Two yeasts, composite leavening agent of two yeasts and method for preparing low-alcohol yellow peach beverage

Abstract

The invention discloses two yeasts, a composite leavening agent of the two yeasts and a method for preparing a low-alcohol yellow peach beverage, and belongs to the technical field of microorganisms. The method solves the problems that the fermented beverage is unbalanced in flavor, insufficient in fragrance and too high in acidity due to the fact that the saccharomyces cerevisiae is independently used for fermentation. After the non-saccharomyces cerevisiae is added, the hansenula polymorpha strain HT106 and the saccharomyces cerevisiae JM9 in the grape juice promote growth mutually, meanwhile, aroma substances richer than those of single-bacterium fermentation can be generated, generation of bad flavor can be reduced, and the fermented beverage is endowed with more coordinated taste and flavor. The invention provides a new method for deep processing of yellow peaches and development of low-alcohol beverages.

Description

Technical Field

[0001] This invention relates to two yeast strains and their combined fermentation agent, and a method for preparing low-alcohol yellow peach beverages, belonging to the field of microbial technology. Background Technology

[0002] As consumers become increasingly focused on healthy drinking, low-alcohol beverages are gradually becoming an alternative to traditional alcoholic drinks. In recent years, the low-alcohol beverage market has experienced rapid growth, and the global market size is projected to reach US$1.89 billion by 2030, with a compound annual growth rate of 4.32%.

[0003] Yellow peaches are rich in vitamins, phenolic compounds, and various trace elements, and their pleasant aroma is loved by consumers. However, yellow peaches have a short shelf life after harvest and are highly susceptible to spoilage. Due to their rich fruity aroma, yellow peaches are an ideal raw material for low-alcohol beverages. Processing yellow peaches into low-alcohol beverages not only makes full use of this high-quality resource but also increases the added value of the product.

[0004] Patent CN111621430A discloses a brewing yeast strain suitable for making yellow peach wine and its application. This strain can adapt to the fermentation environment of yellow peach juice, has a fast fermentation speed, and complete fermentation. Furthermore, while ensuring strong alcohol production (10.71% vol), the resulting yellow peach wine has a harmonious taste and distinct yellow peach characteristics. Most reported literature on yellow peach wine focuses on fruit wines with alcohol contents between 3.10% vol and 14.50% vol (Food Industry Technology, 2018. 39(14); CN116670262A; Brewing Technology, 2024. 11(365)). De-alcoholization is usually carried out after fermentation, using techniques such as reverse osmosis (RO), nanofiltration (NF), or vacuum distillation (VD) to remove ethanol from alcoholic beverages. However, these treatments may negatively impact the flavor compounds and aroma quality of the product.

[0005] To improve the aroma and quality of low-alcohol peach beverages, patent CN120442409A discloses a low-ethanol, high-aroma strain of *Hansenula polymorpha* from grape juice and its applications. The provided high-aroma strain ferments a low-alcohol peach beverage with an alcohol content of 0.4% vol. Sensory evaluation shows that the beverage fermented by this strain exhibits prominent floral and fruity aromas, with improved richness and balance. However, single-strain fermentation suffers from a limited range of aroma characteristics. This invention utilizes mixed-strain fermentation to synthesize a more diverse range of esters, alcohols, and other aroma compounds, resulting in a more harmonious and layered flavor profile. Summary of the Invention

[0006] To address the shortcomings of the existing technology, this invention provides a low-alcohol yellow peach beverage and its preparation method, aiming to solve the technical problem of insufficient flavor in low-alcohol beverages prepared from a single strain.

[0007] The first technical solution provided by this invention is a grape juice yeast strain with low ethanol production and high aroma production (Hansenula polymorpha). Hanseniaspora grapes The grape juice containing Hansenula polymorpha HT106 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 15, 2025, with accession number GDMCC NO.66957.

[0008] The second technical solution provided by this invention is a low-ethanol-producing brewing yeast strain ( Saccharomyces yeast The brewing yeast JM9 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 15, 2025, with accession number GDMCC NO.66956.

[0009] The third technical solution provided by the present invention is a compound fermentation agent, which contains the grape juice Hansenula polysaccharide HT106 described in the first technical solution and the wine yeast JM9 described in the second technical solution.

[0010] In some embodiments, the ratio of live bacteria of Hansenula polymorpha HT106 to Saccharomyces cerevisiae JM9 in the grape juice is 1:1.

[0011] In some embodiments, the grape juice contains a mixed bacterial concentration of Hansenula polymorpha HT106 and Saccharomyces cerevisiae JM9 of not less than 1 × 10⁻⁶. 8 cfu / mL or 1×10 8 cfu / g.

[0012] The fourth technical solution provided by the present invention is a method for preparing the compound fermentation agent described in the third technical solution. The method involves mixing the grape juice Hansenula polymorpha HT106 described in the first technical solution and the wine yeast JM9 described in the second technical solution at a ratio of 1:1 and inoculating them into a liquid culture medium. After continuous activation for two generations, the activated culture is inoculated into a liquid culture medium at a volume percentage of 2-3% and cultured. The cell precipitate is obtained by centrifugation and resuspending to obtain the compound fermentation agent.

[0013] In some embodiments, activation is performed at 25-28°C for 10-12 hours.

[0014] The fifth technical solution provided by this invention is the application of the grape juice containing Hansenula polysaccharide HT106 as described in the first technical solution, the brewing yeast JM9 as described in the second technical solution, or the compound fermentation agent as described in the third technical solution in fermented foods.

[0015] In some embodiments, the fermented food is a low-alcohol yellow peach beverage.

[0016] In some embodiments, the fermented food also contains additives selected from one or more combinations of flavorings, fruit and vegetable juices, herbal tea juices, colorings, acidity regulators, preservatives, antioxidants, thickeners, and sweeteners.

[0017] The sixth technical solution provided by the present invention is a method for preparing a low-alcohol yellow peach beverage, wherein the method involves incorporating the compound fermenting agent described in the third technical solution into a fermentation system containing yellow peach juice to obtain a low-alcohol yellow peach beverage.

[0018] In some embodiments, the inoculum amount of the compound fermenting agent in the fermentation system is 2-3%, the fermentation temperature is 25-28°C, and the fermentation time is at least 2 days.

[0019] The seventh technical solution provided by the present invention is the application of the grape juice containing Hansenula polymorpha HT106 as described in the first technical solution, the wine yeast JM9 as described in the second technical solution, or the compound fermentation agent as described in the third technical solution in improving the content of flavor compounds in yellow peach beverages.

[0020] In some embodiments, the flavor compounds include phenethyl acetate, isoamyl acetate, benzyl acetate, furfuryl acetate, ethyl palmitate, cis-3-hexenol, phenethyl alcohol, γ-decyl lactone, and sulphalaldehyde.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: Fermentation using only Saccharomyces cerevisiae can lead to unbalanced flavors, insufficient aroma, and excessive acidity in fermented beverages. However, adding non-Saccharomyces cerevisiae strains, such as Hansenula polymorpha HT106 and Saccharomyces cerevisiae JM9, promotes the mutual growth of these two yeasts, resulting in a richer array of aroma compounds compared to single-strain fermentation. It also reduces the production of undesirable flavors, giving the fermented beverage a more harmonious taste and flavor. This invention provides a new method for the deep processing of yellow peaches and the development of low-alcohol beverages.

[0022] Preservation of biological materials A grape vine's juice contains Hansenula polymorpha ( Hanseniaspora grapes HT106, taxonomically named *Hansenula polymorpha* (grape juice spores). Hanseniaspora grapes The specimen was deposited on September 15, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong, China, with accession number GDMCC NO.66957.

[0023] A low-ethanol-producing strain of brewer's yeast ( Saccharomyces cerevisiae JM9, taxonomically named *Saccharomyces cerevisiae* (JM9), is a type of yeast. Saccharomyces cerevisiae The sample was deposited on September 15, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong, China, with accession number GDMCC NO.66956. Attached Figure Description

[0024] Figure 1 Sensory evaluation of low-alcohol yellow peach beverages fermented with different non-brewing yeasts and brewing yeasts. Detailed Implementation

[0025] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0026] Test method: 1. The physicochemical indicators were determined in accordance with the "GBT-2006 General Analytical Methods for Wines and Fruit Wines" for the analysis of the physicochemical indicators of low-alcohol yellow peach beverages. 2. Determination of flavor compounds Flavor compounds in low-alcohol yellow peach beverages were extracted using headspace solid-phase microextraction (HS-SPME). 5g of sample was accurately weighed and placed in a 20mL headspace vial, along with 0.2g of NaCl, and the vial was sealed. The mixture was equilibrated in a 55℃ water bath for 5 min, and then the aged extraction head was inserted into the headspace vial for 40 min of extraction. The sample was then analyzed by GC-MS after 5 min of resolution at the GC-MS inlet. GC-MS conditions: HP-INNOWax capillary column (60 μm × 0.25 mm × 0.25 μm); Temperature program: 40 °C for 3 min, ramp to 120 °C at 4 °C / min, ramp to 210 °C at 6 °C / min, hold for 9 min, ramp to 240 °C at 25 °C / min, hold for 3 min. Injector and ion source temperatures were 250 °C and 230 °C, respectively; helium flow rate was 1 mL / min; EI source energy was 70 eV; mass spectrometry scan range was 30–450 amu. Qualitative analysis of all volatile compounds was performed using the NIST20 mass spectrometry library.

[0027] 3. Sensory evaluation The sensory evaluation panel consisted of 12 trained evaluators who underwent standardized training to ensure accurate descriptions of sensory attributes and differentiation of intensity levels between samples. Prior to the formal experiments, panel members first determined high-frequency descriptors by evaluating randomly coded samples. They trained using reference samples to establish consensus on the intensity of each characteristic. All attributes, including fruit, floral, peach, sweet, acidic, and likability, were rated using a 9-point scale (e.g., 1 = very weak, 9 = very strong). Samples were placed in ISO standard tasting glasses, labeled with three-digit random codes, and then placed on white plates, presented in a random order. Discussion was prohibited during the evaluation process, and participants rinsed their mouths with water after each sample. The evaluations were conducted in separate chambers at room temperature (22°C–24°C) under white light.

[0028] Raw materials used in the examples: 1. Strains Information Table 1. Strain Information

[0029] 2. Preparation of culture medium YPD medium (g / L): glucose 20.0, peptone 20.0, yeast extract 10.0.

[0030] YPD agar medium (g / L): glucose 20.0, peptone 20.0, yeast extract 10.0, agar powder 20.0.

[0031] Example 1 1. Preparation of seed liquid Saccharomyces cerevisiae JM9 seed culture: Saccharomyces cerevisiae JM9 stored in glycerol tubes were taken from a -80℃ freezer and inoculated into YPD liquid medium at a 3% inoculation rate. Each generation was cultured for 24 hours, and the culture was subcultured twice. The culture was centrifuged at 4500 r / min for 10 min, washed three times with distilled water, and the bacterial sludge was resuspended in an equal volume of sugar- and acid-adjusted yellow peach juice for later use. Grape juice Hansenula spores HT106 seed culture: Grape juice Hansenula spores HT106 stored in glycerol tubes was inoculated into YPD liquid medium at a 3% inoculation rate. Each generation was cultured for 12 hours, and the culture was subcultured twice. The culture was centrifuged at 4500 r / min for 10 min, washed three times with distilled water, and the bacterial sludge was resuspended in an equal volume of sugar- and acid-adjusted yellow peach juice for later use. 2. Preparation of low-alcohol yellow peach beverage The specific steps are as follows: (1) Preparation of yellow peach juice: Adjust the sugar content of concentrated yellow peach juice to 15°Brix using distilled water; (2) Sterilization: Sterilize the prepared yellow peach juice at 75℃ for 30 minutes; (3) Inoculation: Add Saccharomyces cerevisiae JM9 seed liquid and Saccharomyces spp. Hansenula spp. HT106 seed liquid to sterilized yellow peach juice for mixed fermentation. The inoculation amount of Saccharomyces cerevisiae JM9 seed liquid is 1.5%, and the inoculation amount of Saccharomyces spp. Hansenula spp. HT106 seed liquid is 1.5%. (4) Fermentation: Ferment the inoculated yellow peach juice at 25℃ for 48 hours; (5) Centrifugation: Centrifuge the fermented low-alcohol yellow peach beverage at 10,000 rpm and 4°C for 10 min. (6) Storage: The supernatant of the fermented beverage after centrifugation is placed into a sterile beverage bottle and stored at 4°C.

[0032] Example 2 Low-alcohol yellow peach beverage was prepared by single-strain fermentation of Saccharomyces cerevisiae JM9. The inoculum size for single-strain fermentation was 3%, and the preparation method was the same as in Example 1.

[0033] Example 3 Low-alcohol yellow peach beverage was prepared by fermenting grape juice with Hansenula polymorpha HT106. The inoculum size for single-strain fermentation was 3%, and the preparation method was the same as in Example 1.

[0034] Comparative Example 1 Low-alcohol yellow peach beverage was prepared by single-strain fermentation of commercial brewing yeast AS1394. The inoculum size for single-strain fermentation was 3%, and the preparation method was the same as in Example 1.

[0035] Comparative Example 2 The culture was mixed with commercial brewing yeast AS1394 and grape juice Hansenula spores HT106 seed culture for fermentation. The inoculum amount of commercial brewing yeast AS1394 was 1.5%, and the inoculum amount of grape juice Hansenula spores HT106 was 1.5%. The preparation method was the same as in Example 1.

[0036] Comparative Example 3 Low-alcohol yellow peach beverage was prepared by single-strain fermentation of grape juice with Hansenula polymorpha Y112. The inoculum size for single-strain fermentation was 3%, and the preparation method was the same as in Example 1.

[0037] Effect Example 1: Comparison of physicochemical properties of low-alcohol beverages prepared in Examples 1-3 and Comparative Examples 1-3 The physicochemical properties of low-alcohol yellow peach beverages were analyzed according to GB / T-2006 General Analytical Methods for Wines and Fruit Wines. The results are shown in Table 2.

[0038] As shown in Table 2, the pH values ​​of the fermented beverages from different embodiments and comparative examples vary significantly. Compared to the comparative example, the pH of the beverage fermented with the strain of the present invention is significantly increased, resulting in a smoother taste. The alcohol content of the beverage fermented with the strain of the present invention is lower, meeting the standards for general low-alcohol beverages.

[0039] Table 2 Physicochemical properties of yellow peach beverages fermented using different implementation methods

[0040] Effect Example 2: Comparison of the ability to generate flavor compounds in low-alcohol beverages prepared in Examples 1-3 and Comparative Example 3 Flavor compounds in low-alcohol yellow peach beverages were extracted using headspace solid-phase microextraction (HS-SPME). 5g of sample was accurately weighed and placed in a 20mL headspace vial, along with 0.2g of NaCl, and the vial was sealed. The mixture was equilibrated in a 55℃ water bath for 5 min, and then the aged extraction head was inserted into the headspace vial for 40 min of extraction. The sample was then analyzed by GC-MS after 5 min of resolution at the GC-MS inlet. GC-MS conditions: HP-INNOWax capillary column (60 μm × 0.25 mm × 0.25 μm); Temperature program: 40 °C for 3 min, ramped to 120 °C at 4 °C / min, then ramped to 210 °C at 6 °C / min for 9 min, ramped to 240 °C at 25 °C / min for 3 min. Injector and ion source temperatures were 250 °C and 230 °C, respectively; helium flow rate was 1 mL / min; EI source energy was 70 eV; mass spectrometry scan range was 30–450 amu. Qualitative analysis of all volatile compounds was performed using the NIST20 mass spectrometry library. Results are shown in Table 3.

[0041] Table 3. Volatile compound content of yellow peach beverages fermented using different methods

[0042] Compared with Examples 2, 3, and Comparative Example 3, the mixed-culture fermentation in Example 1 significantly increased the diversity and concentration of volatile compounds. Example 1 contained a total of 34 volatile compounds, representing an increase of three compound types and an 81.2% increase in total content compared to Comparative Example 3 (patent CN120442409A).

[0043] Compared to Examples 2 and 3, Example 1 introduces new ester compounds through the interaction of HT106 and JM9: phenethyl acetate, isoamyl acetate, benzyl acetate, furfuryl acetate, and ethyl palmitate; alcohol compounds: cis-3-hexenol, phenethyl alcohol, and myristol; and lactone compounds: γ-decyl lactone and peach aldehyde. Phenethyl acetate contributes fruit and banana aromas, isoamyl acetate enhances banana and pear aromas, while benzyl acetate, furfuryl acetate, and ethyl palmitate contribute rich fruity aromas. Alcohols are the second largest component in low-alcohol beverages and significantly influence their flavor. Cis-3-hexenol imparts fresh green leaf and floral aromas to the low-alcohol yellow peach beverage, while myristol provides a subtle, mild waxy odor. γ-decyl lactone and peach aldehyde are characteristic aroma compounds of the low-alcohol yellow peach beverage, contributing a rich peach flavor. Compared to Examples 2 and 3, Example 1, through the interaction of HT106 and JM9, resulted in a 333.58% increase in the content of phenethyl acetate and a 249.15% increase in the content of isoamyl acetate, with other esters also showing significant increases. The presence of more types or amounts of these volatile compounds in Example 1 compared to the comparative example enhanced the aroma richness of the low-alcohol yellow peach beverage, producing a technical effect unexpected by those skilled in the art.

[0044] Effect Example 3: Sensory evaluation comparison of low-alcohol beverages prepared in Examples 1-3 and Comparative Examples 1-3 Sensory indicators included aroma and liking, as shown in Table 4. Sensory rating criteria: 0 indicates none, 1-3 indicates weak, 4-6 indicates moderate, and 7-9 indicates strong. The sensory evaluation panel consisted of 12 researchers from Shanghai University of Applied Technology, and the results were averaged. The sensory evaluation results are as follows: Figure 1 .

[0045] Table 4 Sensory indicators and their reference samples

[0046] As shown in Figure 1, the sensory evaluation results indicate that the fruit and peach aroma scores of Example 1 are significantly higher than those of the comparative example. The low-alcohol yellow peach beverage fermented in Example 1 exhibits the most prominent peach aroma and significantly reduces the unpleasant acidic odor. Compared to Comparative Example 3 (Patent CN120442409A), the aroma of Example 1 is more harmonious and has the highest liking. Combining volatile matter determination and sensory analysis, the mixed culture of *Hansenula polymorpha* HT106 and *Saccharomyces cerevisiae* JM9 strains in grape juice significantly enhances the flavor and sensory qualities of the low-alcohol yellow peach beverage, demonstrating their great potential in low-alcohol yellow peach production.

[0047] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A grape vine's juice contained Hansenula polymorpha ( ) Hanseniaspora uvarum HT106, characterized in that, The grape juice containing *Hansenula polymorpha* HT106 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 15, 2025, with accession number GDMCC NO.66957.

2. A low-ethanol-producing strain of brewing yeast ( Saccharomyces cerevisiae JM9, characterized in that, The brewing yeast JM9 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 15, 2025, with accession number GDMCCNO.66956.

3. A compound fermentation agent, characterized in that, The compound fermentation agent contains the grape juice Hansenula polysaccharide HT106 as described in claim 1 and the brewing yeast JM9 as described in claim 2.

4. The compound fermentation agent according to claim 3, characterized in that, The ratio of live bacteria of *Hansenula polymorpha* HT106 and *Saccharomyces cerevisiae* JM9 in the grape juice is 1:1; the mixed bacterial concentration of *Hansenula polymorpha* HT106 and *Saccharomyces cerevisiae* JM9 in the grape juice is not less than 1×10⁻⁶. 8 cfu / mL or 1×10 8 cfu / g.

5. A method for preparing the composite fermentation agent according to claim 3 or 4, characterized in that, The method involves mixing the grape juice Hansenula polysaccharide HT106 of claim 1 and the brewing yeast JM9 of claim 2 in a 1:1 ratio and inoculating them into a liquid culture medium. The mixture is then activated at 25-28°C for 10-12 hours, and activated for two generations. The activated culture is then inoculated into a liquid culture medium at a volume percentage of 2-3%, centrifuged to obtain cell precipitate, and resuspended to obtain the compound fermentation agent.

6. The application of the grape juice containing Hansenula polymorpha HT106 as described in claim 1, the brewing yeast JM9 as described in claim 2, or the compound fermenting agent as described in claim 3 or 4 in fermented foods.

7. A method for preparing a low-alcohol yellow peach beverage, characterized in that, The method involves inoculating the compound fermenting agent described in claim 3 or 4 into a fermentation system containing yellow peach juice to obtain a low-alcohol yellow peach beverage.

8. The method according to claim 7, characterized in that, The inoculum amount of the compound fermentation agent in the fermentation system is 2-3%, the fermentation temperature is 25-28℃, and the fermentation time is at least 2 days.

9. The application of the grape juice containing Hansenula polymorpha HT106 as described in claim 1, the brewing yeast JM9 as described in claim 2, or the compound fermenting agent as described in claim 3 or 4 in improving the content of flavor compounds in yellow peach beverages.

10. The application according to claim 9, characterized in that, The flavor compounds include phenethyl acetate, isoamyl acetate, benzyl acetate, furfuryl acetate, ethyl palmitate, cis-3-hexenol, phenethyl alcohol, γ-decyl lactone, and sulphalaldehyde.

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