Yeast and application thereof
By using the Ludwig yeast Saccharomycodes ludwigii to ferment blackberry glandular rib and European plum wine, the problems of monotonous flavor and poor adaptability to low-alcohol wine caused by commercial yeast were solved, efficient fermentation and flavor enhancement were achieved, and the sensory quality of the wine was improved.
Patent Information
- Application Number
- CN202510972705.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-15
- Publication Date
- 2025-10-28
AI Technical Summary
Existing commercial brewing yeasts result in monotonous flavors and difficulty in showcasing the unique characteristics of fruit flavors in fruit wine production. Furthermore, they exhibit poor adaptability and low fermentation efficiency when brewing low-alcohol fruit wines, affecting the sensory quality and safety of the fruit wines.
Saccharomycodes ludwigii (CGMCC No. 33752) was used to ferment Aronia nigra and Prunus mume wines to enhance antioxidant capacity, reduce tannin content, lower alcohol and methanol content, and increase the variety of volatile flavor compounds.
It improves fermentation efficiency, enhances the flavor richness and functionality of fruit wine, reduces alcohol content and methanol content, improves the antioxidant capacity and aroma complexity of fruit wine, and avoids the production of adverse by-products.
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Figure CN120843306A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fermentation technology, and more specifically to a strain of Ludwig's yeast and its applications. Background Technology
[0002] Fruit wine is a beverage made from fruit through yeast fermentation. Its flavor, taste, and nutritional value are highly dependent on the characteristics of the yeast used. Currently, commercial brewing yeasts are commonly used in fruit wine production, due to their strong fermentation capacity and high alcohol tolerance. However, long-term industrial application of these yeasts has gradually revealed problems: the fruit wines have a monotonous flavor, insufficient aroma complexity, and difficulty in fully showcasing the unique flavors of different fruits.
[0003] Non-saccharifying yeasts can synthesize large amounts of volatile substances such as esters and alcohols. Some non-saccharifying yeasts can secrete enzymes such as pectinase and glucosidase, which play a positive role in shaping the aroma characteristics of fruit wines. In recent years, many non-saccharifying yeasts have attracted attention due to their potential in aroma production, acid reduction, and antioxidant effects. However, non-saccharifying yeasts are greatly affected by the environment. When fermenting certain special fruits (such as fruits with high acidity, high tannins, or high sugar), they may experience poor adaptability, low fermentation efficiency, or the production of undesirable byproducts, which seriously affect the sensory quality and safety of fruit wines.
[0004] Alcoholic beverages with an alcohol content of less than 8% vol are considered low-alcohol drinks. Currently, based on health-conscious consumption trends and market demand, providing low-alcohol-risk beverages for specific groups has become a major trend. Furthermore, low-alcohol fruit wines, while reducing alcohol production, can maximize the preservation of the natural flavor and nutrients of the fruit (such as polyphenols and vitamins), thus solving problems such as flavor masking caused by excessively high alcohol concentrations in traditional fermented fruit wines. This expands the application of fruit wines in a wider range of fields such as catering and sports rehabilitation. However, there are currently few reports on yeasts used for preparing low-alcohol fruit wines.
[0005] Therefore, discovering novel non-brewing yeast strains with unique fermentation characteristics suitable for brewing refreshing fruit wines with low alcohol content, especially those that can adapt to diverse fruit substrates and enhance the flavor richness and functionality of fruit wines, has become an important research direction for the fruit wine industry and is of great significance for promoting the high-quality development of the fruit wine industry. Summary of the Invention
[0006] In view of this, the present invention provides a Ludwig yeast strain and its applications.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions:
[0008] A strain of Saccharomycodes ludwigii was deposited on March 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33752, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0009] A fruit wine fermentation agent comprising the aforementioned Ludwig yeast.
[0010] The above-mentioned application of Ludwig yeasts in fruit wine brewing.
[0011] Furthermore, the fruit wine is fermented wine of black chokeberry and fermented wine of European plum.
[0012] Furthermore, it can be used to improve antioxidant capacity, reduce tannin content, reduce alcohol content in fermented wines, reduce methanol content, and increase the variety of volatile flavor compounds.
[0013] A method for preparing fruit wine, using the aforementioned Ludwig yeast.
[0014] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows: the Lude yeast LNJ-S-1 of the present invention has strong fermentation ability. Compared with the existing commercially available fruit and vegetable brewing yeast, it has high fermentation efficiency of raw materials, reduces tannin content, improves antioxidant capacity, reduces alcohol content of fermented wine, reduces methanol content, and increases the types of volatile flavor substances. Attached Figure Description
[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0016] Figure 1 This is a colony morphology diagram of strain LNJ-S-1 in Example 1 of the present invention;
[0017] Figure 2 The results of microscopic examination of the LNJ-S-1 strain in Example 1 of this invention;
[0018] Figure 3 This is a homology comparison between the LNJ-S-1 strain in Example 1 of the present invention and Saccharomycodes ludwigii;
[0019] Figure 4 The tolerance of the LNJ-S-1 strain to tannins in Example 2 of this invention;
[0020] Figure 5 This refers to the transformation ability of the LNJ-S-1 strain to tannins in Example 2 of the present invention;
[0021] Figure 6 The images show the fermented black chokeberry wine (left) and European plum wine (right) from Example 3 of this invention.
[0022] Figure 7 The volatile components of black chokeberry wine fermented by Angel Yeast in Example 3 of this invention;
[0023] Figure 8 These are the volatile components of black chokeberry wine fermented by LNJ-S-1 of Ludwig's yeast in Example 3 of this invention;
[0024] Figure 9 The volatile components of the fruit wine yeast fermented by Angel in Example 3 of this invention are:
[0025] Figure 10 These are the volatile components of the fermented European plum wine made by the Ludwig yeast LNJ-S-1 in Example 3 of this invention. Detailed Implementation
[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0027] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.
[0028] Example 1
[0029] Isolation, identification, and preservation of Lude's yeast LNJ-S-1
[0030] I. Separation
[0031] In January 2025, the strain was isolated and purified from naturally fermented apple pulp in Shenyang, Liaoning Province. Colonies from the fermented apple pulp were picked using an inoculation loop and streaked continuously onto PDA agar plates. The streaked plates were incubated at 30°C for 48 hours. Colonies were observed, and morphologically sound, dispersed single colonies were picked and streaked continuously onto PDA agar plates for further incubation. The purified strain was obtained after repeated incubation and named LNJ-S-1.
[0032] II. Identification
[0033] 1. Morphological and physiological biochemical identification
[0034] The LNJ-S-1 strain isolated above was cultured on PDA solid medium at 30℃ for 48 h, forming round, regular, smooth white colonies. Figure 1 ).
[0035] Spread a small amount of LNJ-S-1 bacterial suspension evenly onto a glass slide and quickly fix it with an alcohol lamp; add crystal violet stain and hold for 1 min, then wash with water; cover with iodine solution for 1 min, then wash with water; quickly decolorize with 95% ethanol and immediately wash with water; add safranin stain and hold for 30 s, wash with water, then blot dry with filter paper; observe under an oil immersion microscope. Single bacteria appear blue-purple, indicating Gram-positive bacteria. Figure 2 ).
[0036] 2. Molecular biological identification
[0037] The activated LNJ-S-1 strain was inoculated into PDA liquid medium and cultured at 30℃ for 24 h. 10 mL of the culture medium was then centrifuged at 10000 r / min for 15 min at 4℃, and the bacterial pellet was collected. The strain was identified by a third party, and the 16S rRNA sequencing results were as follows:
[0038] , SEQ ID No.1.
[0039] Sequence alignment showed that its homology with the 16S rRNA sequence of *Yeastraea rudae* was higher than 99%. Figure 3 The identified yeast was named *Saccharomycodes ludwigii*.
[0040] III. Preservation
[0041] Strain LNJ-S-1, classified as Saccharomycodes ludwigii, was deposited on March 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33752, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0042] Example 2
[0043] Tolerance of tannins to LNJ-S-1 (a type of yeast)
[0044] Gallic acid (1%, 3%, and 5%) was added to PDA medium (different concentrations of gallic acid medium were used, with gallic acid equivalents replacing tannins). Lude's yeast LNJ-S-1 and commercially purchased Saccharomyces cerevisiae (Angel's fruit wine yeast) were simultaneously inoculated into different concentrations of gallic acid medium to assess tannin tolerance. Cell growth was expressed as cell concentration and measured using OD. 600 Methods for determination. Tannin content was determined using a tannin content test kit (Suzhou Grees Biotechnology Co., Ltd.)
[0045] Depend on Figure 4 It was found that a 1% gallic acid concentration promoted the growth of Ludwig's yeast LNJ-S-1. Although the cell concentration of Ludwig's yeast LNJ-S-1 decreased with increasing gallic acid concentration, Ludwig's yeast LNJ-S-1 showed higher tolerance to tannins compared to commercially available yeasts.
[0046] Depend on Figure 5 It is known that, compared with commercially available yeasts, Lude yeast LNJ-S-1 has a stronger ability to degrade tannins, thus improving the taste of fermented fruit wine and reducing bitterness.
[0047] Example 3
[0048] Functional study of Lude's yeast LNJ-S-1
[0049] 1. Preparation of fermented wine from black chokeberry (pH: 3.55, sugar content 13.3%)
[0050] (1) Wash, dry and crush the black chokeberry fruit.
[0051] (2) Activate LNJ-S-1 of the Lude yeast in PDA liquid medium. Activate commercially purchased brewing yeast (Angel's fruit wine yeast) according to the instructions: add 10 times the amount of warm water at 40℃ to the fruit wine yeast, stir well, and let stand for 20 minutes.
[0052] (3) Using the same bacterial concentration (10 9 Inoculate crushed black chokeberry with 3% of Ludwig's yeast LNJ-S-1 (CFU / mL) and Angel's fruit wine yeast, respectively. Add crushed rock sugar equal to 35% of the weight of the black chokeberry and mix well. Ferment at 22-25℃ for 10 days, stirring and degassing once a day.
[0053] (4) After fermentation for 10 days, filter the filtrate and let it stand at 22-25℃ in the dark for 1 month. If gas is found to be produced during the period, exhaust the gas in time.
[0054] (5) After aging, filter and sterilize the wine at 60℃ for 30 minutes to produce black chokeberry fermented wine. Figure 6 Left).
[0055] 2. Preparation of fermented wine from European plum (pH: 3.02, sugar content: 13.8%)
[0056] (1) Wash, dry, pit, and crush the plums for later use.
[0057] (2) Activate LNJ-S-1 of the Lude yeast in PDA liquid medium. Activate commercially purchased brewing yeast (Angel's fruit wine yeast) according to the instructions: add 10 times the amount of warm water at 40℃ to the fruit wine yeast, stir well, and let stand for 20 minutes.
[0058] (3) Using the same bacterial concentration (10 9 Inoculate crushed European plums with 3% of Ludwig's yeast LNJ-S-1 (CFU / mL) and Angel's fruit wine yeast, add 20% crushed rock sugar (by weight of the European plums), mix well, and ferment at 22-25℃ for 2 weeks, stirring and degassing once a day.
[0059] (4) After fermentation for 2 weeks, filter the filtrate and let it stand at 22-25℃ in the dark for 1 month. If gas is found to be produced during the period, exhaust the gas in time.
[0060] (5) After aging, filter the wine and sterilize it at 60°C for 30 minutes to produce European plum fermented wine. Figure 6 right).
[0061] 3. Determination of alcohol content and methanol content
[0062] The alcohol content was measured using a fruit wine alcohol meter at 20°C.
[0063] The methanol content was determined using a rapid methanol detection tube.
[0064] The black chokeberry wine and European plum wine fermented by Ludwig yeast LNJ-S-1 were compared with those fermented by Angel Yeast's fruit wine yeast.
[0065] Table 1. Determination of alcohol content and methanol content in fermented wine
[0066]
[0067] Note: "-" indicates not detected.
[0068] As shown in Table 1, comparing the berry wine fermented with Ludwig's yeast LNJ-S-1 with the berry wine fermented with Angel Yeast, it can be seen that fermenting high-acid, low-sugar berries with Ludwig's yeast LNJ-S-1 of the present invention can significantly reduce the alcohol content and methanol content of the wine.
[0069] 4. Determination of total antioxidant capacity and tannin content
[0070] Total antioxidant capacity was determined using a total antioxidant capacity kit.
[0071] Tannin content was determined using a tannin content test kit (Suzhou Gres Biotechnology Co., Ltd.).
[0072] The black chokeberry wine and European plum wine fermented by Ludwig yeast LNJ-S-1 were compared with those fermented by Angel Yeast's fruit wine yeast.
[0073] Table 2. Determination of antioxidant capacity and tannin content in fermented wine
[0074]
[0075] As shown in Table 2, comparing the berry wine fermented with Ludwig's yeast LNJ-S-1 with the berry wine fermented with Angel Yeast, it can be seen that fermenting high-acid, low-sugar berries with Ludwig's yeast LNJ-S-1 of the present invention can effectively improve the total antioxidant capacity and reduce the tannin content.
[0076] 5. Determination of volatile flavor components
[0077] Volatile flavor components in fermented wine were determined using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS). 4-Methyl-2-pentanol was used as an internal standard. 5 mL of wine sample was extracted with headspace solid-phase microextraction at 40 °C for 30 min, followed by desorption at 250 °C for 2 min. Gas chromatography was performed at an initial temperature of 50 °C, held for 5 min, then increased to 100 °C at a rate of 2 °C / min, and then increased to 230 °C at a rate of 4 °C / min, held for 10 min. Helium was used as the carrier gas at a flow rate of 1.5 mL / min. Mass spectrometry was performed using an EI ion source with an electron impact energy of 70 eV, an ion source temperature of 230 °C, a quadrupole temperature of 150 °C, and a full scan with a mass scan range of 40–600 aum.
[0078] The wines of black chokeberry and European plum fermented with LNJ-S-1 *Lactobacillus rudius* were compared with those fermented with Angel Yeast's fruit wine yeast. The results are as follows: Figures 7-10 As shown in Table 3.
[0079] Table 3. Volatile Flavor Components (Types) of Fermented Wines
[0080]
[0081] Specifically:
[0082] The unique components produced by fermenting black chokeberry fruit wine with Lude's yeast LNJ-S-1 include:
[0083] 2-Phenylethyl hexanoate: It has a fresh, fruity aroma, similar to pineapple, and a pleasant taste;
[0084] Ethyl 3-hydroxyoctanoate: It has fruity, floral and wine-like aromas, and is often described as a mixture of pineapple, apple, brandy or wine aromas;
[0085] Methyl decanoate: It has a unique green aroma, a coolness, a fresh and rich balsamic aroma, a sweet woody aroma, and a pine needle-like aroma;
[0086] Ethyl 5-methylnonanoate: It has a health beer aroma that is a mixture of rose and fruity notes, with slight oily, fruity and nutty aroma characteristics;
[0087] Diethyl succinate: Sweet fruity aroma, with the sweet scent of fruits (such as apples and strawberries), accompanied by a subtle and pleasant fragrance;
[0088] 1,3-Propanediol diacetate: has a distinctive fruity aroma and a hint of mint flavor;
[0089] In addition, it produces a variety of unique alcohols, including 1,2,3-butanetriol, 5-methyl-2-heptanol, 1,9-nonanediol, 2,3-butanediol, 2,6,8-trimethyl-4-nonanediol, nerol, etc., all of which contribute significantly to aroma.
[0090] The unique components produced by fermenting European plum wine with Lude's yeast LNJ-S-1 include:
[0091] Cyclohexanol acetate: It has floral, woody and iris aromas, with a delicate and long-lasting fragrance;
[0092] Hexyl acetate: It has a fruity aroma, specifically a mixed aroma of pear, apple, banana or pineapple, with a sweet and refreshing taste;
[0093] In addition, it produces a variety of unique alcohols, including 5-methoxy-1-pentanol, 3-methyl-2-butanol, 3-methylthiopropanol, 1,2,3,4,5-cyclopentanol, and 1,3,5-cyclohexanetriol, all of which contribute significantly to aroma.
[0094] In summary, comparing berry wines fermented with Luder yeast LNJ-S-1 with berry wines fermented with Angel Yeast's fruit wine yeast, it is evident that fermentation of high-acid, low-sugar berry wines using the Luder yeast LNJ-S-1 of this invention increases aromatic esters, alcohols, and aldehydes that promote aroma, while reducing toxic substances from alcohol metabolism. In conclusion, fermentation with Luder yeast LNJ-S-1 effectively improves the complexity of the wine's aroma while avoiding symptoms such as headaches caused by undesirable substances.
[0095] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0096] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A strain of Ludwig's yeast, characterized in that, Its classification name is Saccharomycodesludwigii, and it was deposited on March 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33752. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.
2. A fruit wine fermentation agent, characterized in that, Includes the Lude yeast as described in claim 1.
3. The application of the Ludwig yeast as described in claim 1 in fruit wine brewing.
4. The application as described in claim 3, characterized in that, The fruit wine is fermented wine made from black chokeberry and European plum.
5. The application as described in claim 3, characterized in that, It is used to improve antioxidant capacity, reduce tannin content, reduce alcohol content in fermented wine, reduce methanol content, and increase the variety of volatile flavor compounds.
6. A method for preparing fruit wine, characterized in that, Use the Luder yeast as described in claim 1.