Screening ester-producing yeasts to fortify the brewing of rice-flavor Baijiu for enhanced aromas

ABSTRACT

To enhance the aromas in Guangdong rice-flavor Baijiu, ester-producing yeast was selected to fortify Baijiu brewing. Among eight kinds of ester-producing yeasts selected, Saccharomyces cerevisiae CM15 (CM15) that showed both the stronger ability to utilize substrates to produce esters and the excellent tolerance to industrially relevant stress factors was chosen. When CM15 was synergistically fermented with six kinds of Kojis from distilleries of rice-flavor liquor in Guangdong, the enhanced total esters had happened to the liquors brewing with the fortified four kinds of Kojis, especially with Koji F. When Koji F was fortified with CM15, the resultant Baijiu showed a higher esters proportion and a lower higher alcohol ratio than that of Baijiu brewed only with Koji F, with the content of ethyl acetate and ethyl lactate increasing by 25% and 214%, respectively. This study suggested that CM15 can be used as a functional microorganism to fortify Baijiu brewing, which might also be suitable for other traditional fermented foods.

KEYWORDS:

• Ester-producing yeast
• higher alcohol
• rice-flavor liquor
• ethyl acetate
• ethyl lactate

1. Introduction

Baijiu, as the traditional fermented flavor spirit, has become a vital contributor to the global liquor industry [1]. Nowadays, the liquor can be sorted by four basic flavors, including sauce flavor, strong flavor, light flavor, and rice flavor. Wherein, the rice flavor liquor is made from rice with Xiaoqu sending out the elegant and sweet rice aromas with ethyl acetate and β– phenylethyl alcohol as the main body [2]. Among esters in rice-flavor liquor, the most abundant ester is ethyl lactate, then followed by ethyl acetate [2]. However, the overall content of flavor components in rice-flavor Baijiu is low, resulting in a thin flavor in taste [2]. Esters are important aromas, the content of which is an important index to distinguish the quality of Baijiu [3,4]. Isolation of aroma-producing yeasts to fortify Baijiu brewing is a good choice to enhance the sensory qualities.

The source of samples to screen aroma-producing yeasts depends on the characteristics of the target yeasts expected. Osmotolerant yeasts could be screened from high sugar niches, such as molasses, grape must, sugar juice, and syrup, under media with high sugar concentrations [5,6]. The low-methanol and high aroma-producing yeasts could be obtained from samples of Maotai (Guizhou) and Xifeng distilleries (Shaanxi, China) after enrichment under YEPD medium containing apple squares, purification on the YEPD agar plates, and sensory evaluation of resultant cider [7]. Both Daqu and Xiaoqu are the Kojis and widely applied for the production of fermented foods because of a lot of microorganisms involved that can produce enzymes for starch hydrolysis and aromas production [8]. Indigenous yeasts with high ethyl acetate production and high ethanol yield were screened from Daqu by purification on YEPD plates [9,10] and were used to fortify the solid-state fermentation of Baijiu with ethyl acetate improved, suggesting that Kojis is a good source for the isolation of ester-producing yeasts.

Yeasts, as one of the most important microorganisms for ethanol fermentation, are closely related to the quality of liquors [11]. It is well known that the liquor brewing process is a dynamic progress, during which the acidity and temperature are dynamically changed [12]. In some Xiaoqu Baijiu fermentation, the temperature increased fast along with acids and ethanol produced quickly, due to the action of Saccharomyces cerevisiae, which would inhibit the growth and metabolites of yeasts in turn [13,14] and influence the constitution of flavor compounds. It is reported that Zygosaccharomyces bailii BCV 08 isolated from red wine barrels in Brazil could tolerate low pH values (3.5), the addition of which could enhance the content of ethyl esters in wine at acidic condition [15]. Esters in the wort could be enhanced under relative higher fermentation temperature [16] or with high specific gravity fermentation [17]. However, high sugar content would cause high osmotic pressures and inhibit the growth and reproduction of yeasts, resulting in the bad quality of wine [18]. In a word, ester-producing yeasts that showed strong abilities to tolerate stress factors would be an excellent co-starter to enhance the content of esters in wine. So, yeasts that can tolerate high-temperature, high osmotic pressure, low acidity, and high alcohol concentration are badly needed.

Nowadays, the Guangdong rice-flavor Baijiu is thin in taste, owing to the low content of esters. Daqu liquor fortified by ester-producing yeasts showed the improved quality and flavor, which might be also suitable for the rice-flavor Baijiu brewing with Xiaoqu. It is quite urgent to obtain yeasts with good aroma producing performance to fortify rice-flavor Baijiu brewing. In this study, eight ester-producing yeasts were screened from Xiaoqus of Guangdong’s distilleries, and the fermentation abilities among yeasts were evaluated by fermentation test. Then, yeast with good fermentation traits and the excellent ability to produce volatile flavor compounds was selected for further studies, such as the tolerance to industrially relevant stress factors and the capability to fortify Baijiu brewing. This study not only suggests an excellent ester-producing yeast, which can improve the quality of Guangdong rice-flavor Baijiu, but also provides methods to enhance the flavor of other traditional fermented foods by screening functional microorganisms as the co-starter.

2. Material and methods

2.1. Isolation of ester-producing yeasts and phylogenetic analysis

Eight ester-producing yeasts used in this study were isolated from nine kinds of Guangdong’ Kojis after primary and secondary screening (Table 1). The primary screening was performed under rose bengal agar medium (Solarbio, Beijing, China), which could inhibit the growth of bacteria. Firstly, 5.0 g Koji were added into 250 mL conical flask containing 45 mL sterile water and shaken for 30 min at 200 rpm to be fully mixed. Then, 1 mL of sample solution was added into a tube containing 9 mL YEPD medium and cultured at 28°C, 120 rpm for 24 h. And then 1 mL cell culture was diluted to 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴ in sequence with sterile water. Finally, 200 μL diluted cells were pipetted on the rose bengal agar medium plate, separately, and cultured at 28°C for 48 h to select a typical yeast colony. The secondary screening was performed under YEPD medium containing 10 g/L glyceryl tributyrate (Aladdin, Shanghai, China) to select ester-producing yeast with hydrolytic circle on the plate.

Table 1. Eight ester-producing yeasts isolated from Guangdong rice flavor liquor’s Kojis.

Koji	Source	Ester-producing yeast obtained	Similar strains	Homology similarity of 26S rDNA (%)
Koji A	Guangdong Jiujiang Distillery Co., Ltd.	JJ19	Clavispora lusitaniae 32 M	100
Koji B	Guangdong Changleshao Co., Ltd.	CM15	Saccharomyces cerevisiae BYB-2	99.65
Koji C	Guangdong Changleshao Co., Ltd.	—	—	—
Koji D	Guangzhou Yingjinqian Conghua Sanhua distillery	YJ18,YJ26 YJ26	Clavispora lusitaniae YM26322, Clavispora lusitaniae LMICRO133	100, 100
Koji E	Guangzhou Yingjinqian Conghua Sanhua distillery	CZ13	Saccharomyces cerevisiae ITS_9	100
Koji F	Pingyuan laobachi distillery	—	—	—
Koji G	Meizhou bazheniang Wine Co., Ltd	—	—	—
Koji H	Heyuan jinhuangtian Liquor Co., Ltd	—	—	—
Koji J	Meizhou Hakka courtyard Wine Co., Ltd	JX02,JX07,JX19	Saccharomyces cerevisiae 106-K6, Saccharomyces cerevisiae, F2-Y333-8, Saccharomyces cerevisiae N215	100, 99.63 100

Note: ‘—’ indicated that no excellent ester-producing yeasts had been isolated.

The clone of each yeast was cultured under YEPD medium, respectively. Samples were collected at 12 h cultivation. Genomic DNA (gDNA) of yeasts was extracted using yeast Genomic DNA Extraction Kit (Solarbio, Beijing, China). 26S rDNA D1/D2 was amplified with primers NL-1 (5’-GCATATCAATAAGCGGAGGAAAAG-3’) and NL-4 (GGTCCGTGTTTCAAGACGG) by Primer STAR®HS (Takara, Beijing, China). The PCR was performed as follows: 94°C for 5 min and then 28 cycles of 98°C for 10 s, 55°C for 30 s, 72°C for 45 s, and then followed by a final extension at 72°C for 10 min. The PCR products were sent to Shenggong (Shanghai, China) for sequencing. The homology similarity of 26S rDNA was analyzed with Nucleotide Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) by finding the closest homolog of the isolates from databases and exhibited with the phylogenetic tree as previous study described [6].

2.2. Medium and culture conditions for esters formation

Glucose medium (w/v): Glucose (Aladdin, Shanghai, China) 10%, peptone (Oxoid, UK) 2%, yeast extract (Oxoid, UK) 1%.

Sugarcane molasses medium: Sugarcane molasses (Zhongfuxin, Guangxi, China) 20 BX°, (NH₄)₂SO₄ (Macklin, Shanghai, China) 0.1%, KH₂PO₄ (Macklin, Shanghai, China) 0.1%, MgSO₄ (Macklin, Shanghai, China) 0.04% (w/v).

Nitrogen-rich medium (w/v): Soybean meal (Macklin, Shanghai, China) 5%, peptone 2%, yeast extract 1%.

YEPD medium (w/v): Glucose 2%, peptone 2%, yeast extract 1%.

GPYM medium (w/v): Glucose 4%, peptone 0.5%, yeast extract 0.5%, MgSO₄·7 H₂O 0.1%, malt extract (Solarbio, Beijing, China) 0.5%.

About 100 mL of glucose medium, sugarcane molasses medium, and nitrogen-rich medium were prepared, respectively, in 250 mL conical flask, and 10% (v/v) yeast seed liquids were inoculated, respectively, and cultivated at 28°C, 150 rpm for 3 days.

2.3. Evaluation of the fermentation ability among ester-producing yeasts

The metabolism rate of ester-producing yeasts was monitored by weight loss of CO₂ emission as previous study described [19]. Briefly, the yeast seed liquid was inoculated (10%, v/v) into GPYM medium, and cultured at 28°C with three replications. Before fermentation, the initial weight of the fermentation broth was measured and then was weighted and recorded at every 12 h after sufficiently shaking. When the weight loss between two adjacent 12 h was less than 0.2 g, the fermentation was over.

Both alcoholic strength and total acid were measured based on the Chinese standard (GB/T 10,345–2007, 2007) [20]. Residual sugar was determined according to the Chinese standard (GB/T 5009.7–2016, 2016) [21].

2.4. The tolerance test of ester-producing yeasts under industrially relevant stresses

The yeast seed liquids about 10⁷ colony forming unit (CFU)/mL were separately inoculated (10%, v/v) into the YEPD medium with different tolerance conditions and cultured at 28°C, 150 rpm for 48 h. The tolerance of yeasts to sugar (150 g/L, 250 g/L, 350 g/L, 450 g/L, 550 g/L, 650 g/L), acid (pH 5, pH 4.5, pH 4, pH 3.5, pH 3, pH 2.5), ethanol (0%, 6%, 8%, 10%, 12%, 14%, 16% (%vol)), temperature (36°C, 38°C, 40°C, 42°C, 44°C, 46°C), and NaCl (0 g/L, 40 g/L, 80 g/L, 120 g/L, 160 g/L, 180 g/L) was investigated by counting the number of viable yeasts. The data are the means and standard deviations of two replications.

2.5. Rice-flavor liquor brewing with CM15 fortified Xiaoqu

The fermentation process of rice-flavor liquor brewing was performed according to our previous study [22] with some modification. Briefly, the rice (250 g) was washed twice and drained off. Then, 190 mL water was added and steamed at 115°C for 15 min with autoclave (Shenan, Shanghai, China) to obtain the flexible but not sticky, ripe but not rotten rice. When the temperature of the rice dropped to room temperature, various Xiaoqus were added according to the recommended dosage shown in Table 2 and mixed separately. Then, the mixture was put into 2 L fermentation jar (Oiyou, Hebei, China) with a pit in the center for saccharification at 34°C for 40 h in the biochemical incubator (labotery, Tianjing, China). After that, washed cells of 25 mL CM15 (10⁷ CFU/mL) were added to the fermentation jar along with 300 mL water and stirred thoroughly. And then the fermentation jar was sealed with plastic wrap and put into the biochemical incubator at 28°C for 15 days. Finally, the wort was added with 250 mL water and steamed with wine distillation and purification machine (Aoniya, China) to obtain 250 mL liquors. Each experiment had three replications.

Table 2. Types of kojis and their inoculation amount.

Koji	inoculation amount	Koji	inoculation amount
Koji B	1.2%	Koji F	0.5%
Koji C	1.2%	Koji G	0.3%
Koji D	1.0%+0.3% angel yeast	Koji H	2.0%

2.6. Volatile component analysis

Volatile component analysis was conducted by headspace solid-phase micro-extraction (HS-SPME) (Supelco, Bellefonte, PA, USA) coupled with gas chromatography-mass spectrometry (GC-MS) (Agilent, Santa Clara, CA, USA), flame ionization detector (FID) (Agilent, Santa Clara, CA, USA), and a 30 m × 0.25 mm × 0.25 μm fused silica capillary column DB-WAX UI (Agilent, Santa Clara, CA, USA) based on previous study [23] but with some modification. Briefly, the fermentation broth (7 mL) was put into 25 mL sample bottle, containing 1.4 g NaCl and magnetic rotor for headspace solid-phase microextraction (HS-SPME) at 45°C, 220 rpm for 50 min with 50/30 μm DVB/car/PDMS as the SPME fiber (Supelco, Bellefonte, PA, USA). After extraction, the SPME fiber was desorbed at 250°C for 5 min with helium (purity >99.999%) at a constant rate of 1.0 mL/min as the carrier gas. GC-MS condition: electron ion source temperature, 230°C; electronic energy, 70 eV; acquisition mode, full scan, and quality scanning range (m/z): 50 ~ 550. The initial temperature of the oven was 40°C for 5 min and increased to 100°C at a rate of 5°C/min for 2 min. Then, the temperature was increased to 220°C at the rate of 15°C/min and held for 5 min. The n-amyl acetate (Aladdin, Shanghai, China) was used as an interior label to calculate the absolute quality of volatile components by HS-SPME-GC-MS. Proteo Wizard software was used to convert the original mass spectrometry data, and the XCMS package was used to correct the retention time, peak recognition, peak integration, etc. OSI-SMMS software was applied to identify the substance from the database. The relative content of each component was obtained based on the peak area normalization method. The absolute quality of each volatile component was calculated according to the relative area and absolute content of n-amyl acetate (the ratio of the relative area was equal to the ratio of the absolute content).

Ethyl acetate, ethyl lactate, and β–phenylethyl alcohol in Baijiu were also measured based on gas chromatography [22]. The inlet temperature of the sample was 220°C, and 1 μL sample was analyzed under the condition of 100:1 split ratio, 1 mL/min flow rate, 220°C flame ionization detector temperature, and 40:400:25 ratio of hydrogen (H₂):air (O₂):tail blowing (N₂). The standard samples of ethyl acetate, ethyl lactate, and β– phenylethyl were used for qualitative and quantitative analysis.

2.7. Statistical analysis

The heat map was drawn with Excel 2017. Other figures were drawn with origin nine. All data were analyzed by t-test with p < 0.05 as the threshold of the significant difference.

3. Results

Nowadays, the Guangdong rice-flavor Baijiu is thin in taste, owing to the low ester content. The organoleptic quality of fruit wines [24], grape must [25] and Daqu Baijiu was improved with the fortification of aroma-producing yeasts [9,10], suggesting that it might also be appropriate for rice-flavor Baijiu brewing with Xiaoqu. In this study, eight ester-producing yeasts were screened, and the ability of ester production among yeasts was evaluated. Finally, the yeast with good fermentation traits and excellent ability to produce esters was selected to fortify Baijiu brewing.

3.1. Fermentation traits of 8 isolated ester-producing yeasts

It is reported that lipase, esterase, and alcohol acyltransferase would participate in the formation of eaters [26]. The agar plate containing glyceryl tributyrate could be used to isolate ester-producing yeasts owing to the lipase, which could hydrolyze glyceryl tributyrate to form the hydrolytic circle [27]. The bigger the hydrolytic circle, the stronger the ester formation ability of yeasts will be. Based on this method, the ester-producing yeasts were isolated and identified as Saccharomyces cerevisiae (CM15, JX02, JX07, JX19, and CZ13) and Clavispora lusitaniae (JJ19, YJ18, and YJ26) after phylogenetic tree analysis (Fig. S1).

CO₂ weight loss during the fermentation process was measured as a very important parameter to evaluate the fermentation ability of eight studied strains. As shown in Figure 1A, at the first 3 days, all the stains had reached the highest peak of fermentation and then the fermentation ability gradually decreased. At the 5th day, the CO₂ weight loss between two adjacent measuring points was no more than 0.1 g, suggesting that the main fermentation stage was over. Among the ester-producing yeasts, JX02, JX19, JJ19, CM15, and YJ18 showed both relatively higher fermentation ability with earlier starting fermentation points than that of YJ26, indicating that JX02, JX19, JJ19, CM15, and YJ18 could grow fast enough to produce metabolites. The ability of substrate consumption and alcohol formation among the ester-producing yeasts was also measured (Figure 1B). The results showed that CM15 and JX19 had the stronger ability to utilize substrates to produce low content of alcohols (Figure 1B), suggesting that CM15 and JX19 might be potential candidates to transform substrates into esters, acids, etc. rather alcohols.

Figure 1. The fermentation rate (A), alcohol content and residual sugar content (B) among eight kinds of selected ester-producing yeasts.

To evaluate the potential application of yeasts studied, three kinds of media were used. As shown in Figure 2, more volatile flavor compounds could be formed with glucose and sugarcane molasses as the carbon sources than that with soybean meal, implying that ester-producing yeasts preferred carbon-rich medium to nitrogen-rich medium. Cultured under glucose, CM15 showed the strongest ability to produce volatile flavor compounds, especially esters. As shown in Figure 3A, there were eight kinds of esters produced by CM15, and the amount of phenethyl acetate was the highest one then followed by ethyl acetate. CM15 also showed excellent traits to produce volatile flavor compounds when cultured under sugarcane molasses , and more alcohols were formed then followed by esters, among which, phenethyl acetate and ethyl 3-phenylpropionate were the main esters (Figure 3B). Cultured with nitrogen-rich medium, CM15 showed advantages over other ester-producing yeasts as well, with alcohols and esters as the main volatile flavor compounds. Among the esters produced, ethyl acetate and phenethyl acetate accounted for a dominant position (Figure 3C). Based on the data above, CM15 exhibited the strongest ability to produce total esters from three media among eight studied yeasts (Figure 3) and also showed excellent cell growth with glucose as the carbon source (Fig. S2), suggesting that CM15 might be an excellent ester-producing yeast and could be used to produce esters under different substrates for different applications.

Figure 2. Volatile flavor compounds formation by eight kinds of ester-producing yeasts with glucose (A), sugarcane molasses (B) and nitrogen-rich medium (C) as the carbon source respectively.

Figure 3. Heat map of esters formation by eight kinds of ester-producing yeasts with glucose (A), sugarcane molasses (B) and nitrogen-rich medium (C) as the carbon source respectively. The number in the heat map was the average concentration of esters with the unit mg/L. The significant difference of total esters’ concentration between CM15 and the second excellent yeast under different media was analyzed (p ≤0.001, ***; p ≤0.0001, ****; t-test).

3.2. The tolerance of CM15 to different industrially relevant stress factors

During the brewing, microorganisms, such as yeasts, will confront large numbers of stresses, including osmotic pressure, high ethanol concentration, high temperature, low pH, etc. yeasts that showed excellent resistance to stresses, such as high-temperature, high osmotic pressure, low acidity, and high alcohol concentration, are badly needed for applications.

(i) the performance of CM15 under the osmotic pressure

High osmotic pressure is commonly seen in the large-scale production of fermented food. Therefore, yeast with high tolerance to osmosis is beneficial for high-gravity brewing, both from qualities and economics. The tolerance of CM15 to the high osmosis is exhibited in Figure 4A-B. Results showed that by the increase of glucose content, the growth of CM15 was continuously decreased, and finally CM15 could tolerate 450 g/L glucose with the viable cell at 8 × 10⁷ CFU/mL (Figure 4A). As shown in Figure 4B, the growth of yeasts was inhibited to a different degree with the increase of NaCl concentration, which caused both the osmotic stress and the ion toxicity to cells [28]. When NaCl concentration was lower than 80 g/L, the viable cells of CM15 was still at a higher level. Finally, it could be concluded that CM15 could tolerate up to 120 g/L NaCl.

Figure 4. The tolerant performance of CM15 under glucose (A), NaCl (B), ethanol (C), temperature (D) and pH (E).

(ii) the performance of CM15 under the ethanol stress

Ethanol is one of the main products of the most yeasts. High ethanol concentration is toxic to yeasts and affects cell metabolism, thereby inhibiting the growth and activity of yeasts [29]. The higher the alcohol content is, the more obvious the inhibition effects will be. Yeasts with high alcohol tolerance could survive in the main fermentation phase and synthesis aroma substances during the post-fermentation period. When the alcohol content was increased to 8% vol, the number of viable cells of CM15 was higher than that under 0–6% vol and then decreased sharply with the increase of ethanol content (Figure 4C). Eventually, CM15 could tolerate an alcohol content of 12% vol.

(iii) the performance of CM15 under the temperature stress

During the brewing, much energy could be formed and released, which in turn affects the metabolism of microbiota. Therefore, high temperature-tolerant strains are beneficial for the accumulation of metabolic products during the fermentation. As shown in Figure 4D, CM15 could withstand the temperature of 42°C, and at that point, the number of viable cell was 5 × 10⁷ CFU/mL.

(iv) the performance of CM15 under the low pH stress

In general, yeasts can grow well in the acidic environment at pH 5.0 ~ 6.0. In the early stage of brewing, the pH is quite suitable for yeasts to grow along with organic acids produced, which will change the pH value of the fermented mash and seriously affect the growth and metabolism of yeasts in turn. The selection of yeasts that grow well under acidic stress is extremely important. As shown in Figure 4E, the optimal growth of CM15 could be obtained at pH 4 and finally, CM15 could tolerate pH value at 2.5 with 1 × 10⁷ CFU/mL viable cell.

3.3. The performance of CM15 on Baijiu brewing with Kojis

CM15 was used to fortify six kinds of Kojis from different distilleries in Guangdong to brew Baijius, and the results are shown in Figure 5A. Compared with the control, CM15 could fortify Koji D to improve the content of ethyl acetate in liquors by 93%; Cooperated with Koji B, CM15 could increase the content of ethyl acetate, ethyl lactate, and β-phenylethyl alcohol in liquors by 34%, 72%, and 54%, respectively. When Koji F was fortified with CM15, the ethyl acetate and ethyl lactate content in liquor were increased by 25% and 214%, respectively, along with the content of β- phenylethyl alcohol reduced by 13% insignificantly. As shown in Figure 5B, CM15 can fortify four kinds of Kojis, such as Koji B, D, F, and H, for enhanced total esters, along with the alcohol content ranging from 37% to 44% vol and total acid content decreased.

Figure 5. Effects of CM15 addition on the flavor substances (A), total acid, total ester and alcohol (B) of rice-flavor liquor in Guangdong. The data are the means and standard deviations of three replications (p ≤0.05, *; p ≤0.01, **; t-test).

Brewing with CM15 fortified Koji F, the resultant Baijiu showed richest volatile flavor substances and was further analyzed by HS-SPME-GC-MS (Table 3). Baijiu brewing only with Koji F had 13 kinds of esters and 5 kinds of alcohols, with a relative percentage of 29% and 35%, separately. Brewing with CM15 fortified Koji F, the rice-flavor liquor contained six kinds of esters and four kinds of alcohols, with a relative percentage of 37% and 28%, respectively. Ten kinds of substances were detected in both liquors, such as ethyl acetate, methyl benzoylformate, ethyl caprylate, ethyl caprate, ethyl myristate, ethyl palmitate, 2-methyl-1-propanol, 2-methyl-1-butanol, 3-buten-1-ol, and phenylethyl alcohol, suggesting that these substances are the main aromas in this type of Baijiu. Compared with the control, the relative content of esters in the liquors fortified with CM15 was increased by 28%, along with the content of alcohol reduced by 19%.

Table 3. Effect of CM15 on the formation of volatile esters and alcohols in rice-flavor liquor.

Compound identified	Retention index	Relative percentage (%)
		Koji F	Koji F+CM15
Esters
Ethyl acetate	-	3.06 ± 0.42	5.68 ± 0.00
Ethyl caproate	1216	0.30 ± 0.01	-
Pentyl acetate	-	-	12.05 ± 0.85
Methyl benzoylformate	-	9.15 ± 0.00	6.83 ± 0.75
Ethyl caprylate	1439	0.92 ± 0.01	2.16 ± 1.53
Ethyl caprate	1647	3.75 ± 0.57	6.54 ± 0.44
Ethyl laurate	1850	0.68 ± 0.01	-
Ethyl myristate	2035	1.53 ± 0.08	0.96 ± 0.14
Pentadecanoic acid, ethyl ester	2041	0.02 ± 0.00	-
Ethyl palmitate	2246	7.78 ± 0.42	2.43 ± 0.40
9-Hexadecenoic acid, ethyl ester	2260	0.07 ± 0.01	-
Octadecanoic acid, ethyl ester	2400	0.21 ± 0.03	-
Ethyl oleate	2479	0.76 ± 0.07	-
9,12-Octadecadienoic acid (Z, Z)-, ethyl ester	2501	0.34 ± 0.01	-
Percentage of esters		28.56 ± 0.53	36.65 ± 3.81
Alcohols
2-Methyl-1-propanol	1113	4.20 ± 0.21	3.22 ± 0.25
2-Methyl-1-butanol	1220	23.26 ± 1.91	20.90 ± 0.00
3-Buten-1-ol	-	0.15 ± 0.01	0.11 ± 0.03
3-Methyl-2-buten-1-ol	-	0.04 ± 0.00	-
Phenylethyl alcohol	1924	7.29 ± 1.50	4.17 ± 1.12
Percentage of alcohol		34.94 ± 3.64	28.39 ± 1.40

Note: bold letters indicated the classification of main components and its proportion.

4. Discussion

4.1. CM15 isolated from exhibited the Kojis excellent ability for ester production

Nowadays, the overall content of flavor components in rice-flavor Baijiu is low, resulting in thin flavor in taste. Esters are the most important components of aromas, the content and proportion of which are the important index to distinguish the quality of Baijiu [3,4]. To make up for this insufficiency, much research was performed and focused on the fortified starters to brew Baijiu. It is reported that aroma-producing yeasts such as Pichia and Saccharomyces were isolated from Daqu Baijiu fermenting material to fortify the Koji to increase the content of volatile compounds [10,30] and the resultant Baijiu was much mellower and more aromatic [31]. In this study, isolation of ester-producing yeasts from various Guangdong’s Xiaoqu was performed. CM15 that showed the excellent ability to utilize substrates to produce highest content of esters among eight studied yeasts was selected (Figure 3). The poor ability of CM15 to produce alcohol shown in Figure 1B suggested that CM15 might be the potential candidate to transform substrates into volatile flavor compounds such as esters and acids. As shown in Figure 2, CM15 definitely exhibited the advantages of producing volatile flavor compounds under different media over other strains. Different media also led to different compositions of flavor compounds. With glucose as the carbon source, esters were the main aromas then followed by acids and alcohols. However, under sugarcane molasses or nitrogen-rich matrix, alcohols were the main volatile flavor compounds, followed by esters and acids, which might be ascribed to proteins, vitamins, and trace elements in sugarcane molasses [32] and soybean meals [33], facilitating the production of higher alcohols via the Ehrlich pathway [34].

4.2. CM15 isolated from Koji exhibited excellent stresses tolerance

During the Baijiu brewing, ester is mainly produced from alcohol and organic acid or acyl-CoA under the catalysis of enzymes, such as lipase, esterase, and alcohol acyltransferase [26]. Factors that affect the growth and metabolism of microorganisms will affect the formation of esters, including the titer of alcohol, organic acid, and sugar in the medium and the level of temperature and oxygen [9]. So, the isolated ester-producing yeasts should show both the excellent ability to produce aromas and the outstanding capability to tolerate the harsh conditions during brewing.

(i) To assess the tolerance of CM15 to the osmotic pressure

High-density fermentation exhibits many advantages when compared with that of the traditional fermentation, such as the enhanced production of products, high efficiency of equipment utilization, and reduced labor and energy [6]. However, high sugar content would cause high osmotic pressure, and inhibit the growth and metabolism of the yeast, resulting in the bad quality of wine [18]. In order to adapt to this situation, yeasts might selectively absorb cations by plasma membrane, and accumulate some compatible substances such as glycerol and trehalose to maintain the osmotic pressure to resist the outflow of intracellular water molecules at the first stage [35]. So, a long lag period was observed, followed by the relief of osmotic pressure after the growth and metabolism of yeasts. When the yeast was cultured under 200 g/L sugar at 28°C, the lag phases took –7 h, and took 11 h when at 280 g /L sugar [36]. CM15 could grow well under 150 g/L glucose and was inhibited significantly at 250 g/L. Eventually, CM15 could tolerate 450 g/L glucose with the viable cell at 8 × 10⁷ CFU/mL (Figure 4A), suggesting that during the brewing, CM15 was inhibited by the high osmotic pressure at the first stage and with the time going on, CM15 that could tolerate 450 g/L glucose would thrive to produce flavor compounds at post fermentation period.

It is reported that the cessation of growth and fermentation under 70 g/L xylose seemed to be attributed to salt formed by neutralizing organic acid rather than the toxicity of ethanol [37]. The growth of yeasts was significantly inhibited under 100 g/L NaCl [38]. During the fermentation, the cell growth and glucose consumption were significantly improved when the salt tolerance of Torulopsis mogii was enhanced under 140 g/L NaCl with biotin addition [39]. In this study, the viable cell number of CM15 began to decrease at 80 g/L NaCl and finally CM15 could tolerate 120 g/L NaCl with 2 × 10⁶ CFU/mL viable cell and 160 g/L NaCl with 4 × 10⁵ CFU/mL viable cell (Figure 4B). Data above indicated that CM15 with excellent high osmotic tolerance could be used in Baijiu or soy sauce fermentation to generate more flavor substances.

(ii) To evaluate the tolerance of CM15 to ethanol stress

Once the yeast steps into the log phase, sugar will be consumed fast and osmotic pressure is released along with ethanol produced, which is also toxic to microorganisms at high concentration. In some yeasts, when the alcohol concentration is over 4% vol, the uptake rate of sugar and amino acids can be reduced by 50% [40]. When the alcohol content is 12% vol, the number of viable cells decreases significantly [41], owing to the inhibited plasma membrane ATPase of yeasts caused by ethanol [42]. In this study, the number of viable CM15 treated with 8% vol ethanol was higher than that under 0–6% vol and then decreased sharply with the increase of ethanol content, indicating that the alcohol tolerance of CM15 might be stimulated at 8% vol, with the changed composition of the cell wall and membrane [43]. It is reported that the doubling time of Saccharomyces cerevisiae was significantly prolonged under ethanol stress (11% vol), which could be significantly reduced by improving the ethanol tolerance of yeasts [44]. The indigenous Saccharomyces cerevisiae, such as CHO2 and FF07, isolated from mezcal distilleries in Oaxaca State was able to grow in YEPD medium with 12% vol ethanol after 72 h cultivation [45]. In this study, CM15 could tolerate 12% vol ethanol stress, and the number of viable cells was about 1.4 × 10⁷ CFU/mL after 48 h cultivation (Figure 4C), suggesting that CM15 had the excellent tolerance to ethanol stresses.

(iii) To appraise the tolerance of CM15 to the temperature

Ester-producing yeasts should also withstand high temperature. The optimum temperature of most yeasts to produce ethanol is 30°C–35°C, and in summer, the room temperature of most regions can easily reach to 40°C [28]. Meanwhile, heat can also be produced during fermentation, which would influence both the cell activity and enzyme activity. High temperature can also cause oxidative stress by generating reactive oxygen species in yeasts [46] and change the physical and chemical properties of cell components, resulting in cell membrane damage and metabolic activities reduction [47]. Therefore, high temperature-tolerant strains are beneficial for the accumulation of metabolic products during the fermentation. As shown in Figure 4D, CM15 could withstand the high temperature of 42°C, and at that point, the number of viable cell was 5 × 10⁷ CFU/mL, which exhibited a better temperature tolerance than that of cachaca yeast resisted 37°C [48] and showed comparable temperature tolerance to indigenous Saccharomyces cerevisiae CH7 and PA18 under 42°C [45].

(iv) To determine the tolerance of CM15 to low pH

The low pH of the fermented mash can not only inhibit the contamination of bacteria but also be beneficial for the formation of esters [49]. However, low pH can change the electrolytes in cells [50] and affect the growth and metabolism of microbes, resulting in insufficient utilization of substrates. It is reported that with acetic acid concentrations ≥0.8% w/v, ethanol fermentation was completely inhibited in mashes at pH 4 [51]. The selection of yeasts that grow well under acidic stress is extremely important. As shown in Figure 4E, the optimal growth of CM15 could be obtained at pH 4. When pH value decreased to 2.5, the number of viable CM15 was about 1 × 10⁷ CFU/mL, which was comparable to pectinolytic yeast isolated from fermented cocoa beans with optimal growth at pH 5 and a very low growth rate at pH 2 [52].

4.3. CM15 isolated from Koji could fortify Baijiu brewing with enhanced quality

Nowadays, one of the problems that hinders the development of rice-flavor Baijiu is the lack of aromas. To make up for this insufficiency, CM15 that exhibited both the excellent ability to produce volatile flavor compounds and the good quality to tolerate various stresses was synergistically fermented with six kinds of Kojis from different distilleries in Guangdong. CM15 could significantly fortify Koji B and F to improve the content of ethyl acetate and ethyl lactate in both rice-flavor Baijius (Figure 5A), along with improved total esters and reduced total acids produced (Figure 5B), especially with Koji F. When Koji F was fortified with CM15, the resultant Baijiu showed higher contents of esters and lower titer of higher alcohols than that of Baijiu brewing only with Koji F. It is reported that the proper content of higher alcohol can increase the mellowness of wine, but too high content can easily lead to hangover after drinking [53]. The fortification of Koji F with CM15 could increase ester content and reduce alcohol titer during the Baijiu brewing, which might be related to the changed microbial community composition and the metabolic activity of microbiota reported in Baijiu brewing with the addition of functional strains [4,30].

As stated above, CM15 was the best one among eight ester-producing yeasts for ester production and also exhibited the excellent tolerance to high-temperature, high osmotic pressure, high alcohol content, and the low acidity. Finally, CM15 was used to fortify six kinds of Guangdong’s Kojis for rice-flavor Baijiu brewing, with which the total content of ethyl acetate and ethyl lactate as the main body of aroma had been improved in Koji B, Koji C, Koji D, and Koji F, separately. Especially with Koji F, the content of esters had been significantly improved, along with the proportion of higher alcohols in liquor decreased.

4.4. Practical application and future research prospects of CM15

The aroma-producing yeast was widely used during the production of traditional fermented foods, such as wine [54], Baijiu [55], and soy sauce [56]. CM15 that showed both the stronger ability to utilize substrates to produce esters and the excellent tolerance to industrially relevant stress factors could improve the quality of rice-flavor Baijiu, which might be also appropriate to be used as the potential functional microorganism to improve the quality of some other fermented foods. Meanwhile, phenethyl acetate is the highly desirable aroma chemical with honey-aroma flavor but with high price and limited availability [57]. As shown in Figure 3, the amount of phenethyl acetate had ranked top two among esters produced by CM15 under glucose, sugarcane molasses, or nitrogen-rich medium, suggesting that CM15 might be the excellent phenethyl acetate producer and could be used for phenethyl acetate from renewable feedstock, such as L-phenylalanine.

To make CM15 to be the chassis microorganism for industrial applications, the genome and pathways of ester formation in CM15 should be revealed, from which, some valuable genes could be obtained for genetic engineering and metabolic engineering. At the same time, the genetic operation platforms should be constructed, by which, CM15 would be engineered to be an excellent producer of phenethyl acetate or other desirable aroma chemicals. CM15 as the functional microorganism could improve the quality of Baijiu, and the mechanisms behand, such as the change of the microbial community composition and metabolism activity during Baijiu brewing caused by CM15, are deserved to be further researched by meta-genomics, meta-transcriptome, and metabolomics analyses.

In conclusion, CM15 selected from Kojis showed both the stronger ability to utilize substrates to produce esters and the excellent tolerance to industrially relevant stress factors. When synergistically fermented with six kinds of Kojis from industries of rice-flavor liquor in Guangdong, the enhanced total esters had happened to the liquors brewing with the fortified four kinds of Kojis, especially with Koji F. When Koji F was fortified with CM15 for Baijiu brewing, the resultant Baijiu had higher ester proportion and lower higher alcohol ratio than that of Baijiu brewing only with Koji F, with the content of ethyl acetate and ethyl lactate increased by 25% and 214%, respectively. This study suggested that CM15 selected from Kojis can be used as a functional microorganism to fortify Baijiu brewing and the change of the microbial community composition and metabolism activity during Baijiu brewing caused by CM15 deserved for further research.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Acknowledgments

This research was supported by the Research Capacity Enhancement Project of Key Discipline in Guangdong Province (2021ZDJS005), Guangdong Provincial Agricultural Science and Technology Innovation and Extension Project (2023KJ101), Excellent doctoral research funds (KA22016B778), and Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology (2021B1212040013).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21655979.2023.2255423.

Additional information

Notes on contributors

Chunyun Qu

Chunyun Qu analysis and interpretation of the data, drafting the work, final approval of the version to be published

Liying Peng

Liying Peng acquisition of data for the work, reviewing the work critically for important intellectual content, final approval of the version to be published

Yongtao Fei

Yongtao Fei analysis and interpretation of the data, reviewing the work critically for important intellectual content, final approval of the version to be published

Jinglong Liang

Jinglong Liang acquisition of data, reviewing the work critically for important intellectual content, final approval of the version to be published

Weidong Bai

Weidong Bai design of the work, reviewing the work critically for important intellectual content, final approval of the version to be published

Gongliang Liu

Gongliang Liu design of the work, reviewing the work critically for important intellectual content, final approval of the version to be published, funding acquisition.