Identification and selection of non-Saccharomyces strains isolate from brazilian apple must

Identificação e seleção de leveduras não-Saccharomyces isoladas de mostos de maçãs brasileiras

Giovana Arruda Moura Pietrowski Juliana Vitória Messias Bittencourt Luciana Rocha Brandão Carlos Augusto Rosa Aline Alberti Alessandro Nogueira About the authors

ABSTRACT:

This study aimed to know the biodiversity of non-Saccharomyces yeasts in Brazilian apples must with potential to improve of the aromatic quality of ciders. The strains were isolated from thirty-five (35) Gala and Fuji apple musts from different locations from south region of Brazil. Forty-five (45) strains were isolate and identified by PCR analysis. Results indicated ten (10) species: Candida oleophila, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Lodderomyces elongisporus, Pichia anomala, Pichia fermentans and Rhodotorula mucilaginosa. The genus Rhodotorula sp., Lodderomyces sp. and Clavispora sp. constituted 71.2% of the strains identified. The following strains, C. oleophila, R. mucilaginosa, P. fermentans, H. uvarum and H. guilliermondii were selected in qualitative tests due the fruity aroma production by trained team in the aromatic assessment of cider.

Key words:
apiculate yeast; biodiversity; fruity aroma; PCR analysis

RESUMO:

Este estudo teve como objetivo conhecer a biodiversidade de leveduras não-Saccharomyces em maçãs com potencial para a melhoria da qualidade aromática da sidra brasileira. As cepas foram isoladas de trinta e cinco (35) mostos de maçã Gala e Fuji de diferentes locais da região Sul do Brasil. Quarenta e cinco (45) cepas foram isoladas e identificadas por análise de PCR. Os resultados indicaram dez (10) espécies: Candida oleophila, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Lodderomyces elongisporus, Pichia anomala, Pichia fermentans e Rhodotorula mucilaginosa. Três desses gêneros (Rhodotorula sp., Lodderomyces sp. e Clavispora sp.) juntos constituíram 71,2% das cepas identificadas. Entre estas cepas, C. oleophila, R. mucilaginosa, P. fermentans, H. uvarum e H. guilliermondii foram selecionadas em teste qualitativo devido a produção de aroma frutado, indicando potencial para a produção de compostos aromáticos na sidra.

Palavras-chave:
levedura apiculada; biodiversidade; aroma frutado; análise por PCR

Cider is a beverage obtained by the total or partial alcoholic fermentation (1.5 to 8.0°GL) of varietal apple musts or blends of table and/or industrial apples. Fermentation can be natural or with inoculum of S. cerevisiae in the apple must (SANTOS et al., 2015SANTOS, C. M. E. et al. Apple aminoacid profile and yeast strains in the formation of fusel alcohols and esters in cider production. Journal of Food Science, v. 80, p.1170-1177, 2015. Available from: https://doi.org/10.1111/1750-3841.12879>. Accessed Mar. 09, 2018. doi: 10.1111/1750-3841.12879.
https://doi.org/10.1111/1750-3841.12879...
). However, in both cases cider-making is not a sterile process. Many other yeast species belonging to several non-Saccharomyces genera occur in apple must and can contribute, at the beginning of fermentation, to the sensory characteristics of cider, mainly with fruity aroma, one of the most important indicators of its quality (VALLES et al., 2007VALLES, B.S. et al. Yeast species associated with the spontaneous fermentation of cider. Food Microbiology , v. 24, p. 25-31, 2007. Available from: <http://dx.doi.org/10.1016/j.fm.2006.04.001>. Accessed: Oct. 23, 2017. doi: 10.1016/j.fm.2006.04.001.; PIETROWSKI et al., 2012PIETROWSKI, G.A.M. et al. Influence of fermentation with Hanseniaspora sp. yeast on the volatile profile of fermented apple. Journal of Agricultural andFood Chemistry , v. 60, p. 9815-21, 2012. Available from: http://dx.doi.org/10.1021/jf302290k>. Accessed: Jun. 28, 2017. doi: 10.1021/jf302290k.
http://dx.doi.org/10.1021/jf302290k...
; COUSIN et al., 2017COUSIN, F.J. et al. Microorganisms in fermented apple beverages: current knowledge and future directions. Microorganisms, v. 5, n. 3, p. 1-22, 2017. Available from: http://dx.doi.org/10.3390/microorganisms5030039>. Accessed: Aug. 15, 2017. doi: 10.3390/microorganisms5030039.
http://dx.doi.org/10.3390/microorganisms...
).

However, in sulphited crushed apple or/and apple must (>50mg SO2.L-1) or apple must inoculated with a high population of Saccharomyces cerevisiae (>107 cfu.mL-1) and high temperatures (>20oC, processing in the southern hemisphere) cider has neutral sensory notes or a slightly yeast aroma (SANTOS et al., 2015SANTOS, C. M. E. et al. Apple aminoacid profile and yeast strains in the formation of fusel alcohols and esters in cider production. Journal of Food Science, v. 80, p.1170-1177, 2015. Available from: https://doi.org/10.1111/1750-3841.12879>. Accessed Mar. 09, 2018. doi: 10.1111/1750-3841.12879.
https://doi.org/10.1111/1750-3841.12879...
). Conversely, apple musts without the addition of sulphite, with a low initial population of Saccharomyces sp. and control of temperature, “fruity” or “floral” notes appear due to the formation of esters and fusel alcohols by non-Saccharomyces strains (NOGUEIRA et al., 2008NOGUEIRA, A. et al. Slow fermentation in French cider processing due to partial biomass reduction. Journal of the Institute of Brewing, v. 114, p. 102-110, 2008. Available from: http://dx.doi.org/10.1002/j.2050-0416.2008.tb00313.x>. Accessed: Sept. 15, 2017. doi: 10.1002/j.2050-0416.2008.tb00313.x.
http://dx.doi.org/10.1002/j.2050-0416.20...
; PIETROWSKI et al., 2012PIETROWSKI, G.A.M. et al. Influence of fermentation with Hanseniaspora sp. yeast on the volatile profile of fermented apple. Journal of Agricultural andFood Chemistry , v. 60, p. 9815-21, 2012. Available from: http://dx.doi.org/10.1021/jf302290k>. Accessed: Jun. 28, 2017. doi: 10.1021/jf302290k.
http://dx.doi.org/10.1021/jf302290k...
).

Studies have shown a growing interest in the industrial application of non-conventional yeasts, due to their ability to positively contribute to the flavour and aroma of wines, ciders, fermented fruit beverages and distillates (PADILLA et al., 2016PADILLA, B. et al. Past and future of non-Saccharomyces yeasts: from spoilage microorganisms to biotechnological tools for improving wine aroma complexity. Frontiers in Microbiology, v.7, p. 1-20 (article 411), 2016. Available from: http://dx.doi.org/10.3389/fmicb.2016.00411>. Accessed: Sept. 15, 2017. doi: 10.3389/fmicb.2016.00411.
http://dx.doi.org/10.3389/fmicb.2016.004...
, HU et al., 2018HU, K. et al. Increase of medium-chain fatty acid ethyl ester content in mixed H. uvarum/S. cerevisiae fermentation leads to wine fruity aroma enhancement. Food Chemistry, v. 239, p. 495-501, 2018. Available from: https://doi.org/10.1016/J.FOODCHEM.2017.06.151>. Accessed: Mar. 08, 2018. doi: 10.1016/J.FOODCHEM.2017.06.151.
https://doi.org/10.1016/J.FOODCHEM.2017....
). Thus, the objective of this study was to know the biodiversity of non-Saccharomyces yeasts in Brazilian apple must and to verify the presence of strains with technological potential for cidermaking.

Isolation of non-Saccharomyces yeasts was performed on freshly extracted apple musts. For the experiments, thirty-five (35) samples of Gala and Fuji apples (5.0kg) were obtained from several producers from southern states of Brazil (Paraná, Santa Catarina and Rio Grande do Sul).

Fruits were selected but they were not washed and sanitized. The apple musts were obtained by milling, which was followed by centrifugation in a juicer (Philips Walita, 700W, Rio de Janeiro, BR). From each apple must, serial dilutions up to 10-4 were prepared in 0.1% peptone water that had been previously sterilized in autoclave (Phoenix, model AV75, 4726 Series, São Paulo) at 121°C for 15min. Dilutions were plated on the surface of YA-LYS (11.75g/L of yeast extract and 20.0g/L of agar-agar from HiMedia Laboratories Pvt. Ltd, India and 2.3g/L of lysine, Biotec, Brazil) and incubated at 25°C for 48h (Quimis oven, model 316B24, Series 6520, Ohio, US) (KURTZMAN et al., 2003KURTZMAN, C.P. et al. Methods to identify yeast. In: BOEKHOUT, T.; ROBERT, V. Yeast in Food: beneficial and detrimental aspects. England: CRC Press & Woodhead Publishing Limited, 2003, p. 69-121.). In order to obtain pure cultures, the colonies were visually differentiated and then inoculated in the same culture medium for purification and isolation. The colonies isolated were grown in rosa bengala chloranphenicol (Merck) medium, which inhibits the growth of bacteria by the presence of 0.1% of the antibiotic chloramphenicol (ZOTT et al., 2008ZOTT, K. et al. Dynamics and diversity of non-Saccharomyces yeasts during the early stages in winemaking. International Journal ofFood Microbiology , v. 125, n. 2, p.197-203, 2008. Available from: http://dx.doi.org/10.1016/j.ijfoodmicro.2008.04.001>. Accessed: Sept. 15, 2017. doi: 10.1016/j.ijfoodmicro.2008.04.001.
http://dx.doi.org/10.1016/j.ijfoodmicro....
). Conservation of the isolated yeasts occurred in inclined tubes with YMA medium (Yeast Malt Agar, HiMedia Laboratories Pvt. Ltd, India) at a temperature of 7-10°C; recovery for selection of the strains occurred in GPYB medium (Merck, Germany) that consists of 40g/L D-glucose, 5g/L yeast extract, 5g/L peptone and 15g/L agar (KURTZMAN et al., 2003).

Total DNA was extracted using the technique described by BRANDÃO et al. (2011BRANDÃO, L.R. et al. Yeasts from an oligotrophic lake in Patagonia (Argentina): diversity, distribution and synthesis of photoprotective compounds and extracellular enzymes. FEMS Microbiology Ecology, v. 76, p. 1-13, 2011. Available from: http://dx.doi.org/10.1111/j.1574-6941.2010.01030.x>. Accessed: Jul. 10, 2017. doi: 10.1111/j.1574-6941.2010.01030.x.
http://dx.doi.org/10.1111/j.1574-6941.20...
). To obtain PCR fingerprinting, was used the Micro/mini satellite-primed PCR (MSP-PCR) fingerprinting method. The PCR reactions were performed according to LIBKIND et al. (2003LIBKIND, D. et al. Molecular characterization of carotenogenic yeasts from aquatic environments in Patagonia, Argentina. Antonie van Leeuwenhoek Journal, v. 84, p. 313-322, 2003.). Isolated yeast with identical patterns of DNA bands were grouped and considered as the same species. Isolates of each molecular group formed by PCR-fingerprinting were identified by sequencing the D1/D2 domains of the largest sub-unit of the rRNA gene. The D1/D2 domains of the largest sub-unit of the rRNA gene were amplified according to the protocol previously described by LACHANCE et al. (1999LACHANCE, M.A. et al. Kodamaea kakaduensis and Candida tolerans, two new ascomycetous yeast species from Australian Hibiscus flowers. Canadian Journal of Microbiology, v. 45, p. 172-177, 1999. Available from <hAvailable from ttp://dx.doi.org/10.1139/cjm-45-2-172 >. Accessed: Oct. 27, 2017. doi: 10.1139/cjm-45-2-172.
ttp://dx.doi.org/10.1139/cjm-45-2-172...
) using the primers NL-1 (5´-GCATATCAATAAGCGGAGGAAAAG-3’) and NL-4 (5´-GGTCCGTGTTTCAAGACGG-3’). Products obtained after the PCR reactions were purified and sequenced using a MegaBacetm automated sequencer (Amersham Biosciences, USA). Sequences obtained were compared with those deposited in the GenBank database using the Basic Local Alignment Search Tool (ALTSCHUL et al., 1997ALTSCHUL, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, v. 25, p. 3389-3402, 1997.).

For the selection of isolated yeast a pasteurized apple must (80-85oC/20min) was processed according to ALBERTI et al. (2016ALBERTI, A. et al. Impact on chemical profile in apple juice and cider made from unripe, ripe and senescent dessert varieties. LWT - Food Science and Technology, v. 65, p. 436-443, 2016. Available from: https://doi.org/10.1016/j.lwt.2015.08.045>. Accessed: Mar. 08, 2018. doi: 10.1016/j.lwt.2015.08.045
https://doi.org/10.1016/j.lwt.2015.08.04...
). A colony of the pure cultures was inoculated in 250mL of apple must and incubated for 72h at 20°C. Turbidity and production of carbon dioxide were evaluated as indicators of fermentative activity. Selection of strains that produced fruity/floral aromas was performed by simple olfactory analysis by trained team (ten cider experts) using the free profile descriptive sensory technique (STONE et al., 2012STONE, H., BLEIBAUM, R., THOMAS, H.A. Sensory Evaluation Practices. 4 ed., USA: Academic press, Elsevier, 4 ed., p. 278-80, 2012.). The evaluated attributes of aroma, after smelling the sample twice, were fruity, floral, yeast, pasteurized apple must (control) and atypical cider aroma. The yeast strains that show fruity and floral aroma were selected.

Identification of isolated yeasts (non-Saccharomyces strains) obtained from thirty-five (35) samples of apple must, can be observed in table 1. 84.9% of the strains were identified by molecular taxonomy, corresponding to forty-five (45) selected strains. Some strains were not identified, due to problems with amplification of the extracted DNA. Identified yeast strains were: thirteen (13) belonged to the genus Rhodotorula sp., 9 to Lodderomyces sp., 10 to Clavispora sp., 5 to Candida sp., 4 to Hanseniaspora sp. and 4 to Pichia sp. (Table 1). Three of these genus (Rhodotorula sp., Lodderomyces sp. and Clavispora sp.) together they constituted 71.2% of the strains identified.

Table 1
Identification and diversity of non-Saccharomyces yeasts isolated and selected of fresh apple must.

Three species of Candida sp. were reported in the present study. Candida tropicalis and Candida oleophila had already been detected in apple juice and cider (COTON et al., 2006COTON, E. et al. Yeast ecology in French cider and black olive natural fermentations. International Journal of Food Microbiology , v. 108, p. 130-135, 2006. Available from: http://dx.doi.org/10.1016/j.ijfoodmicro.2005.10.016>. Accessed: Jul. 10, 2017. doi: 10.1016/j.ijfoodmicro.2005.10.016.
http://dx.doi.org/10.1016/j.ijfoodmicro....
) and the Candida parapsilosis in cider (BEDRIÑANA et al., 2010BEDRIÑANA, R.P. et al. Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias. Food Microbiology, v. 27, p. 503-508, 2010. Available from: http://dx.doi.org/10.1016/j.fm.2009.11.018>. Accessed: Oct. 20, 2017. doi: 10.1016/j.fm.2009.11.018.
http://dx.doi.org/10.1016/j.fm.2009.11.0...
). Clavispora lusitanea and Candida parapsilosis are human pathogens. The Lodderomyces elongisporus strain is associated with bloodstream infections (DÖĞEN et al., 2017DÖĞEN, A. et al. MTL genotypes, phenotypic switching, and susceptibility profiles of Candida parapsilosis species group compared to Lodderomyces elongisporus. Plos ONE, v. 12, n. 8, p. 1-16, 2017. Available from: http://dx.doi.org/10.1371/journal.pone.0182653>. Accessed: Aug. 15, 2017. doi: 10.1371/journal.pone.0182653.
http://dx.doi.org/10.1371/journal.pone.0...
). Thus, the presence of these species may pose a risk to people who consume unpasteurized apple juice.

Hanseniaspora uvarum has been isolated from apple must, cider and wine (COTON et al., 2006COTON, E. et al. Yeast ecology in French cider and black olive natural fermentations. International Journal of Food Microbiology , v. 108, p. 130-135, 2006. Available from: http://dx.doi.org/10.1016/j.ijfoodmicro.2005.10.016>. Accessed: Jul. 10, 2017. doi: 10.1016/j.ijfoodmicro.2005.10.016.
http://dx.doi.org/10.1016/j.ijfoodmicro....
; VALLES et al., 2007VALLES, B.S. et al. Yeast species associated with the spontaneous fermentation of cider. Food Microbiology , v. 24, p. 25-31, 2007. Available from: <http://dx.doi.org/10.1016/j.fm.2006.04.001>. Accessed: Oct. 23, 2017. doi: 10.1016/j.fm.2006.04.001.; BEDRIÑANA et al., 2010BEDRIÑANA, R.P. et al. Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias. Food Microbiology, v. 27, p. 503-508, 2010. Available from: http://dx.doi.org/10.1016/j.fm.2009.11.018>. Accessed: Oct. 20, 2017. doi: 10.1016/j.fm.2009.11.018.
http://dx.doi.org/10.1016/j.fm.2009.11.0...
; VARELA, 2016VARELA, C. The impact of non-Saccharomyces yeasts in the production of alcoholic beverage. Applied Microbiology and Biotechnology, v.100, p. 9861-9874, 2016. Available from: http://dx.doi.org/10.1007/s00253-016-7941-6>. Accessed: Sept. 15, 2017. doi: 10.1007/s00253-016-7941-6.
http://dx.doi.org/10.1007/s00253-016-794...
). In this study, the Hanseniaspora guilliermondii (Kloeckera apiculata or Kloeckera apis) species was isolated from apple must, but it had been previously isolated from grapes, grape must and wine (VARELA, 2016VARELA, C. The impact of non-Saccharomyces yeasts in the production of alcoholic beverage. Applied Microbiology and Biotechnology, v.100, p. 9861-9874, 2016. Available from: http://dx.doi.org/10.1007/s00253-016-7941-6>. Accessed: Sept. 15, 2017. doi: 10.1007/s00253-016-7941-6.
http://dx.doi.org/10.1007/s00253-016-794...
; ÇELIK et al., 2017ÇELIK, Z. D. et al. Molecular characterization and technological properties of wine yeasts isolated during spontaneous fermentation of Vitis vinifera L. cv. ‘Narince’ grape must grow in ancient wine making area Tokat, Anatolia. BIO Web of Conferences, v. 9, p. 1-7, 2017. Available from: https://doi.org/10.1051/bioconf/20170902017>. Accessed: Mar. 08, 2018. doi: 10.1051/bioconf/20170902017.
https://doi.org/10.1051/bioconf/20170902...
). The genus Hanseniaspora sp. has been successfully used to improve the aromatic quality of wine (PADILLA et al., 2016PADILLA, B. et al. Past and future of non-Saccharomyces yeasts: from spoilage microorganisms to biotechnological tools for improving wine aroma complexity. Frontiers in Microbiology, v.7, p. 1-20 (article 411), 2016. Available from: http://dx.doi.org/10.3389/fmicb.2016.00411>. Accessed: Sept. 15, 2017. doi: 10.3389/fmicb.2016.00411.
http://dx.doi.org/10.3389/fmicb.2016.004...
) and cider (PIETROWSKI et al., 2012PIETROWSKI, G.A.M. et al. Influence of fermentation with Hanseniaspora sp. yeast on the volatile profile of fermented apple. Journal of Agricultural andFood Chemistry , v. 60, p. 9815-21, 2012. Available from: http://dx.doi.org/10.1021/jf302290k>. Accessed: Jun. 28, 2017. doi: 10.1021/jf302290k.
http://dx.doi.org/10.1021/jf302290k...
; COUSIN et al., 2017COUSIN, F.J. et al. Microorganisms in fermented apple beverages: current knowledge and future directions. Microorganisms, v. 5, n. 3, p. 1-22, 2017. Available from: http://dx.doi.org/10.3390/microorganisms5030039>. Accessed: Aug. 15, 2017. doi: 10.3390/microorganisms5030039.
http://dx.doi.org/10.3390/microorganisms...
), indicating the importance of this discovery in Brazilian apple must. In addition, H. uvarum can be preserved lyophilized for up to 12 months without losing viability (percentage of living cells) and vitality (fermentative capacity) (PIETROWSKI et al., 2015PIETROWSKI, G. A. M. et al. Viability of Hanseniaspora uvarum yeast preserved by lyophilization and cryopreservation. Yeast, v. 32, p. 559-565, 2015. Available from: https://doi.org/10.1002/yea.3079>. Accessed Mar. 09, 2018. doi: 10.1002/yea.3079.
https://doi.org/10.1002/yea.3079...
).

The yeast strains that not modify the primary aroma of the pasteurized apple must or produced an unpleasant aroma (atypical cider aroma), were discarded. The five (5) non-Saccharomyces and non-pathogenic yeasts (Candida oleophila [No5]; Rhodotorula mucilaginosa [No32]; Pichia fermentans [No39]; Hanseniaspora uvarum [No52] and Hanseniaspora guilliermondii [Nº 53]) were selected in qualitative tests due the fruity or floral aroma production by trained team in the aromatic assessment of cider. Therefore, this study indicated new microorganisms that can be used to improve the aroma quality of cider.

ACKNOWLEDGEMENTS

The authors are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação Araucária (FA), for financial support and scholarships.

REFERENCES:

  • 0
    CR-2017-0886.R1

Publication Dates

  • Publication in this collection
    2018

History

  • Received
    06 Oct 2017
  • Accepted
    01 Apr 2018
  • Reviewed
    26 Apr 2018
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