Lactobacillus plantarum capable of efficiently metabolizing azo pigment as well as preparation method and application of lactobacillus plantarum

Abstract

The invention discloses a lactobacillus plantarum mutant strain capable of tolerating and efficiently metabolizing azo pigment, the strain is named as lactobacillus plantarum E4103 in taxonomy, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.31189. The strain is subjected to adaptive domestication in a continuous culture mode of continuously increasing the concentration of azopigment allura red, and is obtained through natural selection. The obtained strain can normally grow under the condition of the allura red with the concentration of 0.52 g / L, compared with an original strain, the decolorization rate of the allura red is increased by 10.66%, theoretical basis and guidance can be provided for follow-up development of azo dye decolorization strains, and application and popularization of the strain in the industrial field are promoted.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a plant lactobacillus capable of efficiently metabolizing azo pigments, and a preparation method and application thereof. Background Art

[0002] With the growth of population and the development of industry, more than 100,000 commercial dyes are produced every year in the world, with an output of more than 700,000 tons. More than 80% of the dyes are complex organic compounds containing azo bonds and aromatic rings, which are mainly used in printing and dyeing, textiles, plastics, papermaking, food, cosmetics and pharmaceutical industries, generating a large amount of colored wastewater. Dyes can still make the color of water darker even at very low concentrations. Many synthetic dyes are difficult to degrade due to their complex structures. In aquatic ecosystems, improper treatment of textile dye wastewater reduces light penetration, which in turn affects photosynthesis, dissolved oxygen concentration, water quality, and the survival of aquatic plants and animals, leading to serious ecological problems. In addition, many artificially synthesized azo dyes are toxic, carcinogenic, mutagenic, and have potential health hazards to humans.

[0003] The treatment method of traditional dyes mainly includes physical and chemical methods such as oxidation, flocculation, hydrogen peroxide and ultraviolet irradiation. But due to its high cost, the production scale is limited, and even the removal effect of some dyes is not obvious. Many bacteria, fungi and algae have the ability to adsorb or degrade azo dyes, and water-soluble azo dyes can be converted into stable and nontoxic final products by biological methods, so biological methods are considered to be the treatment methods with strong specificity, low resource consumption, high efficiency and environmental protection at present. Plant lactobacillus is a kind of lactic acid bacteria, which is widely distributed in nature, and because it has good environmental adaptability, stability and safety, it is also highly valuable in important fields closely related to human life such as food and medicine. However, artificially synthesized azo pigments are not traditional carbon sources and nitrogen sources of plant lactobacillus, and the metabolic efficiency of bacterial strains to such compounds is generally lower, and such compounds also have certain toxicity to bacterial strains, causing bacterial strains to be unable to grow and reproduce normally, and being difficult to realize industrial application. Therefore, the bacterial strains that develop and efficiently utilize azo pigments are problems to be solved in this area. Summary of the invention

[0004] The invention aims to provide a plant lactobacillus capable of efficiently metabolizing azo pigments and a preparation method and application thereof.

[0005] The invention relates to a mutant strain of Lactobacillus plantarum, which is obtained by taking wild-type Lactobacillus plantarum as a starting bacterium and performing adaptive domestication on the starting strain in a manner of increasing the concentration of azo pigment (allura red) in a culture medium in a continuous culture stepwise manner.

[0006] The first aspect of the present invention provides a plant lactobacillus that efficiently metabolizes azo pigments, the taxonomic name of which is plant lactobacillus E4103, and the deposit number is CGMCC No. 31189 at the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0007] The second aspect of the present invention provides the use of the above-mentioned Lactobacillus plantarum in decolorizing food or the environment;

[0008] The third aspect of the present invention provides a method for preparing the above-mentioned Lactobacillus plantarum, comprising the following steps:

[0009] (1) Preparation of preliminary liquid culture medium: 9-11 g / L peptone, 9-11 g / L beef extract, 4-6 g / L yeast powder, 19-21 g / L glucose, 0.9-1.1 g / L Tween-80, 1-3 g / L dipotassium hydrogen phosphate, 4-6 g / L sodium acetate, 1-3 g / L sodium citrate, 0.15-0.25 g / L magnesium sulfate heptahydrate, 0.05-0.06 g / L anhydrous manganese sulfate, adjust the pH value to 6-7, sterilize at 120-134°C for 10-20 min, and cool for use;

[0010] (2) Activating the bacterial colony: streak the starting strain stored in a glycerol tube at -90 to -70°C onto an MRS agar medium, culture it anaerobically at 30 to 38°C for 46 to 50 hours, obtain a single colony, and then transfer it into the liquid culture medium described in (1), and culture it anaerobically at 30 to 38°C for 16 to 24 hours;

[0011] (3) First stage of domestication: Control the OD of the strain activated in step (2) above 600 The medium in step (1) is inoculated with 0.75-0.85, the initial concentration of the azo pigment allura red is 0.05-0.07 g / L, the concentration is increased by 0.02 g / L each generation until the allura red concentration reaches 0.5-0.6 g / L, and anaerobically cultured at 30-38° C. for 24-48 hours;

[0012] (4) Second stage acclimation: The bacteria acclimated in the first stage in step (3) are inoculated with an OD600 of 0.75 to 0.85 into the medium with half the glucose content in step (1), and step (3) is repeated to obtain the result;

[0013] Further, in step (1), the preparation of the liquid culture medium includes: taking 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 20 g / L glucose, 1 g / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L sodium citrate, 0.2 g / L magnesium sulfate heptahydrate, 0.054 g / L anhydrous manganese sulfate, adjusting the pH value to 6-7, sterilizing at 121° C. for 15 min, and cooling for standby use;

[0014] Furthermore, in step (2), the storage temperature is -80°C;

[0015] Furthermore, in step (2), the anaerobic culture is carried out at 37° C. for 48 h;

[0016] Further, in step (3), the OD 600 is 0.8;

[0017] Furthermore, in step (3), the initial allure red concentration of the azo pigment is 0.06 g / L, and the concentration is increased by 0.02 g / L each generation until the concentration of the azo pigment allure red reaches 0.52 g / L, and the mixture is anaerobically acclimated at 37° C. for 24-48 h;

[0018] Further, in step (3), the OD 600 is 0.8;

[0019] Beneficial Effects

[0020] The present invention is domesticated in stages, in which the azo pigment decolorizing bacteria are first activated in the first stage to improve the vitality of the microorganisms, so that the microorganisms gradually adapt to the high concentration of allure red environment; and then domesticated in the second stage, by reducing the glucose content in the culture medium to reduce the carbon source, the microorganisms are promoted to utilize the carbon source provided by the azo pigment allure red, thereby improving the adaptability of the microorganisms to allure red and the decolorization efficiency. The mutant strain can be used as a decolorizing strain in food or the environment, which is conducive to reducing the cost of sewage decolorization and promoting its wide application in the industrial field. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the decolorization rate of the starting strain and mutant strain in culture medium with different glucose content

[0022] Figure 2 is the decolorization rate of the starting strain in culture medium with different allura red concentrations DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with specific embodiments and accompanying drawings, but it should be understood that the examples given are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used can be obtained through commercial channels.

[0024] Example 1

[0025] (1) Preparation of preliminary liquid culture medium: 9 g / L peptone, 9 g / L beef extract, 4 g / L yeast powder, 19 g / L glucose, 0.9 g / L Tween-80, 1 g / L potassium dihydrogen phosphate, 4 g / L sodium acetate, 1 g / L sodium citrate, 0.15 g / L magnesium sulfate heptahydrate, 0.05 g / L anhydrous manganese sulfate, adjust the pH value to 6-7, sterilize at 120°C for 10 min, and cool for use;

[0026] (2) The starting strain stored in a glycerol tube at -85°C was streaked onto MRS agar medium and cultured anaerobically at 30°C for 46 h to obtain single colonies; 10 single colonies were picked from the plate and inoculated into MRS medium at pH 6-7 and cultured at 30°C for 16 h;

[0027] (3) First stage of acclimatization: The starting point of acclimatization is when the decolorization rate of the starting bacteria reaches more than 85% within 24 hours (0.05 g / L). The initial OD 600 The strain was cultured with an inoculum size of 0.75. The strain OD was observed every 6 hours. 600 When the strain enters the stationary phase, subculture to a medium with an increased concentration of 0.02 g / L azo pigment allura red. If the strain hysteresis period increases significantly during this period, reduce the concentration of 0.02 g / L allura red and repeat the subculture process.

[0028] Repeat the above steps until the strain reaches the biological limit, that is, the growth rate and hysteresis period of the strain cannot be restored after repeated cultivation in a certain concentration of Allura Red medium for more than 5 generations. The domesticated strain is cultivated in the maximum domestication concentration Allura Red (0.5 g / L) medium for 5 generations to obtain the first stage domesticated strain.

[0029] (4) Second stage acclimatization: By comparing the decolorization rate of the strain in culture medium with the same allura red concentration but different glucose content, it was found that the decolorization rate of the strain was faster in the culture medium with half the glucose content. Therefore, step (3) was repeated in the MRS culture medium with half the glucose content.

[0030] Example 2

[0031] (1) Preparation of preliminary liquid culture medium: 11 g / L peptone, 11 g / L beef extract, 6 g / L yeast powder, 21 g / L glucose, 1.1 g / L Tween-80, 3 g / L dipotassium hydrogen phosphate, 6 g / L sodium acetate, 3 g / L sodium citrate, 0.25 g / L magnesium sulfate heptahydrate, 0.06 g / L anhydrous manganese sulfate, adjust the pH value to 6-7, sterilize at 134°C for 20 min, and cool for use;

[0032] (2) The starting strain stored in a -70°C glycerol tube was streaked onto MRS agar medium and cultured anaerobically at 38°C for 50 h to obtain a single colony.

[0033] Ten single colonies were picked from the plate and inoculated into MRS medium with a pH of 6-7 and cultured at 38°C for 48 h.

[0034] (3) First stage of acclimatization: The starting point of acclimatization is when the decolorization rate of the starting bacteria reaches more than 85% within 24 hours (0.07 g / L). The initial OD 600 The strain was cultured with an inoculum size of 0.85. The strain OD was observed every 6 hours. 600 When the strain enters the stationary phase, subculture to a medium with an increased concentration of 0.02 g / L azo pigment allura red. If the strain hysteresis period increases significantly during this period, reduce the concentration of 0.02 g / L allura red and repeat the subculture process.

[0035] Repeat the above steps until the strain reaches the biological limit, that is, the growth rate and hysteresis period of the strain cannot be restored after repeated cultivation in a certain concentration of Allura Red medium for more than 5 generations. The domesticated strain is cultivated in the medium with the maximum domestication concentration of Allura Red (0.6 g / L) for 5 generations to obtain the first stage domesticated strain.

[0036] (4) Second stage acclimatization: By comparing the decolorization rates of the strain in culture media with the same allura red concentration but different glucose contents, it was found that the decolorization rate of the strain was faster in the culture medium with half the glucose content, so the above step (3) was repeated in the MRS culture medium with half the glucose content.

[0037] Embodiment 3:

[0038] (1) Preparation of preliminary liquid culture medium: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 20 g / L glucose, 1 g / L Tween-80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L sodium citrate, 0.2 g / L magnesium sulfate heptahydrate, 0.054 g / L anhydrous manganese sulfate, adjust the pH value to 6-7, sterilize at 121°C for 15 min, and cool for use;

[0039] (2) The starting strain stored in a -80°C glycerol tube was streaked onto MRS agar medium and cultured anaerobically at 37°C for 48 h to obtain a single colony.

[0040] Ten single colonies were picked from the plate and inoculated into MRS medium with a pH of 6-7 and cultured at 37°C for 48 h.

[0041] (3) First stage of acclimatization: The starting point of acclimatization is when the decolorization rate of the starting bacteria reaches more than 85% within 24 hours (0.06 g / L). The initial OD 600 The strain was cultured with an inoculum size of 0.8. The strain OD was observed every 6 hours. 600 When the strain enters the stationary phase, subculture to a medium with an increased concentration of 0.02 g / L azo pigment allura red. If the strain hysteresis period increases significantly during this period, reduce the concentration of 0.02 g / L allura red and repeat the subculture process.

[0042] Repeat the above steps until the strain reaches the biological limit, that is, the growth rate and hysteresis period of the strain cannot be restored after repeated cultivation in a certain concentration of Allura Red medium for more than 5 generations. The domesticated strain was cultivated in the medium with the maximum domestication concentration of Allura Red (0.52 g / L) for 5 generations to obtain the first stage domesticated strain.

[0043] (4) Second stage acclimatization: By comparing the decolorization rates of the strain in culture media with the same allura red concentration but different glucose contents, it was found that the decolorization rate of the strain was faster in the culture medium with half the glucose content, so the above step (3) was repeated in the MRS culture medium with half the glucose content.

[0044] After testing, the decolorization rate of low concentration (0.06-0.1 g / L) Allura Red in this example reached more than 90% within 48 hours after acclimation; in a high concentration (0.3 g / L or more) Allura Red environment, the growth condition of the mutant bacteria was significantly better than that of the original bacteria.

[0045] Embodiment 4:

[0046] The only difference between Example 4 and Example 1 is that during the first stage of acclimation, the glucose content of the culture medium is 20 g / L; during the second stage of acclimation, the glucose content of the culture medium is 10 g / L.

[0047] After testing, the decolorization rate of low concentration (0.06-0.1 g / L) azo pigment allura red in this example reached more than 90% within 30 hours after acclimation.

[0048] Embodiment 3:

[0049] Take 250 μl of culture medium every 6 hours, centrifuge at 10000 rpm for 10 minutes, and take the supernatant. Use a spectrophotometer to measure the absorbance value at the characteristic absorption wavelength (504 nm) of allura red. The decolorization rate is calculated using the following formula:

[0050] Decolorization rate (%) = ((A0-A1) / A0) × 100%

[0051] In the formula, A0 represents the absorbance value of the culture medium containing allura red at 0 h, and A1 represents the absorbance value of the culture medium containing allura red after a period of culture.

[0052] like Figure 1 As shown, when the concentration of allure red is 0.12 g / L and the glucose content of the culture medium is 20 g / L, the decolorization rate of the starting strain is 84%, and the decolorization rate of the mutant strain is 86%, which is 2% higher than that of the starting strain; when the glucose content of the culture medium is 10 g / L, the decolorization rate of the starting strain is 87%, and the decolorization rate of the mutant strain is 93%, which is 5% higher than that of the starting strain. Therefore, the domestication method of Example 3 is the best embodiment of the present invention.

[0053] In summary, the present invention provides a method for acclimating and cultivating plant lactobacillus that efficiently metabolizes azo pigments, wherein the acclimation medium formula is simple and the cost is low. The strain obtained by the method can significantly improve the decolorization rate of the dye and improve the degradation efficiency of the dye.

Claims

1. A plant lactobacillus that efficiently metabolizes azo pigments, characterized in that: The taxonomic name of the Lactobacillus plantarum is Lactobacillus plantarum E4103, and its preservation number is CGMCC No. 31189 in the General Microbiological Center of China Microorganism Culture Collection Administration.

2. An application of the plant lactobacillus described in claim 1 in decolorizing food or the environment.

3. A method for preparing Lactobacillus plantarum according to claim 1, characterized in that: The following steps are involved: (1) Preparation of preliminary liquid culture medium: 9-11 g / L peptone, 9-11 g / L beef extract, 4-6 g / L yeast powder, 19-21 g / L glucose, 0.9-1.1 g / L Tween-80, 1-3 g / L dipotassium hydrogen phosphate, 4-6 g / L sodium acetate, 1-3 g / L sodium citrate, 0.15-0.25 g / L magnesium sulfate heptahydrate, 0.05-0.06 g / L anhydrous manganese sulfate, adjust the pH value to 6-7, sterilize at 120-134°C for 10-20 min, and cool for use; (2) Activating the bacterial colony: streak the starting strain stored in a glycerol tube at -90 to -70°C onto an MRS agar medium, culture it anaerobically at 30 to 38°C for 46 to 50 hours, obtain a single colony, and then transfer it into the liquid culture medium described in (1), and culture it anaerobically at 30 to 38°C for 16 to 24 hours; (3) First stage of domestication: Control the OD of the strain activated in step (2) above 600 The medium in step (1) is inoculated with 0.75-0.85, the initial concentration of the azo pigment allura red is 0.05-0.07 g / L, the concentration is increased by 0.02 g / L each generation until the allura red concentration reaches 0.5-0.6 g / L, and anaerobically cultured at 30-38° C. for 24-48 hours; (4) Second stage of domestication: Control the OD of the strain that has undergone the first stage of domestication in step (3) above 600 The medium with the glucose content reduced by half in step (1) is inoculated with 0.75-0.85, and the culture step (3) is repeated to obtain the product.

4. The method for preparing plant lactobacillus according to claim 3, wherein: Step (1), the preparation of the liquid culture medium comprises: taking 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 20 g / L glucose, 1 g / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L sodium citrate, 0.2 g / L magnesium sulfate heptahydrate, 0.054 g / L anhydrous manganese sulfate, adjusting the pH value to 6-7, sterilizing at 121° C. for 15 min, and cooling for standby use.

5. The method for preparing plant lactobacillus according to claim 3, wherein: In step (2), the storage temperature is -80°C.

6. The method for preparing plant lactobacillus according to claim 3, wherein: In step (2), the anaerobic culture is carried out at 37° C. for 48 hours.

7. The method for preparing plant lactobacillus according to claim 3, wherein: In step (3), the OD 600 is 0.

8.

8. The method for preparing plant lactobacillus according to claim 3, characterized in that: In step (3), the initial allure red concentration of the azo pigment is 0.06 g / L, and the concentration is increased by 0.02 g / L each generation until the concentration of the azo pigment allure red reaches 0.52 g / L, and the mixture is anaerobically acclimated at 37° C. for 24-48 hours.

9. The method for preparing plant lactobacillus according to claim 3, characterized in that: In step (3), the OD 600 is 0.8.

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