Debaryomyces Hansenii Strains from Valle De Los Pedroches Iberian Dry

J. Microbiol. Biotechnol. (2017), 27(9), 1576–1585 https://doi.org/10.4014/jmb.1704.04045 Research Article Review jmb

Debaryomyces hansenii Strains from Valle De Los Pedroches Iberian Dry Meat Products: Isolation, Identification, Characterization, and Selection for Starter Cultures José Ramos1, Yessica Melero1, Laura Ramos-Moreno1, Carmen Michán2*, and Lourdes Cabezas3

1Departamento de Microbiología, Escuela Técnica Superior de Ingeniería Agronómica y de Montes, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, Córdoba 14071, Spain 2Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, Córdoba 14071, Spain 3Departamento de Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Córdoba, Campus de Rabanales, Edificio Darwin, Córdoba 14071, Spain

Received: April 19, 2017 Revised: June 13, 2017 Yeasts, filamentous fungi, and bacteria colonize the surface of fermented sausages during the Accepted: June 26, 2017 ripening process. The source of this microbiota is their surrounding environment, and is First published online influenced by the maturing conditions and starter cultures. Debaryomyces hansenii was July 7, 2017 previously isolated from several dry-cured meat products and associated with the lipolytic

*Corresponding author and proteolytic changes that occur in these products, influencing their taste and flavor. Phone: +34-957-218082; Therefore, this study isolated the yeast microbiota present in the casing from different meat Fax: +34-957-218592; E-mail: [email protected] products (“lomo,” “chorizo,” and “salchichón”) from the Valle de los Pedroches region in southern Spain. D. hansenii was by far the most abundant species in each product, as all 22 selected isolates were identified as D. hansenii by biochemical and/or molecular methods. In contrast, no yeasts were found in the meat batter. These data constitute the first study of the yeasts present in “lomo” sausages and particularly the highly appreciated Valle de los Pedroches “lomo” sausages. Furthermore, the resistance of these isolates to different pHs, temperatures, and saline stress was studied, together with their catabolic characteristics. Based on the results, certain isolates are proposed as valuable candidate starter cultures that could improve both the manufacture and the flavor of such dry-cured meat products, and provide an understanding of new mechanisms involved in stress tolerance. Applied medium- scale industrial tests are currently in progress. pISSN 1017-7825, eISSN 1738-8872

Introduction the microbiota growing on these products. Their origins are mainly related to the environment and the meat used as Yeasts and lactic acid bacteria are two predominant raw material. In these products, the highest counts of groups of microorganisms usually found in fermented yeasts are obtained at the end of the fermentation stage [2, sausages, yet whereas lactobacilli are mainly in the meat 3], although they are greatly reduced in the final product batter, yeasts are most frequently found on the sausage [2]. Although Debaryomyces hansenii is the most commonly surface [1]. Studies on the yeast flora of fermented sausages isolated yeast from fermented sausages, an important are very limited, compared with those focused on bacteria, diversity of other yeast species has also been found in these even though they are considered important components of meat products, but with less occurrence, such as Trichosporon

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ovoides, Yarrowia lipolytica, Citeromyces matritensis, and Candida are produced by both large and small factories in Spain. spp. [1, 2, 4, 5]. The generation of volatile and aroma The manufacture of these products is essentially very compounds by D. hansenii is considered an important similar: mixing mince pork meat in the case of “chorizo” contribution to the ripening process and has been found to and “salchichón” or removing the fat from loin pork in be strongly dependent on the clones, strains, or biotypes “lomo,” followed by the addition of spices and seasoning present during the maturation [6, 7]. D. hansenii can prevent (garlic, oregano, sodium chloride, paprika in “chorizo,” black the formation of lipid oxidation products in fermented pepper in “salchichón,” etc). In general, the qualitative sausages, and contributes to improve sensory components, characteristics of naturally fermented sausages are known principally flavors, such as ethyl esters. D. hansenii also to be largely dependent on (i) the quality of the ingredients helps with the catabolism of organic acids (lactic and acetic and raw materials, (ii) the specific conditions of the acids), reduction of nitrite, and ammonia production [8, 9]. processing and ripening, and (iii) the composition of the Notwithstanding, and as mentioned above, D. hansenii is microbial populations. a highly heterogeneous species with wide phenotypic Accordingly, this study attempted to isolate and identify differences between strains as regards the biochemical the indigenous yeast microbiota present in three types activities that contribute to the sensorial characteristics of of Iberian fermented sausage (“lomo,” “chorizo,” and the final product [10]. The introduction of starter cultures “salchichón”) from the Valle de los Pedroches region in the industrial production of fermented sausages allows a (Córdoba, Spain). These fermented sausages are increasingly shortened ripening period, ensures color development, known worldwide for their high quality, making them enhances flavor, and improves product safety [11]. However, pivotal for the economy of the region. However, there has this can also provoke a loss of the unique organoleptic been no previous study on the yeast microbiota present in characteristics of naturally fermented sausages [12]. The these products. In addition, the identified isolates were most common starter cultures used in the manufacture of tested under different stress conditions and enzymatically fermented sausage products belong to different species of characterized. As a result, the strains with putative lactic acid bacteria and Staphylococcus [13, 14], but in some technological properties of interest for industrial production fermented sausages from Southern European countries, are proposed as components for starter cultures that will molds and yeasts are also being included as starter cultures help to both conserve the specific local characteristics and for their positive effects on flavor and texture [2, 6, 14, 15]. increase the quality standards of the products. D. hansenii is able to grow in the pH, water content, temperature, and salt concentration found in fermented Materials and Methods sausages [16, 17]. The D. hansenii genome was already completely sequenced in 2004 [18], and consists of 12.2 Mb Meat Product Samples and Culture Conditions distributed in seven chromosomes with almost 7,000 Three different Iberian dry meat products (“salchichón,” predicted open reading frames (http://www.genolevures.org). “chorizo,” and “lomo”) presented in slices and vacuum packaged Among yeasts, D. hansenii seems to have the highest coding with a weight of about 100 g were acquired directly from the capacity, amounting to 79.2% of the genome with a putative factory. Samples were taken in duplicate. Portions of 5-10 g of casing and batter, respectively, were number of 6,906 detected coding DNA sequences, and an obtained aseptically from the three different products and overall redundancy of almost 50% [19]. Overrepresented homogenized with 45 or 90 ml of a sterile peptone water solution genes encode for such pathways as the metabolism of (1/10) in a Stomacher (IUL Instruments, Spain) for 2 min. Further cyclic and unusual nucleotides, β-oxidation of fatty acids, decimal dilutions of the homogenate were made with the same amine metabolism, energy generation, phosphate utilization, diluent and 1 ml portions spread on Plate Count Agar (Oxoid, and peroxisomal organization (for a recent review see [20]), UK) to enumerate the total viable microorganisms (TVC, total which can explain the high enzymatic potential of D. hansenii. viable count). The plates were incubated at 30ºC for 72 h. To Furthermore, a new family of genes with no homology to enumerate the yeasts, 0.1 ml was spread on the surface of Rose any other known sequences has been recently identified in Bengal Chloramphenicol Agar (Applichem Panreac, Spain) and D. hansenii ([20]; the Ramos group, unpublished results). the plates were incubated at 26°C for 48-72 h. Spanish fermented sausages, like “salchichón,” “chorizo,” The plates on which the number of colony forming units (CFU) and loin pork (“lomo”), are appreciated for their organoleptic ranged from 30 to 300 were selected for counting, which was expressed as the log of colony forming units per gram of sample. characteristics. Many varieties of these fermented sausages 10 The enumeration of the microorganisms was carried out using

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duplicate samples. The cells were routinely grown in a complex CAC GGC ATA CGT-3’). Forty PCR cycles were performed, with YPD medium (1% Yeast Extract, 2% Peptone, 2% Glucose, pH 5.8; each cycle consisting of 45 sec of denaturation at 95°C, 45 sec of Applichem Panreac). annealing at 50°C, and 120 sec of extension at 72°C. Gel electrophoresis. The amplification products were resolved Isolation and Identification of Yeasts using 2% agarose electrophoresis in a TBE (89 mM Tris-borate, Yeasts isolated from the three fermented sausage products were 2 mM EDTA, pH 8.3) buffer and stained with ethidium bromide randomly selected from the Sabouraud Chloramphenicol Agar [23]. After the electrophoresis, images were captured using Chemi (Scharlau Chemie, Spain) and incubated at 26ºC for 48-72 h. The Doc and analyzed with Image Lab software (Bio-Rad, USA). pure cultures were kept on agar slants on the same medium and conserved at 4ºC for short periods. The samples were stored at Serial Dilution Experiments for Evaluating Resistance to Different -80ºC with 20% (v/v) glycerol as the cryoprotector. Stresses The isolated strains were characterized in YPD. Growth was Biochemical Methods and Enzymatic Activity of Strains tested when adjusting the medium to different pH values (4.5, 5.8, D. hansenii was biochemically identified by growing cells in a and 7.6), with different concentrations of NaCl (0, 0.5, 1, 2, and selective and differential solid medium, Debaryomyces Differential 2.7 M), or incubating at different temperatures (22°C, 26°C, 31°C, Medium (DDM). This medium allows detection of the beta- and 36°C) [24]. Growth was determined by plating 10-fold serial glucuronidase enzyme using the chromogenic substrate magenta- dilutions of each culture. The plates were incubated under the glucuro CHA (5Br-6Cl-3indolyl-beta-D-glucuronide), cyclohexyl required conditions for approximately 48 h. ammonium salt [21]. Drop suspensions (7 µl) were inoculated in duplicate and incubated at 26ºC for 48 h. Statistics The extracelullar lipolytic activity was evaluated on plates of Statistical significance was evaluated using ANOVA, followed tributyrin agar (VWR International, USA) by measuring the by a post hoc multiple comparison according to Student–Newman– diameter of the hydrolysis zone [22]. The cell suspensions were Keuls. A value of p ≤ 0.01 was considered significant, using InStat dropped and the plates were incubated at 26ºC for 4 days. software. Api ZYM strips 25200 plus reagents ZYM A and ZYM B (bioMérieux, France) were used to test the enzymatic activities of the Results isolated strains. The strips were incubated at 30ºC for 3 h. The results were interpreted according to the manufacturer’s specifications. Total Microbe and Yeast Viable Count Determination To start this study, the total microorganisms and yeast Molecular Methods viable counts from the casing and batter meat of “lomo,” DNA isolation. Genome DNA was extracted using the hot- “chorizo,” and “salchichón” from the Valle de los Pedroches phenol method previously described [23]. Briefly, the cells were region were isolated. The total microbial viable counts were grown in YPD for 24 h. Three milliliters of this culture was then centrifuged and the cells obtained were washed with water and significantly higher (p < 0.01) for “lomo” (7.04 log CFU/g) centrifuged again. Next, the pellet was disrupted by adding 0.2 ml when compared with “chorizo” and “salchichón,” which of a lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM showed similar counts (6.01 and 5.93 log CFU/g, respectively). Tris-HCl pH 8.0, 1 mM EDTA pH 8.0), plus approximately 0.2 ml These differences may have been related to the manufacture of glass beads and 0.2 ml of phenol:chloroform:isoamyl alcohol of the products, as “lomo” is made from a loin joint, whereas (24:24:1). The cells were then vortexed 5 times for 1 min with an “chorizo” and “salchichón” are made from minced meat interval of at least 1 min on ice. After breaking the cells, 0.2 ml of a and have a higher fat/lean meat ratio. In any case, the TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) was added and counts obtained were in the same range as those described the mixture was centrifuged again. Thereafter, the supernatant previously for similar products from different locations containing genomic DNA was precipitated with ethanol, and the [25-29]. final pellet was dissolved in 0.1 ml of the TE buffer. The gDNA In this study, the focus was on the yeast strains found in sample quality was checked electrophoretically on an agarose gel these sausages. The presence of yeasts during the ripening and the quantification was performed spectrophotometrically to determine the absorbance at 260 and 280 nm. process has a positive effect as they contribute to the flavor PCR. The PCR amplification was carried out in a mixture (25 µl with their enzymatic activities (proteolytic, lipolytic, etc.) final volume) of a BIO-X-ACT short polymerase (Bioline, UK) and and prevent rancidity, whereas molds usually have negative 1 µg of gDNA from the isolated strains, plus 0.2 µM of the specific consequences mainly related to mycotoxin production [30- primers for the common region of the D. hansenii exclusive gene 33]. However, little has been published on the presence and family (Forward: 5’-GTC GAC TCA GTC TGC TAT AGC CAA functions of yeast strains in “salchichón” and “chorizo” CTT CAA GAA ATA G-3’; Reverse: 5’-GAA TTC ATG GAA GCA sausages, and, to our best knowledge, there are no such

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previous studies on “lomo” [34, 35]. Therefore, this study fully characterized: 12 from “lomo,” 7 from “chorizo,” and investigated the yeasts in their casings. The counts were 3 from “salchichón.” significantly different among the three products tested (p < As D. hansenii has already been identified as the main 0.01), the top being “lomo” (6.6 log CFU/g) and the lowest contributor to the flavor and quality of similar sausages “salchichón” (4.4 log CFU/g). As with the TVC, the yeast [6, 8, 34, 39], the selected yeasts were grown on DDM levels were higher in “lomo” than in the other two products, plates and biochemically identified (Fig. 1) based on their and also more elevated than those previously reported for β-glucuronidase activity in the presence of the chromogenic fermented sausages from other locations (up to 6.0 log CFU/g) substrate magenta-glucuro [21]. Drop suspensions from the [1, 2, 34]. individually cultivated isolates were inoculated in duplicate The presence of microorganisms was investigated both and compared with two Saccharomyces cerevisiae strains on the surface of the three products and in the batter meat. (BY4741 and W303.1A) and one D. hansenii wild-type All presented a TVC between 6.5 and 7.7 log CFU/g. These laboratory strain (CBS767) that were used as negative and values agree roughly with previous counts for similar dry positive controls, respectively. Almost all the isolated cured meat products [36-38]. However, when investigating yeasts showed the characteristic purple color related to the the yeast presence in the meat batter, none of the three β-glucuronidase activity of D. hansenii strains. It is worth products exhibited any significant presence, even though noting that the isolated LRB1, LRB4, and SRC2 strains yeasts have been isolated from other sausage batters [4]. exhibited a particularly intense purple color, which may be Consequently, the yeasts present in the casing of these dry explained on the basis of a higher enzymatic activity meat products were further studied. (Fig. 1). Although only D. hansenii produces violet colonies [21], the absence of color does not necessarily mean the Isolation and Identification of Yeasts absence of D. hansenii, as two white isolates (LB1 and SRC1) From a total of around 100 independent casing yeast colonies, diverse isolates were selected according to their different morphologies, sizes, and/or colony colors. Among the most representative, 22 clearly different strains were

Fig. 2. Amplification of specific genes for D. hansenii isolates. Fig. 1. Growth of isolated yeasts in Debaryomyces differential gDNA was extracted from the isolated yeasts and the D. hansenii- medium (DDM). specific genes were amplified. The figure shows the amplified All the cultures were spotted in duplicate. Sc NC1 and Sc NC2 fragments obtained after the PCR and separation on a 2% agarose gel. correspond to the S. cerevisiae negative controls; Dh PC is the NC corresponds to the S. cerevisiae negative control, and PC is the D. hansenii positive control. D. hansenii positive control.

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were also possibly affiliated with this yeast. Therefore, tested ([20]; Ramos et al., unpublished). A set of primers their affiliation was confirmed using molecular methods. that amplify the common sequence for these genes (approximately 1,000 base pairs) was designed and used to Molecular Identification amplify the gDNA from all the strains isolated in this study To confirm whether the current isolates belonged to the (see Materials and Methods). A positive and a negative D. hansenii species, the presence of a gene family that has control (D. hansenii CBS767 and S. cerevisiae BY4741, been identified in silico exclusively in this species was respectively) were also included. All the strains previously

Fig. 3. Resistance of D. hansenii isolates to different stresses. Ten-fold dilutions were spotted on YPD supplemented with different stressors. The plates were incubated for 48 h at 26ºC unless otherwise stated. The control plate corresponds to YPD agar plates, pH 5.8, without added NaCl and incubated at 26ºC. In all cases, two control laboratory strains (S. cerevisiae and D. hansenii) were included. The effects of changes in pH (A), temperature (B), and NaCl presence (C) on growth were tested.

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biochemically identified as D. hansenii contained the specific putatively interesting technological characteristics. A 10 μl genes and were therefore molecularly identified as D. hansenii volume of the cell dilutions was spotted onto YPD agar (Fig. 2 and data not shown). Moreover, the SRC1 and LB1 plates adjusted to different pHs or a medium supplemented strains that did not produce a purple color in the DDM with high NaCl. In other cases, the plates were incubated at were also molecularly identified as D. hansenii. different temperatures. The effect of the different stresses on cell growth was examined after 48 h of incubation. Evaluation of Resistance of Isolated Yeast Strains under Growth under all these conditions was compared with the Different Stress Conditions control plate of YPD at pH 5.8 without additional NaCl and The effects of pH, temperature, and salt stress on the incubated at 26ºC. The stress tests included the two control yeast growth was determined by spotting serial 10-fold laboratory strains (S. cerevisiae BY4741 and D. hansenii 767) diluted cells onto YPD agar plates, in order to evaluate and 22 yeasts isolated from the fermented sausages.

Fig. 3. Continued.

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pH variations during maturation are usually related to LR2 were not affected at all). bacterial lactic acid production [29, 40]. Very recently, a Several reasons prompted the investigation of the effect positive correlation between pyrazine formation, which of temperature and the salt concentration on the isolated contributes to aroma, and alkaline pH, was reported [41]. strains. (i) Toxic biogenic amines can be found in dry Meanwhile, a low pH during fermentation is usually fermented sausages at relevant concentrations, and the favored to inhibit growth of unwanted bacteria [42]. amounts of these amines have been previously related to Therefore, yeast strains that can grow in a wide range of both temperature and NaCl in salami due to their influence pHs are preferred as they can improve the organoleptic on the lactic acid bacteria populations [45]. (ii) Although properties of the sausages and prevent spoilage. Thus, the Debaryomyces has been reported as a halotolerant yeast, this growth of the current isolates was investigated in acidic character significantly varies between strains belonging to and alkaline conditions (pH 4.5 and 7.6, respectively) the same species [46]. (iii) Finally, D. hansenii is relatively (Fig. 3A and Table 1). The behavior of the control strains sensitive to high temperatures. Therefore, to implement agreed well with previous reports, which showed that robust starter cultures, it was considered crucial to search S. cerevisiae yeast growth is optimal at pH 6, but does not for tolerant strains in an ecosystem (Valle de los Pedroches) grow at a higher pH [43], whereas D. hansenii grows better that reaches high temperatures during summer time. in an alkaline medium [44]. In the case of acidic conditions, Consequently, this study tested the effect of both increasing a wide range of growth was observed among the isolates; and decreasing the temperature from the standard (26ºC) that is, LR1 and CRC2 grew very well, but LRC1 and LCR2 on the growth of the current isolates. Only the S. cerevisiae were significantly affected by a low pH. In an alkaline control strain was able to grow at 36ºC (data not shown). medium, all the isolates showed more robust growth than Surprisingly, several of the current isolates (viz., strains S. cerevisiae, although with some variability (i.e., LR1 and LR1, LR2, LRF1, and LRF2) were unaffected when increasing the temperature, exhibiting more growth than the D. hansenii Table 1. Resistance of selected isolates to different abiotic stresses. control (Fig. 3B and Table 1). As regards resistance to lower Strain pH 4.5 pH 7.6 22°C 31°C NaCl temperatures, the growth of S. cerevisiae was impaired LRB1 + + + when decreasing the temperature to 22ºC, whereas most of LRB2 + the D. hansenii strains tested grew fairly well. Nonetheless, LRB3 + certain isolates showed a degree of variability, where CRF1 LRB4 + and CRF2 were clearly more sensitive to a temperature reduction (Fig. 3B and Table 1). Furthermore, it is important LR1+++++ to highlight that, although D. hansenii is more sensitive LR2+++++ than S. cerevisiae at temperatures above 28ºC, the current LRF1+++++ authors previously reported that the presence of salt in the LRF2+++++ medium contributed to protect Debaryomyces cells against LB1+++ different abiotic stress conditions, which may be specially LB2+++ relevant in the case of high temperatures [47]. Thus, the LRC1 + growth was also tested with several concentrations of LRC2 + + + added salt. Fig. 3C shows the performances of the isolates CRC1 when 2.7 M of NaCl was added to the growth medium. As CRC2 + + expected, the S. cerevisiae control strain was clearly more CRF1 + sensitive to NaCl than the D. hansenii laboratory strain, as CRF2 + originally reported by Norkrans and Kylin [48] and later CRF3 + confirmed by several other authors. In the present study, CB1 all the isolates grew better than both control strains, and CB2+++++ some showed a remarkably high salt tolerance, such as SRC1 + + LR1, LR2, and LRF1. SRC2 + Strains LR1, LR2, LRF1, LRF2, and CB2 were able to SRF1 + + + resist pH variations, both high and low temperatures, and Strains more resistant to different stresses tested (pH, temperature, salt content) a high salt content (Table 1) and thus were selected for than the wild type are marked with +. further studies of their lytic enzymatic properties.

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Table 2. Enzymatic activities of strains isolated from Valle de los Pedroches Iberian dry meat products, determined by growth on tributyrin agar (a) or by API ZYM test (b). (a) Strain Halo size Dh 767 + LR1 ++ LR2 ++ LRF1 ++ LRF2 ++ CB2 ++ +, Enzymatic activity.

(b) Fats Amino acids/Proteins Phosphatases Esterase Esterase Lipase Leu Val or Cys α-Chemo- Acid Naphthol-AS-BI- Strain Trypsin APa (C4) lipase (C14) arylamidase arylamidase trypsin phosphatase hydrolase Dh767 - -/+ ------/+ + + LR1 - + - + - -/+ - - + + LR2 -/+ -/+ - + - -/+ - - + ++ LRF1 + + - + -/+ - -/+ - + ++ LRF2 ++ ++ + + -/+ + -/+ -/+ ++ +++ CB2 -/+ + - + - -/+ - - + + +, Enzymatic activity. aAP, alkaline phosphatase.

Metabolic Analysis of Selected Strains molecular analyses of D. hansenii-specific genes previously Microorganisms can alter the sausage flavor by degrading identified by the current authors (manuscript in preparation). amino acids and lipids [39]. The selected strains were grown The results show that although both methods usually overlap, on tributyrin agar, and their lipase activity was estimated a low percentage of strains lacking β-glucuronidase activity based on the clearance halo around the fungal colony. All were positively identified as D. hansenii using the molecular the tested strains generated a larger halo than that observed approach (Figs. 1 and 2, data not shown), highlighting the with the control D. hansenii strain CBS767, meaning that need for complementary analyses when using enzymatic their lipolytic activities were higher than those of the identification. control, although no differences were observed among Next, to select the best candidates for starter cultures, the them (Table 2A). Next, a semiquantitative API ZYM test yeast isolates were tested with different environmental (bioMérieux) was applied to study the presence of specific stressors and metabolically characterized. Some isolates lipolytic and proteolytic enzymes in the selected strains presented an enhanced catalytic potential under the (Table 2B). Strain LRF2 presented an exceptional catalytic different conditions studied. It is important to note that, in potential with increased levels of enzymes related to both addition to contributing to the sausage flavor, an increased fats and protein catabolism plus elevated phosphatase lipolytic activity in natural fermented sausages has been activities. Moreover, strains LR2 and LRF1 showed enhanced related to a decrease in lipid peroxidation and thus toxic enzymatic capacities (Table 2B). aldehyde content [49]. Metabolic products from lipolytic enzymes, as stated above, together with proteolytic Discussion enzymes, influence flavor development in dry fermented sausages [50]. In particular, the amine content has been This study isolated 22 yeast strains from sausages reported to increase during the ripening process [51]. produced in Valle de los Pedroches. The growth of these Moreover, adding external catalytic enzymes has been yeast strains was then identified using a Debaryomyces proposed to accelerate flavor development in these meat Differential Medium, as previously reported [21], and by products, although with mixed results [50, 51]. Therefore, a

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slow process using selected starter strains with a high Debaryomyces spp. on aroma formation and sensory quality lipolytic potential is preferred to improve flavor and avoid of dry-fermented sausages. Meat Sci. 68: 439-446. lipid peroxidation. 9. Ozturk I, Sagdic O. 2014. Biodiversity of yeast mycobiota in Using these characteristics as a guide, the LRF2, LRF1, “sucuk,” a traditional Turkish fermented dry sausage: and LR2 strains were selected as potential starter cultures, phenotypic and genotypic identification, functional and technological properties. J. Food Sci. 79: M2315-M2322. and are currently being tested for industrial production. 10. Cocolin L, Dolci P, Rantsiou K. 2011. Biodiversity and Furthermore, these strains are also being studied molecularly dynamics of meat fermentations: the contribution of molecular to improve their stress resistance. methods for a better comprehension of a complex ecosystem. 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September 2017 ⎪ Vol. 27⎪ No. 9