Determination of killer activity in yeasts isolated from the elaboration of seasoned green table olives

Determination of killer activity in yeasts isolated from the elaboration of seasoned green table olives Paper details: Read through the paper and produce a 1-2 paragraph summary or abstract. Determination of killer activity in yeasts isolated from the elaboration of seasoned green table olives Alejandro Hernández, Alberto Martín, María G. Córdoba, María José Benito, Emilio Aranda, Francisco Pérez-Nevado ? Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Ctra. de Cáceres s/n. 06071 Badajoz, Spain Received 10 January 2007; received in revised form 31 July 2007; accepted 6 November 2007 Abstract In this work 51 yeasts strains isolated from seasoned green table olives and belonging to the Candida , Debaryomyces , Kluyveromyces , Pichia , and Saccharomyces genera were characterized by their killer activity in different conditions. Killer activity of isolates was analyzed in a medium with different pH's (3.5 to 8.5) and NaCl concentrations (5, 8, and 10%). At every pH tested, all the genera studied had killer strains, although the smallest percentages of killer yeasts were found at the highest pH (8.5). The presence of 5 and 8% NaCl increased the detected killer percentage, but the highest salt concentration (10%) decreased it. The interaction between the reference killer yeasts and yeasts isolated from olives was analyzed . Most isolates were killer-sensitive to one or more killer reference strains. Only 2 of the 51 strains tested were considered killer-neutral. Cross- reaction trials between isolates and spoilage yeasts showed that, of the isolates, nine killer strains, belonging to Debaryomyces hansenii , Kluy- veromyces marxianus , Pichia anomala , Pichia guilliermondii , and Saccharomyces cerevisiae , had the broadest spectra of action against yeasts that cause spoilage. These killer yeasts and the toxins that they produce are candidates for further investigation as suppressors of indigenous olive tab le yeast growth. The results confirmed the highly polymorphic expression of the killing activity, with each strain showing different killer activitie s. This method may thus be very useful for simple and rapid characterization of yeast strains of industrial interest. © 2007 Elsevier B.V. All rights reserved. Keywords: Killer; Yeast; Brine; Fermentation; Olives 1. Introduction The presence of yeasts in different kinds of table olive fermentation is common ( Marquina et al., 1992; Llorente et al., 1997; Kotzekidou, 1997; Tassou et al., 2002; Durán Quintana et al., 2003; Hernández et al., 2006 ). The predominant yeast species that have been isolated from Greek-style black olives are Torulaspora delbrueckii , Debaryomyces hansenii , and Crypto- coccus laurentii ( Kotzekidou, 1997 ). Other workers ( Marquina et al., 1992 ) have isolated Pichia membranifaciens and related species as the dominant yeasts from spontaneous fermentations of olive brines from Portugal as the dominant yeasts. In studies performed by Marquina et al. (1997) with olive brines from seven locations in Morocco, the most ubiquitous and abundant species were T. delbrueckii , Candida boidinii ,and P. membra- nifaciens . Various studies have attributed to the yeast population the role of contributing to the sensorial characteristics of table olives ( Garrido et al., 1995; Sánchez et al., 2000 ). Since these micro-organisms could have an important effect on the quality of this product, their study could help in the selection of starter cultures to be used in the elaboration of table olives. Yeasts such as Candida krusei , and P. membranifaciens (originally Candida valida ), for example, are not considered spoilage yeasts and could be used as starter cultures ( Durán Quintana et al., 1979 ). However, other surveys have associated the presence of yeasts with different kinds of olive spoilage ( Vaughn et al., 1969 and 1972; Durán Quintana et al., 1979 and 1986 ). If film- forming yeasts are not controlled, they can rapidly oxidize the desirable acidity in the storage brines of Sicilian-style and Spanish-type olives. Vaughn et al. (1972) found that Saccharo- A vailable online at www.sciencedirect.com International Journal of Food Microbiology 121 (2008) 178 – 188 www.elsevier.com/locate/ijfoodmicro ? Corresponding author. Tel.: +34 924 286200; fax: +34 924 286201. E-mail address: [email protected] (F. Pérez-Nevado). URL: http://eia.unex.es (F. Pérez-Nevado). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi: 10.1016/j.ijfoodmicro.2007.11.044 myces cerevisiae (originally identified as S. oleaginosus ), Sac- charomyces kluyveri , and Pichia anomala (originally identified as Hansenula anomala ) can cause softening and gas-pocket formation in olives. Also, pink yeasts identified as Rhodotorula glutinis , Rhodotorula minuta , and Rhodotorula rubra cause slow softening of olive tissue ( Vaughn et al., 1969 ). The pre- sence of various yeast strains of S. cerevisiae and P. anomala has been related to “ alambrado ” (bloater) spoilage in spontaneous fermentation in black olives ( Durán Quintana et al., 1979 ). Other species that have been related to this alteration are Pichia sub- pelliculosa (originally Hansenula subpelliculosa ), Kluyvero- myces thermotolerans (originally Kluyveromyces veronae ), Candida saitoana (originally Torulopsis candida ), Candida norvegica (originally Torulopsis norvegica ), D. hansenii , and Pichia fermentans . Different methods have been used to control spoilage pro- duced by yeasts in table olives. The most commonly used practice in this industry of controlling the pH and salt level of the brine is insufficient to avoid these problems ( Lamzira et al., 2005 ). Studies with an essential oil prepared from garlic had a major effect in controlling the yeast population, but the orga- noleptic characteristics of the product were affected ( Asehraou et al., 1997 ). Trials with pH adjusted to 4, added potassium sorbate, and Lactobacillus plantarum inoculation have found that bloater spoilage was reduced drastically ( Asehraou et al., 2002; Lamzira et al., 2005 ). The use of yeasts as starter culture could protect the product against spoilage yeasts, selecting, for example, killer yeasts which are known to be able to control spoilage in the preservation of food. Killer yeasts can produce toxic proteins or glycoproteins (so-called killer toxins) that can cause death in other sensitive (killer-sensitive) yeast strains. The killer phenotype appears to be widely distributed within many yeast genera ( Schmitt and Breinig, 2002 ) some of them isolated from a great variety of fermentation food processes ( Llorente et al., 1997; Regodón et al., 1997; Gulbiniene et al., 2004 ). It is affected by diverse ambient conditions like pH, including temperature, and the presence of salt ( Woods and Bevan, 1968; Llorente et al., 1997; Marquina et al., 2001; Buzzini et al., 2004; Izgü and Altinbay, 2004 ). Moreover, its detection depends strongly on the sensitive strains used — the killing ability of different compounds may be underestimated or may even remain unnoticed depending on the selection of the appropriate sensitive strain and other experimental conditions. For this reason, the high variability of the killer phenomenon in nature provides an exceptional tool for the discrimination of yeasts at the strain level. Also, the use of these yeasts as a biocontrol method may improve table olives by reducing the requirement for salt or such chemical preservatives as sorbic acid or similar. The aim of the present work was to study the killer activity of yeast strains isolated from seasoned green table olives. Since salt addition is a common practice in the production of tra- ditional fermented table olives, the effect of NaCl addition on the killer expression was analyzed, as also was the effect of pH. In addition, the killer activity and sensitivity of isolates to different killer reference and wild yeast strains was analyzed with a view to the potential use of these yeasts as biocontrol agents against spoilage yeasts. 2. Materials and methods 2.1. Yeast strains isolated from green table olives Seventy-four indigenous yeast strains isolated from seasoned table olive fermentation by Hernández et al. (2006) were tested. Table 1 List of pre-selected strains used in the survey Species        Strain  Origin      Species        Strain  Origin Candida                         Pichia C. inconspicua FM47  Olive brine P. anomala FM5   Olive brine C. lusitaniae FM59  Olive brine P. anomala FM14  Olive brine C. maris FM15  Olive brine P. anomala FM23  Olive brine C. maris FM46  Olive brine P. anomala FM25  Olive brine C. maris FM66  Olive brine P. anomala FM30  Olive brine C. maris FM67  Olive brine P. anomala FM31  Olive brine C. zeylanoides FM10  Olive brine P. anomala FM34  Olive brine C. zeylanoides FM71  Olive brine P. anomala FM36  Olive brine Cryptococcus                     P. anomala FM37  Olive brine C. humicola FM29  Olive brine P. anomala FM38  Olive brine C. humicola MP11  Fresh olives P. anomala FM40  Olive brine Debaryomyces                    P. anomala FM52  Olive brine D. hansenii FM1   Olive brine P. anomala FM58  Olive brine D. hansenii FM32  Olive brine P. anomala FM62  Olive brine D. hansenii FM72  Olive brine P. anomala FM73  Olive brine Kluyveromyces Olive brine P. anomala FM74  Olive brine K. marxianus FM2   Olive brine P. anomala FM79  Olive brine K. marxianus FM6   Olive brine P. guilliermondii MP6   Fresh olives K. marxianus FM7   Olive brine Saccharomyces K. marxianus FM11  Olive brine S. cerevisiae FM8   Olive brine K. marxianus FM12  Olive brine S. cerevisiae FM9   Olive brine K. marxianus FM19  Olive brine S. cerevisiae FM20  Olive brine K. marxianus FM24  Olive brine S. cerevisiae FM39  Olive brine K. marxianus FM26  Olive brine S. cerevisiae FM41  Olive brine K. marxianus FM27  Olive brine S. cerevisiae FM43  Olive brine K. marxianus FM69  Olive brine S. cerevisiae FM44  Olive brine K. marxianus FM78  Olive brine S. cerevisiae FM51  Olive brine S. cerevisiae FM68  Olive brine Table 2 List of potential spoilage strains used in the survey Species        Strain  Origin       Species       Strain  Origin Candida                          Rhodotorula C. albicans FM70  Olive brine R. glutinis FM21  Olive brine C. glabrata FM50  Olive brine R. glutinis MP9   Fresh olives C. parapsilosis FM57  Olive brine R. minuta FM17  Olive brine C. rugosa FM4   Olive brine Trichosporon C. rugosa FM16  Olive brine T. cutaneum FM3   Olive brine C. rugosa FM18  Olive brine T. cutaneum FM13  Olive brine Cryptococcus                      T. cutaneum FM45  Olive brine C. albidus FM22  Olive brine T. cutaneum FM54  Olive brine C. albidus FM33  Olive brine C. albidus MP5   Fresh olives C. albidus MP7   Fresh olives C. laurentii FM60  Olive brine C. laurentii FM63  Olive brine C. laurentii FM75  Olive brine C. laurentii MP2   Fresh olives C. laurentii MP8   Fresh olives C. laurentii MP10  Fresh olives 179 A. Hernández et al. / International Journal of Food Microbiology 121 (2008) 178 – 188