Ester-producing aroma type heat-resistant lamoney yeast strain and application thereof

Abstract

The invention relates to an ester-producing aroma type heat-resistant saccharomyces lajanus strain, which is separated and screened from loquat peel in Yonghenzhen town in Hangzhou City, Zhejiang Province, and has the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.34289 and the preservation date of April 21, 2025. According to the invention, the liquid microbial inoculum of the heat-resistant ramose yeast is added in the brewing process of the loquat fruit wine, so that the fermentation rate can be increased, flavor substances (ethyl acetate, phenethyl alcohol, isoamyl alcohol, isobutanol, ethanol, isoamyl alcohol, phenethyl alcohol, isoamyl acetate and phenethyl acetate) in the loquat fruit wine are increased, and especially the contents of isoamyl acetate, phenethyl acetate and phenethyl alcohol are increased; the method has important significance on improving the quality of the loquat fruit wine, and has a very good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit wine brewing, and particularly relates to an ester-aroma-producing heat-resistant Lachancea yeast strain and application thereof. Background Art

[0002] Loquat is a fruit native to my country, with production accounting for 70% of the world's total. As a typical respiratory climacteric fruit, its post-harvest physiological characteristics make the fruit extremely susceptible to softening and rotting. It is also susceptible to mechanical damage during transportation and storage, making it difficult to preserve. At the same time, the concentrated ripening period of loquat leads to high sales pressure and significant price fluctuations. Although deep-processing loquat wine can extend the industrial chain and increase added value, the flavor substances (such as polyphenols, esters, etc.) in loquat wine are limited by the metabolic capacity of the fermentation bacteria, and the aroma complexity is insufficient, making it difficult to meet consumers' demand for the coordination of fruit aroma and wine aroma.

[0003] In recent years, the collaborative fermentation of non-Saccharomyces yeasts with Saccharomyces cerevisiae has become a research hotspot for enhancing the flavor of fruit wine. Non-Saccharomyces yeasts secrete extracellular enzymes such as β-glucosidase, releasing bound aroma precursors and synthesizing flavor compounds such as esters and higher alcohols, thereby improving the aroma of fruit wine and imparting its unique flavor. Fruit wine flavor compounds are primarily produced through yeast metabolism, so the screening and application of aroma-producing yeasts is of great practical significance for improving the quality of fruit wine.

[0004] Existing loquat wine brewing mostly relies on wine yeast or a single brewing yeast, but these strains have low metabolic efficiency for loquat-specific carbon sources (such as pectin and sorbitol) and lack the synergistic effect of the natural bacterial flora attached to the loquat peel. Traditional brewing yeast has insufficient ability to degrade malic acid, resulting in excessive acidity in the finished wine. Therefore, screening indigenous strains with aroma-producing characteristics and adapted to the loquat matrix from the loquat orchard environment and naturally fermented mash has become a key technical path to break through the flavor bottleneck, and can provide strong support for the brewing of high-quality loquat wine with rich floral and fruity aroma, sweet taste, and refreshing taste. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention aims to provide a heat-resistant Lachancea yeast strain producing ester aroma and a technical solution for its application.

[0006] The present invention is specifically implemented through the following technical solutions:

[0007] A thermotolerant Lachancea yeast strain producing an ester aroma was isolated and screened from loquat peel in Tangqi Town, Linping District, Hangzhou City, Zhejiang Province. The strain was deposited on April 21, 2025, at the General Microbiology Center of the China Microorganism Collection Committee (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with a deposit number of CGMCC No. 34289 and a suggested taxonomic name: thermotolerant Lachancea yeast Lachancea athermotolerans.

[0008] Application of ester-aroma-producing thermotolerant Lachancea strain in loquat wine brewing.

[0009] Furthermore, the application is to inoculate the strain during loquat wine brewing to increase the flavor components of the loquat wine.

[0010] Furthermore, the brewed loquat wine has an alcohol content of 9.3% vol, a sensory score of 91.5 points, and the inoculated strain is a liquid bacterial agent of the strain.

[0011] Furthermore, the liquid bacterial agent is prepared by inoculating the seed liquid of the bacterial agent into a sterile liquid culture medium and culturing it at 28°C for 20-48 hours. The loquat wine is brewed using a loquat fruit fermentation medium, which is prepared by removing the core, cutting, protecting the color, squeezing the juice, enzymolyzing, pasteurizing, cooling, and then inoculating the activated seed liquid.

[0012] Compared with the prior art, the invention adds a liquid inoculum of heat-resistant Lachancea yeast during the loquat wine brewing process, thereby increasing the fermentation rate and the flavor substances (ethyl acetate, phenylethyl alcohol, isoamyl alcohol, isobutanol, ethanol, isoamyl alcohol, phenylethyl alcohol, isoamyl acetate, and phenylethyl acetate) in the loquat wine, especially increasing the content of isoamyl acetate, phenylethyl acetate, and phenylethyl alcohol, which is of great significance for improving the quality of loquat wine and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 GC-MS total ion patterns (a) natural fermentation; b) PP4X fermentation);

[0014] Figure 2 Figures of PP4X colony morphology and cell morphology ((a) PP4X colony morphology; (b) PP4X cell morphology);

[0015] Figure 3 This is the PP4X evolutionary tree. DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0017] Example 1

[0018] This example provides an ester-fragrance-producing, heat-resistant Lachancea yeast strain, which was isolated and screened from loquat peel in Tangqi Town, Linping District, Hangzhou City, Zhejiang Province. DNA sequence comparison showed that the strain had a high similarity with Lachancea bacteria and was identified as Lachancea. Its deposit number is CGMCC NO.34289, and the deposit date is April 21, 2025.

[0019] The isolation method of the ester-producing heat-resistant Lachancea yeast strain is as follows:

[0020] (1) YPD culture medium isolation and microscopic observation: Add sterile water to loquat peel and orchard soil samples and shake at 145 rpm for 20 min. After standing, collect the supernatant, dilute it, and spread it on YPD culture medium. Incubate at 28-30°C for 48 h. Pick out single colonies and purify them 3-4 times by streaking. Record the characteristics of the colonies. At the same time, pick out the colonies to make slides and observe the cell morphology under a 400× microscope.

[0021] (2) The yeast strains initially screened were amplified using ITS1 and ITS4 universal primers. Fungal DNA was then extracted and sequenced. The resulting strain gene sequences were compared with the GenBank database using the BLAST tool in the National Center of Biotechnology Information (NCBI). Strain sequences with high homology were selected as references. A phylogenetic tree was constructed using MEGA7 software, and a bootstrap test was performed to determine the strain species.

[0022] Strain PP4X appears as spherical projections with neat edges on YPD solid medium. Its surface is glossy, creamy, and somewhat sticky. Microscopically, its cells appear round. PCR amplification of the yeast was performed using the upstream primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and the downstream primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and the amplified product was sequenced. The gene sequence of the strain was successfully obtained. The strain was identified using the NCBI BLAST tool and compared with the GenBank database. A phylogenetic tree was constructed using MEGA7 software for identification. The strain showed high similarity to Lachancea species, confirming its identity.

[0023] Example 2: Strain purification and screening

[0024] Sample solution preparation

[0025] Loquat fermentation liquid: crush and pulp the loquats, take a certain amount of pulp, put it into a sterile conical flask, expose it to air and ferment it naturally at room temperature for several days until bubbles are generated, and take the supernatant.

[0026] Loquat (peel) and orchard soil: Take a certain amount of sample, add it to a conical flask with sterile water, shake it well, let it stand, and collect the supernatant.

[0027] The sample solution was diluted with sterile saline to different concentrations (10 -1 to 10 -5 ) suspension. Take 100 μL of the suspension of each sample at each concentration, plate three replicates for each concentration and sample onto YPD solid medium, and incubate in a 28°C constant-temperature incubator for 48 hours. After 48 hours, colonies will grow. Initial screening is performed based on morphology, size, and color. Single colonies with typical yeast morphology are selected and streaked onto fresh YPD solid medium for 48 hours. After multiple streaking purifications, single colonies are isolated. The screening results are shown in Table 1.

[0028] Table 1: Yeast fermentation flavor

[0029]

[0030] As shown in Table 1, loquat wines brewed with PP1 and TR5 have off-flavors, loquat wines brewed with PP4 have a more obvious wine aroma but a lighter fruit aroma, loquat wines brewed with PP8, PPJ3, and PPJ6 have a fruit aroma but a lighter wine aroma. In comparison, loquat wines fermented with PP3, PP4X, PP9, TR1, and PPJX have obvious wine and fruit aromas.

[0031] Strain performance test

[0032] Yeast alcohol production ability test: The alcohol production ability test method refers to the method of Wu Zhuofan et al. and is slightly modified. The separated and screened yeast is spread on the YPD solid culture medium and cultured in a constant temperature incubator at 28°C for 48 hours. Prepare TTC solid culture medium (pay attention to the light and high temperature protection of TTC reagent), pour the TTC solid culture medium into the upper layer of the solid culture medium of the original yeast culture to completely cover the original colony, and observe the color of the colony after culturing in the dark for 2 hours. Yeast with alcohol production ability appears red on the TTC plate. The stronger the alcohol production ability, the darker the red color. This can be used to judge the size of the yeast's alcohol production ability. TTC can be used to identify the alcohol production ability of yeast and screen out yeast that does not produce alcohol or has weak alcohol production ability, thereby reducing the number of strains entering the next screening stage and reducing the workload.

[0033] Yeast Fermentation Flavor Test: After the TTC test, yeasts with a certain alcohol-producing capacity were used for fermentation experiments. The yeast was inoculated at a 2% inoculum into loquat pulp at 23°Bx and fermented at 28°C for 7 days. After fermentation, the alcohol content was measured and the flavor was evaluated (primarily focusing on aroma, fruity flavor, and the presence of off-flavors).

[0034] Yeast Gas Production Test: Alcohol-producing yeasts identified through alcohol production and fermentation flavor tests were tested for gas production using the Dulbecco's tubule fermentation method. After liquid culture, the yeast strains were inoculated at a 2% inoculum into tubes of YPD liquid culture medium containing Dulbecco's tubules. The cells were then incubated at 28°C for 48 hours. Gas production and odor were recorded at 12, 24, and 48 hours. The fermentation capacity and odor of each strain were compared based on gas production and odor, allowing strains with strong fermentation capacity and a favorable fermentation odor to be selected. The results are shown in Table 2.

[0035] Table 2: Gas production by yeast Dulbecco's tubule fermentation

[0036]

[0037] As shown in Table 2, all five strains produced some gas during fermentation within 12 hours, indicating that all five strains possessed a certain degree of fermentation capacity. After 24 hours, the Dulbecco's tubules of strains PP4X and PPJX were filled with gas, indicating strong fermentation capacity. After 48 hours, the Dulbecco's tubules of strain PP9 were filled with gas, indicating strong fermentation capacity. However, the Dulbecco's tubules of strains PP9 and TR1 were less gas-rich, indicating weaker fermentation capacity.

[0038] Yeast tolerance test: The strains selected from the gas production test are subjected to sugar tolerance, alcohol tolerance, and acid tolerance tests. The Dulbecco's tubule fermentation method is used to observe the gas production in the Dulbecco's tubules to screen out strains with stronger tolerance.

[0039] Sugar tolerance test: Using sucrose as the raw material, the sugar concentration of YPD liquid medium was adjusted to 17°Bx, 20°Bx, 23°Bx, 26°Bx, and 29°Bx, respectively. The yeast to be tested was then inoculated at a 2% inoculum into the medium containing Dulbecco's tubules. The culture was incubated in a 28°C incubator for 48 hours. Gas production in the Dulbecco's tubules was observed and recorded at 12, 24, and 48 hours of incubation. The results are shown in Table 3.

[0040] Table 3: Yeast sugar tolerance

[0041]

[0042] Table 3 shows that at sugar concentrations of 17°Bx and 20°Bx, all three yeast strains produced sufficient gas to fill the Dulbecco's tubules. When the sugar concentration exceeded 23°Bx, PP3's gas production decreased, while PP4X and PPJX still produced enough gas to fill the Dulbecco's tubules, indicating that PP4X and PPJX have greater sugar tolerance.

[0043] Acid tolerance test: YPD liquid medium was adjusted to pH 2, 2.5, 3, 3.5, and 4 using trifluoroacetic acid. The test yeast was then inoculated at a 2% inoculum and cultured in a 28°C incubator for 48 hours. Gas production in the Dulbecco's tubules was observed and recorded at 12, 24, and 48 hours. The results are shown in Table 4.

[0044] Table 4: Yeast acid tolerance

[0045]

[0046] Table 4 shows that gas production by the three yeast strains gradually decreased with decreasing pH. At pH 4, all three yeast strains produced sufficient gas to fill the Dulbecco's tubules. However, PP3's gas production decreased significantly after the pH dropped below 3, and PP4X and PPJX's gas production also decreased significantly after the pH dropped below 2.

[0047] Alcohol tolerance test: YPD liquid medium was adjusted to 10%, 12%, 14%, 16%, and 18% anhydrous ethanol by volume. The test yeast and lactic acid bacteria were inoculated at a 2% inoculum. The cultures were incubated in a 28°C incubator for 48 hours. Gas production in the Dulbecco's tubules was observed and recorded at 12, 24, and 48 hours. The results are shown in Table 5.

[0048] Table 5: Alcohol tolerance of yeast

[0049]

[0050] Table 5 shows that gas production by all three yeast strains gradually decreases with increasing alcohol content. At 10% alcohol content, all three yeast strains produce sufficient gas to fill the Dulbecco's tubules. However, when the alcohol content rises above 14%, gas production by PP3 and PPJX decreases significantly. PP4X's gas production also decreases significantly at 18% alcohol content, demonstrating that PP4X has strong alcohol tolerance and can be used in low-alcohol fruit wine brewing.

[0051] Fermentation Flavor Test with Lactobacillus Plantarum: Five strains of Lactobacillus plantarum were mixed with selected yeasts for fermentation. Lactobacillus plantarum and yeast were inoculated at a 1:1 ratio into loquat pulp at 23°C and fermented at 28°C for 7 days. Flavor evaluation was performed after fermentation.

[0052] 2. Fermentation process

[0053] Select fresh and intact loquats as raw materials, wash them, drain the water and set aside. Remove the core of the loquats and cut them into small pieces. Put them into a 0.5% ascorbic acid and 0.5% sodium citrate solution to protect the color. Mix the loquats with sterile water in a ratio of 3:1 and beat them into pulp. Add 120 mg / kg of pectinase to the pulp, hydrolyze it in a 38°C water bath for 3 hours, and then heat treat it at 80°C for 5 minutes to inactivate the enzyme. Add a certain amount of activated yeast to the treated pulp, add white sugar to adjust the initial sugar content of the pulp to 23°Bx, and then ferment the loquat wine. During the fermentation period, the fermentation temperature is controlled at around 28°C, and ventilation and stirring are carried out every 12 hours. After 7 days of fermentation, the new wine is separated by centrifugation (10,000 rpm, 10 minutes), the supernatant is taken, and after clarification and sterilization, a sample is taken and refrigerated at 4°C for storage.

[0054] Determination of Volatile Flavor Compounds: Volatile flavor compounds were analyzed using headspace solid-phase microextraction and gas chromatography-mass spectrometry. 5 mL of loquat wine sample was added to a 10 mL headspace vial. Before extraction, 1.25 g of NaCl and a small rotor were added to the headspace vial. A magnetic stirring water bath was used, magnetic stirring was activated, and the sample was heated in a 45°C water bath to equilibrate for 20 minutes. The extraction tip was then inserted into the injection vial, and the fiber tip was pushed out so that its tip was approximately 1 cm above the sample liquid surface. Extraction was then performed at 45°C for 40 minutes, with continuous magnetic stirring. Immediately after extraction, the extraction tip was inserted into the GC-MS inlet and analyzed at 250°C for 5 minutes. Note that the extraction tip should be aged at 270°C for 10 minutes between each sample extraction to prevent cross-contamination between samples.

[0055] The content of volatile substances was analyzed using an Agilent 7890B-5977B gas chromatograph-mass spectrometer with an Agilent DB-WAX (30m×0.25mm×0.25μm) capillary column. The gas chromatograph program and parameter settings were as follows: starting temperature 40°C, maintained for 5 minutes, increased to 120°C at 4°C / min, maintained for 2 minutes, and then increased to 220°C at 5°C / min, maintained for 5 minutes. The injection port temperature was 250°C, and 1mL / min high-purity helium was used as the carrier gas for non-split injection. The transmission line and ion source were operated at 250°C and 230°C, respectively, with an electric shock energy of +70eV, a scan range of 35m / z-450m / z, and a scan frequency of 3.00 times / second. The results are as follows. Figure 1 shown.

[0056] Depend on Figure 1 It can be seen that the peak conditions of the two loquat wines, naturally fermented loquat wine ZR and yeast fermented loquat wine PP4X, are different, which means that the aroma components of the three groups of loquat wines are different.

[0057] GC-MS analysis of the two loquat wine samples revealed over 70 volatile compounds, which were categorized into seven chemical structures: alcohols, acids, aldehydes, hydrocarbons, esters, ketones, and others. Approximately 20 volatile flavor compounds were detected in naturally fermented loquat wine ZR, and approximately 30 volatile flavor compounds were detected in yeast-fermented loquat wine PP4X. This suggests that PP4X fermentation and MX mixed-bacteria fermentation can increase the diversity of volatile flavor compounds in loquat wine. The main volatile flavor components in naturally fermented loquat wine ZR included ethanol, ethyl acetate, acetic acid, isoamyl acetate, phenylethyl acetate, phenylethanol, isoamyl alcohol, nonanoic acid, and isobutyric acid. The main volatile flavor components in loquat wine fermented with yeast PP4X are ethanol, isoamyl acetate, phenylethyl acetate, ethyl acetate, phenylethyl alcohol, isoamyl alcohol, and isobutanol. While the types of volatile flavor components are largely consistent with those in naturally fermented loquat wine ZR, the relative content of isoamyl acetate, phenylethyl acetate, and phenylethyl alcohol is higher, resulting in a richer aroma than naturally fermented loquat wine ZR, where the majority of the volatile aroma components are ethyl acetate. Furthermore, the levels of nonanoic acid and isobutyric acid in PP4X loquat wine are lower than in ZR loquat wine. Nonanoic acid may bring a faint fat or waxy aroma to fruit wine, and isobutyric acid may increase the sense of rancidity or odor. Their presence may make ZR loquat wine have some unpleasant odor, while PP4X loquat wine reduces their relative content. The above results show that the volatile aroma composition of loquat wine fermented by PP4X yeast is richer than that of naturally fermented loquat wine, and it can produce more esters and other substances, enriching the flavor of fruit wine.

[0058] Example 3: Study on aroma production of Lachancea thermotolerans in laboratory small-scale loquat wine brewing

[0059] During the brewing of small-scale loquat wine in the laboratory, the strain of the present application was inoculated into the loquat fermentation medium. Samples were taken on the seventh day of fermentation to test the alcohol content of the fermented wine and conduct sensory evaluation. The content of volatile flavor substances in the final fermentation liquid was detected by gas chromatography-mass spectrometry. The specific protocol is as follows:

[0060] 1) Selection and pretreatment of loquats: Select fresh and intact loquats, wash and drain them, remove the core and cut them into small pieces, and put them into 0.5% ascorbic acid and 0.5% sodium citrate solution to protect the color.

[0061] 2) Preparation of Fermentation Medium: Pulp the loquat fruits with sterile water in a 3:1 ratio. Add 120 mg / kg of pectinase to the pulp and incubate in a 38°C waterbath for 3 hours. Heat-treat the pulp at 80°C for 5 minutes to inactivate the enzyme. Adjust the sugar content of the pulp to 23°Bx with white sugar and pasteurize at 65°C for 20 minutes.

[0062] 3) Laboratory-scale loquat wine brewing experiment: The thermotolerant Lachancea atheromolerans strain was inoculated into loquat fermentation medium at a concentration of 0.5 g / L. The culture was kept in a closed chamber at a constant temperature of 28°C. During fermentation, the medium was aerated and stirred every 12 hours. After 7 days of fermentation, the new wine was separated by high-speed centrifugation. The supernatant was collected and the alcohol content of the fermented wine was measured and sensory evaluation was performed.

[0063] 4) Detection of flavor substances in fermentation broth:

[0064] Headspace conditions: 5 mL of loquat wine sample was added to a headspace vial. 1.25 g of NaCl was added to the vial before extraction. The sample was equilibrated in a water bath at 45°C for 20 minutes. The extraction head was then inserted into the injection vial and extracted at 45°C for 40 minutes with continuous magnetic stirring. Desorption was performed at 250°C for 5 minutes before injection.

[0065] Gas chromatography-mass spectrometry conditions: The column was an Agilent DB-WAX (30 m × 0.25 mm × 0.25 μm). The initial temperature was 40°C, held for 5 min, then increased at 4°C / min to 120°C, held for 2 min, then increased at 5°C / min to 220°C, held for 5 min. The inlet temperature was 250°C, and high-purity helium carrier gas was used at 1 mL / min, with splitless injection. The transfer line and ion source were operated at 250°C and 230°C, respectively. The shock energy was +70 eV, and the scan range was 35 m / z to 450 m / z, with a scan rate of 3.00 scans / second.

[0066] The fermentation cycle of the strain is 7 days, and the final loquat wine has an alcohol content of 9.3% vol and a sensory score of 91.5 points.

[0067] Example 4: Study on flavor substances in laboratory small-scale loquat wine brewing compared with natural fermentation

[0068] When brewing small-scale loquat wine in the laboratory, commercial yeast was inoculated into the loquat fermentation medium. After seven days of fermentation, the types of flavor substances in the final product were detected using a gas chromatography-mass spectrometer. The specific scheme is as described in Example 3.

Claims

1. A heat-resistant Lachancea yeast strain producing ester aroma, characterized in that The strain was isolated and screened from loquat peel in Tangqi Town, Linping District, Hangzhou City, Zhejiang Province. Its preservation number is: CGMCC NO.34289, and the preservation date is: April 21, 2025.

2. Use of the ester-producing heat-resistant Lachancella strain as claimed in claim 1 in loquat wine brewing.

3. The use according to claim 2, characterized in that The application is to inoculate the strain during loquat wine brewing to increase the flavor components of the loquat wine.

4. The use according to claim 3, characterized in that The brewed loquat wine had an alcohol content of 9.3% vol and a sensory score of 91.5 points. The inoculated strain was the liquid inoculum of this strain.

5. The use according to claim 4, characterized in that The liquid bacterial agent is prepared by inoculating the seed liquid of the bacterial agent into a sterile liquid culture medium and culturing at 28°C for 20-48 hours. The loquat wine is brewed using a loquat fruit fermentation culture medium, which is prepared by removing the core, cutting the loquats into pieces, protecting the color, squeezing the juice, enzymolyzing, pasteurizing, cooling, and then inoculating the activated seed liquid.

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