CN115125169A - Enterococcus faecium and application thereof as feed additive in pigeon breeding - Google Patents
Enterococcus faecium and application thereof as feed additive in pigeon breeding Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/205—Bacterial isolates
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Abstract
The invention discloses enterococcus faecium and application thereof as a feed additive in pigeon breeding. The Enterococcus faecium (Enterococcus faecium) is separated from the intestinal tract of healthy piglets, belongs to gram-positive bacteria, and has the microbial preservation number of CGMCC No. 24651. The enterococcus faecium separated by the invention is prepared into bacterial powder and then added into a basal feed of a pigeon to feed the pigeon, so that the average daily gain, the breast muscle brightness, the IgG content and the superoxide dismutase content in serum of the pigeon are obviously improved, and the GH and IGF-1 gene expression level in liver tissues is obviously increased. Therefore, the enterococcus faecium provided by the invention can be used as a feed additive for healthy breeding of young pigeons, and has application prospects in the aspects of promoting the growth of young pigeons, improving the meat quality of pigeons, improving the immunity and oxidation resistance of young pigeons and the like.
Description
Technical Field
The invention relates to lactic acid bacteria, in particular to Enterococcus faecium (Enterococcus faecium) separated from intestinal tracts of healthy piglets and application thereof as a feed additive in pigeon breeding or growth, and belongs to the field of Enterococcus faecium and application thereof.
Background
The pigeon meat is rich in nutrition, delicious in taste and rich in protein, has the functions of nourishing and tonifying qi, and dispelling wind and removing toxicity, and is deeply loved by consumers. However, in recent years, the problem of poultry intestinal tract has been highlighted because of the prohibition of use of feed antibiotics, and therefore, the development of antibiotic substitutes has become an important research direction. The probiotics is a kind of active microorganism beneficial to a host, and can effectively inhibit the colonization of intestinal pathogenic bacteria, improve the immunity of the organism and promote the growth of poultry by improving and shaping the intestinal microenvironment.
The probiotics which can be used as feed additives and are listed in the feed additive variety catalog (2013) issued by the Ministry of agriculture in China comprise enterococcus faecium. Enterococcus faecium is a lactic acid bacterium with strong stress resistance such as heat resistance, acid resistance, cholate resistance and the like, has good drug resistance, can resist several clinically common antibiotics, and can be rapidly planted and expanded to form dominant flora after entering an organism to play a probiotic role. However, in the breeding and production of meat pigeons, the influence of enterococcus faecium on the growth of the young pigeons is less reported, and the application of the enterococcus faecium in the feeding of the young pigeons is not reported. Therefore, the research on the regulation and control functions of the enterococcus faecium as the feed additive in the aspects of the growth and development and the immune function of the young pigeons has important significance.
Disclosure of Invention
One of the objects of the present invention is to provide a strain of enterococcus faecium;
the invention also aims to apply the enterococcus faecium as a feed additive to the breeding or production of pigeons;
the above object of the present invention is achieved by the following technical solutions:
one aspect of the invention provides an Enterococcus faecium (Enterococcus faecalis) separated from intestinal tracts of healthy piglets, belonging to gram-positive bacteria, and the microorganism preservation number of the Enterococcus faecium is as follows: CGMCC No.24651, and the classification name is: enterococcus faecium is Enterococcus faecium, the preservation date is 2022, 4 months and 7 days, and the preservation unit is: the China general microbiological culture Collection center (CGMCC) has the following preservation addresses: beijing, Chaoyang district, Beichen Xilu No.1 institute, institute of microbiology, China academy of sciences.
The invention also provides a bacterial powder containing the enterococcus faecium HB01, and the effective viable count of the enterococcus faecium in the bacterial powder is 3 multiplied by 10 9 CFU/g。
Another aspect of the present invention provides the use of enterococcus faecium as a feed additive in the breeding or production of pigeons, including but not limited to: (1) the feed additive can be used for promoting the growth of pigeons; (2) the pigeon meat quality is improved by using the pigeon meat quality improving agent as a feed additive; (3) the feed additive can improve the immunity of pigeons; (4) the antioxidant performance of the pigeons is improved by using the pigeons as a feed additive.
The enterococcus faecium separated by the invention is prepared into bacterial powder, and then is added into a basal feed of a pigeon to feed a young pigeon with the age of 12 days, and the young pigeon is continuously fed for 16 days, so that the average daily gain, the breast muscle brightness, the IgG content and the superoxide dismutase content in serum of the young pigeon are obviously improved, and the GH and IGF-1 gene expression level in liver tissues is obviously increased. Therefore, the enterococcus faecium strain provided by the invention can be prepared into bacterial powder, and the bacterial powder can be used as a feed additive to be added into a young pigeon feeding feed to prepare a probiotic young pigeon feeding feed for healthy breeding of meat pigeons.
As a reference feeding method, for example, the enterococcus faecium is prepared into bacterial powder, and then added with 1% of water to be uniformly mixed with the basal feed for young pigeons to obtain the mixed feed, and the effective viable count of the enterococcus faecium in the mixed feed is 1.11 × 10 7 CFU/ml. Feeding the young pigeons with the mixed feed probiotics containing the enterococcus faecium for 2 times every day, feeding 70ml of the mixed feed probiotics for each feeding, and continuously feeding for 16 days.
As a preferred embodiment, the basic feed consists of corn, pea, cooked soybean, granular material and active beer yeast, and the mass percentages of the components are as follows: 25% of corn, 20% of pea, 20% of cooked soybean, 34% of granular material and 1% of active beer yeast; wherein the granular material comprises the following components: 59.29 percent of corn, 30 percent of soybean meal, 4.9 percent of cottonseed meal, 1.49 percent of soybean oil, 1.32 percent of stone powder, 0.25 percent of choline chloride, 1.98 percent of calcium hydrophosphate and 0.77 percent of premix.
The invention further provides a preparation method of the basic feed, which comprises the following steps: mixing corn 25%, pea 20%, cooked soybean 20%, granule 34% and active beer yeast 1% uniformly, and pulverizing in a pulverizer.
The mixed feed added with the enterococcus faecium prepared by the invention is fed to young pigeons of 12 days old, and is continuously fed for 16 days, so that the average daily gain, the breast muscle brightness, the IgG content and the superoxide dismutase content in serum of the young pigeons are obviously improved, and the GH and IGF-1 gene expression level in liver tissues is obviously increased. Therefore, the strain provided by the invention has the probiotic effect, can improve the production performance and the immune function of young pigeons, and can be used as a feed additive for the healthy breeding of meat pigeons.
Drawings
FIG. 1 is a colony morphology of Enterococcus faecium (Enterococcus faecium) HB01 CGMCC No. 24651.
FIG. 2 is a gram stain pattern of Enterococcus faecium (Enterococcus faecium) HB01 CGMCC No. 24651.
FIG. 3 is a graph showing the effect of enterococcus faecium HB01 feeding on average daily gain of young pigeons.
FIG. 4 shows the effect of enterococcus faecium HB01 feeding on the relative expression level of GH, GHR and IGF-1 genes in young pigeon livers.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 isolation and characterization of Enterococcus faecium (Enterococcus faecalis) HB01
1. Isolation culture of strains
Healthy piglets (without pathogens and without antibiotics) are purchased from a certain pig farm in Cangzhou city in Hebei province, the neck of the piglets is bled to die, the whole bodies of the piglets are soaked and disinfected by benzalkonium bromide, after dissection, duodenum and large intestine are immediately collected in an aseptic operation table in an aseptic mode, the intestines are cut open by sterilized scissors, and normal saline is washed clean to remove the content of the intestines. Scraping intestinal mucosa with sterilized glass slide, grinding in mortar, diluting with normal saline, sequentially diluting to 10 deg.C 3 、10 4 、10 5 And (3) gradient, taking 0.1mL of each dilution, coating the dilution on an MRS agar medium containing calcium carbonate, and carrying out inverted culture in an incubator at 37 ℃ for 24-48 h. Performing primary screening according to phenotypic differences of colony morphology, color, size and the like, selecting a bacterial colony with good growth vigor, inoculating the bacterial colony to a liquid culture medium at 37 ℃, shaking at 150r/min overnight, using 25% glycerol to freeze at-80 ℃, and naming the separated strain as HB 01.
2. Identification of strains
The strain HB01 is streaked on an MRS agar culture medium, and is cultured for 24-48h at 37 ℃, the bacterial colony is round, is gray, has a smooth surface and a convex middle part, has the diameter of about 2mm, is gram-positive, has a spherical cell shape, and is mostly arranged in pairs or short chains.
The 16S rDNA sequence of the strain HB01 is shown in SEQ ID No. 1.
Test example 1 application test of strain HB01 as feed additive in young pigeon production
1 preparation of enterococcus faecium feeding feed
1.1 basic feed
The basic feed used in the test had the following composition and content: 25% of corn, 20% of pea, 20% of cooked soybean, 34% of granular material and 1% of active beer yeast. Wherein the compositions and contents of the granules are as follows: 59.29 percent of corn, 30 percent of soybean meal, 4.9 percent of cottonseed meal, 1.49 percent of soybean oil, 1.32 percent of stone powder, 0.25 percent of choline chloride, 1.98 percent of calcium hydrophosphate and 0.77 percent of premix.
1.2 enterococcus faecium gavage feed
Adding 1% enterococcus faecium powder into the basic feed, adding water, stirring, wherein the ratio of water to feed is 7:3, and the effective viable count of enterococcus faecium contained in the final feed is 1.11 × 10 7 CFU/ml。
Grouping and feeding management of young pigeons
Randomly selecting 120 white-feather king pigeons with 12 days of age, close weight and health, wherein each half of the male and female pigeons are randomly divided into 2 groups, a control group (group A) and an enterococcus faecium group (group B), each group has 6 repetitions, and each repetition has 10 young pigeons for artificial feeding. The control group is fed with basal diet, and the enterococcus faecium group is fed with enterococcus faecium feeding material with effective viable count of 1.11 × 10 7 CFU/ml. The young pigeons are manually fed at 8:00 am and 15:00 pm every day, the feeding amount of each young pigeon is 70mL every time, and the test period is 16 days.
3 results of the test
3.1 Effect on growth Performance
The young pigeons were weighed and recorded at 12 and 28 days of age, and the average daily gain was counted in units of each repetition. Compared with a control group, the average daily gain of the young pigeons aged 12 to 28 days in the enterococcus faecium group is obviously improved.
3.2 Effect on meat quality
When young pigeons are slaughtered at the age of 28 days, the left pectoral muscles are collected, the flesh color, the pH value and the drip loss of the pectoral muscles are determined, and the brightness of the pectoral muscles of the young pigeons is remarkably improved by the enterococcus faecium group (Table 1).
TABLE 1 influence of enterococcus faecium on the breast muscle quality of young pigeons
3.3 Effect on immune and antioxidant indices
When young pigeons are slaughtered at 28 days of age, the levels of immunoglobulin a (iga) and immunoglobulin g (igg) in the serum are determined (table 2), and the superoxide dismutase (SOD), total antioxidant capacity (T-AOC), Catalase (CAT) activity and the contents of Malondialdehyde (MDA), glutathione peroxidase (GSH-Px) in the liver are determined (table 3).
TABLE 2 influence of enterococcus faecium on serum immunoglobulin levels in young pigeons
TABLE 3 influence of enterococcus faecium on the antioxidant index of young pigeon liver
3.4 Effect on Young pigeon growth-related Gene expression level
The young pigeons are slaughtered at the age of 28 days, and the relative expression levels of growth related genes, namely Growth Hormone (GH), Growth Hormone Receptor (GHR) and insulin-like growth factor 1(IGF-1) genes in the livers of the young pigeons are determined by a qRT-PCR method. The expression level of GH and IGF-1 genes in the liver of the enterococcus faecium group is obviously higher than that of the control group.
SEQUENCE LISTING
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> enterococcus faecium and application thereof as feed additive in pigeon breeding
<130> BJ-2001-220419A
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1423
<212> DNA
<213> Enterococcus faecium
<400> 1
ttctttttcc accggagctt gctccaccgg aaaaagagga gtggcgaacg ggtgagtaac 60
acgtgggtaa cctgcccatc agaaggggat aacacttgga aacaggtgct aataccgtat 120
aacaatcgaa accgcatggt tttgatttga aaggcgcttt cgggtgtcgc tgatggatgg 180
acccgcggtg cattagctag ttggtgaggt aacggctcac caaggccacg atgcatagcc 240
gacctgagag ggtgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 300
cagcagtagg gaatcttcgg caatggacga aagtctgacc gagcaacgcc gcgtgagtga 360
agaaggtttt cggatcgtaa aactctgttg ttagagaaga acaaggatga gagtaactgt 420
tcatcccttg acggtatcta accagaaagc cacggctaac tacgtgccag cagccgcggt 480
aatacgtagg tggcaagcgt tgtccggatt tattgggcgt aaagcgagcg caggcggttt 540
cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa actgggagac 600
ttgagtgcag aagaggagag tggaattcca tgtgtagcgg tgaaatgcgt agatatatgg 660
aggaacacca gtggcgaagg cggctctctg gtctgtaact gacgctgagg ctcgaaagcg 720
tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaagt 780
gttggagggt ttccgccctt cagtgctgca gctaacgcat taagcactcc gcctggggag 840
tacgaccgca aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 900
gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ttgaccactc 960
tagagataga gcttcccctt cgggggcaaa gtgacaggtg gtgcatggtt gtcgtcagct 1020
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttattg ttagttgcca 1080
tcattcagtt gggcactcta gcaagactgc cggtgacaaa ccggaggaag gtggggatga 1140
cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg ggaagtacaa 1200
cgagttgcga agtcgcgagg ctaagctaat ctcttaaagc ttctctcagt tcggattgca 1260
ggctgcaact cgcctgcatg aagccggaat cgctagtaat cgcggatcag cacgccgcgg 1320
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1380
gaagtcggtg aggtaacctt tttggagcca gccgcctaag gtg 1423
Claims (10)
1. An Enterococcus faecium (Enterococcus faecium) is characterized in that the preservation number of the microorganism is CGMCC No. 24651.
2. The use of enterococcus faecium according to claim 1 as a feed additive in pigeon breeding or growth.
3. The use according to claim 1, comprising: enterococcus faecium is used as a feed additive for promoting the growth of pigeons.
4. Use according to claim 1, characterized in that it comprises: enterococcus faecium is used as feed additive to improve pigeon meat quality.
5. The use of enterococcus faecium according to claim 1 for preparing a feed additive for enhancing immunity of pigeon.
6. The use of enterococcus faecium according to claim 1 for preparing a feed additive for improving the antioxidant properties of pigeons.
7. A mixed feed for promoting the growth of pigeons or improving the quality of pigeon meat, which consists of a basal feed and a feed additive, wherein the feed additive is the enterococcus faecium as claimed in claim 1.
8. A mixed feed for improving the immunity or the antioxidation performance of pigeons, which consists of a basal feed and a feed additive, and is characterized in that the feed additive is the enterococcus faecium as claimed in claim 1.
9. The mixed feed according to claim 7 or 8, wherein the ratio of the basal feed to the feed additive is 99: 1.
10. the mixed feed as claimed in claim 7 or 8, wherein the basic feed comprises corn, pea, cooked soybean, granulated material and active beer yeast, and the mass percentages of the components are as follows: 25% of corn, 20% of pea, 20% of cooked soybean, 34% of granular material and 1% of active beer yeast; wherein the granular material comprises the following components: 59.29 percent of corn, 30 percent of soybean meal, 4.9 percent of cottonseed meal, 1.49 percent of soybean oil, 1.32 percent of stone powder, 0.25 percent of choline chloride, 1.98 percent of calcium hydrophosphate and 0.77 percent of premix;
the preparation method of the basic feed comprises the following steps: mixing semen Maydis, semen Pisi Sativi, cooked semen glycines, granule and active beer yeast, and pulverizing in pulverizer.
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