Composite freeze-dried bacterial powder for protecting broiler liver damage and its application

Abstract

The present invention belongs to the technical field of composite bacterial powder used for healthy breeding of broiler chickens, and in particular relates to a composite freeze-dried bacterial powder for protecting broiler chickens from liver damage (especially caused by aflatoxin B1) and its application. The composite bacterial powder contains not less than 1.0×10 Lactobacillus casei per gram. 6 CFU / g, Enterococcus faecalis not less than 2.0×10 6 CFU / g and skimmed milk powder, trehalose, sodium glutamate and silicon dioxide are prepared by vacuum drying; the composite probiotic powder provided by the present invention can significantly alleviate the inflammatory response of chicken embryo primary hepatocytes induced by aflatoxin B1 (AFB1) and reduce inflammatory factors. Adding not less than 1kg / ton of composite bacterial powder to moldy feed can significantly improve the growth performance of broilers and protect the health of broiler liver tissue. The composite bacterial powder of the invention can be used to prevent and treat broiler liver damage induced by mycotoxin pollution, environmental stress and other factors, and improve the production performance and breeding benefits of broilers.

Description

Technical Field

[0001] The invention belongs to the technical field of composite bacterial powder used in broiler breeding, and in particular relates to a composite freeze-dried bacterial powder for protecting broiler liver damage induced by aflatoxin B1 and an application thereof. Background Art

[0002] Mycotoxins are a class of toxic fungal secondary metabolites mainly produced by Aspergillus, Penicillium and Fusarium, which pose a great threat to human and animal health. Aflatoxin is the most toxic and widely polluted fungal toxin, and is widely present in various foods such as nuts, corn and rice. The main toxic structure of aflatoxin is the coumarin ring, which can be divided into four different types of mycotoxins, namely B1, B2, G1 and G2; the toxicity is divided into AFB1>AFG1>AFB2>AFG2. The most toxic aflatoxin B1 (AFB1) is classified as a Class 1 carcinogen by the International Agency for Research on Cancer, and has hepatotoxicity, immunotoxins and teratogenicity. The present invention aims to screen a group of composite probiotics that can effectively alleviate AFB1-induced liver damage in broilers. Summary of the invention

[0003] The purpose of the present invention is to provide a composite freeze-dried bacterial powder for protecting broiler liver damage and application thereof.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] A composite bacterial solution for promoting the proliferation of chicken embryo liver cells, wherein the live bacterial counts of Lactobacillus casei and Enterococcus faecalis are adjusted to 1.0×10 6 CFU / mL, and after compounding at a ratio of 1:2, the supernatant of the composite bacteria could significantly activate the Nrf2 antioxidant pathway, increase the proliferation of chicken embryo primary hepatocytes, and reduce the inflammatory response induced by AFB1.

[0006] A composite freeze-dried powder for protecting broiler liver damage, wherein the bacterial count of Lactobacillus casei and Enterococcus faecalis fermentation liquid is adjusted to 1.0×10 7 CFU / mL, mixed in a volume ratio of 1:2, then added 3-20% skim milk powder, 1-10% trehalose, 1-10% sodium glutamate and 1-10% silicon dioxide, mixed and freeze-dried to obtain freeze-dried bacterial powder. The composite bacterial powder contains no less than 1.0×10 Lactobacillus casei per gram. 6 CFU / g, Enterococcus faecalis not less than 2.0×10 6 CFU / g and skimmed milk powder, trehalose, sodium glutamate and silicon dioxide.

[0007] Adding no less than 1kg / ton of the above-mentioned composite freeze-dried bacterial powder to moldy feed can significantly reduce mycotoxin-induced liver tissue pathological damage, especially aflatoxin-induced broiler liver damage, reduce inflammatory response, and improve production performance. It is used to alleviate mycotoxin-induced broiler liver damage.

[0008] The present invention has the advantages that the composite probiotic powder provided by the present invention significantly reduces the expression of inflammatory factors in liver cells, and the molecular mechanism analysis at the cellular level shows that AFB1 mediates the NOD / MAPK pathway to promote the inflammatory response of chicken embryo liver cells and promote cell apoptosis; the composite probiotic supernatant can activate the Nrf2 antioxidant pathway of chicken embryo liver cells, promote cell proliferation, and reduce apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a diagram showing the effect of CPS4 on the viability of primary cells induced by AFB1 in the present invention.

[0010] Figure 2 Graph showing the effects of different combinations of the present invention on the cell cycle of primary chicken embryo liver cells, wherein B1: control group, B2: AFB1 group, B3: CPS4 group and B4: CPS4+AFB1 group.

[0011] Figure 3 This is a graph showing the percentage of CPS4 and AFB1 in the four cell cycles of primary chicken embryo hepatocytes; CPS4: supernatant of compound probiotics.

[0012] Figure 4 This is the result of RNA-seq in chicken embryo primary hepatocytes induced by CPS4 and AFB1; Note: P<0.05 was used as the threshold between the two groups, and |log2(fold change)|>1 was used as the differentially expressed gene.

[0013] Figure 5 The figure shows the effects of CPS4 and AFB1 on the mRNA and protein expression of inflammation-related genes in primary cells; Figure A: mRNA expression of inflammation-related genes; Figure B: Western Blot inflammation-related protein expression; Figure C: Western Blot grayscale quantitative value.

[0014] Figure 6 The figure is the effect of adding CPS4 and AFB1 on the expression of genes and proteins related to the Nrf2 antioxidant pathway in hepatocytes; Figure A: the expression level of mRNA of antioxidant-related genes; Figure B: Western Blot protein expression diagram; Figure C: Western Blot grayscale quantitative value.

[0015] Figure 7 This is a microscopic observation of broiler livers treated with the composite bacterial powder and toxin group.

[0016] Figure 8 This is the immunohistochemistry result of inflammatory factors in broiler liver tissue by the composite bacterial powder and toxin group. DETAILED DESCRIPTION

[0017] 1.1.1 Activation of strains

[0018] Lactobacillus casein and Enterococcus faecalis were preserved by the Microbial Resource Development and Utilization Laboratory of Huanghuai University. Lactobacillus casein and Enterococcus faecalis were inoculated in MRS medium (g / L): peptone 10, yeast extract 10, glucose 20, Tween 80 1mL, K2HPO42, sodium acetate 5, sodium citrate 2, MgSO40.2, MnSO40.05, pH 6.20-6.60; cultured at 37℃ for 24h. After 24h of microbial culture, the plate count method was used to count the number of viable bacteria, and the number of viable bacteria was adjusted to 1.0×10 6 AFB1 was purchased from Sigma-Aldrich (St. Louis, MO, USA), and diluted with 50% ethanol to a content of 8000 μg / L. Before use, it was filtered with filter paper and then filtered with a 0.22 μm filter membrane. The filtrate was stored at 4°C for subsequent experiments.

[0019] According to the test results, different volume ratios of Lactobacillus casei and Enterococcus faecalis viable counts were prepared:

[0020] CP1: Lactobacillus casei and Enterococcus faecalis mixed in a volume ratio of 1:0

[0021] CP2: Lactobacillus casei and Enterococcus faecalis mixed in a volume ratio of 0:1

[0022] CP3: Lactobacillus casei and Enterococcus faecalis mixed in a volume ratio of 2:1

[0023] CP4: Lactobacillus casei and Enterococcus faecalis mixed in a volume ratio of 1:2

[0024] CP5: Lactobacillus casei and Enterococcus faecalis mixed in a volume ratio of 1:1

[0025] At the same time, the corresponding supernatants were named CPS1, CPS2, CPS3, CPS4 and CPS5.

[0026] 1.1.2 In vitro culture of primary chicken hepatocytes and plotting of growth curve

[0027] 14-day-old fertilized eggs (purchased from Kaifeng Chicken Breeding Co., Ltd., China) were cleaned with 75% alcohol, placed on a vertical flow clean bench, and treated with ultraviolet light for 20 min. The air chamber of the chicken embryo was carefully broken with tweezers, and the embryo was quickly decapitated after removal. The liver tissue was then peeled out and rinsed in phosphate buffer containing 1% penicillin (10,000 U / mL)-streptomycin (10 mg / mL) (Beijing Solar Biotechnology Co., Ltd., Beijing, China).

[0028] The method for preparing hepatocytes is as follows: add 1 mL of collagenase and 1 mL of neutral protease, digest at 37°C for 30 minutes, and shake once every 3 minutes. Then add 2 mL of M199 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. After shaking up and down, collect the filtrate with a 200-mesh sieve, centrifuge at 1000 r / min for 10 minutes, and remove the supernatant. Add 1.5 mL of M199 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin to the centrifuge tube, add 3 mL of 50% Percoll separation solution, mix evenly, and centrifuge at 3000 r / min for 15 minutes. After centrifugation, wash out the upper layer, take the middle layer and put it into a new centrifuge tube, add an equal amount of M199 medium, and centrifuge at 1000 r / min for 10 minutes. Remove the supernatant, and finally resuspend the hepatocytes with M199 medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin, and adjust the cell concentration to 5.0×10 5 Cells were cultured in 96-well or 12-well culture plates at 37°C and 5% CO2 incubator. The cell culture medium was replaced every 2 days.

[0029] 1.2 Effects of different concentrations of aflatoxin B1 on the viability of chicken embryo hepatocytes

[0030] Primary hepatocytes were inoculated in 96-well plates. Cell viability was determined by MTT method every 2 days. The growth curves of the three types of cells were plotted with time as the horizontal axis and absorbance as the vertical axis. The three types of cells were tested uniformly on the 4th day after culture. The hepatocytes were adjusted to a density of 5.0×105 / mL and inoculated into 96-well plates. They were cultured until the 4th day, the culture medium was removed, washed twice with PBS, and acted with different concentrations of aflatoxin B1 for 6, 12, 24, and 48 hours, respectively. The AFB1 concentrations of hepatocytes were 0, 10, 20, 40, and 80 μg / L, respectively. AFB1 was diluted with the corresponding cell culture medium without serum and antibiotics.

[0031] After the cell action was completed, 10 μL of 5 mg / mL MTT was added to each well for 4 h, the cell supernatant was removed, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the plate was shaken at room temperature for 10 min. The absorbance (A) was measured at a wavelength of 490 nm using an ELX800 microplate reader (Bio-TEK Instruments Inc., Winooski, VT, USA), and the wavelength was set to 630 nm. Relative cell viability (%) = (A490 nm to A630 nm of the test group) / (A490 nm to A630 nm of the control group) × 100%.

[0032] Table 1 Effects of different aflatoxin B1 concentrations and action time on primary cell viability (%)

[0033]

[0034]

[0035] 1.3 Effects of composite probiotics (CP) and supernatant (CPS) on the viability of chicken embryo hepatocytes

[0036] Table 2 Effects of different CP or CPS concentrations and action time on primary hepatocyte viability (%)

[0037]

[0038] 1.4 Verification of CPS4 alleviating AFB1-induced chicken embryo hepatocyte toxicity

[0039] According to the previous experiment, the optimal CPS and AFB1 treatment concentrations were determined. The optimal CPS concentration was selected as CPS4 (Lactobacillus casei and Enterococcus faecalis were mixed in a volume ratio of 1:2), the AFB1 treatment concentration was 40μg / mL, and the treatment time was 12h. The experiment was divided into 4 groups: the control group, the AFB1 group, the CPS4 group, and the CPS4+AFB1 group, and the cell viability was measured.

[0040] 1.5 RNA-seq sequencing

[0041] The RNA-seq results of the control group, AFB1 group, CPS4 group and CPS4+AFB1 group were compared. Figure 4. A total of 1835 genes changed compared with the control group, of which 1441 genes were downregulated. A total of 1456 genes changed compared with the AFB1 and CPS4+AFB1 groups, of which 373 genes were downregulated. Figures A and B show the enrichment pathway statistics of the control group VS AFB1 group and the AFB1 group VS CPS4+AFB1 group, respectively. The results suggest that AFB1 mediates the NOD / MAPK pathway to promote the inflammatory response of hepatocytes, and the relief effect of CPS4 may be related to the improvement of the antioxidant capacity of hepatocytes.

[0042] 1.6 Verification of AFB1-mediated NOD / MAPK pathway promoting inflammatory response in chicken embryo hepatocytes

[0043] According to the results of KEGG enrichment pathway experiments, the control group, AFB1, CPS4 and CPS4+AFB14 groups were selected to verify the NOD / MAPK inflammatory pathway. Figure 5 The results showed that AFB1 significantly upregulated the expression of NOD / MEKA inflammatory pathway and TNF-α related genes and protein genes, while CPS significantly reduced the expression of the above genes (P<0.05).

[0044] 1.7CPS4 mediates Nfr2 pathway to alleviate AFB1-induced hepatotoxicity

[0045] Based on the results of the KEGG enrichment pathway experiment, the Nrf2 antioxidant pathway was verified.

[0046] 2. Screening of the optimal dosage of compound probiotics in broiler animal experiments

[0047] 2.1 Effect of adding compound probiotics to moldy diets on broiler production performance

[0048] 2.1.1 Preparation of composite probiotic powder

[0049] According to the results of cell experiments, the number of live bacteria of Lactobacillus casei and Enterococcus faecalis was 1.0×10 6 CFU / mL, the best effect was achieved when the volume ratio was 1:2. The bacterial count of the two probiotics, lactic acid bacteria and Enterococcus faecalis, was adjusted to 1.0×10 7 CFU / mL, (3-20%) skimmed milk powder, (1-10%) trehalose, (1-10%) sodium glutamate and (1-10%) silicon dioxide were added in a weight / volume ratio for freeze-drying to obtain freeze-dried bacterial powder.

[0050] 2.1.2 Design of broiler animal experiment

[0051] A two-stage experiment was conducted, divided into two stages: early stage (1-21d) and late stage (22-42d). In the first stage, 300 healthy AA broilers (half male and half female) at 0 days of age with no difference in weight were selected and divided into 6 treatment groups, with 5 replicates in each treatment group and 10 chickens in each replicate. In the second stage (22-42d), chickens that were too large or too small were removed on the basis of the original grouping, and each group was divided into 8 replicates, with 5 chickens in each replicate. There was no significant difference in weight between the groups (P>0.05). The experimental design and grouping are as follows:

[0052] Group A: The control group was fed with a basic diet

[0053] Group B: Moldy corn group (AFB1 final measured 42.15ug / kg)

[0054] Group C: basic diet + 1kg compound probiotic powder / ton of feed

[0055] Group D: Moldy corn + 0.5kg compound probiotics / ton feed (final AFB1 content 43.16ug / kg)

[0056] Group E: Moldy corn + 1kg compound probiotics / ton feed (final AFB1 content 42.18ug / kg)

[0057] Group F: Moldy corn + 1.5kg compound probiotics / ton feed (final AFB1 content 42.59ug / kg)

[0058] The moldy corn was naturally moldy corn, collected from farmers in the suburbs of Zhengzhou. The moldy corn was crushed and tested for AFB1 toxin content of 70μg / kg, which was used to replace normal corn. The test was conducted at the experimental base of Henan Agricultural University in Xuchang City, Henan Province, and AA broiler chicks were purchased from Kaifeng Xingda Breeding Farm. They were raised in multi-layer cages, with 24h light, free access to food and water, and natural ventilation. The immunization program was: 7-day-old new-branch bivalent vaccine, 21-day-old Newcastle disease live vaccine. The experimental diet was fed regularly at 7:30 and 18:30 every day, and free access to water.

[0059] 2.1.3 Indicator Statistics

[0060] Record the daily feed intake and recover the spilled feed; observe and record the health status, mortality and diarrhea of ​​the broilers. Weigh the broilers after fasting for 12 hours on 21d and 42d, calculate the average daily weight gain (ADG) and average daily feed intake (ADF) of each replicate, and calculate the feed conversion rate (F / G) and mortality rate. Mortality rate = number of deaths / total number of chickens,

[0061] The effect of adding probiotics to moldy diets on the production performance of 1-42d broilers is shown in Table 11. In the first stage, the feed intake of the six treatment groups was not significantly different (P>0.05). In terms of daily weight gain, group C was significantly higher than the other five treatment groups (P<0.05), group B was significantly lower than the other five treatment groups (P<0.05), and there was no significant difference between the other four groups. In terms of feed-to-meat ratio, group D was the highest, and group B was the lowest, which was significantly different from the other groups (P<0.05). There was no significant difference between the other four treatment groups (P>0.05). In the early stage of the entire experiment, deaths occurred in groups B and D, with mortality rates of 2% and 4%, respectively. Comprehensive analysis showed that group F had the best effect in alleviating the toxic and side effects of toxins in the first stage, and there was no significant difference from the control group.

[0062] In the second stage: the three groups D, E and F containing moldy corn and different doses of compound probiotics were compared with group B, showing good effects on increasing daily weight gain and reducing feed conversion rate, among which group F was significantly different from group B (P<0.05). The three compound probiotic groups had no significant difference in feed-to-meat ratio and daily weight gain compared with the control group (P>0.05). This shows that compound probiotics have a good effect in alleviating the toxicity of toxins. Group C does not contain toxins, and has the best effect in feed-to-meat ratio and daily weight gain, indicating that compound probiotics have a good promoting effect on improving the production performance of broilers. Comprehensive analysis shows that in the second stage, groups E and F performed well in alleviating the effects of toxins on broiler growth performance.

[0063] Table 3 Effect of adding probiotics to moldy diets on the production performance of 1-42d broilers

[0064]

[0065] HE staining

[0066] Microscopic damage of toxin group to broiler liver Figure 7 From the figure, we can observe that there are vacuoles in the cytoplasm of the cells, indicating an increase in fat particles in the hepatocytes, lymphocyte infiltration (and diffuse microvascular fatty degeneration. Group C has no lesions, and Group F has basically no obvious lesions, but enlarged intercellular spaces can still be seen.

[0067] Conclusion: Toxins increased liver damage caused by toxins, while probiotics reduced the damage.

[0068] 2.1.4 Immunohistochemistry results

[0069] Table 4 Liver tissue related protein positive cell rate and integrated optical density

[0070]

[0071]

[0072] Conclusion: Toxins increased the expression of three inflammatory factor proteins in the liver, and probiotic powder reduced the expression of these inflammatory factors; finally, adding no less than 1kg of composite probiotic powder to moldy feed can effectively alleviate mycotoxin-induced broiler liver damage and improve production performance. Molecular mechanism analysis at the cellular level showed that AFB1 mediated NOD / MAPK pathway to promote inflammatory response in chicken embryo hepatocytes and promote cell apoptosis; composite probiotic supernatant can activate the Nrf2 antioxidant pathway in chicken embryo hepatocytes, promote cell proliferation, and reduce apoptosis.

Claims

1. Use of a composite bacterial supernatant for promoting the proliferation of chicken embryo liver cells in the preparation of an agent for reducing liver damage in broilers caused by aflatoxin, characterized in that: The preparation method of the composite bacterial supernatant is as follows: Lactobacillus casei and Enterococcus faecalis are inoculated into MRS culture medium respectively, and cultured until the number of viable bacteria is 1.0×10 6 CFU / mL, the bacterial solution was mixed at a volume ratio of 1:2, and the composite bacterial supernatant was obtained by centrifugation. The composite bacterial supernatant activated the Nrf2 antioxidant pathway, increased the proliferation of chicken embryo primary hepatocytes, and reduced the inflammatory response induced by AFB1.

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