CN111281970A

CN111281970A - Composite probiotic fermentation composition and application thereof in preparation of preparation for preventing and treating mycotoxin-induced epithelial cell injury

Info

Publication number: CN111281970A
Application number: CN201811502245.9A
Authority: CN
Inventors: 尹清强; 黄玮玮; 常娟; 王平; 卢富山; 王潇; 宋安东; 刘超齐; 王国强; 耿启泉
Original assignee: HENAN PUAI FEED CO Ltd; Henan Agricultural University
Current assignee: HENAN PUAI FEED CO Ltd; Henan Agricultural University
Priority date: 2018-12-10
Filing date: 2018-12-10
Publication date: 2020-06-16

Abstract

The invention provides a composite probiotic fermented composition and application thereof in preparation of a preparation for relieving epithelial cell damage caused by mycotoxin, belonging to the technical field of mycotoxin degradation, wherein the composite probiotic fermented composition comprises a composite probiotic fermented supernatant and an aflatoxin B1 degrading enzyme-zearalenone degrading enzyme complex enzyme solution; the volume ratio of the composite probiotic fermentation supernatant to the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution is (2.6-3.4): (1.5-2.5). The aflatoxin B1 degrading enzyme activity of the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme compound enzyme liquid is 250-350U/L, and the zearalenone degrading enzyme activity is 25-35U/L. The composite probiotic fermentation composition can effectively relieve epithelial cell injury caused by aflatoxin B1(AFB1) and Zearalenone (ZEA).

Description

Composite probiotic fermentation composition and application thereof in preparation of preparation for preventing and treating mycotoxin-induced epithelial cell injury

Technical Field

The invention belongs to the technical field of mycotoxin degradation, and particularly relates to a composite probiotic fermented composition and application thereof in preparation of a preparation for preventing and treating epithelial cell damage caused by mycotoxin.

Background

Mycotoxins are ubiquitous in human food, animal feed and animal products, as well as in soil. The mycotoxin is derived from secondary metabolites of Aspergillus fungi, Aspergillus fungi and Fusarium, and has high chemical stability. Currently, about 100 fungi are reported to produce about 300 potentially toxic fungal metabolites. Mycotoxins pose a hazard to world human and animal food safety. Among the numerous mycotoxins, aflatoxin B1(AFB1) and Zearalenone (ZEA) are considered to be the major mycotoxin contaminants in feed, agricultural products and by-products. Among them, AFB1 is the most lethal, hepatotoxic, teratogenic and carcinogenic, and is identified by IARC as a class I carcinogen. ZEA has estrogen-like effect, 1/10 is the intensity of estrogen, which can cause the increase of estrogen level of female animals, thereby affecting the reproductive physiology of animals, producing the symptoms like excessive estrogen, such as false heat, abortion, dead fetus, the function and morphological change of reproductive organs, and the like, and further producing cytotoxicity and genetic toxicity, damaging liver and kidney and reducing immune function. Thus, mycotoxin contamination in food and feed has attracted a great deal of attention. At present, the coexistence probability of more than two toxins in production practice is more than 70%, and the harm generated by a plurality of toxins is more than that of one toxin. However, the evaluation of mycotoxin hazards has often been limited to one mycotoxin or class, and the additive and synergistic hazard effects between mycotoxins have been ignored.

Disclosure of Invention

In view of the above, the present invention aims to provide a composite probiotic composition and its application in preparation of a preparation for preventing and treating mycotoxin-induced epithelial cell damage; the composite probiotic composition can effectively relieve epithelial cell injury caused by aflatoxin B1(AFB1) and Zearalenone (ZEA).

In order to achieve the above purpose, the invention provides the following technical scheme: a composite probiotic fermented composition comprises composite probiotic fermented supernatant and aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution; the volume ratio of the composite probiotic fermentation supernatant to the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution is (2.6-3.4): (1.5-2.5);

the composite probiotic fermentation supernatant is obtained by the following method:

fermentation liquor of Lactobacillus casei, Bacillus subtilis and Candida utilis is prepared by mixing the following raw materials in percentage by volume (0.9-1.1): (0.9-1.1): (0.9-1.1) obtaining mixed fermentation liquor by mixing in proportion, and centrifuging the mixed fermentation liquor to obtain composite probiotic fermentation supernatant;

the bacterial concentration of the fermentation liquor of the lactobacillus casei, the bacillus subtilis and the candida utilis is independently (0.1-9) multiplied by 10⁷CFU/mL；

The aflatoxin B1 degrading enzyme activity of the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme compound enzyme liquid is 250-350U/L;

the zearalenone degrading enzyme activity of the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme compound enzyme liquid is 25-35U/L.

Preferably, the preservation number of the Lactobacillus casei is CGMCC 1.2884; the preservation number of the Bacillus subtilis is CGMCC 1.0504; the preservation number of the Candida utilis Candidautilis is CGMCC 2.0615.

Preferably, the volume ratio of the composite probiotic fermentation supernatant to the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution is (2.8-3.2): (1.8-2.2).

Preferably, the ratio of the bacterial concentration of the fermentation liquor of the lactobacillus casei, the bacillus subtilis and the candida utilis is (0.9-1.1): (0.9-1.1): (0.9-1.1).

Preferably, the bacterial concentration of the fermentation liquor of the lactobacillus casei, the bacillus subtilis and the candida utilis is independently (0.8-1.2) multiplied by 10⁷CFU/mL。

Preferably, the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme complex enzyme solution is prepared by the following method:

1) performing solid state fermentation on Aspergillus oryzae at 28-32 ℃ for 6-8 days to obtain an Aspergillus oryzae fermentation product;

2) and mixing and oscillating the aspergillus oryzae fermentation product and DMEM/F12, standing, and filtering to obtain the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme complex enzyme solution.

Preferably, the preservation number of the aspergillus oryzae is CGMCC 5817.

Preferably, the temperature of the mixing oscillation in the step 2) is 28-32 ℃, the time of the mixing oscillation is 50-70 min, and the rotating speed of the mixing oscillation is 100-200 rpm; the standing time is 50-70 min.

Preferably, the filtration in the step 2) sequentially comprises gauze filtration, filter paper filtration and filter membrane filtration.

The invention also provides application of the composition in preparing a preparation for relieving epithelial cell injury caused by mycotoxin.

The invention has the beneficial effects that: the invention provides a composite probiotic fermentation composition, which comprises composite probiotic fermentation supernatant and aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme liquid, wherein the composite probiotic fermentation composition can remarkably up-regulate the relative mRNA abundance (P <0.05) of B lymphocytoma-2 (Bcl-2), aspartic acid proteolytic enzyme 3(caspase-3) containing cysteine, and intestinal tight junction protein (ZO-1 and occludin) genes, and remarkably down-regulate the relative mRNA abundance (P <0.05) of apoptosis genes (Bax), glutamine transporter (ASCT2) and interleukin-6 (IL6) genes; the complex probiotic fermented composition can be seen to effectively relieve epithelial cell damage caused by aflatoxin B1(AFB1) and Zearalenone (ZEA).

The composite probiotic fermented composition can be used for preparing a preparation for relieving epithelial cell injury caused by mycotoxin, and provides a novel biological preparation for reducing the harm of various mycotoxins to animal intestinal tracts and health.

Drawings

FIG. 1 is a graph of the effect of different treatments on the relative LDH release from IPEC-J2 cells in example 1;

FIG. 2 is a graph showing the effect of CFSCP + MDE on the abundance of Bax, Bcl-2 and Caspase-3mRNA in example 1;

FIG. 3 is a graph of the effect of CFSCP + MDE on the abundance of Ocplus and ZO-1mRNA in example 1;

FIG. 4 is a graph of the effect of CFSCP + MDE on the abundance of SGLT1, GLUT2, ASCT2 and PepT1mRNA in example 1;

FIG. 5 is a graph of the effect of CFSCP + MDE on IL6mRNA abundance in example 1.

Detailed Description

The invention provides a composite probiotic fermented composition, which comprises composite probiotic fermented supernatant and aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme liquid; the volume ratio of the composite probiotic fermentation supernatant to the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution is (2.6-3.4): (1.5-2.5);

mixing fermentation liquids of Lactobacillus casei, Bacillus subtilis and Candida utilis to obtain a mixed fermentation liquid, and centrifuging the mixed fermentation liquid to obtain a composite probiotic fermentation supernatant;

In the invention, the composite probiotic fermented composition comprises composite probiotic fermented supernatant. The composite probiotics comprise Lactobacillus casei, Bacillus subtilis and Candida utilis; in the invention, the Lactobacillus casei, the Bacillus subtilis and the Candida utilis are preferably purchased from China general microbiological culture collection center (CGMCC); the preservation number of the Lactobacillus casei is CGMCC 1.2884; the preservation number of the Bacillus subtilis is CGMCC 1.0504; the preservation number of the Candida utilis is CGMCC 2.0615.

In the invention, the composite probiotic fermentation supernatant is prepared by the following method: mixing fermentation liquids of lactobacillus casei, bacillus subtilis and candida utilis to obtain a mixed fermentation liquid, and centrifuging the mixed fermentation liquid to obtain a composite probiotic fermentation supernatant.

The fermentation liquid of the lactobacillus casei is preferably obtained by inoculating the lactobacillus casei into an MRS culture medium, standing and culturing for 22-26 h at 36-38 ℃. The bacillus subtilis fermentation liquor is preferably obtained by inoculating the bacillus subtilis into an LB culture medium and performing shake culture at 36-38 ℃ for 22-26 h, wherein the rotation speed of the shake culture is preferably 160-200 rpm, and more preferably 180 rpm. The Candida utilis fermentation liquor is preferably obtained by inoculating the Candida utilis into a YPD culture medium and performing shake culture at the temperature of 28-32 ℃ for 22-26 h; the rotation speed of the shaking culture is preferably 160-200 rpm, and more preferably 180 rpm.

After the three fermentation liquors are obtained by respective culture, the concentration of the fermentation liquor bacteria of the lactobacillus casei, the bacillus subtilis and the candida utilis is preferably adjusted to (0.1-9) multiplied by 10⁷CFU/mL, more preferably (0.8 to 1.2). times.10⁷CFU/mL, most preferably 1.0X 10⁷And after CFU/mL, mixing the three fermentation liquors to obtain a mixed fermentation liquor, wherein the volume ratio of the three fermentation liquors to be mixed is preferably (0.9-1.1): (0.9-1.1): (0.9-1.1), more preferably 1:1: 1.

After the mixed fermentation broth is obtained, the mixed fermentation broth is centrifuged to obtain composite probiotic fermentation supernatant. In the invention, the rotation speed of the centrifugation is preferably 2500-3500 rpm, more preferably 2800-3200 rpm, and most preferably 3000 rpm; the time for centrifugation is preferably 10-20 min, more preferably 13-17 min, and most preferably 15 min. In the invention, after the centrifugation, a centrifugation supernatant is collected, preferably, the centrifugation supernatant is subjected to filter membrane filtration, and a filtrate is collected to be the composite probiotic fermentation supernatant (hereinafter referred to as CFSCP). In the present invention, the pore size of the filter is preferably 0.22. mu.m.

In the invention, the composite probiotic fermentation composition comprises an aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution; the aflatoxin B1 degrading enzyme activity of the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme compound enzyme liquid is 250-350U/L, and preferably 280-320U/L; the zearalenone degrading enzyme activity of the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme compound enzyme liquid is preferably 25-35U/L, and more preferably 28-32U/L. The source of the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme complex enzyme solution is not specially limited, and the complex enzyme solution only has enzyme activities corresponding to the two enzymes; the enzyme solution may be obtained by mixing two commercially available enzyme solutions, or may be prepared from a fermentation product of a microorganism producing the two enzymes.

In the specific implementation process of the invention, the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme complex enzyme solution is prepared by the following method: 1) performing solid state fermentation on Aspergillus oryzae at 28-32 ℃ for 6-8 days to obtain an Aspergillus oryzae fermentation product; 2) and mixing and oscillating the aspergillus oryzae fermentation product and DMEM/F12, standing, and filtering to obtain the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme complex enzyme solution.

In the invention, the Aspergillus oryzae is preferably purchased from China general microbiological culture Collection center (CGMCC), and the preservation number of the Aspergillus oryzae is CGMCC 5817. Inoculating the aspergillus oryzae into a solid culture medium to perform solid fermentation to obtain an aspergillus oryzae fermentation product; the solid culture medium preferably comprises bran, bean pulp, corn and water; the mass ratio of the bran to the bean pulp to the corn to the water is preferably (6-8): 1.5-2.5): 1: (5.5-6.5), and more preferably 7:2:1: 6. In the invention, the solid culture medium is preferably inoculated with the aspergillus oryzae spore suspension after high-temperature sterilization, and the concentration of the aspergillus oryzae spore suspension is preferably 10⁷～10⁹spore/mL, more preferably 10⁸spore/mL; the inoculated Aspergillus oryzae sporeThe mass-to-volume ratio of the suspension to the solid medium is preferably 1 ml: (10-14) g, more preferably 1 ml: 12 g. The high-temperature moist heat sterilization parameters are not particularly limited, and the conventional parameters in the field can be adopted; the temperature of high temperature moist heat sterilization is preferably 121 deg.C, and the pressure is preferably 1.034 × 10⁵Pa, time is preferably 20 min. The temperature of the solid state fermentation in the present invention is preferably 30 ℃ and the time is preferably 7 days.

According to the invention, after the solid state fermentation is finished, an Aspergillus oryzae fermentation product is obtained, and the Aspergillus oryzae fermentation product and DMEM/F12 are mixed, vibrated and then kept stand. In the invention, the mass ratio of the Aspergillus oryzae fermentation product to DMEM/F12 is preferably 1: (2.5 to 3.5), more preferably 1 (2.9 to 3.1). In the present invention, the DMEM/F12(w/v) is a conventional commercially available product. The mixing and oscillating temperature is preferably 28-32 ℃, more preferably 29-31 ℃, and most preferably 30 ℃; the mixing and oscillating time is preferably 50-70 min, more preferably 55-65 min, and most preferably 60 min; the rotation speed of the mixing oscillation is preferably 100-200 rpm, and more preferably 120-180 rpm; the standing time is preferably 50-70 min, more preferably 55-65 min, and most preferably 60 min.

After standing, the feed liquid after standing is filtered to obtain the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme complex enzyme liquid. In the present invention, the filtration preferably includes gauze filtration, filter paper filtration, and membrane filtration in this order. In the invention, the gauze filtration preferably adopts 6-10 layers of gauze filtration, and more preferably 8 layers; the filter paper filtration is preferably carried out using Whatman No.4 filter paper. After the filter paper is filtered and before the filter paper is filtered, the method preferably further comprises the step of centrifuging the filtrate filtered by the filter paper; the rotation speed of the centrifugation is preferably 2500-3500 rpm, more preferably 2800-3200 rpm, and most preferably 3000 rpm; the time for centrifugation is preferably 10-20 min, more preferably 13-17 min, and most preferably 15 min. The supernatant after centrifugation is collected and filtered by a filter membrane, and the pore diameter of the filter membrane for filtering the filter membrane is preferably 0.22 mu m.

In the invention, the volume ratio of the composite probiotic fermentation supernatant to the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution is (2.6-3.4): (1.5-2.5); the selection is (2.8-3.2): (1.8-2.2), more preferably 3: 2. In the invention, the composite probiotic fermentation supernatant has the function of degrading aflatoxin B1 and zearalenone; the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme compound enzyme liquid also has the effect of degrading aflatoxin B1 and zearalenone, and the compound probiotic fermentation supernatant and the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme compound enzyme liquid are mixed according to the proportion, so that the synergistic effect of degrading aflatoxin B1 and zearalenone is increased.

The invention also provides application of the composition in preparing a preparation for relieving epithelial cell injury caused by mycotoxin. In the invention, the preparation for relieving epithelial cell injury caused by mycotoxin preferably also comprises auxiliary materials, and the preparation has no special requirement on the types of the auxiliary materials and only needs the auxiliary materials acceptable by the conventional compound probiotic fermented composition; the dosage form of the preparation is not particularly required by the invention, and the preparation can be prepared by the conventional dosage form in the field. In the present invention, the epithelial cell damage caused by mycotoxins is preferably epithelial cell damage caused by a plurality of mycotoxins, more preferably epithelial cell damage caused by aflatoxin B1(AFB1) and Zearalenone (ZEA); the epithelial cells are preferably animal epithelial cells, more preferably porcine epithelial cells.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1.1 chemical reagents

Aflatoxin B1(AFB1) and Zearalenone (ZEA) were purchased from Sigma, USA, with a purity of > 99%, and dissolved in DMSO and absolute ethanol, respectively, to give stock solutions with concentrations of 1mg/mL and 10mg/mL, respectively. The stock was diluted to toxin solutions of different concentrations according to the experimental design with the cell culture medium DMEM/F12 containing 10% fetal bovine serum. Pancreatin, PBS and MTT were all purchased from shanghai solibao. The final DMSO concentration and the absolute ethanol concentration in the cell culture medium were 0.1% and 0.5% (v/v), respectively.

The IPEC-J2 cell line (cat # C0668, available from Shanghai Feite laboratory science and technology Co., Ltd.) is a continuously cultured intestinal epithelial cell line derived from the jejunal epithelium of newborn piglets. The cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum at 5X 10⁵one/mL was inoculated at 25cm²Culturing in a cell culture flask at 37 deg.C and 5% CO₂Passage was performed twice weekly in a cell incubator.

1.2 preparation of the Strain

Aspergillus oryzae (A. oryzae) CGMCC 5817, Lactobacillus casei (L.casei) CGMCC1.2884, Bacillus subtilis CGMCC1.0504 and Candida utilis (C.utilis) CGMCC2.0615 are purchased from China general microbiological culture Collection center (CGMCC). Culturing subtilis in LB culture medium, shaking at 37 deg.c and 180rpm for 24 hr; and L, culturing casein in an MRS culture medium, and performing static culture at 37 ℃ for 24 h. Utilis was cultured in YPD medium, shaking at 30 ℃, 180rpm for 24h, and three strains were kept after 24h of culture. The number of viable bacteria of the three probiotics is adjusted to 1 × 10⁷CFU/mL, mixing at a ratio of 1:1:1, centrifuging at 3000rpm for 15min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain sterile composite probiotic supernatant (CFSCP).

Solid state fermentation a. oryzae preparation of AFB1 and ZEA degradation crude enzyme solutions: adding 15g of solids into bran, soybean meal and corn according to the mass ratio of 7:2:1, and then adding the solid: distilled water (5: 3 by weight) 9mL of distilled water was added, the mixture was stirred in a 250mL Erlenmeyer flask, and the mixture was heated at 121 ℃ and 1.034X 10⁵Sterilizing with high pressure steam under Pa for 20min, cooling, inoculating 2mL of 1 × 10⁸spores/mL of spore suspension. Culturing at 30 deg.C for 7 d. Extracting the fermented crude enzyme solution, adding 75ml of MEM/F12(w/v) into the solid fermented product, shaking at 150rpm at 30 deg.C for 1h, and standing for 1 h. Then filtering with 8 layers of gauze, collecting filtrate, filtering with Whatman No.4 filter paper, collecting filtrate, centrifuging at 3000rpm for 15min to remove precipitate. And finally, filtering and centrifuging by using a 0.22-micron filter membrane to obtain supernatant, collecting filtrate filtered by the filter membrane to obtain crude aspergillus oryzae enzyme solution, and storing at 4 ℃ for later use. The measured AFB1 degrading enzyme activity of the crude Aspergillus oryzae enzyme solution is 284.3U/L, and the measured ZEA degrading enzyme activity is 31.0U/L.

1.3 cell viability assay

Pancreatizing cells in logarithmic growth phase (80% confluency, about 48h), preparing cell suspension with DMEM, adjusting cell concentration, inoculating to 96-well plate to ensure that the number of cells in each well is 4000, and placing the cell culture plate in CO₂Culturing in incubator at 37 deg.C and 70% humidity for 24 hr, discarding culture medium, adding 100 μ L culture medium containing aflatoxin B1 alone or zearalenone or two mixed mycotoxins, and culturing in cell culture incubator for 24 hr. Then 10. mu.L of MTT (5mg/mL) was added to each well, left to stand at 37 ℃ for 4 hours, 150. mu.L of DMSO was added, and the mixture was shaken in a shaker for 10 min. Absorbance was measured at 490nm and IPEC-J2 relative cell viability was expressed as the ratio of OD of treated cells to OD of untreated cells.

1.4 design of the experiment

The test was divided into five groups:

control group (toxin-free group)

Toxin addition group Z500 or Z1000 (addition of ZEA:500 or 1000. mu.g/L broth);

toxin addition groups A40 or A80 (addition of AFB1:40 or 80. mu.g/L);

toxin addition group Z500+ A40 or Z1000+ A80 (addition of AFB1+ ZEA:500+40 or 1000+ 80. mu.g/L, respectively);

the toxin addition group Z500+ A40+ CFSCP + MDE (AFB1+ ZEA:500+ 40. mu.g/L), the volume of the whole reaction is 100. mu.L, wherein the addition amount of CFSCP + MDE is 5. mu.L, and the two are combined according to the volume ratio of 3: 2.

1.5 apoptosis assay

After 24h of mycotoxin treatment, the cells were washed 2 times with 1mL of PBS, then the cells were digested with trypsin without EDTA, centrifuged at 800rpm for 5min at 4 ℃, and the supernatant was discarded to collect the cells. Resuspending the cells in PBS, collecting the cell suspension, centrifuging at 4 deg.C and 800rpm for 5min, discarding the supernatant, and adding 500. mu.L of 1 × bindingbuffer (10 × biningbuffer diluted with distilled water 1: 9), 5 μ L of Annexin-FITC and 10 μ L of PI were added in order and mixed gently. The reaction was carried out at room temperature in the dark for 30min, and a flow cytometer BD FACSCAnto was used^TMAnd II, performing flow detection. The detection conditions are as follows: the excitation wavelength Ex is 488nm, and the emission wavelength Em is 530 nm.

1.6 LDH assay

LDH assay was performed using Biyuntian LDH assay kit, cells were plated in 96-well plates (4000 cells/well), cultured for 24h, then treated with mycotoxins for 24h, cell supernatants (for detecting LDH in supernatants) were collected, 100. mu.L of a medium containing a lysate was added to the cells at the bottom of the wells, and 5% CO was added at 37 ℃ to assay₂Incubate for 45min in the incubator. After collecting the cells and cell lysate, the cells were centrifuged at 250rpm for 4min, and 50. mu.L of supernatant (for detecting LDH in the cells) was collected. Transferring the cell supernatant and the cell lysis supernatant to a new 96-well plate respectively, adding 50 mu L of substrate mixture into each well, covering the 96-well plate with tinfoil paper to avoid light, incubating at room temperature for 30min, adding 50 mu L of stop solution into each well, and detecting the light absorption value on a microplate reader, wherein the detection wavelength is 492 nm. LDH activity-treated/control LDH release.

1.7 fluorescent quantitative PCR assay of apoptosis, intestinal barrier function, nutrient transport function, inflammatory factors and function-related genes

IPEC-J2 cells were seeded in 6-well plates (Costar, Corning, NY, USA) ensuring 3X 10 cells per well⁵Cells, after 24h cell adherence, were washed once with PBS and then treated with serum-free AFB1 and ZEA containing media for 24 h. Total RNA extraction was performed by Trizol (Invitrogen, Carlsbad, Calif., USA). The extracted RNA was dissolved in 30. mu.L of RNase-free water and stored at-80 ℃ for future use. RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE). Reverse transcription of RNA into cDNA, the reverse transcription procedure was performed according to Bao bioengineering (Dalian) Co., Ltd

RT reagentKit reverse transcription kit instruction, reverse transcription of cDNA at-20 ℃ storage. The primers are composed ofSynthesized by Shanghai bioengineering technology, Inc. (see Table 1).

1.8 data analysis

Each treatment was tested in 6 replicates. Experimental data were analyzed using statistical ANOVA variance using SPSS20.0 software, with results expressed as mean ± standard deviation and significant differences expressed as P < 0.05.

TABLE 1 RT-PCR primer design

2. Test results

2.1 Effect relationship between AFB1 and ZEA on IPEC-J2 cell viability time and concentration

The optimal concentration and action time of AFB1 and ZEA for causing cell damage are determined, and the test is prepared for the next step. The time and concentration relationship results of AFB1 and ZEA on the cell viability are shown in Table 2, and the data in the table show that the damage of two toxins on cells is larger than that of a single toxin, and the optimal concentration of AFB1 and ZEA for causing cell damage is Z500+ A40, and the action time is 24 h.

TABLE 2 time-dose Effect of two mycotoxins on the relative viability of cells

Note: the same lower case letters in the same column indicate no significant difference (p >0.05), and the different lower case letters in the same column indicate significant difference (p < 0.05). Capital letters in the same row are the same and mean no significant difference (p >0.05), and capital letters in the same row are different and mean significant difference (p < 0.05). IPEC-J2 relative cell viability (%) (treatment group a 490-treatment group a 630)/(control group a 490-control group a630) × 100.

2.2 mitigating Effect of CFSCP + MDE on cellular injury caused by AFB1 and ZEA

The results of necrotic cells (Q1), late apoptotic cells (Q2), early apoptotic cells (Q3), and viable cells (Q4) in table 3 indicate that the necrotic cell rate, late apoptotic cell rate, early apoptotic cell rate, and viable cell rate were significantly lower for the a40, Z500+ a40, CFSCP + MDE, and Z500+ a40+ CFSCP + MDE groups than for the control group (P <0.05), with the exception of the late apoptotic cell rate and early apoptotic cell rate for the Z500 group. Cell mortality increased with the addition of AFB1 and ZEA (P <0.05), but after addition of CFSCP + MDE, cell mortality could be significantly reduced (P < 0.05). The early and late apoptosis rates of cells were significantly higher in the a40, Z500+ a40, and Z500+ a40+ CFSCP + MDE groups than in the control and Z500 groups (P < 0.05). Compared with the control group, the viable cell rate is reduced with the addition of AFB1 and ZEA (P <0.05), but after the addition of CFSCP + MDE, cytotoxicity caused by AFB1 and ZEA can be remarkably reduced, and the viable cell rate is increased (P < 0.05).

2.3 Effect of CFSCP + MDE on IPEC-J2 cell LDH

In general, IPEC-J2 cell membrane integrity is determined by LDH released into the medium, with higher LDH representing more cell damage. Figure 1 shows that when AFB1 concentration reached 40 μ g/L, LDH release was significantly increased relative to other groups (P <0.05), indicating that AFB1 is prone to cause cell damage. However, when 500. mu.g/L of ZEA was combined with 40. mu.g/LAFB 1, LDH release was significantly reduced (P < 0.05). Also, the addition of CFSCP + MDE had less effect on decreasing LDH activity (P > 0.05).

TABLE 3 Effect of CFSCP + MDE on IPEC-J2 apoptosis (n ═ 6)

Note: the data is the same as the lower case letters in the same column and indicates no significant difference (p >0.05), and the lower case letters in the same column and indicates significant difference (p < 0.05). Q1, Q2, Q3 and Q4 indicate cell death rate, late apoptosis rate, early apoptosis rate and viable cell rate, respectively.

2.4 Effect of CFSCP + MDE on AFB1 and ZEA causing IPEC-J2 cytokine mRNA abundance

Compared with other groups, the relative mRNA abundances of the B-lymphocytoma-2 (Bcl-2) and the cysteine-containing aspartate proteolytic enzyme 3(caspase-3) of the Z500+ A40+ CFSCP + MDE group are significantly up-regulated, and the expression level of the apoptosis gene (Bax) is significantly down-regulated (p < 0.05). Bax relative mRNA abundance of a40 and Z500+ a40 was significantly up-regulated (p <0.05), while Bcl-2 was significantly down-regulated. Caspase-3 was significantly downregulated (p <0.05) for the Z500 and A40 groups (see FIG. 2). In general, Bcl-2 inhibits apoptosis, while Bax and Caspase-3 promote apoptosis, suggesting that the addition of CFSCP + MDE may play a role in inhibiting apoptosis.

FIG. 3 shows that the relative mRNA abundances of ZO-1 and occludin were significantly up-regulated in the Z500+ A40+ CFSCP + MDE group relative to the other groups (p <0.05), and the relative mRNA expression of occludin was significantly lower in the A40 group than in the Z500+ A40 and Z500+ A40+ CFSCP + MDE groups (p < 0.05). The relative amounts of ZO-1mRNA expression were significantly lower in groups a40 and Z500+ a40 than in the other three groups (p < 0.05). ZO-1 and occludin are 2 intestinal tight junction proteins, and the addition of CFSCP + MDE can up-regulate the expression genes of the two proteins, thus indicating that the intestinal structure becomes tighter under the stimulation of mycotoxin to resist the harm of the mycotoxin.

Fig. 4 shows that the expression level of the glutamine transporter (ASCT2) mRNA was significantly lower in the Z500+ a40+ CFSCP + MDE group than in the other groups (p <0.05), whereas the expression level of the mRNA in SGLT1 was significantly higher in the Z500+ a40 group than in the other groups (p <0.05), although the addition of CFSCP + MDE restored the mRNA expression of sodium glucose co-transporter 1(SGLT1) to the normal level in the control group. The mRNA expression level of peptide transporter 1(PepT1) was significantly lower in the three groups, all containing AFB1, than in the control group (p < 0.05). The glucose transporter 2(GLUT2) mRNA expression levels in the five groups differed insignificantly (p > 0.05). The down-regulation of ASCT2 helped to inhibit tumor cell proliferation, indicating that the addition of CFSCP + MDE was beneficial in mitigating AFB1 and ZEA-induced damage to cells. SGLT1 plays an important role in the active absorption of sodium ions and glucose by intestinal epithelial cells, and the addition of CFSCP + MDE restores the normal level of sodium ions and glucose absorption by the intestinal tract. PepT1 is a peptide vector with low affinity and high capacity, and the down regulation of the PepT1 indicates that AFB1 seriously influences the absorption of nutrients such as peptide in the intestinal tract.

FIG. 5 shows that AFB1 alone or in combination with ZEA significantly up-regulated the expression level of interleukin-6 (IL6) mRNA (p < 0.05). However, addition of CFSCP + MDE significantly down-regulated IL6mRNA expression (p < 0.05). Since IL6 is the main signal factor of intestinal immune and inflammatory reaction, mycotoxin can improve the expression level of IL6 by stimulating the immune system of the body, and the addition of CFSCP + MDE can reduce or eliminate the stress caused by mycotoxin, thereby having important significance for protecting intestinal epithelial cells.

The invention shows that the high-efficiency combination of the composite probiotics and the mycotoxin can relieve the damage and necrosis of ZEA and AFB1 to porcine intestinal cells, and improve the immune response of an organism and the transportation and absorption of nutrient substances. Provides a new solution for the biodegradation of mycotoxin and protecting the body from the harm of mycotoxin.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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HENAN AGRICULTURAL University

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Claims

1. A composite probiotic fermented composition comprises composite probiotic fermented supernatant and aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution; the volume ratio of the composite probiotic fermentation supernatant to the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution is (2.6-3.4): (1.5-2.5);

fermentation liquor of Lactobacillus casei, Bacillus subtilis and Candida utilis is prepared by mixing the following raw materials in a volume ratio of (0.9-1.1): (0.9-1.1): (0.9-1.1) mixing in proportion to obtain mixed fermentation liquor, and centrifuging the mixed fermentation liquor to obtain composite probiotic fermentation supernatant;

The activity of the aflatoxin B1 degrading enzyme in the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme compound enzyme liquid is 250-350U/L;

the activity of the zearalenone degrading enzyme in the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme compound enzyme liquid is 25-35U/L.

2. The composition of claim 1, wherein the Lactobacillus casei has a deposit number of CGMCC No. 1.2884; the preservation number of the Bacillus subtilis is CGMCC No. 1.0504; the preservation number of the Candida utilis is CGMCC No. 2.0615.

3. The composition according to claim 1 or 2, wherein the volume ratio of the composite probiotic fermentation supernatant to the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme composite enzyme solution is (2.8-3.2): (1.8-2.2).

4. The composition as claimed in claim 1 or 2, wherein the ratio of the bacterial concentration of the fermentation liquor of lactobacillus casei, bacillus subtilis and candida utilis is (0.9-1.1): (0.9-1.1): (0.9-1.1).

5. The composition of claim 4, wherein the fermentation broth of Lactobacillus casei, Bacillus subtilis, and Candida utilis has a bacterial concentration of (0.8-1.2) x 10⁷CFU/mL。

6. The composition as claimed in claim 1, wherein the aflatoxin B1 degrading enzyme-zearalenone degrading enzyme complex enzyme solution is prepared by a method comprising the following steps:

7. The composition of claim 6, wherein the Aspergillus oryzae has a accession number of CGMCC 5817.

8. The composition according to claim 6 or 7, wherein the temperature of the mixing oscillation in the step 2) is 28-32 ℃, the time of the mixing oscillation is 50-70 min, and the rotation speed of the mixing oscillation is 100-200 rpm; the standing time is 50-70 min.

9. The composition according to claim 6 or 7, wherein the filtration in step 2) comprises gauze filtration, filter paper filtration and membrane filtration in sequence.

10. Use of a composition according to any one of claims 1 to 9 for the preparation of a formulation for the prevention and treatment of epithelial cell damage caused by mycotoxins.