A Lactiplantibacillus plantarum NXU0013 and its fermented wolfberry wine

Abstract

The present invention belongs to the field of microbial technology, and particularly relates to a Lactobacillus plantarum NXU0013 and its uses. The Lactobacillus plantarum NXU0013 is preserved in the China General Microbiological Culture Collection Center with the preservation number CGMCC NO: 28437. When this bacterium co-ferments alcoholic beverages with yeast, due to the synergistic effect, various different metabolites are produced respectively, and these substances endow the fruit wine beverage with a unique taste and flavor. The lactic acid produced by the metabolism of Lactobacillus plantarum NXU0013 undergoes an esterification reaction with ethanol under the action of an enzyme to form ethyl lactate, which plays an important role in the aroma formation of various wines.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus plantarum NXU0013 and a use thereof. Background Art

[0002] In recent years, the mixed fermentation of lactic acid bacteria and yeast to brew fruit wine has attracted much attention. When yeast and lactic acid bacteria are fermented together, they are in a biological mixed fermentation system, and there is a coordination effect between the metabolites in the system. Studies have shown that when yeast and lactic acid bacteria co-ferment alcoholic beverages, due to the synergistic effect of yeast and lactic acid bacteria, each produces a variety of different metabolites, which give fruit wine beverages a unique taste and flavor. In traditional brewing, they often complement each other and play a perfect role. The lactic acid produced by the metabolism of lactic acid bacteria reacts with ethanol under the action of enzymes to produce ethyl lactate. It plays an important role in the aroma of many kinds of wine. Therefore, it is often used for mixed bacteria fermentation in traditional brewing. Summary of the invention

[0003] In view of the above characteristics, the present invention provides a plant lactobacillus NXU0013, which has strong high sugar and acid resistance, and its OD value is still greater than 0.5 when the glucose concentration reaches 350g / L; it can grow significantly in the pH range of 3.5 to 5.5. In terms of temperature tolerance, the strain can grow well when it is greater than 20°C, and grows slowly when the temperature is lower than 20°C, but the sustainability is good.

[0004] In a first aspect, the present invention provides a Lactobacillus plantarum NXU0013, whose deposit number is CGMCCNO.28437.

[0005] In a second aspect, the present invention provides an optimal ratio of the aforementioned Lactobacillus plantarum NXU0013 and Saccharomyces cerevisiae.

[0006] The third aspect of the present invention provides a method for fermenting wolfberry wine with the plant lactobacillus NXU0013, comprising the following steps: mixing the plant lactobacillus NXU0013 with saccharomyces cerevisiae in a ratio of 1:3, inoculating at a rate of 3.5% and fermenting at a temperature of 21°C.

[0007] In a fourth aspect, the present invention provides an effect of the plant lactobacillus NXU0013 on the flavor substances of wolfberry wine.

[0008] Impact:

[0009] (1) In terms of volatile components, the participation of Lactobacillus plantarum NXU0013 in fermentation increases the types and contents of volatile flavor substances in the fruit wine, giving the wolfberry wine a unique fruity and winey aroma, making its taste more harmonious, mellow and soft, and improving the quality of the wolfberry wine;

[0010] (2) Compared with the single-strain fermentation of Saccharomyces cerevisiae, the wolfberry wine fermented with Lactobacillus plantarum NXU0013 contains more aroma substances.

[0011] (3) Compared with the single-strain fermentation of Saccharomyces cerevisiae, the wolfberry wine fermented with Lactobacillus plantarum NXU0013 has a rich, complex, and harmonious fruity aroma, with a mellow taste and a unique wolfberry wine aroma. Although the single-strain fermentation results in a harmonious wine body, the aroma is single-layered and lacks fermentation aroma.

[0012] Biological material preservation information:

[0013] Lactobacillus plantarum NXU0013, classified as Lactobacillus plantarum, with the Latin name Lactobacillus plantarum. This strain NXU0013 was deposited at the China General Microbiological Culture Collection Center on September 12, 2023, with the deposit number CGMCC NO. 28437 and the deposit address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Description of the drawings

[0014] Figure 1 shows the colony morphology of Lactobacillus plantarum NXU0013 on MRS solid medium;

[0015] Figure 2 shows the results of Lactobacillus plantarum NXU0013's tolerance to high sugar and acidity;

[0016] Figure 3 shows the results of Lactobacillus plantarum NXU0013's tolerance to ethanol and low temperature;

[0017] Figure 4 shows the growth curve of Lactobacillus plantarum NXU0013;

[0018] Figure 5 shows the phylogenetic tree constructed from the 16S rDNA of Lactobacillus plantarum NXU0013;

[0019] Figure 6 shows the determination of the optimal ratio of Lactobacillus plantarum NXU0013 to Saccharomyces cerevisiae;

[0020] Figure 7 shows the determination of the optimal inoculation amount of Lactobacillus plantarum NXU0013 to Saccharomyces cerevisiae;

[0021] Figure 8 shows the determination of the optimal fermentation temperature of Lactobacillus plantarum NXU0013 and Saccharomyces cerevisiae;

[0022] Figure 9 shows the effect of Lactobacillus plantarum NXU0013 on the production of organic acids by Saccharomyces cerevisiae;

[0023] Figure 10Effects of Lactobacillus plantarum NXU0013 on Lycium barbarum polysaccharides, flavonoids and total esters in Lycium barbarum wine Detailed implementation manners

[0024] The present invention will be further explained in combination with embodiments and corresponding drawings. The following embodiments are only for illustrative purposes and do not limit the scope of the present invention.

[0025] Example 1

[0026] Isolation and screening of Lactobacillus plantarum NXU0013

[0027] 1. Preliminary screening of samples

[0028] Take samples of natural fermentation, perform gradient dilution in sequence, and coat them on MRS medium respectively. Make 3 parallels for each gradient on the medium, and culture at 37°C for 48h - 72h. Select colonies with the characteristics of lactic acid bacteria, as Figure 1 shown. Through Gram staining and enzyme touch test, lactic acid strains are preliminarily identified. Inoculate the preliminarily screened strains into the solid medium of Lycium barbarum juice, and select the lactic acid bacteria with better growth and inoculate them into the MRS slant medium for low-temperature preservation at 4°C.

[0029] 2. Re-screening of samples

[0030] Pick suspected lactic acid strains of different categories or different isolation sources of the same category, activate them in MRS culture solution for 24h, inoculate them into the sterilized Lycium barbarum juice medium respectively, place them in a constant temperature incubator at 37°C for 3d and then take samples. Measure the acidity and sensory score, screen the better lactic acid strains, and conduct tolerance experiments.

[0031] 3. Tolerance experiment

[0032] Conduct experiments on the tolerance of lactic acid bacteria to high sugar, low acid, ethanol and low temperature. Inoculate each strain into the medium with glucose concentrations of 150g / L, 200g / L, 200g / L, 250g / L, 300g / L; acidities of pH = 3.5, pH = 4, pH = 4.5, pH = 5, pH = 5.5; ethanol concentrations of 3%, 6%, 9%, 12%, 15% at 37°C, and in a constant temperature incubator at temperatures of 16°C, 20°C, 24°C, 28°C, 32°C for 24h. Measure the OD value at 600nm, compare the tolerance of each strain, and repeat the experiment 3 times. The results are as Figure 2 、 Figure 3 shown, and select the strains with good tolerance from them.

[0033] Example 2

[0034] Identification of Lactobacillus plantarum NXU0013

[0035] The selected excellent lactic acid bacteria were cultured at 37°C for 24 h, centrifuged at 8000 r / min for 10 min at 4°C, and the cells were collected after washing twice with sterile normal saline. The genomic DNA of the strains was extracted with reference to the Bacterial Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.). The 16S rRNA of the selected lactic acid strains was amplified by PCR using universal primers (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-CTACGGCTACCTTGTTACGA3′). PCR amplification system: ddH 2 O 9.5 μL, 1 μL each of 10 μmol / L 27F primer and 1492R primer, 0.2 μL of 5 U / μL Taq polymerase, 2.5 μL of 10× Buffer (containing Mg 2+ ) and 0.5 μL of 50 U / μL genomic DNA template. PCR program: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 90 s, for 30 cycles; extension at 72°C for 10 min, and stored at 4°C. After detecting the PCR products by 1% agarose gel electrophoresis, sequencing was performed, and the sequencing results were subjected to homology analysis with the sequences in the NCBI BLAST database.

[0036] Through the sequencing of the amplified products of 16S rDNA, as shown in SEQ ID NO: 1, the specific sequence is as follows:

[0037]

[0038] GCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGAA

[0039] The 16S rDNA gene sequence results of the strain were BLAST-aligned in the NCBI database. As Figure 4 The similarity of the obtained DNA fragment to Lactobacillus plantarum was 99%. This strain could be identified as Lactobacillus plantarum and was named Lactobacillus plantarum NXU0013.

[0040] Example 3

[0041] Determination of the growth curve of Lactobacillus plantarum NXU0013

[0042] After activating and culturing the excellent Lactobacillus plantarum NXU0013 obtained by screening, it was inoculated into MRS liquid medium at an inoculation amount of 1%, cultured at 20 °C, sampled every 12 h until the bacterial cells died, and finally the OD600nm value was measured to plot its growth curve as Figure 5 shown.

[0043] The growth curve graph shows that the growth of Lactobacillus plantarum NXU0013 was slow within 0 h - 24 h, the logarithmic growth phase was from 24 h to 72 h during which Lactobacillus plantarum NXU0013 grew rapidly, the stationary phase was from 72 h to 122 h, and the decline phase started after 122 h.

[0044] Example 4

[0045] Process optimization of co-fermenting wolfberry wine with Lactobacillus plantarum NXU0013 and Saccharomyces cerevisiae JY2.

[0046] 1. Determination of the optimal ratio of Saccharomyces cerevisiae JY2 to Lactobacillus plantarum NXU0013:

[0047] Adjust the initial sugar concentration of wolfberry juice to 22 Brix, pH to 3.5, and the total inoculation amount of the strains to 3%. Different inoculation ratios were set and asynchronous inoculation fermentation was carried out at 20 °C until fermentation terminated. The total ester and sensory score were measured to determine the optimal ratio. As Figure 6 can be seen, for the wolfberry wine fermented with different ratios of Saccharomyces cerevisiae JY2 to Lactobacillus plantarum NXU0013, there were significant differences in the total ester and sensory score. When NXU0013:JY2 = 1:3, the sensory score was the highest at 92 points and the total ester content was 1.39 g / L.

[0048] 2. Determination of the optimal inoculation amount

[0049] Adjust the initial sugar concentration of wolfberry juice to 22 Brix and the pH to 3.5. Select Lactobacillus plantarum NXU0013:Saccharomyces cerevisiae JY2 = 1:3 for inoculation, and set different inoculation amounts as 2%, 3%, 4%, 5%, and 6%. Ferment at 20 °C until fermentation terminates. Determine the total ester and sensory score to determine the optimal inoculation amount. As Figure 7 can be seen, with the increase of the inoculation amount, the total ester and sensory score show a trend of increasing first and then decreasing. When the inoculation amount is 3%, the total ester content is 1.39 g / L, the sensory score is the highest, and the optimal inoculation amount is 3%.

[0050] 3. Determination of the optimal fermentation temperature

[0051] Adjust the initial sugar concentration of wolfberry juice to 22 Brix and the pH to 3.5. Inoculate and ferment according to the ratio of Lactobacillus plantarum NXU0013:Saccharomyces cerevisiae JY2 of 1:3, and the inoculation amount is 3%. Ferment at 16 °C, 20 °C, 24 °C, 28 °C, and 32 °C respectively until fermentation terminates. Determine the total ester and sensory score to determine the optimal fermentation temperature. As Figure 8 shown, the optimal fermentation temperature is 20 °C.

[0052] Example 5

[0053] Effect of Lactobacillus plantarum NXU0013 on Saccharomyces cerevisiae JY2 and the flavor of wolfberry wine

[0054] 1. Effect of Lactobacillus plantarum NXU0013 on organic acid production by JY2

[0055] As Figure 9 shown, in addition to the increase in lactic acid in the co-fermentation group, other acids are lower than those in the single-strain fermentation group of JY2, indicating that the participation of Lactobacillus plantarum NXU0013 produces a large amount of lactic acid. At the same time, Lactobacillus plantarum NXU0013 utilizes more reducing sugars, affecting the content of the metabolites of yeast, so there is also a decrease in the content in terms of acids. These tartaric acid, acetic acid, capric acid, and malic acid all have a certain degree of sour and astringent taste, and their reduction is beneficial to balancing the taste of wolfberry wine. At the same time, the increase in lactic acid enhances the softness.

[0056] 2. Effect of Lactobacillus plantarum NXU0013 on wolfberry polysaccharide, flavonoid, and total ester in wolfberry wine

[0057] As Figure 10As shown in the figure, compared with the JY2 single-strain fermentation group, the content of Lycium barbarum polysaccharide in the co-fermentation group was lower. It may be that in the later stage of fermentation, the enzymes produced by Lactobacillus plantarum NXU0013 can degrade it into small-molecule substances or use it as a carbon source, resulting in a decrease in the content of Lycium barbarum polysaccharide. However, the total flavonoid content increased. It may be that the glycosidases secreted by Saccharomyces cerevisiae JY2 and Lactobacillus plantarum NXU0013 released the polysaccharides and phenolic substances combined with proteins, and the bound flavonoids were decomposed into free flavonoids, resulting in an increase in the total flavonoid content.

Claims

1. A Lactobacillus plantarum NXU0013, characterized in that The strain NXU0013 was deposited in the China General Microbiological Culture Collection Center on September 12, 2023, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.28437.

2. An application of Lactobacillus plantarum NXU0013 as claimed in claim 1 in fermenting wolfberry wine.

3. A method for preparing wolfberry wine, comprising the following steps: 1) adding sterile water to wolfberry pulp, performing enzymatic hydrolysis, adjusting sugar, adjusting acidity, and sterilizing to obtain wolfberry juice; 2) The plant lactobacillus NXU0013 and saccharomyces cerevisiae JY2 described in claim 1 are respectively inoculated into wolfberry juice for fermentation to obtain wolfberry wine; the volume ratio of the plant lactobacillus NXU0013 to saccharomyces cerevisiae JY2 is 1:3.

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