Hansenula polymorpha with low ethanol yield and high aroma yield and application of hansenula polymorpha

Abstract

The invention discloses hansenula polymorpha with low ethanol yield and high aroma yield and application of hansenula polymorpha, and belongs to the technical field of microorganisms. According to the invention, a strain of high-aroma-yield saccharomycetes, which is hanseniaspora uvarium, is separated from fruits, and the preservation number of the saccharomycetes is CCTCC (China Center for Type Culture Collection) NO: M 20242562. The quality of the low-alcohol yellow peach beverage fermented by the high-aroma-yield strain provided by the invention is improved, and the alcoholic strength after fermentation is well reduced and is only 0.4% vol. Through flavor substance detection before and after fermentation, flavor substances generated by the strain are rich in variety, for example, 90.73 mu g / kg of phenethyl alcohol is newly generated. Sensory evaluation shows that the low-alcohol yellow peach beverage fermented by the strain is outstanding in flower fragrance and fruit fragrance, the richness and the balance degree are improved, it is proved that the overall flavor characteristics of the low-alcohol beverage can be remarkably improved, and more pleasant sensory experience is brought to consumers.

Description

Technical Field

[0001] The invention relates to a Hansenula sporogenes strain of grape juice with low ethanol production and high aroma production and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Alcoholic beverages possess a unique flavor. With improvements in living standards and shifts in public health awareness, the harmful effects of high-alcohol beverages on human health have become increasingly recognized. Reducing the alcohol content in alcoholic beverages satisfies consumers' pursuit of health, leading to a growing demand for low-alcohol beverages. However, due to limitations in production processes and the lack of suitable fermentation strains, low-alcohol beverages often lack aroma and flavor. Current research focuses on developing low-alcohol-yielding yeasts suitable for fermentation.

[0003] Flavor is a key factor influencing the quality of low-alcohol beverages and a key indicator influencing consumer acceptance. The production process for low-alcohol beverages primarily includes juice blending, fermentation, dealcoholization, sterilization, and canning. However, the dealcoholization step requires high temperatures, which significantly damages aroma components, resulting in a poor flavor profile for the dealcoholized low-alcohol beverage, hindering its development. Currently, most research on low-alcohol beverages focuses on preparation methods and dealcoholization process optimization. A low-alcohol grape sparkling wine preparation process uses dry grape wine as a raw material and adds water to the blend. This significantly reduces the nutrient content of the low-alcohol beverage and dilutes the flavor, resulting in a weak flavor. A low-alcohol coconut water and its preparation method utilize a mixed fermentation of Saccharomyces cerevisiae and Lactobacillus plantarum, but the resulting low-alcohol coconut water beverage still has a high alcohol content. A low-alcohol fermented fig beverage and its preparation method utilize intermittent oxygenation during the fermentation phase to promote yeast proliferation and produce a unique flavor. However, this method results in a high alcohol content after fermentation and requires dealcoholization, which still cannot avoid flavor loss. Therefore, low-alcohol beverages produced by existing technologies still have problems such as flavor loss and high alcohol content.

[0004] During the fermentation process, fermentation strains produce a rich variety of flavor substances, such as esters, acids, alcohols, and terpenes. Therefore, fermentation strains have a significant impact on the aroma of low-alcohol beverages. Aroma-rich strains can significantly enhance the flavor of low-alcohol beverages. Given the current lack of low-alcohol, high-aroma-yielding strains suitable for fermenting low-alcohol yellow peach beverages, the present invention has developed a low-ethanol, high-aroma-yielding yeast through multiple screening processes. This reduces the ethanol content of fermented beverages from the source, increases the flavor of low-alcohol beverages, and improves the quality of low-alcohol beverages. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a low-ethanol-yielding and high-aroma-yielding Hansenula grape juice yeast and its application, aiming to solve the technical problem in the prior art of lacking a strain suitable for the fermentation of low-alcohol yellow peach beverages.

[0006] The first technical solution provided by the present invention is a low-ethanol-yielding and high-aroma-yielding Hanseniaspora uvarum Y112 strain of grape juice. The grape juice Hanseniaspora Y112 has been deposited in the China Center for Type Culture Collection on November 15, 2024, with the deposit number CCTCC NO: M 20242562.

[0007] The second technical solution provided by the present invention is a microbial preparation containing the Hansenula sporangiophora vitis vinifera Y112 described in the first technical solution.

[0008] In certain embodiments, the concentration of Hansenula vitis spores Y112 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0009] Furthermore, the concentration of Hansenula sporangiophora vitis vinifera Y112 in the microbial preparation is at least 1×10 7 CFU / mL or 1×10 7 CFU / g.

[0010] The third technical solution provided by the present invention is a starter culture, which contains the Hansenula sporangiophora Y112 described in the first technical solution or the microbial preparation described in the second technical solution.

[0011] In certain embodiments, the amount of Hansenula sporangiophora Y112 added to the starter is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0012] Furthermore, the amount of Hansenula sporangiophora vitis vinifera Y112 added to the starter is at least 1×10 7 CFU / mL or 1×10 7 CFU / g.

[0013] The fourth technical solution provided by the present invention is a food, which is a fermented food obtained by fermenting with the Hansenula sporangiophora Y112 described in the first technical solution, the microbial preparation described in the second technical solution, or the starter described in the third technical solution.

[0014] In certain embodiments, the fermented food includes dairy products, soy products, fruit and vegetable products, or other fermented foods containing Hansenula vitis sporangia Y112.

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

[0016] In certain embodiments, the fermented food further contains additives, and the additives are selected from one or a combination of two or more of spices, fruit and vegetable juices, scented tea juices, colorants, acidity regulators, preservatives, antioxidants, thickeners, and sweeteners.

[0017] The fourth technical solution provided by the present invention is the use of the Hansenula sporangiophora Y112 described in the first technical solution, or the microbial preparation described in the second technical solution, or the starter described in the third technical solution in yellow peach fermentation.

[0018] The fifth technical solution provided by the present invention is a method for preparing a low-alcohol yellow peach beverage, which comprises introducing the Hansenula sporangiophora Y112 described in the first technical solution, or the microbial preparation described in the second technical solution, or the starter described in the third technical solution into a fermentation system containing yellow peach juice for fermentation to obtain a low-alcohol yellow peach beverage.

[0019] In certain embodiments, the inoculation amount of the Hansenula sporangiophora vitis Y112 in the fermentation system is 2-3%, the fermentation temperature is 25-28° C., and the fermentation time is at least 2 days.

[0020] The sixth technical solution provided by the present invention is the use of the Hansenula sporangiophora Y112 described in the first technical solution, or the microbial preparation described in the second technical solution, or the starter culture described in the third technical solution in increasing the content of flavor compounds in yellow peach beverages.

[0021] In certain embodiments, the flavor compounds include ethyl acetate, isoamyl acetate, gamma-decalactone, phenylethyl alcohol, geraniol, acetic acid, octanoic acid, and furfural.

[0022] Beneficial effects

[0023] The present invention isolated a high-aroma yeast strain from fruit, Hanseniaspora uvarum, with a deposit number of CCTCC NO: M 20242562. This high-aroma yeast strain fermented with this strain improves the quality of a low-alcohol yellow peach beverage and significantly reduces the alcohol content after fermentation to only 0.4% vol. Sensory evaluation before and after fermentation showed that this strain produces a good fermentation aroma and a rich variety of flavor substances. Sensory evaluation also revealed that this strain enhances the fruity and floral aromas of the low-alcohol beverage.

[0024] Biomaterial Deposit

[0025] A strain of Hanseniaspora uvarum Y112, taxonomically named Hanseniaspora uvarum, was deposited in the China Center for Type Culture Collection on November 15, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 20242562. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the phylogenetic (developmental) tree of Hansenula vitis vinifera Y112 based on 26 rDNA genes.

[0027] Figure 2 This is a microscope observation of Hansenula sporangiophora Y112 in grape juice.

[0028] Figure 3 This is the growth curve of Hansenula sporangiophora Y112.

[0029] Figure 4 This is a sensory evaluation chart of a low-alcohol beverage containing Hansenula sporogenes Y112. DETAILED DESCRIPTION

[0030] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0031] The raw materials used in the embodiment are:

[0032] 1. YPD medium (g / L): glucose 20.0%, peptone 20.0%, yeast powder 10.0%.

[0033] 2. YPD medium plate (g / L): glucose 20.0, peptone 20.0, yeast powder 10.0, agar powder 20.0.

[0034] 3. TTC medium (g / L): peptone 10.0, beef extract powder 3.0, sodium chloride 5.0, agar 15.0, TTC 0.01.

[0035] 4. Hansenula sporogenes HT2-28 is derived from the natural fermentation liquid of yellow peach.

[0036] Example 1 Isolation, screening and identification of yeast

[0037] (1) Sample pretreatment

[0038] Take several portions of 20g of fruit, remove the core and chop them, then put them into a fermentation tank, add 50ml of sterile water and 5g of white sugar, stir evenly, and then put them into a 25℃ incubator to ferment for 2 days.

[0039] (2) Yeast isolation

[0040] Sterile water was used to dilute the treated samples to different concentrations, with the gradient set at 10 -1 ~10 -6 Each gradient was set up in triplicate. Appropriate amounts of the dilutions were spread onto YPD solid medium and incubated in a 25°C incubator for 2 days. Strains of varying sizes, milky white colonies, and typical yeast morphology were selected using sterile toothpicks and placed into liquid YPD medium and incubated in a 25°C incubator for 1 day. These strains were then streaked onto YPD solid medium for purification to obtain purified single colonies. The purified strains were stored in YPD liquid medium supplemented with 30% glycerol as a protective agent and frozen at -80°C.

[0041] A total of 327 bacterial strains were isolated from different batches of samples on YPD solid medium.

[0042] (3) Screening of low-ethanol-producing yeast

[0043] TTC plate color development: Activate the 327 isolated strains, then pipette 2 μL of the bacterial solution onto TTC medium. Incubate inverted at 25°C for 2 days. Observe the color reaction. Yeast colonies with strong ethanol production will appear dark red, followed by pink. Non-ethanol-producing yeast will show no color. Strain Y112 will show a light pink color, indicating weak ethanol production during fermentation.

[0044] (4) Screening of high-aroma yeast

[0045] Olfactory analysis: The strains that have passed the initial screening were transferred to YPD liquid culture medium and cultured in a 25℃ incubator for 1 day. The blank YPD liquid culture medium without inoculation of the strains was used as a control, and the fermentation liquid was subjected to olfactory analysis to screen out yeasts with rich aroma production.

[0046] (5) Biological identification of Hansenula viticola

[0047] PCR amplification of the 26S rDNA D1 / D2 region was performed using yeast genomic DNA as a template and primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′). The PCR reaction system (25 μL) consisted of 1 μL of template, 0.5 μL each of primers NL1 and NL4, 10.5 μL of 2× Rapid Taq Master Mix, and 12.5 μL of ddH2O. The PCR reaction conditions were: initial denaturation at 95°C for 3 minutes, denaturation at 95°C for 10 seconds, annealing at 52°C for 1 minute, and extension at 72°C for 1 minute 30 seconds, followed by 35 cycles of extension at 72°C for 5 minutes. 5 μL of PCR amplification product was analyzed by 1% agarose gel electrophoresis, and then the PCR amplification solution was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The DNA sequence of the strain obtained by sequencing was compared with the GenBank sequence homology from the National Center for Biotechnology Information (NCBI) database. A phylogenetic tree was constructed using MEGA10.0, and 1000 bootstrap tests were performed. Figure 1 It can be seen that all the screened strains formed a cluster with the Hanseniaspora uvarum model strain QTX D5 at a 100% step test confidence level, and thus the Y112 strain could be identified as Hanseniaspora uvarum.

[0048] (6) Microbiological characteristics of Hansenula sporeifera in grape juice

[0049] Characteristics of colonies on YPD plates: The colonies are round, with a convex center, a smooth and moist surface, regular edges, and are milky white and opaque.

[0050] Colony characteristics under microscope: Figure 2 As shown, the strain appears oval or rod-shaped under a microscope.

[0051] Growth curve: The growth curve of Hansenula sporogenes Y112 was determined by measuring OD600. Figure 3 The results of growth curve determination showed that the logarithmic growth period of Hansenula sporangiophora vitis vinifera Y112 was 2 to 8 h, the strain grew rapidly and had strong growth activity, making it suitable for making a high-activity starter culture.

[0052] Example 2: Fermentation of low-alcohol beverages with Hansenula sporogenes Y112 in grape juice

[0053] The grape juice spore Hansenula Y112 strain was inoculated into YPD liquid medium and activated for two generations. Then, the activated culture was inoculated into the liquid medium at a volume percentage of 2-3%, and the cell pellet was obtained by centrifugation. The pellet was suspended in sterile yellow peach juice to obtain a starter. In the starter, the number of viable bacteria was 10 7 The concentrated yellow peach juice was diluted and prepared to a sugar content of 15 Brix, sterilized by pasteurization, and after cooling, a working starter was added, with the starter accounting for 2% of the volume of the fermented beverage. After mixing, the mixture was fermented at 25°C for 2 days to obtain a low-alcohol yellow peach beverage.

[0054] Example 3

[0055] The grape juice spore Hansenula Y112 strain was inoculated into a liquid culture medium and activated for two generations. Then, the activated culture was inoculated into the liquid culture medium at a volume percentage of 2-3%, and the cell precipitate was obtained by centrifugation. The precipitate was suspended in sterile yellow peach juice to obtain a working starter. In the starter, the number of viable bacteria was 10 7 The concentrated yellow peach juice was diluted and prepared to a sugar content of 15 Brix, sterilized by pasteurization, and after cooling, a working starter culture was added, wherein the working starter culture accounted for 5% of the volume of the fermented beverage. After mixing, the mixture was fermented at 25°C for 2 days to obtain a low-alcohol yellow peach beverage.

[0056] Example 4

[0057] The grape juice spore Hansenula Y112 strain was inoculated into a liquid culture medium and activated for two generations. Then, the activated culture was inoculated into the liquid culture medium at a volume percentage of 2-3%, and the cell precipitate was obtained by centrifugation. The precipitate was suspended in sterile yellow peach juice to obtain a working starter. In the starter, the number of viable bacteria was 10 7 The concentrated yellow peach juice was diluted and prepared to a sugar content of 10 Brix, sterilized by pasteurization, and after cooling, a working starter culture was added, wherein the working starter culture accounted for 2% of the volume of the fermented beverage. After mixing, the mixture was fermented at 25°C for 2 days to obtain a low-alcohol yellow peach beverage.

[0058] Comparative Example 1

[0059] Preparation of yellow peach juice without Hansenula vitis spores Y112 specifically comprises the following steps: diluting concentrated yellow peach juice to 15 Brix, and performing the other operations according to the steps of Example 2 except that the strain is not inoculated.

[0060] Comparative Example 2

[0061] The preparation of Hansenula vitis spores HT2-28 comprises the following steps: the steps in Example 2, except that Y112 is replaced by HT2-28.

[0062] Test Example 1

[0063] Comparison of the flavor compound production capabilities of the low-alcohol beverages prepared in Examples 2 to 4 and Comparative Examples 1 and 2.

[0064] Headspace solid-phase microextraction (HS-SPME) was used to extract flavor compounds from a low-alcohol yellow peach beverage. A 5g sample was accurately weighed and placed in a 20mL headspace vial. 0.2g of NaCl was added and the vial was sealed. The vial was equilibrated in a 55°C waterbath for 5 minutes. An aging extraction tip was then inserted into the headspace vial and extracted for 40 minutes. The sample was then desorbed in the GC-MS inlet for 5 minutes before GC-MS analysis.

[0065] GC-MS conditions: An HP-INNOWax capillary column (60 μm × 0.25 mm × 0.25 μm) was used; the temperature program was: 40°C for 3 min, then increased at 4°C / min to 120°C, then increased at 6°C / min to 210°C, held for 9 min, then increased at 25°C / min to 240°C, held for 3 min. The inlet and ion source temperatures were 250°C and 230°C, respectively. The helium flow rate was 1 mL / min, and the EI source energy was 70 eV. The mass spectrometer scan range was m / z 30 to 450 amu. All volatile compounds were identified using the NIST20 mass spectral library. The results are shown in Table 1.

[0066] As shown in Table 1, GC-MS detected a total of 60 volatile flavor compounds, including 20 esters, 13 alcohols, 8 acids, 6 ketones, 10 aldehydes, and 3 aromatic compounds. Fermentation of a low-alcohol yellow peach beverage with Hansenula sporogenes Y112 significantly impacted the flavor components. After fermentation with Hansenula sporogenes Y112, 12 more ester compounds were found compared to Comparative Example 1 and 6 more compared to Comparative Example 2, with the total ester content increasing by 246.4%. Three more alcohol compounds were found, with the total alcohol content increasing by 351.1%, a 67.29% increase compared to Comparative Example 2. The Examples exhibited a richer content and variety of ester compounds (ethyl acetate, propyl acetate, isoamyl acetate, benzyl acetate, etc.) and alcohol compounds (isobutanol, isoamyl alcohol, phenylethyl alcohol, etc.). Among various aromatic compounds, esters are the largest aromatic compound in low-alcohol yellow peach beverages. Different esters can increase the complexity of the aroma of low-alcohol beverages. Ethyl acetate can bring fruity aroma. Compared with the comparative example, the new example produced 826.23μg / kg of ethyl acetate. Isoamyl acetate produces a banana-like aroma in low-alcohol beverages. Compared with the comparative example, the new example produced 60.06μg / kg of isoamyl acetate. Lactones are considered to be the main contributors to peach aroma, especially γ-decalactone, which is considered to be the characteristic aroma compound of yellow peach. Compared with the comparative example, the new example produced 38.55μg / kg of γ-decalactone. These substances are abundant in the examples, demonstrating the high aroma-producing ability of the strain. The second largest aromatic compound is higher alcohols, among which phenylethanol can bring rose aroma to low-alcohol beverages. Compared with the comparative example, the new example produced 90.73μg / kg of phenylethanol. Trace volatile compounds such as linalool (citrus aroma) and geraniol can bring a refreshing taste of citrus, making people feel happy. Compared with the comparative example, the examples contain richer species; acetic acid and caprylic acid have high aroma intensity in low-alcohol beverages, and give low-alcohol beverages a sweet, fruity and cheesey aroma, so that the low-alcohol beverages fermented by this strain have both floral and fruity aromas. The content of the examples is higher than that of the comparative example, which corresponds to the sensory evaluation. Terpene compounds are related to floral and lemon aromas, and furfural can contribute "sweet aroma", "baked aroma", "woody aroma" and "toasted bread" aromas in low-alcohol beverages. The types of these compounds in the examples are more than those in the comparative example, which increases the richness of the aroma of the low-alcohol yellow peach beverage.

[0067] Table 1 Content of volatile compounds

[0068]

[0069]

[0070]

[0071] Test Example 2

[0072] The low-alcohol beverages prepared in Examples 2 to 4 and Comparative Examples 1 and 2 were subjected to sensory evaluation.

[0073] The sensory index includes aroma and taste. The specific indexes are shown in Table 2. Sensory scoring standard: 0 means no, 1-3 means weak, 4-6 means medium, 7-9 means strong. The sensory evaluation team consists of 12 experimenters from Shanghai University of Technology, and the results are averaged. Figure 4 .

[0074] Table 2 Sensory indicators

[0075]

[0076] Depend on Figure 4 As can be seen, the control sample scored low for the sensory attributes of "balance" and "richness," and its aroma was poorly rich. Compared to the control sample, the example sample scored higher for "richness" and achieved the highest scores for "fruity" and "floral." This demonstrates that the addition of Hansenula sporangiophora Y112 significantly improved the aroma and flavor of the low-alcohol beverage. Furthermore, significant differences were observed between the example sample and the control sample in indicators such as fruity, floral, peachy, and richness, demonstrating the significant impact of Hansenula sporangiophora Y112 on low-alcohol beverages.

[0077] Test Example 3

[0078] Table 3 shows a comparison of the physical and chemical properties of the low-alcohol beverages prepared in Examples 2-4 and Comparative Examples 1 and 2. The pH and sugar content of Example 2 are similar to those of Comparative Example 2. The low-alcohol beverage fermented with Hansenula sporangiophora Y112 had an alcohol content of only 0.4% vol. While the alcohol content increased slightly with increasing inoculum level, it remained below 1.5% vol. The low-alcohol beverage fermented with yellow peach juice, which has a lower sugar content, had an even lower alcohol content. Compared to the comparative examples, the low-alcohol beverage prepared in the examples had an alcohol content reduced by 52.17%.

[0079] Table 3 Comparison of physical and chemical indicators

[0080] Alcohol content (vol%) pH Brix Example 2 0.4±0.1 3.15+0.01 12.76±0.06 Example 3 1.1±0.2 3.06±0.03 11.76±0.15 Example 4 0.3±0.1 3.21±0.02 8.8±0.1 Comparative Example 1 0 3.51+0.02 15.23±0.05 Comparative Example 2 2.3+0.1 2.95+0.06 11.46±0.14

[0081] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Hanseniaspora uvarum Y112, characterized in that: It was deposited in the China Center for Type Culture Collection on November 15, 2024, with the deposit number CCTCC NO:M 20242562.

2. A microbial preparation containing the Hansenula sporangiophora Y112 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The concentration of Hansenula vitis spores Y112 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A leavening agent, characterized in that The fermentation agent contains the Hansenula sporangiophora Y112 according to claim 1 or the microbial preparation according to claim 2 or 3.

5. A food, characterized in that The food is a fermented food obtained by fermenting the Hansenula sporangiophora Y112 according to claim 1, the microbial preparation according to claim 2 or 3, or the starter according to claim 4.

6. Use of the Hansenula sporogenes Y112 of claim 1, or the microbial preparation of claim 2 or 3, or the starter of claim 4 in yellow peach fermentation.

7. A method for preparing a low-alcohol yellow peach beverage, characterized in that: The method comprises introducing the Hansenula sporangiophora Y112 of claim 1, or the microbial preparation of claim 2 or 3, or the starter of claim 4 into a fermentation system containing yellow peach juice for fermentation to obtain a low-alcohol yellow peach beverage.

8. The method according to claim 7, characterized in that The inoculation amount of the Hansenula sporogenes Y112 in the fermentation system is 2-3%, the fermentation temperature is 25-28° C., and the fermentation time is at least 2 days.

9. Use of the Hansenula sporogenes Y112 of claim 1, or the microbial preparation of claim 2 or 3, or the starter culture of claim 4 in increasing the content of flavor compounds in yellow peach beverages.

10. The use according to claim 9, characterized in that The flavor compounds include ethyl acetate, isoamyl acetate, γ-decalactone, phenylethyl alcohol, geraniol, acetic acid, octanoic acid, and furfural.

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