CN110241055B - Pseudomonas putida for degrading coumarin and application thereof - Google Patents

Pseudomonas putida for degrading coumarin and application thereof Download PDF

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CN110241055B
CN110241055B CN201910654904.9A CN201910654904A CN110241055B CN 110241055 B CN110241055 B CN 110241055B CN 201910654904 A CN201910654904 A CN 201910654904A CN 110241055 B CN110241055 B CN 110241055B
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coumarin
pseudomonas putida
degrading
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CN110241055A (en
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黄申
毛多斌
杨鹏飞
韩丽
杨靖
杨峰
马宁
周利峰
魏涛
闫美玲
卢慧
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Zhengzhou University of Light Industry
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C12R2001/40—Pseudomonas putida

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Abstract

The invention relates to the field of coumarin degrading bacteria, in particular to a coumarin degrading bacteria and application thereof. The classification of the coumarin degrading bacteria is named as pseudomonas putida (pseudomonas putida)Pseudomonas putidaHS-C2), which has been preserved in 2019, 5 and 13 days to China general microbiological culture Collection center (CGMCC), with the preservation number of CGMCC No. 17758. The pseudomonas putida can degrade coumarin and generate dihydrocoumarin, and the degradation rate of the coumarin is 99.94%.

Description

Pseudomonas putida for degrading coumarin and application thereof
Technical Field
The invention belongs to the field of bioengineering, relates to a pseudomonas putida for degrading coumarin and application thereof, and particularly relates to a pseudomonas putida, a culture medium thereof and application thereof for degrading coumarin and generating dihydrocoumarin.
Background
Coumarin was found in 1820 and is found in a wide range of higher plants, especially in the leguminosae, rutaceae, orchids, compositae, solanaceae, umbelliferae, and saxifragaceae families, and even in animals and in microbial metabolites. Coumarin can be regarded as lactone of o-hydroxy cinnamic acid, has aromatic odor, and is often classified into simple coumarin, pyranocoumarin, furocoumarin and other coumarins according to the difference of substituents and positions on rings.
Coumarin can be used as perfume and perfume fixative, and can be added into cosmetics. In order to prevent the occurrence of pinholes due to the unevenness of the metal plating layer, coumarin is often added to the metal plating solution. Most of natural coumarin compounds containing coumarin parent nucleus have agricultural activities of sterilization, weeding, deinsectization, plant growth regulation and the like, and osthole is widely applied as an agricultural pesticide with bactericidal and bacteriostatic activities. In addition, the coumarin has a special chemical structure, so that the coumarin is widely applied to medical treatment, and the coumarin active ingredients extracted from some plants can resist HIV and inhibit cancer cell proliferation, thereby bringing possibility for curing intractable diseases. It can be used together with vanillin as flavoring agent for candy and cake, or added into tobacco to improve natural fragrance of tobacco leaf, so as to make tobacco leaf fragrant, fine and dense, but since being classified as carcinogenic substance in the 50 s of 20 th century, it has been used in food and cosmetics industry for a short time. As coumarin is listed as a carcinogen, dihydrocoumarin belongs to a substitute of coumarin at present and is generally used as an additive for preparing spices such as coconut fragrance, cinnamon fragrance and the like.
The sweet and fragrant black tonka bean tincture is a tobacco additive, and is beneficial to increasing tobacco fragrance and covering up miscellaneous gas by being added into tobacco shreds, so that the fragrance type of the tobacco shreds is changed into strong fragrance type. Coumarin is used as a main component of the black tonka bean tincture, and the strong carcinogenic effect on human bodies greatly limits the development and the use of coumarin in the fields of food, cosmetics and cigarettes. Therefore, there is a need in the market to find a safe and effective method for degrading coumarin so that the coumarin does not lose all aspects of excellent biological activity and the carcinogenic effect is eliminated. The black tonka bean tincture can be used in the fields of food, tobacco and the like, so that the cost is reduced to the maximum extent and the development is promoted.
Disclosure of Invention
The invention provides pseudomonas putida, a culture medium thereof and application of the pseudomonas putida in degrading coumarin and generating dihydrocoumarin, aiming at solving the technical problem of coumarin degradation.
In order to solve the technical problem, the following technical scheme is adopted:
pseudomonas putida for degrading coumarin, and pseudomonas putida (A), (B) and (C)Pseudomonas putida) Has been preserved in 2019, 5, 13 th month to China general microbiological culture Collection center, the preservation address is: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing; the preservation number is CGMCC No. 17758.
The size of the pseudomonas putida thallus is 0.6-0.8 Mum multiplied by 1.5-2.0 Mum, is thin rod-shaped, has no flagellum, is milky round spot-shaped on a flat plate, and has bright surface, neat edge, non-transparency and raised center.
The culture method of the pseudomonas putida comprises the following steps: inoculating the pseudomonas putida bacterial liquid into a culture medium according to the inoculation amount of 2% for culture under the conditions of pH 5-9, temperature 30 ℃, rotation speed 120 rpm and culture for 12 hours.
The formula of the culture medium is as follows: k2HPO4 1 g/L，MgSO4•7H2O 0.5 g/L，FeSO4•7H2O 0.005 g/L，NaCl 0.5 g/L，KH2PO4 0.65 g/L，MnSO4 0.001 g/L，(NH4)2•SO4 0.5 g/L，CaCl2•2H2O 0.1g/L，Na2MoO4•2H2O0.005 g/L, 2 g/L coumarin.
The pseudomonas putida is used for degrading coumarin and generating dihydrocoumarin.
The pseudomonas putida is used for degrading coumarin and generating dihydrocoumarin, and the degradation rate of the coumarin is 99.94%.
The invention has the beneficial effects that:
(1) the engineering bacteria screened by the application can degrade coumarin in a culture medium which takes coumarin as a unique carbon source, the degradation rate is 99.94%, and the coumarin can be efficiently degraded to generate dihydrocoumarin; dihydrocoumarin is colorless or yellowish liquid, and can be used as substitute of coumarin, and as a perfume in food and cosmetic industry, such as: preparing cinnamon, coconut, cream and essence for cigarettes.
(2) The screened pseudomonas putida can degrade more than 99% of coumarin within 72 hours, and the bacterium can act on the black tonka bean tincture in the future and eliminate carcinogenic factors.
Drawings
FIG. 1 is a diagram showing the colony and cell morphology of Pseudomonas putida, wherein a is a diagram showing the colony of Pseudomonas putida, and b is a diagram showing the cell morphology.
FIG. 2 is a graph showing the growth curve of Pseudomonas putida and the degradation curve of coumarin.
FIG. 3 is a GC-MS total ion flow diagram of the degradation of coumarin by Pseudomonas putida to yield dihydrocoumarin.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described below with reference to the following examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The experimental materials of the present application are as follows:
the formula of the coumarin culture medium is as follows:
k2HPO 41 g/L, MgSO4.7H2O 0.5 g/L, FeSO4.7H2O 0.005 g/L, NaCl 0.5 g/L, KH2PO40.65 g/L, MnSO40.001 g/L, (NH4)2, SO 40.5 g/L, CaCl2.2H2O 0.1g/L, Na2MoO4.2H2O 0.005 g/L, 2 g/L coumarin is added on the basis of the culture medium.
Disclosed formula of bacterial LB medium:
10g tryptone, 5g yeast extract, 10g NaCl, 1L distilled water, pH to 7.0.
Screening of coumarin degrading bacteria
Selecting 1-3 representative walnuts, persimmons and corn plants in Zhang village of Buffalo in high and new areas of Zhengzhou city, Henan province, adopting a five-point sampling method, respectively collecting fertile soil of 5-10cm from perennial walnut forest, perennial persimmon forest and perennial farmland, storing the fertile soil in a sealed bag, making a record, and immediately taking the soil back to a laboratory. Respectively and randomly taking 5g of perennial walnut forest, persimmon forest and farmland soil into a 100mL triangular flask, adding 30mL of sterilized water, and uniformly shaking the mixture for one hour at the conditions of 30 ℃ of a shaking table and 120 r/min of rotation speed. Inoculating a soil solution into an LB culture medium, culturing for one day under the same conditions, then inoculating into a coumarin liquid culture medium according to the inoculum size of 2%, and culturing for one week to obtain a bacterial source.
Transferring 1 mL of the bacterium source obtained by enrichment culture into 30mL of a fermentation culture medium, taking a coumarin culture medium without the bacterium source as a control, culturing for 12h, detecting by GC-MS, and comparing with a GC-MS detection graph of a blank control. Selecting bacteria source capable of degrading coumarin, transferring 1 mL of bacteria source, sequentially diluting in gradient, and selecting 10-6Concentration gradient plating on carbon-deficient coumarin-rich medium is shown in figure 1, a. And (3) culturing the coated plate in an incubator at 30 ℃ for 2-3 d, selecting a colony with a better form, repeatedly streaking, separating and purifying until no foreign colony appears on the plate.
Second, preparation of seed liquid
Pseudomonas putida to be preserved in a slant (Pseudomonas putidaHS-C2) was inoculated into 30mL of LB medium and cultured for 12 hours.
Thirdly, bacterial degradation characteristics of coumarin
Inoculating 2% of Pseudomonas putida seed solution into 2 g/L coumarin culture medium, culturing for 72h at 30 deg.C and 120 rpm of shaking table. Blank control was also performed. Sampling 30mL of the solution every 6 h, and determining the content of coumarin in the fermentation liquor by using GC-MS.
After 72h of culture, the residual concentration of coumarin in the original 1890.12 mg/L coumarin culture medium was 52.61 mg/L, and the control was 1890.12 mg/L.
Fourth, preparation before sample measurement
Taking 30mL of fermentation liquor, centrifuging for 10 min under the condition of 10000 r/min, taking 20mL of CH2Cl2Extracting in a separating funnel, slowly shaking to mix the two phases, standing for 10 min, collecting the lower organic phase,repeating the above steps for 3 times, mixing organic phases, adding anhydrous sodium sulfate, drying, standing overnight, filtering, and rotary evaporating to remove CH2Cl2Thereafter, 1 mL of chromatographically pure CH was added2Cl2Dissolving, filtering with 0.45 μm organic filter membrane, and detecting by GC-MS analysis.
The physicochemical test results of the degrading bacteria are shown in fig. 2, the residual amount of coumarin gradually decreases with the lapse of culture time, and the detection results prove that the degradation rate of the pseudomonas putida to coumarin is 99.94%. As shown in the GC-MS total ion flow diagram of fig. 3, pseudomonas putida degrades coumarin to produce dihydrocoumarin.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (4)

1. The pseudomonas putida for degrading coumarin is characterized by comprising the following components in percentage by weight: the pseudomonas putida is preserved in 2019, 5 and 13 days to China general microbiological culture Collection center, and the preservation number is CGMCC No. 17758.
2. The method for culturing Pseudomonas putida according to claim 1, comprising the steps of: inoculating the pseudomonas putida bacterial liquid into a culture medium according to the inoculation amount of 2% for culture under the conditions of pH 5-9, temperature 30 ℃, rotation speed 120 rpm and culture for 12 hours.
3. The culture method of pseudomonas putida according to claim 2, wherein the culture medium is prepared from: k2HPO4 1 g/L，MgSO4•7H2O 0.5 g/L，FeSO4•7H2O 0.005 g/L，NaCl 0.5 g/L，KH2PO4 0.65 g/L，MnSO4 0.001 g/L，(NH4)2•SO4 0.5 g/L，CaCl2•2H2O 0.1g/L，Na2MoO4•2H2O 0.005 g/L，2 g/LCoumarin is provided.
4. The use of pseudomonas putida according to claim 1 for degrading coumarin to produce dihydrocoumarin.
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