A strain of Lactobacillus and its application in fruit wine

Abstract

This invention discloses a strain of *Lactobacillus shelveticus* HSCY2050, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221830, and deposited on November 28, 2022. *Lactobacillus shelveticus* HSCY2050 was cultured at a concentration of 1 × 10⁻⁶... 9 When a concentration of CFU / mL was added to fruit wine and stored at 4°C for 30 days, the viable count of HSCY2050 in the fruit wine could still be as high as 7.43 × 10⁻⁶. 6 The Lactobacillus helveticus HSCY2050 of this invention, with a concentration of CFU / mL, has extremely high application potential in the preparation of fruit wines rich in active lactic acid bacteria.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus helveticus HSCY2050 that can tolerate fruit wine and survive in fruit wine for a long time. Background Technology

[0002] Fruit wine not only retains the flavor and nutrients of the fruit, but also has effects such as relieving fatigue, relaxing muscles and promoting blood circulation, strengthening the body, refreshing the mind and dispelling cold, lowering cholesterol, anti-aging, and reducing the incidence of cardiovascular and cerebrovascular diseases, making its development prospects very broad. With social development and the improvement of people's living standards, modern people are paying more and more attention to quality of life and health preservation. Therefore, while ensuring the flavor and quality of fruit wine, improving its beneficial properties will greatly enhance the product's market competitiveness.

[0003] In recent years, my country's microbiology research has developed rapidly, effectively leveraging the benefits of probiotics in promoting disease prevention and maintaining health. People are increasingly valuing the role of probiotics, making research and exploration of their application in food of great practical significance. Currently, lactic acid bacteria are a widely discovered and used type of probiotic, named for their ability to ferment carbohydrates into lactic acid. Numerous studies have shown that lactic acid bacteria promote digestion and absorption, regulate intestinal flora balance, improve gut health, and modulate immunity. Therefore, preparing a fruit wine rich in active lactic acid bacteria is very helpful in enhancing its probiotic properties.

[0004] However, fruit wines are rich in substances such as ethanol, acetic acid, and higher alcohols that inhibit the survival or growth of lactic acid bacteria, making it difficult for them to survive for extended periods in fruit wine. Therefore, there is an urgent need to find a strain of lactic acid bacteria that can tolerate fruit wine and survive there for a long time, making it possible to prepare fruit wines rich in active lactic acid bacteria. Summary of the Invention

[0005] To solve the above-mentioned technical problems, this invention provides a strain of lactic acid bacteria that can tolerate fruit wine and survive in fruit wine for a long time, making it possible to prepare fruit wine rich in active lactic acid bacteria, thereby enhancing the probiotic properties of fruit wine.

[0006] Specifically, the first aspect of the present invention provides a strain of Lactobacillus helveticus HSCY2050, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221830 and deposit date of November 28, 2022.

[0007] A second aspect of the present invention provides a microbial agent comprising Lactobacillus helveticus HSCY2050 as described in the first aspect 1 of the present invention.

[0008] The third aspect of this invention provides the application of Lactobacillus helveticus HSCY2050 described in the first aspect of this invention and the microbial inoculant described in the second aspect of this invention in winemaking.

[0009] In some embodiments, the wine is fruit wine.

[0010] A fourth aspect of the present invention provides a wine containing *Lactobacillus helveticus* HSCY2050 as described in the first aspect of the present invention.

[0011] In some embodiments, the viable bacteria content of HSCY2050 in the wine is not less than 7 × 10⁻⁶. 6 CFU / mL.

[0012] In some embodiments, the wine is fruit wine.

[0013] The fifth aspect of the present invention provides a method for preparing fruit wine, the method comprising adding Lactobacillus helveticus HSCY2050 described in the first aspect of the present invention and the microbial agent described in the second aspect of the present invention to the wine.

[0014] In some embodiments, the wine is fruit wine.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] This invention provides a strain of Lactobacillus helveticus HSCY2050 that can tolerate fruit wine. This Lactobacillus helveticus HSCY2050 was used at a rate of 1×10⁻⁶. 9 When a concentration of CFU / mL was added to fruit wine and stored at 4°C for 30 days, the viable count of Lactobacillus helveticus HSCY2050 in the fruit wine could still be as high as 7.43 × 10⁻⁶. 6 With a concentration of CFU / mL, the Lactobacillus helveticus HSCY2050 of this invention has extremely high application potential in the preparation of fruit wines rich in active lactic acid bacteria. Attached Figure Description

[0017] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0018] Figure 1 The plate culture colony morphology of Lactobacillus helveticus HSCY2050 of the present invention;

[0019] Figure 2The cell morphology of Lactobacillus helveticus HSCY2050 stained with crystal violet is shown in the figure. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains. The terms “first,” “second,” and similar terms used in the specification and claims of this patent application do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Similarly, the terms “an” or “a” and similar terms do not indicate a limitation of quantity, but rather indicate the presence of at least one.

[0022] Example 1: Isolation, purification and identification of Lactobacillus helveticus HSCY2050

[0023] (1) Isolation and purification of strains

[0024] Weigh 10g of Zhenjiang vinegar mash sample and place it in 90mL of sterilized MRS liquid culture medium. Incubate at 37℃ and 200r / min for 20min with shaking. Then, perform a 10-fold serial dilution of the sample, selecting a dilution gradient of 10... -3 10 -4 10 -5 and 10 -6 For each serial dilution, 100 μL of each dilution was spread onto MRS agar plates and incubated upside down at 37°C for 48 h. Colonies with large clear zones were picked from the MRS agar plates and streaked three times to obtain pure cultures, which were then stored at 4°C for testing. The MRS liquid medium consisted of: 10.0 g tryptone, 10.0 g beef extract, 5.0 g yeast extract, 2.0 g diammonium citrate, 20.0 g glucose, 1.0 mL Tween 80, 5.0 g sodium acetate, 2.0 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 20.0 g calcium carbonate, and 1000 mL distilled water, pH 6.2–6.6, sterilized at 115°C for 20 min. The MRS solid medium was supplemented with 18.0 g / L agar.

[0025] (2) Alcohol tolerance test of the strain

[0026] The activated bacterial culture was inoculated at a ratio of 5% into sterilized MRS media with alcohol concentrations of 0, 3%, 6%, 9%, 12%, and 15%, and cultured statically for 5 days. The OD value of the bacterial culture was measured at 600 nm using a spectrophotometer. The strain could still grow in MRS media with an alcohol concentration of 15%, and the OD value was [not specified]. 600 =0.428.

[0027] (3) Morphological identification of strains

[0028] Figure 1 The colony morphology of *Lactobacillus helveticus* HSCY2050 of this invention is shown in the plate culture. After being cultured at 37°C for 48 hours, the colonies of strain HSCY2050 on the plate were milky white, round, raised, smooth, opaque, moist, and easy to pick up. Figure 2 The image shows the crystal violet staining of *Lactobacillus helveticus* HSCY2050, the present invention. Under a microscope, the cells appear as long rods, arranged singly, in pairs, or in short chains. Based on the colony and cell morphology, it is preliminarily identified as a type of *Lactobacillus*.

[0029] (4) Molecular biological identification of strains

[0030] The bacteria obtained through purification and screening were collected by centrifugation of fresh bacterial culture in the exponential growth phase, and genomic DNA was extracted using a genomic DNA extraction kit. The full-length 16S rDNA sequence was amplified using universal bacterial primers P0-P6 (China Master's Thesis Full-text Database, Zhu Qihan, "Microbial Isolation and Acid Production Characteristics during Zhenjiang Vinegar Fermentation", 2008). Sequencing of the PCR amplification products was performed by Shanghai Sangon Biotech Co., Ltd. The obtained 16S rDNA sequence was then compared with the NCBI database using BLAST to determine its species. The full-length 16S rDNA sequence of the screened strain was 1432 bp, and the strain with the highest homology was *Lactobacillus helveticus*, with a similarity of 99.72% (16S rDNA sequence shown in SEQ ID NO: 1). This strain was named *Lactobacillus helveticus* HSCY2050.

[0031] The 16S rDNA nucleotide sequence of Lactobacillus helveticus HSCY2050:

[0032]

[0033] The above-mentioned strain was deposited at the China Center for Type Culture Collection (CCTCC) on November 28, 2022, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit registration number is CCTCC NO: M 20221830, and its taxonomic name is Lactobacillus helveticus.

[0034] Example 2: Tolerance of Lactobacillus helveticus HSCY2050 to fruit wine at different storage temperatures

[0035] Add Lactobacillus helveticus HSCY2050 to commercially available fruit wine (purchased from Yantai Changyu Grape Wine Co., Ltd., 13% vol) until the viable count of Lactobacillus helveticus HSCY2050 in the fruit wine is 1×10⁻⁶. 9 CFU / mL was used to prepare fruit wine rich in lactic acid bacteria. The fruit wine sample was then divided into two equal portions and stored simultaneously at 4℃ and 25℃ for 30 days. The viable count of *Lactobacillus helveticus* HSCY2050 in the fruit wine samples was determined by plate counting at 7, 10, 20, and 30 days of storage. The results are shown in Table 1. Table 1 shows that *Lactobacillus helveticus* HSCY2050 at a concentration of 1×10⁻⁶ CFU / mL was significantly increased. 9 When a concentration of CFU / mL was added to fruit wine and stored at 4°C for 30 days, the viable count of *Lactobacillus helveticus* HSCY2050 in the fruit wine could still be as high as 7.43 × 10⁻⁶. 6 CFU / mL. This indicates that *Lactobacillus helveticus* HSCY2050 exhibits strong resistance to fruit wine. Furthermore, compared to room temperature (25℃), refrigerating *Lactobacillus helveticus* HSCY2050 at 4℃ maintains a higher concentration of viable bacteria in the fruit wine.

[0036] Table 1. Viable counts of *Lactobacillus helveticus* HSCY2050 in fruit wine at different storage temperatures.

[0037]

[0038] Example 3: Tolerance of different lactic acid bacteria to fruit wine

[0039] Lactobacillus helveticus HSCY2050, Lactobacillus casei CICC25095 (purchased from CICC), and Lactobacillus plantarum CICC22195 (purchased from CICC) were added separately to fruit wine (purchased from COFCO Great Wall Wine Co., Ltd., 13.5% vol), with a viable count of 1×10⁻⁶ in each. 9CFU / mL was used to obtain fruit wines GJ1, GJ2, and GJ3 rich in lactic acid bacteria. The fruit wine samples were stored at 4℃ for 30 days. At 7, 10, 20, and 30 days of storage, the viable counts of *Lactobacillus helveticus* HSCY2050, *Lactobacillus casei* CICC25095, and *Lactobacillus plantarum* CICC22195 in the fruit wine samples were determined by plate counting. The results are shown in Table 2. Table 2 shows that when *Lactobacillus casei* CICC25095 and *Lactobacillus plantarum* CICC22195 were stored in fruit wine at 4℃, the viable counts of *Lactobacillus casei* CICC25095 and *Lactobacillus plantarum* CICC22195 decreased significantly with prolonged storage time, eventually reaching zero. However, when *Lactobacillus helveticus* HSCY2050 was stored in fruit wine at 4℃, the viable count of *Lactobacillus helveticus* HSCY2050 remained as high as 5.02 × 10⁻⁶ after 30 days of storage. 6 CFU / mL. It is evident that among *Lactobacillus helveticus* HSCY2050, *Lactobacillus casei* CICC25095, and *Lactobacillus plantarum* CICC22195, *Lactobacillus helveticus* HSCY2050 exhibits excellent wine-tolerance properties.

[0040] Table 2. Viable bacterial counts of different lactic acid bacteria in fruit wine

[0041]

[0042] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0043] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This description is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A strain of Lactobacillus helveticus ( Lactobacillus helveticus HSCY2050, characterized in that, The Lactobacillus helveticus is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221830 and deposit date of November 28, 2022.

2. A microbial inoculant, characterized in that, The microbial agent comprises Lactobacillus helveticus HSCY2050 as described in claim 1.

3. The application of Lactobacillus helveticus HSCY2050 as described in claim 1 and the microbial agent as described in claim 2 in winemaking.

4. The application according to claim 3, characterized in that, The wine in question is fruit wine.

5. A type of wine, characterized in that, The wine contains Lactobacillus helveticus HSCY2050 as described in claim 1.

6. The wine according to claim 5, characterized in that, The viable HSCY2050 content in the wine is not less than 7×10⁻⁶. 6 CFU / mL.

7. The wine according to claim 5 or 6, characterized in that, The wine in question is fruit wine.

8. A method for preparing fruit wine, characterized in that, The method includes adding the Lactobacillus helveticus HSCY2050 of claim 1 or the microbial agent of claim 2 to the wine.

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