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Potential production of 2-phenylethanol and 2-phenylethylacetate by non-Saccharomyces yeasts from Agave durangensis

Potential production of 2-phenylethanol and 2-phenylethylacetate by non-Saccharomyces yeasts from... Introduction The participation of non-Saccharomyces yeasts in fermentation processes is of great importance due to their participation in the formation of esters and superior alcohols, which confer characteristic aromas to beverages such as wine and mescal. The aim The aim of this study was identify and evaluate the potential aroma production of yeast native of Agave fermentation by the mescal production in Durango, Mexico. Isolated yeasts were molecularly identified by 5.8s ribosomal gene; the potential production of aromas was carried out in fermentations with the addition of L-phenylalanine and evaluated after 24 h of fermen- tation. Extraction and quantification of aromatic compounds by headspace solid-phase micro-extraction (HS-SPME) and gas chromatograph mass spectrometry (GC-MS). Results The isolated non-Saccharomyces yeasts could be classified into six different genera Saccharomyces cerevisiae, Clavispora lusitaniae, Torulaspora delbrueckii, Kluyveromyces dobzhanskii, Kluyveromyces marxianus,and Kluyveromyces sp. All probed strains presented a potential aroma production (ethyl acetate, isoamyl acetate, isoamyl alcohol, benzaldehyde, 2-phenylethyl butyrate, and phenylethyl propionate), particularly 2-phenylethanol and 2-phenylethylacetate; the levels found in the Kluyveromyces marxianus ITD0211 yeast have the highest 2-phenylethylacetate production at 203 mg/L and Kluyveromyces marxianus ITD0090 with a production of 2-phenylethanol at 1024 mg/L. Conclusion Non-Saccharomyces yeasts were isolated from the mescal fermentation in Durango; the Kluyveromyces genus is the most predominant. For the production of aromas, highlighting two strains of Kluyveromyces marxianus produces competitive quantities of compounds of great biotechnological interest such as 2-phenylethanol and 2-phenylethylacetate, without resorting to the genetic modification of yeasts or the optimization of the culture medium. . . . . Keywords Mescal Bioconversion Aroma L-Phenylalanine Kluyveromyces marxianus * Olga Miriam Rutiaga-Quiñones Departamento de Ingenierías Química y Bioquímica, Tecnológico omrutiaga@itdurango.edu.mx Nacional de México/ Instituto Tecnológico de Durango, Felipe Pescador 1803 Ote, Colonia Nueva Vizcaya, C.P. Pablo Jaciel Adame-Soto 34080 Durango, Durango, Mexico jaciel_as@hotmail.com Departamento de Microbiología e Inmunología, Unidad de Elva Teresa Aréchiga-Carvajal Manipulación Genética del Laboratorio de Micología y elva.arechigacr@uanl.edu.mx Fitopatología. Unidad C. Facultad de Ciencias Biológicas, Mercedes G López Universidad Autónoma de Nuevo León, C.P. 66451 San Nicolás de mercedes.lopez@cinvestav.mx Los Garza, Nuevo León, Mexico Silvia Marina González-Herrera 3 Departamento de Biotecnología y Bioquímica, Centro de smgonzalez@itdurango.edu.mx Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Apartado Postal 629, C.P, 36821 Irapuato, Guanajuato, Mexico Martha Rocio Moreno-Jiménez mrmoreno@itdurango.edu.mx Facultad de Ciencias Químicas-Laboratorio de Genética molecular, Norma Urtiz-Estrada Universidad Juárez del Estado de Durango, Av. Veterinaria S/N Col. urtizn@hotmail.com Valle del Sur. C.P., 34120 Durango, Durango, Mexico 990 Ann Microbiol (2019) 69:989–1000 Introduction 2006). 2-PE can also be metabolized to 2-PEA by a trans- esterification reaction, which involves the transfer of a group The non-Saccharomyces yeasts are well recognized for their of acetyl-coenzyme A acetate to the hydroxyl group of 2-PE contribution to the aroma of fermentative beverages (Cordente (Hazelwood et al. 2008; Pires et al. 2014). When L-Phe is the et al. 2012; Ciani et al. 2016; Masneuf-Pomarede et al. 2016), sole nitrogen source in the medium, large amounts of 2-PE are especially wine. Their presence has also been reported in mescal accumulated. Several biotechnological processes are known and tequila. In Mexico, these alcoholic beverages are distin- for producing 2-PE, based on this pathway, and considerable guished from each other, based on the agave species used in their progress has been made on the development of this process. In production. For example, Agave tequilana Weber var. Azul (blue this context, yeast biodiversity may be greatly impacted by the variety) is used for tequila, whereas Agave salmiana and Agave production of different aroma products derived from primary durangensis,(or Agave duranguensis) among others, are used and secondary metabolism. The diversity of non- for mescal production in various regions of Mexico (Lappe- Saccharomyces yeasts responsible for many of the volatile Oliveras et al. 2008; Páez-Lerma et al. 2010; De los Rios- compounds found in mescal, in the state of Durango, Deras et al. 2015; Kirchmayr et al. 2017). Mescal has elevated Mexico, has not yet been evaluated. This research aimed to its economic importance in the last years (Kirchmayr et al. 2017). identify the non-Saccharomyces microbiota present in fer- During the mescal production process, agave juice is naturally mentations in three different mescal-producing regions and fermented by native yeasts, such as Saccharomyces, Pichia, assess the production potential of aromatic compounds the Kluyveromyces, Candida, Debaryomyces, Hanseniaspora, addition of L-Phe as an inductor. Kloeckera, Schizosaccharomyces, Torulaspora,and Zygosaccharomyces (Lachance 1995; Díaz-Montaño et al. 2008; Escalante-Minakata et al. 2008). Previously published re- Materials and methods search on fermentations of agaves suggests that non- Saccharomyces yeasts have an important role in the initial fer- Yeast strains mentation process and influence the production of the volatile compounds (Lappe-Oliveras et al. 2008; Narváez-Zapata et al. Thirty-four native strains, identified as non-Saccharomyces 2010; Martell Nevárez et al. 2011). The potential use of these from Agave durangensis fermentation and obtained from the yeasts as inoculants has been described (Rodríguez-Sifuentes Collection of the Instituto Tecnologico de Durango, were iso- et al. 2014; Nuñez-Guerrero et al. 2016), as well as their partic- lated from three mescal-producing regions of Durango State, ipation in generating the volatile compounds in mescal, mainly Mexico: Mezquital (23° 28′ 22″ N, 104° 24′ 40″ W), Nombre esters (Martell Nevárez et al. 2011; Rutiaga-Quiñones et al. 2012; de Dios (23° 51′ 00″ N, 104° 14′ 00″ W), and Durango (24° Hernández-Carbajal et al. 2013). Despite the increasing use of 01′ N, 104° 40′ W). All yeast strains were conserved, as cul- non-Saccharomyces yeasts in biotechnology, there are still many ture stock at − 20 °C in 30% (v/v) glycerol. opportunities to improve native yeast exploration. These pros- pects have led to a great interest in further enhancing the number Molecular identification of non-Saccharomyces yeasts available, by selecting or develop- ing strains with novel and attractive properties. Growth conditions Flavor has a major impact on the quality perception of food and beverages, and fragrances are highly valued in the Yeast cells preserved in glycerol were first activated on YDP cosmetic and perfume industry. For natural aroma compounds solid medium (glucose 20 g/L, casein peptone 20 g/L, yeast that exist at low concentrations in their original sources, bio- extract 10 g/L, and agar 20 g/L). DNA was then extracted at technological processes represent an attractive alternative to 24-h growth, using the method detailed by Sambrook and the traditional preparation by extraction (Schrader et al. 2004). Russell (2001). Due mainly to its sweet and rose-like taste and odor 2- phenylethanol (2-PE) and its more fruit-like form, acetate ester Polymerase chain reaction and amplification 2-phenylethylacetate (2-PEA), find use in various flavor com- positions (Fabre et al. 1998). For food applications, the rising Polymerase chain reaction (PCR) was carried out in 50-μL demand for natural products means natural flavor compounds volumes, using 2.0 μL of DNA with ITS1 (5′–TCC GTA are increasingly becoming a necessity (Etschmann and GGT GAA CCT GCG G–3′) and ITS4 (5′–TCC TCC GCT Schrader 2006, Morrissey et al, 2015). TAT TGATAT GC–3′) primers to amplify the rDNA repeat unit Both 2-PE and 2-PEA can be produced by de novo that includes the 5.8S rRNA gene and the two non-coding synthesis or from L-phenylalanine (L-Phe) by non- regions designated the internal transcribed spacers (ITS1 and Saccharomyces yeast whole-cell biocatalysis via the Ehrlich ITS4) (White et al. 1990). Amplification began with an initial- pathway (Etschmann et al. 2003), (Etschmann and Schrader izationstep ofonecycleat95°Cfor 5min, then35cyclesof Ann Microbiol (2019) 69:989–1000 991 95 °C for 1 min, 52 °C for 2 min, and 72 °C for 2 min, followed Genomics for Biodiversity Laboratory (Langebio) of by a final elongation at 72 °C for 10 min (White et al. 1990). Cinvestav (Irapuato, Mexico). The PCR product was electrophoresed on 1% agarose gel with TAE 0.5× buffer (Promega, Madison, WI, USA), at 95 V for 45 min, stained with ethidium bromide (Sigma–Aldrich. St. Phylogenetic analysis Louis, MO, USA) and visualized under UV light (Benchtop UV transilluminator, Upland, CA, USA); DNA fragment sizes The obtained sequences were aligned using the MUSCLE were determined using a 100-bp DNA ladder (Promega, USA). program (https://www.ebi.ac.uk/Tools/msa/muscle), and The PCR product was purified using C H NO and C H O(> regions of local similarity between sequences were identified 2 7 2 2 6 99%) (Sigma–Aldrich). The rDNA sequences were acquired from the National Center for Biotechnology Information using an ABI PRISM Model 3730XL sequencer (Applied (NCBI) database of GenBank using the BLAST program Biosystems, Inc., Foster City, CA, USA) at the National (https://blast.ncbi.nlm.nih.gov/Blast). Phylogenetic analyses Table 1 Strains used in this study Species Strain Locality Accession no. Clavispora lusitaniae ITD 0132 Mezquital MH282797 Kluyveromyces marxianus ITD 0002 Mezquital MH282778 Kluyveromyces marxianus ITD 0003 Mezquital MH282779 Kluyveromyces marxianus ITD 0090 Mezquital MF797638.1 Kluyveromyces marxianus ITD 0091 Mezquital MH282784 Kluyveromyces marxianus ITD 0092 Mezquital MH282785 Kluyveromyces marxianus ITD 0093 Mezquital MH282786 Kluyveromyces marxianus ITD 0128 Mezquital MH282787 Kluyveromyces marxianus ITD 0141 Mezquital MH282790 Kluyveromyces marxianus ITD 0142 Mezquital MH282791 Kluyveromyces marxianus ITD 0145 Mezquital MH282792 Kluyveromyces marxianus ITD 0211 Mezquital MH282793 Kluyveromyces sp. ITD 0040 Mezquital MH282781 Kluyveromyces sp. ITD 0041 Mezquital MH282782 Kluyveromyces sp. ITD 0089 Mezquital MH282783 Kluyveromyces sp. ITD 0136 Mezquital MH282788 Kluyveromyces sp. ITD 0137 Mezquital MH282789 Torulaspora delbrueckii ITD 0110 Mezquital MH282795 Torulaspora delbrueckii ITD 0129 Mezquital MH282796 Saccharomyces cerevisiae ITD 0109 Mezquital MH282794 Clavispora lusitaniae ITD 0095 Nombre de Dios MH282804 Clavispora lusitaniae ITD 0099 Nombre de Dios MH282805 Clavispora lusitaniae ITD 0103 Nombre de Dios MH282806 Clavispora lusitaniae ITD 0104 Nombre de Dios MH282807 Clavispora lusitaniae ITD 0107 Nombre de Dios MH282808 Kluyveromyces marxianus ITD 0102 Nombre de Dios MH282801 Kluyveromyces marxianus ITD 0264 Nombre de Dios MH282802 Kluyveromyces marxianus ITD 0268 Nombre de Dios MH282803 Kluyveromyces sp. ITD 0046 Nombre de Dios MH282798 Kluyveromyces sp. ITD 0048 Nombre de Dios MH282799 Kluyveromyces sp. ITD 0049 Nombre de Dios MH282800 Kluyveromyces marxianus ITD 0069 Durango MH282810 Kluyveromyces sp. ITD 0062 Durango MH282809 Kluyveromyces dobzhanskii ITD 0157 Durango MH282811 Kluyveromyces marxianus CBS 600 Reference KY103809.1 992 Ann Microbiol (2019) 69:989–1000 were conducted in MEGA7 Program. The sequences were at a linear flow of 2 mL/min. The injector and detector temper- deposited in GenBank. atures were 230 and 260 °C, respectively. The oven temperature was increased from 40 to 240 °C, using the following program: Production of volatile organic compounds the initial temperature was maintained for 3 min, ramped at 4 °C/min to 100 °C, held for 1 min, and then ramped at Chemicals and reagents 4 °C/min to 240 °C and held for 10 min. The ionization voltage was 70 eV. All the assays were performed twice. The analyzed L-Phe (< 98%), 2-PE (> 99%), and 2-PEA (> 99%) were pur- compounds were identified by comparing their mass spectra with chased from Sigma–Aldrich. Na HPO .2H O, MgSO .7H O those in the NIST database (Calvo-Gómez et al. 2004). In addi- 2 4 2 4 2 (Caisson Laboratory In., Smithfield, UT, USA), and citric acid tion, the volatile compounds of interest (2-PE and 2-PEA) were were obtained from Fermont (Mexico City, Mexico). Glucose, quantified by standard curves. yeast extract and casein peptone came from BD Bioxon (Mexico City, Mexico). Statistical analysis Bioconversion Data of the volatile compounds, 2-PE and 2-PEA, were eval- uated by the HSD–Tukey–Kramer comparison test, at α = The strains were pre-grown in 125-mL baffled Erlenmeyer 0.01 All statistical analyses were done using JMP software flasks (Corning, Inc., USA) with vented top, containing a version 13.2 (SAS Institute, Inc., NC, USA). 50-mL operative volume of standard yeast medium YPD broth (20 g/L glucose, 20 g/L casein peptone, and 10 g/L yeast extract), at 30 °C for 12 h and 120 rpm. For fermentation, the Result and discussion strains were inoculated at a concentration of 10 cells/mL and incubated at 30 °C for 24 h and 120 rpm. Duplicate experi- Molecular identification and phylogenetic analyses ments were done for induction with L-Phe (9 g/L), in which the culture medium contained 30 g/L glucose, 35 g/L Table 1 indicates the molecular identification of the studied Na HPO .2H O, 10.5 g/L citric acid, 0.5 g/L MgSO .7H O, 2 4 2 4 2 strains, for each geographic region. These strains corresponded and 0.17 g/L yeast extract, in a 50-mL medium, in a 125-mL to six different genera: Clavispora lusitaniae, Kluyveromyces sp., Erlenmeyer flask (Etschmann et al. 2004). The yeast Kluyveromyces dobzhanskii, Kluyveromyces marxianus, Kluyveromyces marxianus CBS 600 (KY103809.1) was in- Saccharomyces cerevisiae,and Torulaspora delbrueckii. cluded as a reference. Previous investigations of the yeasts associated with mescal pro- duction in Mexico, described the presence of non- Gas chromatography–mass spectrometry analysis Saccharomyces strains, such as K. marxianus, C. lusitaniae, and Pichia fermentans from Agave salmiana fermentation, in The volatile organic compounds were extracted by headspace San Luis Potosí State (Escalante-Minakata et al. 2008). In anoth- solid-phase micro-extraction (HS-SPME) with a er Vinata, from the same region, the non-Saccharomyces yeasts divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco, were: K. marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Bellefonte, PA, USA). One milliliter of the sample was taken C. lusitaniae, T. delbrueckii,and Candida ethanolica (Verdugo- from each fermentation at 24 h, placed inside a 4-mL vial, sealed Valdez et al. 2011). In mescal produced using the species Agave tightlywithascrew-topseptum-containing cap, and allowed to durangensis in Durango, the predominant non-Saccharomyces stand at 35 °C for 1 h. The SPME needle was then inserted yeasts belonged to Candida genus, including Candida lusitaniae, through the septum, the holder was secured, and the fiber was Candida kefir, Candida glabrata, Candida laurentii,and exposed to the headspace. After 1 h of sampling at 35 °C, the fiber was retracted and immediately inserted into the inlet of a HP Fig. 1 Neighbor-joining trees were constructed from the evolutionary„ 5890 Series II GC instrument directly coupled to an HP 5972 distance data for ITSI-5.85 rDNA-ITS2. The percentage of replicate mass-selective detector (Hewlett–Packard, Palo Alto, CA, USA) trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). a Kluyveromyces marxianus tree of group one. b and equipped with an HP-FFAP capillary column (25 m × Kluyveromyces dobhzankii tree. c Kluyveromyces sp of group two tree. 0.320 mm i.d., film thickness 0.50 m; Hewlett–Packard), for The accession numbers of reference sequences used in this tree are as thermal desorption. The injection was accomplished by desorp- follows: K. nonfermentans (AB011512.1), K. lactis (AB011515.1), K. wickerhamii (AB011521.1), K. aestuarii (AB011513.1), tion of the fiber at 230 °C for 6 min with the injector operated in K. marxianus (AB011518.1), K. marxianus (MH045720.1), the splitless mode for 1 min. An additional 5-min exposure in the K. marxianus (MH045721.1), K. marxianus (MH045719.1), injection port allowed the fiber to be cleaned of any compound K. marxianus (MG966429.1), K. marxianus (JX174415.1), that may not have been desorbed during the initial minute K. dobzhanskii (ABO11514.1), and D. hansenii (JQ912667.1). Evolutionary analyses were conducted in MEGA7 (Calvo-Gómez et al. 2004). Helium was used as the carrier gas, Ann Microbiol (2019) 69:989–1000 993 ITD0093(MH282786) K-marxianus(AB011518.1) ITD0002(MH282778) ITD0102(MH282801) ITD0091(MH282784) ITD0003(MH282779) ITD0141(MH282790) ITD0145(MH282792) ITD0069(MH282810) ITD0092(MH282785) ITD0128(MH282787) ITD0142(MH282791) ITD0211(MH282793) ITD0264(MH282802) ITD0268(MH282803) ITD0090(MF797638.1) Kmarxianus-CHP7(MH045719.1) Km-KDLYH1-1(JX174415.1) Km-CDA2(MG966429.1) Kmarxianus-CDB5(MH045721.1) Kmarxianus-CDB1(MH045720.1) K-dobzhanskii(AB011514.1) K-nonfermentans(AB011512.1) K-lactis(AB011515.1) K-wickerhamii(AB011521.1) K-aestuarii(AB011513.1) Debaryomyces-hansenii(JQ912667.1) K-nonfermentans(AB011512.1) K-aestuarii(AB011513.1) K-lactis(AB011515.1) K-wickerhamii(AB011521.1) K-marxianus(AB011518.1) K-dobzhanskii(AB011514.1) ITD0157(MH282811) Debaryomyces-hansenii(JQ912667.1) ITD0049(MH282800) ITD0062(MH282809) ITD0048(MH282799) ITD0046(MH282798) ITD0041(MH282782) ITD0040(MH282781) ITD0037(MH282780) 75 ITD0089(MH282783) ITD0136(MH282788) ITD0137(MH282789) K-nonfermentans(AB011512.1) K-marxianus(AB011518.1) K-dobzhanskii(AB011514.1) Kmarxianus-CDB1(MH045720.1) Kmarxianus-CDB5(MH045721.1) Kmarxianus-CHP7(MH045719.1) Km-CDA2(MG966429.1) K-lactis(AB011515.1) Km-KDLYH1-1(JX174415.1) K-wickerhamii(AB011521.1) K-aestuarii(AB011513.1) Debaryomyces-hansenii(JQ912667.1) 2 994 Ann Microbiol (2019) 69:989–1000 Candida tropicalis (Páez-Lerma et al. 2010). Equally, in fourcroydes Lem. There are numerous accounts of this species Durango State, Páez-Lerma et al. (2013) observed diverse micro- during the different stages of processing and fermentation of organisms at the beginning of fermentation: S. cerevisiae, Agave to obtain traditional Mexican beverages, such as T. delbrueckii, K. marxianus, Candida diversa, P. fermentans, Bpulque,^ mescal, and tequila (Rodrigues de Miranda 1979; and Hanseniaspora uvarum, but only T. delbrueckii and Lachance 1995; Lappe et al. 2004; De León Rodríguez et al. S. cerevisiae were found at the end of the fermentations. 2008; Lappe-Oliveras et al. 2008; Páez-Lerma et al. 2010; Recently, Kirchmayr et al. (2017) mentioned K. marxianus, Kurtzman et al. 2011; Verdugo-Valdez et al. 2011), where its Zygosaccharomyces rouxii, Z. bisporus, T. delbrueckii,and presence has been associated with the sensory qualities of these Pichia membranifaciens as the main microbiota present, after beverages (Escalante-Minakata et al. 2008). S. cerevisiae, during mescal production in Oaxaca State. In our The species present in relatively low quantity was study, Mezquital region presented the greatest number and diver- T. delbrueckii, found only in the region of the Mezquital. sity of isolated non-Saccharomyces, which included Figure 2 b shows the phylogenetic tree for strains ITD0110 K. marxianus, T. delbrueckii,and C. lusitaniae.Both and ITD0129. These strains have been linked to a high pro- K. marxianus and C. lusitaniae were also detected in mescal duction of volatile compounds that impart unique characteris- from Nombre de Dios. In fermentation of agave in Durango, tics to beverages, such as mescal, and also other flavor com- the species identified were K. marxianus and K. dobzhanskii. pounds, including terpenoids, esters, higher alcohols, glycerol This article is the first report where the strain K. dobzhanskii acetaldehyde, acetic acid, and succinic acid (Moreira et al. hasbeenfoundinnatural fermentation processes. This genus 2005; Jolly et al. 2014). Rutiaga-Quiñones et al. (2012)pro- has been cataloged as the closest Kluyveromyces lactis relative filed the volatile compounds in Agave duranguensis juice of wild or native strains, so it has been used for modeling popu- supplemented with NH Cl and fermented with the yeast lation genetics (Belloch et al. 1997, 2002; Sukhotina et al. 2006; T. delbrueckii ITD0110. However, the genetic diversity pres- Lane and Morrissey 2010). ent in this genus was not established. Nuñez-Guerrero et al. In phylogenetic studies of Kluyveromyces strains (Fig. 1), (2016)isolated S. cerevisiae, T. delbrueckii,and K. marxianus three groups were recognized. The first two groups comprised from A. duranguensis fermentation and proposed the use of a strains directly related to the genus K. marxianus and mixture of 75% S. cerevisiae and 25% T. delbrueckii as an K. dobzhanskii, respectively (Fig. 1a, b). The third group had inoculant to make mescal. direct relationship to the genera of the Kluyveromyces family (Fig. 1c). These strains were present in all the regions, accounting Production of volatile organic compounds for 35% (Mezquital), 50% (Nombre de Dios), and 30% (Durango) of the total of the isolated Kluyveromyces strains and Table 2 presents the volatile compounds produced by the non- can represent a particular genetic diversity for K. marxianus Saccharomyces yeasts studied in this work. In general, all strains isolated from the fermentation process during the produc- strains were producers of esters, fatty acids esters, and higher tion of mescal. In a recent study of the genetic diversity of the alcohols. Esters are key flavor compounds in fermented bev- genus K. marxianus, all the isolates from a lactic environment erages, like mescal. Among the acetate esters, the synthesis of were either diploid or triploid, whereas non-lactic isolates were ethyl acetate, which is responsible for the bouquet and desir- haploid (Ortiz-Merino et al. 2018). Additionally, the authors dis- able fruity flavors, depends on the ethanol concentration while tinguished three clades, of which the strain UFS-Y2791, isolated the synthesis of isoamyl acetate, isobutyl acetate and 2-PEA, from American agave juice and representing the third clade, relies on the concentration of their corresponding higher alco- proved to be more diverse than the others (Ortiz-Merino et al. hol, by the action of an alcohol acetyltransferase (Gethins 2018). So far, only the presence of K. marxianus strains from et al. 2015; Loser et al. 2014). different mescal production has been reported in the literature, The ethyl esters of short-chain fatty acids present are 2- indicating that the current work is the first to show that phenylethyl butyrate and Phenylethyl propionate, synthesized K. marxianus strains isolated from agave fermentation (mescal from 2-PE and short-chain fatty acids. Phenylethyl propionate or tequila) have distinct genetic differences between them. Páez- is an ester desirable in wine, due to its floral aroma (Beckner Lerma et al. (2013) noted these differences with S. cerevisiae Whitener et al. 2015; Padilla et al. 2016). The formation of benz- strains in wine. aldehyde from Phe has been studied in several microorganisms, Additionally, phylogenetic analysis among the Clavispora such as Pseudomonas putida and the white rot fungi, Tremetes lusitaniae yeasts from this study (Fig. 2a) evidenced the genetic suaveolens, Polyporus tuberaste, Bierkandera adusteand,and variability between strains of Clavispora lusitaniae.These yeasts Phanerochaete chrysosporium (Rojas et al. 2001; Liu et al. were found predominantly in fermentations from Nombre de 2004). Hence, these yeasts seem to have benzaldehyde produc- Dios, with 45% of the strains identified as C. lusitaniae. Pérez- tion potential. The principal volatile compounds found were 2- Brito et al. (2015) characterized the great genetic diversity of PE, which is considered to be one of the most important aromatic C. lusitaniae strains isolated from the fermentation of Agave alcohols, and 2-PEA. The higher alcohols are predominantly Ann Microbiol (2019) 69:989–1000 995 Clavisporalusitaniae-CBS:5094(AY321465.1) Clavisporalusitaniae-PMM10-1024036L-isolate-ISHAM-ITS ID-MITS815(KP131848.1) Clavisporalusitaniae-CNRMA10.800-isolate-ISHAM-ITS ID-MITS841(KP131835.1) Clavisporalusitaniae-H27507-04(HQ693786.1) Clavisporalusitaniae-ATCC-34449(KU729100.1 ) Clavisporalusitaniae-CNRMA11.496-isolate-ISHAM-ITS ID-MITS845(KP131836.1) Clavisporalusitaniae-MTCC1001(AY174102.1) Clavisporalusitaniae-CBS:5299(KY102564.1) Clavisporalusitaniae-IWG-DG-o(MG518174.1) ITD0099(MH282805) ITD0095(MH282804) ITD0104(MH282807) ITD0132(MH282797) ITD0103(MH282806) ITD0107(MH282808) Clavisporalusitaniae-WTS2D(MG183704.1) Debaryomyces-hansenii(JQ912667.1) Saccharomyces-cerevisiae-F2(JX141369.1) Saccharomyces-cerevisiae-F1(JX141368.1) ITD0109(MH282794) ITD0129(MH282796) ITD0110(MH282795) Torulaspora-delbrueckii-T6(FJ838774.1) Debaryomyces-hansenii(JQ912667.1) 0.050 Fig. 2 Neighbor-joining trees were constructed from the evolutionary (HQ693786.1), C. lusitaniae (KU729100.1), C. lusitaniae distance data for ITSI-5.85 rDNA-ITS2. The percentage of replicate (KP131836.1), C. lusitaniae (AY174102.1), C. lusitaniae trees in which the associated taxa clustered together in the bootstrap test (KY102564.1), C. lusitaniae MG518174.1), C. lusitaniae (1000 replicates). a Clavispora lusitaniae tree. b S. cerevisiae–T. (MG183704.1), S. cerevisiae (JX141369.1), S. cerevisiae (JX141368.1), delbrueckii tree. The accession numbers of reference sequences used in T. delbrueckii (FJ838774.1), and D. hansenii (JQ912667.1). Evolutionary these trees are as follows: C. lusitaniae (AY321465.1), C. lusitaniae analyses were conducted in MEGA7 (KP131848.1), C. lusitaniae (KP131835.1), C. lusitaniae formed by yeastduringfermentationbytheEhrlichpathway, The production of volatile organic compounds (2-PE and involving amino acid degradation, particularly Phe (Hazelwood 2-PEA) presented significant statistical differences between et al. 2008;Styger etal. 2011) but can also be synthesized from by non-Saccharomyces yeasts is shown in Table 3.The most glucose via pyruvate (Cordente et al. 2012). The Ehrlich pathway productive yeasts, in terms of 2-PE, were as follows: involves three steps: phenylpyruvate is decarboxylated to Kluyveromyces marxianus (ITD0090, ITD0091), as well as phenylacetaldehyde, then reduced to 2-PE (Etschmann and the yeasts Kluyveromyces sp. (ITD0046, ITD0089 and Schrader 2006), and finally esterified to 2-PEA. ITD0040), Kluyveromyces dobzhanskii ITD0157, and 996 Ann Microbiol (2019) 69:989–1000 Table 2 Volatile metabolites Metabolite RTm/z Sensorial description produced by non-Saccharomyces strains Ethyl acetate 0.95 61-70-73-88 Pineapple, sweet, and fruit Isoamyl acetate 4.04 55-70-87 Banana, sweet and fruit Isoamyl alcohol 6.24 55-70-87 Alcohol, banana, and malt Benzaldehyde 16.36 51-77-106 Almond, burnt sugar, cherry, and sweet 2-Phenylethylacetate 22.72 121-104-91-77 Floral, fruit, honey, and roses 2-Phenylethyl butyrate 23.68 117-104-91-77-71-65 Yeast, strawberry, floral, and sweet Phenylethyl propionate 23.79 104-91-77-57 Floral, red fruit, and honey 2-Phenylethanol 24.22 103-91-77-65-51 Roses, fresh, and leafy Clavispora lusitaniae ITD0107. K. marxianus yeast ITD0090 biotransformation in standard medium for 72-h batch cultures. can be classified as the largest producer of 2-PE, even when Clavispora lusitaniae WUT17 strain reached the levels of compared with the most studied yeast K. marxianus CBS600 2.04 g/L of 2-PE in a standard medium and 0.95 g/L of 2- (Table 3). The concentrations obtained were similar to that PE in an organic waste-based medium, which is superior to reported by Etschmann et al. (2003) (890 mg/L in a the one reported by Etschmann et al. (2003)of 0.33 g/L. It is molasses-based medium). Eshkol et al. (2009) evaluated the well known that 2-PE synthesis is carried out by the Ehrlich potential of stress-tolerant Saccharomyces strains to produce pathwayinyeast,suchas K. marxianus and Yarrowia 2-PE under inductive conditions (Phe addition) and detected lipolytica (Fabre et al. 1998; Celińska et al. 2013). In a recent the concentrations between 340 and 460 mg/L at 48-h incuba- study, González et al. (2018) screened the 2-PE production tion, but these concentrations increased to 540 and 850 mg/L potential of some non-Saccharomyces yeasts and discovered with selected yeast, when conditions were optimized with a2-PEproductiveyeast (T. delbrueckii). However, in all 10 g/L Phe addition, which were very similar quantities to cases, non-Saccharomyces species produce lower quantities those reported here, without the optimization process. In a than S. cerevisiae, indicating that the Ehrlich pathway may related study, with the K. marxianus strain CBS6556, the op- not be as active in non-Saccharomyces species as in timization of the grape must culture medium with 3 g/L of L- Saccharomyces, at least under nitrogen-limiting conditions. Phe improved the 2-PE titer of 0.39 g/L after 84 h of culture to Rutiaga-Quiñones et al. (2012) revealed the non- 0.47 g/L (Garavaglia et al. 2007). Mei et al. (2009)alsoused a Saccharomyces yeasts potential for volatile compounds, par- yeast Saccharomyces cerevisiae BD and reported in situ prod- ticularly in A. duranguensis juice for mescal production; in uct adsorption techniques, to obtain a better performance re- this study, the strains T. delbrueckii ITD0110 and garding the biotransformation of L-phenylalanine to 2- K. marxianus ITD0211 showed to be more productive of 2- phenylethanol, reaching a concentration of 4.65 g/L of 2-PE PE under nitrogen-limiting conditions that the strain with a content of 10 g/L of L-Phe in the medium. Chreptowicz S. cerevisiae ITD0109. A possible theory for our observations, et al. (2016) with yeast not genetically modified strain when the fermentations of different strains induced with L-Phe Saccharomyces cerevisiae JM2014 was isolated from a as the only source of nitrogen were evaluated, is that the fermented milk drink (Turkey), producing a total concentration Ehrlich route is working on these strains, but the metabolic of 3.60 g/L of 2-PE after 72-h incubation at 30 °C batch culture plasticity differs for each of the strains studied. These results with a medium containing 5 g/L of L-Phe in a 4-L bioreactor at allow to raise genetic and biochemical differences between the laboratory scale. Recently, De Lima et al. (2018) evaluated the strains of wine production and mescal, but additional studies potential of yeast strain K. marxianus CCT 7735 in the 2-PE are required to elucidate and describe them. production and reported a production of 2.47 g/L of 2-PE, with The Table 3 illustrates that 2-PEA production presents sig- the optimization in the medium through the optimal conditions nificant difference for each strain where that highlighting the achieving thus a production of 3.44 g/L of 2-PE. Lu et al. K. marxianus strains (ITD0040, ITD0090, ITD0102, and (2016) showed the 2-PE titer in a batch fermentation with ITD0211). A previous research by Rojas et al. (2001) de- the stress-tolerant yeast Candida glycerinogenes WL2002-5, scribed a very productive H. guilliermondii yeast, with a 2- reaching 5 g/L from L-Phe, under optimized culture condi- PEA production of 83.83–163.8 mg/L, when using 2-PE as tions. Genetic modification strategies have also been consid- induction conditions and in the presence of extraction solvent. ered, to further increase 2-PE production, such as ARO8 and The present results describe a difference in the production ARO10 overexpression in S. cerevisiae SPO810 yeast the 2-PE potential from L-Phe induction, among all the strains studied, reached 2.61 g/L after 60 h of cultivation (Yin et al. 2015). highlighting two strains, K. marxianus ITD0090 and Chreptowicz et al. (2018) reported yeast strains capable of K. marxianus ITD0211, due to the potential to overproduce producing over 2 g/L 2-PE through the L-Phe 2-PE and 2-PEA, respectively. Etschmann et al. (2005) Ann Microbiol (2019) 69:989–1000 997 Table 3 Production 2- Species ID strains Production of 2-PE (mg/ ID Strains Production of 2-PEA phenylethanol and 2- L) (mg/L) phenylethylacetate obtained by different yeast strains non- Mean S.d Mean S.d Saccharomyces by HS-SPME a–f h–j Clavispora lusitaniae ITD0107 764.80 62.64 ITD0095 22.43 2.55 b–g h–j ITD0095 636.60 47.41 ITD0107 17.17 4.52 d–j i,j ITD0132 511.89 2.23 ITD0104 13.16 1.14 g–j i,j ITD0104 394.74 42.54 ITD0099 12.14 0.29 g–j i,j ITD0099 370.89 10.13 ITD0132 11.89 0.19 g–j j ITD0103 357.54 29.41 ITD0103 9.20 1.06 a a Kluyveromyces marxianus ITD0090 1024.46 306.38 ITD0211 203.53 3.52 a–c a ITD0091 848.37 112.28 ITD0102 202.07 26.28 b–h a–c ITD0211 630.81 7.13 CBS600 166.24 0.40 b–h a–d ITD0102 618.88 46.90 ITD0091 136.49 28.48 b–i a–e ITD0093 596.41 26.16 ITD0128 134.27 13.52 c–i b–f ITD0092 564.33 1.90 ITD0264 108.66 5.95 e–j b–f ITD0069 507.95 33.82 ITD0002 108.54 24.08 f–j d–h ITD0268 446.14 29.03 ITD0069 89.36 2.79 g–j d–i ITD0145 436.08 28.34 ITD0142 86.96 11.79 g–j d–j ITD0142 391.87 3.21 ITD0092 79.61 0.47 g–j d–j ITD0041 383.35 2.05 ITD0268 71.79 12.81 g–j d–j ITD0128 380.32 3.41 ITD0145 71.30 4.80 g–j e–j ITD0003 371.87 19.70 ITD0141 60.54 2.56 h–j f–j ITD0141 310.26 5.96 ITD0093 56.74 4.87 i,j i,j ITD0002 273.18 63.30 ITD0003 12.10 1.85 j b–f ITD0264 217.89 3.97 ITD0041 107.02 4.44 a–e a,b CBS600* 806.30 55.86 ITD0090 177.36 64.88 a,b a–c Kluyveromyces sp. ITD0046 901.78 126.87 ITD0040 165.34 28.25 a–d a–e ITD0089 832.62 60.34 ITD0089 133.57 22.87 a–e a–f ITD0040 804.55 9.92 ITD0062 131.25 2.70 b–g b–f ITD0137 643.05 22.86 ITD0046 116.48 6.13 c–i c–g ITD0049 559.25 47.84 ITD0136 98.46 9.66 d–j c–h ITD0037 516.94 51.03 ITD0037 91.82 15.70 e–j d–j ITD0048 492.63 4.10 ITD0049 67.32 9.22 f–j d–j ITD0136 448.58 19.08 ITD0048 62.72 7.84 f–j e–j ITD0062 442.38 24.29 ITD0137 59.89 6.05 a–e b–f Kluyveromyces dobzhanskii ITD0157 789.36 55.07 ITD0157 122.41 9.00 e–j j Saccharomyces cerevisae ITD0109 491.38 70.73 ITD0109 8.97 0.37 b–g g–j Torulaspora delbruekii ITD0110 676.55 27.27 ITD0110 25.50 7.37 e–j h–j ITD0129 506.33 23.75 ITD0129 20.18 2.48 Media with the same letter are not significantly different according to the HSD–Tukey–Kramer comparison test (α =0.01) described that the yeast K. marxianus CBS600 produced aroma threshold is around 0.25 mg/L (Viana et al. 2011). Also, 1.3 g/L of 2-PEA, and a maximum of 4 g/L, using an a patent has been granted for the production of 2-PEA in organophilic pervaporation technique for continuous in situ anaerobic conditions with the Kluyveromyces marxianus product removal (ISPR) in a previously optimized medium. KY3 strain with a production of 435 mg/L (Chang et al. The production of 2-PEA by starter of Hanseniaspora vinae– 2014). Recently, it has been reported solid-state fermentation Saccharomyces cerevisiae has been reported, where the con- processes (SSF) for 2-PEA and 2-PE production using centration was 0.81 to 1.70 mg/L in wine; 2-phenylethyl ace- agroindustrial residue sugarcane bagasse as sole carbon tate levels in wine vary from traces to 0.96 mg/L whereas its source for the biotransformation of L-phenylalanine using 998 Ann Microbiol (2019) 69:989–1000 Biotechnol 40:389–392. https://doi.org/10.1007/s10295-013-1240- Kluyveromyces marxianus strain as inoculum, showing effec- tive results as in other systems submerged fermentation Chang JJ, Ho CY, Huang CC, et al. (2014) Flavor compound-producing (Martínez et al. 2018). yeast strains. US Patent No. 8703474 B2 Guo et al. (2017) designed and expressed a 2-PEA biosyn- Chreptowicz K, Wielechowska M, Główczyk-Zubek J, Rybak E, Mierzejewska J (2016) Production of natural 2-phenylethanol: from thetic pathway in E. coli and in shake flask cultures with L-Phe biotransformation to purified product. Food Bioprod Process 100: (1 g/L) and recorded the generation of 268 mg/L of 2-PEA. 275–281. https://doi.org/10.1016/j.fbp.2016.07.011 This amount is very similar to the one reported in yeast ITD Chreptowicz K, Sternicka MK, Kowalska PD, Mierzejewska J (2018) 00211, highlighting that the production by the strains of this Screening of yeasts for the production of 2-phenylethanol (rose aro- study has not gone through the process of optimization. ma) in organic waste-based media. Lett Appl Microbiol 66:153–160 Ciani M, Morales P, Comitini F et al (2016) Non-conventional yeast species for lowering ethanol content of wines. Front Microbiol 7: 1–13. https://doi.org/10.3389/fmicb.2016.00642 Cordente AG, Curtin CD, Varela C, Pretorius IS (2012) Flavour-active Conclusions wine yeasts. Appl Microbiol Biotechnol 96:601–618. https://doi. org/10.1007/s00253-012-4370-z This study presents preliminary evidence of differences be- De León Rodríguez A, Escalante Minakata MDP, Jiménez García MI, tween non-Saccharomyces yeasts found during fermentation Ordoñez Acevedo LG, Flores Flores JL, Barba de la Rosa AP (2008) of A. durangensis for the production of mescal. Particularly, Characterization of volatile compounds from ethnic Agave alcoholic beverages by gas chromatography-mass spectrometry. Food Kluyveromyces yeasts have a high variability among them Technol Biotechnol 46:448–455 with respect to the production of volatile organic compounds, de Lima LA, Diniz RHS, de Queiroz MV, Fietto LG, Silveira WB (2018) where it was evidenced that these have the extraordinary po- Screening of yeasts isolated from Brazilian environments for the 2- tential to produce aromas, particularly, 2 PE and 2 PEA. phenylethanol (2-PE) production. Biotechnol Bioprocess Eng 23: 326–332. https://doi.org/10.1007/s12257-018-0119-6 Funding This work was supported by the Tecnologico Nacional de De los Rios-Deras GC, Rutiaga-Quiñones OM, López-Miranda J, Páez- México [grant number 4551.12-P] and the Consejo Nacional de Ciencia Lerma J, López MG, Soto-Cruz NO (2015) Improving Agave y Tecnología (CONACyT) scholarship awarded to Pablo Jaciel Adame- durangensis must for enhanced fermentation. Effects on mezcal Soto 435680. composition and sensory properties. 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Potential production of 2-phenylethanol and 2-phenylethylacetate by non-Saccharomyces yeasts from Agave durangensis

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Springer Journals
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Copyright © 2019 by Università degli studi di Milano
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-019-01489-0
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Abstract

Introduction The participation of non-Saccharomyces yeasts in fermentation processes is of great importance due to their participation in the formation of esters and superior alcohols, which confer characteristic aromas to beverages such as wine and mescal. The aim The aim of this study was identify and evaluate the potential aroma production of yeast native of Agave fermentation by the mescal production in Durango, Mexico. Isolated yeasts were molecularly identified by 5.8s ribosomal gene; the potential production of aromas was carried out in fermentations with the addition of L-phenylalanine and evaluated after 24 h of fermen- tation. Extraction and quantification of aromatic compounds by headspace solid-phase micro-extraction (HS-SPME) and gas chromatograph mass spectrometry (GC-MS). Results The isolated non-Saccharomyces yeasts could be classified into six different genera Saccharomyces cerevisiae, Clavispora lusitaniae, Torulaspora delbrueckii, Kluyveromyces dobzhanskii, Kluyveromyces marxianus,and Kluyveromyces sp. All probed strains presented a potential aroma production (ethyl acetate, isoamyl acetate, isoamyl alcohol, benzaldehyde, 2-phenylethyl butyrate, and phenylethyl propionate), particularly 2-phenylethanol and 2-phenylethylacetate; the levels found in the Kluyveromyces marxianus ITD0211 yeast have the highest 2-phenylethylacetate production at 203 mg/L and Kluyveromyces marxianus ITD0090 with a production of 2-phenylethanol at 1024 mg/L. Conclusion Non-Saccharomyces yeasts were isolated from the mescal fermentation in Durango; the Kluyveromyces genus is the most predominant. For the production of aromas, highlighting two strains of Kluyveromyces marxianus produces competitive quantities of compounds of great biotechnological interest such as 2-phenylethanol and 2-phenylethylacetate, without resorting to the genetic modification of yeasts or the optimization of the culture medium. . . . . Keywords Mescal Bioconversion Aroma L-Phenylalanine Kluyveromyces marxianus * Olga Miriam Rutiaga-Quiñones Departamento de Ingenierías Química y Bioquímica, Tecnológico omrutiaga@itdurango.edu.mx Nacional de México/ Instituto Tecnológico de Durango, Felipe Pescador 1803 Ote, Colonia Nueva Vizcaya, C.P. Pablo Jaciel Adame-Soto 34080 Durango, Durango, Mexico jaciel_as@hotmail.com Departamento de Microbiología e Inmunología, Unidad de Elva Teresa Aréchiga-Carvajal Manipulación Genética del Laboratorio de Micología y elva.arechigacr@uanl.edu.mx Fitopatología. Unidad C. Facultad de Ciencias Biológicas, Mercedes G López Universidad Autónoma de Nuevo León, C.P. 66451 San Nicolás de mercedes.lopez@cinvestav.mx Los Garza, Nuevo León, Mexico Silvia Marina González-Herrera 3 Departamento de Biotecnología y Bioquímica, Centro de smgonzalez@itdurango.edu.mx Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Apartado Postal 629, C.P, 36821 Irapuato, Guanajuato, Mexico Martha Rocio Moreno-Jiménez mrmoreno@itdurango.edu.mx Facultad de Ciencias Químicas-Laboratorio de Genética molecular, Norma Urtiz-Estrada Universidad Juárez del Estado de Durango, Av. Veterinaria S/N Col. urtizn@hotmail.com Valle del Sur. C.P., 34120 Durango, Durango, Mexico 990 Ann Microbiol (2019) 69:989–1000 Introduction 2006). 2-PE can also be metabolized to 2-PEA by a trans- esterification reaction, which involves the transfer of a group The non-Saccharomyces yeasts are well recognized for their of acetyl-coenzyme A acetate to the hydroxyl group of 2-PE contribution to the aroma of fermentative beverages (Cordente (Hazelwood et al. 2008; Pires et al. 2014). When L-Phe is the et al. 2012; Ciani et al. 2016; Masneuf-Pomarede et al. 2016), sole nitrogen source in the medium, large amounts of 2-PE are especially wine. Their presence has also been reported in mescal accumulated. Several biotechnological processes are known and tequila. In Mexico, these alcoholic beverages are distin- for producing 2-PE, based on this pathway, and considerable guished from each other, based on the agave species used in their progress has been made on the development of this process. In production. For example, Agave tequilana Weber var. Azul (blue this context, yeast biodiversity may be greatly impacted by the variety) is used for tequila, whereas Agave salmiana and Agave production of different aroma products derived from primary durangensis,(or Agave duranguensis) among others, are used and secondary metabolism. The diversity of non- for mescal production in various regions of Mexico (Lappe- Saccharomyces yeasts responsible for many of the volatile Oliveras et al. 2008; Páez-Lerma et al. 2010; De los Rios- compounds found in mescal, in the state of Durango, Deras et al. 2015; Kirchmayr et al. 2017). Mescal has elevated Mexico, has not yet been evaluated. This research aimed to its economic importance in the last years (Kirchmayr et al. 2017). identify the non-Saccharomyces microbiota present in fer- During the mescal production process, agave juice is naturally mentations in three different mescal-producing regions and fermented by native yeasts, such as Saccharomyces, Pichia, assess the production potential of aromatic compounds the Kluyveromyces, Candida, Debaryomyces, Hanseniaspora, addition of L-Phe as an inductor. Kloeckera, Schizosaccharomyces, Torulaspora,and Zygosaccharomyces (Lachance 1995; Díaz-Montaño et al. 2008; Escalante-Minakata et al. 2008). Previously published re- Materials and methods search on fermentations of agaves suggests that non- Saccharomyces yeasts have an important role in the initial fer- Yeast strains mentation process and influence the production of the volatile compounds (Lappe-Oliveras et al. 2008; Narváez-Zapata et al. Thirty-four native strains, identified as non-Saccharomyces 2010; Martell Nevárez et al. 2011). The potential use of these from Agave durangensis fermentation and obtained from the yeasts as inoculants has been described (Rodríguez-Sifuentes Collection of the Instituto Tecnologico de Durango, were iso- et al. 2014; Nuñez-Guerrero et al. 2016), as well as their partic- lated from three mescal-producing regions of Durango State, ipation in generating the volatile compounds in mescal, mainly Mexico: Mezquital (23° 28′ 22″ N, 104° 24′ 40″ W), Nombre esters (Martell Nevárez et al. 2011; Rutiaga-Quiñones et al. 2012; de Dios (23° 51′ 00″ N, 104° 14′ 00″ W), and Durango (24° Hernández-Carbajal et al. 2013). Despite the increasing use of 01′ N, 104° 40′ W). All yeast strains were conserved, as cul- non-Saccharomyces yeasts in biotechnology, there are still many ture stock at − 20 °C in 30% (v/v) glycerol. opportunities to improve native yeast exploration. These pros- pects have led to a great interest in further enhancing the number Molecular identification of non-Saccharomyces yeasts available, by selecting or develop- ing strains with novel and attractive properties. Growth conditions Flavor has a major impact on the quality perception of food and beverages, and fragrances are highly valued in the Yeast cells preserved in glycerol were first activated on YDP cosmetic and perfume industry. For natural aroma compounds solid medium (glucose 20 g/L, casein peptone 20 g/L, yeast that exist at low concentrations in their original sources, bio- extract 10 g/L, and agar 20 g/L). DNA was then extracted at technological processes represent an attractive alternative to 24-h growth, using the method detailed by Sambrook and the traditional preparation by extraction (Schrader et al. 2004). Russell (2001). Due mainly to its sweet and rose-like taste and odor 2- phenylethanol (2-PE) and its more fruit-like form, acetate ester Polymerase chain reaction and amplification 2-phenylethylacetate (2-PEA), find use in various flavor com- positions (Fabre et al. 1998). For food applications, the rising Polymerase chain reaction (PCR) was carried out in 50-μL demand for natural products means natural flavor compounds volumes, using 2.0 μL of DNA with ITS1 (5′–TCC GTA are increasingly becoming a necessity (Etschmann and GGT GAA CCT GCG G–3′) and ITS4 (5′–TCC TCC GCT Schrader 2006, Morrissey et al, 2015). TAT TGATAT GC–3′) primers to amplify the rDNA repeat unit Both 2-PE and 2-PEA can be produced by de novo that includes the 5.8S rRNA gene and the two non-coding synthesis or from L-phenylalanine (L-Phe) by non- regions designated the internal transcribed spacers (ITS1 and Saccharomyces yeast whole-cell biocatalysis via the Ehrlich ITS4) (White et al. 1990). Amplification began with an initial- pathway (Etschmann et al. 2003), (Etschmann and Schrader izationstep ofonecycleat95°Cfor 5min, then35cyclesof Ann Microbiol (2019) 69:989–1000 991 95 °C for 1 min, 52 °C for 2 min, and 72 °C for 2 min, followed Genomics for Biodiversity Laboratory (Langebio) of by a final elongation at 72 °C for 10 min (White et al. 1990). Cinvestav (Irapuato, Mexico). The PCR product was electrophoresed on 1% agarose gel with TAE 0.5× buffer (Promega, Madison, WI, USA), at 95 V for 45 min, stained with ethidium bromide (Sigma–Aldrich. St. Phylogenetic analysis Louis, MO, USA) and visualized under UV light (Benchtop UV transilluminator, Upland, CA, USA); DNA fragment sizes The obtained sequences were aligned using the MUSCLE were determined using a 100-bp DNA ladder (Promega, USA). program (https://www.ebi.ac.uk/Tools/msa/muscle), and The PCR product was purified using C H NO and C H O(> regions of local similarity between sequences were identified 2 7 2 2 6 99%) (Sigma–Aldrich). The rDNA sequences were acquired from the National Center for Biotechnology Information using an ABI PRISM Model 3730XL sequencer (Applied (NCBI) database of GenBank using the BLAST program Biosystems, Inc., Foster City, CA, USA) at the National (https://blast.ncbi.nlm.nih.gov/Blast). Phylogenetic analyses Table 1 Strains used in this study Species Strain Locality Accession no. Clavispora lusitaniae ITD 0132 Mezquital MH282797 Kluyveromyces marxianus ITD 0002 Mezquital MH282778 Kluyveromyces marxianus ITD 0003 Mezquital MH282779 Kluyveromyces marxianus ITD 0090 Mezquital MF797638.1 Kluyveromyces marxianus ITD 0091 Mezquital MH282784 Kluyveromyces marxianus ITD 0092 Mezquital MH282785 Kluyveromyces marxianus ITD 0093 Mezquital MH282786 Kluyveromyces marxianus ITD 0128 Mezquital MH282787 Kluyveromyces marxianus ITD 0141 Mezquital MH282790 Kluyveromyces marxianus ITD 0142 Mezquital MH282791 Kluyveromyces marxianus ITD 0145 Mezquital MH282792 Kluyveromyces marxianus ITD 0211 Mezquital MH282793 Kluyveromyces sp. ITD 0040 Mezquital MH282781 Kluyveromyces sp. ITD 0041 Mezquital MH282782 Kluyveromyces sp. ITD 0089 Mezquital MH282783 Kluyveromyces sp. ITD 0136 Mezquital MH282788 Kluyveromyces sp. ITD 0137 Mezquital MH282789 Torulaspora delbrueckii ITD 0110 Mezquital MH282795 Torulaspora delbrueckii ITD 0129 Mezquital MH282796 Saccharomyces cerevisiae ITD 0109 Mezquital MH282794 Clavispora lusitaniae ITD 0095 Nombre de Dios MH282804 Clavispora lusitaniae ITD 0099 Nombre de Dios MH282805 Clavispora lusitaniae ITD 0103 Nombre de Dios MH282806 Clavispora lusitaniae ITD 0104 Nombre de Dios MH282807 Clavispora lusitaniae ITD 0107 Nombre de Dios MH282808 Kluyveromyces marxianus ITD 0102 Nombre de Dios MH282801 Kluyveromyces marxianus ITD 0264 Nombre de Dios MH282802 Kluyveromyces marxianus ITD 0268 Nombre de Dios MH282803 Kluyveromyces sp. ITD 0046 Nombre de Dios MH282798 Kluyveromyces sp. ITD 0048 Nombre de Dios MH282799 Kluyveromyces sp. ITD 0049 Nombre de Dios MH282800 Kluyveromyces marxianus ITD 0069 Durango MH282810 Kluyveromyces sp. ITD 0062 Durango MH282809 Kluyveromyces dobzhanskii ITD 0157 Durango MH282811 Kluyveromyces marxianus CBS 600 Reference KY103809.1 992 Ann Microbiol (2019) 69:989–1000 were conducted in MEGA7 Program. The sequences were at a linear flow of 2 mL/min. The injector and detector temper- deposited in GenBank. atures were 230 and 260 °C, respectively. The oven temperature was increased from 40 to 240 °C, using the following program: Production of volatile organic compounds the initial temperature was maintained for 3 min, ramped at 4 °C/min to 100 °C, held for 1 min, and then ramped at Chemicals and reagents 4 °C/min to 240 °C and held for 10 min. The ionization voltage was 70 eV. All the assays were performed twice. The analyzed L-Phe (< 98%), 2-PE (> 99%), and 2-PEA (> 99%) were pur- compounds were identified by comparing their mass spectra with chased from Sigma–Aldrich. Na HPO .2H O, MgSO .7H O those in the NIST database (Calvo-Gómez et al. 2004). In addi- 2 4 2 4 2 (Caisson Laboratory In., Smithfield, UT, USA), and citric acid tion, the volatile compounds of interest (2-PE and 2-PEA) were were obtained from Fermont (Mexico City, Mexico). Glucose, quantified by standard curves. yeast extract and casein peptone came from BD Bioxon (Mexico City, Mexico). Statistical analysis Bioconversion Data of the volatile compounds, 2-PE and 2-PEA, were eval- uated by the HSD–Tukey–Kramer comparison test, at α = The strains were pre-grown in 125-mL baffled Erlenmeyer 0.01 All statistical analyses were done using JMP software flasks (Corning, Inc., USA) with vented top, containing a version 13.2 (SAS Institute, Inc., NC, USA). 50-mL operative volume of standard yeast medium YPD broth (20 g/L glucose, 20 g/L casein peptone, and 10 g/L yeast extract), at 30 °C for 12 h and 120 rpm. For fermentation, the Result and discussion strains were inoculated at a concentration of 10 cells/mL and incubated at 30 °C for 24 h and 120 rpm. Duplicate experi- Molecular identification and phylogenetic analyses ments were done for induction with L-Phe (9 g/L), in which the culture medium contained 30 g/L glucose, 35 g/L Table 1 indicates the molecular identification of the studied Na HPO .2H O, 10.5 g/L citric acid, 0.5 g/L MgSO .7H O, 2 4 2 4 2 strains, for each geographic region. These strains corresponded and 0.17 g/L yeast extract, in a 50-mL medium, in a 125-mL to six different genera: Clavispora lusitaniae, Kluyveromyces sp., Erlenmeyer flask (Etschmann et al. 2004). The yeast Kluyveromyces dobzhanskii, Kluyveromyces marxianus, Kluyveromyces marxianus CBS 600 (KY103809.1) was in- Saccharomyces cerevisiae,and Torulaspora delbrueckii. cluded as a reference. Previous investigations of the yeasts associated with mescal pro- duction in Mexico, described the presence of non- Gas chromatography–mass spectrometry analysis Saccharomyces strains, such as K. marxianus, C. lusitaniae, and Pichia fermentans from Agave salmiana fermentation, in The volatile organic compounds were extracted by headspace San Luis Potosí State (Escalante-Minakata et al. 2008). In anoth- solid-phase micro-extraction (HS-SPME) with a er Vinata, from the same region, the non-Saccharomyces yeasts divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco, were: K. marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Bellefonte, PA, USA). One milliliter of the sample was taken C. lusitaniae, T. delbrueckii,and Candida ethanolica (Verdugo- from each fermentation at 24 h, placed inside a 4-mL vial, sealed Valdez et al. 2011). In mescal produced using the species Agave tightlywithascrew-topseptum-containing cap, and allowed to durangensis in Durango, the predominant non-Saccharomyces stand at 35 °C for 1 h. The SPME needle was then inserted yeasts belonged to Candida genus, including Candida lusitaniae, through the septum, the holder was secured, and the fiber was Candida kefir, Candida glabrata, Candida laurentii,and exposed to the headspace. After 1 h of sampling at 35 °C, the fiber was retracted and immediately inserted into the inlet of a HP Fig. 1 Neighbor-joining trees were constructed from the evolutionary„ 5890 Series II GC instrument directly coupled to an HP 5972 distance data for ITSI-5.85 rDNA-ITS2. The percentage of replicate mass-selective detector (Hewlett–Packard, Palo Alto, CA, USA) trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). a Kluyveromyces marxianus tree of group one. b and equipped with an HP-FFAP capillary column (25 m × Kluyveromyces dobhzankii tree. c Kluyveromyces sp of group two tree. 0.320 mm i.d., film thickness 0.50 m; Hewlett–Packard), for The accession numbers of reference sequences used in this tree are as thermal desorption. The injection was accomplished by desorp- follows: K. nonfermentans (AB011512.1), K. lactis (AB011515.1), K. wickerhamii (AB011521.1), K. aestuarii (AB011513.1), tion of the fiber at 230 °C for 6 min with the injector operated in K. marxianus (AB011518.1), K. marxianus (MH045720.1), the splitless mode for 1 min. An additional 5-min exposure in the K. marxianus (MH045721.1), K. marxianus (MH045719.1), injection port allowed the fiber to be cleaned of any compound K. marxianus (MG966429.1), K. marxianus (JX174415.1), that may not have been desorbed during the initial minute K. dobzhanskii (ABO11514.1), and D. hansenii (JQ912667.1). Evolutionary analyses were conducted in MEGA7 (Calvo-Gómez et al. 2004). Helium was used as the carrier gas, Ann Microbiol (2019) 69:989–1000 993 ITD0093(MH282786) K-marxianus(AB011518.1) ITD0002(MH282778) ITD0102(MH282801) ITD0091(MH282784) ITD0003(MH282779) ITD0141(MH282790) ITD0145(MH282792) ITD0069(MH282810) ITD0092(MH282785) ITD0128(MH282787) ITD0142(MH282791) ITD0211(MH282793) ITD0264(MH282802) ITD0268(MH282803) ITD0090(MF797638.1) Kmarxianus-CHP7(MH045719.1) Km-KDLYH1-1(JX174415.1) Km-CDA2(MG966429.1) Kmarxianus-CDB5(MH045721.1) Kmarxianus-CDB1(MH045720.1) K-dobzhanskii(AB011514.1) K-nonfermentans(AB011512.1) K-lactis(AB011515.1) K-wickerhamii(AB011521.1) K-aestuarii(AB011513.1) Debaryomyces-hansenii(JQ912667.1) K-nonfermentans(AB011512.1) K-aestuarii(AB011513.1) K-lactis(AB011515.1) K-wickerhamii(AB011521.1) K-marxianus(AB011518.1) K-dobzhanskii(AB011514.1) ITD0157(MH282811) Debaryomyces-hansenii(JQ912667.1) ITD0049(MH282800) ITD0062(MH282809) ITD0048(MH282799) ITD0046(MH282798) ITD0041(MH282782) ITD0040(MH282781) ITD0037(MH282780) 75 ITD0089(MH282783) ITD0136(MH282788) ITD0137(MH282789) K-nonfermentans(AB011512.1) K-marxianus(AB011518.1) K-dobzhanskii(AB011514.1) Kmarxianus-CDB1(MH045720.1) Kmarxianus-CDB5(MH045721.1) Kmarxianus-CHP7(MH045719.1) Km-CDA2(MG966429.1) K-lactis(AB011515.1) Km-KDLYH1-1(JX174415.1) K-wickerhamii(AB011521.1) K-aestuarii(AB011513.1) Debaryomyces-hansenii(JQ912667.1) 2 994 Ann Microbiol (2019) 69:989–1000 Candida tropicalis (Páez-Lerma et al. 2010). Equally, in fourcroydes Lem. There are numerous accounts of this species Durango State, Páez-Lerma et al. (2013) observed diverse micro- during the different stages of processing and fermentation of organisms at the beginning of fermentation: S. cerevisiae, Agave to obtain traditional Mexican beverages, such as T. delbrueckii, K. marxianus, Candida diversa, P. fermentans, Bpulque,^ mescal, and tequila (Rodrigues de Miranda 1979; and Hanseniaspora uvarum, but only T. delbrueckii and Lachance 1995; Lappe et al. 2004; De León Rodríguez et al. S. cerevisiae were found at the end of the fermentations. 2008; Lappe-Oliveras et al. 2008; Páez-Lerma et al. 2010; Recently, Kirchmayr et al. (2017) mentioned K. marxianus, Kurtzman et al. 2011; Verdugo-Valdez et al. 2011), where its Zygosaccharomyces rouxii, Z. bisporus, T. delbrueckii,and presence has been associated with the sensory qualities of these Pichia membranifaciens as the main microbiota present, after beverages (Escalante-Minakata et al. 2008). S. cerevisiae, during mescal production in Oaxaca State. In our The species present in relatively low quantity was study, Mezquital region presented the greatest number and diver- T. delbrueckii, found only in the region of the Mezquital. sity of isolated non-Saccharomyces, which included Figure 2 b shows the phylogenetic tree for strains ITD0110 K. marxianus, T. delbrueckii,and C. lusitaniae.Both and ITD0129. These strains have been linked to a high pro- K. marxianus and C. lusitaniae were also detected in mescal duction of volatile compounds that impart unique characteris- from Nombre de Dios. In fermentation of agave in Durango, tics to beverages, such as mescal, and also other flavor com- the species identified were K. marxianus and K. dobzhanskii. pounds, including terpenoids, esters, higher alcohols, glycerol This article is the first report where the strain K. dobzhanskii acetaldehyde, acetic acid, and succinic acid (Moreira et al. hasbeenfoundinnatural fermentation processes. This genus 2005; Jolly et al. 2014). Rutiaga-Quiñones et al. (2012)pro- has been cataloged as the closest Kluyveromyces lactis relative filed the volatile compounds in Agave duranguensis juice of wild or native strains, so it has been used for modeling popu- supplemented with NH Cl and fermented with the yeast lation genetics (Belloch et al. 1997, 2002; Sukhotina et al. 2006; T. delbrueckii ITD0110. However, the genetic diversity pres- Lane and Morrissey 2010). ent in this genus was not established. Nuñez-Guerrero et al. In phylogenetic studies of Kluyveromyces strains (Fig. 1), (2016)isolated S. cerevisiae, T. delbrueckii,and K. marxianus three groups were recognized. The first two groups comprised from A. duranguensis fermentation and proposed the use of a strains directly related to the genus K. marxianus and mixture of 75% S. cerevisiae and 25% T. delbrueckii as an K. dobzhanskii, respectively (Fig. 1a, b). The third group had inoculant to make mescal. direct relationship to the genera of the Kluyveromyces family (Fig. 1c). These strains were present in all the regions, accounting Production of volatile organic compounds for 35% (Mezquital), 50% (Nombre de Dios), and 30% (Durango) of the total of the isolated Kluyveromyces strains and Table 2 presents the volatile compounds produced by the non- can represent a particular genetic diversity for K. marxianus Saccharomyces yeasts studied in this work. In general, all strains isolated from the fermentation process during the produc- strains were producers of esters, fatty acids esters, and higher tion of mescal. In a recent study of the genetic diversity of the alcohols. Esters are key flavor compounds in fermented bev- genus K. marxianus, all the isolates from a lactic environment erages, like mescal. Among the acetate esters, the synthesis of were either diploid or triploid, whereas non-lactic isolates were ethyl acetate, which is responsible for the bouquet and desir- haploid (Ortiz-Merino et al. 2018). Additionally, the authors dis- able fruity flavors, depends on the ethanol concentration while tinguished three clades, of which the strain UFS-Y2791, isolated the synthesis of isoamyl acetate, isobutyl acetate and 2-PEA, from American agave juice and representing the third clade, relies on the concentration of their corresponding higher alco- proved to be more diverse than the others (Ortiz-Merino et al. hol, by the action of an alcohol acetyltransferase (Gethins 2018). So far, only the presence of K. marxianus strains from et al. 2015; Loser et al. 2014). different mescal production has been reported in the literature, The ethyl esters of short-chain fatty acids present are 2- indicating that the current work is the first to show that phenylethyl butyrate and Phenylethyl propionate, synthesized K. marxianus strains isolated from agave fermentation (mescal from 2-PE and short-chain fatty acids. Phenylethyl propionate or tequila) have distinct genetic differences between them. Páez- is an ester desirable in wine, due to its floral aroma (Beckner Lerma et al. (2013) noted these differences with S. cerevisiae Whitener et al. 2015; Padilla et al. 2016). The formation of benz- strains in wine. aldehyde from Phe has been studied in several microorganisms, Additionally, phylogenetic analysis among the Clavispora such as Pseudomonas putida and the white rot fungi, Tremetes lusitaniae yeasts from this study (Fig. 2a) evidenced the genetic suaveolens, Polyporus tuberaste, Bierkandera adusteand,and variability between strains of Clavispora lusitaniae.These yeasts Phanerochaete chrysosporium (Rojas et al. 2001; Liu et al. were found predominantly in fermentations from Nombre de 2004). Hence, these yeasts seem to have benzaldehyde produc- Dios, with 45% of the strains identified as C. lusitaniae. Pérez- tion potential. The principal volatile compounds found were 2- Brito et al. (2015) characterized the great genetic diversity of PE, which is considered to be one of the most important aromatic C. lusitaniae strains isolated from the fermentation of Agave alcohols, and 2-PEA. The higher alcohols are predominantly Ann Microbiol (2019) 69:989–1000 995 Clavisporalusitaniae-CBS:5094(AY321465.1) Clavisporalusitaniae-PMM10-1024036L-isolate-ISHAM-ITS ID-MITS815(KP131848.1) Clavisporalusitaniae-CNRMA10.800-isolate-ISHAM-ITS ID-MITS841(KP131835.1) Clavisporalusitaniae-H27507-04(HQ693786.1) Clavisporalusitaniae-ATCC-34449(KU729100.1 ) Clavisporalusitaniae-CNRMA11.496-isolate-ISHAM-ITS ID-MITS845(KP131836.1) Clavisporalusitaniae-MTCC1001(AY174102.1) Clavisporalusitaniae-CBS:5299(KY102564.1) Clavisporalusitaniae-IWG-DG-o(MG518174.1) ITD0099(MH282805) ITD0095(MH282804) ITD0104(MH282807) ITD0132(MH282797) ITD0103(MH282806) ITD0107(MH282808) Clavisporalusitaniae-WTS2D(MG183704.1) Debaryomyces-hansenii(JQ912667.1) Saccharomyces-cerevisiae-F2(JX141369.1) Saccharomyces-cerevisiae-F1(JX141368.1) ITD0109(MH282794) ITD0129(MH282796) ITD0110(MH282795) Torulaspora-delbrueckii-T6(FJ838774.1) Debaryomyces-hansenii(JQ912667.1) 0.050 Fig. 2 Neighbor-joining trees were constructed from the evolutionary (HQ693786.1), C. lusitaniae (KU729100.1), C. lusitaniae distance data for ITSI-5.85 rDNA-ITS2. The percentage of replicate (KP131836.1), C. lusitaniae (AY174102.1), C. lusitaniae trees in which the associated taxa clustered together in the bootstrap test (KY102564.1), C. lusitaniae MG518174.1), C. lusitaniae (1000 replicates). a Clavispora lusitaniae tree. b S. cerevisiae–T. (MG183704.1), S. cerevisiae (JX141369.1), S. cerevisiae (JX141368.1), delbrueckii tree. The accession numbers of reference sequences used in T. delbrueckii (FJ838774.1), and D. hansenii (JQ912667.1). Evolutionary these trees are as follows: C. lusitaniae (AY321465.1), C. lusitaniae analyses were conducted in MEGA7 (KP131848.1), C. lusitaniae (KP131835.1), C. lusitaniae formed by yeastduringfermentationbytheEhrlichpathway, The production of volatile organic compounds (2-PE and involving amino acid degradation, particularly Phe (Hazelwood 2-PEA) presented significant statistical differences between et al. 2008;Styger etal. 2011) but can also be synthesized from by non-Saccharomyces yeasts is shown in Table 3.The most glucose via pyruvate (Cordente et al. 2012). The Ehrlich pathway productive yeasts, in terms of 2-PE, were as follows: involves three steps: phenylpyruvate is decarboxylated to Kluyveromyces marxianus (ITD0090, ITD0091), as well as phenylacetaldehyde, then reduced to 2-PE (Etschmann and the yeasts Kluyveromyces sp. (ITD0046, ITD0089 and Schrader 2006), and finally esterified to 2-PEA. ITD0040), Kluyveromyces dobzhanskii ITD0157, and 996 Ann Microbiol (2019) 69:989–1000 Table 2 Volatile metabolites Metabolite RTm/z Sensorial description produced by non-Saccharomyces strains Ethyl acetate 0.95 61-70-73-88 Pineapple, sweet, and fruit Isoamyl acetate 4.04 55-70-87 Banana, sweet and fruit Isoamyl alcohol 6.24 55-70-87 Alcohol, banana, and malt Benzaldehyde 16.36 51-77-106 Almond, burnt sugar, cherry, and sweet 2-Phenylethylacetate 22.72 121-104-91-77 Floral, fruit, honey, and roses 2-Phenylethyl butyrate 23.68 117-104-91-77-71-65 Yeast, strawberry, floral, and sweet Phenylethyl propionate 23.79 104-91-77-57 Floral, red fruit, and honey 2-Phenylethanol 24.22 103-91-77-65-51 Roses, fresh, and leafy Clavispora lusitaniae ITD0107. K. marxianus yeast ITD0090 biotransformation in standard medium for 72-h batch cultures. can be classified as the largest producer of 2-PE, even when Clavispora lusitaniae WUT17 strain reached the levels of compared with the most studied yeast K. marxianus CBS600 2.04 g/L of 2-PE in a standard medium and 0.95 g/L of 2- (Table 3). The concentrations obtained were similar to that PE in an organic waste-based medium, which is superior to reported by Etschmann et al. (2003) (890 mg/L in a the one reported by Etschmann et al. (2003)of 0.33 g/L. It is molasses-based medium). Eshkol et al. (2009) evaluated the well known that 2-PE synthesis is carried out by the Ehrlich potential of stress-tolerant Saccharomyces strains to produce pathwayinyeast,suchas K. marxianus and Yarrowia 2-PE under inductive conditions (Phe addition) and detected lipolytica (Fabre et al. 1998; Celińska et al. 2013). In a recent the concentrations between 340 and 460 mg/L at 48-h incuba- study, González et al. (2018) screened the 2-PE production tion, but these concentrations increased to 540 and 850 mg/L potential of some non-Saccharomyces yeasts and discovered with selected yeast, when conditions were optimized with a2-PEproductiveyeast (T. delbrueckii). However, in all 10 g/L Phe addition, which were very similar quantities to cases, non-Saccharomyces species produce lower quantities those reported here, without the optimization process. In a than S. cerevisiae, indicating that the Ehrlich pathway may related study, with the K. marxianus strain CBS6556, the op- not be as active in non-Saccharomyces species as in timization of the grape must culture medium with 3 g/L of L- Saccharomyces, at least under nitrogen-limiting conditions. Phe improved the 2-PE titer of 0.39 g/L after 84 h of culture to Rutiaga-Quiñones et al. (2012) revealed the non- 0.47 g/L (Garavaglia et al. 2007). Mei et al. (2009)alsoused a Saccharomyces yeasts potential for volatile compounds, par- yeast Saccharomyces cerevisiae BD and reported in situ prod- ticularly in A. duranguensis juice for mescal production; in uct adsorption techniques, to obtain a better performance re- this study, the strains T. delbrueckii ITD0110 and garding the biotransformation of L-phenylalanine to 2- K. marxianus ITD0211 showed to be more productive of 2- phenylethanol, reaching a concentration of 4.65 g/L of 2-PE PE under nitrogen-limiting conditions that the strain with a content of 10 g/L of L-Phe in the medium. Chreptowicz S. cerevisiae ITD0109. A possible theory for our observations, et al. (2016) with yeast not genetically modified strain when the fermentations of different strains induced with L-Phe Saccharomyces cerevisiae JM2014 was isolated from a as the only source of nitrogen were evaluated, is that the fermented milk drink (Turkey), producing a total concentration Ehrlich route is working on these strains, but the metabolic of 3.60 g/L of 2-PE after 72-h incubation at 30 °C batch culture plasticity differs for each of the strains studied. These results with a medium containing 5 g/L of L-Phe in a 4-L bioreactor at allow to raise genetic and biochemical differences between the laboratory scale. Recently, De Lima et al. (2018) evaluated the strains of wine production and mescal, but additional studies potential of yeast strain K. marxianus CCT 7735 in the 2-PE are required to elucidate and describe them. production and reported a production of 2.47 g/L of 2-PE, with The Table 3 illustrates that 2-PEA production presents sig- the optimization in the medium through the optimal conditions nificant difference for each strain where that highlighting the achieving thus a production of 3.44 g/L of 2-PE. Lu et al. K. marxianus strains (ITD0040, ITD0090, ITD0102, and (2016) showed the 2-PE titer in a batch fermentation with ITD0211). A previous research by Rojas et al. (2001) de- the stress-tolerant yeast Candida glycerinogenes WL2002-5, scribed a very productive H. guilliermondii yeast, with a 2- reaching 5 g/L from L-Phe, under optimized culture condi- PEA production of 83.83–163.8 mg/L, when using 2-PE as tions. Genetic modification strategies have also been consid- induction conditions and in the presence of extraction solvent. ered, to further increase 2-PE production, such as ARO8 and The present results describe a difference in the production ARO10 overexpression in S. cerevisiae SPO810 yeast the 2-PE potential from L-Phe induction, among all the strains studied, reached 2.61 g/L after 60 h of cultivation (Yin et al. 2015). highlighting two strains, K. marxianus ITD0090 and Chreptowicz et al. (2018) reported yeast strains capable of K. marxianus ITD0211, due to the potential to overproduce producing over 2 g/L 2-PE through the L-Phe 2-PE and 2-PEA, respectively. Etschmann et al. (2005) Ann Microbiol (2019) 69:989–1000 997 Table 3 Production 2- Species ID strains Production of 2-PE (mg/ ID Strains Production of 2-PEA phenylethanol and 2- L) (mg/L) phenylethylacetate obtained by different yeast strains non- Mean S.d Mean S.d Saccharomyces by HS-SPME a–f h–j Clavispora lusitaniae ITD0107 764.80 62.64 ITD0095 22.43 2.55 b–g h–j ITD0095 636.60 47.41 ITD0107 17.17 4.52 d–j i,j ITD0132 511.89 2.23 ITD0104 13.16 1.14 g–j i,j ITD0104 394.74 42.54 ITD0099 12.14 0.29 g–j i,j ITD0099 370.89 10.13 ITD0132 11.89 0.19 g–j j ITD0103 357.54 29.41 ITD0103 9.20 1.06 a a Kluyveromyces marxianus ITD0090 1024.46 306.38 ITD0211 203.53 3.52 a–c a ITD0091 848.37 112.28 ITD0102 202.07 26.28 b–h a–c ITD0211 630.81 7.13 CBS600 166.24 0.40 b–h a–d ITD0102 618.88 46.90 ITD0091 136.49 28.48 b–i a–e ITD0093 596.41 26.16 ITD0128 134.27 13.52 c–i b–f ITD0092 564.33 1.90 ITD0264 108.66 5.95 e–j b–f ITD0069 507.95 33.82 ITD0002 108.54 24.08 f–j d–h ITD0268 446.14 29.03 ITD0069 89.36 2.79 g–j d–i ITD0145 436.08 28.34 ITD0142 86.96 11.79 g–j d–j ITD0142 391.87 3.21 ITD0092 79.61 0.47 g–j d–j ITD0041 383.35 2.05 ITD0268 71.79 12.81 g–j d–j ITD0128 380.32 3.41 ITD0145 71.30 4.80 g–j e–j ITD0003 371.87 19.70 ITD0141 60.54 2.56 h–j f–j ITD0141 310.26 5.96 ITD0093 56.74 4.87 i,j i,j ITD0002 273.18 63.30 ITD0003 12.10 1.85 j b–f ITD0264 217.89 3.97 ITD0041 107.02 4.44 a–e a,b CBS600* 806.30 55.86 ITD0090 177.36 64.88 a,b a–c Kluyveromyces sp. ITD0046 901.78 126.87 ITD0040 165.34 28.25 a–d a–e ITD0089 832.62 60.34 ITD0089 133.57 22.87 a–e a–f ITD0040 804.55 9.92 ITD0062 131.25 2.70 b–g b–f ITD0137 643.05 22.86 ITD0046 116.48 6.13 c–i c–g ITD0049 559.25 47.84 ITD0136 98.46 9.66 d–j c–h ITD0037 516.94 51.03 ITD0037 91.82 15.70 e–j d–j ITD0048 492.63 4.10 ITD0049 67.32 9.22 f–j d–j ITD0136 448.58 19.08 ITD0048 62.72 7.84 f–j e–j ITD0062 442.38 24.29 ITD0137 59.89 6.05 a–e b–f Kluyveromyces dobzhanskii ITD0157 789.36 55.07 ITD0157 122.41 9.00 e–j j Saccharomyces cerevisae ITD0109 491.38 70.73 ITD0109 8.97 0.37 b–g g–j Torulaspora delbruekii ITD0110 676.55 27.27 ITD0110 25.50 7.37 e–j h–j ITD0129 506.33 23.75 ITD0129 20.18 2.48 Media with the same letter are not significantly different according to the HSD–Tukey–Kramer comparison test (α =0.01) described that the yeast K. marxianus CBS600 produced aroma threshold is around 0.25 mg/L (Viana et al. 2011). Also, 1.3 g/L of 2-PEA, and a maximum of 4 g/L, using an a patent has been granted for the production of 2-PEA in organophilic pervaporation technique for continuous in situ anaerobic conditions with the Kluyveromyces marxianus product removal (ISPR) in a previously optimized medium. KY3 strain with a production of 435 mg/L (Chang et al. The production of 2-PEA by starter of Hanseniaspora vinae– 2014). Recently, it has been reported solid-state fermentation Saccharomyces cerevisiae has been reported, where the con- processes (SSF) for 2-PEA and 2-PE production using centration was 0.81 to 1.70 mg/L in wine; 2-phenylethyl ace- agroindustrial residue sugarcane bagasse as sole carbon tate levels in wine vary from traces to 0.96 mg/L whereas its source for the biotransformation of L-phenylalanine using 998 Ann Microbiol (2019) 69:989–1000 Biotechnol 40:389–392. https://doi.org/10.1007/s10295-013-1240- Kluyveromyces marxianus strain as inoculum, showing effec- tive results as in other systems submerged fermentation Chang JJ, Ho CY, Huang CC, et al. (2014) Flavor compound-producing (Martínez et al. 2018). yeast strains. US Patent No. 8703474 B2 Guo et al. (2017) designed and expressed a 2-PEA biosyn- Chreptowicz K, Wielechowska M, Główczyk-Zubek J, Rybak E, Mierzejewska J (2016) Production of natural 2-phenylethanol: from thetic pathway in E. coli and in shake flask cultures with L-Phe biotransformation to purified product. Food Bioprod Process 100: (1 g/L) and recorded the generation of 268 mg/L of 2-PEA. 275–281. https://doi.org/10.1016/j.fbp.2016.07.011 This amount is very similar to the one reported in yeast ITD Chreptowicz K, Sternicka MK, Kowalska PD, Mierzejewska J (2018) 00211, highlighting that the production by the strains of this Screening of yeasts for the production of 2-phenylethanol (rose aro- study has not gone through the process of optimization. ma) in organic waste-based media. Lett Appl Microbiol 66:153–160 Ciani M, Morales P, Comitini F et al (2016) Non-conventional yeast species for lowering ethanol content of wines. Front Microbiol 7: 1–13. https://doi.org/10.3389/fmicb.2016.00642 Cordente AG, Curtin CD, Varela C, Pretorius IS (2012) Flavour-active Conclusions wine yeasts. Appl Microbiol Biotechnol 96:601–618. https://doi. org/10.1007/s00253-012-4370-z This study presents preliminary evidence of differences be- De León Rodríguez A, Escalante Minakata MDP, Jiménez García MI, tween non-Saccharomyces yeasts found during fermentation Ordoñez Acevedo LG, Flores Flores JL, Barba de la Rosa AP (2008) of A. durangensis for the production of mescal. Particularly, Characterization of volatile compounds from ethnic Agave alcoholic beverages by gas chromatography-mass spectrometry. Food Kluyveromyces yeasts have a high variability among them Technol Biotechnol 46:448–455 with respect to the production of volatile organic compounds, de Lima LA, Diniz RHS, de Queiroz MV, Fietto LG, Silveira WB (2018) where it was evidenced that these have the extraordinary po- Screening of yeasts isolated from Brazilian environments for the 2- tential to produce aromas, particularly, 2 PE and 2 PEA. phenylethanol (2-PE) production. Biotechnol Bioprocess Eng 23: 326–332. https://doi.org/10.1007/s12257-018-0119-6 Funding This work was supported by the Tecnologico Nacional de De los Rios-Deras GC, Rutiaga-Quiñones OM, López-Miranda J, Páez- México [grant number 4551.12-P] and the Consejo Nacional de Ciencia Lerma J, López MG, Soto-Cruz NO (2015) Improving Agave y Tecnología (CONACyT) scholarship awarded to Pablo Jaciel Adame- durangensis must for enhanced fermentation. Effects on mezcal Soto 435680. composition and sensory properties. Rev Mex Ing Quím 14:363– 371 http://www.redalyc.org/articulo.oa?id=62041194013 Díaz-Montaño DM, Délia ML, Estarrón-Espinosa M, Strehaiano P Compliance with ethical standards (2008) Fermentative capability and aroma compound production by yeast strains isolated from Agave tequilana Weber juice. Enzym Conflict of interest The authors declare that they have no conflict of Microb Technol 42:608–616. https://doi.org/10.1016/j.enzmictec. interest. 2007.12.007 Escalante-Minakata P, Blaschek HP, Barba De La Rosa AP et al (2008) Research involving human participants and/or animal This article does Identification of yeast and bacteria involved in the mezcal fermen- not contain any studies with human or animal. tation of Agave salmiana. Lett Appl Microbiol 46:626–630. https:// doi.org/10.1111/j.1472-765X.2008.02359.x Informed consent Not applicable. 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Annals of Microbiology – Springer Journals

Published: Jun 17, 2019

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