distinct advantage. Numerous cold-adapted yeasts species have been isolated from artificial cold environments, as well as cold-stored fruits. Recently, we isolated and identified epiphytic yeasts during the cold postharvest storage of pears and fine fruits from packinghouses in Argentinean Patagonia, and we tested their efficacy in controlling the postharvest diseases of different fruits caused by several pathogens. Additionally, killer yeasts as producers of mycocins or killer toxins that can neutralize the activities of spoilage yeasts in wines represent an interesting biocontrol strategy. Several screening studies focused to determine the occurrence of killer yeasts in winemaking environments have been carried out, and they have demonstrated the presence of killer phenotypes in yeasts from wines, cellar surfaces, and winery equipment. In previous studies carried out in our laboratory, most yeasts isolated from spontaneously fermenting grape musts evidenced killer character. These studies provide an exceptional source of potential antagonist yeasts to be used in biocontrol of undesired microorganisms in wine. Nonetheless, it is necessary to continue to identify new potential microorganisms and to develop a better understanding of the biology of yeast biocontrol systems to increase the potential of postharvest biocontrol as a viable alternative to synthetic postharvest fungicides and chemical preservatives against wine spoilage yeast.