Preparation method and application of kluyveromyces marxianus crude extract metabolite
By fermenting Max Kluviere and preparing crude metabolites, the problem of difficult to effectively control foodborne pathogenic bacteria in the prior art is solved, and the broad-spectrum antibacterial effect on a variety of pathogenic bacteria is achieved.
Patent Information
- Application Number
- CN202410621758.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-20
- Publication Date
- 2025-03-11
AI Technical Summary
The prior art is difficult to effectively control foodborne pathogenic bacteria, such as E. coli, Salmonella, etc., resulting in food safety problems.
By fermenting Maxkluvia yeast, a crude extract metabolite of Maxkluvia was prepared, and a gel-like crude extract was obtained by extraction, rotary evaporation, nitrogen blowing and other steps. It was further separated and purified by a normal phase silica gel chromatography column to obtain an antibacterial activity-2-(1H-indole-3-yl)-ethyl acetic acid.
This method can effectively inhibit a variety of foodborne pathogenic bacteria, show a broad-spectrum antibacterial effect, and has a wide application prospect in the control of pathogenic bacteria in the food industry.
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Figure CN119592641A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms and relates to a preparation method and application of a crude metabolite of Kluyveromyces marxianus. Background Art
[0002] In the prior art, Gram-negative bacteria such as Escherichia coli, Salmonella, Vibrio fluvialis, Enterobacter sakazakii, and Vibrio parahaemolyticus are all common foodborne pathogens. When the pathogens reach a certain number, they can seriously endanger human health; Gram-positive bacteria such as Staphylococcus aureus, Listeria, and Bacillus cereus are also foodborne pathogens. Once the pathogens invade the body, they will cause diseases and damage human health; in recent years, microbial agents have been considered to be environmentally friendly, and the effective bacteria therein can metabolize to produce a variety of active enzymes or antibiotic substances, which can inhibit the growth and reproduction of pathogenic fungi or bacteria. Therefore, screening and separating potential pathogens to control microorganisms and their metabolites are of great significance to the development of the food industry. Summary of the invention
[0003] In view of the above problems, the present invention aims to provide a method for preparing crude metabolites of Kluyveromyces marxianus and its application.
[0004] The technical solution of the present invention is: a method for preparing a crude metabolite of Kluyveromyces marxianus according to the present invention, wherein the preparation steps are as follows:
[0005] Step (1): inoculating the activated Kluyveromyces marxianus into NYDB medium for fermentation, thereby preparing a fermentation broth;
[0006] Step (2): centrifuging the obtained fermentation liquid in a centrifuge to precipitate, and then discarding the precipitate to obtain the supernatant;
[0007] Step (3): extract the obtained supernatant with an equal volume of ethyl acetate, combine the extracts, rotary evaporate to dryness, re-dissolve with methanol, and blow nitrogen to obtain a gel-like crude extract, i.e., crude metabolites of Kluyveromyces marxianus.
[0008] Furthermore, in step (1), the specific preparation steps of the fermentation broth are as follows: 100 μL of Kluyveromyces marxianus is activated in 5 mL of NYDB test tube culture medium, and the culture is placed at 28° C. and 180 rpm for 20 hours;
[0009] Take 100 μL of activated Kluyveromyces marxianus in 5 mL of NYDB test tube culture medium, place it at 28°C and 180 rpm and shake it for 20 hours to use as seed liquid;
[0010] The prepared seed liquid was transferred into NYDB medium at a volume ratio of 2% for fermentation, and the culture was placed at 28° C. and 180 rpm for 2 days to obtain a fermentation liquid.
[0011] Furthermore, the preparation method of the NYDB culture medium is as follows: taking 1 L as an example, 8 g of beef extract, 5 g of yeast powder, 10 g of glucose, and 1000 L of distilled water are required for mixing and preparing.
[0012] Furthermore, in step (2), the rotation speed of the centrifuge is 8000 rpm and the centrifugation time is 10 min.
[0013] Furthermore, in step (3), the specific preparation steps of the gel-like crude extract are as follows: the supernatant is extracted with an equal volume of ethyl acetate, the aqueous phase is removed, the organic phase is extracted to dryness using a rotary evaporator at 40°C and 120rpm, and then dissolved with 10mL of ethyl acetate, and nitrogen blown at 35°C to obtain a gel-like crude extract, i.e., a crude metabolite of Kluyveromyces marxianus, which is then placed in a -20°C refrigerator for use.
[0014] Furthermore, in step (3), the prepared Kluyveromyces marxianus crude metabolites are separated, purified and eluted to finally prepare 2-(1H-indol-3-yl)-ethyl acetate; specifically:
[0015] The obtained gelatinous crude extract was separated and purified by normal phase silica gel chromatography, and petroleum ether was used to load the column. After loading, it was eluted with a gradient mixture of petroleum ether and ethyl acetate, and the elution gradient was 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively, to obtain six components Fr.1 to Fr.6;
[0016] Component Fr.5 of the six components was separated and purified using a chromatographic column to obtain three components, Fr.5A to Fr.5C;
[0017] The components Fr.5B and Fr.5C in the combined fractions were separated and purified on a chromatographic column to obtain two components, Fr.5B1 and Fr.5B2;
[0018] Fr.5B2 is 2-(1H-indol-3-yl)-ethyl acetate obtained from a gelatinous crude extract;
[0019] The structural formula of the acetic acid-2-(1H-indol-3-yl)-ethyl ester is shown in formula (1):
[0020]
[0021] Furthermore, the conditions for separating and purifying the gelatinous crude extract by normal phase silica gel chromatography are: the filler is 200-300 mesh silica gel powder, and an equal mass of 100-200 mesh silica gel powder and the gelatinous crude extract are mixed evenly to serve as the separation sample.
[0022] Further, in the separation and purification of component Fr.5 among the six components using a chromatographic column, the chromatographic column is a semi-preparative high performance liquid chromatography C18 column, 250 mm×10 mm, 10 μm, the mobile phase volume ratio is MeOH:H2O=45:55, the flow rate is 3 mL / min, and the detection wavelength is 254 nm;
[0023] In the separation and purification of components Fr.5B and Fr.5C in the combined components, the chromatographic column is an analytical HPLC C18 column, 4.6mm×10mm, 5μm, the mobile phase is a gradient elution of 10% to 90% of methanol in water, the flow rate is 1mL / min, and the detection wavelength is 254nm.
[0024] Furthermore, the crude metabolites of Kluyveromyces marxianus prepared by the preparation method are used in controlling pathogenic bacteria.
[0025] Furthermore, the pathogens described in the application include Escherichia coli, Salmonella, Vibrio fluvialis, Enterobacter sakazakii, Vibrio parahaemolyticus, Staphylococcus aureus, Listeria and Bacillus cereus.
[0026] The method for inhibiting pathogenic bacteria by crudely extracting metabolites of Kluyveromyces marxianus is specifically as follows:
[0027] Taking 2L of Kluyveromyces marxianus crude metabolites as an example, 300 μL of methanol was dissolved in the Kluyveromyces marxianus crude metabolites (XZD); 15 mL of LB solid culture medium was added to a culture dish with a sterile Oxford cup in the center to solidify; 5 mL of LB semi-solid culture medium was drawn into a sterilized 10 mL centrifuge tube, and 60 μL of activated pathogenic bacteria solution was added to the above centrifuge tube;
[0028] After shaking the above centrifuge tube evenly in a vortex instrument, pour it into a solidified culture dish with a sterile Oxford cup. After solidification, take out the Oxford cup; aspirate 60 μL of crude metabolites of Kluyveromyces marxianus into the middle well of the culture medium as the treatment group, and add 60 μL of methanol into the middle well of the culture medium as the control group; culture all the above samples at 37°C for 24 hours, and measure the diameter of the inhibition zone; each treatment was repeated 3 times, and the experiment was repeated 2 times.
[0029] The beneficial effects of the present invention are as follows: the present invention obtains crude metabolites of Kluyveromyces marxianus by fermenting Kluyveromyces marxianus, and the new potential antibacterial active substances mined by the present invention are shown to have antibacterial activity in inhibiting pathogenic bacteria. The crude metabolites of Kluyveromyces marxianus obtained have antibacterial activity and have broad application prospects in the development of antibacterial agents. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 It is a preparation flow chart of the present invention;
[0031] Figure 2 It is a bar graph showing that the crude metabolites of Kluyveromyces marxianus inhibit 8 pathogenic bacteria in the examples of the present invention. DETAILED DESCRIPTION
[0032] The specific technical scheme of the present invention is further described in detail below with reference to specific examples.
[0033] Kluyveromyces marxianus XZ1, which was deposited in China Center for Type Culture Collection on January 5, 2022, with the deposit address at Wuhan University, China, and the deposit number is CCTCC NO: M2022014;
[0034] As shown in the figure, (a) the fermentation method of Kluyveromyces marxianus is as follows: the fermentation process of the strain mainly includes three steps: activation of the strain, preparation of the seed liquid of the strain to be fermented, and large-scale fermentation;
[0035] 1. Take out 100 μL of Kluyveromyces marxianus from the preservation tube and activate it in 5 mL of NYDB test tube culture medium, and shake and culture it at 28°C and 180 rpm for 20 hours;
[0036] 2. Take 100 μL of the activated strain (Kluyveromyces marxianus) and put it into 5 mL of new test tube culture medium, place it at 28°C and 180 rpm for 20 hours, and use it as seed liquid;
[0037] 3. Transfer the prepared seed solution into NYDB medium at 2% for large-scale fermentation, place in a shaker at 28°C and ferment at 180 rpm for 48 hours;
[0038] (II) The method for extracting crude metabolites of Kluyveromyces marxianus is as follows:
[0039] 4. Centrifuge the fermentation broth at 8000 rpm / min for 10 min, discard the precipitate, and keep the supernatant;
[0040] 5. Filter the supernatant twice through a 0.22 mm filter membrane to obtain a sterile supernatant; extract the sterile supernatant with ethyl acetate (v / v=1:1), remove the aqueous phase, and extract the organic phase to dryness using a rotary evaporator at 40°C, 120 rpm, then dissolve it with 10 mL of methanol, transfer the extract to a 10 mL vial, and place the fermentation metabolites (crude metabolites of Kluyveromyces marxianus) obtained after nitrogen blowing at 35°C in a -20°C refrigerator for use.
[0041] (III) The method for separating and purifying crude metabolites of Kluyveromyces marxianus by normal phase silica gel chromatography is as follows:
[0042] After the crude metabolites of Kluyveromyces marxianus are completely dissolved in ethyl acetate, 100-200 mesh silica gel powder equivalent to the mass of the sample is weighed and mixed with the crude extract evenly, and the silica gel powder is evaporated to dryness by a rotary evaporator for use as the sample to be separated; petroleum ether is loaded into the column by the wet column packing method, and the lower piston is opened to allow the eluent to flow out slowly; then 200-300 mesh silica gel is slowly and continuously poured into the column until the silica gel is added and settled in the column without changing; the sample to be separated is slowly poured onto the silica gel surface in the chromatography column, and the upper surface of the silica gel is always kept flat, and finally a layer of quartz sand is placed; gradient elution is performed with petroleum ether / ethyl acetate (PE / EtOAc), and the elution gradient is 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and a total of six components from Fr.1 to Fr.6 are obtained, and 5 column volumes are collected for each gradient;
[0043] (IV) The method for separating and purifying crude metabolites of Kluyveromyces marxianus by reverse phase silica gel chromatography is as follows:
[0044] The component Fr.5 was dissolved in chromatographic grade methanol, filtered through a 0.22 mm filter membrane, and then separated and purified by semi-preparative high performance liquid chromatography C18 column to obtain three components Fr.5A to Fr.5C; the chromatographic column was Mercury C18, 250 mm × 10 mm, 10 μm, the mobile phase was MeOH: H2O = 45:55, the flow rate was 3 mL / min, and the detection wavelength was 254 nm;
[0045] The components Fr.5B and Fr.5C were combined and dissolved in chromatographic grade methanol, filtered through a 0.22 mm filter membrane, and then separated and purified by a high performance liquid chromatography C18 column to obtain two components, Fr.5B1 and Fr.5B2, wherein Fr.5B2 is 2-(1H-indol-3-yl)-ethyl acetate; the chromatographic column was Agilent C18, 4.6 mm × 10 mm, 5 μm, the mobile phase was methanol-water 10% to 90% gradient elution, the flow rate was 1 mL / min, and the detection wavelength was 254 nm;
[0046] (V) The method for inhibiting pathogenic bacteria by crude extraction of Kluyveromyces marxianus metabolites is as follows:
[0047] Taking 2L of crude metabolites of Kluyveromyces marxianus as an example, 300 μL of methanol (MeOH) is needed to be dissolved in crude metabolites of Kluyveromyces marxianus (XZD); 15 mL of LB solid culture medium is added to a culture dish with a sterile Oxford cup in the center to solidify; 5 mL of LB semi-solid culture medium is taken into a sterilized 10 mL centrifuge tube, and 60 μL of activated pathogenic bacteria liquid is added to the above centrifuge tube, including Escherichia coli (E c), Salmonella (S s), Vibrio fluvii (V f), Enterobacter sakazakii (E s), Vibrio parahaemolyticus (V p), Staphylococcus aureus (S a), Listeria monocytogenes (L m), and Bacillus cereus (B c);
[0048] After shaking the centrifuge tube evenly in a vortex instrument, pour it into the solidified culture dish with the sterile Oxford cup. After solidification, take out the Oxford cup; pipette 60 μL of crude metabolites of Kluyveromyces marxianus into the middle well of the culture medium as the treatment group, and add 60 μL of methanol into the middle well of the culture medium as the control group; culture all the above samples at 37°C for 24 hours, and measure the diameter of the inhibition zone; each treatment was repeated 3 times, and the experiment was repeated 2 times.
[0049] The results are as follows Figure 2 As shown in the figure, by measuring the diameter of the inhibition zone, the antibacterial effect on different pathogenic bacteria was compared in a statistical sense. It was found that the diameters of the inhibition zones of the crude metabolites of Kluyveromyces marxianus against Escherichia coli (E c), Salmonella (S s), Vibrio fluvialis (Vf), Enterobacter sakazakii (E s), Vibrio parahaemolyticus (V p), Staphylococcus aureus (S a), Listeria monocytogenes (L m), and Bacillus cereus (B c) were 2.5 cm, 2.1 cm, 2 cm, 2.1 cm, 2.3 cm, 1.7 cm, 2.9 cm, and 1.5 cm, respectively. It can be seen that the inhibitory effect on Listeria monocytogenes (L m) was the best, and the crude metabolites of Kluyveromyces marxianus showed a broad-spectrum antibacterial effect on pathogenic bacteria.
Claims
1. A method for preparing crude metabolites of Kluyveromyces marxianus, characterized in that: The preparation steps are as follows: Step (1): inoculating the activated Kluyveromyces marxianus into NYDB medium for fermentation, thereby preparing a fermentation broth; Step (2): centrifuging the obtained fermentation liquid in a centrifuge to precipitate, and then discarding the precipitate to obtain the supernatant; Step (3): extract the obtained supernatant with an equal volume of ethyl acetate, combine the extracts, rotary evaporate to dryness, re-dissolve with methanol, and blow nitrogen to obtain a gel-like crude extract, i.e., crude metabolites of Kluyveromyces marxianus.
2. The method for preparing a crude metabolite of Kluyveromyces marxianus according to claim 1, characterized in that: In step (1), the specific preparation steps of the fermentation broth are as follows: 100 μL of Kluyveromyces marxianus is activated in 5 mL of NYDB test tube culture medium; 100 μL of activated Kluyveromyces marxianus was cultured in 5 mL of new NYDB test tube culture medium and used as seed solution; The prepared seed solution was transferred into NYDB medium at a volume ratio of 2% for fermentation, thereby preparing a fermentation solution.
3. The method for preparing a crude metabolite of Kluyveromyces marxianus according to claim 2, characterized in that: The activation, culture and fermentation conditions are: shaking and fermenting at 28° C. and 180 rpm for 20 hours.
4. The method for preparing a crude metabolite of Kluyveromyces marxianus according to claim 2, characterized in that: The preparation method of the NYDB culture medium is as follows: taking 1 L as an example, 8 g of beef extract, 5 g of yeast powder, 10 g of glucose, and 1000 L of distilled water are mixed and prepared.
5. The method for preparing a crude metabolite of Kluyveromyces marxianus according to claim 1, characterized in that: In step (2), the rotation speed of the centrifuge is 8000 rpm and the centrifugation time is 10 min.
6. The method for preparing a crude metabolite of Kluyveromyces marxianus according to claim 1, characterized in that: In step (3), the specific preparation steps of the gel-like crude extract are as follows: the supernatant is extracted with an equal volume of ethyl acetate, the aqueous phase is removed, the organic phase is extracted to dryness using a rotary evaporator at 40°C and 120rpm, then dissolved with 10mL of ethyl acetate, and nitrogen blown at 35°C to obtain a gel-like crude extract, i.e., a crude metabolite of Kluyveromyces marxianus, which is then placed in a -20°C refrigerator for use.
7. The method for preparing a crude metabolite of Kluyveromyces marxianus according to claim 1, characterized in that: In step (3), the prepared Kluyveromyces marxianus crude metabolites are separated, purified and eluted to finally prepare 2-(1H-indol-3-yl)-ethyl acetate.
8. Use of the crude metabolite of Kluyveromyces marxianus prepared by the preparation method according to any one of claims 1 to 7 in controlling pathogenic bacteria.
9. The use according to claim 8, characterized in that: The pathogenic bacteria include Escherichia coli, Salmonella, Vibrio fluvii, Enterobacter sakazakii, Vibrio parahaemolyticus, Staphylococcus aureus, Listeria and Bacillus cereus.
10. The use according to claim 7, wherein the method for inhibiting pathogenic bacteria by extracting Kluyveromyces marxianus metabolites is as follows: Taking 2L of crude metabolites of Kluyveromyces marxianus as an example, 300 μL of methanol was dissolved in the crude metabolites of Kluyveromyces marxianus; 15 mL of LB solid culture medium was added to a culture dish with a sterile Oxford cup in the center to solidify; 5 mL of LB semi-solid culture medium was drawn into a sterilized 10 mL centrifuge tube, and 60 μL of activated pathogenic bacteria solution was added to the above centrifuge tube; After shaking the above centrifuge tube evenly in a vortex instrument, pour it into a solidified culture dish with a sterile Oxford cup. After solidification, take out the Oxford cup; aspirate 60 μL of crude metabolites of Kluyveromyces marxianus into the middle well of the culture medium as the treatment group, and add 60 μL of methanol into the middle well of the culture medium as the control group; culture all the above samples at 37°C for 24 hours, and measure the diameter of the inhibition zone; each treatment was repeated 3 times, and the experiment was repeated 2 times.