CN111528396B - Application of candida guilliermondii in degradation of antibiotics - Google Patents

Application of candida guilliermondii in degradation of antibiotics Download PDF

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CN111528396B
CN111528396B CN202010383506.0A CN202010383506A CN111528396B CN 111528396 B CN111528396 B CN 111528396B CN 202010383506 A CN202010383506 A CN 202010383506A CN 111528396 B CN111528396 B CN 111528396B
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meat
antibiotics
candida guilliermondii
bone meal
degradation
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CN111528396A (en
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孙力军
孙东方
王雅玲
房志家
邓旗
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Guangdong Ocean University
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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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry

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  • Microbiology (AREA)
  • Health & Medical Sciences (AREA)
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Abstract

The invention discloses candida guilliermondii (Candida guilliermondii) The application in degrading antibiotics or the application in improving the degradation removal rate of the antibiotics. According to the invention, the Candida guilliermondii is researched and found for the first time to obviously degrade antibiotics, particularly sulfonamide antibiotics, and the Candida guilliermondii is applied to meat and bone meal of sick animals, and the result shows that the degradation rate of sulfadiazine in the meat and bone meal of the sick animals is close to 90%, and the degradation rate of protein is only about 2%, so that the utilization rate of the meat and bone meal of the sick animals is greatly improved, and the pollution of the antibiotics to the environment is reduced.

Description

Application of candida guilliermondii in degradation of antibiotics
Technical Field
The invention belongs to the technical field of animal husbandry and veterinary medicine. More particularly, it relates to the use of Candida guilliermondii for the degradation of antibiotics.
Background
At present, the harmless treatment of sick livestock and poultry forms a quite large scale, and corresponding harmless treatment mechanisms are established in counties and cities all over the country due to the national requirements. The most widely applied treatment method is to sterilize the sick and dead livestock and poultry at high temperature and control the livestock and poultry by a humidifying machine. The waste residue after chemical treatment is dried to obtain a large amount of meat and bone meal, wherein the protein content is about 40-50%, and the mineral content is about 30%, so that the meat and bone meal is a good nutritional resource for feeding. However, because the sick animals use a large amount of antibiotics in the treatment process, the antibiotic residues in the meat and bone meal seriously exceed the standard, particularly the sulfonamide antibiotics are seriously remained, and the concentration of the antibiotic residues in part of samples is extremely high. Therefore, the further utilization of meat and bone meal of the sick animals is limited, most of the meat and bone meal are directly discarded, and the great waste of resources is caused.
The research literature on antibiotic degradation mainly focuses on antibiotic sewage treatment, mainly including gamma-radiation degradation method and ultrasonic waveMethod, palladium-gold catalyst method, Fe +3 Carrier oxidation degradation and microbial degradation. The gamma-radiation degradation method needs special radiation equipment, has great hidden danger to public safety, and has strict limitation on the application range. The degradation efficiency of the ultrasonic method is low. Palladium-gold catalyst method and Fe +3 The carrier oxidation degradation method needs to add exogenous chemicals into the matrix, and simultaneously, the catalytic oxidation process can oxidize protein and other nutrient substances into harmful substances, so that secondary pollution can be caused to meat and bone meal. The microbial degradation method is a low-cost and mild antibiotic degradation method, but relatively few degradation technologies for sulfonamide antibiotics are provided, and particularly, no technology suitable for degrading sulfonamide antibiotics in meat and bone meal of sick animals is reported.
Disclosure of Invention
In view of the deficiencies of the prior art, the present invention aims to provide Candida guilliermondii (Candida guilliermondii) The strain is derived from standard strains of China general microbiological culture collection management center, the collection address is No. 3 of No. 1 Hospital, North Chenlu, the south Korean district, Beijing, and the collection number is CGMCC 2.1801. The research finds that the candida guilliermondii has obvious degradation effect on antibiotics, particularly sulfonamide antibiotics, and can obviously degrade the antibiotics in the meat and bone meal of the sick animals, almost does not damage protein in a matrix, and improves the utilization rate of the meat and bone meal of the sick animals.
The invention aims to provide application of Candida guilliermondii in degrading antibiotics.
Another object of the present invention is to provide a method for degrading antibiotics in meat and bone meal of diseased animals by using Candida guilliermondii.
The above purpose of the invention is realized by the following technical scheme:
the present inventors have found that Candida guilliermondii (C.guilliermondii) (C.guilliermondii)Candida guilliermondii) Has good degradation effect on antibiotics, so the application of the candida guilliermondii in degrading the antibiotics is within the protection scope of the invention.
The Candida guilliermondii and the Pichia pastoris used in the patent 201710515467.3 are the same strain, are derived from a preservation mode strain of the China general microbiological culture Collection center, have the preservation address of No. 3 Hospital No. 1 of the Kyoho area in Beijing and have the preservation number of CGMCC 2.1801.
Preferably, the antibiotic is a sulfonamide antibiotic.
More preferably, the antibiotic is sulfadiazine.
Preferably, the antibiotic is derived from meat and bone meal of the diseased animal.
Preferably, the diseased animal meat and bone powder is subjected to harmless treatment.
In addition, the research result shows that the Candida guilliermondii can be used for remarkably degrading antibiotics in the meat and bone meal of the sick animals, almost not destroying proteins in the meat and bone meal of the sick animals, and greatly improving the utilization possibility of the meat and bone meal of the sick animals. Therefore, the method for degrading the antibiotics in the meat and bone meal of the sick animals by using the Candida guilliermondii also belongs to the protection scope of the invention.
Preferably, the antibiotics in the meat and bone meal of the sick animals are sulfanilamide antibiotics.
More preferably, the antibiotic in the meat and bone meal of the sick animal is sulfadiazine.
Preferably, the diseased animal meat and bone powder is subjected to harmless treatment.
Preferably, the method comprises in particular the steps of: mixing meat and bone powder of diseased animals with water, adding adjuvants, adjusting pH to 5.5-7.5 to obtain fermentation culture medium, sterilizing, inoculating 2-5% Candida guilliermondii strain liquid into the fermentation culture medium, and fermenting at 25-32 deg.C for 2-4 days.
Preferably, the concentration of sulfadiazine in the meat and bone meal of the sick animal is not higher than 20 mg/kg. When the antibiotic is degraded according to the steps, when the concentration of sulfadiazine in the meat and bone meal of the sick animal is not higher than 20mg/kg, the prepared fermentation liquor has no inhibition effect on candida guilliermondii and does not influence the normal growth of the strain.
More preferably, the inoculum size is 3%.
More preferably, the water amount is 2-4 times of the mass of the meat and bone meal of the sick animals.
More preferably, the adjuvant is molasses or glucose.
More preferably, the molasses or glucose is 1-3% by weight of the total.
More preferably, the fermentation temperature is 28 ℃ and the fermentation time is 3 days.
The invention has the following beneficial effects:
the invention researches and discovers that the Candida guilliermondii has obvious degradation effect on the antibiotics for the first time, and the Candida guilliermondii is applied to the antibiotics for degrading the meat and bone meal of the sick animals. Compared with the existing antibiotic degradation method, such as the gamma-radiation degradation method, the method has high safety; compared with the ultrasonic method, the degradation rate is obviously improved, compared with palladium-gold catalysis and Fe +3 The carrier oxidation method does not bring secondary pollution to the water body. Meanwhile, the invention has the effects of low cost, low protein loss and high antibiotic degradation speed, well solves the problem of degrading sulfonamide antibiotics in the meat and bone meal of the sick animals, simultaneously ensures that the protein in the matrix is not damaged to the maximum extent, not only greatly improves the utilization rate of the meat and bone meal of the sick animals, but also reduces the pollution to the environment.
Drawings
FIG. 1 is a comparison of inhibition zones of fermentation substrates before and after degradation of antibiotics in meat and bone meal of diseased animals.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The diseased animal meat and bone powder is subjected to harmless treatment, is purchased from Zhejiang He and environmental protection science and technology Limited company, and meets the technical specification of harmless treatment on animals died of illness (No. 2013 34 of agricultural medicine) issued by Ministry of agriculture.
Example 1 degradation experiment of Candida guilliermondii sulfadiazine
1. Experimental methods
The invention uses LC/MS method to detect the residual sulfadiazine, calculates the degradation rate, and has the following concrete operations:
mass spectrum conditions: spraying voltage: 4.5 kv; atomization flow rate: 3.0L/min; flow rate of drying gas: 15L/min; collision gas pressure (CID): 230 kPa.
Liquid chromatography conditions: a chromatographic column: gold C18, 5 μm, 150 mm × 2.1 mm (inner diameter); mobile phase: methanol A; ammonium acetate 5 mmol/L (0.1% formic acid) in water B, elution gradient as shown in Table 1, flow rate: 300 muL/min; column temperature: 30 ℃; sample introduction amount: 10 muL.
TABLE 1 liquid chromatography mobile phase gradient elution for separating drugs
Figure 94265DEST_PATH_IMAGE001
Sulfadiazine is dissolved in an aqueous solution, the obtained solution is diluted by wort of 5 ℃ Be until the final concentrations of sulfadiazine are 25mg/L, 5mg/L and 1mg/L, each concentration is set to Be 3 in parallel, and sterilized wort of 5 ℃ Be + sulfadiazine with the same concentration are used as a control group. Adding 2% of Candida guilliermondii strain liquid, and fermenting at 28 deg.C for 2 days. And detecting the amount of sulfadiazine in the fermentation liquor by an LC/MS method.
2. Results of the experiment
The results show that the degradation rates of sulfadiazine at the end of fermentation are 76.8%, 85.7% and 92.4%, respectively.
EXAMPLE 2 sulfadiazine degradation in meat and bone meal of sick animals
Mixing meat and bone meal of sick animals with 3 times of water, adding molasses accounting for 1.5% of the total weight, adjusting pH to 6.8 to obtain a fermentation culture medium, adding into a triangular flask, sterilizing, inoculating 3% of Candida guilliermondii strain liquid into the fermentation culture medium, and fermenting at 28 deg.C for 3 days.
Taking 0.2g of fermentation substrate on a nutrient agar plate containing escherichia coli indicator bacteria, measuring the diameter of a bacteriostatic zone, taking the substrate which is not fermented as a blank control, and comparing the degradation conditions of sulfadiazine before and after fermentation. As shown in figure 1, the unfermented meat and bone powder has obvious inhibition effect on Escherichia coli, and the inhibition zone shows that the meat and bone powder has high antibiotic residue and can inhibit the growth of Escherichia coli. After the Candida guilliermondii is added for fermentation, the meat and bone meal has no inhibition effect on escherichia coli, which shows that the antibiotics in the meat and bone meal are significantly degraded after fermentation.
The residual sulfadiazine content of the substrate before and after fermentation was measured by LC-MS method (same as example 1) to calculate the degradation rate. Protein and free amino acid before and after matrix fermentation are respectively measured according to a first method full-automatic Kjeldahl method in GB 50095-2010 food safety national standard food protein measurement and a GB 5009.235-2016 food safety national standard food amino acid nitrogen measurement method, and the protein degradation rate is calculated. The result shows that the residual sulfadiazine content in the meat and bone meal of the sick animal before fermentation is 8.08mg/kg, the sulfadiazine content at the end of fermentation is 0.85 mg/kg, the sulfadiazine degradation rate is 89.5 percent, and the protein degradation rate is 2.1 percent.
EXAMPLE 3 sulfadiazine degradation in meat and bone meal of sick animals
Mixing meat and bone meal of sick animals with 4 times of water, adding molasses 3% of the total weight, adjusting pH to 5.5 to obtain a fermentation culture medium, sterilizing, inoculating 2% Candida guilliermondii strain liquid into the fermentation culture medium, and fermenting at 25 deg.C for 4 days.
The measurement mode is as example 2, and the result shows that the residual sulfadiazine in meat and bone powder of the sick animals before fermentation is 8.08mg/kg, the sulfadiazine content at the end point of fermentation is 0.94 mg/kg, the sulfadiazine degradation rate is 88.3 percent, and the protein degradation rate is 2.6 percent.
Example 4 sulfadiazine degradation in meat and bone meal of diseased animals
Mixing meat and bone meal of sick animals with 2 times of water, adding 1% of glucose based on the total weight, adjusting pH to 7.5 to obtain a fermentation culture medium, sterilizing, inoculating 5% of Candida guilliermondii strain liquid into the fermentation culture medium, and fermenting at 32 deg.C for 2 days.
The measurement mode is as in example 2, and the result shows that the residual sulfadiazine content in the meat and bone meal of the sick animal before fermentation is 8.08mg/kg, the sulfadiazine content at the end of fermentation is 1.19 mg/kg, the sulfadiazine degradation rate is 85.2 percent, and the protein degradation rate is 2.3 percent.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (6)

1. Candida guilliermondii (Candida guilliermondii) The application in the aspect of degrading antibiotics or in improving the degradation removal rate of antibiotics is characterized in that the antibiotics are sulfadiazine, and the Candida guilliermondii is Candida guilliermondii CGMCC 2.1801.
2. The use of claim 1, wherein the antibiotic is derived from meat and bone meal of a diseased animal.
3. A method for degrading antibiotics in meat and bone meal of sick animals is characterized in that Candida guilliermondii (Candida guilliermondii) (A. guilliermondii (A.)) is utilizedCandida guilliermondii) Degrading antibiotics in meat and bone powder of the sick animals, wherein the antibiotics in the meat and bone powder of the sick animals are sulfadiazine, and the Mongolian candida guilliermondii is CGMCC 2.1801.
4. The method according to claim 3, characterized in that it comprises in particular the steps of: mixing meat and bone powder of diseased animals with water, adding adjuvants, adjusting pH to 5.5-7.5 to obtain fermentation culture medium, sterilizing, inoculating 2-5% Candida guilliermondii strain liquid into the fermentation culture medium, and fermenting at 25-32 deg.C for 2-4 days.
5. The method of claim 4, wherein the amount of water is 2-4 times the mass of the meat and bone meal of the diseased animal.
6. The method according to claim 4, wherein the auxiliary material is molasses or glucose, and the amount added is 1-3% by weight.
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CN105861385A (en) * 2016-05-23 2016-08-17 中国科学院城市环境研究所 Sulfadimidine degrading bacterium S-07 and screening method thereof
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