Staphylococcus for producing enzyme and aroma, and application thereof in preparation of sauce-flavor high-temperature Daqu

Abstract

The application discloses a zymogenic and aroma-producing staphylococcus NHY-25, which is preserved in the Guangdong Microbial Culture Collection Center on May 26, 2025, and has a preservation number of GDMMC NO:66396. The strain is applied to the strengthening of Daqu, and can improve the enzyme activity in high-temperature Daqu, for example, the activities of protease, saccharifying power, fermenting power and liquefying power of the Daqu can be improved. Through detection of volatile compounds in the strengthened Daqu and the un-strengthened Daqu, it is found that the type and content of the volatile compounds in the strengthened Daqu are improved. The strain makes the sauce-flavor of the high-temperature Daqu more prominent, and can be applied to the strengthening of Daqu, thereby providing a new thought for improving the quality of Daqu.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, specifically to an enzyme- and aroma-producing Staphylococcus aureus NHY-25 and its application in the preparation of soy sauce-flavored high-temperature koji. Background Technology

[0002] Maotai-flavor liquor is renowned for its unique Maotai aroma, and high-temperature Daqu (a type of starter culture) serves as the core saccharification and fermentation agent, directly influencing the quality of the liquor. Traditional high-temperature Daqu uses wheat as its raw material and ferments through natural inoculation with environmental microorganisms, reaching temperatures as high as 60-65℃. During this process, the microbial community produces enzymes such as amylase and protease through metabolic activities, breaking down the starch and protein in the raw materials, while simultaneously generating aroma compounds such as pyrazines, esters, and phenols.

[0003] Natural inoculation significantly influences the microbial community of Daqu (a type of starter culture), resulting in substantial differences in enzyme activity and aroma compounds between different batches. For example, while Bacillus is the dominant genus in traditional processes, fluctuations in its abundance can lead to unstable saccharification and fermentation power. Furthermore, natural inoculation makes it difficult to selectively introduce specific heat-resistant, high-enzyme-activity strains, limiting improvements in Daqu quality. The amylase and protease activities in existing Daqu are generally low; for instance, the saccharification power of yellow Daqu is only 74.67 U, significantly lower than that of white Daqu. This is mainly due to the insufficient proportion of enzyme-producing strains in naturally inoculated microorganisms and the lack of synergistic effects. For example, while Bacillus can produce amylase, its enzyme activity enhancement is limited when used alone.

[0004] Staphylococci belong to the genus *Staphylococcus* in the family Micrococciaceae, and are Gram-positive bacteria. Based on their ability to coagulate plasma, they can be divided into two main categories: coagulase-positive and coagulase-negative. Studies have shown that Staphylococci are ubiquitous in soil, air, water sources, dust, and various fermented foods. Most are non-pathogenic and can produce various enzymes that promote the accumulation of small-molecule metabolites, thereby improving the color, flavor, and taste of food. In recent years, a large number of Staphylococci have also been found in Daqu (a type of starter culture). For example, a positive correlation has been found between Staphylococci and saccharification power in high-temperature Daqu, and nine flavor compounds produced in high-temperature Daqu are positively correlated with Staphylococci. Staphylococci significantly contribute to the enzyme activity of high-temperature Daqu, including saccharification power, fermentation power, and esterification power. However, the distribution, functional characteristics, and application potential of Staphylococci in high-temperature Daqu are still unclear, and related reports are lacking. Summary of the Invention

[0005] This invention aims to provide a Staphylococcus aureus strain NHY-25 that produces enzymes and aromas, and its application in the preparation of high-temperature Daqu (a type of starter culture) for soy sauce aroma. This strain can metabolize and produce proteases and lipases, with a k-value of 2.00 for the protease and 2.33 for the lipase. Simultaneously, through simulated fermentation, it can synthesize volatile compounds related to the flavor of Baijiu, such as acetoin (1244.32 ± 242.06 μg / L), acetic acid (731.78 ± 168.00 μg / L), and 3-methylbutyric acid (495.84 ± 69.55 μg / L). This invention also discloses that the application of this strain in Daqu fortification can improve enzyme activity in high-temperature Daqu, such as increasing the activity of proteases, saccharification power, fermentation power, and liquefaction power. Furthermore, by detecting volatile compounds in fortified and unfortified Daqu, it was found that this strain increases both the types and contents of volatile compounds in fortified Daqu. This strain enhances the soy sauce aroma of high-temperature koji and can be used for koji strengthening.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] Firstly, an enzyme- and aroma-producing Staphylococcus aureus, NHY-25, was deposited at the Guangdong Microbial Culture Collection Center on May 26, 2025, with accession number GDMMC NO: 66396.

[0008] The nucleotide sequence of the 16S rDNA of the above-mentioned Staphylococcus aureus is shown in SEQ ID NO.1.

[0009] SEQ ID NO.1

[0010]

[0011] The morphological characteristics of the Staphylococcus aureus are as follows: after being cultured on LB medium at 37°C for 48 hours, the strains were observed to have a smooth, milky-white surface and did not form spores. Under a microscope, the strains were observed to aggregate into grape-like clusters.

[0012] The enzyme production characteristics of the above-mentioned Staphylococcus on LB solid medium are: protease (k=2.00) and lipase (k=2.33).

[0013] The aroma-producing characteristics of the above-mentioned Staphylococcus on wheat liquid culture medium are as follows: it produces flavor compounds such as acetoin, tetramethylpyrazine, 2,3-butanediol, 3-methyl-1-butanol, (E)-2-heptanal, nonanal, acetic acid, furfural, decanal, 1-(2-furanyl)-ethyl ketone, 3-furanethanol, 3-methylbutyric acid, hexanoic acid, benzyl alcohol, phenylethanol, and phenol.

[0014] The flavor substance is obtained by fermenting the above-mentioned Staphylococcus aureus in wheat liquid culture medium.

[0015] Secondly, the present invention provides the use of the above-mentioned Staphylococcus aureus in the preparation of soy sauce-flavored high-temperature koji.

[0016] The use of Staphylococcus aureus in high-temperature koji for soy sauce aroma includes improving enzyme activity and flavor substance content in the koji.

[0017] The enzymes include proteases, saccharification power, esterification power, liquefaction power, and fermentation power; the flavor substances include tetramethylpyrazine, trimethylpyrazine, methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 3-ethyl-2,5-dimethylpyrazine, 2,6-diethylpyrazine, 3,5-diethyl-2-methylpyrazine, 3-methylbutyric acid, 2-methylpropionic acid, acetic acid, (R,R)-2,3-butanediol, (S,S)-2,3-butanediol, acetoin, and phenol.

[0018] Beneficial Effects: This invention isolated and screened a Staphylococcus aureus strain NHY-25 from high-temperature koji (fermentation starter) of soy sauce aroma type, with the preservation number GDMMC NO: 66396. This strain possesses protease and lipase activities and can produce various flavor compounds such as acetoin, acetic acid, and 3-methyl-1-butanol under fermentation conditions. Using this strain in the preparation of high-temperature koji for soy sauce aroma type can enhance the saccharification power, liquefaction power, fermentation power, and protease activity of the koji, and increase the content of various flavor compounds such as tetramethylpyrazine, phenol, and 3-methyl-butyric acid. This strain meets the requirements of soy sauce aroma type baijiu brewing, providing a new option for enhancing the flavor of baijiu during brewing, and has broad application prospects and great application value.

[0019] Attached Figure Description Figure 1 This is a phylogenetic tree of Staphylococcus NHY-25 constructed using the Neighbor-Joining method based on the 16S sequence, as described in this invention.

[0020] Figure 2 This is a microscopic morphological image of Staphylococcus aureus NHY-25 of the present invention;

[0021] Figure 3 This is a colony morphology diagram of Staphylococcus NHY-25 of the present invention;

[0022] Figure 4 The image shows the results of the assay for the activity of staphylococcal NHY-25 enhanced koji enzyme in this invention. Detailed Implementation

[0023] The following detailed description illustrates the specific implementation method:

[0024] A type of staphylococcus that can produce enzymes and aromas, named NHY-25, is classified as follows: Staphylococcus saprophyticus It was deposited at the Guangdong Microbial Culture Collection Center on May 26, 2025, with accession number GDMMC NO: 66396.

[0025] It was isolated from high-temperature Daqu (a type of starter culture) in a winery in Renhuai. Its biological characteristics are as follows: after culturing on LB medium at 37 °C for 48 h, the strain surface was observed to be smooth and milky white, without spore formation. Under a microscope, the strain could be observed to aggregate into grape-like clusters. The nucleotide sequence of the 16S rDNA of the Staphylococcus aureus described in this invention is shown in SEQ ID NO:1.

[0026] SEQ ID NO.1

[0027]

[0028] Example 1: Strain Screening

[0029] 1. Initial screening of strains

[0030] Materials: High-temperature Daqu (fermented starter culture) from a winery in Renhuai

[0031] Culture medium: Mannitol high salt agar (MSA) Culture medium: Commercial synthetic culture medium, purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., product model HB4128.

[0032] 2. Test methods:

[0033] High-temperature Daqu (a type of starter culture) from a winery in Renhuai was selected, pulverized, and 25 g of the sample was weighed into 225 mL of sterile physiological saline. Glass beads were added and the mixture was shaken to mix. 1 mL of this sample solution was then diluted to 10 mL. －2 10 －3 10 －4 10 －5 Four gradients were applied, and each sample dilution was then spread onto mannitol high-salt agar (MSA) plates. The plates were inverted and incubated at 37°C for 48 hours. After colonies appeared, they were observed, and the purified strains with good morphology were selected and stored at 4°C. Figure 2 As shown.

[0034] Example 2: Morphological characteristics of the Staphylococcus aureus of the present invention

[0035] In all the following experiments, freshly activated bacterial cells were used. A small amount of bacterial cells was picked and inoculated into LB liquid medium and incubated at 37°C for 48 h. Colony and cell morphology observation: The activated Staphylococcus bacteria were streaked onto LB solid medium using an inoculation loop and incubated at 37°C for 48 h. Colony morphology was observed. A small amount of bacterial cells was picked up with an inoculation needle and observed under a microscope. Cell observation results: Under the microscope, Staphylococcus strains were observed to often cluster in grape-like patterns. Figure 2 As shown. Colony observation results: The surface of the strain was observed to be smooth, milky white, and it did not form spores. Figure 3 As shown.

[0036] Example 3: Molecular biological identification of the strain

[0037] DNA was extracted from the target strain using a bacterial genomic DNA extraction kit. Using this DNA as a template, 16S rDNA amplification was performed using universal primers 27F and 1492R. PCR amplification conditions were: 95 ℃ for 30 s; 58 ℃ for 30 s, 72 ℃ for 90 s, 35 cycles; 72 ℃ for 5 min. The amplified products were purified and sequenced (Beijing Qingke). The sequencing results were compared with the NCBI database using BLAST. By comparing the 16S sequence with the strains exhibiting the highest sequence homology, the *Staphylococcus* species were determined, and a phylogenetic tree was constructed using MEGA11. The PCR amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the NCBI database using BLAST. By comparing the 16S sequence with the strains exhibiting the highest sequence homology, the *Staphylococcus* species were determined, and a phylogenetic tree was constructed using MEGA11. Staphylococcus saprophyticus The strains showed 100% homology, and their 16S sequence phylogenetic tree ( Figure 1 Display and Staphylococcus saprophyticus Based on homology, combined with colony morphology, bacterial cell characteristics, and 16S rDNA gene sequence analysis, it was identified as Staphylococcus aureus and named NHY-25.

[0038] Its biological characteristics are as follows: after being cultured on LB medium at 37 °C for 48 h, the surface of the strain is smooth and milky white, and no spores are formed. Under a microscope, the strain can be observed to aggregate into grape-like clusters.

[0039] Example 4: Enzyme-producing properties of strain NHY-25 of the present invention

[0040] Culture media: protease medium, lipase medium, LB liquid medium (1 g sodium chloride, 1 g peptone, 0.5 g yeast extract, 100 mL sterile water).

[0041] Protease activity assay: The screened pure colonies were activated and inoculated into protease medium, and cultured at 37 °C for 2 days. The presence or absence of a clear zone around the colonies was observed on the protease medium to determine the presence of protease activity. Protease medium: 15% skim milk powder was added to the original LB liquid medium, and then sterilized at 121 °C for 20 min.

[0042] Lipase activity assay: The screened pure colonies were activated and inoculated into lipase medium, and cultured at 37 ℃ for 2 days. The presence or absence of a clear zone around the colonies was then observed to determine lipase activity. Lipase medium: Based on the original LB liquid medium, the medium was sterilized at 121 ℃ for 20 min. After cooling to 55 ℃, a well-mixed mixture of glyceryl tartrate and polyvinyl alcohol was added to the medium, and then poured into plates. Referring to Table 1, the activities of the strain's protease (k=2.00) and lipase (k=2.33) were observed.

[0043] Table 1 shows the enzyme production characteristics of Staphylococcus NHY-25 in this invention.

[0044]

[0045] This indicates that strain NHY-25 can secrete proteases and lipases with significant enzyme activity, possessing the ability to decompose proteins and fats, thus laying the foundation for material transformation during Daqu fermentation.

[0046] Example 5: Analysis of the main flavor components in the fermentation broth of strain NHY-25 of the present invention

[0047] Culture medium: Wheat liquid culture medium. Take crushed wheat from the koji-making workshop, add water at a ratio of 1:6, boil for 10 min, cool to approximately 70 ℃, add α-amylase at a ratio of 15.2%, stir well, cool to 55 ℃, add acidic protease at a ratio of 1%, incubate at 55 ℃ for 30 min, filter, adjust pH to 6.2, and adjust sugar content to 5°Bx. Pretreatment method for flavor substances in the fermentation broth: Accurately pipette 1.0 mL of fermentation broth into a headspace vial, and add 10 μL of 2-octanol (concentration 6.2 mg / L).

[0048] Flavor compounds in the fermentation broth were extracted after pretreatment and then analyzed by HS-SPME. Separation was performed according to the following program: GC-MS conditions: The instrument was an Agilent 6890N-5975B equipped with a DB-WAX (60 m × 250 μm, 0.25 μm) column; the injection port temperature was 250 ℃; the carrier gas was high-purity helium (>99.999%), with a constant flow rate of 1 mL / min, splitless mode; the temperature program was 40 ℃ for 5 min, increasing to 100 ℃ at 4 ℃ / min, increasing to 230 ℃ at 6 ℃ / min, and holding for 10 min; the ion source temperature was 250 ℃, the transfer line temperature was 300 ℃, the ionization mode was EI (70 eV), and the scan range was 35-400 μm.

[0049] As can be seen from the above examples, the NHY-25 strain of the present invention can be used to ferment and produce a variety of flavor compounds, especially acetoin (1244.32 ± 242.06 μg / L), acetic acid (731.78 ± 168.00 μg / L), and 3-methylbutyric acid (495.84 ± 69.55 μg / L). Acetoniin has a pleasant buttery and sweet taste and is also a necessary precursor for the synthesis of tetramethylpyrazine and 2,3-butanediol, which has a significant impact on the flavor of baijiu (Chinese liquor). See Table 2.

[0050] Table 2 shows the results of flavor compound production by simulated fermentation of Staphylococcus aureus NHY-25 in this invention.

[0051]

[0052] This indicates that strain NHY-25 can produce a variety of flavor compounds under simulated Daqu fermentation conditions, especially acetoin (a sweet precursor in sauce-flavored baijiu) and esters, confirming that its aroma-producing ability is directly related to the formation of Daqu flavor.

[0053] Example 6: Determination of enzyme activity of the enhanced Daqu strain NHY-25 of the present invention

[0054] Strengthened Daqu: Pure wheat is moistened with hot water and then pulverized according to the preparation process requirements of high-temperature Daqu for soy sauce aroma, and mixed with water to form Daqu embryos. The target strain NHY-25 is activated using an activation medium. After confirming the absence of contamination, it is then processed according to a 10... 6 The inoculum was inoculated into the Daqu (a type of starter culture) at a rate of cells / mL and fermented for 40 days.

[0055] Saccharification power determination: Weigh 10 g of Daqu powder into a 250 mL beaker, add 20 mL of water and acetate-sodium acetate buffer solution, adjust the water temperature to 35 ℃, soak for 1 h, and then filter. Take a test tube, add 25.0 mL of soluble starch solution and 5.0 mL of Daqu sample solution, shake well, add 1 mL of 20% NaOH solution, and pipette 5.0 mL as a blank solution. Titrate with glucose standard solution and record the volume V1. Take another test tube, repeat the above sample addition operation, place in a 35 ℃ constant temperature water bath for saccharification for 1 h, add 1 mL of 20% NaOH solution, take 5.0 mL of saccharified solution and 5.0 mL each of Fehling's solution A and B into an Erlenmeyer flask, add 10 mL of water, titrate, and record the volume V2. Saccharification power of the blank group: 265.90 U, saccharification power of the experimental group: 369.05 U. Figure 4 As shown.

[0056] Liquefaction power determination: Weigh 10 g of Daqu powder into a 250 mL beaker, add 90 mL of water and 10 mL of pH 4.6 acetate-sodium acetate buffer, adjust the water temperature to 35 ℃, and soak for 3 h (stirring every 15 min). Filter with defatted cotton and set aside. Take 20 mL of 2% soluble starch solution and 5 mL of water into a 50 mL colorimetric tube, accurately add 10 mL of Daqu leaching filtrate in a 35 ℃ constant temperature water bath, shake thoroughly, and start timing. At regular intervals, add approximately 0.25 mL of the reaction solution to a colorimetric tube containing dilute iodine solution (approximately 3 mL), observe the color, and record the time when the solution no longer changes color (close to the original color of iodine solution). Liquefaction power of the blank group: 0.29 U, liquefaction power of the experimental group: 0.55 U. Figure 4 As shown.

[0057] Esterification power determination: 25 g of Daqu powder was added to a 250 mL Erlenmeyer flask containing 1.5 mL of hexanoic acid, 25 mL of anhydrous ethanol, and 75 mL of distilled water. The esterification was carried out in an incubator at 35 ℃ for 7 days. The esterified solution was then transferred to a 250 mL distillation flask and heated until the distilled water reached 50 mL. After saponification with standard NaOH (0.1 mol / L) solution, the total ester content was determined by titration, and the esterification power could be calculated. The esterification power of the blank group was 561.41 U, and that of the experimental group was 602.81 U. Figure 4 As shown.

[0058] Fermentation power determination: 0.5 g of Daqu powder was added to a 100 mL Erlenmeyer flask containing 50 mL of sterilized saccharification solution (7° Bé). The flask was then filled and sealed with 5 mL of sulfuric acid solution (2.61 mol / L), and the total weight was recorded. The mixture was then incubated at 30 ℃ for 72 h. The fermentation tank was removed to remove CO2, and the mixture was weighed again. Finally, the fermentation power of the Daqu was calculated based on the mass difference before and after fermentation. Fermentation power of the control group: 0.51 U; Fermentation power of the experimental group: 1.13 U. Figure 4 As shown.

[0059] Protease activity assay: 5 g of Daqu powder was dispersed in 100 mL of distilled water and stirred in a 40 ℃ water bath for 60 min. After filtration, the solution was diluted 10-fold with PBS buffer (0.1 mol / L, pH 7.2). The amount of tyrosine hydrolyzed from casein by 1 mL of the filtrate was then measured at 40 ℃ for 10 min to calculate the protease activity. Compared with the control group, the strain increased protease activity in Daqu. The protease activity in the blank group was 0.38 g / 100 g·3 h, and the protease activity in the experimental group was 0.65 g / 100 g·3 h. Figure 4 As shown.

[0060] This indicates that strain NHY-25 can significantly enhance the activity of hydrolytic enzymes (protease, amylase) and alcohol-producing enzymes in Daqu (a type of starter culture), optimize the saccharification and fermentation capacity of Daqu, and provide a highly efficient enzyme system for Baijiu (Chinese liquor) brewing.

[0061] Example 7: Detection of flavor substances in Daqu (a type of Chinese liquor) enhanced by strain NHY-25 of the present invention.

[0062] 1.00 g of Daqu (a type of Chinese liquor) and 10 µL of internal standard (2-octanol, 6.2 mg / L) were accurately added to a 20 mL headspace vial. Extraction was performed using a 50 / 30 µm DVB / CAR / PDMS fiber at 60 °C for 50 min, followed by desorption at 250 °C for 5 min. GC conditions: inlet temperature 250 °C; carrier gas: high-purity helium (99.999%); flow rate: 1 mL / min; column temperature: 40 °C, held at 40 °C for 5 min, ramped up to 100 °C at 4 °C / min, ramped up to 230 °C at 6 °C / min, held for 10 min. MS conditions: ion source temperature, 250 °C; transfer line temperature, 300 °C; ionization mode, EI (70 eV); scan range: 35–400 μ-m. After comparison with the NIST 2023 library, compounds with ≥80% similarity were retained for further analysis.

[0063] Table 3 shows the results of flavor compounds produced by the Staphylococcus NHY-25 enhanced Daqu (a type of starter culture) of this invention.

[0064]

[0065] The flavor detection results of Daqu (a type of Chinese koji) with and without inoculation with strain NHY-25 are shown in Table 3. The blank group did not produce 2-ethyl-5-methylpyrazine, 3-methylbutyric acid, acetoin, or phenol. In Daqu inoculated with strain NHY-25, the contents of these substances were as follows: 2-ethyl-5-methylpyrazine (68.04±12.15 mg / L), 3-methylbutyric acid (18.41±3.33 mg / L), acetoin (291.25±33.50 mg / L), and phenol (131.68±38.96 mg / L).

[0066] The concentrations of tetramethylpyrazine increased from 44.53±7.84 mg / L to 99.81±32.05 mg / L, trimethylpyrazine from 16.98±3.27 mg / L to 42.30±17.56 mg / L, 2,6-diethylpyrazine from 2.50±0.95 mg / L to 6.48±3.93 mg / L, 2,3-dimethylpyrazine from 1.92±0.21 mg / L to 8.13±4.16 mg / L, 3,5-diethyl-2-methylpyrazine from 0.81±0.20 mg / L to 5.58±1.83 mg / L, methylpyrazine from 1.62±0.53 mg / L to 2.10±0.75 mg / L, and 3-ethyl-2,5-dimethylpyrazine from 0.57±0.11 mg / L. The concentrations of acetic acid, 2-methylpropionic acid, (R,R)-2,3-butanediol, (S,S)-2,3-butanediol increased from 0.32±0.09 mg / L to 5.09±2.89 mg / L, acetic acid from 0.63±0.38 mg / L to 2.89±1.21 mg / L, (R,R)-2,3-butanediol from 3.10±1.28 mg / L to 7.77±2.46 mg / L, and (S,S)-2,3-butanediol from 1.95±0.64 mg / L to 7.35±3.09 mg / L.

[0067] This indicates that strain NHY-25 significantly enriches the aroma components of Daqu (a type of Chinese koji) by producing key flavor compounds such as pyrazines, phenols, and esters through metabolism, especially enhancing characteristic flavors such as soy sauce aroma and roasted aroma, thus verifying its core role in improving the flavor of Daqu.

[0068] In summary, Staphylococcus NHY-25 ( Staphylococcus saprophyticus This strain has a clear ability to produce enzymes (proteases, lipases) and aromas (acetoin, tetramethylpyrazine, etc.). Through simulated fermentation and Daqu enhancement experiments, it has been confirmed that this strain can enhance the enzyme activity (saccharification power, esterification power, etc.) and flavor substance diversity (especially pyrazines) of Daqu. It provides a highly efficient functional strain for the quality improvement of soy sauce-flavored high-temperature Daqu and has significant industrial application value.

Claims

1. A Staphylococcus aureus NHY-25 that produces enzymes and aromas, characterized in that: It was deposited at the Guangdong Microbial Culture Collection Center on May 26, 2025, with accession number GDMMC NO: 66396.

2. The Staphylococcus NHY-25 according to claim 1, characterized in that: The Staphylococcus NHY-25 strain was screened from Moutai high-temperature Daqu (a type of Chinese liquor), and its 16S rDNA is shown in SEQ ID NO.

1.

3. The use of Staphylococcus NHY-25 according to any one of claims 1 to 2 in the production of protease and lipase.

4. The application of Staphylococcus aureus NHY-25 as described in claim 3 in improving the content of flavor substances, characterized in that: The flavoring substances include tetramethylpyrazine, trimethylpyrazine, methylpyrazine, 2,3-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 3-ethyl-2,5-dimethylpyrazine, 2,6-diethylpyrazine, 3,5-diethyl-2-methylpyrazine, 3-methylbutyric acid, 2-methylpropionic acid, acetic acid, (R,R)-2,3-butanediol, (S,S)-2,3-butanediol, acetoin, and phenol.

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