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Identification and selection of non-Saccharomyces strains isolate from brazilian apple must/Identificacao e selecao de leveduras nao-Saccharomyces isoladas de mostos de macas brasileiras.

Cider is a beverage obtained by the total or partial alcoholic fermentation (1.5 to 8.0[degrees]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., 2015). 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., 2007; PIETROWSKI et al., 2012; COUSIN et al., 2017).

However, in sulphited crushed apple or/ and apple must (>50mg S[O.sub.2].[L.sup.-1]) or apple must inoculated with a high population of Saccharomyces cerevisiae (>[10.sup.7] cfu.m[L.sup.-1]) and high temperatures (>20[degrees]C, processing in the southern hemisphere) cider has neutral sensory notes or a slightly yeast aroma (SANTOS et al., 2015). 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., 2008; PIETROWSKI et al., 2012).

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., 2016, HU et al., 2018). 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 (Parana, 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, Sao Paulo) at 121[degrees]C for 15min. Dilutions were plated on the surface of YALYS (11.75g/L of yeast extract and 20.0g/L of agaragar from HiMedia Laboratories Pvt. Ltd, India and 2.3g/L of lysine, Biotec, Brazil) and incubated at 25[degrees]C for 48h (Quimis oven, model 316B24, Series 6520, Ohio, US) (KURTZMAN et al., 2003). 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., 2008). 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[degrees]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 BRANDAO et al. (2011). 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. (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 subunit 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. (1999) 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 MegaBaceta 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., 1997).

For the selection of isolated yeast a pasteurized apple must (80-85oC/20min) was processed according to ALBERTI et al. (2016). A colony of the pure cultures was inoculated in 250mL of apple must and incubated for 72h at 20[degrees]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., 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.

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., 2006) and the Candida parapsilosis in cider (BEDRINANA et al., 2010). Clavispora lusitanea and Candida parapsilosis are human pathogens. The Lodderomyces elongisporus strain is associated with bloodstream infections (DOGEN et al., 2017). 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., 2006; VALLES et al., 2007; BEDRINANA et al., 2010; VARELA, 2016). 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, 2016; QELIK et al., 2017). The genus Hanseniaspora sp. has been successfully used to improve the aromatic quality of wine (PADILLA et al., 2016) and cider (PIETROWSKI et al., 2012; COUSIN et al., 2017), 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., 2015).

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 [No. 5]; Rhodotorula mucilaginosa [No. 32]; Pichia fermentans [No. 39]; Hanseniaspora uvarum [No. 52] and Hanseniaspora guilliermondii [N[degrees] 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.

http://dx.doi.org/10.1590/0103-8478cr20170886

ACKNOWLEDGEMENTS

The authors are grateful to the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and the Fundaqao Araucaria (FA), for financial support and scholarships.

DECLARATION OF CONFLICTS OF INTEREST

All the co-authors reported that they have no conflict of interest with the publication of this manuscript.

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Received 10.06.17

Approved 04.01.18

Giovana Arruda Moura Pietrowski (1) Juliana Vitoria Messias Bittencourt (1) Luciana Rocha Brandao (2) Carlos Augusto Rosa (2) Aline Alberti (3) Alessandro Nogueira (3)*

(1) Departamento de Alimentos, Universidade Tecnologica Federal do Parana (UTFPR), Ponta Grossa, PR, Brasil.

(2) Departamento de Microbiologia, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil;

(3) Departamento de Engenharia de Alimentos, Universidade Estadual de Ponta Grossa (UEPG), Av. Carlos Cavalcanti, n. 4748, CEP 84030-900, Ponta Grossa, PR, Brasil. E-mail: alessandronog@yahoo.com.br.

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

Code of isolated yeasts      Size of         Name of species
                            fragment (a)

2/5/37                         613         Candida oleophila
45                             570         Candida parapsilosis
46                             570         Candida tropicalis
3/4/6/9/41                     517         Clavispora lusitaniae
10/12                          517         Clavispora lusitaniae
7/11/15                        541         Clavispora lusitaniae
42/43                          578         Hanseniaspora guilliermondii
53                             564         Hanseniaspora guilliermondii
52                             590         Hanseniaspora uvarum
20/38/40/44/4748/49/50/51      588         Lodderomyces elongisporus
1/8                            561         Pichia anomala
                                             (Wickerhanomyces anomalus)
22/39                          581         Pichia fermentans
24                             571         Rhodotorula mucilaginosa
28/29/30/31/33/34              564         Rhodotorula mucilaginosa
23/36                          564         Rhodotorula mucilaginosa
25/26/32                       614         Rhodotorula mucilaginosa
35                             561         Rhodotorula mucilaginosa

Code of isolated yeasts      GenBank    Similarity (b) (%)
                              Number

2/5/37                       EU326130           98
45                           AB617999           95
46                           HM589856           95
3/4/6/9/41                   AB617983          100
10/12                        AB617983           98
7/11/15                      GU460176           99
42/43                        AB618029           99
53                           EU386743           93
52                           GU080043           99
20/38/40/44/4748/49/50/51    HM357469           99
1/8                          HQ199214          100

22/39                        GQ121617           99
24                           EF174506           99
28/29/30/31/33/34            HQ116535           99
23/36                        HQ116535           98
25/26/32                     EU563932           97
35                           HQ199212           99

Note: (a) Value for the number of base pairs per fragment;
(b) Percentage of identical nucleotides in the sequence
obtained from the D1/D2 region of the 26S rRNA gene and
sequence found in Genbank.
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Author:Pietrowski, Giovana Arruda Moura; Bittencourt, Juliana Vitoria Messias; Brandao, Luciana Rocha; Rosa
Publication:Ciencia Rural
Date:May 1, 2018
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