Isolation, identification, and characterization of wild budding yeasts from rose flowers in Fukuyama city, Hiroshima, Japan, and their application in bread and wine production

Abstract

In this study, we isolated 741 wild budding yeast strains from the flowers of 45 rose cultivars growing in Fukuyama city, Hiroshima, Japan. Of these 741 strains, 21 were found to have high fermentation abilities in yeast extract-peptone-dextrose (YPD) medium. Four of the 21 strains were able to ferment bread dough to make bread. These yeasts were identified as Saccharomyces cerevisiae, Lachancea fermentati, Lachancea kluyveri, and a Torulaspora sp. based on DNA sequences from the 26S rDNA D1/D2 regions. The CO₂ production profiles of the bread dough generated by the rose yeasts were evaluated using a Fermograph. Saccharomyces cerevisiae FRY2915 exhibited the highest fermentation capacity. Furthermore, FRY2915 was able to ferment grape juice to produce wine, yielding an alcohol concentration of more than 12%. The four rose yeasts isolated during this study have the potential to produce various types of unique fermented foods, thus enhancing the value of the microbiota associated with rose flowers.

1. Introduction

Yeasts are eukaryotic unicellular microorganisms that have been extensively studied because of their utility for alcoholic fermentation and CO₂ production, biochemical analyses, and biomaterial production, and as genetic model organisms and molecular biology vectors (Botstein & Fink, 2011; Walker & Walker, 2018; Nielsen, 2019). The genome sequence of Saccharomyces cerevisiae, used universally for making bread and alcohol, was the first genome-sequenced eukaryote (Goffeau et al., 1996; Mewes et al., 1997; Wolfe & Shields, 1997). Subsequently, genome sequences from many yeast species have been determined and compared to elucidate the evolutionary mechanisms leading to yeast diversification (Delneri et al., 2003; Wolfe, 2003; Byrne & Wolfe, 2005; Wolfe et al., 2015; Drinnenberg et al., 2019).

Many fermented foods, including bread, wine, and beer, are produced by various yeast species such as Saccharomyces and non-Saccharomyces budding yeasts (Bely, Stoeckle, Masneuf-Pomarède, & Dubourdieu, 2008; Breda, Jolly, & Wyk, 2013; Eldarov, Kishkovskaia, Tanaschuk, & Mardanov, 2016; Eder & Rosa, 2019; Li, Song, Li, Ma, & Cui, 2019; Porter, Divol, & Setati, 2019; Bellut, Krogerus, & Arendt, 2020). Although a postzygotic reproductive isolation mechanism resides among Saccharomyces yeast species based on the criterion of an interspecific F1 block (Toyomura & Hisatomi, 2021), many interspecific hybrids in fermented foods were determined to be domesticated yeasts (González, Barrio, Gafner, & Querol, 2006; Sipiczki, 2008; González, Barrio, & Querol, 2008; Peris, Lopes, Belloch, Querol, & Barrio, 2012; Gallone et al., 2018; Langdon et al., 2019). Characteristics such as fragrance, flavor, and taste in fermented foods are often attributed to the yeasts used in the fermentation process and the starting materials. Thus, the yeasts used in fermentation processes are crucial to confer unique characteristics to the fermented foods.

Since yeasts are heterotrophic microorganisms that require organic compounds as carbon sources, they are frequently found in relatively nutrient-rich environments such as tree sap, decayed leaves, fruit skins, insects, and plant flowers (Phaff, Miller, & Mrak, 1978). Although yeasts associated with plant flowers have been reported (Kevan, Eisikowitch, Fowle, & Thomas, 1988; Herrera, de Vega, Canto, & Pozo, 2009; Herrera, & Pozo, 2010; Herrera, Pozo, & Bazaga, 2011; Pozo & Jacquemyn, 2019; Pozo et al., 2020; Jacquemyn, Pozo, Álvarez-Pérez, Lievens, & Fukami, 2021; Pozo, Mariën, van Kemenade, Wäckers, & Jacquemyn, 2021; Rering, Rudolph, & Beck, 2021), a report detailing yeasts living in rose flowers has not been published. In this study, we isolated wild budding yeasts from rose flowers in Fukuyama city, Hiroshima, Japan, and systematically identified and characterized these rose yeasts in relation to their application in bread and wine production.

Fukuyama city of Hiroshima Prefecture in Japan is known as a rose city where more than one million rose plants are cultivated; in fact, the city holds a large rose festival every year in mid-May. The World Federation of Rose Societies (WFRS) 20th World Rose Convention will also be held in Fukuyama in 2025. Rose flowers, whose colors and fragrances are wonderful and rich in variety are globally loved and have been bred to produce more than 40,000 cultivars. It was expected that various wild yeasts reside in rose flowers because the latter contain nutritious materials such as pollen that allow yeast to grow. In this study, we isolated 741 strains of wild budding yeasts from the flowers of 45 rose cultivars. Four of these strains were found to produce unique fermented foods and were extensively studied from the viewpoint of molecular taxonomy and fermentation abilities to make bread and wine. The rose yeasts isolated in this study would thus be helpful in generating new types of fermented products, contributing further to the image of the elegance of rose flowers.

2. Materials and methods

2.1. Yeast strains

The yeast strains used in this study are listed in Table 1. The rose yeasts identified in this study were deposited at the NITE Biological Resource Center (NBRC, Chiba, Japan) with the assigned NBRC numbers shown in Table 1.

Table 1. Yeast strains used in this study

Yeast strain	Source (Rose cultivar)	Year of isolation	DNA accession numbera	Scientific name	NBRC number	Nickname
FRY994	Aromatherapy	2013	LC617397	Torulaspora sp.	114950	Aroma yeast
FRY1029	Secret Perfume	2013	LC617398	Lachancea fermentati	114951	Perfume yeast
FRY1158	Damascena	2013	LC617399	Lachancea kluyveri	114952	Damask yeast
FRY2915	Mister Lincoln	2015	LC617400	Saccharomyces cerevisiae	114953	Lincoln yeast
Strain Ab	Baker's yeast			Saccharomyces cerevisiae
Strain Bb	Baker's yeast			Saccharomyces cerevisiae
OC-2c	Wine yeast			Saccharomyces cerevisiae
Strain Cc	Wine yeast			Saccharomyces cerevisiae
DBVPG 6173d	Brewer's yeast			Saccharomyces cerevisiae	10217

^aDNA sequence from D1/D2 region of 26S rDNA.

^bControl strain from commercial baker's yeast.

^cControl strain from commercial wine yeast.

^dNeotype strain of Saccharomyces cerevisiae.

2.2. Isolation of wild budding yeasts from rose flowers

The roses were cultivated at the horticulture center of Fukuyama city, Hiroshima, Japan. Rose flowers were collected at the end of the blossoming period to allow full development of the yeast in the flowers. Pesticides were not applied from the second week before rose blooming to the time the flowers were collected. Single large-sized flowers and several small-sized flowers were collected for each rose cultivar, preventing microbial contamination from outside the flowers. The rose flowers were then immersed in 250 mL of yeast extract-peptone-dextrose (YPD) liquid medium in a 500-mL Erlenmeyer flask, separately per cultivar. Yeast enrichment cultures were achieved by incubation at room temperature for 1 wk. Aliquots from the above cultures were spread onto YPD solid medium in 90-mm Petri dishes and incubated at 26 °C for 2 d to allow the yeast to form colonies. Yeast-like colonies exhibiting creamy, glossy, and pearlescent characteristics were picked and microscopically examined to distinguish budding yeasts from other types of microorganisms. The wild budding yeasts were qualified as rose yeasts and were stored in 25% aqueous glycerol at −80 °C.

2.3. Examination of fermentation abilities via the Durham method

The fermentation capacities of the strains were measured using the standard Durham tube test (van Dijken, van den Bosch, Hermans, de Miranda, & Scheffers, 1986). The yeast cells were cultured overnight in YPD liquid medium. For each strain, a 1-mL yeast culture was added to 7 mL YPD medium in a 10-mL Spitz tube containing a Durham test tube filled with YPD medium. The samples were incubated at 26 °C for 6 h to examine the generation of CO₂ in the Durham test tube. The strains whose fermentation was confirmed by the Durham method were tested in the bread production experiments described in section 2.4.

2.4. Making bread using a domestic bread-making machine

Five grams of wet yeast was collected by centrifugation from YPD cultures that had been incubated at 26 °C over two nights. The cells were mixed with 190 mL tepid water, 280 g hard flour, 20 g sucrose, and 4 g salt. The samples were then placed in a domestic bread-making machine (HB-100, MK Seiko, Nagano, Japan). Nine minutes after start of the fast-baking mode, 20 g of butter was added. After a 3-h period of kneading, first fermentation, second fermentation, and venting, the stirring rod was removed. The dough was appropriately shaped and fermented for the final time and baked. These procedures were performed according to the manufacturer's instructions.

2.5. Identification of yeast species based on data from D1/D2 regions of 26S rDNA

The D1/D2 regions of 26S rDNA were PCR-amplified using NL-1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL-4 (5′-GGTCCGTGTTTCAAGACGG-3′) primers with EX-Taq Hot Start DNA polymerase (TaKaRa Bio, Shiga, Japan), using a previously reported PCR method for yeast colonies (Dunham, Gartenberg, & Brown, 2015). The PCR conditions were as follows: 94 °C for 3 min (hot start), 94 °C for 20 s (denaturing), 58 °C for 20 s (annealing), 72 °C for 30 s (extension), and 72 °C for 5 min 40 s (final treatment). There were 30 cycles of denaturing, annealing, and extension. The PCR products were purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). DNA sequencing of the PCR products was conducted using the NL-1 and NL-4 primers with the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Massachusetts, USA) on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). DNA sequencing was performed according to the supplier's standard protocol. The sequence data were processed using SnapGene software (GSL Biotech LLC, California, USA). The DNA sequences were deposited in the DDBJ/EMBL/GenBank databases with the DNA accession numbers assigned as indicated in Table 1. The yeast species names were inferred by subjecting the DNA sequence data to BLASTn searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) (Table 1).

2.6. Microscopic observation of yeast cells

Yeast cells suspended in distilled water were applied to glass slides and covered with coverslips. The preparations were observed under a bright-field microscope with a ×100 oil immersion objective lens and photographed using a CCD camera with a micrometer calibrated to the same view. The yeast cells were classified into three size categories: small, approximately 3 µm in diameter; medium, approximately 5 µm in diameter; and large, approximately 7.5 µm in diameter.

2.7. Electrophoretic karyotyping of yeast strains with a contour-clamped homogeneous electric field (CHEF) apparatus

The strains were electrophoretically karyotyped using a pulsed-field gel electrophoresis technique with the CHEF-DRII system (Bio-Rad Laboratories, California, USA) (Sugihara, Hisatomi, Kodama, & Tsuboi, 2011).

2.8. Fermentation profiles of bread dough by rose yeasts using Fermograph equipment

Aliquots (0.25 g) of wet yeast that had been cultured in YPD media at 26 °C over two nights were collected by centrifugation and mixed with 9.5 mL water, 14 g hard flour, 0.5 g sucrose, and 0.2 g salt. The dough mixtures were placed in 200-mL glass vials and CO₂ production was measured with a Fermograph II-W (ATTO, Tokyo, Japan) at 37 °C, measuring the generation of CO₂ every 5 min for 24 h. Fermentation profiles were analyzed twice by a computer and processed to visualize the data averages.

2.9. Fermentation profiles of grape juice by rose yeasts using Fermograph equipment

Sucrose (10.7 g) and 0.14-g tartaric acid were added to 100 mL of 100% grape juice (Welch's Grape 100, Asahi Soft Drinks, Japan) to adjust the final sugar concentration and pH to 23% and 3.3, respectively. The samples were poured into 200-mL glass vials. Total sulfurous acid was adjusted to 150 mg/L with potassium disulfite and the samples were kept at room temperature for 24 h to stabilize them. Then, 3.3 mL of yeast culture grown in YPD medium at 26 °C over two nights was added to each sample and then evaluated with the Fermograph II-W at 26 °C, measuring the generation of CO₂ every 5 min for 10 d. Fermentation profiles were analyzed twice by a computer and processed to visualize the data averages.

2.10. Analyses of alcohol concentrations in wine

Wine samples (75 mL) that had been fermented in the Fermograph for 10 d were poured into round bottom flasks, and 25 mL of distilled water was added. The wine samples (100 mL) were heated and left to evaporate until the volume was reduced to 70 mL. Thereafter, another 5 mL of distilled water was added (final volume of 75 mL). Then, 15 mL was sampled and analyzed using a mechanical oscillator densitometer for alcohol concentration (DA-155; Kyoto Electronics, Kyoto, Japan) to measure the concentrations of alcohol. Measurements were performed twice and averaged.

3. Results

3.1. Isolation and identification of wild budding yeasts from rose flowers

A total of 741 strains of wild budding yeasts were obtained from 45 rose cultivars that represented a wide range of flower colors (Table 2). A total of 21 strains of rose yeasts were determined to have high fermentation abilities through a standard Durham method using YPD medium (Table 2). Out of the 21 strains, four were found to effectively ferment dough to make satisfactory bread using a domestic bread-making machine (Table 2 and Fig. 1). DNA sequences of the D1/D2 regions of the 26S rDNA of these rose yeasts were determined and submitted to the BLASTn search engine to identify the yeast species (Table 1). FRY1029, FRY1158, and FRY2915 derived from the Secret Perfume, Damascena, and Mister Lincoln cultivars were Lachancea fermentati, L. kluyveri, and Saccharomyces cerevisiae, respectively. Although FRY994 derived from the Aromatherapy cultivar was inferred to be Torulaspora delbrueckii, six of the 589 nucleotides were different between FRY994 and the type strain of T. delbrueckii (CBS 1146). Thus, FRY994 was temporarily designated as a Torulaspora sp. These strains were deposited in the NBRC (NITE, Chiba, Japan), accompanied by NBRC numbers (Table 1). Because these rose yeasts will be commercially available for fermenting foods, we have given these strains nicknames associated with the rose names from which the corresponding yeasts were isolated (Table 1).

Table 2. Isolation and characterization of wild budding yeasts from rose flowers cultivated in Fukuyama city, Hiroshima, Japan

Name of rose cultivara	Color of rose flowers	Number of yeasts isolated	Number of fermenting yeastsb	Number of bread-making yeastsc	Yeast code
Aromatherapy	Pink	20	5	1	FRY994
Ayumi	Yellow	8	0	-
Beautiful Fukuyama	Red	26	1	0
Charmy Fukuyama	Red	2	0	-
Damascena	Pink	40	1	1	FRY1158
Dog Rose	White	12	0	-
Duftgold	Yellow	15	2	0
Elle	Pink	38	2	0
Fen Zhang Lou	White	14	0	-
Fragrant Apricot	Orange	20	0	-
French Perfume	Pink	10	0	-
Fukuyama Castle	Purple	21	0	-
Gekko	Yellow	8	0	-
Goldmarie	Yellow	14	0	-
Harmonie	Orange	13	0	-
Herz Ass Climbing	Red	25	0	-
Honoka	Pink	6	0	-
Houjun	Pink	18	0	-
Jasmina	Pink	11	0	-
Koki	Pink	29	0	-
Lemon & Ginger	Yellow	9	0	-
Lovely Fukuyama	Red	12	0	-
Masayuki	White	29	0	-
Mister Lincoln	Red	9	1	1	FRY2915
Miwaku	Pink	17	1	0
Momoka	Pink	18	2	0
Oklahoma	Red	4	0	-
Parole	Red	3	0	-
Perennial Blush	White	16	0	-
Pope John Paul Ⅱ	White	13	1	0
Prestige de Lyon	Pink	37	0	-
Princess Fukuyama	Yellow	14	2	0
Rakuen	Orange	29	0	-
Rose Fukuyama	Pink	18	0	-
Secret Perfume	Pink	25	3	1	FRY1029
Shunpoh	Pink	23	0	-
Smile Fukuyama	Pink	3	0	-
Souvenir d'Anne Frank	Orange	17	0	-
Speelwark	Yellow	2	0	-
Stephanie de Monaco	Pink	3	0	-
Stroberry Daikiri	Pink	19	0	-
Sweet Melina	Pink	4	0	-
White Christmas	White	33	0	-
Yua	Pink	32	0	-
Yumeka	Pink	2	0	-
Total	45	741	21	4

^aRose cultivars are listed alphabetically.

^bFermentation abilities were judged after incubation for 6 h at 26 °C in YPD medium by means of Durham method.

^cBread-making abilities were judged by means of a domestic bread-making machine.

Fig. 1. Morphology of yeasts isolated from rose flowers and characteristics of breads made by rose yeasts. Photographs in the upper row represent rose flowers from which yeasts were isolated. Micrographs in the middle row represent yeast cells derived from the above rose flowers. Photographs in the lower row represent characteristics of breads made by the above rose yeasts.

3.2. Morphology of the four rose yeasts exhibiting the ability to make bread

The characteristics of the four rose yeasts that were able to produce bread from dough are summarized in Figure 1. The cells of strain FRY994 isolated from one of the pink rose cultivars, Aromatherapy, were medium in size and produced round-headed bread. The cells of strain FRY1029 isolated from one of the pink rose cultivars, Secret Perfume, were small in size and produced flat-headed bread. The cells of strain FRY1158 isolated from one of the pink rose cultivars, Damascena, were large in size and produced slightly burned bread. The cells of strain FRY2915 isolated from one of the red rose cultivars, Mister Lincoln, were large in size and produced exceedingly expanded bread. The odors and tastes of these breads varied, reflecting the distinct characteristics of the yeast used.

3.3. Electrophoretic karyotyping of rose yeasts and related yeast strains

Electrophoretic karyotypes of the four strains of rose yeast, commercial yeast strains for fermented foods, and the taxonomical neotype strain of S. cerevisiae were analyzed using a CHEF apparatus. Because FRY2915, strain A, strain B, OC-2, strain C, and DBVPG 6173 are all S. cerevisiae strains, their banding patterns were similar (Table 1 and Fig. 2). FRY994, FRY1029, and FRY1158 possessed a small number of relatively large chromosomes (Table 1 and Fig. 2). Notably, the strains of S. cerevisiae can be distinguished when their band patterns are compared in detail.

Fig. 2. Electrophoretic karyotypes of rose yeasts, commercial strains, and the neotype strain of Saccharomyces cerevisiae. Chromosome-sized DNA was separated at 14 °C using a CHEF apparatus with 60 s pulses for 22 h and 120 s pulses for 10 h at 200 V.“Marker”indicates the S. cerevisiae size marker (Bio-Rad Laboratories, California, USA). Yeast strains used in the CHEF analysis are listed in Table 1.

3.4. Bread making: fermentation profiles of bread dough by rose yeasts and related yeast strains

We examined the dough fermentation profiles of the four strains of rose yeast, commercial baker's yeast, and the taxonomical neotype strain of S. cerevisiae using a Fermograph. Strains A and B from commercial baker's yeast were confirmed to have higher fermentation abilities than FRY2915, FRY1158, FRY1029, FRY994, and DBVPG 6173 (Table 1 and Fig. 3). Because the four strains of rose yeast have the ability to ferment dough to make bread satisfactorily with bread-making techniques (Fig. 1), their capacity to ferment dough could be elevated to the level of strains A and B to obtain 2-deoxyglucose-resistant mutants that assimilate and ferment maltose in flour more effectively (Orikasa, Mikumo, & Ohwada, 2018).

Fig. 3. Fermentation of bread dough by rose yeasts and control yeast strains. In total, 24.5 g of bread dough was fermented by each yeast strain at 37 °C with the accumulation of CO₂ monitored using a Fermograph. Yeasts used in this analysis are listed in Table 1.

3.5. Winemaking: fermentation profiles of grape juice by rose yeasts and related yeast strains

We also examined whether the four rose yeasts are useful for winemaking. Thus, 100% grape juice supplemented with sucrose (23% of total sugar concentration) was fermented by the four strains of rose yeast, commercial wine yeasts, and the neotype strain of S. cerevisiae. CO₂ output was monitored with a Fermograph. Under 150 mg/L total sulfurous acid, strain FRY2915 exhibited high fermentation ability at almost the same level as the commercial wine yeasts, OC-2, and strain C, with other three rose yeast strains showing lower fermentation abilities (Table 1 and Fig. 4). As expected from the CO₂ output profiles (Fig. 4), the alcohol concentration produced by strain FRY2915 was almost at the same as that of the commercial wine yeasts, OC-2, and strain C (Fig. 5), suggesting that the sugar in the grape juice samples was completely converted to ethanol and CO₂ in each batch. Strain FRY2915, in particular, was shown to be highly tolerant to sulfurous acid, promoting efficient ethanol fermentation (Figs. 4, 5). Wine made by strain FRY2915 was distinct in terms of aroma and taste, indicating the unique wine characteristics possible with rose yeasts.

Fig. 4. Fermentation of grape juice by rose yeasts and control yeast strains. Grape juice (100 mL) supplemented with sucrose (23% sugar concentration) was fermented by yeasts at 26 °C with the accumulation of CO₂ monitored using a Fermograph. Total sulfurous acid added in each batch was 150 mg/L. Yeasts used in this analysis are listed in Table 1.

Fig. 5. Alcohol production in winemaking by rose yeasts and control yeast strains. Alcohol concentrations were measured according to a conventional method. Yeasts used in this analysis are listed in Table 1.

4. Discussion

Yeasts have long been known to be associated with wild and cultivated flowers and floral nectar (Phaff el al., 1978; Kevan et al., 1988; Herrera et al., 2009; Herrera, & Pozo, 2010; Herrera et al., 2011; Pozo & Jacquemyn, 2019; Pozo et al., 2020; Jacquemyn et al., 2021; Pozo et al., 2021; Rering et al., 2021), yet they have not been intensively studied for commercial applications. Thus, in this study, we isolated 741 strains of wild budding yeasts from 45 rose cultivars cultured in Fukuyama city, Hiroshima, Japan. This success could be attributed to the characteristics of rose flowers, as they give off fragrance to attract various insects and bloom for longer periods compared to other flowers, allowing yeast strains to proliferate efficiently. We suggest that insects such as small scarab beetles, bees, and fruit flies prefer to eat the pollen in rose flowers, and yeasts carried by the insects proliferate as they can obtain more nutrients from the pollen. It is plausible that most yeast species are associated with the particular pollen contained in rose flowers of each cultivars because the demand for nutrients obtained from pollen is different among yeast species.

Of the 741 rose yeast strains examined by Durham fermentation tests, 21 strains exhibited high fermentation abilities after incubation for 6 h at 26 °C in YPD medium. Moreover, four of these 21 strains were able to produce acceptable bread using a domestic bread-making machine. Various wild yeasts have been reported to produce fermented foods such as bread, wine, and beer (Bely et al, 2008; Breda et al, 2013; Eldarov et al., 2016; Eder & Rosa, 2019; Li et al., 2019; Porter et al., 2019; Bellut et al., 2020). Therefore, the rose yeasts isolated in this study have the potential to be useful in making unique fermented foods. The rose yeasts that were used to produce bread were also utilized to produce wines with distinct characteristics, which further suggests that rose yeasts can produce various types of unique fermented foods.

Rose flowers are renowned for their elegant image and esthetics, and the originality of the rose yeasts isolated in this study could potentially have worldwide appeal. Various types of unique fermented foods could then be produced under a material transfer agreement (MTA), and the pedigree of the rose yeasts could be verified through molecular identification using karyotyping, which could distinguish the rose yeasts from other yeast stains. The rose yeasts could be bred to ferment target materials more efficiently through selection under various assimilation and fermentation conditions (Orikasa et al, 2018). Further study of the life cycles, breeding systems, and whole-genome sequences of these rose yeasts will be necessary to obtain sufficient data for effective breeding.

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country in which they were performed.

Acknowledgments

The authors are grateful to Takuya Hanaoka, Chisa Sugihara, Yuuki Kamegawa, Satomi Oohara, Mizuki Karakawa, Kouhei Kiso, Ryotaro Hara, Ryutaro Sugihara, and Ren Watanabe for their helpful assistance. We are also thankful to Motoharu Nakashima, Minoru Yukiyasu, and Takamasa Tsuchida for their encouragement. We thank Dr. Takayuki Yoshizaki for his kind guidance during winemaking. We greatly appreciate support from the horticulture center (Fukuyama city, Hiroshima, Japan) to collect rose flowers used in this study. Lastly, we are deeply grateful for many valuable suggestions of the Reviewers to improve our manuscript. This work was supported by the Takaki Shunsuke Foundation for Science and Technology of Bread (2017).

References

• Bellut, K., Krogerus, K., & Arendt, E.K. (2020). Lachancea fermentati strains isolated from Kombucha: Fundamental insights, and practical application in low alcohol beer brewing. Frontiers in Microbiology, 11, 764. https://doi.org/10.3389/fmicb.2020.00764
• Bely, M., Stoeckle, P., Masneuf-Pomarède, I., & Dubourdieu, D. (2008). Impact of mixed Torulaspora delbrueckii-Saccharomyces cerevisiae culture on high-sugar fermentation. International Journal of Food Microbiology, 122, 312-320. https://doi.org/10.1016/j.ijfoodmicro.2007.12.023
• Botstein, D., & Fink, G.R. (2011). Yeast: An experimental organism for 21st century biology. Genetics, 189, 695-704. https://doi.org/10.1534/genetics.111.130765
• Breda, V., Jolly, N., & Wyk, J. (2013). Characterisation of commercial and natural Torulaspora delbrueckii wine yeast strains. International Journal of Food Microbiology, 163, 80-88. https://doi.org/10.1016/j.ijfoodmicro.2013.02.011
• Byrne, K.P., & Wolfe, K.H. (2005). The yeast gene order browser: combining curated homology and syntenic context reveals gene fate in polyploid species. Genome Research, 15, 1456-1461. https://doi.org/10.1101/gr.3672305
• Delneri, D., Colson, I., Grammenoudi, S., Roberts, I.N., Louis, E.J., & Oliver, S.G. (2003). Engineering evolution to study speciation in yeasts. Nature, 422, 68-72. https://doi.org/10.1038/nature01418
• Drinnenberg, I.A., Berger, F., Elsässer, S.J., Andersen, P.R., Ausió, J., Bickmore, W.A., Blackwell, A.R., Erwin, D.H., Gahan, J.M., Gaut, B.S., Harvey, Z.H., Henikoff, S., Kao, J.Y., Kurdistani, S.K., Lemos, B., Levine, M.T., Luger, K., Malik, H.S., Martín-Durán. J.M., ... Wolfe, K.H. (2019). EvoChromo: Towards a synthesis of chromatin biology and evolution. Development, 146, dev178962. https://doi.org/10.1242/dev.178962
• Dunham, M.J., Gartenberg, M.R., & Brown, G.W. (2015). Methods in yeast genetics and genomics: A Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press.
• Eder, M.L.R., & Rosa, A.L. (2019). Yeast diversity in Vitis non-vinifera ecosystems. Revista Argentina de Microbiología, 51, 278-283. https://doi.org/10.1016/j.ram.2018.09.004
• Eldarov, M.A., Kishkovskaia, S.A., Tanaschuk, T.N., & Mardanov, A.V. (2016). Genomics and biochemistry of Saccharomyces cerevisiae wine yeast strains. Biochemistry (Mosc), 81, 1650-1668. https://doi.org/10.1134/S0006297916130046
• Gallone, B., Mertens, S., Gordon, J.L., Maere, S., Verstrepen, K.J., & Steensels, J. (2018). Origins, evolution, domestication and diversity of Saccharomyces beer yeasts. Current Opinion in Biotechnology, 49, 148-155. https://doi.org/10.1016/j.copbio.2017.08.005
• Goffeau, A., Barrell, B.G., Bussey, H., Davis, R.W., Dujon, B., Feldmann, H., Galibert, F., Hoheisel, J.D., Jacq, C., Johnston, M., Louis, E.J., Mewes, H.W., Murakami, Y., Philippsen, P., Tettelin, H., & Oliver, S.G. (1996). Life with 6000 genes. Science, 274, 563-567. https://doi.org/10.1126/science.274.5287.546
• González, S.S., Barrio, E., Gafner, J., & Querol, A. (2006). Natural hybrids from Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations. FEMS Yeast Research, 6, 1221-1234. https://doi.org/10.1111/j.1567-1364.2006.00126.x
• González, S.S., Barrio, E., & Querol, A. (2008). Molecular characterization of new natural hybrids of Saccharomyces cerevisiae and S. kudriavzevii in brewing. Applied and Environmental Microbiology, 74, 2314-2320. https://doi.org/10.1128/AEM.01867-07
• Herrera, C.M., de Vega, C., Canto, A., & Pozo, M.I. (2009). Yeasts in floral nectar: a quantitative survey. Annals of Botany, 103, 1415-1423. https://doi.org/10.1093/aob/mcp026
• Herrera, C.M., & Pozo, M.I. (2010). Nectar yeasts warm the flowers of a winter-blooming plant. Proceedings of the Royal Society, 277, 1827-1834. https://doi.org/10.1098/rspb.2009.2252
• Herrera, C.M., Pozo, M.I., & Bazaga, P. (2011). Clonality, genetic diversity and support for the diversifying selection hypothesis in natural populations of a flower-living yeast. Molecular Ecology, 20, 4395-4407. https://doi.org/10.1111/j.1365-294X.2011.05217.x
• Jacquemyn, H., Pozo, M.I., Álvarez-Pérez, S., Lievens, B., & Fukami, T. (2021). Yeast-nectar interactions: metacommunities and effects on pollinators. Current Opinion in Insect Science, 44, 35-40. https://doi.org/10.1016/j.cois.2020.09.014
• Kevan, P.G., Eisikowitch, D., Fowle, S., & Thomas, K. (1988). Yeast-contaminated nectar and its effects on bee foraging. Journal of Apicultural Research, 27, 26-29. https://doi.org/10.1080/00218839.1988.11100777
• Langdon, Q.K., Peris, D., Baker, E.C.P., Opulente, D.A., Nguyen, H-V., Bond, U., Gonçalves, P., Sampaio, J.P., Libkind, D., & Hittinger, C.T. (2019). Fermentation innovation through complex hybridization of wild and domesticated yeasts. Nature Ecology & Evolution, 3, 1576-1586. https://doi.org/10.1038/s41559-019-0998-8
• Li, Z., Song, K., Li, H., Ma, R., & Cui, M. (2019). Effect of mixed Saccharomyces cerevisiae Y10 and Torulaspora delbrueckii Y22 on dough fermentation for steamed bread making. International Journal of Food Microbiology, 303, 58-64. https://doi.org/10.1016/j.ijfoodmicro.2019.05.009
• Mewes, H.W., Albermann, K., Bähr, M., Frishman, D., Gleissner, A., Hani, J., Heumann, K., Kleine, K., Maierl, A., Oliver, S.G., Pfeiffer, F., & Zollner, A. (1997). Overview of the yeast genome. Nature, 387, 7-65. https://doi.org/10.1038/42755
• Nielsen, J. (2019). Yeast systems biology: model organism and cell factory. Biotechnology Journal, 14, e1800421. https://doi.org/10.1002/biot.201800421
• Orikasa, Y., Mikumo, D., & Ohwada, T. (2018). A 2-Deoxyglucose-resistant mutant of Saccharomyces cerevisiae shows enhanced maltose fermentative ability by the activation of MAL genes. Foods, 7, 52-61. https://doi.org/10.3390/foods7040052
• Peris, D., Lopes, C.A., Belloch, C., Querol, A., & Barrio. E. (2012). Comparative genomics among Saccharomyces cerevisiae × Saccharomyces kudriavzevii natural hybrid strains isolated from wine and beer reveals different origins. BMC Genomics, 13, 407. https://doi.org/10.1186/1471-2164-13-407
• Phaff, H.J., Miller, M.W., & Mrak, E.M. (1978). The life of yeasts. 2nd ed. Revised and enlarged. Harvard University Press.
• Porter, T.J., Divol, B., & Setati, M.E. (2019). Lachancea yeast species: Origin, biochemical characteristics and oenological significance. Food Research International, 119, 378-389. https://doi.org/10.1016/j.foodres.2019.02.003
• Pozo, M.I., & Jacquemyn, H. (2019). Addition of pollen increases growth of nectar-living yeasts. FEMS Microbiology Letters, 366, fnz191. https://doi.org/10.1093/femsle/fnz191
• Pozo, M.I., Mariën, T., van Kemenade, G., Wäckers, F., & Jacquemyn, H. (2021). Effects of pollen and nectar inoculation by yeasts, bacteria or both on bumblebee colony development. Oecologia, 195, 689-703. https://doi.org/10.1007/s00442-021-04872-4
• Pozo, M.I., van Kemenade, G., van Oystaeyen, A., Aledón-Catalá, T., Benavente, A., Van den Ende, W., Wäckers, F., & Jacquemyn, H. (2020). The impact of yeast presence in nectar on bumble bee behavior and fitness. Ecological Monographs, 90, e01393. https://doi.org/10.1002/ecm.1393
• Rering, C.C., Rudolph, A.B., & Beck, J.J. (2021). Pollen and yeast change nectar aroma and nutritional content alone and together, but honey bee foraging reflects only the avoidance of yeast. Environmental Microbiology, 23, 4141-4150. https://doi.org/10.1111/1462-2920.15528
• Sipiczki, M. (2008). Interspecies hybridization and recombination in Saccharomyces wine yeasts. FEMS Yeast Research, 8, 996-1007. https://doi.org/10.1111/j.1567-1364.2008.00369.x
• Sugihara, C., Hisatomi, T., Kodama, T., & Tsuboi, M. (2011). The GAL10 gene is located 40 kbp away from the GAL7-GAL1 region in the yeast Kazachstania naganishii. Current Microbiology, 63, 366-371. https://doi.org/10.1007/s00284-011-9988-z
• Toyomura, K., & Hisatomi, T. (2021). Postzygotic reproductive isolation among three Saccharomyces yeast species. Yeast, 38, 326-335. https://doi.org/10.1002/yea.3549
• van Dijken, J.P., van den Bosch, E., Hermans, J.J., de Miranda, L.R., & Scheffers, W.A. (1986). Alcoholic fermentation by‘non-fermentative’yeasts. Yeast, 2, 123-127. https://doi.org/10.1002/yea.320020208
• Walker, G.M., & Walker, R.S.K. (2018). Enhancing yeast alcoholic fermentations. Advances in Applied Microbiology, 105. 87-129. https://doi.org/10.1016/bs.aambs.2018.05.003
• Wolfe, K. (2003). Evolutionary biology: Speciation reversal. Nature, 422, 25-26. https://doi.org/10.1038/422025a
• Wolfe, K.H., Armisén, D., Proux-Wera, E., ÓhÉigeartaigh, S.S., Azam, H., Gordon, J.L., & Byrne, K.P. (2015). Clade- and species-specific features of genome evolution in the Saccharomycetaceae. FEMS Yeast Research, 15, fov035. https://doi.org/10.1093/femsyr/fov035
• Wolfe, K.H., & Shields, D.C. (1997). Molecular evidence for an ancient duplication of the entire yeast genome. Nature, 387, 708-713. https://doi.org/10.1038/42711