Characterization of the Yeast Hanseniaspora vineae Isolated from the Wine Grape ‘Yamasachi’ and Its Use for Bread Making

Abstract

Fermentative yeast strain TW15 was isolated from the grape cultivar Yamasachi. It was identified as Hanseniaspora vineae based on the ribosomal D1/D2 domain, but the sequence of its 18S-26S rDNA spacer region was not identical to those retrieved from the H. vineae database. TW15 showed higher fermentation ability of medium containing a fructose and glucose mixture (55:45) compared with five other H. vineae strains and four Saccharomyces cerevisiae baking strains. The baking tests revealed that TW15 produced greater volume in baked products than did the baking strain. The preliminary sensory evaluation showed similarities between the two baked products, but the product of TW15 had a more distinct and desirable flavor. Compared with bread samples produced with the baking strain, the amount of acetoin was much higher in bread samples made with TW15. H. vineae TW15 is thus suitable for production of baked goods with palatable quality.

Introduction

The quality of alcoholic beverages is usually affected by the raw materials, fermentation conditions, and yeast strain used in their production. This phenomenon is due to the fact that yeast cells synthesize various metabolites including alcohols, organic acids, and esters through the conversion of sugars to ethanol. During the manufacturing processes, pure yeast cultures from the genus Saccharomyces are inoculated to stabilize the quality of the products. At least eight species have been included in the genus Saccharomyces (Alsammar et al., 2019), among which the strains of S. cerevisiae are used extensively in fermentation industries of beverages and foods (Hittinger et al., 2018). In the natural winemaking process, which does not utilize sterilization, S. cerevisiae, characterized by high ethanol productivity and tolerance to external ethanol, dominates in the later stage of fermentation, while various yeasts other than S. cerevisiae grow at early stages of fermentation (Raymond Eder et al., 2017). Simultaneous inoculation with these non-Saccharomyces yeasts and S. cerevisiae alters the amount of volatile compounds, contributing to diversification and improvement of final products (Liu et al., 2019; Morales et al., 2019; Zhang et al., 2018a).

The principal role of yeast in bread making is to leaven the dough by releasing CO₂ gas through fermentation. The yeast strains used in bread making are exclusively strains of S. cerevisiae, and sugar metabolism happening during this process follows the fundamental pathway occurring in alcoholic beverages. However, the use of only S. cerevisiae strains limits the flavor of baked products.

During experiments conducted to explore alternative baking yeast strains from natural resources, we identified a yeast species, Hanseniaspora vineae, in wine grapes that could potentially be used in bread making. The present paper reports on species identification of the isolated strain and its use for bread making.

Materials and Methods

Yeast strain isolation    The wine grape Yamasachi is a cryotolerant cultivar that was constructed by crossing the cultivar Kiyomi, selected for vinification, with a wild grape at the Ikeda-cho Grape and Wine Research Institute, Japan. Yamasachi grapes were collected from a vineyard of the Institute in October 2016. To isolate yeast strains from the grapes, three samples were prepared, each containing 7.0 mL of squeezed grape juice mixed with 1.0 mL of 10.0% maltose, 1.0 mL of 2.0% sodium propionate, and 1.0 mL of ethanol containing 1.0 mg chloramphenicol. Each mixture was incubated at 30 °C for 3 days and used as the first enrichment culture. A 0.05-mL volume of this broth was transferred to a medium composed of 20% sucrose, 1.0% yeast extract (Nakalai Tesque Inc., Kyoto, Japan), 2.0% polypeptone (Nippon Seiyaku Co., Tokyo, Japan), 0.2% sodium propionate, and 0.01% chloramphenicol; this was used as the second enrichment culture medium. During incubation for 6 days, film appeared on the surface of two cultures, and the broth of a culture which formed no visible film was streaked on yeast-extract peptone dextrose agar containing 1.0% yeast extract, 2.0% polypeptone, 2.0% glucose, and 2.0% agar. Finally, a large colony was isolated as strain TW15 and deposited as NITE P-02881 in the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary.

Species identification    Physiological properties of the isolated yeast and analysis of the ribosomal D1/D2 domain and 18S-26S rDNA spacer region were conducted by methods described elsewhere (Kurtzman et al., 2011; Oda et al., 2010). The 18S-26S sequence was deposited under DDBJ/EMBL/GenBank Accession Number LC474406.

Yeast strains    Seven strains of Hanseniaspora vineae (NBRC 1412, NBRC 1415^T, NBRC 1416, NBRC 1753, NBRC 1754, NBRC 10226, NBRC 100790) and three baking strains of Saccharomyces cerevisiae (NBRC 2043, NBRC 2044, NBRC 2375) were obtained from the NITE Biological Resource Center. S. cerevisiae strain HP467 was isolated from the commercial compressed yeasts (Oda et al., 2010).

Culture    Yeast cells were grown aerobically in 10.0 mL of seed medium containing 1.0% yeast extract, 2.0% polypeptone, and 2.0% glucose, and 0.6 mL of this seed medium was inoculated onto 60 mL of a production medium containing 1.0% Bacto-yeast extract (Difco), 2.0% Bacto-peptone (Difco), 2.0% glucose, 0.2% KH₂PO₄, 0.1% MgSO₄·7H₂O, and 0.05% Adekanol LG-294 as an antifoaming agent in a 300-mL baffled Erlenmeyer flask. Both cultures were incubated for 24 h at 30 °C with shaking (150 rpm).

Liquid fermentation ability    Cultured cells harvested from the production medium were washed with deionized water and suspended in deionized water to a volume of 7.0 mL; 1.0 mL of this suspension was used for determination of cell weight dry matter.

The fermentation medium (20.0 mL) contained 2.5 g of fructose and glucose mixture (55:45), 0.25 g asparagine monohydrate, 5.0 mL of mineral solution (15.0 g/L NaH₂PO₄· 2H₂O, 10.0 g/L MgSO₄ · 7H₂O, 4.0 g/L KCl), 5.0 mL of vitamin solution (1.0 mg/L thiamine-HCl, 1.0 mg/L pyridoxine-HCl, 1.0 mg/L nicotinic acid), and 3.0 mL of citrate buffer (10.0 g trisodium citrate dihydrate was dissolved with deionized water to a volume of 100 mL and its pH was adjusted to 5.5 by adding 10% citrate monohydrate). Reaction was started by adding 5.0 mL of cell suspension, which contained 200 mg cell dry matter, in a 100 mL Erlenmeyer flask equipped with a fermentation tube, and the mixture was incubated for 3 h at 30 °C with shaking (80 rpm). The reduction in mixture weight due to evolved CO₂ was recorded as liquid fermentation ability (mg/3 h).

Baking test    Yeast cells were grown as described above except that 1.0 mL of the seed culture was inoculated onto 100 mL of the production medium in a 500-mL baffled Erlenmeyer flask. Cultured cells were harvested by centrifugation, washed twice with distilled water, and placed on a porous plate to create a yeast cake containing 33% (w/w) of cells as dry matter.

Breads were prepared in a bread machine SD-BMT1000 (Panasonic Corp., Osaka, Japan) in about 4 h using the setting for standard white bread preparation. Dough formula was as follows: 250 g of strong wheat flour (Camellia, Nisshin Flour Milling Co., Ltd., Tokyo, Japan), 10.0 g of unsalted butter (Yotsuba Milk Products Co., Ltd., Sapporo, Japan), 17.0 g of sugar containing fructose (Fruits Sugar, Nissin Sugar Co., Ltd., Tokyo, Japan) and glucose (Total Grape Sugar, Japan Garlic Co., Ltd., Takasaki, Japan) used for the fermentation medium described above, 6.0 g of skim milk (Hokkaido Skim Milk, Megmilk Snow Brand Co., Ltd., Tokyo, Japan), 5.0 g of salt, 7.0 g of yeast cake, and 170 mL of water. Specific volume (mL/g) of the baked product was calculated from the weight and volume measured after cooling to room temperature.

Analysis of volatile compounds    A bread sample (1.0 g) was cut from each of the three loaves, and placed in a 15-mL glass vial sealed with a silicone septum. Volatile compounds in headspace were adsorbed to solid-phase microextraction fiber (DVB/Carboxen/PDMS 50/30 µm, Supelco Co., Bellefonte, PA, USA) for 30 min at 40 °C and analyzed with gas chromatography mass spectrometry GCMS-QP 2010 Plus (Shimadzu Corp., Kyoto, Japan) (Aslankoohi et al., 2016).

Evaluation by taste sensor system and analysis of soluble compounds    Bread samples (each 5.0 g) removed from each of the three loaves were homogenized by a mill mixer in 20 mL of deionized water for 1 min. The resulting slurries were centrifuged, filtered, and used for the following experiments as bread extracts.

To determine the taste of each extract, outputs from five sensors with an artificial lipid membrane were recorded by the taste sensory system TS-5000Z (Intelligent Sensor Technology, Inc., Kanagawa, Japan). The results were converted to scores for eight tastes (sourness, acidic bitterness, astringency, umami, saltiness, aftertaste from acidic bitterness, aftertaste from astringency, and richness) according to the manual provided by the supplier (Kobayashi et al., 2010). Bread extracts may affect umami and saltiness, taking into consideration that water-insoluble compounds were not extracted very efficiently.

Free amino acids were determined after the removal of protein by the trichloroacetic acid treatment and the conversion to derivatives of phenyl isothiocyanate (Zhu et al., 2016). Organic acids and fermentable sugars in the extract were analyzed with high performance liquid chromatography (Oda et al., 2003; Santiago et al., 2015).

Reproducibility    All experiments were carried out three times independently and the obtained data are shown as average values ± standard deviations. Student's t-tests were conducted to detect significant differences between the two baked products.

Results

Taxonomic characteristics of TW15    The cells were apiculate, 4 to 6 µm by 8 to 12 µm, reproduced by bipolar budding (Fig. 1), and grew at 20 °C and 30 °C, but not at 37 °C. The strain fermented glucose vigorously, but not galactose, sucrose, maltose, lactose, raffinose, or trehalose. Assimilated carbon compounds were limited to glucose, maltose, cellobiose, and salicin. According to these phenotypic characters and the sequence identity of the ribosomal D1/D2 domain (DDBJ/EMBL/GenBank Accession Number KY107860), strain TW15 was classified as Hanseniaspora vineae (Cadez and Smith, 2011). The phylogenetic tree based on the sequences of the 18S-26S rDNA spacer region resolved the strain within the H. vineae clade (Fig. 2), but its sequence differed at five nucleotide positions when compared to the sequence of the type strain CBS 2171.

Fig. 1.

Cell morphology of the yeast strain TW15 isolated from wine grape ‘Yamasachi’.

Cells were grown in yeast-extract peptone dextrose medium for 24 h at 30 °C with shaking. The bar indicates 10 µm.

Fig. 2.

A phylogenetic tree constructed by the neighbor-joining method from the sequences of the 18S-26S rDNA spacer region.

Hanseniaspora osmophila was used as outgroup. DDBJ/EMBL/GenBank accession numbers are given in parentheses. The bar indicates 0.01 substitutions per nucleotide position. Bootstrap values were calculated from 1000 trees.

Some properties required for baking strains    Eight strains of H. vineae and four baking strains of S. cerevisiae were cultured to test the growth and liquid fermentation ability of the strains, which is a simple method to estimate leavening ability in dough. Sucrose, which is usually used in the medium, was replaced with a mixture of fructose and glucose (55:45) because H. vineae strains cannot ferment sucrose. The mixture of fructose and glucose can be obtained as a cheap commercial isomerized sugar syrup sweetener.

The cell yield of H. vineae strains was low and about half the cell yield of the S. cerevisiae strains, while TW15 showed higher fermentation ability than the other strains (Table 1). These observations suggested that TW15 can be used as a potential alternative baking strain.

Table 1. Cell yield and liquid fermentation abilities of yeast strains

Species	Strain	Cell yield (mg dry matter/ 60 mL medium)	Liquid fermentation ability (mg/3 h)
Hanseniaspora vineae	TW15	315 ± 7	484 ± 18
	NBRC 1412	233 ± 11	-
	NBRC 1415T	322 ± 7	420 ± 32
	NBRC 1416	271 ± 8	322 ± 33
	NBRC 1753	299 ± 4	418 ± 25
	NBRC 1754	273 ± 0	372 ± 7
	NBRC 10226	327 ± 4	363 ± 55
	NBRC 100790	243 ± 11	-
Saccharomyces cerevisiae	HP467	681 ± 11	372 ± 23
	NBRC 2043	670 ± 15	359 ± 15
	NBRC 2044	679 ± 0	340 ± 18
	NBRC 2375	663 ± 4	364 ± 9

The cell yields of NBRC 1412 and NBRC 100790 were too low to determine liquid fermentation ability.

Data are shown as the average values ± standard deviations from three independent experiments.

Characteristics of baked breads    TW15 was used in bread making to compare the physical and sensory properties of the bread with those made with S. cerevisiae HP467. The specific volumes of the bread produced by TW15 (4.62 ± 0.03 mL/g) was significantly larger than that of the bread produced with HP467 (4.35 ± 0.02 mL/g) (P = 0.0001) (Fig. 3).

Fig. 3.

Appearance of baked breads prepared from dough leavened by Hanseniaspora vineae TW15 and Saccharomyces cerevisiae HP467.

The results of three independent baking tests are shown from top to bottom.

Preliminary sensory evaluation by three panels made up of consumers revealed that both products were similar in appearance and texture, and the bread produced using TW15 showed a distinct, more desirable flavor and slightly different taste (data not shown). A total of 17 volatile compounds were identified in the bread samples. Of those, acetoin, 2-phenylethyl acetate, and acetic acid were present in bread samples produced with TW15 in more than twice the levels recorded in samples produced with HP467 (Table 2). Evaluation of bread extracts by the taste sensor system revealed a significant difference in four tastes, umami, richness, saltiness, and acidic bitterness (Table 3), which may not be detectable by human taste sense because humans cannot register a taste difference of less than 1.0 score unit (Kobayashi et al., 2010). In the extracts, citric acid and lactic acid were undetectable and the contents of total free amino acids, succinic acid, and acetic acid differed significantly between the two breads (Table 4). Strain TW15 fermented fructose more rapidly than strain HP467, but did not consume endogenous sucrose and maltose, the products of starch degradation (Table 4). The total amounts of sugars consumed by TW15 were lower than those consumed by HP467.

Table 2. Volatile compounds isolated from bread samples prepared by using Hanseniaspora vineae TW15 and Saccharomyces cerevisiae HP467

Compound	Relative peak area (%)		Ratio of area (TW15/HP467)	P-values
	TW15	HP467
Acetic acid	1.834 ± 0.160	0.650 ± 0.151	2.82	<0.00001
Acetoin	9.985 ± 0.928	0.434 ± 0.132	23.01	<0.00001
Benzaldehyde	0.456 ± 0.058	0.229 ± 0.086	1.99	0.00001
Diacetyl	0.124 ± 0.038	0.000 ± 0.000	-	<0.00001
Dodecane	0.155 ± 0.038	0.093 ± 0.029	1.67	0.002
Furfural	0.022 ± 0.006	0.018 ± 0.009	1.23	0.289
Hexanoic acid	0.136 ± 0.066	0.107 ± 0.024	1.27	0.250
Hexanol	0.230 ± 0.110	0.185 ± 0.076	1.24	0.344
Isoamyl alcohol	5.615 ± 0.328	7.681 ± 1.114	0.73	0.001
Isoamyl butyrate	0.057 ± 0.008	0.057 ± 0.009	1.01	0.861
Isobutyric acid	0.147 ± 0.055	0.242 ± 0.047	0.61	0.002
Octanoic acid	0.079 ± 0.055	0.048 ± 0.031	1.64	0.184
2-Pentylfuran	0.097 ± 0.014	0.058 ± 0.010	1.66	0.00001
2-Phenylethyl acetate	0.928 ± 0.055	0.165 ± 0.155	5.62	<0.00001
2-Phenylethyl alcohol	5.440 ± 0.459	8.087 ± 0.827	0.67	<0.00001
Valeric acid	0.053 ± 0.016	0.055 ± 0.013	0.97	0.809

Table 3. Taste sensory evaluation of the extracts from bread samples prepared by using Hanseniaspora vineae TW15 and Saccharomyces cerevisiae HP467

Taste information	Scores		P-values
	TW15	HP467
Umami	5.37 ± 0.04	5.45 ± 0.03	0.0001
Richness	2.07 ± 0.06	1.92 ± 0.09	0.001
Saltiness	2.91 ± 0.04	2.97 ± 0.05	0.014
Acidic bitterness	1.90 ± 0.03	1.96 ± 0.02	0.001

Table 4. Soluble compounds in the extract from bread samples prepared by using Hanseniaspora vineae TW15 and Saccharomyces cerevisiae HP467

Compound	Contents (mg/100 g dry matter)		P-values
	TW15	HP467
Total free amino acids	214 ± 6	208 ± 1	0.015
Organic acid
Acetic acid	167 ± 6	83 ± 6	<0.00001
Succinic acid	17 ± 2	63 ± 7	<0.00001
Sugar
Fructose	662 ± 196	800 ± 126	0.096
Glucose	41 ± 80	0 ± 0	0.15
Lactose	1,370 ± 84	1,327 ± 68	0.246
Maltose	3,423 ± 163	3,235 ± 155	0.023
Sucrose	237 ± 42	0 ± 0	<0.00001

Discussion

Because the present study aimed to isolate yeast strains capable of vigorous fermentation of maltose and sucrose as candidate novel baking strains, the enrichment cultures were conducted on the medium containing these sugars and propagated cells were purified on the glucose-containing medium. The isolated strain TW15 is however unable to ferment maltose or sucrose, indicating that its cells grew on glucose derived from squeezed juice in the first enrichment culture even with the addition of maltose, and it successively multiplied on monosaccharides hydrolyzed from sucrose by the action of coexisting film-forming yeasts in the second enrichment culture. High concentration of sucrose in the second enrichment culture after conversion to monosaccharides might cause efficient selective pressure to obtain TW15 with high liquid fermentation ability.

The use of TW15 as a commercial baking yeast may be limited by its inability to ferment sucrose and the low cell yield. Sucrose in dough formulation can be replaced by high fructose corn syrup as an economical substrate of fermentation. Cell growth depends on optimal growth temperature, which for TW15 might be lower than that for conventional baking strains. TW15 grew at 30 °C but not at 37 °C, while baking strains usually grow at both temperatures. Therefore, for commercial propagation, it may be necessary to investigate culture conditions such as temperature, type of raw materials (monosaccharide hydrolyzed from corn starch or sucrose contained in molasses), and the feeding method for sugar solution when fed-batch culture is applied.

As expected, TW15 was applicable for the production of bread using dough containing a mixture of the sugars fructose and glucose. If the prepared dough retains gas consistently, gas production will determine bread volume. This was not observed in our experiment, as TW15 fermented less sugar compared with HP467, whereas the bread produced with TW15 had a larger volume than the bread leavened with HP467. The cause of this discrepancy between fermentation and volume of baked bread remains questionable. One of the possible explanations is that CO₂ evolved from the conversion of 2-acetolactate to acetoin by TW15 during the final heat treatment of the bread, eliciting greater volume of baked breads.

The differences in taste scores obtained by the taste sensor system and the contents of soluble compounds seem to be too small, and therefore, the results of the preliminary sensory evaluation should be explained by the concentration of the volatile compounds from baked breads.

The bread produced by TW15 contained much more acetoin than the bread made using HP467. Formation of acetoin and diacetyl is reported to change desirable aroma profiles of the sourdough and breads by enhancing buttery flavor (Comasio et al., 2019). Apiculate yeasts from the genera Hanseniaspora and Kloeckera, which are involved in wine fermentation, usually produce much more acetoin compared with Saccharomyces cerevisiae strains when used for vinification (Romano and Suzzi, 1996). These characteristics may be common to apiculate yeasts, which convert pyruvate to acetoin as a manner of detoxification based on less-resistant properties to ethanol.

Other volatile compounds specifically found in bread produced by TW15 were 2-phenylethyl acetate and acetic acid. Certain amounts of acetic acid were suggested to enhance bread aroma (Birch et al., 2013), but its higher concentration may cause negative effects on bread quality by producing an acidic odor or increasing its acidity. Phenylethyl acetate is one of the volatile compounds responsible for the floral aroma of rose and other flowers, and its production was similarly observed in wine fermentation (Wei et al., 2019; Zhang et al., 2018b). Compared with S. cerevisiae, H. vineae possesses a higher ability to synthesize benzenoid compounds, leading to aromatic intermediates (Martin et al., 2016). In the genome of H. vineae, ARO8 and ARO9 (genes encoding aromatic amino acid aminotransferases) and ARO10 (a gene encoding phenylpyruvate decarboxylases) were duplicated to stimulate production of 2-phenylethyl acetate and phenylpropanoids (Giorello et al., 2019). The present experiments suggest that 2-phenylethyl alcohol synthesized from phenylpyruvate by decarboxylases encoded in ARO10 was readily converted into 2-phenylethyl acetate. These characteristic compounds may have affected the flavor in the preliminary sensory evaluation.

Non-conventional yeasts include various non-Saccharomyces yeasts and they are typically not used in alcohol fermentation or baking industries (Mattanovich et al., 2014). In the production of alcoholic beverages, cultures of non-Saccharomyces yeasts alone or mixed with Saccharomyces yeasts are used as starters of fermentation to improve the aroma of the products (Gschaedler, 2017; Jiang et al., 2017; Serra Colomer et al., 2018; van Rijswijck et al., 2017; Varela, 2016). Hanseniaspora species were dominant among non-Saccharomyces yeasts isolated from wine grapes (Mendoza et al., 2019). Among them, H. uvarum exhibited high population growth, but H. vineae was the strain that elevated the aroma the most in laboratory experiments (Carrau et al., 2015). Other reports confirmed that supplementation with the H. vineae strain in wine fermentation elicits a distinct and better quality (Wei et al., 2019; Zhang et al., 2018b).

In the baking industry, Torulaspora delbrueckii has attracted attention for specialized bread making methods as a non-Saccharomyces yeast that is tolerant to high sugar content and freezing (Alves-Araújo et al., 2007). Zhou et al. (2017) identified Kazachstania gamospora and Wickerhamomyces subpelliculosus as alternative baking strains with high stress tolerance and broad aroma profiles. They also reported that bread made with H. vineae had better intensity in taste and aroma, although its gassing power from sucrose was hardly detected.

In conclusion, baked goods with diverse and palatable quality can be produced by using the yeast H. vineae TW15. Additionally, TW15 can be used in combination with conventional baker's yeast as a supplement to enhance the quality of breads.

Acknowledgements    We would like to thank Hiroaki Yamauchi for his helpful advice on the baking experiments and Editage (www.editage.jp) for English language editing.

Abbreviations

NITE

National Institute of Technology and Evaluation

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