CN112401083A - Bile acid composite microbial inoculum and application thereof in preparation of laying hen feed additive - Google Patents
Bile acid composite microbial inoculum and application thereof in preparation of laying hen feed additive Download PDFInfo
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- CN112401083A CN112401083A CN202011306687.3A CN202011306687A CN112401083A CN 112401083 A CN112401083 A CN 112401083A CN 202011306687 A CN202011306687 A CN 202011306687A CN 112401083 A CN112401083 A CN 112401083A
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- 239000003613 bile acid Substances 0.000 title claims abstract description 81
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 title claims abstract description 80
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 45
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
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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
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a bile acid complex microbial inoculum and application thereof in preparing a laying hen feed additive, wherein the bile acid complex microbial inoculum comprises a paradisella dirichi suspension and bile acid, and the paradisella dirichi suspension is obtained by culturing paradisella dirichi LCG-06 with the preservation number of CGMCC No. 20820. The compound microbial inoculum can remarkably promote the deposition of fat-soluble vitamins in the liver and yolk of laying hens, improve the absorption of intestinal tracts of the laying hens on the fat-soluble vitamins, improve the growth performance of the laying hens, promote the growth speed of the laying hens, remarkably improve the quality of laid eggs, and reduce the addition amount of the vitamins in the feed processing process so as to reduce the breeding cost.
Description
Technical Field
The invention belongs to the field of poultry breeding, and particularly relates to a bile acid complex microbial inoculum and application thereof in preparation of a laying hen feed additive.
Technical Field
With the improvement of living standard, people have gradually increased demand for nutrition, especially more and more demand for eggs. In recent years, the laying hen breeding industry in China is rapidly developed, and the scale and the production level are greatly improved. However, due to the existing large-scale livestock breeding mode and the problem of feed in the breeding process, the laying rate of the laying hens is low, the quality of egg products is poor, various diseases are easily produced by the laying hens, and a lot of nutrition in the feed cannot be fully absorbed by the laying hens, so that the growth of the laying hens is influenced. In order to increase the laying rate of laying hens and the quality of eggs, farmers often use some veterinary drugs or other feed additives, but the additives have certain side effects, influence the growth of the laying hens and remain in egg products, so that people can seriously harm the health after eating the additives. Therefore, it is necessary to develop a natural and efficient microbial inoculum capable of improving the nutrient absorption of the laying hens and the egg quality.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a bile acid complex microbial inoculum and application thereof in preparing a laying hen feed additive. The bile acid complex microbial inoculum can promote the absorption of fat-soluble vitamins of laying hens and improve the quality of eggs.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a bile acid complex microbial inoculum which comprises a paradisella dieselii bacterial suspension and bile acid.
Further, the paradisella dirichiana bacterial suspension is obtained by culturing and fermenting paradisella dirichiana LCG-06 with the preservation number of CGMCC No. 20820.
Further, the paradisella dieselii LCG-06 is an anaerobe.
Further, the bacterial content of the bile acid complex microbial inoculum is not less than 5 multiplied by 108CFU/mL。
Further, the preparation steps of the bile acid complex microbial inoculum are as follows:
(1) activating the paradisella dieldii LCG-06 in a nutrient liquid culture medium for 40-48h under an anaerobic environment at 37 ℃; adding a sucrose solution with the mass concentration of 20% into the activated bacterium liquid, uniformly mixing, centrifuging, adding a sucrose solution with the mass concentration of 10% into the precipitate, and uniformly resuspending to obtain a paradise dychii LCG-06 bacterium suspension;
(2) and uniformly mixing the parabacteroides dieldii LCG-06 bacterial suspension with bile acid to obtain the bile acid composite microbial inoculum.
Further, the volume-mass ratio of the paradisella dirichiana LCG-06 bacterial suspension to the bile acid is 8-10: 1.
Further, the bile acid comprises not less than 78% of hyocholic acid and hyodeoxycholic acid and not less than 18% of chenodeoxycholic acid.
Further, the bacterial content of the paradisella dieselii LCG-06 bacterial suspension is not less than 2 multiplied by 109CFU/mL。
The invention also provides application of the bile acid complex microbial inoculum in preparation of a laying hen feed additive.
Further, the use method of the bile acid complex microbial inoculum comprises the following steps: the use method of the bile acid complex microbial inoculum comprises the following steps: the bile acid complex microbial inoculum is evenly mixed in the laying hen feed by the dosage of 500-1000mL/t feed and is continuously fed for 2-8 weeks.
Further, 1000mL/t of bile acid complex bacteria agent is added into the feed for laying hens which are cultured for less than 2 weeks.
Further, 500mL/t of bile acid complex bacteria agent is added into the feed for laying hens which are cultured for more than 4 weeks.
Furthermore, the bile acid complex microbial inoculum can promote the deposition of fat-soluble vitamins in the liver and egg yolk of laying hens.
Further, the bile acid complex microbial inoculum can increase the color of egg yolks and the hardness of egg shells.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention screens out a strain of parabacteroides dieldii LCG-06 from the feces in the intestinal tract of a pig, which does not cause damage to animal and human bodies, and the invention utilizes sucrose solution to prepare the parabacteroides dieldii LCG-06 into bacterial suspension, and then the bacterial suspension is compounded with bile acid in an optimized proportion to prepare the composite microbial inoculum, the composite microbial inoculum has natural components and no toxic and side effects, can remarkably promote the deposition of fat-soluble vitamins in the liver and yolk of a laying hen, improve the absorption of the fat-soluble vitamins by the intestinal tract of the laying hen, and remarkably improve the quality of laid eggs; the yolk color of the egg can be obviously improved by adding 500mL/t of the composite microbial inoculum, the eggshell thickness can be improved by 1000mL/t, the egg quality is further improved, and the eggshell color can be uniform; in addition, in the feed processing process, the dosage of the vitamin A can be reduced by 5%, the dosage of the vitamin D, E, K can be reduced by 10-15%, the breeding cost is reduced, and the growth and egg quality of the laying hens can be better promoted, so that the feed has wide application prospects.
Drawings
FIG. 1 is a photograph of a colony of Parabacteroides diutanensis LCG-06 on TSA medium.
FIG. 2 is a diagram showing the effect of a complex microbial inoculum on egg shells; the left side is test 1 group eggs and the right side is control group eggs.
FIG. 3 is a graph showing the influence of the complex microbial inoculum on the color of egg yolk; control group eggs are on the left and test 1 group eggs are on the right.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Example 1:
screening and identification of Parabacteroides dirichiana LCG-06
1. Screening of Parabacteroides diesei LCG-06
Taking intestinal feces from pig intestinal tract, mixing 10g feces with 100mL sterile water, filtering to remove impurities to obtain feces diluent, and adding sterile water to the diluent to obtain 10-1、10-2、10-3、10-4、10-5Diluting the solution; respectively take 10-2、10-3、10-4、10-5The dilutions were plated on soy casein agar medium (TSA) containing 80% N2、10％CO2And 10% of H2Culturing at 37 deg.C for 4 days in anaerobic environment, selecting obvious colony, purifying for several times to obtain single colony, and named as LCG-06, and storing in soybean casein agar slant culture medium. The formula of the TSA culture medium is as follows: tryptone 15.0g, soytone 5.0g, sodium chloride 5.0g, agar 15.0g, sterile water 1000mL, pH 7.3. + -. 0.2.
As shown in FIG. 1, the colony biology of the strain LCG-06 in TSA medium is characterized in that: round or round-like, milky white or off-white, flat, 0.2-1.8mm in diameter, smooth and lusterless surface, semitransparent and relatively neat edge.
2. Identification of Parabacteroides dieldii LCG-06
Extracting genome DNA of the strain LCG-06, taking the genome DNA as a template, performing PCR amplification by using a 16S rRNA universal primer to obtain a 16S rRNA amplification sequence, sequencing the sequence, and performing Blast comparison on the sequence to show that the homology of the strain LCG-06 and paradises dissonans in a GenBank gene library is the highest, so that the strain LCG-06 is judged to be paradises.
And (3) performing strain preservation on the screened paradisella dirichiana LCG-06, wherein the preservation unit comprises: china general microbiological culture Collection center (CGMCC); address: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 09 month and 25 days 2020; the preservation number of the Parabacteroides dibacteroides distasonis is CGMCC No. 20820.
3. Preparation of a suspension of Parabacteroides dieldii LCG-06 bacteria
(1) Selecting the preserved paradise dirichi LCG-06 into a nutrient liquid culture medium (the formula comprises tryptone 15.0g, soytone 5.0g, sodium chloride 5.0g, sterile water 1000mL, and pH 7.4), and performing activated culture for 48h at 37 ℃ in an anaerobic environment to obtain paradise dirichi LCG-06 bacterial liquid;
(2) adding a sucrose solution with the mass concentration of 20% into the bacterial liquid according to the volume ratio of 1: 1, uniformly mixing, and centrifuging at 10000rpm/min for 5min to obtain a bacterial strain precipitate;
(3) re-dissolving the strain precipitate with 10% sucrose solution at a volume to mass ratio of 2: 1, and mixing to obtain a suspension of Parabacteroides dyad LCG-06 with a bacteria content of 2 × 10 or more9CFU/mL。
Preparation of bile acid
The preparation process of the bile acid comprises the following steps:
(1) saponification: adding 1kg of pig gall powder crushed and sieved by a 40-mesh sieve into a reaction kettle, adding 10L of sodium hydroxide solution with the mass concentration of 10%, heating and stirring until the mixture is boiled, keeping the boiling state and stirring for 14 hours, then cooling saponification liquid generated by the reaction until solid and liquid are layered, removing supernatant, and obtaining the residual solid which is bile acid saponification product;
(2) and (3) decoloring: adding 6L of water into the bile acid saponified product, heating to 80 ℃ to completely dissolve the bile acid saponified product, then pumping the bile acid saponified product into a decoloring tank, adding 300mL of hydrogen peroxide, uniformly stirring, reacting at normal temperature for 24 hours, and then filtering the reaction solution into an acidification tank to obtain a filtrate;
(3) acidifying: cooling the filtrate to room temperature, slowly adding 10% hydrochloric acid solution while stirring, stopping adding acid when the pH value of the solution is 3, and centrifugally filtering to obtain a white solid;
(4) and (3) purification: continuously adding water to wash the white solid matter, removing water-soluble impurities attached to the surface of the white solid matter, and finishing the water washing after the filtered water is detected to be neutral; placing the white solid after centrifugal filtration into a drying oven, and drying at 100 ℃ until the moisture is less than 10% to obtain a bile acid crude product;
(5) and (3) recrystallization: putting the bile acid crude product into an extraction tank, adding ethyl acetate with the volume 10 times of the weight of the bile acid crude product, stirring until the bile acid crude product is completely dissolved, then adding 0.08g of anhydrous sodium sulfate for dehydration, filtering and collecting filtrate, carrying out reduced pressure concentration on the filtrate, and recovering ethyl acetate to obtain liquid bile acid;
(6) and (3) drying: and (3) putting the liquid bile acid into an oven, and drying at 100 ℃ until the water content is not more than 1% to obtain the finished product bile acid.
The finished bile acid contains 78.6% of hyocholic acid and hyodeoxycholic acid and 20.0% of chenodeoxycholic acid.
Preparation of bile acid composite bacterial agent
Mixing the prepared finished product bile acid with the paraBacteroides Diels LCG-06 bacterial suspension at a ratio of 1: 10(g/mL) to obtain bile acid composite bacterial agent with bacterial content of not less than 5 × 108CFU/mL。
Example 2
In a scientific research practice base of Shandong agricultural university, 48 laying hens aged 420 days (laying rate of 80%) are cultured and randomly divided into a control group, an experiment 1 group and an experiment 2 group, wherein each group comprises 16 laying hens, each group comprises 8 replicates, and each replicate comprises 2 laying hens. The control group is fed with basic daily ration, the test 1 group and the test 2 group are respectively added with 500mL/t and 1000mL/t of bile acid complex microbial inoculums, and the three groups are all cultured according to the conventional breeding method of the laying hens. Randomly extracting 12 in each group at 2 weeks, 4 weeks and 8 weeks of the positive test period, and measuring the content of fat-soluble vitamin A, D, E, K in serum; then randomly extracting 8 slaughter test livers in each group for 4 weeks and 8 weeks respectively in the positive test period to determine the A, D, E, K content in the livers, and simultaneously randomly extracting 12 eggs in each group to determine the A, D, E, K content in the yolks; and finally, randomly drawing 16 eggs in each group in 4 weeks of the positive test period, and determining the egg quality.
The results are as follows:
as can be seen from the table 1, the content of vitamin A in serum is not affected when the bile acid complex bacterium agent is fed for 2 weeks and 4 weeks, but the serum vitamin A in a 500mL/t group is obviously lower than that in a control group and a 1000mL/t group when the bile acid complex bacterium agent is fed for 8 weeks; feeding bile acid complex bacteria for 4 weeks, wherein the content of vitamin A in the liver and yolk of the 500mL/t group is obviously higher than that of the control group and the 1000mL/t group; after the bile acid complex bacterium agent is fed for 8 weeks, the content of vitamin A in the liver of the 200g/t group is the highest, and the content of vitamin A in the yolk of the 500mL/t group is not obviously different from that of the control group, but is obviously higher than that of the 1000mL/t group.
TABLE 1 influence of bile acid Complex microbial Agents on vitamin A absorption
As can be seen from Table 2, the content of vitamin D in serum is not affected by feeding the bile acid complex inoculant for 2 weeks and 4 weeks, but the content of vitamin D in serum in a 500mL/t group is obviously lower than that in a control group and a 1000mL/t group after feeding the bile acid complex inoculant for 8 weeks; after feeding for 4 weeks, the content of vitamin D in the liver and yolk of the groups of 500mL/t and 1000mL/t is obviously higher than that of the control group; after feeding for 8 weeks, the vitamin D content in the liver of the 500mL/t and 1000mL/t groups is obviously higher than that of the control group, but the vitamin D content in the yolk of the 1000mL/t group is obviously lower than that of the control group and the 500mL/t group.
TABLE 2 influence of bile acid complex bacteria on vitamin D absorption
As can be seen from the table 3, the content of vitamin E in serum is not affected by feeding the bile acid complex microbial inoculum for 2 weeks, the content of vitamin E in serum is obviously improved by feeding for 4 weeks, but the content of vitamin E in serum is obviously reduced by feeding for 8 weeks; after feeding for 4 weeks, the vitamin E content in the liver and yolk is obviously higher than that in the control group at 500mL/t and 1000 mL/t; after the feed is fed for 8 weeks, the vitamin E content in the liver of 500mL/t and 1000mL/t groups is obviously higher than that of a control group, and the vitamin E content in the yolk of the 500mL/t group is obviously higher than that of the control group and 1000mL/t group.
TABLE 3 influence of bile acid complex bacteria on vitamin E absorption
As can be seen from Table 4, the serum vitamin K content was not affected by the administration of the bile acid complex microbial inoculum for 2 weeks and 4 weeks, and the serum vitamin K content was decreased by the administration of the bile acid complex microbial inoculum for 8 weeks; after the chicken is fed for 4 weeks, the vitamin K content in the liver and the yolk of the groups of 500mL/t and 1000mL/t is obviously higher than that of the control group; after feeding for 8 weeks, vitamin K in liver and egg yolk of 1000mL/t group is significantly lower than that of control and 500mL/t group.
TABLE 4 influence of bile acid complex bacteria on vitamin K absorption
As can be seen from tables 5 and 6, the egg shape index is significantly changed and the color of the yolk is significantly darkened by feeding the bile acid complex microbial inoculum for 4 weeks. The 500mL/t bile acid composite bacterial agent group can obviously improve the color of the yolk, and 1000mL/t can obviously improve the thickness of the eggshell. As can also be seen from the fig. 2-3, the addition of the bile acid complex microbial inoculum can obviously deepen the color of the yolk and can also make the color of the eggshell of the egg more uniform and uniform.
TABLE 5 influence of bile acid complex inoculant on egg quality
TABLE 6 influence of bile acid complex bacteria on eggshell color
Note: big, middle and small respectively indicate the blunt end, middle end and tip end of the egg.
In conclusion, the bile acid complex microbial inoculum added into the laying hen breeding feed can remarkably promote the deposition of fat-soluble vitamins in the liver and yolk of laying hens, improve the absorption of the fat-soluble vitamins by intestinal tracts of the laying hens and remarkably improve the quality of laid eggs; the yolk color can be obviously improved by adding 500mL/t of composite microbial inoculum, and the eggshell thickness can be obviously improved by 1000 mL/t; in the feed processing process, the dosage of the vitamin A is reduced by 5 percent, the dosage of the vitamin D, E, K is reduced by 10-15 percent, 1000mL/t of bile acid complex microbial inoculum can be added into the feed for short-term (within 2 weeks) breeding layers, and 500mL/t of bile acid complex microbial inoculum can be added into the feed for long-term (more than 4 weeks) breeding layers.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A bile acid complex microbial inoculum is characterized by comprising a paradisella dieselii suspension and bile acid.
2. The bile acid complex microbial inoculum of claim 1, wherein the paradisella dieldii bacterial suspension is obtained by culturing and fermenting paradisella dieldii LCG-06 with the preservation number of CGMCC No. 20820.
3. The bile acid complex microbial inoculum according to claim 1, wherein the bacterial content of the bile acid complex microbial inoculum is not less than 5 x 108CFU/mL。
4. The bile acid complex microbial inoculum according to claim 2, which is prepared by the following steps:
(1) activating the paradisella dieldii LCG-06 in a nutrient liquid culture medium for 40-48h under an anaerobic environment at 37 ℃; adding a sucrose solution with the mass concentration of 20% into the activated bacterium liquid, uniformly mixing, centrifuging, adding a sucrose solution with the mass concentration of 10% into the precipitate, and uniformly resuspending to obtain a paradise dychii LCG-06 bacterium suspension;
(2) and uniformly mixing the parabacteroides dieldii LCG-06 bacterial suspension with bile acid to obtain the bile acid composite microbial inoculum.
5. The bile acid complex microbial inoculum of claim 4, wherein the volume mass ratio of the paradisella dieldii LCG-06 bacterial suspension to the bile acid is 8-10: 1.
6. The bile acid complex microbial inoculant according to claim 4, wherein the bile acids comprise not less than 78% of hyocholic acid and hyodeoxycholic acid and not less than 18% of chenodeoxycholic acid.
7. The use of the bile acid complex bacterial agent of claim 1 in the preparation of a layer feed additive.
8. The use of claim 7, wherein the bile acid complex microbial inoculum is used by the method comprising the following steps: the bile acid complex microbial inoculum is evenly mixed in the laying hen feed by the dosage of 500-1000mL/t feed and is continuously fed for 2-8 weeks.
9. The use of claim 7, wherein the bile acid complex microbial inoculum can promote the deposition of fat-soluble vitamins in the liver and egg yolk of laying hens.
10. The use of claim 7, wherein the bile acid complex microbial inoculum is capable of increasing the color of egg yolk and the hardness of egg shell.
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