Two strains of non-saccharomyces and a method for making wine using a composite starter thereof

Abstract

This invention relates to the field of wine production technology, and particularly to two aroma-producing yeasts capable of withstanding environmental stress, their compound fermentation agent, and a method for winemaking. The compound fermentation agent comprises *Hansenula polymorpha* CVE-HU47 (exhibition number CGMCC NO.24818) and *Megamiprid* CVE-MP27 (exhibition number CGMCC NO.24819). During winemaking, the compound fermentation agent is first inoculated, and after 72-80 hours of fermentation, *Saccharomyces cerevisiae* CECA is inoculated. After 200-240 hours of fermentation, a wine with unique local Chinese flavor characteristics can be obtained. This invention can improve the sensory quality of wine, including acidity and aroma quality, effectively enhancing the complexity and diversity of wine aromas.

Description

Technical Field

[0001] This invention relates to the field of wine production technology, and in particular to two non-brewing aroma-producing yeasts and their compound fermentation agent and a method for brewing wine. Background Technology

[0002] Grape wine fermentation is a complex microbial reaction process, in which yeast plays a crucial role. Yeasts convert reducing sugars in grape juice into alcohol and carbon dioxide through respiration, while simultaneously producing various secondary metabolites, such as esters, higher alcohols, organic acids, and aldehydes and ketones, which significantly contribute to the taste and aroma of the wine. Aroma is one of the important indicators for evaluating wine, especially terpenes and esters; wines with complex, balanced aromas and unique flavors are more favored by consumers. Volatile aroma components in wine can enhance its sensory characteristics and improve its sensory quality. Acidity is an important quality of wine, giving it a fresh and crisp taste and forming the sensory framework of wine. Acidity originates from tartaric acid and malic acid in grapes, as well as succinic acid, citric acid, lactic acid, and acetic acid produced during fermentation. However, acetic acid is the main volatile acid in wine, accounting for over 95%. Excessive acetic acid content results in a pungent sour taste, reducing the quality of the wine and even rendering it undrinkable.

[0003] During wine fermentation, non-sacchariculture yeasts primarily appear in the early stages. However, as fermentation progresses, nutrients decrease and ethanol concentration increases, causing non-sacchariculture yeasts to gradually disappear. Meanwhile, Sacchariculture yeasts, with their higher alcohol tolerance, begin to dominate fermentation and ultimately complete the process. Sacchariculture yeast, as a crucial strain for alcoholic fermentation, enables rapid, complete, and efficient fermentation, making it highly favored by wineries. Using Sacchariculture yeast strains with excellent brewing characteristics not only improves wine quality but also shortens the fermentation cycle, reduces production costs, and ultimately enhances the wine's market competitiveness. For a long time, screening for Sacchariculture yeasts with excellent brewing characteristics has been a primary focus of wine research. However, long-term research has revealed that using commercial Sacchariculture yeasts leads to severe homogenization of wines, lacking the aromatic complexity and stylistic uniqueness inherent in natural fermentation. Further research into non-sacchariculture yeasts has shown that they can secrete various hydrolytic enzymes that degrade bound wine aromas into free aromas and produce large amounts of secondary metabolites such as glycerol, esters, and higher alcohols, thus improving the aroma quality of the wine. However, non-brewing yeasts have relatively weak fermentation capabilities and often cannot dominate the entire fermentation process. Therefore, a mixed fermentation method using brewing yeast is generally employed to complete the fermentation process. Selecting different species of non-brewing yeasts for mixed inoculation with brewing yeast can effectively improve the sensory qualities of various wines, and this technique is becoming increasingly widely used.

[0004] Among numerous non-sacchariculture yeasts, *H. uvarum* and *M. pulcherrima* can significantly improve the aroma profile and sensory quality of wine. *H. uvarum* produces high levels of esters, imparting floral and fruity aromas to wine and significantly enhancing the sensory quality of fresh wines; however, its high acetic acid content limits its application. *M. pulcherrima* secretes various hydrolytic enzymes, increasing the aroma of terpenes and isoprene compounds in wine. Due to its low ethanol yield, its greatest application is in the production of low-alcohol wines. During wine fermentation, a certain concentration of SO2 (generally 60 mg / L) is added to inhibit contamination and reproduction of other microorganisms. On the other hand, the high sugar concentration in grape juice inhibits yeast growth. Because *M. pulcherrima* has weak tolerance to SO2 and high sugar concentrations, its application in winemaking is limited. Therefore, screening for non-sacchariculture yeast strains that can tolerate high concentrations of SO2 and sugar is a prerequisite for industrial application. In our preliminary research, we screened two strains of *Hansenula polysaccharide* CVE-HU47 and *Megamich. Maggi* CVE-MP27 from the Helan Mountain East Foothills region of Ningxia. Their most notable characteristics are their tolerance to high concentrations of SO2 and sugar, and their prominent aroma-producing features. HU47 produces high levels of esters but low levels of volatile acids, while MP27 produces high levels of terpenes, as well as succinic acid, citric acid, and lactic acid, showing potential for increasing acidity. To maximize the advantages of combining *Hansenula polysaccharide* and *Megamich. Maggi*, we combined the two strains to obtain a compound fermentation agent suitable for wine fermentation environments with high SO2 and sugar concentrations. This agent enhances the sensory aroma quality and acidity of wine, increasing the complexity and diversity of its aroma. Summary of the Invention

[0005] The purpose of this invention is to provide two aroma-producing yeast strains and their compound fermentation agent, as well as a method for brewing wine, which can withstand the fermentation environment of high concentrations of SO2 and sugar, while improving the sensory aroma quality and acidity of the wine, and increasing the complexity and diversity of the wine aroma.

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

[0007] A non-Sacchariform yeast strain, specifically Hanseniaspora uvarum CVE-HU47, is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCCNO.24818.

[0008] This non-brewing yeast was isolated from the eastern foothills of the Helan Mountains in Ningxia. It can tolerate high concentrations of sulfur dioxide and sugar stress, and produces low levels of volatile acids and high levels of esters, terpenes, and isoprene aroma compounds during wine fermentation.

[0009] A non-Saccharomyces cerevisiae strain, specifically Metschnikowia pulcherrima CVE-MP27, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.24819.

[0010] This non-brewing yeast, isolated from the eastern foothills of the Helan Mountains in Ningxia, can tolerate high concentrations of sulfur dioxide and sugar stress. During wine fermentation, it produces low levels of ethanol but high levels of citric acid, succinic acid, and lactic acid, thereby increasing the acidity of the wine and enhancing its aroma of terpenes.

[0011] A compound fermentation agent comprising *Hansenula polymorpha* CVE-HU47 and *Megamiprid* CVE-MP27, wherein the volume ratio of the two for inoculation is 0.8–1:1, and the total inoculation concentration is 0.1–1 × 10⁻⁶. 7 CFU / mL.

[0012] A method for brewing rosé wine using the aforementioned compound fermentation agent includes the following steps:

[0013] (1) Take out each strain stored in -80℃ glycerol, thaw it, and bring it to room temperature. Inoculate it into YPD liquid medium at a ratio of 10% to 15% (v / v), and place it in a constant temperature shaking incubator for 18 hours to increase its growth. Wait until the strain density reaches 10 7 ~10 8 CFU / mL was used to obtain a single bacterial culture;

[0014] (2) After harvesting, the grapes are sorted by bunch, sorted by berries, and crushed by destemming. The juice is then pressed and introduced into a fermentation tank. A clarifying agent is added to obtain clarified grape juice, and 80-100 mg / L of SO2 is added.

[0015] (3) The compound fermentation agent and wine yeast are sequentially inoculated to complete the following first and second fermentations: The compound fermentation agent is inoculated into the clarified grape juice obtained in step (2) at an inoculation concentration of 0.1 to 1 × 10⁻⁶. 7 The first fermentation was completed at CFU / mL over 72–80 hours; then, commercial brewer's yeast CECA was inoculated at a concentration of 0.1–1 × 10⁻⁶ CFU / mL. 6 CFU / mL, the second fermentation is completed in 200-240 hours;

[0016] The inoculation ratio of the compound fermentation agent to the brewing yeast is 10:1, and the fermentation parameters are: pH 3.0-3.5, fermentation temperature 20-23℃, and residual sugar content below 4g / L, resulting in a dry rosé wine with floral and fruity aromas, suitable acidity, and low volatile acid content.

[0017] In step (2), the SO2 is added in the form of potassium metabisulfite.

[0018] In step (3), the following fermentation parameters are further controlled:

[0019] The sugar concentration is 250-300 g / L, the alcohol concentration is 9-11% (v / v), and the nitrogen source concentration is 200-300 mg / L.

[0020] The resulting wine product contains the following aroma compounds: esters, higher alcohols, volatile fatty acids, and other aroma compounds; among which:

[0021] The esters are ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, phenylethyl acetate, isoamyl acetate, ethyl dodecanoate, and methyl octanoate.

[0022] The higher alcohols are isobutanol, 1-butanol, isoamyl alcohol, 1-octanol, 1-decyl alcohol, 4-methyl-1-pentanol, 3-methyl-1-pentanol, and 2-phenylethanol;

[0023] The fatty acids are hexanoic acid, octanoic acid, and decanoic acid;

[0024] The other aroma compounds are β-damascene, citronellol, 4-terpene alcohol, benzaldehyde, and styrene.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] Currently, the mixed fermentation technology of brewing yeast and non-brewing yeast is widely used in wine fermentation, and the use of non-brewing yeast with excellent fermentation performance has become a research hotspot both domestically and internationally. Because non-brewing yeast has a weaker tolerance to high sugar and SO2 stress, it is necessary to screen for highly tolerant strains, which is a prerequisite for its application. On the other hand, there are many types of non-brewing yeast with distinct characteristics, and different yeasts can be combined to leverage their unique features and comprehensively improve the sensory quality of wine. To enhance the aroma and acidity of wine, this invention uses a mixed fermentation agent (1:1 ratio) of *H. uvarum* CVE-HU47 and *M. pulcherrima* CVE-MP27 from grape juice. This agent is inoculated into rosé wine, and after 3 days of fermentation, commercial brewing yeast *S. cerevisiae* CECA is inoculated, with an inoculation ratio of non-brewing yeast to brewing yeast of 10:1. The grape juice contains *Hansenula polymorpha* HU47, which is tolerant of high concentrations of sulfur dioxide and sugar stress. During fermentation, it produces low levels of volatile acids and high levels of esters, terpenes, and isoprene aromatic compounds, imparting aromas of rose, honey, fruit, and floral notes to the wine and significantly improving its sensory quality. *Megamix* MP27 is also tolerant of high concentrations of sulfur dioxide and sugar stress, producing high levels of citric acid, succinic acid, and lactic acid, increasing the acidity of the wine and enhancing its terpene aroma. The compound fermentation agent and inoculation process obtained in this invention can enhance the floral and fruity aromas of wine, improve the complexity and quality of its aromas, and increase its acidity. Attached Figure Description

[0027] Figure 1 The colony morphology of the yeast strains CECA, HU47, and MP27 as measured in this invention;

[0028] Figure 2 This is a growth curve of the yeast strain tested in this invention;

[0029] Figure 3 The growth curves (CO2 production) of each experimental group in this invention are shown.

[0030] Figure 4 The image shows a radar chart of sensory evaluations for different experimental groups in this invention. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] Examples 1 and 2 are studies on the fermentation capacity and aroma-producing characteristics of two aroma-producing non-sacchariculture yeast strains obtained by screening in this invention. Example 3 is a study on combining the above two strains with Sacchariculture yeast to obtain the compound fermentation agent and brewing method of this invention.

[0033] This invention screened two excellent aroma-producing non-sacchariculture yeast strains: *Hanseniaspora uvarum* CVE-HU47 (a self-screened strain, deposited on April 29, 2022 at the China General Microbiological Culture Collection Center, accession number: CGMCC NO.24818) and *Metschnikowia pulcherrima* CVE-MP27 (a self-screened strain, deposited on April 29, 2022 at the China General Microbiological Culture Collection Center, accession number: CGMCC). Based on NO.24819, a compound fermentation agent consisting of two yeasts, CVE-HU47 and CVE-MP27 (in a 1:1 ratio), was obtained. When used to inoculate rosé wine, commercial wine yeast (S. cerevisiae) CECA was inoculated after 3 days of fermentation. The inoculation ratio of non-wine yeast to wine yeast was 10:1. This compound fermentation agent and inoculation process can enhance the floral and fruity aromas of wine, improve the aroma complexity and quality of wine, and increase the acidity of wine, showing great application potential.

[0034] Specifically, this involved screening native wild non-brewing yeasts to obtain grape juice containing *Hansenula polymorpha* CVE-HU47 and *Megamich* CVE-MP27, both with excellent brewing characteristics. Based on this, *Hansenula polymorpha* CVE-HU47 and *Megamich* CVE-MP27 were blended and inoculated with *Cremastra appendiculata* CECA to comprehensively utilize the brewing and aroma-producing characteristics of various microorganisms. Ultimately, this combined fermentation agent and inoculation process was found to enhance the floral and fruity aromas of wine, improve its aroma quality, increase its diversity and complexity, and increase its acidity.

[0035] Example 1

[0036] The winemaking characteristics of grape juice containing *H. uvarum* CVE-HU47 (accession number CGMCC NO.24818) and *M. pulcherrima* CVE-MP27 (accession number CGMCC NO.24819) were evaluated, with commercial brewing yeast *Saccharomyces cerevisiae* CECA as a control. The specific detection methods are as follows:

[0037] (1) Colony morphology characteristics: Grape juice stored at -80℃ was thawed and inoculated into 50ml centrifuge tubes containing 15mL of YPD liquid medium for activation. The bacterial solution was diluted with sterile distilled water and evenly spread on WLN medium. The culture was carried out at 30℃ until the colonies with complete morphology were formed.

[0038] (2) Growth curve: Grape juice containing Hansenula polymorpha HU47 and Maggi Mage Mage MP27 were respectively at 10 6 CFU / mL was inoculated into 25 mL of YPD liquid medium and incubated statically at 28 °C. OD was measured every 12 h. 600 Absorbance. The growth time of the strain is plotted on the x-axis, and the measured OD value is plotted on the y-axis. 600 The growth curve was plotted with absorbance values ​​on the ordinate, with Saccharomyces cerevisiae CECA as a control, and three biological replicates were set up for each experimental group.

[0039] (3) Tolerance test: After activating grape juice with Hansenula polymorpha HU47 and Magemycosis MP27, respectively, they were subjected to 10 6 Inoculation with CFU / mL was performed in YPD liquid medium to test sugar tolerance, alcohol tolerance, SO2 tolerance, pH tolerance, and Cu content. 2+ The gradient design for the tolerance and temperature tolerance experiments is as follows:

[0040] Sugar gradient: The total sugar concentration of YPD liquid medium was adjusted to 100 g / L, 200 g / L, 250 g / L, 300 g / L, and 350 g / L respectively, with a glucose to fructose ratio of 1:1 (w / w);

[0041] Alcohol content gradient: 3% v / v, 6% v / v, 9% v / v, 12% v / v, 15% v / v;

[0042] SO2 concentration gradient: The SO2 concentration of YPD liquid culture medium was adjusted to 60 mg / L, 120 mg / L, 180 mg / L, 240 mg / L, and 300 mg / L using sulfurous acid;

[0043] pH gradient: The pH of YPD liquid culture medium was adjusted to 2.0, 2.5, 3.0, 3.5, and 4.0 respectively using tartaric acid;

[0044] Metal Cu 2+ Concentration gradient: YPD liquid culture medium Cu was added to a 0.1 mol / L CuSO4 solution. 2+ The concentrations were adjusted to 0.05 mmol / L, 0.1 mmol / L, 0.2 mmol / L, 0.5 mmol / L, and 1 mmol / L, respectively.

[0045] Temperature gradient: 10℃, 15℃, 20℃, 25℃, 30℃.

[0046] All experimental groups were incubated statically at 28℃ for 72 h, and then the OD was measured using a UV spectrophotometer. 600The absorbance was measured using Saccharomyces cerevisiae CECA as a control, and three biological replicates were set up for each experimental group.

[0047] (4) Flocculation test: Grape juice containing Hansenula polymorpha HU47 and Magemy's yeast MP27 was activated and then subjected to a 10-fold increase in concentration. 6 Inoculate the culture medium with a concentration of CFU / mL into YPD liquid medium and incubate statically at 28°C. OD is measured every 12 hours. 600 The absorbance was measured until the strain reached the logarithmic stationary phase. The bacterial pellet was then collected by centrifugation (10000 rpm, 2 min). The pellet was washed twice with deflocculation buffer, followed by two washes with sterile water. The washed pellet was then placed in deflocculation buffer, and its OD value was measured. 600 Absorbance value, denoted as A; then, the flocculation buffer was placed in a 50 mL Erlenmeyer flask and incubated with shaking at 30℃ and 100 rpm for 2 h. 5 mL of the bacterial cell suspension was transferred to a 10 mL test tube, and after standing vertically for 30 min, 350 μL was taken below the concave meniscus and its OD value was measured again. 600 Absorbance value, denoted by B. Using *Saccharomyces cerevisiae* CECA as a control, the experiment was conducted in triplicate.

[0048] The flocculation value is expressed by the following formula: Flo = B / A × 100%

[0049] In the formula, A represents the OD of the bacterial cells suspended in the flocculation buffer before the conical flask shaking culture. 600 Absorbance; B represents the OD of bacterial cells after 30 minutes of flocculation and sedimentation. 600 Absorbance value; flocculation value is represented by Flo. The smaller the flocculation value, the stronger the flocculation ability of the strain. When Flo is greater than 70%, the strain is judged to have low flocculation ability; when Flo is between 30% and 70%, the strain is judged to have medium flocculation ability; when Flo is between 0% and 30%, the strain is judged to have high flocculation ability.

[0050] (5) Foaming test: Add 10 mL of YPD liquid medium to each test tube (15 mm × 150 mm), and then incubate each strain of the test tube with 10 mL of YPD liquid medium. 6Inoculation was performed at a rate of CFU / mL into YPD liquid medium, and the culture was statically incubated at 28°C. Foam height was observed and measured every 4 hours until the maximum foam height was achieved. The foam-producing ability of the strain was classified into three levels: a foam height below 2 mm indicated low foam production (referred to as low foam production); a foam height between 2 mm and 4 mm indicated moderate foam production (referred to as moderate foam production); and a foam height above 4 mm indicated high foam production (referred to as high foam production). *Saccharomyces cerevisiae* CECA was used as a control, and three biological replicates were set up for each experimental group.

[0051] (6) H2S production capacity: Activated grape juice was spotted onto BIGGY medium containing *Hansenula polymorpha* HU47 and *Megmecium mihanovichii* MP27, respectively. The spotted BIGGY plates were incubated at 28°C for 4 days, and the colony color changes were recorded. Colony color was categorized into five levels: white colonies indicate no H2S production; light brown colonies indicate low H2S production; brown colonies indicate moderate H2S production; and dark brown colonies indicate high H2S production. *Saccharomyces cerevisiae* CECA was used as a control, and three biological replicates were set up for each experimental group.

[0052] (7) Carbon source assimilation capacity: Activated grape juice was subjected to a 10-fold increase in the concentration of Hansenula polymorpha HU47 and Maggi Mage MP27. 6 Inoculation at CFU / mL levels was performed in basal media containing glucose, fructose, sucrose, maltose, xylose, and galactose, each with a carbon source concentration of 50 mmol / L. After inoculation, the media were incubated at 25°C for 21 days. Results were determined by thoroughly mixing the culture medium after 21 days using a vortex mixer. A black line approximately 3–4 mm wide was drawn on an A4 white sheet of paper. The paper was then held close to the test tube and observed against natural light to determine the presence and clarity of the black line. A completely invisible black line was marked as "+++", a faint black line as "++", a borderline black line as "+", and a completely clear black line as "-". *Saccharomyces cerevisiae* CECA was used as a control. Three biological replicates were set up for each experimental group.

[0053] (8) Nitrogen source demand capacity: The inorganic ammonium salt and mixed amino acids in the YPD liquid culture medium were adjusted to the same ratio. The initial mass concentration of assimilable nitrogen in the YPD liquid culture medium was adjusted to 100 mg / L, 200 mg / L, 300 mg / L and 400 mg / L, respectively. Grape juice containing Hansenula polymorpha HU47 and Magemycosis MP27 were respectively added at 10 6Inoculate with CFU / mL into YPD medium containing different mass concentrations of yeast-assimilable nitrogen sources as described above, and measure OD every 24 hours. 600 Absorbance values ​​were used to plot yeast growth curves. Saccharomyces cerevisiae CECA was used as a control, and three biological replicates were set up for each experimental group.

[0054] like Figure 1 As shown in the figure, the colony morphology of each strain on WLN medium is as follows: CECA colonies are cream-colored (light yellow) with a light green center, a smooth, opaque surface with spherical protrusions, and neat edges; HU47 colonies are green with spherical protrusions, are glossy, opaque, and have smooth edges; MP27 colonies are white with a smooth surface and flat edges.

[0055] The growth curves of each strain are as follows: Figure 2 As shown in the figure, all tested yeasts maintained a relatively fast growth rate and were in good growth condition before 132 hours; they entered the late growth stage around 156 hours, and entered the death stage after around 168 hours. Based on cell mass, Saccharomyces cerevisiae CECA showed the strongest growth ability, followed by HU47 and MP27.

[0056] Regarding environmental tolerance, the levels of *Hansenula polymorpha* HU47 and *Megmecium mihanovichii* MP27 in grape juice were measured under different sugar concentrations, alcohol volume fractions, sulfur dioxide mass concentrations, pH values, and Cu. 2+ The tolerance of these two non-Saccharomyces cerevisiae strains to different stress environments was characterized by their growth under different concentrations and fermentation temperatures, with Saccharomyces cerevisiae CECA used as a control strain. The results showed that:

[0057] (1) Sugar tolerance: The two non-Saccharomyces cerevisiae strains were less tolerant to sugar than Saccharomyces cerevisiae CECA, but both were able to tolerate a sugar concentration of 250 g / L. HU47 was more tolerant than MP27. When the sugar concentration exceeded 300 g / L, the growth of both strains was inhibited.

[0058] (2) Ethanol tolerance: Ethanol is the main product of grape juice yeast fermentation. High ethanol concentration has a certain inhibitory effect on the growth of all yeasts. As the ethanol concentration increases, the growth ability of each strain gradually weakens. HU47 and MP27 can both grow in a medium with an alcohol concentration of 9% (v / v), but when the alcohol concentration is >12% (v / v), only CECA can survive.

[0059] (3) Acid tolerance (pH): Both HU47 and MP27 can grow well in an environment of pH 3.0 to 3.5, with MP27 growing better than HU47 at pH 3.0. When pH < 2.5, the growth ability of the tested yeasts is weak.

[0060] (4) SO2 tolerance: When the SO2 concentration is 100-120 mg / L, HU47 and MP27 grow well and show good SO2 tolerance, and HU47 is stronger than MP27.

[0061] (5) Tolerance to Cu 2+ Stress: HU47 and MP27 can tolerate 0.5 mmol / L Cu 2+ Both strains of bacteria were able to grow normally. When Cu... 2+ At a concentration of 1 mmol / L, the measured yeast growth activity was significantly reduced, with HU47 showing stronger growth than MP27.

[0062] (6) Suitable growth temperature: HU47 and MP27 grow better at 18℃. When the temperature is >20℃, the growth ability of the tested yeast is significantly enhanced, and HU47 > MP27, but it cannot exceed 30℃.

[0063] (7) Flocculation ability: Good flocculation ability of yeast helps to clarify wine in the later stage of fermentation. The flocculation values ​​of HU47, MP27 and CECA are between 53.86%, 31.26 and 59.71% respectively, all of which are medium flocculation.

[0064] (8) Bubble production capacity: During wine fermentation, yeast produces carbon dioxide gas and heat by using the sugar in grape juice to generate alcohol, which in turn causes bubbles to form during fermentation. If the amount of gas produced is too large, it can easily cause the "overflowing" phenomenon. The brewing yeast CECA has a high gas production capacity, with a maximum foam height of 4.32 mm, which is considered high foam production. The maximum foam height of grape juice Hansenula spore yeast HU47 is 2.75 mm, which is considered medium foam production. The maximum foam height of Maggimycin MP27 is the lowest at 1.12 mm, which is considered low foam production.

[0065] (9) Hydrogen sulfide production capacity: Hydrogen sulfide (H2S) is the most volatile sulfur-containing compound in wine, with a pungent odor similar to rotten eggs. The human sensory threshold for H2S is 0.12–0.37 mg / L, so even very low H2S concentrations can affect the aroma quality of wine. Based on the colony color of the yeasts on BIGGY medium, Saccharomyces cerevisiae CECA was a medium-producing H2S strain (colony color brownish-red), Hansenula polymorpha HU47 was a low-producing H2S strain (light brownish-red), and Saccharomyces cerevisiae MP27 was a medium-producing H2S strain (colony color brownish-red).

[0066] (10) Carbon source assimilation capacity: All tested strains were able to use glucose, fructose and sucrose for fermentation (Table 1), and the assimilation capacity from strong to weak was: CECA > HU47 > MP27; HU47 and MP27 could not use xylose and galactose for fermentation; Saccharomyces cerevisiae CECA had the highest sucrose utilization capacity, followed by HU47, and MP27 had the weakest sucrose utilization capacity.

[0067] Table 1 shows the carbon source assimilation capacity of the yeast strains tested.

[0068]

[0069] (11) Nitrogen source requirement capacity: Grape juice Hansenula polymorpha HU47 has a relatively low nitrogen source requirement and can grow well under a nitrogen source concentration of 200 mg / L, while Magemycosis MP27 has a higher nitrogen source requirement (200-300 mg / L).

[0070] In summary, HU47 and MP27 exhibit good growth and environmental tolerance, including tolerance to SO2 and high sugar concentrations, but have relatively low H2S production capacity. Specific fermentation parameters are shown in Table 2. Their comprehensive indicators meet the basic requirements for winemaking and have the potential for large-scale application.

[0071] Table 2 Summary of brewing characteristics of HU47 and MP27

[0072]

[0073] Example 2

[0074] To further determine the fermentation and aroma-producing capabilities of *Hansenula polymorpha* HU47 and *Megmecium magnum* MP27 in grape juice, separate fermentation experiments were conducted. 'Beibinghong' grape juice was used in the experiments, and the inoculum size of the yeast was 10... 6CFU / mL. Fermentation was carried out in 50mL centrifuge tubes, each containing 30mL of pasteurized grape juice. The activated yeast was cultured in a shaker (30℃, 180r / min) for one day, then centrifuged at low temperature (4℃) (8000r / min, 5min). The bacterial precipitate at the bottom of the centrifuge tube was collected, washed with sterile water, and then inoculated into the grape juice. Each experimental group was statically fermented in a 28℃ incubator, with *Saccharomyces cerevisiae* EC1118 as a control. Three biological replicates were set up for each experimental group. CO2 weight loss was measured every 24 hours. Fermentation was considered complete if the weight change was less than 2g / L for two consecutive days. After fermentation, 10mL of fermentation sample was taken from each experimental group, centrifuged (10000r / min, 3min), and the supernatant was collected and frozen at -20℃ for analysis of the main fermentation products. By comparing the fermentation rate, ethanol yield, ethanol production rate, and volatile aroma component content of different strains, a preliminary analysis and comparison of the aroma production level and brewing characteristics of different non-brewing yeasts was conducted.

[0075] The major metabolites of the fermentation broth were analyzed using the following method: After filtration (PES, 0.22 μm), the major metabolites were analyzed by high-performance liquid chromatography (HPLC) 1200 (Agilent Technologies, USA). The ion-exchange column was an HPX-87H Aminex ion-exchange column (300 × 7.8 mm, Bio-Rad Laboratories, USA), the mobile phase was 5 mM H₂SO₄ solution, isocratic elution was performed, and the flow rate was 0.6 mL / min. Glucose, fructose, ethanol, and glycerol were determined using a refractive index detector (RID, G1362A, Agilent Technologies, USA) with an injection volume of 20 μL, a column temperature of 45 °C, and an analysis time of 30 min. Organic acids (tartaric acid, malic acid, citric acid, lactic acid, succinic acid, and acetic acid) were determined using a photodiode array detector (DAD, G1315D, Agilent Technologies, USA) with an injection volume of 10 μL, a column temperature of 60 °C, and an analysis time of 25 min.

[0076] Determination of volatile aroma compounds such as esters, higher alcohols, and organic acids in the fermentation broth: 5 mL of fermentation sample was added to a 15 mL sample bottle, along with 1 g NaCl and 10 μL of internal standard (4-methyl-2-pentanol). The bottle was then quickly sealed with a sample bottle cap fitted with a polytetrafluoroethylene (PTFE) septum. The mixture was kept at a constant temperature of 40℃ and equilibrated at 180 rpm for 30 min. After the gas-liquid phase aroma compounds in the sample bottle reached equilibrium, an activated or thermally desorbed polydimethylsiloxane / carbon sieve / divinylbenzene (PDMS / CAR / DVB) extraction head was inserted into the headspace of the sample bottle. Extraction was carried out at a constant temperature of 40℃ with stirring for 30 min to achieve gas-solid and gas-liquid equilibrium. The types and contents of various volatile aroma compounds in the obtained wine were detected using an Agilent 6890 gas chromatograph (GC) and an Agilent 5975 mass spectrometer (MS) (Agilent, USA). Specific conditions were as follows: HP-INNOWAX Polyethylene Glycol capillary column (60m × 0.25mm × 0.25μm, J&W Scientific, USA); carrier gas: high-purity helium; flow rate: 1 mL / min; headspace solid-phase microextraction (HS-SPME) with splitless injection, inserted into the gas chromatograph injector at 250℃, followed by thermal desorption for 25 min. The column oven temperature program was: 40℃ for 5 min, then increased to 200℃ at a rate of 3℃ / min and held for 2 min. The mass spectrometer interface temperature was 280℃, the ion source temperature was 230℃, ionization mode was EI, ion energy was 70 eV, and the mass scan range was 20-350 m / z. For substances with existing standards, qualitative analysis was performed using full-ion mass spectrometry (Scan) based on retention time, retention index, and mass spectrometry information under the same chromatographic conditions established in this experiment. Quantitative analysis was then performed using a standard curve in a simulated wine solution (the synthesized wine solution was an aqueous solution of 2 g / L glucose, 7 g / L tartaric acid, and 12% alcohol, with pH adjusted to 3.3 using NaOH. Mixed aroma standards were prepared in 15 gradients). For substances without standards, semi-qualitative analysis was performed using the retention index of the compound under similar chromatographic conditions reported in the literature and comparison results with the NIST 11 standard library (NIST Chemistry WebBook).

[0077] To determine the individual fermentation characteristics of *Hansenula polymorpha* HU47 and *Megrichtomyces magna* MP27 in grape juice, we measured the contents of major metabolites and volatile aroma compounds in each experimental group after fermentation. The results are shown in Table 3. It can be seen that neither *Hansenula polymorpha* HU47 nor *Megrichtomyces magna* MP27 could complete alcoholic fermentation alone, with residual sugar contents of 54.60 g / L and 54.55 g / L, respectively. Compared to the other two strains, *Megrichtomyces magna* MP27 produced a high amount of glycerol (5.72 g / L), improving the taste and palatability of the wine. *Megrichtomyces magna* MP27 had the lowest ethanol yield, 16.7% lower than *Saccharomyces cerevisiae*, indicating that MP27 has the potential to reduce alcohol content. Furthermore, MP27 also had the highest production capacity for citric acid, succinic acid, and lactic acid, exceeding *Saccharomyces cerevisiae* by 38.7%, 105%, and 50%, respectively, which has a positive effect on increasing the acidity of the wine. HU47 and MP27 exhibit low acetic acid production, decreasing by 70.6% and 54.9% respectively compared to *Saccharomyces cerevisiae* CECA. This characteristic is crucial for HU47, as *Hansenula polymorpha*, a previously reported yeast strain in grape juice, generally possesses high acetic acid production capabilities, limiting its application in winemaking. Regarding aroma (Table 4), HU47 and MP27 show lower synthesis of higher alcohols and fatty acids than *Saccharomyces cerevisiae* CECA, but both non-*Saccharomyces cerevisiae* strains exhibit higher ester synthesis capabilities. Concentrations of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl decanoate, and phenylethyl acetate are all higher in HU47 than in *Saccharomyces cerevisiae*. Furthermore, HU47 has the highest β-damascene content. High ester and β-damascene production can enhance the floral and fruity aromas of wine. In conclusion, HU47 and MP27 possess excellent brewing, fermentation, and aroma-producing characteristics when fermented alone. However, since they cannot ferment independently, they require co-inoculation with *Saccharomyces cerevisiae*. In addition, since MP27 has a high capacity for producing citric acid, succinic acid and lactic acid, and also has a high ester synthesis capacity, we hope to obtain a compound fermentation agent by combining HU47 and MP27. By delaying inoculation with wine yeast (inoculating non-wine yeast first, and then inoculating wine yeast after fermentation for a period of time), we can simultaneously increase the acidity and aroma content of wine.

[0078] Table 3 Content of major metabolites in each experimental group after alcoholic fermentation

[0079]

[0080]

[0081] Note: Data in the table (mean ± relative standard deviation); significance analysis of variance was performed using independent samples t-test. Different letters after the content of the same compound indicate significant differences between different treatment groups (p<0.05).

[0082] Table 4. Aroma substance content of each experimental group after alcoholic fermentation

[0083]

[0084]

[0085] Note: Data in the table (mean ± relative standard deviation). ANOVA was performed using an independent samples t-test. Different letters indicating the concentration of the same compound represent significant differences between treatment groups (p < 0.05).

[0086] Example 3

[0087] The above results indicate that *H. uvarum* HU47 and *M. pulcherrima* MP27 possess excellent brewing, fermentation, and aroma-producing characteristics in grape juice, with significant differences and distinct advantages, thus allowing for blending. Since non-sacchariculture yeasts cannot independently complete alcoholic fermentation, a mixed inoculation method with *Sacchariculture yeast* is necessary. Therefore, we blended HU47 and MP27 at a 1:1 volume ratio, first inoculating them into the grape juice, fermenting for 3 days, and then inoculating with commercial *Sacchariculture yeast* (CECA) for alcoholic fermentation. We investigated the effects of this dual-strain compound fermentation agent and inoculation method on the main metabolites and aroma compounds of wine. As a control, we also studied the fermentation of single CECA strain, HU47+CECA dual-strain, and MP27+CECA. Specific inoculation methods are shown in Table 5.

[0088] Table 5. Mixed fermentation scheme of the strains used in the research.

[0089]

[0090] Note: HMC represents a mixture of three strains of bacteria fermented together.

[0091] Clarified 'Beibinghong' grape juice was used, with 100 mg / L SO2 (added in the form of sulfite) added before inoculation and fermentation. The experimental group used sequential inoculation: first, a non-Saccharomyces yeast mixed culture was inoculated at a 1:1 ratio, with an inoculation volume of 10... 7 CFU / mL, and after 72 hours, inoculate with Saccharomyces cerevisiae CECA at an inoculum size of 10. 6CFU / mL. The fermentation vessel was a 250ml Erlenmeyer flask, each containing 200mL of grape juice. A vertical fermentation plug was fitted to the flask opening, and the gas outlet of the apparatus was sealed with sterile water. The activated yeast was cultured on a shaker (30℃, 180r / min) for one day, then centrifuged at low temperature (4℃) (8000r / min, 5min). The bacterial precipitate at the bottom of the centrifuge tube was collected, washed with sterile water, and transferred to the Erlenmeyer flask. Each experimental group was statically fermented in a 25℃ incubator, with three biological replicates per group. CO2 weight loss was measured every 24 hours for each experimental group. If the weight loss was less than 2g / L for two consecutive days, the fermentation was considered complete. After fermentation, 20mL of fermentation sample was taken from each experimental group, centrifuged (10000r / min, 3min), and the supernatant was collected and frozen at -20℃ for analysis of the main fermentation product. The entire fermentation process was monitored by measuring CO2 weight loss in each experimental group. After fermentation, the main fermentation metabolites, volatile aroma compounds and color indicators in each experimental group were measured; the remaining fermentation broth was centrifuged at low temperature and stored at 4°C for sensory evaluation.

[0092] The determination of the main metabolites and volatile aroma substances in the fermentation broth was carried out in accordance with Example 2.

[0093] Wine Sensory Evaluation: The sensory evaluation panel consisted of 10 members aged 22-26 who had undergone rigorous wine sensory evaluation training; 5 men and 5 women. The evaluation assessed the wine samples based on three aspects: appearance, aroma, and overall evaluation. The maximum score was 10 points, with appearance (color, clarity) accounting for 20%, aroma (floral, fruity, harmony, and intensity) accounting for 50%, and overall evaluation accounting for 30%. Before conducting the sensory analysis, the evaluation panel members received training on the standard aroma compounds in wine. A blind tasting was conducted, with the four experimental groups of wine samples evaluated for aroma in a randomized order and scored on a 10-point scale. Finally, the sensory evaluation results from the panel members were summarized and statistically analyzed to determine the wine quality.

[0094] Depend on Figure 3 It can be seen that before inoculation with Saccharomyces cerevisiae CECA, the fermentation rate of all experimental groups was lower than that of Saccharomyces cerevisiae CECA. After inoculation with Saccharomyces cerevisiae CECA, the fermentation rate of each experimental group increased significantly. CECA fermentation ended after day 8, at which point HC fermentation slowed down, while the fermentation process of the other experimental groups was still in progress. MC and HMC fermentation ended on day 10.

[0095] As shown in Table 7, HC had the highest residual sugar content (25.55 g / L), indicating that HC did not complete alcoholic fermentation, possibly related to the interaction between HU47 and CECA strains during delayed inoculation. The other groups completed alcoholic fermentation, indicating that the addition of MP27 can eliminate the antagonistic effect between HU47 and CECA, allowing fermentation to proceed smoothly. This is a major reason for our combination of MP27 and HU47. Since the alcohol yield of MP27 and HU47 was lower than that of CECA during single-strain fermentation, they both showed the potential to reduce alcohol content when mixed with CECA. The ethanol concentrations in the HC and MC groups were reduced by 15.6% and 7.7% respectively compared to the CECA group. However, the HMC group, which combined all three strains, did not show any alcohol-reducing ability. The acetic acid concentrations in all treatment groups (0.65–0.80 g / L) were lower than those in the control group fermented with Saccharomyces cerevisiae alone (1.18 g / L). HC and HMC were 44.9% and 37.3% lower than CECA, respectively, indicating that HU47 has a strong ability to reduce acetic acid content, which is crucial for the future use of HU47 in wine fermentation production. Due to the involvement of MP27, the concentrations of citric acid and succinic acid in the HMC group were increased by 17.2% and 17.3% compared to the CECA control group, respectively; by 36% and 90.1% compared to the HC group; and by 9.7% and 6.5% compared to the MC group. Regarding lactic acid synthesis, the final lactic acid concentration in the HMC group was 18.1% and 29.2% higher than that in the CECA control group and the MC group, respectively. Increased acidity lowers pH; therefore, changes in wine acidity can be characterized by measuring the pH value of the wine during production. The results showed that the final pH values ​​of wines fermented with CECA alone and with a mixture of HU47 and CECA were 3.62 and 3.60, respectively; the pH value of wines fermented with the MP27 and CECA mixture was 3.55; and the pH value of wines fermented with a mixture of HU47, MP27, and CECA was 3.47, a decrease of 0.15 compared to CECA fermentation alone. This indicates that inoculation with the MP27 and HU47 combined fermentation agent can increase the acidity of the wine.

[0096] Table 6 shows the content of major metabolites in each experimental group during mixed fermentation after alcoholic fermentation (where HU47+MP27+CECA represents mixed fermentation of three strains, and HU47+CECA and MP27+CECA both represent mixed inoculation of two strains).

[0097]

[0098]

[0099] Note: Data in the table (mean ± relative standard deviation); significance analysis of variance was performed using independent samples t-test. Different letters after the content of the same compound indicate significant differences between different treatment groups (p<0.05).

[0100] In terms of aroma, a total of 28 key flavor compounds were detected, mainly including 12 esters, 8 higher alcohols, 3 fatty acids and 5 other substances (β-damascene, citronellol, 4-terpene alcohol, benzaldehyde and styrene).

[0101] Table 7 shows that the total amount of higher alcohols in all experimental groups was below the threshold of 300 mg / L. Compared with the fermentation of Saccharomyces cerevisiae CECA alone (31.50 mg / L), the mixed inoculation of two strains of HC (23.74 mg / L), and MC (34.48 mg / L), the mixed inoculation of three strains of HMC had the highest higher alcohol content, at 35.06 mg / L. The higher alcohols included isoamyl alcohol and 2-phenylethanol, which enhanced the aromas of jasmine, rose, and honey in the wine. The 2-phenylethanol content in all three experimental groups was significantly higher than that in the fermentation of Saccharomyces cerevisiae alone, ranging from 6688.89 to 8660.76 μg / L. The MC experimental group had the highest 2-phenylethanol content, followed by HMC, which was 64.8% higher than that of Saccharomyces cerevisiae CECA, indicating that the inoculation of MP27 increased the 2-phenylethanol content.

[0102] Esters are one of the most important classes of substances in wine, imparting rich floral and fruity aromas. The HMC group had the highest total ester content (27.8 mg / L), which was 15.8%, 13.0%, and 16.3% higher than that of wine yeast fermentation alone (24.0 mg / L), HC (24.6 mg / L), and MC (23.9 mg / L), respectively. The most abundant substances in the HMC group were ethyl acetate, ethyl butyrate, and ethyl octanoate, while the most abundant were ethyl hexanoate, isoamyl acetate, and methyl octanoate. These three esters were found in the highest concentration in the MP27 group, indicating that MP27 promotes the production of these two substances in HMC. It is particularly noteworthy that the sum of methyl octanoate, ethyl octanoate, and ethyl hexanoate in the MC and HMC experimental groups were 215.87 μg / L and 214.47 μg / L, respectively, which are 1.36 times and 1.35 times that of the CECA of Saccharomyces cerevisiae (158.42 μg / L). The increase in the content of these esters enhanced the citrus and fruity aromas in the wine.

[0103] Three volatile fatty acids were detected: hexanoic acid, caprylic acid, and capric acid. These three acids, when below their threshold values, can increase the complexity of wine aromas. The total fatty acid concentration in the HMC group was higher than that in the CECA control group and the HC group, by 30.9% and 18.2%, respectively, but there was no significant difference compared to the MC group. β-Damastone is an important isoprene morphology product that can provide excellent floral and sweet aromas to wine. Due to its low threshold (50 ng / L), even slight changes in its content can significantly affect the quality of the wine. The β-damascene content in the HC experimental group was higher than that of CECA fermentation alone, which is consistent with the results of other studies. Our study found that the combined inoculation of MP27 and HU47 can further increase the β-damascene content. Ultimately, the β-damascene content in the HMC group was increased by 42.7% compared with that of CECA fermentation alone, which improved the aroma quality of the wine. This indicates that the interaction between MP27 and HU47 has a synergistic promoting effect on β-damascene synthesis. Therefore, the combined inoculation of MP27 and HU47 with delayed inoculation fermentation of Saccharomyces cerevisiae is an effective method to increase the concentration of β-damascene in wine.

[0104] Table 7. Aroma substance content of each experimental group after alcoholic fermentation

[0105]

[0106]

[0107] Note: Data in the table (mean ± relative standard deviation); significance analysis of variance was performed using independent samples t-test. Different letters after the content of the same compound indicate significant differences between different treatment groups (p<0.05).

[0108] The differences between the different experimental groups were visually demonstrated through sensory evaluation, and the results are as follows: Figure 4 As shown. In terms of floral aroma scores, HC and HMC scored significantly higher than other experimental groups, while MC and CECA were similar; HMC scored higher in fruit aroma, followed by CECA and HC. In terms of clarity, CECA scored the highest, followed by HMC, and MC scored the lowest. In terms of aroma intensity, HMC scored the highest and CECA scored the lowest in the experimental groups. In addition, regarding acidity in the harmony aspect, HMC and MC scored higher overall, and HMC had the best overall sensory score.

[0109] Therefore, by combining locally sourced grape juice with excellent stress resistance, such as Hansenula polymorpha CVE-HU47 and Maggi Mage CVE-MP27, a mixed fermentation agent is prepared. This agent is then used in conjunction with wine yeast in a delayed fermentation process (inoculating with wine yeast at a ratio of 10:1 after 3 days of fermentation). This method can fully leverage the advantages of both HU47 and MP27, effectively improving the sensory quality of the wine, including acidity and the concentration of aroma compounds (including esters, higher alcohols, and β-damascene). This increases the complexity and diversity of the wine's aroma and enhances the unique local flavor characteristics of Chinese wines.

[0110] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A non-Saccharomyces cerevisiae compound fermenting agent, characterized in that: The compound fermentation agent is *Hansenula polysaccharide* from grape juice (… Hanseniaspora uvarum CVE-HU47 and Maggi yeast ( Metschnikowia pulcherrima CVE-MP27 and *Hansenula polymorpha* CVE-HU47, deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO.24818, and *Megamiprid* CVE-MP27, also deposited at the same center under CGMCC under accession number CGMCC NO.24819, were mixed in an inoculation at a volume ratio of 0.8–1:1, with a total inoculation concentration of 0.1–1 × 10⁻⁶. 7 CFU / mL.

2. The compound fermentation agent according to claim 1, characterized in that: The grape juice contains Hansenula polymorpha CVE-HU47 isolated from the eastern foothills of Helan Mountain in Ningxia. It can tolerate high concentrations of sulfur dioxide and sugar stress, and produces low levels of volatile acids and high levels of esters, terpenes, and isoprene aroma compounds during wine fermentation.

3. The compound fermentation agent according to claim 1, characterized in that: The Maggi Mickey yeast CVE-MP27 was isolated from the eastern foothills of Helan Mountain in Ningxia. It can tolerate high concentrations of sulfur dioxide and sugar stress, and produces low levels of ethanol and high levels of citric acid, succinic acid and lactic acid during wine fermentation, thereby increasing the acidity of the wine and enhancing the aroma of terpenes.

4. A method for brewing rosé wine using the compound fermenting agent according to any one of claims 1-3, characterized in that: Includes the following steps: (1) Take out each strain stored in -80℃ glycerol, thaw it, and bring it to room temperature. Inoculate it into YPD liquid medium at a ratio of 10% to 15% (v / v), and place it in a constant temperature shaking incubator for 18 h to increase its growth. Wait until the strain density reaches 10 7 ~10 8 CFU / mL was used to obtain a single bacterial culture; (2) After harvesting, the grapes are sorted by bunch, sorted by berries and destemmed and crushed. The grape juice is then pressed and introduced into a fermentation tank. A clarifying agent is added to obtain clarified grape juice, and 80-100 mg / L of SO2 is added. (3) The compound fermentation agent and wine yeast are sequentially inoculated to complete the first and second fermentations as follows: The compound fermentation agent is inoculated into the clarified grape juice obtained in step (2) at an inoculation concentration of 0.1~1×10⁻⁶. 7 The first fermentation was completed at CFU / mL over 72–80 hours; then, commercial brewing yeast CECA was inoculated at a concentration of 0.1–1 × 10⁻⁶ CFU / mL. 6 CFU / mL, the second fermentation is completed in 200-240 hours; The inoculation ratio of the compound fermentation agent to the brewing yeast is 10:1, and the fermentation parameters are: pH 3.0~3.5, fermentation temperature 20~23°C, and residual sugar content is less than 4g / L, resulting in a dry rosé wine with floral and fruity aromas, suitable acidity, and low volatile acid content.

5. The method as described in claim 4, characterized in that: In step (2), the SO2 is added in the form of potassium metabisulfite.

6. The method as described in claim 4, characterized in that: In step (3), the following fermentation parameters are further controlled: The sugar concentration is 250-300 g / L, the alcohol concentration is 9-11% (v / v), and the nitrogen source concentration is 200-300 mg / L.

7. The method as described in claim 4, characterized in that: The resulting wine product contains the following aroma compounds: esters, higher alcohols, volatile fatty acids, and other aroma compounds; among which: The esters are ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, phenylethyl acetate, isoamyl acetate, ethyl dodecanoate, and methyl octanoate. The higher alcohols are isobutanol, 1-butanol, isoamyl alcohol, 1-octanol, 1-decyl alcohol, 4-methyl-1-pentanol, 3-methyl-1-pentanol, and 2-phenylethanol; The fatty acids are hexanoic acid, octanoic acid, and decanoic acid; The other aroma compounds are β-damascene, citronellol, 4-terpene alcohol, benzaldehyde, and styrene.

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