CN112641032A

CN112641032A - Application of cryptococcus rhodochrous Y3-based intracellular enzyme in degradation of ochratoxin A

Info

Publication number: CN112641032A
Application number: CN202011504094.8A
Authority: CN
Inventors: 张红印; 魏美林; 张晓云; 杨其亚; 赵利娜
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-04-13
Anticipated expiration: 2040-12-18
Also published as: CN112641032B

Abstract

The invention belongs to the technical field of biological control, and relates to a use for degrading ochratoxin A based on the intracellular enzyme of Cryptococcus Y3; the steps are as follows: firstly, the Cryptococcus Y3 is inserted into a NYDB medium, and after culturing and activation, a bacterial solution is taken. Coat on NYDA medium; then, select a single colony and inoculate it into PM medium. After culturing, centrifuge to obtain bacterial cells. After washing with sterile distilled water, dilute it into a bacterial suspension of 1×10 ⁸ cells/mL, and inoculate it in PM. After culturing in the medium, the cells were collected by centrifugation, liquid nitrogen was added, and ground to obtain powdered cells; Tris-HCl buffer was added to resuspend, and after shaking, the cells were subjected to ice bath treatment and centrifugation, and the supernatant was collected and passed through an organic The pinhole filter membrane is filtered and sterilized to obtain an intracellular enzyme solution; the ochratoxin A is added to the intracellular enzyme solution, and the ochratoxin A can be degraded by shaking and culturing in a dark environment; the invention is safe and environmentally friendly. , which can efficiently and rapidly degrade ochratoxin A.

Description

Application of cryptococcus rhodochrous Y3-based intracellular enzyme in degradation of ochratoxin A

Technical Field

The invention belongs to a method for biologically degrading toxin, and particularly relates to an application of degrading ochratoxin A based on cryptococcus rhodochrous Y3 intracellular enzyme.

Background

Mycotoxins are secondary metabolites produced by fungal microorganisms, and so far, at least 300 types of mycotoxins have been found. Of the five most toxic mycotoxins in the world, ochratoxin A is ranked the second, the most obvious toxic effect is nephrotoxicity, and then liver and immunotoxicity are listed as 2B human carcinogens by the international cancer research institution.

Besides serious toxic effects, ochratoxin a is heavily contaminated in food and feed. There are two main types of moulds which produce ochratoxin A in nature, the first type is Aspergillus ochracea and Penicillium verrucosus, the hosts of the two types of moulds are mainly crops including corn, wheat, rice and the like, and the moulds are seriously ill in tropical and subtropical regions; another type of mould is Aspergillus carbonarins, the host of which is mainly grapes, which is more severe in cold regions. To date, ochratoxin a has been found in a variety of food products, such as soy sauce, grapes and their preparations, and meat products and coffee. The presence of ochratoxin a in humans is also an alarming problem, and it was found in 2019 based on studies in the portugal region that the toxin was detected in the urine of over 90% of children.

Because of the extremely strong toxicity and wide pollution of ochratoxin A, the control of the content of ochratoxin A in food and feed is an important measure for ensuring the food safety. Ochratoxin A is a polyketone compound formed by connecting carboxyl-5-chloro-8-hydroxy-3, 4-dihydro-3-R-methylisothiazolin (ochratoxin alpha) and L-beta-phenylalanine together through amide bonds, is resistant to acid and high-temperature environments, and is difficult to degrade in natural environments. Detoxification is generally achieved by cleaving amide bonds to produce ochratoxins alpha and L-beta-phenylalanine.

The current control methods for ochratoxin A comprise a physical mode, a chemical mode and a biological mode. Physical degradation of ochratoxin A comprises adsorption and thermal degradation; adsorption is a common method for removing toxins, but can not completely degrade the toxins, so that the toxins remain in the environment, and secondary pollution is caused; heating is also an effective way to remove ochratoxin a, but is prone to problems of reduced product quality. Chemical degradation of ochratoxin A is also a common detoxification method, and the chemical degradation of ochratoxin A which is researched is diversified, such as degradation of ochratoxin A by Peroxidase (POD), protein and saccharide, but the operation is complex and the cost is high.

In recent years, the use of microorganisms and enzymes produced by the microorganisms to degrade toxins has become a focus of research, and although many yeasts, lactic acid bacteria and molds have been found to have the efficacy of degrading ochratoxin a, the types of the discovered degrading enzymes are limited and the degrading effect is not good, so that the discovery of new efficient degrading enzymes is very necessary.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the invention provides a strain of Cryptococcus podzolius Y3 which is selected from soil and is separated and screened, and the intracellular enzyme based on Cryptococcus Y3 degrades ochratoxin A, so that the strain has high safety and extremely high degradation effect.

The cryptococcus yeast system for degrading ochratoxin A provided by the invention is screened and separated from soil collected from orchards in the sentence-volume city of Zhenjiang, and is cultured on an NYDA solid medium plate at 28 ℃ for 3d for morphological observation; the 5.8S rDNA-ITS region sequence of this strain has been analyzed and identified molecularly. Meanwhile, the yeast strain provided by the invention is currently preserved in China Center for Type Culture Collection (CCTCC) of the university of Wuhan, China, and the preservation number is as follows: CCTCC NO: m2017505, a proposed classification named Cryptococcus podzolicus Y3. In addition, the applicant of the strain utilized in the present invention has applied for chinese patent invention, CN201711183834.0, with the name: a yeast for degrading citrinin and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

(1) preparing a bacterial suspension: inoculating cryptococcus rhodochrous Y3 into an NYDB culture medium, culturing for 20-24 h under the shaking conditions of 28-30 ℃ and 180rpm, and activating to obtain activated bacterial liquid; then coating the activated bacterial liquid on an NYDA solid culture medium, and culturing at 28-30 ℃ for 48-60 h; then, selecting a single colony of cryptococcus rhodochrous Y3 growing in an NYDA solid culture medium, inoculating the single colony into a PM culture medium for culture, centrifuging after culture to obtain thalli, cleaning the thalli for a plurality of times by using sterile distilled water, centrifuging again after cleaning, collecting the thalli, and diluting the thalli into a bacterial suspension by using the sterile distilled water;

(2) inoculating the bacterial suspension prepared in the step (1) into a PM culture medium, culturing for 20-24 h at 28-30 ℃ under a shaking condition of 180rpm, centrifugally collecting yeast, cleaning with sterile distilled water, centrifugally collecting the yeast again, adding liquid nitrogen to quick-freeze the yeast, and grinding to obtain powdery bacteria; then adding the powdery thalli into Tris-HCl buffer solution (Tris hydrochloride, 50mM, pH 7.1) for heavy suspension, oscillating, then carrying out ice bath treatment, centrifuging after treatment, and collecting supernatant, namely intracellular crude enzyme solution;

(3) filtering and sterilizing the crude intracellular enzyme solution obtained in the step (2) by a pinhole filter membrane to obtain an intracellular enzyme solution; the ochratoxin A is added into the intracellular enzyme solution, and the application of the ochratoxin A in rapid degradation can be realized by shake culture in a dark environment.

Preferably, the rotation speed of the centrifugation in the step (1) is 7000r/min, and the time is 10 min.

Preferably, the composition of the NYDA culture medium in the step (1) is as follows: calculated by 1000mL, 8g of beef extract, 5g of yeast extract, 10g of glucose, 20g of agar and the balance of distilled water, and carrying out moist heat sterilization at 115 ℃ for 15 min.

Preferably, the NYDB culture medium in the step (1) comprises the following components: and (2) counting by 1000mL, 5g of yeast extract, 10g of glucose, 8g of beef extract and the balance of distilled water, wherein the pH is natural, and moist heat sterilization is carried out for 15min at 115 ℃.

Preferably, the components of the PM medium in the step (1) are as follows: 2g of maltose, 10g of cane sugar, 5g of yeast extract, 5g of peptone and 1000mL of primary distilled water, and carrying out moist heat sterilization at 115 ℃ for 15 min.

Preferably, the single colony in the step (1) is inoculated into a PM culture medium for culturing at the temperature of 28-30 ℃ for 48 hours.

Preferably, the washing with sterile distilled water in the step (1) is carried out for 2-3 times; the concentration of the bacterial suspension is 1 x 10⁸cells/mL。

Preferably, the inoculation amount of the bacterial suspension inoculated into the PM culture medium in the step (2) is 1-2%; namely the bacterial suspension is 1 to 2 percent of the volume of the PM culture medium.

Preferably, the oscillation time in the step (2) is 1-2 min; the ice-bath treatment time is 30-35 min; the centrifugation conditions were: 15-20 min at 4 ℃, 11000 Xg.

Preferably, the pore size of the pinhole filter membrane in the step (3) is 0.22 μm, and the filter membrane is an organic filter membrane; the conditions of shake culture are as follows: at 28 ℃, 180rpm, and the time is 0-3 h.

The invention has the advantages that:

(1) the intracellular enzyme of cryptococcus rhodochrous Y3 provided by the invention can efficiently and quickly degrade ochratoxin A, the intracellular enzyme and ochratoxin A can be immediately degraded after being mixed, and the degradation effect is obvious; at 0h, the initial concentration of OTA is 1 mug/mL, and the degradation rate is 83.2%; the initial concentration of OTA is increased to 10 mug/mL, and the degradation rate is still more than 70% in 0 h.

(2) The cryptococcus rhodochrous Y3 used in the invention is determined to have high safety and no harm to human body through an acute toxicity test of mice, so that the cryptococcus rhodochrous Y3 can be applied to controlling ochratoxin A pollution in food, feed and products thereof and the like, and the edible safety of the food and the products thereof is guaranteed.

Drawings

FIG. 1 is a graph showing the effect of intracellular enzymes of Cryptococcus rhodochrous Y3 on the degradation of ochratoxin A in PM medium; wherein: 1. mu.g/mL, 10. mu.g/mL and 20. mu.g/mL represent the initial concentration of OTA after mixing with the intracellular enzyme solution, respectively.

Detailed Description

The invention will be explained in more detail by means of the following examples; the following examples are illustrative only, and the present invention is not limited by these examples.

Example 1:

(1) preparing a bacterial suspension: inoculating cryptococcus Y3 into an NYDB culture medium, culturing for 24h under the shaking conditions of 28 ℃ and 180rpm, and activating to obtain activated bacterial liquid; then coating the activated bacterial liquid on an NYDA solid culture medium, and culturing at 28 ℃ for 48 hours; then, 2-ring NYDA solid medium is selected for growthInoculating Cryptococcus elongatus Y3 single colony to 50mL PM culture medium, culturing, centrifuging to obtain thallus, washing with sterile distilled water for 3 times, centrifuging again after washing, collecting thallus, diluting with sterile water to 1 × 10⁸cell/mL of bacterial suspension;

(2) inoculating the bacterial suspension prepared in the step (1) into a PM culture medium according to the addition of 2%, culturing for 24h under the shaking conditions of 28 ℃ and 180rpm, centrifuging at 7000rpm for 10min, collecting yeast, cleaning with sterile distilled water, and centrifuging again to collect yeast; then adding sterile distilled water for cleaning and centrifuging; the cleaning is repeated for 3 times; collecting thallus, adding liquid nitrogen to quick-freeze the thallus, and grinding to obtain powdered thallus; then adding the powdery thalli into Tris-HCl buffer solution (50mM, pH 7.1) for heavy suspension, shaking for 1min, then carrying out ice bath treatment for 30min, centrifuging for 15min at 11000 Xg after treatment, and collecting supernatant, namely intracellular crude enzyme solution;

(3) filtering and sterilizing the crude intracellular enzyme solution obtained in the step (2) by a 0.22-micron organic pinhole filter membrane to obtain an intracellular enzyme solution; the ochratoxin A is added into the intracellular enzyme solution, and shake culture is carried out at 180rpm and 28 ℃ in a dark environment, so that the purpose of degrading the ochratoxin A can be realized.

Degradation effect:

the sampling time is selected from 0, 1, 2 and 3 h; sterile buffer was used as a control group instead of intracellular enzymes.

Adding ochratoxin A into the intracellular enzyme solution, mixing, and taking a sample, wherein the time is recorded as 0 h:

adding ochratoxin A into an intracellular enzyme solution, mixing, shaking and culturing for 1h, and taking a sample, wherein the mark is 1 h:

adding ochratoxin A into an intracellular enzyme solution, mixing, shaking and culturing for 2h, and taking a sample, wherein the sample is recorded as 2 h:

adding ochratoxin A into an intracellular enzyme solution, mixing, shaking and culturing for 3h, and taking a sample, wherein the mark is 3 h:

adding equal amount of chromatographic grade methanol into the sample, uniformly mixing by vortex oscillation, then passing through a 0.22 mu m organic pinhole filter head, placing in a brown sample introduction bottle, placing in an environment at 4 ℃ and using for HPLC-FLD determination.

The liquid phase detection conditions are as follows: using a Zorbax SB-C18 column (250 mm. times.4.6 mm, 5 μm); mobile phase: acetonitrile (60%): 1% acetic acid (40%); the flow rate is 1.0 mL/min; injecting 20 mu L of sample each time, wherein the column temperature is 30 ℃; the excitation wavelength of the fluorescence detector is 333nm, and the emission wavelength is 460 nm; the peak-off time was about 5.4 min.

As shown in FIG. 1, the degradation rate of ochratoxin A by intracellular enzymes of Cryptococcus rhodochrous Y3 at 0h was 83.2% (initial concentration of 1. mu.g/mL), 70.1% (initial concentration of 10. mu.g/mL) and 52.4% (initial concentration of 20. mu.g/mL);

at 1h, the degradation rate of ochratoxin A is 100% (initial concentration is 1 mu g/mL), 99.5% (initial concentration is 10 mu g/mL) and 98.9% (initial concentration is 20 mu g/mL);

at 2h, no ochratoxin A can be detected under the liquid phase condition; and 3h, no ochratoxin A can be detected under the liquid phase condition.

In summary, the intracellular enzyme of cryptococcus rhodochrous Y3 has excellent degradation effect on the enzyme ochratoxin A.

Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.

Claims

1. The application of degrading ochratoxin A based on cryptococcus pluvialis Y3 intracellular enzyme is characterized by comprising the following specific steps:

(2) inoculating the bacterial suspension prepared in the step (1) into a PM culture medium, culturing for 20-24 h at 28-30 ℃ under a shaking condition of 180rpm, centrifugally collecting yeast, cleaning with sterile distilled water, centrifugally collecting the yeast again, adding liquid nitrogen to quick-freeze the yeast, and grinding to obtain powdery bacteria; then adding the powdery thalli into a Tris-HCl buffer solution for resuspension, shaking, carrying out ice bath treatment, centrifuging after treatment, and collecting supernatant, namely intracellular crude enzyme solution;

(3) filtering and sterilizing the crude intracellular enzyme solution obtained in the step (2) by a pinhole filter membrane to obtain an intracellular enzyme solution; the ochratoxin A is added into the intracellular enzyme solution, and the application of the ochratoxin A in degradation can be realized by shake culture in a dark environment.

2. Use according to claim 1, characterized in that the rotation speed of the centrifugation in step (1) is 7000r/min and the time is 10 min.

3. The use according to claim 1, wherein the NYDA medium in step (1) has the following composition: calculated by 1000mL, 8g of beef extract, 5g of yeast extract, 10g of glucose, 20g of agar and the balance of distilled water, and carrying out moist heat sterilization at 115 ℃ for 15 min.

4. The use according to claim 1, wherein the NYDB medium comprises the following components: and (2) counting by 1000mL, 5g of yeast extract, 10g of glucose, 8g of beef extract and the balance of distilled water, wherein the pH is natural, and moist heat sterilization is carried out for 15min at 115 ℃.

5. The use according to claim 1, wherein the PM medium composition in step (1) is as follows: 2g of maltose, 10g of cane sugar, 5g of yeast extract, 5g of peptone and 1000mL of primary distilled water, and carrying out moist heat sterilization at 115 ℃ for 15 min.

6. The use according to claim 1, wherein the temperature for inoculating the single colony in the PM medium in the step (1) for culturing is 28-30 ℃ and the time is 48 h.

7. Use according to claim 1, wherein the washing with sterile distilled water in step (1) is carried out several times, in particular 2 to 3 times; the concentration of the bacterial suspension is 1 x 10⁸cells/mL。

8. The use according to claim 1, wherein the bacterial suspension is inoculated into the PM medium in the step (2) in an amount of 1-2%.

9. The use according to claim 1, wherein the shaking time in step (2) is 1-2 min; the ice-bath treatment time is 30-35 min; the centrifugation conditions were: 15-20 min at 4 ℃, 11000 Xg.

10. Use according to claim 1, wherein the pore size of the pinhole filter in step (3) is 0.22 μm, the organic phase; the conditions of shake culture are as follows: at 28 ℃, 180rpm, and the time is 0-3 h.