Lactobacillus plantarum FBEL010 and application thereof

Abstract

A strain of *Lactiplantibacillus plantarum*, FBEL010, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 25, 2025, with accession number CGMCC No. 36687. Its 16S rDNA nucleotide sequence is shown in SEQ ID NO. 1. The genome of strain FBEL010 contains a gene encoding the alcohol-acyltransferase AAT, the nucleotide sequence of which is shown in SEQ ID NO. 2. This strain is used in the preparation of fermented fruit and vegetable juices. The prepared fermented fruit and vegetable juices have a total ester content ≥150 μg / L, and significantly increased levels of hexyl acetate and ethyl butyrate fruit aroma components. Testing according to national standards showed no pathogenic bacteria, and the content of contaminating bacteria was within acceptable limits. The content of heavy metals (lead, arsenic, mercury) and additives meets national standards, making it suitable for use as a beverage. This strain can also be used to prepare fermented fruit and vegetable juices such as strawberry, lychee, pineapple, and cucumber.

Description

Technical Field

[0001] This invention belongs to the field of beverage technology, specifically relating to Lactobacillus plantarum fermentation of fruit or vegetable juice. Background Technology

[0002] Fermented fruit and vegetable juices, metabolized by lactic acid bacteria, not only achieve natural preservation but also significantly enhance their nutritional value and bioactivity. With consumers' growing demand for healthy, natural, and diverse products, developing lactic acid bacteria-fermented fruit and vegetable juice beverages that combine probiotic functions with excellent sensory qualities has become an important direction for the food processing industry to increase resource added value and achieve industrial upgrading.

[0003] Existing fermentation processes generally suffer from drawbacks such as poor strain compatibility, low production efficiency, and unsatisfactory flavor. On the one hand, existing commercial strains are insufficient in aroma production under high-acid conditions in fruit juice, resulting in products lacking typical fresh fruit aromas and even easily developing undesirable flavors such as pickled vegetables. On the other hand, due to the lack of effective synergistic fermentation-promoting methods, the fermentation cycle often exceeds 18 hours. This lengthy metabolic process not only limits production efficiency but also easily leads to the accumulation of off-flavor substances such as fusel oils, resulting in products with weak aromas and poor sensory quality.

[0004] Screening superior bacterial strains capable of directionally synthesizing high-aroma esters and establishing corresponding rapid fermentation methods to shorten the process cycle are currently key to improving the quality of fermented fruit and vegetable juices. By leveraging the synergistic effect of strains with specific aroma-producing metabolic characteristics and efficient growth-promoting processes, the precise accumulation of characteristic aroma components can be achieved while suppressing the generation of metabolic off-flavors at the source. This has significant technical implications for the efficient industrial production of high-quality fruit and vegetable juices. Summary of the Invention

[0005] One of the technical problems to be solved by the present invention is to overcome the shortcomings of the prior art and provide a strain of Lactobacillus plantarum FBEL010 that can be applied to the field of fruit and vegetable fermentation food technology.

[0006] Another technical problem to be solved by the present invention is to provide a new use for Lactobacillus plantarum FBEL010.

[0007] The technical solution adopted to solve the above-mentioned technical problems is as follows: A strain of Lactiplantibacillus plantarum, FBEL010, was deposited at the China General Microbiological Culture Collection Center on November 25, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36687. Its 16S rDNA nucleotide sequence is shown in SEQ ID NO.1. The genome of strain FBEL010 contains an alcohol-acyltransferase AAT encoding gene, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] The screening method for the above-mentioned strain FBEL010 consists of the following steps:

[0009] 1. Separation, purification and screening

[0010] Pipette 1 mL of commercially available sauerkraut juice into a centrifuge tube containing 9 mL of physiological saline. Vortex thoroughly, then immediately add 1 mL of the suspension to the centrifuge tube containing 9 mL of physiological saline. Repeat this step to obtain 10 mL of the sauerkraut juice. 6 10 7 10 8 Three dilution gradients were prepared, and 100 μL of each of the three dilution gradients was spread onto MRS solid medium. The plates were then transferred to a 30°C incubator and incubated for 48 hours. Single colonies with different shapes and sizes were picked out, numbered, and inoculated into MRS liquid medium using an inoculation loop. The medium was incubated at 30°C for 24 hours to obtain bacterial cells. The cells were then stored at -80°C using 50% glycerol at a 1:1 ratio.

[0011] MRS medium 1 L is prepared according to the following material ratio: 10 g peptone, 10 g yeast powder, 10 g beef extract, 4 g anhydrous sodium acetate, 2 g diammonium hydrogen citrate, 2 g dipotassium hydrogen phosphate trihydrate, 0.2 g magnesium sulfate heptahydrate, 0.03 g sodium chloride, 0.01 g ferrous sulfate heptahydrate, 0.2 g manganese sulfate monohydrate, 1 mL Tween-80, 40 g glucose, and ultrapure water added to 1 L, pH 6.2-6.4, to prepare liquid MRS medium. Then, 17 g agar powder is added to the MRS medium to prepare solid MRS medium.

[0012] 2. Morphological and molecular identification, strain preservation

[0013] (1) Morphological characteristics

[0014] The bacterial cells were cultured on MRS solid medium at 37°C for 24 hours. Colonies were 1.2-1.5 mm in diameter, milky white, round, with neat edges, and opaque. Under a microscope, they appeared as long rods, arranged singly, Gram-positive, without spore formation, and their morphology was as follows.Figure 1 As shown in Figure 2, the microstructure is as shown in Figure 2.

[0015] (2) Identification and preservation using molecular biological methods

[0016] Genomic DNA was extracted from the bacterial cells according to the instructions of the bacterial genomic DNA extraction kit. Using this DNA as a template, PCR amplification was performed using universal primers (F: 5'-AGAGTTTGATCMTGGCTCAG-3'; R: 5'-GGTTACCTTGTTACGACTT-3') to obtain its 16S rRNA. Sequencing was performed by Beijing Qingke Biotechnology Co., Ltd. The sequence information (SEQ ID NO.1) was compared and analyzed for homology in the NCBI database. The sequence homology was 99.97%. The bacteria were classified and named Lactiplantibacillus plantarum, and further named FBEL010. The DNA was deposited.

[0017] (3) Confirm the AAT gene

[0018] Genomic DNA was extracted from Lactobacillus plantarum FBEL010, and specific primers were designed to amplify the AAT gene. The sequencing results are shown in SEQ ID NO.2 of the sequence listing. The sequence showed up to 99% homology with the known AAT gene sequence with high esterification activity, and the key active sites were conserved.

[0019] The genome of the strain contains a gene encoding an alcohol-acyltransferase AAT (AAT gene) with specific substrate specificity, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0020] Uses of Lactobacillus plantarum FBEL010 in the preparation of fermented fruit juice.

[0021] In the fermented fruit juice, the viable count of *Lactobacillus plantarum* FBEL010 was 1 × 10⁻⁶. 8 CFU / mL ~ 3 × 10 8 The fermented fruit juice contains ≥10 μg / L hexyl acetate and ≥10 μg / L ethyl butyrate, and the total ester content, calculated as ethyl acetate, is ≥150 μg / L.

[0022] The fermented fruit juice is any one of fermented apple juice, fermented pear juice, fermented prune juice, or fermented hawthorn juice.

[0023] Uses of Lactobacillus plantarum FBEL010 in the preparation of fermented vegetable juice.

[0024] In the fermented vegetable juice, the viable count of *Lactobacillus plantarum* FBEL010 was 1 × 10⁻⁶. 8 CFU / mL ~ 3 × 108 CFU / mL, in fermented vegetable juice, hexyl acetate content ≥10μg / L, ethyl butyrate content ≥10μg / L, and total ester content ≥150μg / L (calculated as ethyl acetate).

[0025] The fermented vegetable juice is either fermented carrot juice or fermented tomato juice.

[0026] The beneficial effects of this invention are as follows:

[0027] The strains and methods of this invention can significantly reconstruct the flavor profile of fermented fruit juices, achieving a total ester content (calculated as ethyl acetate) ≥150 μg / L, and significantly increasing characteristic fruit aroma components such as hexyl acetate and ethyl butyrate. It effectively inhibits the formation of undesirable flavor substances such as long-chain alcohols, and during fermentation, it can directionally catalyze the synthesis of key fruit aroma substances such as hexyl acetate and ethyl butyrate, resulting in a total ester content of ≥150 μg / L in the product. The use of tomato juice or its filtrate as a fermentation promoter significantly shortens the fermentation endpoint time by 3-4 hours. This method is suitable for the production of fermented fruit and vegetable juices such as apple juice, pear juice, prune juice, hawthorn juice, carrot juice, and tomato juice, and can also be used for the production of fermented fruit and vegetable juices such as strawberry, lychee, pineapple, and cucumber. Attached Figure Description

[0028] Figure 1 This is a morphological image of Lactobacillus plantarum strain FBEL010.

[0029] Figure 2 This is a microscopic morphological diagram of Lactobacillus plantarum strain FBEL010. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the following embodiments.

[0031] Example 1

[0032] A strain of Lactiplantibacillus plantarum, FBEL010, was deposited on November 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36687. Its 16S rDNA nucleotide sequence is shown in SEQ ID NO. 1. The strain's genome contains an alcohol-acyltransferase AAT encoding gene, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0033] The use of the above-mentioned Lactobacillus plantarum strain FBEL010 in the preparation of fermented apple juice.

[0034] In fermented apple juice, the viable count of Lactobacillus plantarum strain FBEL010 was 1×10⁻⁶. 8 CFU / mL ~ 3 × 10 8 The fermented apple juice contains ≥10 μg / L hexyl acetate and ≥10 μg / L ethyl butyrate, with a total ester content of ≥150 μg / L (calculated as ethyl acetate).

[0035] Taking the preparation of 1000 mL of fermented pear juice as an example, the preparation method is as follows:

[0036] Using 1000 mL of commercially available 70% apple juice as a substrate, the initial pH was adjusted to 5.6. A 6% inoculum of activated Lactobacillus plantarum strain FBEL010 was then inoculated, resulting in an initial bacterial count of 10⁻⁶ in the juice. 6 CFU / mL ~10 7 CFU / mL was added tomato juice as a fermentation promoter at a concentration of 2% of the substrate mass. The mixture was then placed in a constant temperature incubator and statically incubated at 37°C for 17 hours. pH changes were monitored periodically and plate counts were performed until the bacterial concentration in the fermentation broth reached 1×10⁻⁶ CFU / mL. 8 CFU / mL ~ 3 × 10 8 When the CFU / mL and pH drop to the fermentation endpoint of 3.5, stop the fermentation. After fermentation, place the mixture in a refrigerator at ±1℃ for 4–8 hours for post-fermentation ripening to prepare fermented apple juice.

[0037] The prepared fermented apple juice was tested according to the standards of the State Administration for Market Regulation, and the test results are shown in Table 1.

[0038] Table 1. Detection Results of Fermented Apple Juice

[0039]

[0040] As shown in Table 1, the test results meet national health standards. No pathogenic bacteria were detected, and the content of miscellaneous bacteria was within acceptable limits. The content of heavy metals (lead, arsenic, mercury) and additives also meets national standards.

[0041] Example 2

[0042] The use of Lactobacillus plantarum FBEL010 in the preparation of fermented pear juice in this embodiment.

[0043] The nucleotide sequence of Lactobacillus plantarum strain FBEL010 is the same as that in Example 1.

[0044] Use of Lactobacillus plantarum strain FBEL010 in the preparation of fermented pear juice.

[0045] In fermented pear juice, the viable count of Lactobacillus plantarum strain FBEL010 was 1×10⁻⁶. 8CFU / mL ~ 3 × 10 8 CFU / mL.

[0046] The preparation method of fermented pear juice is as follows:

[0047] Using commercially available 70% pear juice as a substrate, and with the same inoculation amount and fermentation conditions as in Example 1 for preparing fermented apple juice, fermented pear juice was prepared.

[0048] The prepared fermented pear juice was tested according to the standards of the State Administration for Market Regulation, and the test results are shown in Table 2.

[0049] Table 2 Detection results of fermented pear juice

[0050]

[0051] As shown in Table 2, the test results meet national health standards. No pathogenic bacteria were detected, and the content of miscellaneous bacteria was within acceptable limits. The content of heavy metals (lead, arsenic, mercury) and additives also meets national standards.

[0052] Example 3

[0053] The use of Lactobacillus plantarum FBEL010 in the preparation of fermented prune juice in this embodiment.

[0054] The nucleotide sequence of Lactobacillus plantarum strain FBEL010 is the same as that in Example 1.

[0055] Use of Lactobacillus plantarum strain FBEL010 in the preparation of fermented prune juice.

[0056] In fermented prune juice, the viable count of Lactobacillus plantarum strain FBEL010 was 1×10⁻⁶. 8 CFU / mL ~ 3 × 10 8 CFU / mL.

[0057] The preparation method of fermented prune juice is as follows:

[0058] Using commercially available 70% prune juice as a substrate, and with the same inoculation amount and fermentation conditions as in Example 1 for preparing fermented apple juice, fermented prune juice was prepared.

[0059] The prepared fermented plum juice was tested in accordance with the standards of the State Administration for Market Regulation, and the test results are shown in Table 3.

[0060] Table 3. Detection Results of Fermented Prune Juice

[0061]

[0062] As shown in Table 3, the test results meet national health standards. No pathogenic bacteria were detected, and the content of miscellaneous bacteria was within acceptable limits. The content of heavy metals (lead, arsenic, mercury) and additives also meets national standards.

[0063] Example 4

[0064] The use of Lactobacillus plantarum FBEL010 in the preparation of fermented hawthorn juice in this embodiment.

[0065] The nucleotide sequence of Lactobacillus plantarum strain FBEL010 is the same as that in Example 1.

[0066] Use of Lactobacillus plantarum strain FBEL010 in the preparation of fermented hawthorn juice.

[0067] In fermented hawthorn juice, the viable count of Lactobacillus plantarum strain FBEL010 was 1×10⁻⁶. 8 CFU / mL ~ 3 × 10 8 CFU / mL.

[0068] The preparation method of fermented hawthorn juice is as follows:

[0069] Using commercially available 70% hawthorn juice as a substrate, and with the same inoculation amount and fermentation conditions as the fermented apple juice prepared in Example 1, fermented hawthorn juice was prepared.

[0070] The prepared fermented hawthorn juice was tested in accordance with the standards of the State Administration for Market Regulation.

[0071] The test results are shown in Table 4.

[0072] Table 4. Detection Results of Fermented Hawthorn Juice

[0073]

[0074] As shown in Table 4, the test results meet national health standards. No pathogenic bacteria were detected, and the content of miscellaneous bacteria was within acceptable limits. The content of heavy metals (lead, arsenic, mercury) and additives also meets national standards.

[0075] Example 5

[0076] The use of Lactobacillus plantarum FBEL010 in the preparation of fermented carrot juice in this embodiment.

[0077] The nucleotide sequence of Lactobacillus plantarum strain FBEL010 is the same as that in Example 1.

[0078] Use of Lactobacillus plantarum strain FBEL010 in the preparation of fermented carrot juice.

[0079] In fermented carrot juice, the viable count of Lactobacillus plantarum strain FBEL010 was 1×10⁻⁶. 8CFU / mL ~ 3 × 10 8 CFU / mL.

[0080] The preparation method of fermented carrot juice is as follows:

[0081] Using commercially available 70% carrot juice as a substrate, fermented carrot juice was prepared by inoculating Lactobacillus plantarum strain FBEL010 with the same inoculation amount and fermentation conditions as the fermented apple juice prepared in Example 1.

[0082] The prepared fermented carrot juice was tested in accordance with the standards of the State Administration for Market Regulation.

[0083] The test results are shown in Table 5.

[0084] Table 5. Detection results of fermented carrot juice

[0085]

[0086] As shown in Table 5, the test results meet national health standards. No pathogenic bacteria were detected, and the content of miscellaneous bacteria was within acceptable limits. The content of heavy metals (lead, arsenic, mercury) and additives also meets national standards.

[0087] Example 6

[0088] The use of Lactobacillus plantarum FBEL010 in the preparation of fermented tomatoes in this embodiment.

[0089] The nucleotide sequence of Lactobacillus plantarum strain FBEL010 is the same as that in Example 1.

[0090] Use of Lactobacillus plantarum strain FBEL010 in the preparation of fermented tomato juice.

[0091] In fermented tomato juice, the viable count of Lactobacillus plantarum strain FBEL010 was 1×10⁻⁶. 8 CFU / mL ~ 3 × 10 8 CFU / mL.

[0092] The preparation method for fermented tomato juice is as follows:

[0093] Using commercially available 70% tomato juice as a substrate, and with the same inoculation amount and fermentation conditions as the fermented apple juice prepared in Example 1, fermented tomato juice was prepared.

[0094] The prepared fermented tomato juice was tested according to the standards of the State Administration for Market Regulation, and the test results are shown in Table 6.

[0095] Table 6. Detection Results of Fermented Tomato Juice

[0096]

[0097] As shown in Table 6, the test results meet national health standards. No pathogenic bacteria were detected, and the content of miscellaneous bacteria was within acceptable limits. The content of heavy metals (lead, arsenic, mercury) and additives also meets national standards.

[0098] To verify the beneficial effects of Lactobacillus plantarum strain FBEL010, the inventors conducted numerous laboratory research experiments using the fermented apple juice from Example 1 of this invention. The various experimental details are as follows:

[0099] 1. Gas chromatography-mass spectrometry analysis of volatile ester components in fermented fruit juice

[0100] 0.005 L of apple juice fermented by strain FBEL010 from Example 1 (hereinafter referred to as FBEL010) was taken, with a total ester content of 210.66±3.49 μg / L. The juice was divided into three groups and compared with common Lactobacillus plantarum (hereinafter referred to as ATCC 14917) and commercial Saccharomyces cerevisiae (hereinafter referred to as DY-10). Headspace solid-phase microextraction-gas chromatography-mass spectrometry was used to perform qualitative and quantitative analysis of esters in the three groups of samples. The experimental results are shown in Table 7.

[0101] Table 7. Ester content of fermented apple juice

[0102]

[0103] Note: "—" indicates not detected.

[0104] As shown in Table 7, the total ester content of the fermentation broth from strain FBEL010 reached 210.66±3.49 μg / L, which was 7.3 times and 11.2 times higher than that of common Lactobacillus plantarum ATCC 14917 (28.97 μg / L) and commercial Saccharomyces cerevisiae DY-10 (18.89 μg / L), respectively. This significant difference demonstrates that strain FBEL010 possesses excellent ester-producing performance in the juice system, indicating that the AAT gene in its genome is efficiently expressed, leading to a biosynthesis rate of esters that is much greater than the hydrolysis rate, thus achieving effective accumulation of total esters.

[0105] The content of hexyl acetate, characterized by green apple and pear aromas, is as high as 12.57 μg / L, which is 5.6 times that of ATCC 14917 group, while it was not detected in commercial strains, giving the fermented juice a fresh original fruit aroma. Strain FBEL010 accumulated high levels of ethyl butyrate (21.61 μg / L) and 2-methylbutyl acetate (101.68 μg / L). These two low-threshold compounds have a sweet aroma of pineapple, banana, and tropical fruits, enriching the aroma of the product.

[0106] Unlike the control strain, which tends to produce long-chain heavy esters such as heptyl acetate and methyl palmitate, which have oily, waxy, or aged flavors, no such undesirable flavor compounds were detected in the fermentation broth of strain FBEL010. This indicates that the strain of this invention, combined with a rapid fermentation process, selectively enriches key aroma compounds while avoiding the generation of undesirable flavors, resulting in a purer aroma and upgrading the flavor quality of fermented fruit juice from traditional sourness to a fresh fruit aroma.

[0107] 2. Sensory evaluation of fermented apple juice

[0108] Ten professionally trained sensory evaluators, half male and half female, were invited to conduct sensory evaluations of three groups of fermented fruit juice samples (FBEL010, ATCC 14917, and DY-10) using a double-blind method. Based on the characteristics of the product of this invention, a percentage-based evaluation standard focusing on "typical fruit aroma" and "purity of taste" was established, as shown in Table 8. The presence of characteristic hexyl acetate (green apple / pear aroma) and ethyl butyrate (tropical fruit aroma) aromas, as well as the presence of fusel oil off-flavors caused by excessively long fermentation times, were examined.

[0109] Table 8 Evaluation Criteria for Fermented Apple Juice

[0110] The sensory score statistics for each group of samples are shown in Table 9.

[0111] Table 9. Scoring Results of Fermented Apple Juice

[0112]

[0113] As shown in Table 9, the FBEL010 group achieved a total score of 90.5, significantly better than all control groups. Its aroma performance was highly consistent with the GC-MS results of high levels of hexyl acetate (green apple aroma) and ethyl butyrate (sweet fruit aroma) in the fermented apple juice of Example 1. Furthermore, thanks to the rapid fermentation process facilitated by tomato juice, the fermentation liquid retained the bright color of the original juice, and effectively avoided the spiciness or bitterness caused by long-chain alcohols, presenting a pure and harmonious fresh fruit flavor. In contrast, the commercial strain DY-10 group scored 74.6, exhibiting a distinct fermented wine and yeast aroma that masked the fruity fragrance. The common strain ATCC 14917 group scored 62.2, exhibiting a lack of aroma and a sharp acidity due to its weak ester production capacity.

[0114] In summary, the FBEL010 strain of this invention, combined with a rapid fermentation process, successfully produced a fermented fruit juice product with excellent sensory quality and typical fresh fruit aroma characteristics.

Claims

1. A strain of Lactiplantibacillus plantarum, FBEL010, was deposited on November 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36687. Its 16S rDNA nucleotide sequence is shown in SEQ ID NO.

1. The genome of strain FBEL010 contains an alcohol-acyltransferase AAT encoding gene, the nucleotide sequence of which is shown in SEQ ID NO.

2.

2. The use of Lactobacillus plantarum FBEL010 of claim 1 in the preparation of fermented fruit juice.

3. The use of *Lactobacillus plantarum* FBEL010 according to claim 2 in the preparation of fermented fruit juice, characterized in that: The fermented fruit juice is any one of fermented apple juice, fermented pear juice, fermented prune juice, or fermented hawthorn juice.

4. The use of *Lactobacillus plantarum* FBEL010 according to claim 2 or 3 in the preparation of fermented fruit juice, characterized in that: The fermented fruit juice contained 1×10 live bacteria of *Lactobacillus plantarum* FBEL010. 8 CFU / mL ~ 3 × 10 8 The fermented fruit juice contains ≥10 μg / L hexyl acetate and ≥10 μg / L ethyl butyrate, and the total ester content, calculated as ethyl acetate, is ≥150 μg / L.

5. The use of Lactobacillus plantarum FBEL010 of claim 1 in the preparation of fermented vegetable juice.

6. The use of *Lactobacillus plantarum* FBEL010 according to claim 5 in the preparation of fermented vegetable juice, characterized in that: The fermented vegetable juice is either fermented carrot juice or fermented tomato juice.

7. The use of *Lactobacillus plantarum* FBEL010 according to claim 5 or 6 in the preparation of fermented vegetable juice, characterized in that: The fermented vegetable juice contained 1×10 live bacteria of *Lactobacillus plantarum* FBEL010. 8 CFU / mL ~ 3 × 10 8 CFU / mL, in fermented vegetable juice, hexyl acetate content ≥10μg / L, ethyl butyrate content ≥10μg / L, and total ester content ≥150μg / L (calculated as ethyl acetate).

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