阅读说明：本技术 甘草酸与益生菌配伍作为缓解呕吐毒素危害的饲料添加剂 (Glycyrrhizic acid and probiotics are combined to be used as feed additive for relieving vomitoxin harm ) 是由 尹清强 许小向 常娟 王平 卢富山 王潇 刘超齐 党晓伟 朱群 李茂龙 张瑞 于 2020-06-18 设计创作，主要内容包括：本发明属于饲料添加剂技术领域,具体涉及甘草酸和益生菌配伍作为缓解呕吐毒素的饲料添加剂。本发明通过研究呕吐毒素诱导肠上皮细胞炎症和凋亡的分子机制,阐明了甘草酸对呕吐毒素诱导的细胞炎症和凋亡的修复机制,在此基础上本发明提出了甘草酸在制备缓解呕吐毒素危害的药物或饲料添加剂中的应用,并提供了一种甘草酸和益生菌配伍的饲料添加剂,为缓解呕吐毒素对动物的毒性、改善畜禽健康状况奠定基础。(The invention belongs to the technical field of feed additives, and particularly relates to a feed additive for relieving vomitoxin, which is prepared by mixing glycyrrhizic acid and probiotics. The invention clarifies a repairing mechanism of glycyrrhizic acid on the vomitoxin-induced cell inflammation and apoptosis by researching a molecular mechanism of vomitoxin-induced intestinal epithelial cell inflammation and apoptosis, provides an application of glycyrrhizic acid in preparing a medicine or feed additive for relieving the harm of vomitoxin on the basis, provides a feed additive prepared by mixing glycyrrhizic acid and probiotics, and lays a foundation for relieving the toxicity of vomitoxin to animals and improving the health condition of livestock and poultry.)

1. Application of glycyrrhizic acid in preparing medicine or feed additive for relieving vomitoxin harm is provided.

2. The use of claim 1, wherein: glycyrrhizic acid relieves cellular inflammation or cellular premature aging caused by vomitoxin by regulating the relative expression of genes IL-6, IL-8, TNF-alpha, COX-2, NF-kb, Bax, Caspase3, or Bcl-2.

3. The use of claim 1, wherein: glycyrrhetinic acid can relieve cellular oxidative stress reaction caused by vomitoxin by regulating the content of lactate dehydrogenase, malondialdehyde, catalase and superoxide dismutase.

4. Use according to claim 2 or 3, characterized in that: the cell is a porcine intestinal cell.

5. The use of claim 1, wherein: the addition amount of glycyrrhizic acid is 50-800 mug/mL.

6. A feed additive for relieving harm of vomitoxin comprises glycyrrhizic acid, yeast and enterococcus.

7. The feed additive according to claim 6, wherein the feed additive is added in an amount of 200-400 mg/kg glycyrrhizic acid feed and 0.5 × 10 yeast⁹~5×10⁹CFU/kg fodder, enterococcus 0.5 × 10⁹~5×10⁹CFU/kg feed.

8. The feed additive according to claim 7, wherein the feed additive is added in an amount of 400mg/kg glycyrrhizic acid feed and 1 × 10 mg yeast⁹CFU/kg fodder, enterococcus 1 × 10⁹CFU/kg feed.

9. The feed additive of claim 6, wherein: the yeast is candida utilis; the enterococcus is enterococcus faecalis.

10. Use of the feed additive according to claim 6 for alleviating the decline in growth performance of piglets caused by vomitoxin.

Technical Field

The invention belongs to the technical field of feed additives, and particularly relates to a feed additive for relieving vomitoxin, which is prepared by mixing glycyrrhizic acid and probiotics.

Background

Deoxynivalenol (DON), also known as vomitoxin, is a type B trichothecene toxin produced by gibberella zeae and mainly exists in cereals such as wheat, corn, oat, barley and the like. DON has toxic effects on humans and many animals, with pigs being one of the most sensitive to DON, causing vomiting, diarrhea, slow growth, immune system disorders and economic losses. The intestinal tract of animals is the first barrier for the body to resist the invasion of external harmful substances, and the intestinal mucosa is an important barrier for absorbing and transporting various nutrient substances such as glucose, amino acid, trace elements and the like. DON can cause increased intestinal permeability, damaged and necrotic mucosa, apoptosis, decreased antioxidant capacity and mitochondrial dysfunction, leading to damage to porcine intestinal tract. Tight junction proteins are connective multiprotein complexes that primarily maintain the intestinal epithelial barrier, causing body injury and inflammation, even affecting transport and absorption of nutrients, when they are disrupted by environmental conditions (mycotoxins, environmental changes, feed replacement, etc.).

The licorice is a perennial herb and has good detoxification function, the licorice extract is widely applied to animal husbandry to promote animal growth, improve meat quality and prevent and treat various livestock and poultry diseases, and in addition, the licorice extract is reported in documents to be clinically used for treating liver injury. Glycyrrhizic Acid (GA) is an extract of liquorice, has various pharmacological activities including anti-inflammatory, immunity regulating, antioxidant, antiviral, anticancer, blood lipid reducing and the like, and can be used for treating or relieving intestinal toxicity and inflammation caused by DON, so that an effective and economical veterinary drug or feed additive is provided.

The probiotics are live bacteria preparations or metabolites thereof which play a beneficial role by improving the micro-ecological balance of the gastrointestinal tract of a host and achieve the purpose of improving the health level and the health state of the host, and beneficial bacteria or fungi in an animal body mainly comprise: lactobacillus, bifidobacterium, actinomycetes, yeast, enterobacter and the like. The probiotics have the effects of regulating the animal gastrointestinal microflora, degrading mycotoxin, relieving immunosuppression, inhibiting the growth and reproduction of harmful bacteria, promoting animal production and the like. The invention provides a feed additive for effectively relieving harm of vomitoxin to intestinal cells, which is prepared by matching glycyrrhizic acid with probiotics, and the feed additive is not reported in the prior art, so that the invention is a main technical problem to be solved.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide glycyrrhizic acid and probiotics which are compatible to be used as a feed additive for relieving DON; the invention provides a feed additive prepared by mixing glycyrrhizic acid and probiotics on the basis of clarifying a repair mechanism of DON-induced cell inflammation and apoptosis by researching a molecular mechanism of DON-induced intestinal epithelial cell inflammation and apoptosis, thereby laying a foundation for relieving the toxicity of DON to animals and improving the health condition of livestock and poultry.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

application of glycyrrhizic acid in preparing medicament or feed additive for relieving DON harm is provided.

Preferably, glycyrrhizic acid relieves cell inflammation or cell premature senility caused by DON by regulating the relative expression of genes IL-6, IL-8, TNF-alpha, COX-2, NF-Kb, Bax, Caspase3 or Bcl-2.

Preferably, glycyrrhizic acid relieves cellular oxidative stress caused by DON by adjusting the levels of lactate dehydrogenase, malondialdehyde, catalase and superoxide dismutase.

Further preferably, the cell is a porcine intestinal cell.

Preferably, the addition amount of the glycyrrhizic acid is 50-800 mug/mL.

Based on a general inventive concept, the present invention further comprises a feed additive for alleviating the hazards of DON, the feed additive comprising glycyrrhizic acid, yeast and enterococcus.

Preferably, the addition amount of the feed additive is 200-400 mg/kg of glycyrrhizic acid feed and 0.5 × 10 of yeast⁹～5× 10⁹CFU/kg fodder, enterococcus 0.5 × 10⁹～5×10⁹CFU/kg feed.

More preferably, the feed additive is added with glycyrrhizic acid 400mg/kg feed and yeast 1 × 10⁹CFU/kg fodder, enterococcus 1 × 10⁹CFU/kg feed.

Preferably, the yeast is candida utilis; the enterococcus is enterococcus faecalis.

Based on a general inventive concept, the invention also comprises the use of said feed additive for alleviating the decline of piglet growth performance caused by DON.

The research result of the invention shows that the DON with the concentration of 0.5 mu g/mL can stimulate for 6 hours to cause the oxidative stress, inflammation or apoptosis of cells; compared with a DON treatment group, the cell activity, the activity of superoxide dismutase (SOD) and Catalase (CAT) can be obviously improved by adding 200-400 mu g/mL of GA while adding DON, and the release of Lactate Dehydrogenase (LDH), the content of Malondialdehyde (MDA) and the apoptosis rate are reduced. GA can also obviously reduce the relative expression of inflammatory genes such as IL-6, IL-8, TNF-alpha, COX-2, NF-Kb, Bax, Caspase3 and the like, obviously increase the relative expression of an anti-inflammatory gene Bcl-2, inhibit the generation of inflammatory factors and chemokines by activating a Tumor Necrosis Factor (TNF) signal pathway, a Toll-like receptor (Toll-like) signal pathway and a nuclear transcription factor (NF-kB) signal pathway, relieve the oxidative stress, the inflammatory reaction, the cytotoxicity and the apoptosis induced by DON, and protect the integrity of intestinal tracts and the health of animals.

The research result of the invention also shows that 400 g/ton glycyrrhizic acid and 1 × 10g candida utilis are added into the feed of the weaned pig¹²The CFU/ton of feed and enterococcus faecalis is 1 × 10¹²CFU/ton of feed can effectively relieve the harm of DON to the growth of piglets. The invention lays a foundation for the glycyrrhizic acid and the probiotics to be used as the feed additive for relieving the DON toxicity and improving the health condition of the livestock and poultry.

Drawings

FIG. 1: the effects of GA and DON on the viability of IPEC-J2 cells; effect of different GA concentrations on IPEC-J2 cell viability at different action times; b, the influence of the co-culture of GA with different concentrations and DON with 0.5 mu g/mL for 6h on the cell viability;

FIG. 2: influence of GA on lactate dehydrogenase release amount and antioxidant parameters in IPEC-J2 cells after DON treatment; a, Lactate Dehydrogenase (LDH) release; b, Malondialdehyde (MDA) content; c, superoxide dismutase (SOD) viability value; d, Catalase (CAT) activity value;

FIG. 3: the effect of GA on DON-induced apoptosis of IPEC-J2 cells; detecting annexin V/FITC/PI stained apoptotic cells by using flow cytometry, wherein Q1, Q2, Q3 and Q4 represent late apoptotic cell rate, necrotic cell rate, viable cell rate and early apoptotic cell rate respectively; b, quantitative analysis of total apoptosis rate of cells; c, Q1, Q2, Q3, Q4 under varying conditions;

FIG. 4: influence of GA on IPEC-J2 cell-related gene expression after DON treatment; a, influence of GA on apoptosis-related genes (Bax, Bcl-2 and Caspase 3); b, the effect of GA on genes associated with cellular inflammation (IL-6, IL-8, TNF-alpha, COX-2 and NF-kappa B);

FIG. 5: the analysis result of the differentially expressed genes of the sample; a, PCA principal component analysis; b, differential expression gene factors between the two treatment groups; c, analyzing clustering heat maps of differentially expressed genes among samples; d, Venn analysis.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

1. Test materials

Glycyrrhizic Acid (GA) is supplied by south-of-the-river, adobe, biologicals ltd. DON was purchased from Sigma-Aldrich (USA); phosphate Buffered Saline (PBS), 0.25% pancreatin-ethylenediaminetetraacetic acid (EDTA), penicillin/streptomycin (10,000 units/10,000. mu.g/ml), dimethyl sulfoxide (DMSO), and thiazolyl tetrazolium bromide (MTT) were purchased from Beijing Soilebao BioLimited; high glucose medium (HGDMEM) and Fetal Bovine Serum (FBS) were purchased from Biological Industries, Inc. (Israel); the Annexin V-FITC/PI kit is purchased from Shanghai Futai Biotech, Inc.; superoxide dismutase (SOD), Catalase (CAT), intracellular Glutathione (GSH) and Malondialdehyde (MDA) kits are purchased from Beijing Solaibao biological Limited company; trizol reagent was purchased from Invitrogen corporation (USA); the reverse transcription kit and the TB GREEN kit were purchased from TaKaRa (Chinese Dalian).

The test pig is weaned ternary hybrid piglet (Duroc, large white and long white). The yeast is candida utilis (Canida utilis, purchased from China general microbiological culture Collection center (CGMCC) with the bacterial number of 2.615); the Enterococcus is Enterococcus faecalis (Enterococcus faecalis, purchased from China general microbiological culture Collection center (CGMCC), with a bacterial number of 1.2135).

2. Test method

1) Cell culture and sample preparation

The IPEC-J2 cell line was provided by the institute of animal science and technology, university of agriculture in Jiangxi. IPEC-J2 cell culture medium is HGDMEM medium containing 10% FBS and 1% penicillin-streptomycin, and is inoculated to 25cm²In a culture flask, and placing at 37 deg.C and 5% CO₂Cultured in an incubator. When the cell fusion rate reaches 80% -90%, the cells are respectively inoculated in a 96-well plate and a 6-well plate for 24h, and then different treatments are carried out. The original DON was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1mg/mL stock solution, which was then sterilized by filtration through a 0.22 μm filter. GA and DON are diluted into different concentrations by adopting culture solution without serum and antibiotics, and are used as the preparation.

2) Data statistics and analysis

All data in the experiment are presented as mean ± standard deviation, and all plots were generated using GraphPad Prism 7. All data were statistically analyzed by one-way analysis of variance (ANOVA) using the SPSS 20.0 general linear model and multiple comparisons were performed using the Duncan test. P <0.05 is significantly different, and P >0.05 is not significantly different.