Aflatoxin b1-degrading strain, compound microbial preparation and application thereof
By co-culturing Enterococcus faecalis HB2-2 with Rhodococcus, a compound microbial preparation was prepared, which solved the problem of poor degradation of aflatoxin B1 in peanut meal and achieved a highly efficient and safe industrial detoxification effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN POLYTECHNIC UNIVERSITY
- Filing Date
- 2023-03-28
- Publication Date
- 2026-07-31
AI Technical Summary
Existing microbial strains have poor degradation effects on aflatoxin B1 in peanut meal and cannot be effectively applied in actual industrial production.
A compound microbial preparation was prepared by co-culturing Enterococcus faecalis HB2-2 and Rhodococcus faecalis. By optimizing culture conditions and fermentation parameters, the efficient degradation of aflatoxin B1 in peanut meal was achieved.
This compound microbial preparation can efficiently degrade aflatoxin B1 in peanut meal under mild conditions, with a degradation rate of up to 89.4%, meeting the needs of industrial production and avoiding material deterioration and nutrient loss.
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Figure CN116426423B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microorganisms and their preparations, specifically relating to a strain that degrades aflatoxin B1, a compound microorganism containing the strain, and the application of the compound microorganism in removing aflatoxin B1 from peanut meal. Background Technology
[0002] Aflatoxin B1 (AFB1) is one of the most widespread and harmful mycotoxins in food and animal feed, exhibiting strong hepatotoxicity, carcinogenicity, teratogenicity, and mutagenicity. AFB1 seriously endangers human and animal health, causing significant economic losses to the food industry, agriculture, and animal husbandry. Currently, methods for degrading AFB1 mainly include physical, chemical, and biological methods. Physical methods include heating, microwave treatment, and ultraviolet irradiation, but these methods are energy-intensive, inefficient, and prone to secondary pollution. Chemical methods mainly use chemical reagents such as acids, alkalis, and oxidants to destroy the toxic sites of AFB1 through acidolysis, alkaline hydrolysis, and oxidation. Chemical methods are highly efficient, but the introduction of chemical reagents raises safety concerns. Microbial methods primarily degrade AFB1 through adsorption on the cell walls of microorganisms or through intracellular or extracellular enzymes secreted by microorganisms. Compared to physical and chemical detoxification methods, biological detoxification methods have advantages such as milder conditions, higher detoxification efficiency, and no secondary pollution.
[0003] In recent years, reported microbial strains that can degrade AFB1 include Bacillus subtilis, lactic acid bacteria, and Aspergillus niger. Oluwafemi et al. isolated five strains of lactic acid bacteria from fermented corn samples: Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, and Lactobacillus plantarum, achieving AFB1 detoxification rates of 32-75% in corn. Prabakaran et al. used coumarin as the sole carbon source to screen a Bacillus subtilis strain, SHT1, from tanneries soil, achieving a 100% AFB1 degradation rate. Sangare et al. screened a Pseudomonas aeruginosa strain, N17-1, from cereal grains and soil, achieving an AFB1 degradation rate of 82.8%. The degradative active substances of this strain were mainly found in the culture supernatant; the degradation activity of the supernatant treated with proteinase K and SDS was significantly reduced, thus proving that the extracellular enzymes of the strain were the degradative agents. Zhang et al. screened a strain of Aspergillus niger capable of degrading AFB1 from feed, and after optimizing fermentation conditions, the degradation rate reached 58.2%. Wang et al. isolated three microbial strains with AFB1 degradation activity from moldy peanuts, among which Fusarium WCQ3361 showed a degradation rate of 95.4% after co-culturing with AFB1 for 24 hours. Although many strains with AFB1 degradation activity have been reported, the degradation ability varies greatly among different microbial species and experimental conditions. Furthermore, most strains only show high degradation activity against pure AFB1 standards, while exhibiting poor degradation effects on AFB1 in peanut meal. Therefore, these strains are currently not suitable for practical industrial production.
[0004] In summary, the currently screened microbial strains only showed degradation activity against pure AFB1, while their degradation effect on AFB1 in peanut meal was poor. This is because AFB1 in peanut meal is encapsulated by cellulose, protein, and other components, and AFB1 has extremely poor water solubility, preventing sufficient contact between microorganisms and AFB1 during fermentation, thus severely reducing the degradation efficiency of AFB1 in peanut meal. Currently, there are no microbial strains suitable for AFB1 detoxification in peanut meal in actual industrial production. Summary of the Invention
[0005] The purpose of this invention is to provide a *Enterococcus faecalis* HB2-2 that degrades AFB1, a compound microbial preparation, and its application. *Enterococcus faecalis* HB2-2 was isolated from a landfill and its preservation number is CGMCC NO.: 26712. *Enterococcus faecalis* HB2-2 can achieve a degradation rate of up to 89.9% for pure AFB1. This invention involves co-culturing *Enterococcus faecalis* HB2-2 with *Rhodococcus* (cell ratio = 1:0.7-1:1.3) to prepare a microbial preparation. This microbial preparation can widely degrade AFB1 in peanut meal, achieving a maximum degradation rate of 89.4% for AFB1 in peanut meal at a material-to-liquid ratio (g / mL) of 1:1-1:40.
[0006] The HB2-2 strain isolated in this invention belongs to the Enterococcus genus. Its biological characteristics are as follows: the colonies are round with neat edges, opaque, milky white on the front, lighter in color, raised in the middle, smooth, moist in texture, easy to pick up, and Gram staining reaction is positive.
[0007] A second aspect of the present invention provides a method for culturing a strain of Enterococcus faecalis, wherein the Enterococcus faecalis strain is activated and then inoculated into a culture medium with an initial pH of 6-8 and a temperature of 25-28°C for cultivation. The strain obtained under these conditions exhibits the optimal degradation rate of AFB1.
[0008] A third aspect of the present invention provides a compound microbial preparation, wherein the compound microbial preparation comprises the above-mentioned Enterococcus faecalis strain and Rhodococcus.
[0009] Furthermore, the cell number ratio of Enterococcus faecalis strain to Rhodococcus is 1:0.7-1:1.3, and in practical applications, they can be mixed in equal proportions.
[0010] The preparation method of the compound microbial preparation of the present invention may include: inoculating the Enterococcus faecalis strain and Rhodococcus into a culture medium (e.g., NB liquid medium) and incubating with shaking at 28-33°C and 180-220 rpm for 8-16 hours.
[0011] A fourth aspect of the present invention provides the application of the aforementioned Enterococcus faecalis HB2-2 or the aforementioned compound microbial preparation in the degradation of AFB1 in peanut meal.
[0012] Further, the removal method may include: inoculating the compound microbial preparation into a fresh culture medium, adding the peanut meal to be treated, and shaking fermentation. The inoculation amount of the compound microbial preparation in the fresh culture medium is 8-12%, and the material-to-liquid ratio of the peanut meal to the fresh culture medium is 1:1-1:40 (g / mL). The shaking fermentation conditions include: a temperature of 31-33℃, a rotation speed of 180-220 rpm, and a time of 90-100 h. More specifically, after activating the Enterococcus faecalis strain, the activated bacterial solution is inoculated into a corresponding liquid culture medium at a certain inoculation amount, and peanut meal containing a certain concentration of AFB1 is added for fermentation culture. The toxin degradation effect is then measured.
[0013] The Enterococcus faecalis HB2-2 strain provided by this invention has a strong ability to degrade AFB1. After fermentation for 48 hours with an inoculum of 10%, the degradation rate can reach about 50%, and after optimization, the degradation rate can be as high as 89.9%. The optimal pH value of this strain is 6-8, the optimal temperature is 26-28℃, and the optimal fermentation time is 96 hours.
[0014] According to a specific embodiment of the present invention, Enterococcus faecalis HB2-2 and Rhodococcus were mixed and cultured (cell ratio = 1:1), inoculated into NB liquid medium, and cultured at 28°C and 200 r / min for 12 h with shaking. Then, 10% of the inoculum (i.e., 1 mL of activated bacterial suspension added to 9 mL of fresh liquid medium) was inoculated into fresh liquid medium, and the peanut meal sample to be treated was added. The mixture was fermented at 32°C and 200 r / min on a shaker for 96 h. Samples were taken to determine the AFB1 content in the samples before and after treatment.
[0015] This invention exhibits highly efficient degradation activity against both pure AFB1 and AFB1 in peanut meal. Therefore, the Enterococcus faecalis HB2-2 of this invention has broad application prospects in AFB1 degradation.
[0016] The beneficial technical effects of this invention are as follows: the *Enterococcus faecalis* HB2-2 screened in this invention can efficiently degrade pure AFB1. The microbial preparation prepared by culturing *Enterococcus faecalis* HB2-2 and *Rhodococcus* strains can efficiently degrade AFB1 in peanut meal; this method operates under mild conditions, does not cause material degradation or nutrient loss, and does not result in pollution or harmful substance residues, thus achieving safe and efficient detoxification of peanut meal and related products contaminated with AFB1.
[0017] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description
[0018] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.
[0019] Figure 1 The colony morphology of Enterococcus faecalis HB2-2 of the present invention is shown.
[0020] Figure 2 This is a liquid chromatogram for detecting AFB1 according to the present invention.
[0021] Figure 3 The effect of fermentation temperature on the degradation of pure AFB1 by Enterococcus faecalis HB2-2 of the present invention is shown.
[0022] Figure 4 The effect of inoculum size on the degradation of pure AFB1 by Enterococcus faecalis HB2-2 of the present invention is shown.
[0023] Figure 5 The effect of pH on the degradation of pure AFB1 by Enterococcus faecalis HB2-2 of the present invention is shown.
[0024] Figure 6 The effect of fermentation time on the degradation of pure AFB1 by Enterococcus faecalis HB2-2 of the present invention is shown.
[0025] Figure 7 The effect of the compound microbial preparation of the present invention on the degradation of AFB1 in peanut meal is shown.
[0026] Figure 8 The residual amount of AFB1 after treating peanut meal with the compound microbial preparation of the present invention is shown.
[0027] Biological Preservation Instructions
[0028] Biological material HB2-2, classified as Enterococcus faecium, was deposited on February 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNO.: 26712. Detailed Implementation
[0029] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.
[0030] This invention uses high performance liquid chromatography to determine the content of AFB1.
[0031] Example 1: Isolation and Identification of Enterococcus faecalis HB2-2
[0032] Isolation of Enterococcus faecalis
[0033] Enterococcus faecalis HB2-2 was isolated from a garbage dump within Wuhan University of Light Industry.
[0034] Primary screening medium: (NH4)2SO4 5.0g, KHPO4 2.5g, MgSO4 1.0g, NaHPO4·12H2O 0.5g, CaCl2 0.1g, agar 20g, pH 6.5, sterilized at 121℃ for 20 minutes. Add 1mL of coumarin solution to 100mL of sterilized medium and pour into plates. Solid medium: Add 1.5-2% agar to the above liquid medium components.
[0035] (2) Isolation and purification of strains
[0036] Take 10g of waste, grind it, and add it to 100mL of the above primary screening medium. Shake and culture on a shaker at 28℃ and 200r / min. After the solution changes from clear to turbid, take 10mL of the supernatant and transfer it to a new 100mL of primary screening medium. Shake and culture on a shaker at 28℃ and 200r / min. Repeat this process 3 times. After the culture medium changes from clear to turbid, collect the supernatant and store it.
[0037] Take 1 mL of the supernatant from the above culture and prepare 10 mL of the solution. -1 10 -3 10 -5 10 -6 10 -7 10 -8 Take 100 μL of the diluted solution and spread it onto the corresponding solid culture medium. Incubate at 28℃ for 12-24 h (3 replicates for each dilution) and observe the growth of colonies on the plates. Select clearly visible single colonies and isolate and purify them multiple times to obtain pure strains.
[0038] Single colonies from the above plates were picked and inoculated into NB liquid medium. 100 μL of 11.3 μg / mL AFB1 standard solution was added to bring the final AFB1 concentration in the fermentation broth to 113 ng / mL. The culture was incubated at 28℃ and 200 rpm for 48 h using a constant temperature shaker. The supernatant was then used to determine the corresponding AFB1 content. After repeated experiments, a strain with highly efficient AFB1 degradation was obtained and named HB2-2. Figure 1 As shown.
[0039] Example 2: Identification of Enterococcus faecalis HB2-2
[0040] (1) Colony morphology of Enterococcus faecalis
[0041] Bacterial morphology study: Gram staining was performed on the bacterial cells, and observation was conducted under a 16×100 eyepiece optical microscope as a control. The colony morphology is as follows: Figure 1 As shown. The main biological characteristics of this strain are: after growing on NB solid medium for 24 hours, the colonies are round, moist, milky white in color, with a smooth surface that is easy to pick up, neat edges, and a positive Gram staining reaction.
[0042] (2) Molecular biological identification of HB2-2 strain
[0043] Total DNA was extracted from strain HB2-2, and PCR amplification was performed using universal primers for bacterial 16S rDNA. The amplified products were sequenced, and homology sequence comparison analysis was performed in the NCBI database. The Query Cover of strain HB2-2 with Enterococcus faecium (CP045602.1) was 99%. This strain was identified as Enterococcus genus, presumably Enterococcus faecium, and named Enterococcus faecium HB2-2.
[0044] Example 3: Method for detecting AFB1
[0045] The detection method for AFB1 in this invention is as follows: Weigh 5g of peanut meal into a 50mL centrifuge tube, add 20mL of methanol-water solution (V / V = 70:30), vortex to mix, place in a water bath shaker at room temperature for 20min, centrifuge at 5000r / min for 10min, and collect the supernatant for later use. Add 23mL of PBS solution containing 1% Tween-20 and mix well. Pass the mixture through an immunoaffinity column at natural flow rate, and finally elute the toxin with 1mL of methanol into a test tube. Filter the filtrate through a 0.22μm organic ultrafiltration membrane. The content of AFB1 in the filtrate is determined by high performance liquid chromatography-post-column photochemical derivatization. Figure 2 As shown.
[0046] Example 4: Optimal growth conditions for Enterococcus faecalis HB2-2
[0047] NB liquid culture medium: 1g yeast extract, 5g polypeptone, 15g sucrose, 3g beef extract, add distilled water to a final volume of 1000mL, pH: 6-8, sterilize at 121℃ for 20 minutes.
[0048] (1) Optimal fermentation temperature of the strain of the present invention
[0049] The effect of different fermentation temperatures on the degradation of AFB1 by Enterococcus faecalis HB2-2 was investigated by controlling the fermentation temperature of the bacterial suspension. Single colonies of HB2-2 were selected and inoculated into NB liquid medium, and cultured at 28℃ and 200 rpm for 12 h with shaking. Then, 10% of the inoculum (1 mL of activated bacterial suspension added to 9 mL of the corresponding liquid medium) was inoculated into fresh NB liquid medium, and 100 μL of 11.3 μg / mL AFB1 standard solution was added to bring the final AFB1 concentration in the fermentation broth to 113 ng / mL. Fermentation was carried out at 20℃, 24℃, 28℃, and 30℃ at 200 rpm for 48 h, with untreated medium serving as a blank control. The fermentation broth was centrifuged, the supernatant was discarded, 4 mL of 70% methanol was added, and the mixture was shaken at room temperature for 20 min. The supernatant was centrifuged again, and the AFB1 content was determined by high-performance liquid chromatography (HPLC) to identify the optimal fermentation temperature.
[0050] from Figure 3 It is known that the optimal fermentation temperature for the strain of the present invention to degrade toxins is 32℃, at which the degradation rate of AFB1 by Enterococcus faecalis HB2-2 is 53.49%.
[0051] (2) Optimal inoculum size of the strain of the present invention
[0052] The effect of different inoculum amounts on the degradation of AFB1 by Enterococcus faecalis HB2-2 was investigated by controlling the inoculum size. With other conditions unchanged, inoculum size gradients of 1%, 5%, 10%, and 20% were set, with untreated culture medium serving as a control group. Fermentation was carried out on a shaker at 28℃ and 200 rpm for 48 h. The fermentation broth was centrifuged, the supernatant was discarded, 4 mL of 70% methanol was added, and the mixture was shaken at room temperature for 20 min. The supernatant was then centrifuged again, passed through a column chromatography, and the AFB1 content was determined by high-performance liquid chromatography (HPLC) to determine the optimal inoculum size.
[0053] from Figure 4 It is known that the optimal inoculum amount of Enterococcus faecalis HB2-2 in this invention is 10%, at which point the degradation rate of AFB1 by the strain is 58.93%.
[0054] (3) Optimal pH value of the strain of the present invention
[0055] The effect of varying initial pH values on the degradation of AFB1 by Enterococcus faecalis HB2-2 was investigated. pH gradients of 2, 4, 6, 7, 8, 9, and 10 were established in the culture medium. Other conditions remained constant: an inoculum size of 10%, a fermentation temperature of 28℃, and a rotation speed of 200 rpm. Fermentation was carried out on a shaker for 48 h, with a control group consisting of untreated medium. The AFB1 content in the fermentation broth was measured using the above method to determine the optimal pH value for fermentation.
[0056] from Figure 5It is known that the optimal pH value of Enterococcus faecalis HB2-2 in this invention is 10, at which point the degradation rate of AFB1 by the strain is 69.28%.
[0057] (4) Optimal fermentation time of the strain of the present invention
[0058] The effect of different fermentation times on the degradation of AFB1 by Enterococcus faecalis HB2-2 was investigated by controlling the fermentation time of the bacterial culture. Under the conditions of initial pH 7, inoculum size 10%, fermentation temperature 32℃, and shaking speed 200 r / min, fermentation times were set at 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, with untreated culture medium serving as the control group. The AFB1 content in the fermentation broth was measured according to the above method to determine the optimal fermentation time.
[0059] from Figure 6 It is known that the optimal fermentation time for the strain of the present invention to degrade toxins is 96 hours, and the degradation rate of AFB1 at this time is 89.98%.
[0060] Example 5: Application of the microbial preparation of the present invention in the degradation of AFB1 in peanut meal
[0061] Because AFB1 in peanut meal is encapsulated by cellulose and protein, traditional microorganisms cannot fully contact AFB1 during fermentation, reducing the degradation efficiency of AFB1 in peanut meal. This invention prepares a highly efficient microbial agent for degrading AFB1 in peanut meal by inoculating Enterococcus faecalis HB2-2 and Rhodococcus faecalis at a 1:1 ratio and culturing at 32°C and 200 rpm with shaking for 12 hours. This agent can effectively disrupt the cellulose and protein structures in peanut meal, allowing the encapsulated AFB1 to be completely released and thus fully degraded by the microbial agent of this invention.
[0062] The effect of the solid-liquid ratio on the degradation of AFB1 by the microbial preparation of the present invention was investigated. Enterococcus faecalis HB2-2 and Rhodococcus were co-cultured (cell ratio = 1:1), inoculated onto NB medium, and cultured at 32℃ and 200 rpm with shaking for 12 h. 10% of the bacterial culture was then inoculated onto fresh liquid medium. Peanut meal samples to be treated were added, with solid-liquid ratios (peanut meal: medium volume, g / ml) of 1:1, 1:5, 1:10, 1:15, 1:20, and 1:40, respectively. Fermentation continued for 96 h, and samples were taken to determine the AFB1 content before and after treatment.
[0063] The results are as follows Figure 7 and Figure 8As shown, with the increase of the system solution, the degradation rate of AFB1 in peanut meal gradually increases. When the material-to-liquid ratio is 1:40, the degradation rate of AFB1 in peanut meal by the microbial agent is 89.4%, and the AFB1 content in the fermented peanut meal is reduced to 11.1 μg / kg. The AFB1 content in the detoxified fermented peanut meal is far lower than the national limit of 50 μg / kg for feed. The microbial agent of the present invention is suitable for industrial detoxification of AFB1 in peanut meal.
[0064] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.
Claims
1. A complex microbial formulation, characterized in that, The compound microbial preparation is composed of a strain that degrades aflatoxin B1 and Rhodococcus, characterized in that the strain that degrades aflatoxin B1 is Enterococcus faecium (…). Enterococcus faecium HB2-2, deposited at CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number CGMCC NO.: 26712; the ratio of the number of aflatoxin B1 degrading strain to Rhodococcus cells is 1:0.7-1:1.
3.
2. The complex microbial formulation according to claim 1, characterized in that, Enterococcus faecium (ATCC 19434) Enterococcus faecium The culture method of HB2-2 includes: after the strain is activated, inoculating into the culture medium with initial pH value of 6-8 and temperature of 25-28 ℃ for culture.
3. The compounded microbial preparation according to claim 1, characterized in that, The preparation method of the compound microbial preparation includes: inoculating the aflatoxin B1 degrading strain and Rhodococcus into the culture medium, and culturing at 28-33℃ and 180-220 rpm for 8-16 hours with shaking.
4. The use of the compound microbial preparation according to any one of claims 1-3 in removing aflatoxin B1 from peanut meal.
5. The application according to claim 4, characterized in that, The removal method includes: inoculating the compound microbial preparation into a fresh culture medium, adding the peanut meal to be treated, and shaking for fermentation.
6. Use according to claim 5, characterized in that, The inoculation amount of the compound microbial preparation in the fresh culture medium is 8-12%, and the material-to-liquid ratio of the peanut meal to the fresh culture medium is 1:1-1:40 (g / mL).
7. Use according to claim 5, characterized in that, The conditions for the oscillating fermentation include: a temperature of 31-33℃, a rotation speed of 180-220 rpm, and a time of 90-100 h.