A high-tyramine-producing Lactobacillus brevis DF-19 and its applications

Abstract

This invention relates to the field of microbial technology and provides a high-tyramine-producing Lactobacillus brevis strain DF-19 and its applications. It was deposited on May 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33896. The strain DF-19 isolated in this invention exhibits high tyramine production capacity, is easy to cultivate, has strong acid and salt tolerance, specific metabolism, genetic stability, and strong biocompatibility, providing a foundation for the efficient production of tyramine and the development of related products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a high-tyramine-producing Lactobacillus brevis DF-19 and its applications. Background Technology

[0002] Tyramine (4-hydroxyphenylethylamine) is a naturally occurring monoamine compound widely found in the metabolic systems of animals, plants, and microorganisms. Its unique amphiphilicity, granted by the benzene ring hydroxyl group and ethylamine side chain, allows it to cross the blood-brain barrier and participate in neural regulation. In the biological world, damaged plant tissues (such as abraded banana peels) accumulate tyramine as a defense signal; animal gut microbiota synthesize tyramine through the tyrosine decarboxylation pathway to regulate energy metabolism balance; and microorganisms such as lactic acid bacteria and yeast use it as a survival strategy in response to acid-base stress.

[0003] Tyramine, as a key bioactive molecule in biological metabolism, plays an irreplaceable role in pharmaceutical preparations, functional foods, and cosmetic products. However, industrial production systems are facing multiple technological constraints. Existing methods for tyramine preparation include chemical synthesis, biocatalysis, and biofermentation. Among these, chemical synthesis uses inexpensive raw materials with high recovery rates, making it suitable for industrial production and currently the main method for tyramine synthesis. However, it generally suffers from the following drawbacks: complex synthesis pathways, numerous reaction steps, and difficulties in product purification leading to low overall yields; the need for corrosive raw materials such as strong acids and alkalis, posing significant environmental hazards; and the requirement for expensive metal catalysts and high-temperature, high-pressure resistant equipment, increasing production costs and operational risks.

[0004] Tyrosine decarboxylase (TDC or TYDC) catalyzes the decarboxylation of tyrosine to produce tyramine and CO2. The Ni Hua research group at Jiangnan University resolved the structure of the TDC protein derived from *Lactobacillus brevis*. Through reaction condition optimization and molecular modification, they were able to achieve the desired tyrosine concentration at 400 mmol / L. -1 When the reaction time was 24 hours, the conversion rate exceeded 99.99%. Enzymatic conversion has the characteristic of relatively specific products and can achieve one-step biotransformation synthesis. Therefore, the preparation of tyramine by biological enzyme method has certain advantages. For example, CN105969755A discloses tyrosine decarboxylase from Lactobacillus brevis CGMCC1.2028 and its S586A mutant catalyzing the decarboxylation reaction of tyrosine to obtain tyramine. CN115678877A discloses tyrosine decarboxylase CtTyDC from Cistanche tubulosa catalyzing the decarboxylation reaction of tyrosine to obtain tyramine. Moreover, the reaction does not depend on the cofactor (pyridoxal phosphate, PLP).

[0005] Studies have found that tyramine can be produced in organisms via the tyrosine metabolic pathway. Plant and insect cells contain low levels of tyramine, making it difficult to obtain high-purity tyramine through isolation and extraction. In contrast, microbial cells have higher tyramine content and tyrosine decarboxylase activity, making microbial fermentation a suitable method for tyramine production. Compared to other methods, bio-fermentation offers milder conditions, simpler operation, and fewer byproducts, but it suffers from drawbacks such as long fermentation cycles, high culture medium costs, and low yields. It is noteworthy that acid stress, a typical environmental pressure in open fermentation, has a dual impact on microbial survival and metabolism: on the one hand, acidic environments can inhibit the growth of most spoilage bacteria, extending food shelf life; on the other hand, low pH conditions may activate the stress response mechanism of tyramine-producing bacteria, promoting tyramine synthesis. Since tyramine is alkaline, it can be used to maintain intracellular pH; therefore, tyramine production is also considered a cellular response to acid stress. However, existing probiotic strains exhibit insufficient activity in acidic environments, limiting their application in high-efficiency tyramine fermentation. Summary of the Invention

[0006] The purpose of this invention is to provide a Lactobacillus brevis DF-19, which aims to solve the problems mentioned in the background art.

[0007] This invention is implemented as follows: a short lactobacillus, named *Lactobacillus brevis* (… Lactobacillus brevis DF-19 was deposited on May 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33896.

[0008] Another objective of this invention is to provide an application of Lactobacillus brevis DF-19 in the synthesis of tyramine.

[0009] Another object of the present invention is to provide a formulation for synthesizing tyramine, the formulation comprising the above-mentioned Lactobacillus brevis DF-19.

[0010] Another objective of this invention is to provide the application of Lactobacillus brevis DF-19 in the preparation of products rich in tyramine.

[0011] In this embodiment of the invention, through targeted isolation, purification, identification, and verification of tyramine production capacity, a high-tyramine-producing strain DF-19 was screened. 16 rDNA sequencing analysis identified this strain as *Lactobacillus brevis*. The strain screened in this embodiment of the invention has a high tyramine production capacity, is easy to cultivate, has strong acid and salt tolerance, specific metabolism, genetic stability, and strong biocompatibility, providing a foundation for the efficient production and application of tyramine. Attached Figure Description

[0012] Figure 1The liquid chromatogram of strain DF-19 provided in Example 1 of this invention;

[0013] Figure 2 The tyramine standard curve provided in Example 1 of this invention;

[0014] Figure 3 The Gram staining results of strain DF-19 provided in Example 1 of this invention;

[0015] Figure 4 The gene phylogenetic tree of strain DF-19 provided in Example 1 of this invention;

[0016] Figure 5 The growth curve of strain DF-19 provided in Example 2 of this invention;

[0017] Figure 6 The results of salt tolerance testing for strain DF-19 provided in Example 2 of this invention;

[0018] Figure 7 The results of the acid resistance test of strain DF-19 provided in Example 2 of this invention;

[0019] Figure 8 This is the result of the effect of lactic acid stress on tyramine production by the strain provided in Example 2 of the present invention;

[0020] Figure 9 This is the result of the effect of acetic acid stress on tyramine production by the strain provided in Example 2 of the present invention;

[0021] Figure 10 The results of the effect of citric acid stress on tyramine production by the strain provided in Example 2 of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0024] Example 1: Isolation and identification of Lactobacillus brevis DF-19:

[0025] 1. Sample source: Naturally fermented tofu from Jilin City, Jilin Province.

[0026] 2. Culture medium:

[0027] MRS liquid culture medium: peptone 10.0 g, beef extract 8.0 g, yeast extract 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 mL, distilled water 1000 mL, pH 5.5-5.9;

[0028] MRS solid medium: Prepared in the same way as MRS liquid medium, with the addition of 15 g of agar powder;

[0029] Tyramine detection solid culture medium: The preparation method is the same as that of MRS solid culture medium, and 7.5 g of L-tyrosine is added.

[0030] 3. Isolation and purification of strains:

[0031] After thoroughly shaking the collected fermentation broth, pipette 0.1 mL of the broth into a test tube containing 0.9 mL of sterile physiological saline and allow it to ferment for 10 minutes. -1 -10 -6 Serial dilution, take 0.1 mL of 10 mL of each solution. -4 10 -5 10 -6 Diluents of different dilution gradients were spread on tyramine detection solid medium and incubated at 37°C for 4 days. If a clear zone appeared, the strain could be preliminarily identified as a tyramine-producing strain. Single colonies of varying morphology and size with large clear zone diameters were then selected and purified three times using the streak plate method. The purified strains were activated three times using MRS liquid medium fortified with 0.1% L-tyrosine to enhance tyrosine decarboxylase activity. The activated strains were then cryopreserved in glycerol tubes at -80°C for later use.

[0032] 4. Verification of tyramine production capacity:

[0033] 4.1 Sample Pretreatment: After thoroughly shaking the activated bacterial suspension (3 times), pipette 0.6 mL of the suspension into 30 mL of MRS liquid medium. Add 10 g / L L-tyrosine as substrate. Incubate at 37℃ and 200 r / min for 72 h. Take 2 mL of the bacterial suspension, add 2 mL of 50 mg / mL trichloroacetic acid solution, shake for a few seconds, and centrifuge at 12000 r / min for 10 min at 4℃. Collect the supernatant. Use sterile MRS liquid medium containing 10 g / L substrate as a control.

[0034] 4.2 Preparation of standard solutions: Weigh 10 mg of tyramine hydrochloride standard, dissolve it in 0.1 mol / L HCl, and make up to 100 mL to prepare a 100 μg / mL tyramine standard stock solution. Then dilute with 0.1 mol / L HCl to prepare a series of standard solutions with final concentrations of 1.0, 2.5, 5, 10, 15, 25, 50, and 100 μg / mL. Store at 4℃ protected from light.

[0035] 4.3 Derivatization of Standards and Samples: The derivatization process was based on the method of Li Ranran et al., with slight modifications. 1 mL of standard solution and sample pretreatment supernatant were placed in separate 10 mL centrifuge tubes. 200 μL of 2 mol / L NaOH solution and 300 μL of saturated NaHCO3 solution were added to make the solution alkaline. After vortexing for 10 s, 1 mL of 10 mg / mL dansyl chloride was added. Layering occurred, indicating that the derivatizing agent was of good quality and usable. The mixture was vortexed for 30 s and then placed in a 40℃ water bath for derivatization in the dark for 45 min. After derivatization, 100 μL of NH3·H2O was added to the mixture, and after mixing, the reaction was terminated in a 25℃ water bath. After 30 min, the mixture was removed and brought to a final volume of 5 mL with acetonitrile. The mixture was then centrifuged at 4℃ (3000 r / min, 10 min). After centrifugation, the supernatant was aspirated using a 2 mL syringe and centrifuged over a 0.45 mL aspirate solution. After filtration through a μm organic filter membrane, the sample is injected into a 1.5 ml vial for testing.

[0036] 4.4 High Performance Liquid Chromatography (HPLC) Conditions: The chromatographic column was a C18 column (4.6 mm × 150 mm, 5 μm). Mobile phase A was water, and mobile phase B was acetonitrile. Isocratic elution was used, and the elution program is shown in Table 1. The flow rate was 1 mL / min, the UV detection wavelength was 254 nm, and the injection volume was 20 μL.

[0037]

[0038] 4.5 Tyramine Detection Results: The tyramine production capacity of the strain (Lactobacillus brevis DF-19) was identified using HPLC. Figure 1 As shown, tyramine exhibits a good chromatographic peak shape with no overlapping peaks, and an elution time of 5.112 min. Tyramine standard samples were determined by high-performance liquid chromatography (HPLC). Figure 2 As shown, within the range of 0-100.0 mg / L, the regression equation for tyramine is y=35.576x-20.95, with a correlation coefficient r=0.9993, indicating a good linear relationship (r>0.999), which shows that the method for determining tyramine content has good reliability.

[0039] Representative tyramine-producing bacteria are shown in Table 2:

[0040]

[0041] a. Strain number explanation: The number represents the strain number;

[0042] The b value represents the mean ± SD (n = 3); different lowercase letters after the number (a, b, c...) indicate that there are significant differences in the tyramine content produced by the strains that produce tyramine in the fermentation samples (LSD, P < 0.05).

[0043] Among them, strain DF-19 had the highest and most stable yield.

[0044] 5. Gram staining experiment of strain DF-19:

[0045] The isolated and purified strain DF-19 was stained according to the Gram staining procedure, including primary staining, mordanting, destaining, and counterstaining. Cell morphology was observed under an oil immersion microscope (4×100). Figure 3 As shown, colony morphology (milky white, round) and Gram staining test (G) were observed. + The rod-shaped bacterium DF-19 was found to be consistent with the characteristics of lactic acid bacteria.

[0046] 6. Molecular biological assays of strain DF-19:

[0047] The isolated and purified strain DF-19 was identified by molecular biology using 16S rDNA analysis. The strain was sequenced by Hangzhou Kaitai Biotechnology Co., Ltd. The test sample sequence was submitted to NCBI, and similarity analysis was performed using the BLAST program on the 16S rDNA gene sequences of known bacterial species.

[0048] Primer information is shown in Table 3:

[0049]

[0050] The sequencing results are shown in SEQ ID NO.3-4, and the assembled result is shown in SEQ ID NO.5. The sequencing results were input into NCBI, and the BLAST function was used to compare the homology of the sequenced gene with the 16S rRNA sequences in the NCBI database to construct a phylogenetic tree, as shown below. Figure 4 As shown, it can be seen that the strain is related to the genus *Lactobacillus brevis* (…). Lactobacillus Based on their close genetic distance, it can be preliminarily determined that they belong to the genus *Lactobacillus brevis*. Lactobacillus ).

[0051] Example 2: Performance characteristics of strain DF-19:

[0052] 1. Growth curve of strain DF-19:

[0053] The isolated and purified strain DF-19 was inoculated into MRS liquid medium at a 1% inoculum and cultured on a shaker at 37℃ and 150 rpm for 48 h. Samples were taken at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, and 48 h to determine the OD of the culture medium. 600nm Values, with incubation time as the x-axis, and bacterial OD values. 600nm Plot a curve with the vertical axis as the ordinate, such as... Figure 5 As shown in the figure, the strain enters the logarithmic growth phase after 5 hours of cultivation and enters the stable growth phase after 30 hours.

[0054] 2. Acid production capacity of strain DF-19:

[0055] The isolated and purified strain DF-19 was inoculated at a rate of 1% into 30 mL of MRS liquid medium and cultured at 37°C for 24 h. The pH value of the culture was measured. After 24 h of culture, the pH value of the isolated strain was found to be between 4.10 and 4.38.

[0056] 3. Determination of salt tolerance of strain DF-19:

[0057] NaCl tolerance is one of the important characteristics of bacterial strains. The tolerance value reflects the osmotic pressure the strain can tolerate. The isolated and purified strain DF-19 was inoculated at a 1% inoculum into MRS liquid medium supplemented with 0%, 2%, 4%, 6%, 8%, and 10% NaCl, and cultured at 37℃ for 24 h. A blank MRS liquid medium was used as a control. The OD value of the culture solution was measured at 600 nm to compare the growth of the strain under different salt concentrations. The results are as follows: Figure 6 As shown, it can be seen that as the NaCl concentration in the culture medium increases, the number of single colonies gradually decreases, compared to the inoculum size at 0 h (1.425 × 10⁻⁶). 7 Compared to the previous comparison, after culturing in 5% NaCl liquid medium for 24 hours, the number of bacteria (1.15 × 10⁻⁶) was significantly lower. 7） The decrease instead reflects that 5% NaCl significantly inhibits the growth of the strain.

[0058] 4. Mucus production experiment:

[0059] The isolated and purified strain DF-19 was inoculated at a rate of 1% on MRS solid medium (with 5% sucrose instead of glucose) and cultured in an incubator at 37°C for 24 h. Colonies were picked up with an inoculation loop to observe whether there was any stringing. The strain did not exhibit stringing, thus indicating that the isolated strain does not produce mucus.

[0060] 5. Nitrite tolerance test:

[0061] The isolated and purified strain DF-19 was inoculated at a rate of 1% into MRS liquid medium supplemented with 0 and 150 mg / L NaNO2, and cultured at 37℃ for 24 h. A blank MRS liquid medium was used as a control. The OD value was measured at 600 nm to compare the tolerance of the strains to nitrite. The results are shown in Table 4.

[0062]

[0063] When cultured under 150 mg / L sodium nitrite, the strain's OD... 600nm A value greater than 0.1 indicates that the strain has good tolerance to nitrite.

[0064] 6. Acid resistance test:

[0065] The isolated and purified strain DF-19 was inoculated at a 2% inoculum into MRS liquid medium at pH 3, 4, 5, 6, and 7, and cultured at 37°C for 24 h. The number of single colonies was measured to verify its acid tolerance. The results are as follows: Figure 7 As shown, the number of single colonies of DF-19 first increases and then decreases as the acidity decreases, indicating that the optimal pH value of the strain is between 5 and 7, suggesting that the strain grows better in a slightly acidic environment.

[0066] 7. Effects of acid stress on tyramine production by the strain:

[0067] The pH of the solid culture medium was adjusted using lactic acid, citric acid, acetic acid, and NaOH to detect acid stress and tyramine production. The pH values ​​were 5.0, 5.5, 6.0, and 7.0 (control group). The bacterial strains were then cultured in these mediums, with three replicates per group. Samples were taken every 12 hours for 48 consecutive hours to determine the tyramine content in each fermentation broth. The effect of pH on tyramine production with increasing culture time was evaluated, and the changes in tyramine content were plotted. The results are shown below. Figures 8 to 10 As shown;

[0068] Under lactic acid stress, the strain rapidly produced a large amount of tyramine within 24–48 h at pH 5.0, pH 5.5.0, and pH 6.0. The strain showed the strongest tyramine production capacity at pH 5.5, reaching 1203.824 ± 27.73 ug / ml.

[0069] Under acetic acid stress, the strain rapidly produced a large amount of tyramine within 24–48 h at pH 5.0, pH 5.5.0, and pH 6.0. The strain showed the strongest tyramine production capacity at pH 5.5, reaching 1059.568 ± 6.56 ug / ml.

[0070] Under citric acid stress, the strain rapidly produced a large amount of tyramine within 24–48 h at pH 5.0, pH 5.5.0, and pH 6.0. The strain showed the strongest tyramine production capacity at pH 5.5, reaching 1102.482 ± 28.42 ug / ml.

[0071] In summary, acidic conditions favor tyramine production by strain DF-19, and the highest tyramine production occurs under lactic acid stress. This suggests that low pH conditions may activate the stress response mechanism of tyramine-producing bacteria, promoting tyramine synthesis.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A high-tyramine-producing Lactobacillus brevis DF-19, characterized in that, It was deposited on May 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33896.

2. The application of the high-tyramine-producing Lactobacillus DF-19 as described in claim 1 in the synthesis of tyramine.

3. The application according to claim 2, characterized in that, Includes the following steps: After shaking the activated, high-tyramine-producing Lactobacillus brevis suspension DF-19, add it to the culture medium, then add L-tyridine as a substrate, culture on a shaker, take the bacterial suspension, add trichloroacetic acid solution, shake, centrifuge, and take the supernatant.

4. The application according to claim 3, characterized in that, The conditions for the shaker culture were 37°C and 200 r / min.

5. The application according to claim 3, characterized in that, The centrifugation operation was carried out at 4°C and at a speed of 12000 r / min.

6. A formulation for synthesizing tyramine, characterized in that, The formulation comprises Lactobacillus brevis DF-19, which is a high-tyramine producer as described in claim 1.

7. The use of the high-tyramine-producing Lactobacillus brevis DF-19 as described in claim 1 in the preparation of tyramine-rich products.

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