CN110050942B - Method for biologically degrading patulin by using saccharomyces cerevisiae - Google Patents
Method for biologically degrading patulin by using saccharomyces cerevisiae Download PDFInfo
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- ZRWPUFFVAOMMNM-UHFFFAOYSA-N Patulin Chemical compound OC1OCC=C2OC(=O)C=C12 ZRWPUFFVAOMMNM-UHFFFAOYSA-N 0.000 title claims abstract description 188
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- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000000593 degrading effect Effects 0.000 title claims abstract description 13
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- 230000003834 intracellular effect Effects 0.000 claims abstract description 30
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- 238000000855 fermentation Methods 0.000 claims description 10
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- 238000002360 preparation method Methods 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
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- 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
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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
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Abstract
The invention discloses a method for biodegrading patulin by using saccharomyces cerevisiae, and relates to the technical field of biology. Culturing the saccharomyces cerevisiae in a culture medium until the cell concentration is about 1.1 to 1.3 multiplied by 10 6 And (3) adding the patulin into the CFU/mL, standing and fermenting to degrade the patulin, wherein the patulin can be completely degraded by 4.0-5.0 mg/L after 96-120h, and under the process conditions, the patulin can stimulate soluble intracellular enzymes generated by the saccharomyces cerevisiae to play a determining role in the degradation process. The method for degrading the patulin by using the saccharomyces cerevisiae has the advantages of high efficiency, simplicity in operation, safety, environmental protection and the like, and provides technical support for controlling patulin pollution in food.
Description
Technical Field
The invention relates to a method for biodegrading patulin by using saccharomyces cerevisiae, belonging to the technical field of biology.
Background
Patulin (PAT), also known as patulin, is a compound mainly made from penicillium (A)Penicillium) Aspergillus (a), (b) and (c)Aspergillus) And genus Aphanizomenon: (Byssochlamys) Polyketone secondary metabolites generated by various fungi are very stable under acidic conditions, can cause teratogenesis, carcinogenesis and mutagenesis due to great harm to human bodies, have the properties of influencing fertility, immunity, heredity and nervous systems, and are widely concerned by countries in the world. The apple and the products thereof are the main pollution source of patulin, so that the patulin content in the products is considered as an important index for evaluating the food safety quality of the products. The content of patulin in apple products is regulated by various authorities in the world similarly, wherein the standards of the European Committee are the strictest, namely that liquid products such as apple juice and the like are less than 50 mu g/kg,Solid products such as apple jam should be less than 25 mug/kg, and infant food should be less than 10 mug/kg. The patulin content in products such as apples and the like is not higher than 50 mug/kg according to the national standard GB2761-2017 in China.
Currently, strategies for the control of patulin include two aspects, namely the reduction of patulin production by inhibiting penicillium expansum infestation from the source, and the reduction or elimination of patulin already present in food products. The former can be controlled by regulating the storage conditions of fruits and products (low-temperature storage, modified atmosphere storage, etc.) or spraying chemical bactericide, etc. Since chemical bactericides cause problems of pesticide residues, generation of drug-resistant strains, environmental pollution and the like, a plurality of developed countries such as Europe and America and the like have strictly regulated the problems, and the application of the bactericides in the fresh keeping of fruits and vegetables is limited. Aiming at the produced patulin, the commonly used detoxification method mainly comprises a physical method and a chemical method, wherein the physical method comprises manual sorting and cleaning, clarification treatment, heating and pressurizing treatment, physical adsorbent treatment, microwave treatment, irradiation treatment or ultrasonic treatment and the like; the chemical method mainly uses ozone, polyphenol or plant extract to degrade patulin in fruits and products thereof. The physical or chemical methods have the defects of incomplete detoxification, complex operation, high technical cost, unknown safety or nutrient loss and the like in the processing process, and the requirements of consumers on green, safe and high-nutritive value products without additives are difficult to meet.
Disclosure of Invention
The invention aims at the problems and provides a method for utilizing saccharomyces cerevisiae (A)Saccharomyces cerevisiae) The fermentation process of the S288C strain is used for realizing the high-efficiency degradation of the patulin in the liquid culture medium.
The method utilizes saccharomyces cerevisiae to biologically degrade the patulin, and has good application prospect in controlling the pollution of the patulin in food.
Technical scheme
A method for degrading patulin by using saccharomyces cerevisiae comprises the following steps: strains of Saccharomyces cerevisiae: (Saccharomyces cerevisiae) S288C is cultured in a culture medium, and is added with patulin to be kept stand and fermented to generate soluble intracellular substancesAn enzyme, thereby degrading the patulin.
Further, the strain of Saccharomyces cerevisiae: (Saccharomyces cerevisiae) S288C, purchased from American Type Culture Collection (ATCC), strain number 204508.
Furthermore, the cell concentration of the saccharomyces cerevisiae cultured in the culture medium is about 1.1 to 1.3 multiplied by 10 6 CFU/mL。
Further, the culture medium is a minimum medium culture medium, and the formula of the culture medium is as follows: 10.0g/L of sucrose, 2.5g/L of dipotassium phosphate, 2.5g/L of monopotassium phosphate, 1.0g/L of diammonium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate and 0.007g/L of manganese sulfate monohydrate, the pH is natural, and the sterilization is carried out for 20min at 121 ℃.
Further, the degradation temperature is 28 to 30 ℃.
Further, the degradation time is 96 to 120h.
Further, the concentration of the patulin is 4.0-5.0 mg/L.
A soluble intracellular enzyme of Saccharomyces cerevisiae for degrading patulin is prepared by stimulating Saccharomyces cerevisiae strain with patulinSaccharomyces cerevisiae) S288C.
Further, the preparation method of the saccharomyces cerevisiae soluble intracellular enzyme comprises the following steps: collecting Saccharomyces cerevisiae fermentation broth, centrifuging at 4 deg.C and 8,000 Xg for 10min, collecting thallus, washing thallus with sterile water, resuspending in Tris-HCl (20mM, pH 7.5), ultrasonically crushing for 30min, breaking cell wall, centrifuging at 4 deg.C and 8,000 Xg for 10min, and collecting supernatant to obtain cell lysate (soluble intracellular enzyme).
The preparation method of the patulin stock solution comprises the following steps: taking out patulin (about 10 mg) from the temperature of minus 20 ℃, weighing and recording the mass of a sample bottle after thawing to room temperature, adding ethyl acetate to dissolve a sample, transferring the sample to a 10mL volumetric flask for constant volume, then subpackaging the sample bottle with 10 samples (brown) and blow-drying the sample bottle with nitrogen for later use, weighing and recording the mass of the sample bottle again after blow-drying the sample bottle in a fume hood, and calculating the sterile ddH required for preparing the patulin stock solution with the concentration of 1000mg/L according to the formula (1) 2 O (pH 4, adjusted with 1% glacial acetic acid) volume,
V = (m 1 -m 0 )/n （1）
wherein V is sterile ddH to be added into the brown bottle 2 The volume of O (pH 4, adjusted with 1% glacial acetic acid), m0 and m1 respectively represent the mass of the pre-solubilization aspergillin vials, and n represents the number of vials dispensed. The stock solution was stored at 4 ℃ until use.
The degradation of the soluble intracellular enzyme to the patulin in the invention has an enzyme reaction system as follows: adding a cell lysate (soluble intracellular enzyme) into Tris-HCl (20mM, pH 7.5) buffer solution, adding a patulin solution (the final concentration is 10 mg/L), uniformly mixing, detecting the concentration of the patulin at different reaction times by UHPLC, and performing heat inactivation on the cell lysate (soluble intracellular enzyme) added into a control group at 95 ℃ for 10min.
Advantageous effects
The method realizes the high-efficiency degradation of the patulin by utilizing the saccharomyces cerevisiae strain to biodegrade the patulin, about 90 percent of the patulin is degraded after 72 hours, and 4.2mg/L of the patulin is completely degraded after 96 hours.
In addition, in the degradation process of the invention, the saccharomyces cerevisiae soluble intracellular enzyme is generated by stimulation of the patulin, about 67 percent of the patulin can be degraded after the saccharomyces cerevisiae soluble intracellular enzyme reacts with the patulin for 1 hour, and the patulin which can react for 8 hours and 10mg/L at most can be completely degraded, thereby playing a decisive role in the degradation process and having good application prospect for controlling the pollution of the patulin in food.
Drawings
FIG. 1 Saccharomyces cerevisiaeSaccharomyces cerevisiae) S288C degradation of patulin;
o indicates the concentration of patulin; and delta represents the cell density of Saccharomyces cerevisiae.
FIG. 2 degradation effect of Saccharomyces cerevisiae soluble intracellular enzymes on patulin;
a. b, c, d, e respectively represent the degradation degree of patulin by the non-inactivated soluble intracellular enzyme in each time period, and the same letter represents no significant difference (significance level: 0.05); A. b, C, D, E, F indicates the degree of degradation of patulin by inactivated soluble intracellular enzymes over various time periods, respectively.
The specific implementation mode is as follows:
example 1: saccharomyces cerevisiae biodegradation of 4.2mg/L patulin
1) Activation and culture of the saccharomyces cerevisiae strain:
taking out the preserved Saccharomyces cerevisiae from the refrigerator at-80 ℃: (Saccharomyces cerevisiae) S288C, inoculating to Potato glucose solid culture medium (PDA) by streaking, and culturing at 28-30 deg.C for 3d. After bacterial colony grows out, taking a single bacterial colony to inoculate into 100mL MM culture medium, adding wine, standing and culturing for 3d at the temperature of 28 to 30 ℃, and when the yeast concentration reaches 1.1 to 1.3 multiplied by 10 7 CFU/mL。
The formula of the MM culture medium is as follows: 10.0g/L of sucrose, 2.5g/L of dipotassium phosphate, 2.5g/L of monopotassium phosphate, 1.0g/L of diammonium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate and 0.007g/L of manganese sulfate monohydrate, the pH is natural, and the sterilization is carried out for 20min at 121 ℃.
2) Preparation method of patulin stock solution
Taking out patulin (about 10 mg) from the temperature of minus 20 ℃, weighing and recording the mass of a sample containing bottle after thawing to room temperature, adding ethyl acetate to dissolve a sample, transferring the sample to a 10mL volumetric flask for constant volume, then subpackaging the sample into 10 sample injection bottles (brown), blow-drying the sample injection bottles with nitrogen for later use, weighing and recording the mass of the sample injection bottles again after blow-drying the sample containing bottles in a fume hood, and calculating the sterile ddH required for preparing the patulin stock solution with the concentration of 1000mg/L according to the formula (1) 2 O (pH 4, adjusted with 1% glacial acetic acid) volume,
V = (m 1 -m 0 )/n （1）
wherein V (mL) is the sterile ddH to be added to the brown bottle 2 The volume of O (pH 4, adjusted with 1% glacial acetic acid), m0 and m1 respectively represent the mass of the pre-solubilization aspergillin vials, and n represents the number of vials dispensed. The stock solution was stored at 4 ℃ until use.
3) The method for biologically degrading patulin by saccharomyces cerevisiae comprises the following steps:
the Saccharomyces cerevisiae culture solution was inoculated into 100mL of fresh MM liquid medium (cell concentration about 1.1 to 1.3X 10) at an inoculum size of 10% 6 CFU/mL), adding wine, standing at 28-30 ℃ for fermentation, completely degrading 4.2mg/L patulin after 96 hours, repeating the experiment for three times, and taking an average value of results.
The experimental results are shown in fig. 1. From the experimental results, it can be seen that: the fermentation process of the saccharomyces cerevisiae can completely degrade the patulin with the initial concentration of about 4.2 mg/L. Growth of the saccharomyces cerevisiae is inhibited after the patulin is added into the culture medium for 24h to 48h, and it is determined that in the process, the saccharomyces cerevisiae soluble intracellular enzyme is generated through stimulation of the patulin, the strain grows logarithmically after adaptation, and about 90% of the patulin is degraded in 72 h.
Example 2: saccharomyces cerevisiae biodegradation of 5.0mg/L patulin
1) Activation and culture of the saccharomyces cerevisiae strain:
taking out the preserved Saccharomyces cerevisiae from the refrigerator at-80 ℃: (Saccharomyces cerevisiae) S288C, inoculating to Potato glucose solid culture medium (PDA) by streaking, and culturing at 28-30 deg.C for 3d. After bacterial colony grows out, taking a single bacterial colony to inoculate into 100mL MM culture medium, adding wine, standing and culturing for 3d at the temperature of 28 to 30 ℃, and when the yeast concentration reaches 1.1 to 1.3 multiplied by 10 7 CFU/mL。
The formula of the MM medium is as follows: 10.0g/L of sucrose, 2.5g/L of dipotassium phosphate, 2.5g/L of monopotassium phosphate, 1.0g/L of diammonium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate and 0.007g/L of manganese sulfate monohydrate, the pH is natural, and the sterilization is carried out for 20min at 121 ℃.
2) Preparation method of patulin stock solution
Taking out patulin (about 10 mg) from the temperature of minus 20 ℃, weighing and recording the mass of a sample containing bottle after thawing to room temperature, adding ethyl acetate to dissolve a sample, transferring the sample to a 10mL volumetric flask for constant volume, then subpackaging the sample into 10 sample injection bottles (brown), blow-drying the sample injection bottles with nitrogen for later use, weighing and recording the mass of the sample injection bottles again after blow-drying the sample containing bottles in a fume hood, and calculating the sterile ddH required for preparing the patulin stock solution with the concentration of 1000mg/L according to the formula (1) 2 O (pH 4, adjusted with 1% glacial acetic acid) volume,
V = (m 1 -m 0 )/n （1）
wherein V (mL) is the sterile ddH that needs to be added to the brown bottle 2 The volume of O (pH 4, adjusted with 1% glacial acetic acid), m0 and m1 respectively represent the mass of the pre-solubilization aspergillin vials, and n represents the number of vials dispensed. The stock solution was stored at 4 ℃ until use.
3) The method for biologically degrading patulin by saccharomyces cerevisiae comprises the following steps:
the Saccharomyces cerevisiae culture solution was inoculated into 100mL of fresh MM liquid medium (cell concentration about 1.1 to 1.3X 10) at an inoculum size of 10% 6 CFU/mL), adding wine, standing at 28-30 ℃ for fermentation, completely degrading 5.0mg/L patulin after 110 hours, repeating the experiment for three times, and taking an average value as a result.
From the experimental results, it can be seen that: the fermentation process of the saccharomyces cerevisiae can completely degrade the patulin with the initial concentration of about 5.0mg/L. Growth of the saccharomyces cerevisiae is inhibited after the patulin is added into the culture medium for 24h to 48h, and it is determined that in the process, the saccharomyces cerevisiae soluble intracellular enzyme is generated through stimulation of the patulin, the strain grows logarithmically after adaptation, and about 90% of the patulin is degraded in 90 h.
Example 3: saccharomyces cerevisiae biodegradation of 4.5mg/L patulin
1) Activation and culture of the saccharomyces cerevisiae strain:
taking out the preserved Saccharomyces cerevisiae from the refrigerator at-80 ℃: (Saccharomyces cerevisiae) S288C, streaking the tube with a glycerol, inoculating the tube to a Potato glucose solid culture medium (PDA), and culturing the tube at 28 to 30 ℃ for 3d. After the bacterial colony grows out, taking a single bacterial colony to inoculate into 100mL MM culture medium, adding wine and standing and culturing for 3d at the temperature of 28 to 30 ℃, until the yeast concentration reaches 1.1 to 1.3 multiplied by 10 7 CFU/mL。
The formula of the MM medium is as follows: 10.0g/L of sucrose, 2.5g/L of dipotassium phosphate, 2.5g/L of monopotassium phosphate, 1.0g/L of diammonium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate and 0.007g/L of manganese sulfate monohydrate, the pH is natural, and the sterilization is carried out for 20min at 121 ℃.
2) Preparation method of patulin stock solution
Taking out patulin (about 10 mg) from the temperature of minus 20 ℃, weighing and recording the mass of a sample containing bottle after thawing to room temperature, adding ethyl acetate to dissolve a sample, transferring the sample to a 10mL volumetric flask for constant volume, then subpackaging the sample into 10 sample injection bottles (brown), blow-drying the sample injection bottles with nitrogen for later use, weighing and recording the mass of the sample injection bottles again after blow-drying the sample containing bottles in a fume hood, and calculating the sterile ddH required for preparing the patulin stock solution with the concentration of 1000mg/L according to the formula (1) 2 O (pH 4, adjusted with 1% glacial acetic acid) volume,
V = (m 1 -m 0 )/n （1）
wherein V (mL) is the sterile ddH to be added to the brown bottle 2 The volume of O (pH 4, adjusted with 1% glacial acetic acid), m0 and m1 respectively represent the mass of the pre-solubilization aspergillin vials, and n represents the number of vials dispensed. The stock solution was stored at 4 ℃ until use.
3) The method for biologically degrading patulin by saccharomyces cerevisiae comprises the following steps:
the Saccharomyces cerevisiae culture solution was inoculated into 100mL of fresh MM liquid medium (cell concentration about 1.1 to 1.3X 10) at an inoculum size of 10% 6 CFU/mL), adding wine, standing and fermenting at 28-30 ℃, completely degrading 4.5mg/L of patulin after 100 hours, repeating the experiment for three times, and taking an average value as a result.
From the experimental results, it can be seen that: the fermentation process of the saccharomyces cerevisiae can completely degrade the patulin with the initial concentration of about 4.5 mg/L. Growth of the saccharomyces cerevisiae is inhibited after the patulin is added into the culture medium for 24h to 48h, and it is determined that in the process, the saccharomyces cerevisiae soluble intracellular enzyme is generated through stimulation of the patulin, the strain grows logarithmically after adaptation, and about 90% of the patulin is degraded in 83 h.
Example 4: degradation of patulin by saccharomyces cerevisiae soluble intracellular enzyme
Collecting Saccharomyces cerevisiae fermentation broth, centrifuging at 4 deg.C and 8,000 Xg for 10min, collecting thallus, washing the thallus with sterile water, resuspending in Tris-HCl (20mM, pH 7.5), ultrasonically crushing for 30min, breaking cell wall, centrifuging at 4 deg.C and 8,000 Xg for 10min, and extracting cell lysate (soluble intracellular enzyme) from the obtained supernatant.
The enzymatic reaction system for the degradation of patulin by cell lysates (soluble intracellular enzymes) is as follows: adding a cell lysate (soluble intracellular enzyme) into a Tris-HCl (20mM, pH 7.5) buffer solution, adding a patulin solution (the final concentration is 10 mg/L), uniformly mixing, detecting the patulin concentration at different reaction times by HPLC, carrying out heat inactivation on the cell lysate (soluble intracellular enzyme) added into a control group at 95 ℃ for 10min, repeating the experiment for three times, and taking an average value according to the result.
The results of the experiment are shown in FIG. 2. Wherein a, b, c, d, e respectively represent the degradation degree of patulin by non-inactivated soluble intracellular enzymes in each time period, and the same letter represents no significant difference (significance level: 0.05); A. b, C, D, E, F indicates the degree of degradation of patulin by inactivated soluble intracellular enzymes in each time period, respectively. From the experimental results, it can be seen that: the soluble intracellular enzyme can degrade about 67% of patulin after reacting with patulin for 1h, and patulin is completely degraded after reacting for 8h or more. In contrast, inactivated intracellular enzymes lose most of their degradation capacity. Therefore, the soluble intracellular enzyme plays a decisive role in the biodegradation of patulin by saccharomyces cerevisiae.
Claims (1)
1. A method for degrading patulin by using saccharomyces cerevisiae is characterized in that a saccharomyces cerevisiae strain S288C is placed in a culture medium for culture, and then is added with patulin, and then is kept stand for fermentation to generate soluble intracellular enzymes, so that the patulin is degraded;
the saccharomyces cerevisiae strain is cultured in a culture medium until the cell concentration is 1.1 to 1.3 multiplied by 10 6 CFU/mL, the culture medium is a basic culture medium, and the formula of the culture medium is as follows: 10.0g/L of sucrose, 2.5g/L of dipotassium phosphate, 2.5g/L of monopotassium phosphate, 1.0g/L of diammonium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate and 0.007g/L of manganese sulfate monohydrate, and sterilizing at 121 ℃ for 20min;
the saccharomyces cerevisiae strain S288C is purchased from American type culture Collection and has the strain number of 204508;
the soluble intracellular enzyme is generated when the saccharomyces cerevisiae strain S288C is stimulated by patulin;
the preparation method of the soluble intracellular enzyme comprises the following steps: collecting Saccharomyces cerevisiae fermentation liquid, centrifuging at 4 deg.C and 8000 Xg for 10min, collecting thallus, washing the thallus with sterile water, suspending in Tris-HCl, ultrasonically crushing for 30min, centrifuging at 4 deg.C and 8,000 Xg for 10min after wall breaking to obtain supernatant, and obtaining soluble intracellular enzyme.
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