Lactobacillus plantarum from the deep sea of the South China Sea and application thereof in bioconversion of curcumin

Abstract

This invention provides a strain of *Lactiplantibacillus plantarum* OLp4 from the deep sea of ​​the South China Sea. This strain was deposited on February 28, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located in Guangzhou, with accession number GDMCCNo: 64378. This invention also provides an application of this *Lactiplantibacillus* strain in converting curcumin to tetrahydrocurcumin. The *Lactiplantibacillus* strain of this invention is easy to culture, grows rapidly, has strong acid-producing capacity, and exhibits extremely strong acid resistance.

Description

A strain of *Lactobacillus plantarum* from the deep sea of ​​the South China Sea and its application in the biotransformation of curcumin. Technical Field

[0001] This invention relates to the fields of food and microbial application technology, and discloses a strain of *Lactobacillus plantarum* derived from the deep sea of ​​the South China Sea and its application in converting curcumin into tetrahydrocurcumin. Background Technology

[0002] Lactobacillus plantarum is a common type of lactic acid bacteria with good ecological and metabolic adaptability, widely found in fermented foods, meat, and the gastrointestinal tract of mammals. Acid-producing capacity is a key characteristic of lactic acid bacteria. The organic acids produced by lactic acid bacteria metabolism, such as lactic acid, acetic acid, propionic acid, citric acid, and succinic acid, not only lower the pH of the gastrointestinal tract, inhibit and kill intestinal pathogens, and regulate the balance of intestinal flora, but also increase the activity of digestive enzymes, promote mineral absorption, alleviate gastrointestinal dysfunction, and reduce the incidence and mortality of diarrhea. Therefore, in practical production, lactic acid bacteria with strong acid-producing capacity have higher application value.

[0003] Generally, in order to produce beneficial health benefits, orally administered probiotics must survive at sufficiently high levels in the gastrointestinal tract. However, the highly acidic environment of the human stomach (pH ≤ 2.5) and the high bile salt environment of the small intestine (0.05-2%) are extremely unfavorable for probiotic survival. Although most lactic acid bacteria can survive at low pH, their acid resistance is strain-specific.

[0004] Curcumin is a polyphenolic compound extracted from the rhizome of turmeric. It is an orange-yellow crystalline powder, poorly soluble in water, and possesses lipid-lowering, anti-tumor, anti-inflammatory, choleretic, anti-atherosclerotic, antioxidant, and anti-Alzheimer's disease effects. While curcumin has significant clinical application value and is an ideal candidate for anti-cancer drugs, its poor water solubility, low in vivo absorption, and low bioavailability greatly limit its application. Therefore, modifying the structure of curcumin to obtain new derivatives through chemical and microbial methods while retaining its original efficacy has become a research hotspot in recent years. By reducing the four double bonds in its linkage chain to different degrees, corresponding hydrogenated derivatives can be obtained. Compared to chemical synthesis, microbial transformation offers advantages such as mild reaction conditions, high selectivity, low cost, and environmental friendliness.

[0005] This study is the first to screen a strain of Lactobacillus plantarum OLp4 from the deep sea of ​​the South China Sea. It has extremely strong acid resistance and can generate tetrahydrocurcumin using curcumin as a substrate, providing a reference for the production of curcumin hydrogenated derivatives by microbial fermentation. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide a strain of Lactobacillus plantarum from the deep sea of ​​the South China Sea. The strain of Lactobacillus plantarum was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on February 28, 2024, with the deposit address in Guangzhou and the deposit number GDMCC No: 64378.

[0007] Optionally, the Lactobacillus plantarum DNA sequence is SEQ ID NO.1.

[0008] Optionally, the *Lactobacillus plantarum* is isolated from deep water in the South China Sea.

[0009] The second technical problem to be solved by this invention is to provide an application of a strain of Lactobacillus plantarum that converts curcumin into tetrahydrocurcumin.

[0010] Optionally, the *Lactobacillus plantarum* has the ability to bioconvert curcumin into its derivative tetrahydrocurcumin, with a conversion rate greater than 40%.

[0011] Optionally, the *Lactobacillus plantarum* converts curcumin into tetrahydrocurcumin using a microbial fermentation method.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. This invention isolates and identifies a strain of Lactiplantibacillus plantarum OLp4. Through genome sequence analysis and phylogenetic tree construction, the strain is identified as Lactiplantibacillus plantarum.

[0014] 2. The *Lactobacillus plantarum* in the invention is easy to cultivate, grows rapidly, has a strong acid-producing capacity, and has extremely strong acid resistance.

[0015] 3. The *Lactobacillus plantarum* in this invention can convert curcumin into tetrahydrocurcumin by microbial fermentation, and the conversion rate is greater than 40%. Attached Figure Description

[0016] Figure 1 is a colony diagram of strain OLp4 in Example 1 of the present invention;

[0017] Figure 2 is a microscopic image of strain OLp4 in Example 1 of the present invention;

[0018] Figure 3 is an agarose gel electrophoresis diagram of the PCR amplification product of strain OLp4 in Example 2 of the present invention;

[0019] Figure 4 is a phylogenetic tree of strain OLp4 in Example 2 of the present invention;

[0020] Figure 5(a) shows the growth curve of strain OLp4 in Example 3 of the present invention;

[0021] Figure 5(b) shows the acid production curve of strain OLp4 in Example 3 of the present invention;

[0022] Figure 6 shows the tolerance of strain OLp4 of Example 4 of the present invention in environments with pH 2, 2.5, and 3.

[0023] Figure 7 shows the HPLC chromatogram of the curcumin standard sample (its retention time is 13.678 min);

[0024] Figure 8 shows the HPLC chromatogram of the tetrahydrocurcumin standard sample (its retention time is 11.695 min);

[0025] Figure 9(a) is the HPLC chromatogram of the blank culture medium (a) in Example 5 of the present invention.

[0026] Figure 9(b) HPLC chromatogram of Lactobacillus plantarum (b) in Example 5 of the present invention.

[0027] Figure 9(c) is an HPLC chromatogram of the extract of *Lactobacillus plantarum* (c) with added curcumin in Example 5 of the present invention (wherein the retention times of curcumin and tetrahydrocurcumin are 11.610 min and 13.573 min, respectively). Detailed Implementation

[0028] Example 1: Obtaining the Lactiplantibacillus plantarum strain OLp4.

[0029] 1. Screening of strains

[0030] Seawater samples were collected from the deep sea of ​​the South China Sea. Each sample was cultured, serially diluted, and plated onto MRS agar plates. The plates were numbered and anaerobically incubated at 37°C for 24 hours. Single colonies with good growth were selected and inoculated into test tubes for further incubation. The colonies were clearly defined, round with raised edges, smooth surfaces, and a milky white color. Microscopically, they appeared as short, thin rods (Figures 1 and 2).

[0031] Example 2: Gene sequence analysis of Lactiplantibacillus plantarum strain OLp4

[0032] 1. Bacterial DNA Genome Extraction

[0033] The culture medium of the strain obtained in Example 1 was centrifuged and the precipitate was collected for DNA extraction.

[0034] 2. Perform PCR amplification of 16S rDNA on this strain:

[0035] PCR amplification: PCR amplification was performed using primers 27F and 1492R. The 27F sequence was 5′-AGAGTTTGATCCTGGCTCAG-3′, and the 1492R sequence was 5′-GGTTACCTTGTTACGACTT-3′. The PCR reaction mixture consisted of 25 μL TAQ enzyme, 2 μL 27F primer, 2 μL 1492R primer, 5 μL bacterial template, and 16 μL sterile water. The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 60 s, 56℃ annealing for 60 s, 72℃ extension for 90 s, 30 cycles; 72℃ extension for 7 min, and termination at 4℃. Gel electrophoresis and PCR product recovery: The PCR products were identified by 1.5% agarose gel electrophoresis. The target PCR products were recovered and purified using a rapid agarose gel DNA recovery kit (Beijing Biotech Biotechnology Co., Ltd.) and then sequenced as 16S rDNA. The PCR products were identified by 1% agarose gel electrophoresis (Figure 3). Observe whether the target band was amplified.

[0036] 3. 16S rDNA sequencing and homology analysis

[0037] The target PCR product was recovered and purified using a rapid agarose gel DNA recovery kit (Beijing Biotech Co., Ltd.). The purified PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the gene sequence is shown in SEQ ID NO: 1. The obtained sequence was submitted to NCBI's GENBANK database to obtain similar sequences. Comparative analysis was performed using BLAST and DNAMAN software, and a phylogenetic tree was constructed using the Neighbor-Joining method, as shown in Figure 4.

[0038] Phylogenetic analysis revealed that this strain is highly related to Lactiplantibacillus plantarum, thus identifying it as a marine-derived Lactobacillus plantarum strain, which we named Lactiplantibacillus plantarum OLp4.

[0039] The *Lactobacillus plantarum* strain obtained from the above screening is named *Lactiplantibacillus plantarum* strain (strain number OLp4). It was deposited on February 28, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) in Guangzhou, with accession number 64378.

[0040] Example 3: Growth curve and acid production capacity of Lactobacillus plantarum OLp4

[0041] Bacillus coagulans, frozen in the laboratory, was activated and passaged multiple times to improve its cell activity. The activated and purified strain was then inoculated onto MRS solid medium for plate selection and purification. The activated and purified strain was inoculated into the medium at a rate of 1% and cultured at 37°C with a shaker at 200 rpm. Odulocyte count (OD) was measured every 4 hours. 600 The pH of the supernatant and total acid were measured. The total acid was determined according to GB 12456-2021. Three sets were repeated, and growth curves and acid production curves were plotted.

[0042] As shown in Figure 5, this strain exhibits a lag phase of 0–4 hours. After 4 hours, it enters the logarithmic growth phase, resulting in a rapid increase in cell number. It enters a stationary phase after 16 hours, where the bacterial concentration gradually levels off. After 24 hours, the cells begin to die and enter the decline phase. The pH decreases rapidly and acid production is high during the first 16 hours due to the rapid growth of the strain, increased viable cell count, and higher acid production. After 16 hours, the acid production capacity gradually weakens and eventually stabilizes, resulting in an acidic bacterial solution with a pH of 3.8. The highest total acid content is 35.47 g / L, indicating that this strain possesses good acid production capacity.

[0043] Example 4: Acid resistance test of Lactobacillus plantarum OLp4

[0044] Select healthy single colonies and culture them in test tubes for 12–18 hours. Adjust the pH of the prepared MRS culture medium to 2, 2.5, and 3 respectively using 1M HCl. Inoculate the culture medium with 1% of the culture medium and culture at 37°C. Take samples at 0, 3, and 6 hours to measure the viable count.

[0045] As shown in Figure 6, strain OLp4 maintained high activity under acidic conditions of pH 2.5 and 3, exhibiting a certain growth trend from 0 to 6 hours. Under extreme conditions of pH 2, strain OLp4 showed signs of death, with the number of viable cells gradually decreasing over time, but still maintaining a survival rate of 49.14% at 6 hours. This indicates that strain OLp4 not only tolerates acid but can also grow under pH 2.5 and 3 conditions, demonstrating strong acid resistance.

[0046] Example 5: Application of Lactobacillus plantarum OLp4 in the conversion of curcumin to tetrahydrocurcumin

[0047] After thawing *Lactobacillus plantarum* OLp4 stored at low temperature on MRS plates, the inoculum was picked up with an inoculation loop and inoculated into MRS liquid seed culture medium. The culture was then incubated at 37°C and 200 rpm for 16 hours to obtain the seed culture solution. In a clean bench, 1 mL of the seed culture solution was inoculated into 9 Erlenmeyer flasks containing 70 mL of liquid culture medium each. Experimental group (c) received 1 mL of 20 mg / mL curcumin dissolved in DMSO, while control group (b) received no curcumin. Each flask received 1 mL of the same volume of deionized water. Blank control (a) showed uninoculated MRS liquid culture medium.

[0048] After 48 hours of conversion, the fermentation product was extracted. An equal volume of ethyl acetate solvent was added to the bacterial culture, and ultrasonic extraction was performed for 30 minutes each time, with the flask shaken every 10 minutes to ensure complete extraction. The extraction was repeated three times in total. Finally, all the collected extracts were rotary evaporated at a temperature below 50°C to obtain a concentrated crude extract, which was then dissolved in methanol for later use.

[0049] The blank group, control group, and experimental group were analyzed by high-performance liquid chromatography (HPLC). The chromatographic column was an Agilent C18 HPLC column, the mobile phase was acetonitrile / 0.5% acetic acid in water (55:45, v / v), the injection volume was 10 μL, the flow rate was 0.8 min / mL, the detection wavelength was 280 nm, and the temperature was 30 °C. The conversion rate was defined as the ratio of the concentration of the product obtained after the reaction to the concentration of the substrate added at the beginning of the reaction.

[0050] As shown in Figure 9(c), a peak appears at 11.6 min in the high performance liquid chromatogram of the experimental group sample. This position is basically consistent with the peak time of the tetrahydrocurcumin standard (Figure 8 shows the HPLC chromatogram of the tetrahydrocurcumin standard sample, with a retention time of 11.695 min), indicating that the product obtained by conversion is tetrahydrocurcumin, and the conversion rate is greater than 40%.

[0051] SEQ ID NO: 1

[0052]

Claims

1. A strain of Lactiplantibacillus plantarum OLp4, characterized by: The Lactobacillus plantarum strain was deposited on February 28, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located in Guangzhou, with accession number GDMCC No: 64378.

2. The *Lactobacillus plantarum* strain according to claim 1, characterized in that: The Lactobacillus plantarum DNA sequence is SEQ ID NO.

1.

3. The *Lactobacillus plantarum* strain according to claim 1, characterized in that: The plant lactobacillus is derived from deep water in the South China Sea.

4. The application of *Lactobacillus plantarum* as described in claim 1 in the conversion of curcumin to tetrahydrocurcumin, characterized in that: The plant lactobacillus converts curcumin into tetrahydrocurcumin through biotransformation.

5. The application of *Lactobacillus plantarum* according to claim 4 in the conversion of curcumin to tetrahydrocurcumin, characterized in that: The *Lactobacillus plantarum* has the ability to bioconvert curcumin into its derivative tetrahydrocurcumin, with a conversion rate greater than 40%.

6. The application of *Lactobacillus plantarum* according to claim 5 in the conversion of curcumin to tetrahydrocurcumin, characterized in that: The plant lactobacillus converts curcumin into tetrahydrocurcumin using a microbial fermentation method.

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