CN113349288A - Application method of L-theanine for relieving vomitoxin damage to pig intestine barrier - Google Patents
Application method of L-theanine for relieving vomitoxin damage to pig intestine barrier Download PDFInfo
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- CN113349288A CN113349288A CN202110759778.0A CN202110759778A CN113349288A CN 113349288 A CN113349288 A CN 113349288A CN 202110759778 A CN202110759778 A CN 202110759778A CN 113349288 A CN113349288 A CN 113349288A
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- A—HUMAN NECESSITIES
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- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
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Abstract
The invention provides an application method of L-theanine in relieving vomitoxin damage to a pig intestinal barrier, and belongs to the technical field of pig feed additive application. Experiments prove that the porcine intestinal epithelial cells have higher concentration tolerance to L-theanine; under the condition of safe dose, the L-theanine can effectively promote the expression of the porcine intestinal epithelial cell tight junction protein, relieve the damage of vomitoxin to the barrier function of the porcine intestinal epithelial cell, reduce the content of bacterial endotoxin in the blood of the piglet and relieve the growth performance reduction caused by the vomitoxin. Therefore, the L-theanine can be used as a pig feed additive, is applied to preventing and treating pig intestinal barrier injury and complications caused by vomitoxin, and has good application prospect.
Description
Technical Field
The invention relates to the technical application field of pig feed additives, and particularly relates to an application method of L-theanine for relieving vomitoxin damage to a pig intestinal barrier.
Background
Vomitoxin (also known as deoxynivalenol) is the most representative B-type trichothecene toxins, and is easily generated in the growth and storage processes of feed raw materials such as corn, wheat and the like. Pollution of vomitoxin (to which pigs are most sensitive) to feed and raw materials is one of the key factors restricting the development of the domestic pig industry.
In recent years, the detection rate of vomitoxin of feed and raw materials in China exceeds 90%, and researches show that: the barrier function of the intestinal epithelium can be damaged when the vomitoxin in the daily ration of the pigs slightly exceeds the standard, so that harmful substances in the intestinal tract enter blood and harm the health and growth of the pigs. Because the vomitoxin is stable in chemical property and high-temperature resistant, the targeted clearing of the vomitoxin in the feed cannot be realized by various current technologies. Therefore, analyzing and controlling the key mechanism of damaging the porcine intestinal epithelial barrier by vomitoxin is the core for promoting the stable development of the live pig industry.
Theanine (Theanine) is a non-protein free amino acid specific to tea leaves, and belongs to a derivative of glutamic acid. Theanine was first discovered in young shoots of tea plants in 1950 and has both L-and D-forms, of which L-theanine (L-theanine) is more readily absorbed by the intestinal tract. The theanine naturally existing in tea tree is L-theanine, and accounts for 30-60% of free amino acid in tea, and is amino acid derivative with the largest content in tea. Research shows that the L-theanine has the effects of protecting the nervous system, reducing blood pressure, relieving fatigue, regulating immunity and the like. Chinese patent CN202010695910.1 proves that theanine can relieve acute alcoholic liver injury by regulating inflammatory factor signal channel through test, and discloses application of theanine in preparation of a product for relieving alcoholic liver injury. Chinese patent CN201880050435.3 discloses a sublingual formulation with water soluble co-crystals of acetylsalicylic acid with citric acid, sodium bicarbonate and L-theanine for the treatment of acute myocardial infarction. Chinese patent CN201810146476.4 discloses the use of theanine in preparing food, health products or medicines for relieving asthenopia. In the aspect of animal intestinal health care, the existing research shows that the L-theanine can improve the jejunum shape of ducks, reduce the depth of crypts, increase the height of villi and improve the intestinal function. However, it is not clear whether L-theanine can alleviate the barrier damage of pig intestinal epithelium caused by vomitoxin, and it is necessary to conduct research.
At present, the extraction and production method of L-theanine is mature day by day, and Chinese patent CN201510304960.1 discloses a method for extracting high-purity theanine from fresh tea leaves, which realizes the high-efficiency extraction of theanine; chinese patent CN201910249563.7 discloses a genetically engineered bacterium for L-theanine production, and construction and application thereof, which can realize large-scale production of theanine, have low cost and potential for being applied to animal intestinal health products. Therefore, the safety of the L-theanine and the prevention and treatment effect of the pig intestinal barrier injury are evaluated, and the L-theanine is developed and utilized as a pig intestinal health-care product, and has important significance for healthy breeding of live pigs.
Disclosure of Invention
Aiming at the problems of damage to intestinal barriers of pigs, complications of the intestinal barriers and the complications of the intestinal barriers caused by overproof of feed vomitoxin in the live pig breeding process, research shows that the L-theanine can effectively improve the daily gain of the pigs and related blood immunity indexes. Therefore, the inventor speculates that the L-theanine can improve the barrier function of the pig intestinal tract and has application potential in protecting the barrier damage of the pig intestinal tract.
Therefore, the inventors have conducted intensive studies to confirm that intestinal cells of swine have good tolerance to L-theanine concentration. Under the condition of safe dosage, the L-theanine can effectively promote the expression of porcine intestinal epithelial cell tight junction protein and relieve the damage of vomitoxin to the barrier function of porcine intestinal epithelial cells. Meanwhile, the L-theanine with a certain dose is fed to the piglets, so that the content of bacterial endotoxin in the blood of the piglets can be reduced, and the growth performance reduction caused by vomitoxin can be relieved. Therefore, the L-theanine can be used as a pig feed additive, is applied to preventing and treating pig intestinal barrier injury and complications caused by vomitoxin, and has a good application prospect.
The invention aims to provide an application method of L-theanine in relieving vomitoxin damage to a pig intestinal barrier.
The mechanism of preventing the barrier injury (intestinal leakage) of the pig intestinal tract caused by vomitoxin by using the L-theanine as the feed additive comprises the following steps: improve the expression of the level of the tight junction protein gene and the protein, and reduce the permeability of the intestinal barrier.
Compared with the traditional feed additive, the application method provided by the invention has the following advantages:
1. the action target point is to improve the tolerance of the intestinal epithelial barrier to vomitoxin and has little influence on the intestinal flora.
2. Has no residue in use and causes no pollution to the environment.
3. The extraction and processing technology of the raw materials is mature, and the cost is low.
Drawings
FIG. 1 shows the effect of different concentrations of L-theanine on the viability of porcine intestinal epithelial cells.
FIG. 2 Effect of L-theanine and vomitoxin on the barrier function of IPEC-J2 in vitro intestinal barrier model.
FIG. 3 Effect of L-theanine and vomitoxin on IPEC-J2 cell claudin.
Detailed Description
The following further explains the development technical scheme of the invention:
experimental materials and methods
(1) Cell: the porcine jejunal epithelial cell line IPEC-J2, provided by the research institute for agricultural ecology in subtropical zone of the chinese academy of sciences.
(2) Medicine preparation: l-theanine (Cat. No. HY-15121) was purchased from MCE; vomitoxin (SML1664-1mL) and fluorescein (46944-100MG-F) were purchased from Shanghai Merck. Consumable material: phosphate Buffered Saline (PBS), DMEM/F12 cell culture medium (11320082), pancreatin digest (25200056), penicillin-streptomycin-glutamine (10378016) and fetal bovine serum (16140071) were purchased from Thermo; six-well transwell plates (pore size 0.4 μm, 3491), 96-well and 6-well cell culture plates were purchased from Corning; black opaque 96-well plates (FCP966-80pcs) were purchased from Shanghai Biyuntian.
(3) Constructing an in vitro intestinal epithelial barrier model based on a Transwell chamber: IPEC-J2 cells were seeded at a density of 1X 10 onto 6-well Transwell plates5One cell/well, change every 24 h. About 14 days later, TEER measurements were performed using a Millipore resistance meter (ESR-2)The amount was measured every 24 h. When the monolayer cells are fully differentiated (differentiation time is about 18 days, typical signs are that the contact inhibition of cell monolayer formation, cell proliferation stop, TEER value is stable and is more than 800 omega cm2) I.e. can be used for subsequent tests.
(4) Constructing a pig intestinal vomitoxin injury model: the porcine intestinal barrier emetic toxin damage model is induced by treating the porcine intestinal barrier emetic toxin with 1.0 mu g/mL of emetic toxin for 48 hours.
(5) Evaluation of porcine intestinal barrier integrity: the test was carried out in an in vitro intestinal epithelial barrier model constructed in a Transwell chamber, and after corresponding treatment of the cells in the intestinal epithelial barrier model, transmembrane resistance value (TEER) values were determined using a Millipore resistance meter (ESR-2), and TEER values were measured every 24h, indicating integrity of the porcine intestinal barrier.
(6) Evaluation of porcine intestinal barrier permeability: the test is carried out in an in vitro intestinal epithelial barrier model of a Transwell chamber, 10 mu L of fluorescein (4kDa) working solution is respectively added into an upper chamber of the Transwell during the test, the upper chamber of the Transwell is incubated for 30 minutes at 37 ℃, 100 mu L of buffer solution is absorbed from a lower chamber of the Transwell, and the fluorescence intensity of the diluent under the condition of Ex/Em (490/525 nm) is respectively detected by a multifunctional microplate reader (TECAN-Infinite M PLEX) for evaluating the paracellular pathway permeability of an IPEC-J2 intestinal epithelial barrier model.
(7) Determination of porcine intestinal barrier tight junction protein expression: extracting total protein in cells by using a total protein extraction kit, determining the protein content by using a BCA method, diluting and balancing the concentration of a protein sample by using sterile water, denaturing the sample at 99 ℃ for 5min, performing SDS-PAGE electrophoresis, sealing by using 5% skimmed milk powder after membrane transfer, then incubating for 2h by using a primary antibody, washing for 5min by using TBST for 5 times, incubating for 2h by using a secondary antibody, washing for 5min by using TBST for 5 times, performing immunochromatography by using horseradish peroxidase (HRP), and analyzing the relative expression amount of the tightly-linked protein in the sample by using an electrophoresis gel imaging system (SYNGENE-G: BOX F3).
Example 1: the method comprises the steps of treating IPEC-J2 cells inoculated in a 96-well plate by adopting L-theanine with different molar concentrations (0-2.0mmol/L), detecting the relative activity of the cells in each treatment group by a cck-8 method, and finding that the L-theanine with the concentration of 0-2.0mmol/L has no significant influence on the activity of porcine intestinal epithelial cells, which indicates that the porcine intestinal cells have higher concentration tolerance to the L-theanine; and the L-theanine with a certain concentration improves the activity of the porcine intestinal epithelial cells in value (figure 1).
Example 2: constructing a pig intestinal barrier model in a transwell chamber by using IPEC-J2 cells, and then treating test groups by respectively adopting 1.0 mu g/mL vomitoxin, 100 mu mol/L-theanine and 1.0 mu g/mL vomitoxin and 100 mu mol/L-theanine for 48h to induce the pig intestinal barrier mycotoxin injury model; detecting the integrity and permeability of the pig intestinal barrier after 48 hours; meanwhile, collecting pig intestinal epithelial cells at the experiment ending stage, and detecting the expression condition of the tight junction protein. The results show that L-theanine ameliorates the emetic toxin-induced impairment of barrier function in porcine intestine (fig. 2), while promoting expression of cellular claudin in the emetic toxin-impaired porcine intestinal barrier model (fig. 3).
Example 3: feeding experiments of vomitoxin superscale ration were carried out on 21-day-old weaned piglets (grown up in Du, 60 piglets, 12 piglets per group). Specifically, basal diet is fed to piglets of a blank control group, fusarium graminearum fermentation liquor is sprayed to piglets of control groups and test groups 1, 2 and 3 (1.0 mL of fermentation liquor is uniformly sprayed to each kilogram of test diet, and the concentration of vomitoxin in the fermentation liquor is 3.64mg/mL), 200mg/kg,400mg/kg and 600mg/kg of L-theanine are additionally added to piglets of the test groups, the test lasts for 14 days, and data such as initial weight and final weight of piglets of the control groups and piglets of each test group are recorded; at the end of the experiment, 15mL of blood was collected via the anterior vena cava of piglets and used for ELISA to determine the content of endotoxin in blood. The results show that the addition of L-theanine increases the daily gain of piglets and reduces the endotoxin content in blood (Table 1).
Table 1: influence of L-theanine on growth of weaned piglets and blood endotoxin content.
Claims (4)
1. An application method of L-theanine in relieving vomitoxin damage to pig intestine barrier is provided.
2. The use of claim 1, wherein: the range of applications includes, but is not limited to, intestinal barrier damage (intestinal leakage) and corresponding complications caused by overproof of the feed vomitoxin.
3. The use as claimed in claim 1, characterized in that: the application mode is that the pig feed is added by pig compound feed, fermented feed and drinking water or directly drenched.
4. The use as claimed in claim 1, characterized in that: the added product has the appearance characteristics of powder, aqueous solvent, enteric coating coated sustained release agent and microcapsule coated agent.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110759778.0A CN113349288A (en) | 2021-07-06 | 2021-07-06 | Application method of L-theanine for relieving vomitoxin damage to pig intestine barrier |
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| CN202110759778.0A CN113349288A (en) | 2021-07-06 | 2021-07-06 | Application method of L-theanine for relieving vomitoxin damage to pig intestine barrier |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115025101A (en) * | 2022-06-29 | 2022-09-09 | 扬州大学 | Lithocholic acid-added medicament for preventing and treating mycotoxin poisoning and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104957433A (en) * | 2015-07-17 | 2015-10-07 | 四川农业大学 | Intestinal tract nutrient capable of alleviating mycotoxicosis of piglets |
| CN105581026A (en) * | 2015-12-16 | 2016-05-18 | 天津市畜牧兽医研究所 | Special stress-resistant feed for weaned piglets and application of special stress-resistant feed |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104957433A (en) * | 2015-07-17 | 2015-10-07 | 四川农业大学 | Intestinal tract nutrient capable of alleviating mycotoxicosis of piglets |
| CN105581026A (en) * | 2015-12-16 | 2016-05-18 | 天津市畜牧兽医研究所 | Special stress-resistant feed for weaned piglets and application of special stress-resistant feed |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115025101A (en) * | 2022-06-29 | 2022-09-09 | 扬州大学 | Lithocholic acid-added medicament for preventing and treating mycotoxin poisoning and preparation method and application thereof |
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