New application of lactobacillus plantarum and method for activating lactobacillus plantarum to produce 6-tp
By fermenting and culturing Lactobacillus plantarum or activating it with the addition of GTP to convert it into 6-PT, the problem of BH4 deficiency in patients with hyperphenylalaninemia has been solved, enabling continuous or rapid production of 6-PT. This has led to the development of drugs and probiotic products for hyperphenylalaninemia, reducing the frequency of medication and economic burden for patients.
Patent Information
- Application Number
- CN202511053427.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-30
- Publication Date
- 2025-11-25
- Estimated Expiration
- 2045-07-30
Smart Images
Figure CN120549989B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of microorganisms, and particularly relates to a new application of lactobacillus plantarum and a method for activating lactobacillus plantarum to produce 6-TP. BACKGROUND
[0002] Patients with hyperphenylalaninemia lack phenylalanine hydroxylase or its coenzyme tetrahydrobiopterin (BH4) in the body, resulting in that phenylalanine cannot be normally converted into tyrosine, and thus is accumulated in the blood. The synthesis of BH4 in the body is as follows: GTP→7,8-Dihydroneopterin 3'-triphosphate→6-Pyruvoyltetrahydropterin (6-Propionyltetrahydropterin)→Tetrahydrobiopterin (BH4); and defects of any enzyme gene in the synthesis path can cause BH4 deficiency.
[0003] Sapropterin is a kind of BH4 analog artificially synthesized and used as a drug for treating hyperphenylalaninemia, which can enter cells, act as a coenzyme of phenylalanine hydroxylase, enhance the activity of phenylalanine hydroxylase, and enable phenylalanine to be normally converted into tyrosine, so as to reduce the level of phenylalanine in the blood. However, the half-life of Sapropterin in the body is about 3-4 hours. Therefore, patients need to take the drug for a long time, which brings inconvenience to the patients and heavy economic burden. SUMMARY
[0004] In view of the above technical problems, the application provides a new application of lactobacillus plantarum and a method for activating lactobacillus plantarum to produce 6-TP; the lactobacillus plantarum can directly produce the precursor 6-Pyruvoyltetrahydropterin (6-Propionyltetrahydropterin) of BH4, and can make up for the defects of related genes in the synthesis path of BH4.
[0005] In order to achieve the above technical purposes, the application provides the following technical solutions.
[0006] The new application of lactobacillus plantarum is the application of the lactobacillus plantarum in preparing a drug for treating hyperphenylalaninemia.
[0007] Further, the lactobacillus plantarum adopts an ATCC standard strain, and the strain number is ATCC14917.
[0008] Further, the precursor 6-Propionyltetrahydropterin of coenzyme tetrahydrobiopterin is obtained by fermentation culture of the lactobacillus plantarum, and the produced 6-Propionyltetrahydropterin is used for preparing a drug for treating hyperphenylalaninemia.
[0009] Further, the Lactobacillus plantarum is used to prepare a probiotic product for supplementing 6-ketopantothenyl tetrahydropterin in vivo.
[0010] A method for activating Lactobacillus plantarum to produce 6-TP, wherein GTP is added in the culture process of Lactobacillus plantarum, so as to activate the Lactobacillus plantarum to convert GTP into 6-ketopantothenyl tetrahydropterin.
[0011] Further, the method specifically comprises:
[0012] (1) Lactobacillus plantarum working strain preparation: Lactobacillus plantarum glycerol strain is cultured by using MRS liquid culture medium, so as to obtain Lactobacillus plantarum working strain;
[0013] (2) Bacterial culture: the Lactobacillus plantarum working strain is inoculated into MRS culture medium, and is cultured at 37°C in a constant temperature incubator for a certain time, so as to obtain Lactobacillus plantarum first generation bacterial liquid;
[0014] (3) GTP and bacterial co-culture: the Lactobacillus plantarum first generation bacterial liquid and GTP aqueous solution are added into the MRS culture medium; and the mixture is cultured in a constant temperature incubator for a certain time, and then the bacterial liquid is centrifuged, so as to obtain supernatant containing 6-ketopantothenyl tetrahydropterin.
[0015] Further, in step (3), the GTP aqueous solution is added into the MRS culture medium, so that the final concentration of GTP is 0.1-10 mg / ml.
[0016] Further, in step (3), the culture time in the constant temperature incubator is 24-48 hours.
[0017] The present application has the following beneficial effects:
[0018] The present application provides a new application of Lactobacillus plantarum, which includes fermenting and culturing the Lactobacillus plantarum to obtain the precursor 6-ketopantothenyl tetrahydropterin of coenzyme tetrahydrobiopterin, and using the produced 6-ketopantothenyl tetrahydropterin to develop and prepare a Hyperphenylalaninemia (high phenylalanine blood disease) rare disease drug; or using the Lactobacillus plantarum to develop a probiotic product, which stably expresses 6-ketopantothenyl tetrahydropterin in human body, so as to supplement the required tetrahydrobiopterin (BH4) in human body. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 The figure is the concentration change curve of 6-ketopantothenyl tetrahydropterin in the culture process of Lactobacillus plantarum and Streptococcus thermophilus in the embodiment of the present application.
[0020] Figure 2 The figure is the concentration change curve of 6-ketopantothenyl tetrahydropterin in the growth process of Lactobacillus plantarum in the embodiment of the present application. Detailed Implementation
[0021] The technical solution of the present invention will be further described below with reference to specific embodiments and accompanying drawings.
[0022] This invention is the first to discover that *Lactobacillus plantarum* possesses the ability to synthesize 6-Pyruvoyltetrahydropterin, which can be directly converted to BH4 by metopterin reductase in vivo. If a patient's BH4 deficiency is caused by a metopterin reductase gene defect, 6-Pyruvoyltetrahydropterin can also be converted to BH4 via the Sepiapterin pathway. Therefore, this novel function of *Lactobacillus plantarum* demonstrates that the bacterium can compensate for defects in related genes along the BH4 synthesis pathway, solving the BH4 deficiency problem and enabling the preparation of drugs for the rare disease hyperphenylalaninemia.
[0023] Example 1: Comparative experiment on the ability of GTP to activate the production of 6-pyruvyltetrahydropterin (i.e., 6-TP) by Lactobacillus plantarum and Streptococcus thermophilus.
[0024] In the human body, GTP can be synthesized into BH4 step by step using different enzymes. The synthetic pathway is GTP → 7,8-Dihydroneopterin 3'-triphosphate → 6-Pyruvoyltetrahydropterin (6-pyruvyltetrahydropterin) → Tetrahydrobiopterin (BH4). Many bacteria, like *Lactobacillus plantarum*, possess enzymes that synthesize GTP into 6-pyruvyltetrahydropterin, but not all bacteria with these enzyme systems can successfully convert GTP to 6-pyruvyltetrahydropterin. Therefore, it is not possible to simply screen for bacteria that produce 6-pyruvyltetrahydropterin through genomic analysis.
[0025] In this invention, two strains of bacteria (Lactobacillus plantarum and Streptococcus thermophilus) containing synthases ranging from GTP to 6-pyruvyltetrahydropterin were cultured. GTP was added during the culture process, and samples were taken at different time points. The changes in the concentration of 6-pyruvyltetrahydropterin during the culture process were detected using an ELISA kit to determine the ability of the two strains to produce 6-pyruvyltetrahydropterin.
[0026] The results show that Lactobacillus plantarum can convert GTP into 6-ketopantothenyl tetrahydropteridine after adding GTP, and the 6-ketopantothenyl tetrahydropteridine in the culture supernatant of Streptococcus thermophilus does not change basically after adding GTP. It is indicated that in the presence of GTP, the same bacteria containing GTP to 6-ketopantothenyl tetrahydropteridine synthesis enzyme do not necessarily have the ability to convert GTP into 6-ketopantothenyl tetrahydropteridine. The following is the strain information and test process and results:
[0027] Strain introduction: Lactobacillus plantarum belongs to Bacilli class, Lactobacillales order, Lactobacillaceae family, Lactobacillus genus, subsp species; it is a common gram-positive probiotic bacteria, widely exists in nature, especially in the oral cavity and digestive organs of humans and other mammals, and is considered as a safe probiotic bacteria with various health benefits; aerobic state, 37 DEG C, cultured in MRS medium.
[0028] Streptococcus thermophilus belongs to Bacilli class, Lactobacillales order, Lactobacillaceae family, Streptococcus genus, S. thermophilus species. It is a necessary material for making yogurt. Studies have shown that this bacterium can only reach the upper half of the small intestine, and scientists believe that live yogurt bacteria (Streptococcus thermophilus) can help people with lactose intolerance digest lactose because the bacteria can produce lactase. Aerobic state, 37 DEG C, cultured in MRS medium.
[0029] In the present application, the Lactobacillus plantarum is purchased from Beijing Bao Bo Wei Biotechnology Co., Ltd.; platform number: bio-53030; strain name: Latin-Lactobacillus plantarum, Chinese-plant Lactobacillus; the strain is numbered ATCC14917 in the American Type Culture Collection (ATCC).
[0030] In the present application, the Streptococcus thermophilus is purchased from Ningbo Taisituo Biotechnology Co., Ltd.; strain number: TS325125; strain name: Latin-Streptococcus thermophilus, Chinese-thermophilic Streptococcus; the strain is numbered ATCC19987 in the American Type Culture Collection (ATCC).
[0031] Experimental process and data statistics:
[0032] Preparation of Lactobacillus plantarum working strain and Streptococcus thermophilus working strain: The purchased Lactobacillus plantarum glycerol strain and Streptococcus thermophilus glycerol strain were stored in a -80°C ultra-low temperature refrigerator, and each glycerol strain was taken out and thawed in a 2-8°C normal temperature refrigerator.
[0033] MRS (manufacturer: Ningbo Mingzhou Biological Technology Co., Ltd., batch number: 240719) liquid medium 110 mL was prepared, and 50 mL and 5 mL were divided into four test tubes of different sizes; 40% glycerol (manufacturer: Wuyi Yanwei Medical Technology Co., Ltd., batch number: 240103) aqueous solution 120 mL was prepared and sterilized at 121°C for 30 min.
[0034] In the biological safety cabinet, 0.2 mL of Lactobacillus plantarum bacterial solution and 0.2 mL of Streptococcus thermophilus bacterial solution were taken with a pipette and inoculated into 5 mL of MRS medium, and incubated at 37°C in a constant temperature incubator for 24-48 h, which was the first generation of bacterial solution.
[0035] In the biological safety cabinet, 5 mL of Lactobacillus plantarum first-generation bacterial solution and 5 mL of Streptococcus thermophilus first-generation bacterial solution were taken with a pipette and inoculated into 50 mL of MRS medium, and incubated at 37°C in a constant temperature incubator for 12-24 h. The culture completed bacterial solution was stained and examined under a microscope, and was identified as no growth of mixed bacteria, which was the second generation of bacterial solution.
[0036] In the biological safety cabinet, the culture completed second-generation bacterial solution was mixed with 40% glycerol aqueous solution at a ratio of 1:1, and was collected in a cryopreservation tube, which was Lactobacillus plantarum working strain and Streptococcus thermophilus working strain, and was stored in a -80°C ultra-low temperature refrigerator.
[0037] Comparison test of GTP-activated Lactobacillus plantarum and Streptococcus thermophilus for 6-ketopantoate tetrahydropterin production:
[0038] Bacterial culture: One Lactobacillus plantarum working strain and one Streptococcus thermophilus working strain were taken out and thawed in a 2-8°C normal temperature refrigerator.
[0039] MRS liquid medium 58 mL was prepared, and 24 mL and 5 mL were divided into four test tubes of different sizes and sterilized at 121°C for 30 min. 25 mg / mL GTP (guanosine-5-triphosphate sodium salt, manufacturer: Shanghai Maikelin Biochemical Technology Co., Ltd., batch number: C16633082) aqueous solution 10 mL,
[0040] In the biological safety cabinet, 0.2 um sterilization filtration was performed.
[0041] In the biological safety cabinet, 0.6 mL of Lactobacillus plantarum working strain and 0.6 mL of Streptococcus thermophilus working strain were each taken with a pipette and inoculated into 5 mL of MRS medium separately, and then incubated at 37°C in a constant temperature incubator for 12-24 h. The harvested bacterial liquid was the first generation bacterial liquid of Lactobacillus plantarum and Streptococcus thermophilus.
[0042] GTP co-culture with bacteria: In the biological safety cabinet, 2 mL of Lactobacillus plantarum and Streptococcus thermophilus first generation bacterial liquid was each taken with a pipette and added to 22 mL of MRS medium, and then 1 mL of 25 mg / mL GTP (guanosine-5-triphosphate sodium salt) aqueous solution was added to the medium to make the final concentration of GTP (guanosine-5-triphosphate sodium salt) aqueous solution 1 mg / mL. The mixture was incubated at 37°C in a constant temperature incubator for 48 h. Before inoculation and after adding GTP (guanosine-5-triphosphate sodium salt) aqueous solution, 2 mL of bacterial liquid sample was taken, and after adding GTP (guanosine-5-triphosphate sodium salt) aqueous solution, 2 mL of bacterial liquid sample was taken at 0 h, 8 h, 24 h, 32 h, and 48 h, respectively.
[0043] After sampling was completed, 1 mL of bacterial liquid was first determined by A600 spectrophotometer for bacterial liquid concentration, and 1 mL of bacterial liquid was centrifuged at 4000 r / min for 10 min, and the supernatant was collected and stored for later use.
[0044] Detection of 6-pyruvoyl tetrahydropterin concentration in each sample by ELISA method:
[0045] ELISA kit (manufacturer: Jiangsu Jingmei Biological Technology Co., Ltd., batch number: 202406)
[0046] Sample addition: standard wells, blank wells (blank control wells do not add sample and enzyme-labeled reagent, and the rest of the steps are the same), and sample wells were set. 50 uL of standard was added to the standard wells on the enzyme-labeled coating plate, and 50 uL of sample diluent was added to the sample wells (the final dilution of the sample was 10 7 times). The sample was added to the bottom of the enzyme-labeled plate well, and the well wall was not touched as much as possible. Shake gently to mix.
[0047] Enzyme addition: 50 uL of enzyme-labeled reagent was added to each well, except for the blank wells.
[0048] Incubation: after the plate was sealed with a sealing film, it was incubated at 37°C for 60 minutes.
[0049] Liquid preparation: 30 times concentrated washing solution was diluted 30 times with distilled water for standby use.
[0050] Washing: the sealing film was carefully removed, and the liquid was discarded. Each well was filled with washing solution, and after standing for 30 seconds, the washing solution was discarded. This was repeated 5 times, and then the plate was dried.
[0051] Color development: Add color reagent A 50 uL to each well, then add color reagent B 50 uL, mix gently, color development at 37°C for 15 minutes in the dark.
[0052] Termination: Add termination solution 50 uL to each well, terminate the reaction (at this time the blue color turns yellow).
[0053] Measurement: Zero with the empty well, measure the absorbance (OD value) of each well in sequence at 450 nm. The measurement should be performed within 15 minutes after adding the termination solution.
[0054] The results of ELISA detection of the 6-pyruvoyl tetrahydropterin concentration of each sample are shown in Table 1 and Figure 1
[0055] Table 1: ELISA detection results of 6-pyruvoyl tetrahydropterin concentration during the cultivation of Streptococcus thermophilus and Lactobacillus plantarum:
[0056]
[0057] From the results of ELISA detection, it can be seen that after adding GTP, the content of 6-PT of Lactobacillus plantarum increased by 15 times, while that of Streptococcus thermophilus remained basically unchanged. This indicates that after adding GTP to Lactobacillus plantarum, the ability to activate the conversion of GTP to 6-PT is activated, and although Streptococcus thermophilus also has corresponding enzymes, it cannot produce 6-PT.
[0058] Example 2: Analysis of the ability of Lactobacillus plantarum to produce 6-pyruvoyl tetrahydropterin without supplementing GTP. The results of this example prove that Lactobacillus plantarum can continuously produce 6-PT without supplementing GTP after entering the stable growth phase. Although the ability of Lactobacillus plantarum to produce 6-PT without supplementing GTP is much lower than that under the condition of supplementing GTP (supplementing GTP, 23 hours of 6-PT production reached 1660.58 ng / L, without supplementing, 24 hours of 6-PT production was 416.1 ng / L, which was 1 / 4 of the condition of supplementing GTP), but it can continuously produce 6-PT, especially after entering the stable growth phase, which simulates the colonization of Lactobacillus plantarum in the intestinal tract, which shows that Lactobacillus plantarum has the potential to develop probiotic products to continuously provide 6-PT for the human body.
[0059] The specific experimental process of this example is as follows:
[0060] Bacterial culture: Take one Lactobacillus plantarum working strain and thaw it in a 2-8°C normal temperature refrigerator.
[0061] Prepare 30 mL of MRS liquid medium, divide 25 mL and 5 mL into two test tubes of different sizes, and sterilize at 121°C for 30 minutes.
[0062] In the biological safety cabinet, 0.6 mL of Lactobacillus plantarum working strain was taken with a pipette and inoculated into 5 mL of MRS medium, and incubated at 37°C in a constant temperature incubator for 12-24 h, which was the first generation of bacterial liquid.
[0063] The second generation of culture: in the biological safety cabinet, 2 mL of the first generation of bacterial liquid was inoculated into 23 mL of MRS medium with a pipette, and incubated at 37°C in a constant temperature incubator for 48 h. The bacterial liquid was sampled 2 mL at 0 h, 8 h, 24 h, 32 h and 48 h of culture, respectively.
[0064] After sampling, 1 mL of bacterial liquid was first determined by A600 spectrophotometer for bacterial liquid concentration, and 1 mL of bacterial liquid was centrifuged at 4000 r / min for 10 min, and the supernatant was frozen for standby.
[0065] ELISA method was used to detect the concentration of 6-keto-pyridine tetrahydropterin in each sample: ELISA kit (manufacturer: Jiangsu Jingmei Biological Technology Co., Ltd., batch number: 202503)
[0066] Sample addition: standard wells, blank wells (blank control wells without sample and enzyme-labeled reagent, and the rest of the operation is the same), and sample wells were set. 50 uL was added to the standard wells on the enzyme-labeled coating plate, and 40 uL of sample diluent was added to the sample wells, followed by 10 uL of sample to be tested (the final dilution of the sample was 5 times). The sample was added to the bottom of the enzyme-labeled plate well, and the well wall was not touched as much as possible, and it was gently shaken to mix.
[0067] Enzyme addition: 50 uL of enzyme-labeled reagent was added to each well, except for the blank wells.
[0068] Incubation: after sealing the plate with a sealing film, it was placed at 37°C for 60 minutes.
[0069] Liquid preparation: 30 times concentrated washing solution was diluted with distilled water 30 times for standby.
[0070] Washing: the sealing film was carefully removed, and the liquid was discarded. After shaking dry, the washing liquid was added to each well, and after standing for 30 seconds, it was discarded. This was repeated 5 times, and then it was tapped dry.
[0071] Color development: 50 uL of color developing agent A was added to each well, followed by 50 uL of color developing agent B, which was gently shaken to mix. Color development was carried out at 37°C for 15 minutes in the dark.
[0072] Termination: 50 uL of termination solution was added to each well to terminate the reaction (at this time, the blue color turned yellow).
[0073] Determination: the zero was adjusted with the blank well, and the absorbance (OD value) of each well was measured in sequence at 450 nm wavelength. The determination should be carried out within 15 minutes after the addition of the termination solution.
[0074] The ELISA detection results are shown in Table 2 andFigure 2
[0075] Table 2: Changes in 6-pyruvoyl tetrahydropterin (6-PT) concentration during the culture process
[0076]
[0077] From the growth curve, it can be seen that before the Lactobacillus plantarum entered the stationary phase, the concentration of 6-PT did not change significantly. When it reached the stationary phase (11 hours of culture), the concentration of 6-PT increased from 220.5 ng / L to 416.1 ng / L, an increase of 1.9 times.
[0078] Summary of 6-PT production by Lactobacillus plantarum at different growth stages: From the test results, it can be seen that Lactobacillus plantarum does not express 6-PT during the rapid propagation period, but starts to express 6-PT continuously after entering the stationary phase. The colonization of microorganisms in the intestinal tract has a state from growth to stability, which simulates the state of Lactobacillus plantarum after stable colonization in the intestinal tract, so this ability of Lactobacillus plantarum is very beneficial for the development of related probiotic products. The ability of Lactobacillus plantarum to continuously express 6-PT after entering the stationary growth phase can be used to develop related probiotic products.
[0079] Example 1 shows that the addition of GTP during the culture of Lactobacillus plantarum can quickly convert GTP to 6-pyruvoyl tetrahydropterin. When GTP is added, the detection value of 6-PT in the culture system is 115.64 ng / L, and after the culture is completed, the concentration of 6-PT is 1757.96 ng / L, which is 15.2 times the initial detection value, indicating that the addition of GTP greatly activates the ability of Lactobacillus plantarum to produce 6-PT. This technical route can be used for high-density fermentation of Lactobacillus plantarum to cultivate and harvest 6-PT. 6-PT is a precursor of BH4 and can be converted to BH4 by human enzymes in the body. Currently, Sepiapterin, as a precursor of BH4, has been identified by FDA as a rare disease drug for the development of high phenylalanine blood disease drugs in the United States. Similarly, as a precursor of BH4, 6-PT also has the potential to be an innovative drug for high phenylalanine blood disease. Therefore, Lactobacillus plantarum has the ability to prepare the innovative drug 6-PT for high phenylalanine blood disease through fermentation.
[0080] Example 2 shows that Lactobacillus plantarum can also produce 6-PT continuously without supplement of GTP. As shown in Table 2 of Example 2, the concentration of 6-PT increased from the initial detection value of 220.5 ng / L at the time of sampling at 0 hours of culture to 416.1 ng / L at the end of 24 hours of culture, which increased by nearly 1.9 times in 24 hours of culture. Therefore, Lactobacillus plantarum also has the ability to be developed into probiotics or live microbial ecological preparations, meeting the needs of continuous supplementation of 6-PT in vivo.
[0081] Example 1 and Example 2 show that Lactobacillus plantarum can not only produce 6-PT by supplementing GTP, but also continuously secrete 6-PT after in vivo colonization, thus having the potential to develop drugs for treating hyperphenylalaninemia and the potential to develop probiotics to supplement 6-PT.
[0082] Example 3: A new application of Lactobacillus plantarum, which is the application of the Lactobacillus plantarum in the preparation of a drug for treating hyperphenylalaninemia. In this embodiment, the Lactobacillus plantarum uses the ATCC standard strain, and the strain number is ATCC14917.
[0083] The application includes two application schemes:
[0084] Scheme 1: The Lactobacillus plantarum is used for fermentation culture to obtain the precursor 6-PT of coenzyme BH4, and the produced 6-PT is used for preparing a drug for treating hyperphenylalaninemia.
[0085] Specifically, Lactobacillus plantarum is fermented and cultured, GTP is supplemented during the culture process, the precursor 6-PT of BH4 is produced, and after purification and freeze-drying, the 6-PT is mixed with a filler, a diluent, a disintegrating agent, etc. in a certain proportion, and then granulated, dried, and filled into capsules, and finally enterically coated to prepare enteric-coated capsules containing 6-PT, which are used as innovative drugs for treating hyperphenylalaninemia. After supplementing GTP, Lactobacillus plantarum can quickly and massively produce 6-PT, and the present application provides a technical route of a drug that can reduce the production cost of the drug.
[0086] Scheme 2: The Lactobacillus plantarum is used for preparing probiotics for supplementing 6-PT in vivo.
[0087] The Lactobacillus fermentum is cultured, after the culture is finished, the Lactobacillus fermentum is collected through centrifugation, the Lactobacillus fermentum freeze-dried bacteria powder after freeze-drying is mixed with fillers, diluents, disintegrants and the like in proportion, and then the mixture is prepared into live bacteria enteric-coated capsules containing the Lactobacillus fermentum through processes such as granulation, drying, whole-grain filling and capsule filling and enteric-coated coating, and the live bacteria enteric-coated capsules are used as probiotics or innovative drugs for the treatment of hyperphenylalaninemia. Since the Lactobacillus fermentum can continuously produce 6-ketopiperidyl tetrahydropteridine without the need for additional GTP, the Lactobacillus fermentum can continuously supplement the precursor 6-ketopiperidyl tetrahydropteridine of BH4 after the Lactobacillus fermentum colonizes in the human intestine, and thus the Lactobacillus fermentum can be used once to achieve the effect of long-term continuous medication.
[0088] Embodiment 4: A method for activating Lactobacillus fermentum to produce 6-TP, wherein GTP is added during the culture of the Lactobacillus fermentum to activate the Lactobacillus fermentum to convert the GTP into 6-ketopiperidyl tetrahydropteridine.
[0089] In this embodiment, the method specifically comprises:
[0090] (1) Preparation of Lactobacillus fermentum working strain: MRS liquid medium is used to culture the Lactobacillus fermentum glycerol strain with the strain number ATCC14917 purchased to obtain a Lactobacillus fermentum working strain. Specifically, the purchased Lactobacillus fermentum glycerol strain is stored in a -80°C ultra-low temperature refrigerator, and one glycerol strain is taken out and thawed in a 2-8°C normal temperature refrigerator.
[0091] Prepare 110 mL of MRS (manufacturer: Ningbo Mingzhou Biological Technology Co., Ltd., batch number: 240719) liquid medium, and divide it into 50 mL and 5 mL in four test tubes of different sizes; prepare 120 mL of 40% glycerol (manufacturer: Wuyi Yanwei Medical Technology Co., Ltd., batch number: 240103) aqueous solution, and sterilize it at 121°C for 30 min.
[0092] In a biological safety cabinet, use a pipette to take 0.2 mL of Lactobacillus fermentum bacterial solution and inoculate it into 5 mL of MRS medium, and then place it in a constant temperature incubator at 37°C for static culture for 24-48 h to obtain a first-generation bacterial solution.
[0093] In a biological safety cabinet, use a pipette to take 5 mL of Lactobacillus fermentum first-generation bacterial solution and inoculate it into 50 mL of MRS medium, and then place it in a constant temperature incubator at 37°C for static culture for 12-24 h. The culture is completed, and the bacterial solution is dyed and examined under a microscope to identify that there is no growth of mixed bacteria, and then the second-generation bacterial solution is obtained.
[0094] In a biological safety cabinet, mix the culture completed second-generation bacterial solution with the 40% glycerol aqueous solution at a ratio of 1:1, and collect it in a cryopreservation tube to obtain the Lactobacillus fermentum working strain, which is stored in a -80°C ultra-low temperature refrigerator.
[0095] (2) Bacterial culture: inoculate the Lactobacillus plantarum working strain into MRS medium, cultivate in a constant temperature incubator at 37°C for a certain period of time, and obtain Lactobacillus plantarum first generation bacterial solution;
[0096] (3) Co-culture of GTP and bacteria: add the Lactobacillus plantarum first generation bacterial solution and GTP aqueous solution to the MRS medium, and incubate in a constant temperature incubator for a certain period of time. Centrifuge the bacterial solution to obtain the supernatant containing 6-ketopantothenate. Specifically, add GTP aqueous solution to the MRS medium to a final GTP concentration of 0.1-10 mg / L (preferably 1 mg / ml); incubate in a constant temperature incubator at 37°C for 24-48 hours.
[0097] In this embodiment, the method further comprises step (4) purification, specifically comprising:
[0098] (4.1) Supernatant preparation: remove the precipitate in the fermentation broth through centrifugation and filtration to obtain a clear fermentation broth supernatant; dilute the supernatant with a suitable buffer to adjust the pH value and ionic strength, which is beneficial to subsequent chromatographic separation.
[0099] (4.2) Preliminary purification: select one or more chromatographic techniques (such as affinity chromatography, ion exchange chromatography or gel filtration chromatography) for preliminary purification; wherein the affinity chromatography can use antibodies or ligands that specifically bind to 6-ketopantothenate as the stationary phase.
[0100] (4.3) Secondary purification: collect the eluate containing 6-ketopantothenate after preliminary purification, and concentrate and dialyze to remove small molecular impurities and salts. If necessary, further purification can be performed again using chromatographic techniques to improve the purity of 6-ketopantothenate.
[0101] Purity detection and verification: use high performance liquid chromatography (HPLC), mass spectrometry (MS) or enzyme-linked immunosorbent assay (ELISA) and other methods to detect and verify the purity of the purified 6-ketopantothenate. At the same time, biological activity determination can also be carried out to ensure that the purified 6-ketopantothenate still has the expected biological function.
[0102] Storage and stability study: store the purified 6-ketopantothenate under appropriate conditions such as low temperature, light protection and moisture protection to ensure its stability and long-term storage. Perform stability studies to evaluate the stability and shelf life of the purified 6-ketopantothenate under different conditions.
[0103] Through the above steps, high-purity 6-ketopantothenate can be effectively purified from the supernatant containing 6-ketopantothenate, providing strong support for subsequent biological research and application.
[0104] Finally, it should be noted that the above only describes the preferred embodiments of the present application, and is not intended to limit the present application. For those skilled in the art, the aforementioned technical solutions can still be modified or deformed and improved, and these all belong to the protection scope of the present application.
Claims
1. A novel application of Lactobacillus plantarum, characterized in that, The new application is the use of *Lactobacillus plantarum* in the preparation of a drug for treating hyperphenylalaninemia; the precursor of coenzyme tetrahydropterin, 6-pyruvyltetrahydropterin, is obtained by fermentation culture of *Lactobacillus plantarum*, and the generated 6-pyruvyltetrahydropterin is used to prepare a drug for treating hyperphenylalaninemia. The *Lactobacillus plantarum* strain used was the ATCC standard strain, with strain number ATCC14917.
2. The novel application of *Lactobacillus plantarum* according to claim 1, characterized in that, A probiotic product for supplementing the body with 6-pyruvyltetrahydropterin was prepared using the aforementioned Lactobacillus plantarum.
3. A method for activating *Lactobacillus plantarum* to produce 6-pyruvyltetrahydropterin, characterized in that, GTP was added during the culture of *Lactobacillus plantarum* to activate the *Lactobacillus plantarum* to convert GTP into 6-pyruvyltetrahydropterin. The generated 6-pyruvyltetrahydropterin was used to prepare a drug for treating hyperphenylalaninemia. The *Lactobacillus plantarum* used was an ATCC standard strain with the strain number ATCC14917.
4. The method for activating *Lactobacillus plantarum* to produce 6-pyruvyltetrahydropterin according to claim 3, characterized in that, The method specifically includes: (1) Preparation of working strain of Lactobacillus plantarum: The glycerol strain of Lactobacillus plantarum was cultured in MRS liquid medium to obtain the working strain of Lactobacillus plantarum; (2) Bacterial culture: The working strain of Lactobacillus plantarum was inoculated into MRS medium and cultured at 37°C in a constant temperature incubator for a certain period of time to obtain the first generation of Lactobacillus plantarum bacterial culture. (3) Co-culture of GTP and bacteria: Add the first generation of Lactobacillus plantarum bacterial culture and GTP aqueous solution to MRS medium; incubate in a constant temperature incubator for a certain period of time, take the bacterial culture and centrifuge to obtain the supernatant containing 6-pyruvyltetrahydropterin.
5. The method for activating *Lactobacillus plantarum* to produce 6-pyruvyltetrahydropterin according to claim 3, characterized in that, In step (3), GTP aqueous solution is added to MRS medium until the final GTP concentration is 0.1~10 mg / ml.
6. The method for activating *Lactobacillus plantarum* to produce 6-pyruvyltetrahydropterin according to claim 3, characterized in that, In step (3), the static incubation time in the constant temperature incubator is 24~48 hours.
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New polypeptide-human 6-acetone acyl tetrahydrogen (bio) pterin synthase (PTPS) 11.88 and polynucleotide for encoding such polypeptide
CN1364883A
Microorganisms for the production of melatonin
US20150024440A1