Screening and Aroma Characteristics of Ester-Producing Yeasts as Starter Cultures for Shanxi Aged Vinegar

CHEN Jia, XING Xiaoying, FENG Zhihong, WANG Rufu*

(Horticulture College, Shanxi Agricultural University, Taigu 030801, China)

Abstract: The purpose of this study was to select starter cultures for the production of Shanxi aged vinegar. We isolated 3 ester-producing yeast strains from fermented grains used in the traditional fermentation process of Shanxi aged vinegar.Two of them were identified as Candida ethanolica, while the remaining one as Pichia manshurica (named as Y14) by morphological characterization and sequence analysis of the 26S rDNA D1/D2 domain. These yeast strains were used in the production of Shanxi aged vinegar, and the resultant vinegar products was subjected to ester composition analysis using headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and sensory evaluation. The results showed that the concentrations of total ester and ethyl acetate in the vinegar fermented by a mixture of P. manshurica Y14 and Daqu (a Chinese traditional fermentation starter used in vinegar production) were increased by 40.5% and 1.3 times compared with the control group. The vinegar was richer esters and fl avor components. P. manshurica Y14 shows promise for use as a new starter culture for Shanxi aged vinegar.

Keywords: Shanxi aged vinegar; ester-producing yeast; aroma production; screening; fermentation

Vinegar is used in food preparation throughout the world[1]. In northern China, the most well-known traditional vinegar is Shanxi post-mature vinegar, which, as for vinegars in general, is treated by the Chinese people as a favorite condiment, health product, and even as a medicine[2]. In contrast to vinegars produced in Italy, France, Germany,and other European countries that utilize the pure-culture submerged fermentation method[3], Shanxi aged vinegar is produced via traditionally solid-state fermentation by multistrains using cereals as raw materials; thus, it has an intense aroma and mellow taste[4]. Accordingly, aroma serves as an important indicator in the quality assessment of Shanxi aged vinegar[5]. Esters, which exhibit an intense aroma, are the important components of the unique aroma of vinegar. Shanxi post-mature vinegar has an exceptionally high ester content,in which, ethyl acetate comprises the highest proportion,accounting for over 60% of the aroma composition,followed by those of butyl acetate, ethyl caproate, and hexyl acetate[6]. In particular, ethyl acetate can be produced by yeast metabolism[7]. During vinegar fermentation, the ester synthesis in yeasts relies on the degradation reaction of acetyl-CoA and alcohols. The main esters synthesized by yeasts include ethyl acetate, isoamyl acetate, ethylbenzene,ethyl isovalerate, ethyl octanoate, azelaic acid ethyl ester,ethyl decanoate, and ethyl hexyl. Therefore, the large quantity of esters synthesized by ester-producing yeasts can overcome the issue of insufficient aroma. Currently, in addition to production of vinegar, ester-producing yeasts are widely used in the production of traditionally fermented foods such as Baijiu liquo[8], wine[9], soy sauce[10]and pickles[11], and are also found in the fermentation starter Daqu[12]. Esterproducing yeasts mainly belong to the genera Candida,Pichia, and Zygosaccharomyces[13], and yeasts of different genera and species synthesize various types and quantities of esters owing to differences in fermentation raw materials and conditions[14].

The purpose of this study was to isolate and purify esterproducing yeast strains from the traditional fermentation of Shanxi aged vinegar and identify these strains via 26S rDNA sequencing. Furthermore, we attempted to enhance the ester contents in Shanxi aged vinegar and thereby improve the product quality by adding these ester-producing yeasts to the fermentation, and then analyzing the esters in the finished vinegar by using headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

1 Materials and Methods

1.1 Materials and reagents

Daqu was provided by Shanxi Zilin Vinegar Industry Co. Ltd. (Taiyuan, Shanxi Province, China).

Rose-Bengal Agar (Qingdao Hopebio-Technology Co. Ltd., Qingdao, China); yeast extract peptone dextrose(YEPD)(Shanghai Guangrui Biotechnology Co. Ltd.,Shanghai, China); YEPD agar (Shanghai Rongchuang Biotechnology Co. Ltd., Shanghai, China); Angel yeast(Angel Yeast Co. Ltd., Hubei, China); DNA Kit (Product No:CW0569, Beijing Comwin Biotech Co. Ltd., Beijing, China);T3-Cloning-Vector, pEASY@-T3 cloning Kit (TransGen Biotech, Inc., Beijing, China). All the standards were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA).

1.2 Instruments and equipment

CX23 Microscope (Olympus Co. Ltd., Tokyo, Japan);ND-1000 spectrophotometer (Nano-Drop Technologies Inc.,Wilmington, DE, USA); ABI-9700 PCR instrument (Applied Biosystems Inc., California, USA).

1.3 Methods

1.3.1 Screening of yeast strains

To screen for yeast strains in the traditional Daqu fermentation culture, we added 10 g of accurately weighed alcohol fermentation mash to 90 mL of physiological saline solution, followed by shaking at 28 ℃ for 30 min. Then,the solution was serially diluted to 10−5and 10−6, and a total of 100 μL of diluent was spread onto Rose-Bengal Agar,followed by incubation at 28 ℃ for 48 h. Single colonies with typical characteristics of yeasts were isolated and purified by streaking. Strains with cellular characteristics of yeasts were inoculated onto YEPD slants and stored at 4 ℃until use. The activated yeasts were inoculated onto YEPD agar and incubated at 28 ℃ for 48 h to observe their colony morphology. Briefly, a single drop of 0.05% methylene blue reagent was mixed with a minute amount of a colony on a glass slide and overlain with a cover slip, and then the cellular morphology was observed under a microscope.

1.3.2 Analysis of ester-producing strain

The isolated yeasts (numbered Y1-Y19) were activated and inoculated into 80 mL of ester-producing fermentation broth (8% glucose, 1% yeast extract, and 2% peptone) at a content of 3% inoculum, followed by static incubation at 28 ℃ for 7 days, thereafter, the total ester content in the fermentation broth was measured.The control strain for ester production was Angel Yeast(Saccharomyces cerevisiae A1).

1.3.3 Yeast genomic DNA extraction

The yeast genomic DNA was extracted from each strain according to the instructions provided with the Yeast Genomic DNA Kit. The DNA purity and concentration were determined using an ND-1000 spectrophotometer. The genomic DNA samples were stored at −80 ℃.

1.3.4 Primer design and synthesis

For species identification of the isolated yeast strains, a pair of specific primers was designed using Primer Premier 5.0 software (PREMIER biological software company,California, USA) based on the sequence of the D1/D2 region near the 5’-end of the 26S rDNA locus of a representative yeast strain in GenBank (Accession No. KC442921.1). The primer sequences were (5’-3’): NL-1 (GCA TAT CAA TAA GCG GAG GAA AAG) and NL-4 (GGT CCG TGT TTC AAG ACG G). The primers were synthesized by the Beijing Genome Institute Co. Ltd. (Liuhe, Beijing, China).

1.3.5 Conventional PCR assay

Genomic DNA samples of different yeasts were used as the templates and a 15 μL PCR reaction system was established as follows: 0.6 μL each of 10 μmol/L forward and reverse primers, 0.8 μL of genomic DNA template,5.5 μL of ddH2O, and 7.5 μL of 2 × Es Taq Master Mix (with dyes, Beijing Comwin Biotech Co. Ltd., Beijing, China).The PCR reaction conditions were set as follows: 94 ℃ for 3 min; 94 ℃ for 30 s, 60 ℃ for 30 s, and 72 ℃ for 30 s for 35 cycles; 72 ℃ for 5 min. The PCR products were analyzed by electrophoresis on a 1.0% agarose gel.

1.3.6 Sequencing

The recovered DNA fragments were ligated to the T3-Cloning-Vector according to the user’s guide provided with the pEASY®-T3 cloning Kit and incubated at 25 ℃for 10 min. Then, the ligation products were transformed into competent cells, followed by blue-white screening on Luria Broth (LB) agar medium. White colonies were picked and cultured in LB broth medium. The broth cultures were sequenced by BGI Life Tech Co. Ltd. (Beijing, China).

1.3.7 Small-scale Shanxi aged vinegar fermentation

To produce Shanxi extra-aged vinegar, 10 kg of ground sorghum was evenly mixed and immersed in 6.5 kg of cold water for 14 h, followed by being steamed for 3 h. When the material was cooled to 25 ℃, 6.3 kg of fi nely ground Daqu and 27 kg of cold water were added and evenly mixed for alcoholic fermentation (termed Lao). Lao was subjected to open fermentation with stirring 4 times daily for the first 3 days at low temperature to improve the air content and reduce the evaporation of ethanol in Lao. This was followed by closed fermentation for 18 days. The fermenting mash was then mixed with 11 kg of bran and 7.5 kg of carvings of millet, which was used to increase the porosity for oxygen uptake, and dispensed into 12 shallow containers for acetic fermentation (termed Pei). Each container was supplemented with 0.7 kg of vinegar “seeds”, which were saved from the previous batch of vinegar Pei. The material was then subjected to acetic fermentation for 9 days and stirred twice daily, at this point, 5% salt was added to prevent further oxidization of the acetic acid. Half of the fermented grains was heated at 90 ℃ for 5 days and stirred once daily (termed Xun Pei or roasting); water was added to the other half of the Pei and immersed for 4 h to dissolve out any soluble components (the leached solution is termed Lin). The Lin was heated to over 90 ℃ and used to immerse the Xun Pei for 12 h.After fi ltration, the resulting fi ltrate is termed “fresh vinegar”.The fresh vinegar was exposed for a year in the aging room to form the fi nished product, Shanxi post-mature vinegar. In total, 4 types of vinegar were produced from the laboratory simulation of the traditional solid-state fermentation of Shanxi aged vinegar. The starter cultures consisted of T1(Daqu, control group); T2 (Y2 + Daqu); T3 (Y14 + Daqu);and T4 (Y18 + Daqu).

The inoculum of the yeast was 7% of the total fermented mash, which was added at the same time as the activated Daqu, on the fi rst day of the alcohol fermentation.

1.3.8 Routine analysis of physical and chemical parameters in vinegar

Total acids, nonvolatile acids, reducing sugar, total nitrogen, and amino nitrogen were determined according to the GB/T 19777-2013 Product of geographical indication:Shanxi extra aged vinegar, as these standards are used for the geographical indication of Shanxi extra aged vinegar product.All the parameters were measured in triplicate.

1.3.9 Determination of volatile aroma components

The vinegar samples were centrifuged at 4 ℃ and 8 000 × g for 10 min, and the supernatant was collected and stored at 4 ℃ until use. A total of 8 mL of supernatant was drawn into a 20 mL vial and supplemented with 0.6 g of NaCl and subjected to the HS autosampler method as follows: 1) HS sampler conditions: oscillation temperature,45 ℃; oscillation time, 2 min, and injection volume, 2 mL;2) GC analysis conditions: TR-5MS column (30 m ×0.25 mm, 0.25 μm), inlet temperature, 300 ℃, carrier gas, He with a fl ow rate of 1 mL/min, injection volume, 350 μL; and split ratio, 20:1. Temperature program: initial temperature,40 ℃ for 3 min; 3 ℃/min increase up to 190 ℃ for 5 min,followed by a further 20 ℃/min increased to 270 ℃ for 5 min;3) MS conditions: electron impact ionization, electron energy,70 eV; ion source temperature, 250 ℃; transfer line temperature, 280 ℃; quad temperature, 180 ℃; and range of m/z, 35–500.

1.3.10 Sensory evaluation method

A 20 cm special cup was used for sensory evaluation;scores were given by 10 specially trained evaluators using a 10-point scale. A sample of 50 mL of Shanxi aged vinegar was dispensed into each cup labeled with only a secret code and was evaluated at 25 ℃. The vinegar was received by sight, smell, and taste in an orderly manner. The average score was used to determine the vinegar fl avor. The scoring weight assigned to appearance is 20%, color is 20%, aroma is 30%, and taste is 30% in the sensory evaluation[15].

1.4 Statistical analysis

All the data were analyzed using SPSS 17.0 software(SPSS Inc., USA) and presented as the ± s. One-way analysis of variance (ANOVA) was used to determine the statistical differences in the data. P < 0.05 was regarded as statistically signi fi cant.

2 Results and Analysis

2.1 Screening of wild yeast strains

Fermented grains from the production line of Shanxi aged vinegar were screened for yeast strains. A total of 19 yeast strains were isolated based on morphological observations (Fig. 1). The screening isolated 3, 8, 5, and 3 yeast strains from the fermented grains sampled at the 3rd, 4th,5th, and 6thdays of fermentation, respectively. These strains were assigned with strain numbers from Y1–Y19.

Fig. 1 Morphological characteristics of wild-type yeast cells

2.2 Ester production by wild yeasts

The ester production capacity of each yeast strain was determined according to the method in 1.3.2 and was shown in Fig. 2. The yeast strains Y14, Y2, and Y18 exhibited a significantly higher ester production capacity than the control group and other yeast strains did (P < 0.05), with ester yields being 36.05, 33.75 and 33.47 g/L, respectively.The fermentation broth was clear with stable fermentation characteristics. Hence, these stains appear to be ideal esterproducing yeasts.

Fig. 2 Ester production capacities of different yeast strains

2.3 Molecular identification of wild yeast strains via 26S rDNA

Fig. 3 Electropherograms of PCR amplified 26S rDNA D1/D2 domain from strains Y2, Y14, and Y18

Agarose gel electrophoresis showed that the size of the target PCR fragments was approximately 500 bp with no trailing or primer dimer (Fig. 3). The specific fragments were sequenced after ligation and transformation, and the lengths of the resulting sequences were 514, 516 and 534 bp,respectively. The results of BLAST analysis (Table 2 and Fig. 4)indicated that both Y2 and Y18 were Candida ethanolica,whereas Y14 was Pichia manshurica.

Fig. 4 Homology analysis of strains Y2, Y14, and Y18

2.4 Routine analysis of physical and chemical parameters in Shanxi aged vinegar

Routine analysis of physicochemical indices in Shanxi aged vinegar (Fig. 5) showed that the addition of yeasts significantly improved the contents of total acid, nonvolatile acid, and amino nitrogen in vinegar (P < 0.05) but did not significantly affect the content of reducing sugars (P > 0.05).The contribution of each strain to the improvement of total ester content in the Shanxi aged vinegar was in the order of Y14 > Y2 > Y18. T3 (Y14 + Daqu) vinegar, which exhibited a significantly higher total ester content than the other groups(P < 0.05).

2.5 Determination of ester composition in the generated Shanxi aged vinegars

Fig. 6 Relative contents of different ester components in the four vinegars

Table 1 GC-MS analysis results of esters in the vinegars

HS-SPME-GC-MS was used to analyze the ester contents in the 4 types of vinegar samples. The addition of these 3 ester-producing yeasts significantly improved the ester content of the resulting vinegars (P< 0.05) (Table 1 and Fig. 6). The fi nished vinegar supplemented with Y14 had abundant esters, which was consistent with the ester content measured in the fermentation broth.

2.6 Sensory evaluation of the generated Shanxi aged vinegars

Table 2 Sensory evaluation scores of the vinegar samples

The sensory evaluation results of the 4 vinegars produced by adding the 4 kinds of strains indicated that Shanxi aged vinegar supplemented with Y14 produced the best aroma (Table 2). Significant differences were obtained in the sensory evaluation scores among the various types of vinegar, which were ordered, from high to low, as Y14 >Y18 > Y2 > the control group.

3 Discussion

The traditional solid-state fermentation of Shanxi aged vinegar is a complex biotransformation process that involves multiple types of microorganisms and in which yeasts play important roles. These yeasts can be generally divided into two categories based on their roles. Specifically,S. cerevisiaeprimarily accomplishes the alcoholic fermentation and exhibits a high fermentation rate and capacity, whereas non-Saccharomycesyeasts that have lower fermentation efficiency but are capable of converting precursors in fermenting mash into fl avoring substances, such as esters, acids, higher alcohols, and aldehydes, play important roles in the formation of fl avors, textures, and colors in the fermented foods[16-21]. In this study, we utilized the 26S rDNA sequence to identify the three ester-producing yeasts isolated from the Shanxi vinegar culture asC. ethanolica(Y2 and Y18) andP. manshurica(Y14). BothCandidaandPichiaare generally considered as non-Saccharomycesyeasts[22].

Pichiayeasts are primary ester-producing yeasts that provide important contributions to fl avors[23].P. manshuricais capable of withstanding extreme conditions, such as low pH, low water activity, high osmotic pressure, and anaerobic states during the production of Shanxi aged vinegar. Oxygenlimiting conditions might induce alcoholic fermentation inP. manshuricaand activate the key enzymes of fermentation pathways that increase the contents of metabolites, such as ethanol, glycerol, and ethyl acetate[24]. Xu Zijing et al.[25]have isolatedP. sydowiorumfrom the distiller’s yeast of Jiannanchun Liquor, which has been shown by GC analysis to produce up to 505.36 mg/100 mL of ethyl acetate. Wang Xuliang et al.[26]have isolated P. anomala from the fermented grains of “light-fragrance” Baijiu liquor, which has been shown by GC to be able to synthesize up to 632.14 mg/L of ethyl acetate. In comparison, the addition of P. manshurica(Y14) isolated in this study into the fermentation culture of Shanxi aged vinegar increased the ethyl acetate content in the fi nished vinegar by 1.3 fold.

Candida yeasts are known to secrete lipase under high osmotic pressure and nutrient-limiting conditions[27]. Feng Jie et al.[9]demonstrated that the addition of C. etchellsii increased the contents of total free amino acids and total content of volatile fl avor compounds in soy sauce by 1.76%and 178.7%, respectively, as compared with that of the control group. In agreement with these fi ndings, the addition of Y2 and Y18 (C. ethanolica) isolated in this study to the fermentation of Shanxi aged vinegar enhanced the aromatic composition of esters by 32.8% and 25.7%, respectively.

Ciani et al.[6-7]found that non-Saccharomyces yeasts could inhibit the fermentation of S. cerevisiae in mixed fermentation. When multiple yeasts are fermented in the same system, their respective maximum biomasses are lower than that of the pure culture owing to the limitation of nutrients and living space[1]. As the starter culture of Shanxi aged vinegar fermentation, Daqu contains enormous microflora that are prepared by the natural inoculation of molds, bacteria,and yeasts. The identification of microorganisms in Daqu by Zheng Xiaowei et al.[28]using the polymerase chain reactiondenaturing gradient gel electrophoresis (PCR-DGGE) technique indicated that both Saccharomyces and non-Saccharomyces yeasts were simultaneously present in Daqu. Furthermore, Wu Jiajia et al.[29]demonstrated that both Saccharomyces and non-Saccharomyces yeasts were simultaneously present in Shanxi aged vinegar during the alcoholic fermentation and acetic fermentation steps. In the current study, we simulated the traditional solid-state fermentation of Shanxi aged vinegar in the laboratory and found that the degree of sugar utilization by yeasts in the mixed fermentation of Daqu and Y14 was equal to that in the control group without inhibiting the overall fermentation capacity of the yeasts.

Here, we employed the GC-MS technique to determine the esters in the resultant vinegars and found that the vinegar from the mixed fermentation of Daqu and Y14 exhibited relatively lower total acid and higher alcohol contents as well as a relatively high ester content, where the total ester content was increased by 40.5% as compared with that of the control group. This might have resulted from the conversion of alcohol and acid into esters by P. manshurica via the esterase synthesis pathway[14]. We further found that ethyl esters were the main esters in the vinegars, producing a fruity and fl oral aroma[4], with the being concentrations above the threshold level in all of the vinegar samples, consistent with the results of the sensory evaluation.

4 Conclusion

Yeasts affect the final chemicals and volatile flavor compositions of Shanxi aged vinegar. Of the identified strains,Y14 (P. manshurica) produced the largest amounts of esters,which are favorable for the fruity aroma of Shanxi aged vinegar. In addition, the use of Y2 and Y18 (C. ethanolica)could increase the formation of aroma compounds and contribute to the aroma complexity of the generated vinegar.Shanxi aged vinegar flavor and quality were successfully developed by adding the Y14 strain. This novel process was demonstrated to be feasible and reproducible for potential industrial application for the improvement of the fl avor and quality of Shanxi aged vinegar production.