CN107279497B - Application of aspartame as additive in animal feed in preventing weaning stress diarrhea of young ruminants - Google Patents
Application of aspartame as additive in animal feed in preventing weaning stress diarrhea of young ruminants Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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Abstract
The invention discloses application of aspartame in promoting the development of small intestines of animals and enhancing the barrier function of the small intestines; the aspartame can be used as an additive in starter foods of young ruminants, stimulates the secretion of GLP-2 through a series of signal paths such as a sweet receptor and the like, and then is combined with a GLP-2R receptor to activate an ERK1/2 signal path, so that the expression of tight junction protein is promoted, the barrier function of small intestine epithelium is enhanced, the cell cycle process is promoted to promote the development of the small intestine epithelium by regulating the expression of cell cycle protein and related dependent kinase of the young ruminants, the barrier function of the small intestine is enhanced, the diarrhea of the young ruminants such as lambs is prevented, the survival rate of the young ruminants such as lambs is improved, the early-weaning intestinal health of the young ruminants is facilitated, and the weaning stress is weakened.
Description
Technical Field
The invention belongs to the field of feed additives, and particularly relates to application of aspartame to promotion of small intestine development and small intestine barrier function enhancement of young ruminants in starter foods.
Background
The digestive tract disease of young ruminants is in a high-incidence stage from postnatal to weaning. Taking lamb as an example, relevant investigation shows that the diarrhea rate of the lamb in lactation period in China is about 50%, the death rate of the diarrhea lamb is up to 20%, and economic loss of more than one billion yuan is caused each year. The cause of diarrhea of lambs is very complex, and the factors of malnutrition of ewes, poor quality of lamb supplementary feeding feeds, stress, pathogenic microorganism infection and the like can be factors inducing the diarrhea of the lambs. It is now possible to treat or alleviate diarrhea during weaning in young ruminants by the use of antibiotics, but with the banning of antibiotics and the problem of antibiotic residues, it is socially desirable to find a safer, effective and economical way. Meanwhile, many studies in recent years show that the intestinal barrier function of young animals which is not developed completely is also an important factor causing the animals to be susceptible to even diarrhea. However, to date, there has been no study of preventing the development of diarrhea in young ruminants from the standpoint of protecting the intestinal epithelial barrier. Therefore, the finding of a corresponding nutrition regulation and control means for promoting the development and improvement of the intestinal barrier function of the young ruminant has important significance for reducing the diarrhea risk of the young ruminant.
The molecular formula of aspartame is: c14H18N2O5The structural formula is as follows:
is a natural functional oligosaccharide, has no dental caries, pure sweet taste, low hygroscopicity, and no stickiness. Can not cause obvious rise of blood sugar, and is suitable for diabetic patients. The aspartame can be used in cake, biscuit, bread, wine, ice cream, popsicle, beverage, and candy according to normal production requirement. In the aspect of feed, the application of aspartame is applied to the aspect of compound sweetener at present, and no report on the protection of intestinal epithelium of young ruminants exists.
Object of the Invention
The invention content is as follows: aiming at the problems in the prior art, the invention provides the application of aspartame in promoting the development of small intestines of animals and enhancing the barrier function of the small intestines. The invention provides a new function of aspartame, which can be used as an additive in starter foods of young ruminants, and can prevent diarrhea of young ruminants such as lambs and the like from the perspective of protecting intestinal epithelial barriers, promote the development of small intestines of the ruminants and enhance the barrier function of the small intestines.
The technical scheme is as follows: in order to achieve the above objects, the use of aspartame according to the present invention for promoting the development of the small intestine and enhancing the barrier function of the small intestine of a young ruminant.
Further, the aspartame is used as an additive in animal feed to promote the development of the small intestine of a young ruminant and enhance the barrier function of the small intestine.
Wherein the feed is a starter feed.
Further, the amount of aspartame added in the starter feed is 0.08-0.16% of the mass of the starter feed. The preferred amount added is 0.08% of the starter mass.
Preferably, the young ruminant is a lamb or a calf. Young ruminants are usually selected as lambs.
Wherein the growth stage of the animal is from postnatal to pre-weaning.
The starting materials and apparatus used in the present invention are commercially available.
Aspartame is available from Neute, Inc. of America
Portable pH meter (HI 9024C; HANNA Instruments, USA)
The blood plasma and blood sugar kit is purchased from Shanghai Rongsheng biological pharmaceutical industry Co Ltd
The GLP-2 fluorescent enzyme immunoassay kit is purchased from Phoenix Europe GmbH Germany
Reverse transcription kit purchased from Takara
Q5 Real-time PCR instrument (Applied Biosystems, USA).
Has the advantages that: compared with the prior art, the invention has the following advantages:
the aspartame used by the invention is an economic and effective sweetening agent, the invention provides a new function of the aspartame, and the aspartame can be used as an additive in starter feed of young ruminants, so that diarrhea of young ruminants such as lambs and the like can be prevented from occurring from protecting intestinal epithelial barriers, and the survival rate of young ruminants such as lambs and the like can be improved; the aspartame used by the invention can stimulate the secretion of GLP-2 through a series of signal pathways such as sweet taste receptors, and then is combined with GLP-2R receptors to activate an ERK1/2 signal pathway, thereby promoting the expression of tight junction protein, enhancing the barrier function of small intestine epithelium, promoting the cell cycle process to promote the development of small intestine epithelium and enhancing the barrier function of small intestine by regulating the expression of cell cycle protein and related dependent kinase of young ruminants, being beneficial to the health of intestinal tracts of young ruminants at the early stage of weaning, and weakening weaning stress.
Drawings
FIG. 1 is a graph of the effect of aspartame addition on weight change in lambs;
FIG. 2 is a graph showing the relationship between the effect of aspartame addition on the relative expression level of lamb small intestine epithelial cell cycle protein mRNA;
FIG. 3 is a graph showing the relationship between the effect of aspartame addition on the relative expression level of mRNA of lamb small intestine epithelial tight junction protein;
FIG. 4 is a graph showing the effect of aspartame addition on the relative expression amounts of mRNA of GCG, GLP-2R, IGF-1 and IGF-1R in lamb small intestine epithelium.
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1
12 newborn lambs (Hu sheep) with good body condition, consistent gestation times and similar weight are selected in the test and randomly divided into a control group and a sweetener (aspartame) treatment group, and each group comprises 6 lambs. At the age of 10 days, the lambs were fed with supplementary starter feed once each at 8 am and 5 pm. At 14 days of age, the test group of lambs began to add aspartame with a starter mass of 0.08% to the starter diet, during the test period, the lambs followed the ewes to feed and drink water freely, fed alfalfa and oat hay freely, and monitored weekly for weight change. There was no significant difference in body weight between the two groups of lambs before the start of the experiment (6.18 ± 0.47vs.5.80 ± 0.26kg, P ═ 0.504). Aspartame was purchased from newt corporation, usa. The starter formula is designed according to the nutritional requirement standard (NY/Y816-2004; Ministry of agriculture, China, 2004) of the starter of Hu sheep, and the composition is shown in Table 1.
Table 1 test starter food composition and nutrient level
Example 2
Example 2 the same starter formula and test method were used except that the test subjects were newborn calves of similar body weight and aspartame 0.16% of the mass of the starter was added to the starter of the treated group.
Example 3
The weight changes of the lambs of the control group and the test group of example 1 were monitored weekly during the test, and the results are shown in fig. 1. There was no significant difference in body weight between the two groups of lambs throughout the study.
Example 4
When the lamb of example 1 is 42 days old, the lamb is fed for two hours in the morning and then slaughtered, the jugular vein is collected before slaughtering, and the blood is centrifuged at 1000g and 4 ℃ for 15min and stored at-20 ℃ for routine blood detection. Immediately separating rumen and small intestine after slaughtering, weighing, collecting representative rumen content, measuring pH value, filtering with four layers of gauze to collect rumen liquid, and storing at-20 deg.C. Representative tissues of duodenum, jejunum and ileum were collected, washed 3 times in ice phosphate buffered saline, and then cut into small 0.5cm × 0.5cm pieces which were stored in a cryovial in liquid nitrogen for subsequent RNA extraction.
Example 5
Detecting the influence of the addition of the sweetening agent aspartame on the weight of each organ of the lamb:
the samples were slaughtered at 42 days of lamb age, and organs were isolated and weighed immediately after lamb slaughter, with results as shown in table 2.
TABLE 2 Effect of sweetener addition on lamb organ weight
Note: data are presented as mean ± sem, n ═ 5
As can be seen from Table 2, the addition of the sweetener aspartame significantly increases the jejunal and caecum weights of the lambs, and has no significant influence on the wet weights of the rumen, the dry weights of the rumen, the wet weights of the valvular stomach, the dry weights of the valvular stomach, the weights of duodenum, ileum, colon and liver. The above results illustrate that: the sweetener aspartame improves jejunum weight, and promotes small intestine development.
Example 6
The blood of example 4 was subjected to routine blood tests and the rumen content was subjected to pH determination to examine the effect of the sweetener aspartame on lamb rumen and blood parameters, and the results are shown in table 3.
The plasma blood glucose kit for routine blood analysis is purchased from Shanghai Rongsheng biological pharmaceutical industry Co., Ltd; the GLP-2 fluorescent enzyme immunoassay kit is purchased from Phoenix Europe GmbH, Germany. The measurement ranges of the blood sugar and GLP-2 kit are 3.89-6.11 mmol/L and 0-10000pg/ml respectively.
Rumen physiological parameters were measured by measuring rumen pH with a portable pH meter (HI 9024C; HANNA Instruments, USA). The concentration of VFA was determined by gas chromatography (GC-14B, Shimadzu, Japan; capillary column: 30 m.times.0.32 mm.times.0.25 mm of membrane thickness) in accordance with the method of Qin (research improvement on the method of measuring rumen volatile fatty acid by gas chromatography, proceedings of Nanjing university of agriculture, 1982(4): 110-.
TABLE 3 Effect of sweetener addition on lamb rumen parameters and blood parameters
As shown in table 3, sweetener addition tended to increase lamb rumen butyrate concentration, other volatile fatty acid concentrations, and plasma GLP-2 concentration; has no significant influence on tumor pH, total volatile fatty acid concentration, acetic acid concentration, propionic acid concentration, ethylene-propylene ratio and plasma glucose concentration. The above results illustrate that: the sweetening agent aspartame promotes the secretion of GLP-2 of lambs per se.
Example 7
The influence of the addition of the sweetening agent aspartame on the relative expression quantity of the mRN on the lamb small intestine epithelium.
RNA extraction and cDNA synthesis: total RNA from small intestine epithelium obtained in example 4 was extracted, and the concentration and purity of RNA were measured using a Nano Drop spectrophotometer. The absorption ratios (260/280nm) of all samples are between 1.8 and 2.0, and the RNA purity is proved to be high. The integrity of the RNA was checked with 1.4% agarose gel. All RNA concentrations were adjusted to 1. mu.g/. mu.L and stored in a freezer at-80 ℃ until use. The cDNA synthesis was carried out using a reverse transcription kit containing genomic RNase purchased from Takara.
Primer synthesis and real-time quantitative PCR: quantitative analysis of target gene and reference genes GAPDH and 18S rRNA with Q5 Real-time PCR instrument (Applied Biosystems, USA), and sequencing of GAPDH primerAccording to Wang et al (Wang A, Gu Z, Heid B, et al, identification and characterization of the bone protein-coupled receptor GPR41 and GPR43 genes 1[ J]Journal of Dairy Science,2009,92(6): 2696-. SYBR was purchased from Takara under the following reaction conditions: pre-denaturation at 95 ℃ for 30s, 5s at 95 ℃ and 30s at 60 ℃, repeating 40 cycles at 95 ℃ and 15 s; 60 ℃ for 1 min; 95, 15 s. Reaction volume 20 μ L: 10.4ul SYBR, 10umol/L upstream and downstream primers 0.4ul, 2ul sample cDNA and 6.8ul sterile water. All samples were in 3 replicates. The relative expression of the target gene is corrected by taking housekeeping gene GAPDH as an internal reference, and data analysis adopts
The method of (1).
TABLE 4 amplification primer sequences
Results are expressed as mean ± standard error (means ± SE). Data were analyzed for significance using independent sample t-test in SPSS 20.0. The mRNA expression level of the target gene was analyzed by using GraphPad Prism 6.01(www.graphpad.com) software. P <0.05 indicates significant difference.
Influence of sweetener aspartame addition on relative expression amount of lamb small intestine epithelial cell cycle protein mRNA: the progression of the cell cycle is mainly regulated by cyclins and their related kinases (CDKs), so that the expression of cyclins and CDKs affects the progression of the cell cycle. As shown in figure 2, the addition of the sweetener aspartame obviously improves the mRNA expression level of duodenum Cyclin D1, CDK1 and CDK6 of lambs; (ii) the amount of mRNA expression of jejunal Cyclin a, Cyclin B1, CDK1, and CDK 6; ileal Cyclin A, Cyclin D1 and CDK 4. The above results show that: the addition of GLP-2 which promotes the synthesis of lambs by aspartame promotes the expression of cell cycle proteins of small intestine epithelium and mRNA of related dependent kinase, promotes the cell cycle process to a certain extent and promotes the proliferation of small intestine epithelium.
Influence of sweetener aspartame addition on relative expression amount of lamb small intestine epithelial tight junction protein mRNA: the integrity of the epithelial barrier is maintained primarily by cell-to-cell connexins and focal adhesion complexes between cells and the matrix anchoring scaffold proteins. As shown in FIG. 3, the addition of aspartame significantly increased the mRNA expression levels of lamb duodenum Claudin-4 and ZO-1; the mRNA expression levels of Claudin-1, Occludin and ZO-1 in the jejunum; ileum Claudin-4 and Occludin mRNA expression level. The Claudin family of proteins are the most prominent transmembrane proteins, whose main function is to close the cell-to-cell gap. Occludin is a transmembrane protein that interacts directly with claudins and actin. Zo-1 is a peripherin that plays a critical role in the distribution and maintenance of tight junctions. The above results show that: the addition of GLP-2 which promotes the secretion of the lamb to aspartame promotes the mRNA expression quantity of the small intestine tight junction protein, is beneficial to the development of the small intestine barrier function of the lamb and maintains the intestinal health.
Influence of sweetener aspartame addition on relative expression amount of lamb small intestine epithelium GCG, GLP-2R, IGF-1 and IGF-1R mRNA: as shown in fig. 4, aspartame addition significantly increased duodenal GCG and IGF-1 compared to the control group; jejunum GCG, IGF-1R and GLP-2R; ileal GCG and IGF-1 mRNA expression levels. The above research results show that: the aspartame promotes the expression of the lamb small intestine epithelial GLP-2 receptor and the expression of IGF-1 and IGF-1 receptors, and the fact that GLP-2 regulates the cell cycle process through an IGF-1 signal channel and promotes the proliferation of the small intestine epithelium is suggested. Meanwhile, the calf of the example 2 is taken as a test object, and the result is similar to that of the above example.
SEQUENCE LISTING
<110> Nanjing university of agriculture
Application of <120> aspartame in promoting small intestine development of young ruminants and enhancing small intestine barrier function
<130>2017
<160>36
<170>PatentIn version 3.3
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Claims (5)
1. Use of aspartame as an additive in animal feed for the prevention of weaning stress diarrhea in young ruminants.
2. Use according to claim 1, wherein the feed is a starter foodstuff.
3. The use according to claim 2, wherein aspartame is added to the starter diet in an amount of 0.08-0.16% by mass of the starter diet.
4. Use according to claim 1, wherein the young ruminant is a lamb or a calf.
5. The use according to claim 1, wherein the animal is grown from postnatal to pre-weaning.
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