CN107236691B - Rex rabbit-derived bacillus subtilis and application thereof in improving growth performance of rex rabbits - Google Patents
Rex rabbit-derived bacillus subtilis and application thereof in improving growth performance of rex rabbits Download PDFInfo
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- CN107236691B CN107236691B CN201710509085.XA CN201710509085A CN107236691B CN 107236691 B CN107236691 B CN 107236691B CN 201710509085 A CN201710509085 A CN 201710509085A CN 107236691 B CN107236691 B CN 107236691B
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Abstract
The invention discloses a Rex rabbit-derived Bacillus subtilis and application thereof in improving growth performance of Rex rabbits, wherein the strain is preserved in China center for type culture Collection in 2017, 5 and 31 months, and the preservation addresses are as follows: eight-path No. 299 in Wuchang district, Wuhan university Collection, 430072, preservation number CCTCC NO: m2017290. Mixing the above Bacillus subtilis at a ratio of 1.0 × 106～107The cfu/g dosage is added into the feed, and the feed has obvious effects of reducing the feed weight ratio of rex rabbits and improving the intestinal enzyme activity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a rex rabbit-derived bacillus subtilis and application thereof in improving the growth performance of rex rabbits.
Background
In recent years, the breeding industry in China faces increasingly serious food safety problems, and although the traditional antibiotics have great advantages in treating animal diseases and promoting animal growth, the traditional antibiotics are disputed due to the defects of easy generation of drug resistance and the like, and are increasingly limited in the breeding industry. Therefore, most countries begin to limit the use of antibiotics and to find alternative antibiotic products to solve the problems faced by the aquaculture industry. The bacillus is an important probiotic, has important significance for promoting the growth and development of animals and maintaining the balance of micro-ecology in the animals, can form endospores with strong stress resistance in severe environment, can tolerate extreme conditions in nature and processing process, has the potential of being developed as a feed probiotic, and has important significance for the healthy breeding of animals. Currently, the bacillus which can be used as a feed comprises the following components: bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus cereus, and the like.
In combination with the current literature reports on bacillus subtilis, the probiotic function of bacillus subtilis on animals is mainly embodied in the following aspects: firstly, bacillus subtilis can improve the immune function of an animal body through various ways; secondly, the bacillus subtilis can reduce the number of harmful bacteria in the intestinal tract, increase the number of beneficial bacteria in the intestinal tract and maintain the balance of intestinal microecology; thirdly, the bacillus subtilis can secrete a plurality of enzymes, promote the digestion and absorption of the animals to the nutrient substances and improve the intestinal form; fourthly, the bacillus subtilis achieves the effect of biological deodorization by inhibiting the conversion of protein into amine and ammonia, reducing the activity of urease, reducing the decomposition of ammonia nitrogen substances, improving the digestibility of nitrogen-containing substances and the like; fifthly, the bacillus subtilis can improve the oxidation resistance of animal organisms and protect the organisms from oxidative stress.
The rex rabbit is a kind of rabbit with both fur and meat, and the skin of the rex rabbit is a successful substitute for fur under the current large background of protecting wild animals. In addition, the rabbit meat is a high-quality meat product with three high, four low recommended by the world health organization, and has wide breeding prospect. Rex rabbits are animals with high sensitivity, and stress and death of the rex rabbits are easily caused by changes of feed raw materials, feed formulas and feeding environments, frightening and the like. The probiotic bacillus is separated from the rex rabbit body and applied to the rex rabbit, stress possibly caused to the rex rabbit after the bacillus is added is reduced, species differences exist among probiotics, bacteria from different host sources are difficult to fix in intestinal tracts of other species of animals, and even can be converted into pathogenic bacteria, and when dominant bacteria from the same host source enter the host body, fixing resistance can be eliminated from complex intestinal flora, and the dominant bacteria can be quickly fixed in the intestinal tracts to play a role. In rex rabbit breeding, diarrhea diseases are high and frequent, the production of the rex rabbits is seriously influenced, the weaned rabbits are easy to have diarrhea due to the fact that the development of an autoimmune system and a digestive system is incomplete, the feed structure is changed after weaning, and the addition of bacillus with high enzyme production can improve the digestive absorption of the rex rabbits, promote the development of intestinal tracts and reduce the occurrence of the diarrhea.
The existing research shows that the death rate of the rex rabbits is abnormally increased by adding a certain dose of non-rex rabbit-derived probiotics in rex rabbit breeding, but the small intestine villus height, crypt depth, mucosal layer thickness and intestinal tract quality of the survival-resistant rex rabbits are obviously increased, the immune organ quality and cytokine level are also obviously improved, the intestinal tract flora structure is more complex, the number of partial harmful bacteria is reduced, the number of beneficial bacteria is increased, and the diarrhea rate is reduced. In addition, the probiotic with a certain dosage can increase the muscle growth speed of rex rabbits. Therefore, the serious problems that part of weak rabbits are intolerant and the death rate is increased in the process of adapting to exogenous probiotics are solved. The probiotics of the rex rabbit source comes from the rex rabbits, intolerance phenomenon cannot occur, and meanwhile, the probiotics effect is achieved, so that the screening and the application of the probiotics of the rex rabbit source are one of effective ways for solving the problem of weaning rabbit cultivation in rex rabbit cultivation at present. But related research is blank at home and abroad. Therefore, the directed screening and application research work of the rex rabbit source high-yield enzyme bacillus is necessary, a reference is provided for using probiotics to replace antibiotic additives in rex rabbit breeding, and a guarantee is provided for producing green and high-quality rabbit meat.
At present, domestic applications for feeding bacillus patents are mostly for feeding bacillus with fattening effect, such as: a method for improving the production performance of Beijing fatty chickens by utilizing bacillus coagulans of neutral protease (application number: 201611014471.3, application date: 2016-11-18, publication number: CN 106520614A, publication date: 2017-03-22) reports that a bacillus coagulans separated from traditional cheese can reduce the death rate of Beijing fatty chickens, remarkably improve the weight and protein utilization rate of the fatty chickens, promote the growth of beneficial bacteria in intestinal tracts, inhibit the growth of harmful bacteria and have good effect on improving the production performance of the fatty chickens; the bacillus coagulans for improving the growth performance of white feather broilers (application number: 201410719197.4, application date: 2014-12-03, publication number: CN 104403972A, publication date: 2015-03-11) reports that the bacillus coagulans separated from the digestive tract of healthy poultry can reduce the cecal colibacillus number and ammonia nitrogen concentration of the white feather broilers, improve the cecal microbial diversity index, reduce the death and culling rate and the material weight ratio, and improve the daily gain of the broilers; the patent bacillus subtilis preparation and the preparation method and application thereof (application number: 201210139547.0, application date: 2012-05-08, publication number: CN 102660534A, publication date: 2012-09-12) report that the bacillus subtilis preparation has the effects of improving digestion function, increasing feed conversion rate and promoting growth after being fed to livestock and poultry by oral administration (drinking water or mixing with feed).
At present, no patent report is found about a method for improving the growth performance of rex rabbits by bacillus. The method for improving the feed conversion rate and the intestinal enzyme activity, reducing the material-to-weight ratio and improving the growth performance of the rex rabbits by feeding the rex rabbits with a bacillus viable preparation of high-yield cellulase, protease and amylase from the rex rabbits is not reported in related literatures and patents at home and abroad.
Disclosure of Invention
In view of the above, the present invention provides a rex rabbit-derived bacillus subtilis and an application thereof in improving the growth performance of rex rabbits.
In order to solve the technical problems, the invention discloses a rex rabbit-derived bacillus subtilis which is preserved in China center for type culture collection in 5-31.2017 at the preservation address: eight-path No. 299 in Wuchang district, Wuhan university Collection, 430072, preservation number CCTCC NO: m2017290.
The invention also discloses an application of the rex rabbit-derived bacillus subtilis in improving the growth performance of rex rabbits.
Further, bacillus subtilis preparation is added into basic ration of rex rabbit, and the content of bacillus subtilis in per gram of basic ration is 1.0 x 106～107cfu。
Further, the bacillus subtilis preparation is bacillus subtilis powder.
Further, the bacillus subtilis powder is prepared by the following method: activating the frozen and preserved bacillus subtilis BSWJ2017003 in a nutrient broth culture medium, inoculating the activated bacillus subtilis BSWJ2017003 into a solid culture medium, culturing for 72-96 h, picking out a colony for microscopic examination, collecting thalli, adding a carrier, and drying at 65 ℃ to prepare bacillus subtilis powder, namely the bacillus subtilis preparation.
Further, the solid medium formula is as follows: 1.3 percent of soybean meal, 1.3 percent of corn flour, 0.5 percent of bran, 0.3 percent of peptone, 2 percent of glucose, 0.1 percent of beef extract, 0.5 percent of sodium chloride, 1.2 percent of agar and the balance of water, wherein the mass percentage of the components is 100 percent, and the pH value is 6.5-7.5.
Furthermore, the content of viable bacillus subtilis in the preparation is 2 multiplied by 1010cfu/g。
Further, the bacillus subtilis preparation feed is added into basic ration of rex rabbits during granulation.
Compared with the prior art, the invention can obtain the following technical effects:
the invention separates to obtain a Rex rabbit source bacillus subtilis, which is mixed with the original bacillus subtilis by 1.0 multiplied by 106～107The cfu/g dosage is added into the feed, so that the feed weight ratio of the rex rabbits can be remarkably reduced, the intestinal enzyme activity is improved, and the like. Wherein the addition amount of 1.0 × 10 is preferably 1.06cfu/g and 1.0X 107The bacillus subtilis effect of cfu/g is most remarkable.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is an electrophoretogram of the Bacillus subtilis BSWJ 201700316S rDNA PCR product of the present invention;
FIG. 2 is a graph showing the effect of different addition amounts on the feed weight ratio of rex rabbits according to the present invention.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
Example 1 isolation and identification of bacteria
First, separation of bacterial strain
Weighing 0.5g of the caecum content of the healthy rex rabbit, adding the caecum content into a conical flask with glass beads filled with 49.5mL of sterile physiological saline, shaking a shaking table for 20min, carrying out water bath at 80 ℃ for 20min, carrying out ten-fold gradient dilution, uniformly coating 100 mu L of each dilution on an NB plate, placing the NB plate in a constant-temperature incubator at 37 ℃ for culturing for 12-24 h, inoculating a strain ring, picking a single strain of suspected bacillus, dropping on the NB plate, repeatedly marking and purifying until no foreign bacteria exist in microscopic examination, and obtaining a strain capable of improving the growth performance of the rex rabbit, wherein the strain is named as BSWJ 2017003.
Secondly, the strain identification (I) identifies the morphological characteristics, culture characteristics and physiological and biochemical characteristics of the strain BSWJ2017003 according to the methods described in Bergey's Manual of bacteria identification (eighth edition) and the manual of common bacteria system identification (edited by Toxiu bead, Chuia Miaoying et al, Beijing: scientific Press, 2001.2), and the specific results are as follows:
morphological and physiological and biochemical characteristics of the strain:
when the bacterial colony is cultured in NB medium at 37 ℃ for 24h, the bacterial colony is light yellow and nearly circular, has dry and dull surface and has ruffled bulges and irregular edges. Gram-positive, straight rod-like under microscope, spore growing in ellipse.
Physiological and biochemical characteristics:
liquefaction of gelatin: +; contacting with enzyme: +; V-P reaction: +; indole formation: -a; citrate utilization: -a; and (3) glucose fermentation: +; glucose gas production: +; and (3) xylose acid production: -a; and (3) gas production by xylose: +; and (3) producing acid by using mannitol: -a; mannitol gassing: +; arabinose produces acid: +; hydrolysis of hippurate: -a; arginine di-hydrolysis: -a; casein hydrolysis: +; hydrolysis of tyrosine: -a; starch hydrolysis: +; nitrate reduction: +; phenylalanine dehydrogenase: -a; growth with 2% NaCl: +; growth with 5% NaCl: +; growth with 7% NaCl: +.
(II) 16S rDNA assay
Selecting a single colony on the plate, carrying out 16S rRNA colony PCR identification on the single colony, sequencing the purified PCR product by virtue of Beijing Huada gene, and carrying out electrophoresis result chart of the PCR product of bacillus subtilis BSWJ 201700316S rDNA (deoxyribonucleic acid) shown in a figure 1 to obtain a sequence SEQ ID NO. 1. According to the sequence homology comparison of Gen-Bank, the strain BSWJ2017003 has 99 percent of homology with Bacillus subtilis GX S-11(GenBank accession number KU904283.1), and the strain is judged to be Bacillus subtilis.
Based on the characteristics, the strain BSWJ2017003 is identified as Bacillus subtilis, and the strain is classified and named as Bacillus subtilis BSWJ 2017003. The strain is preserved in China center for type culture Collection (Wuhan university Collection No. 299, eight-way in Wuchang district, Wuhan City, Hubei province, 430072) in 2017, 5 months and 31 days, and the preservation number is CCTCCNO: m2017290.
EXAMPLE 2 bacterial species cellulose, protease and amylase production Primary selection and selection
1. Primary selection of cellulose, proteinase and amylase produced by strain
1.1 Primary selection of cellulase production
Activating the strain, inoculating the strain into a nutrient broth culture medium, culturing at 37 ℃ for 12h, transferring the strain onto a CMA-Na plate by a dripping method, culturing for 24-48h, dyeing for 30min by adopting 0.2% Congo red, washing off dye liquor by sequentially using distilled water and sodium chloride with the concentration of 1mo/l, fixing the color by using 5% acetic acid solution, respectively measuring the diameter of a dyeing circle and the diameter of a bacterial colony by using a vernier caliper, and calculating the ratio of the two diameters.
1.2 Primary selection of protease
Activating the strain, inoculating to nutrient broth culture medium, culturing at 37 deg.C for 12 hr, transferring to casein plate by dripping method, culturing for 24-48 hr, measuring casein hydrolysis ring diameter and colony diameter with vernier caliper, and calculating the ratio of the two diameters.
1.3 Primary selection of protease
Activating the strain, inoculating to nutrient broth culture medium, culturing at 37 deg.C for 12 hr, transferring onto starch plate by dripping method, culturing for 24-48 hr, staining with Lugol iodine solution, measuring transparent ring diameter and colony diameter with vernier caliper, and calculating the ratio of the two diameters.
The results of the enzyme production preliminary experiments showed that 109 of the 122 isolated strains had the ability to produce cellulase, 100 had the ability to produce protease, and 30 had the ability to produce amylase. The strain BSWJ2017003 has the capability of producing cellulase, protease and amylase simultaneously.
2. Bacterial cellulose, protease and amylase production
2.1 cellulase production Re-selection
2.1.1 preparation of Standard Curve
16 20mL test tubes were sterilized and dried, and glucose solutions of various concentrations were prepared as shown in Table 1. (3 replicates per concentration)
TABLE 1 preparation of cellulase Standard Curve
Adding 1mL of 2mol/L sodium hydroxide solution and 2mL of DNS solution into the glucose solution with different concentrations, shaking uniformly, placing in a boiling water bath for 5min, then cooling with running water, and adding distilled water to reach a constant volume of 20 mL. And respectively measuring the absorbance values of the test tubes by a spectrophotometer at the 485nm position and taking the test tube No. 0 without glucose as a blank control. And establishing a regression equation for solving the glucose through the absorbance value.
2.1.2 measurement of crude enzyme CMC enzyme Activity
Activated bacillusInoculating the bacterial liquid into a seed culture medium I, culturing for 12h at 37 ℃, then inoculating the bacterial liquid into a fermentation culture medium I with the inoculation amount of 2%, culturing for 160r/min by a shaking table, culturing for 48h at 37 ℃, and supplementing CMC-Na after fermenting for 12 h. Centrifuging after fermentation, and collecting supernatant, namely the crude enzyme solution to be detected. The 0.625% CMC-Na solution was preheated to 50 ℃. The following operations were performed in this order: 20ml test tubes with a plug scale are taken, sterilized, dried and numbered in advance. Taking 1.00ml of crude enzyme solution, placing the crude enzyme solution in a50 ℃ water bath kettle for preheating for 2min, adding 4ml of 0.625% CMC-Na solution preheated to 50 ℃, carrying out accurate reaction in a50 ℃ water bath for 5min, taking out, immediately adding an lml2mol/L sodium hydroxide solution and 2ml of DNS color developing solution, placing the sample tube and the control tube after shaking uniformly in a boiling water bath for accurate timing for 5min, taking out, rapidly cooling by flowing water, and adding distilled water to fix the volume to 20 ml. The enzyme was inactivated by placing the blank in a boiling water bath for 10min before adding substrate, and the rest was the same as above. Fixing volume, shaking, and measuring OD with spectrophotometer485The value of (c). One enzyme activity unit (U) was defined as the production of one μmol glucose per minute per ml of crude enzyme solution under the above conditions.
2.1.3 measurement of crude enzyme liquid Filter paper enzyme Activity
The preparation method of the crude enzyme solution is the same as 2.1.2.
Taking 50mg Xinhua filter paper, adding 3mL of 0.2mol/L acetic acid buffer solution with pH4.6, adding 1mL of crude enzyme solution, carrying out accurate reaction in a water bath at 50 ℃ for 60min, adding 2mL of DNS, carrying out a boiling water bath for 5min, and cooling to a constant volume of 20 mL. The enzyme was inactivated by placing the blank in a boiling water bath for 10min before adding substrate, and the rest was the same as above. Fixing volume, shaking, and measuring OD with spectrophotometer485The value of (c). One enzyme activity unit (U) was defined as the production of one μmol glucose per minute per ml of crude enzyme solution under the above conditions.
2.2 protease checking
2.2.1 preparation of Standard Curve
16 tubes with 20mL scales were sterilized and dried, and tyrosine solutions with various concentrations were prepared according to Table 2. (3 replicates per concentration)
TABLE 2 preparation of protease Standard Curve
And (3) taking 1mL of the tyrosine solution with different concentrations, respectively adding 5mL of 0.4mol/mL sodium carbonate solution and 1mL of formalin reagent solution, shaking uniformly, placing in a water bath at 40 ℃ for reacting for 20min, and adding distilled water to reach a constant volume of 20 mL. The absorbance values were determined separately using a spectrophotometer at a wavelength of 660nm, using a test tube No. 0 containing no tyrosine as a blank. And establishing a regression equation for solving tyrosine through the absorbance value.
2.2.2 measurement of protease Activity in crude enzyme solution
Inoculating the activated bacillus liquid into a seed culture medium II, culturing for 12h at 37 ℃, then inoculating into a fermentation culture medium II with the inoculation amount of 2%, performing shake culture for 160r/min, culturing for 48h at 37 ℃, and supplementing glucose after fermenting for 24 h. Centrifuging after fermentation, and collecting supernatant, namely the crude enzyme solution to be detected. Preheating 2% casein solution in 40 deg.C water bath for 5 min. The following operations were performed in this order: 20ml test tubes with a plug scale are taken, sterilized, dried and numbered in advance. Taking 2.50mL of crude enzyme liquid, placing the crude enzyme liquid in a water bath kettle at 40 ℃ for preheating for 2min, adding 2.5mL of 2% casein solution preheated to 40 ℃, carrying out accurate reaction in water bath at 40 ℃ for 10min, taking out, adding 5mL of 0.4mol/L trichloroacetic acid solution to terminate the reaction, immediately shaking up, taking out and standing for 10min, taking 1mL of filtrate, adding 5mL of 0.4mol/L sodium carbonate solution and 1mL of Folin reagent solution, mixing uniformly, placing the mixture in water bath at 40 ℃ for 20min, and adding distilled water to fix the volume to 20 mL. Blank control, the sample was first treated with trichloroacetic acid to inactivate the enzyme, and then with casein solution, followed by the same procedure as above. Determination of OD with spectrophotometer660The value of (c). One unit of enzyme activity (U) was defined as the production of one μmol tyrosine per minute per ml of crude enzyme solution under the above conditions.
2.3 production of Amylase multiple selection
2.3.1 preparation of Standard Curve
19 tubes of 20mL scale were sterilized and dried, and maltose solutions of various concentrations were prepared as shown in Table 1. (3 replicates per concentration)
TABLE 3 preparation of amylase calibration curves
Adding 2mL of DNS solution into the maltose solutions with different concentrations, shaking, placing in a boiling water bath for 5min, cooling with running water, and adding distilled water to reach a constant volume of 20 mL. The absorbance values were determined separately using a spectrophotometer at a wavelength of 520nm, using a No. 0 test tube without maltose as a blank control. And establishing a regression equation for solving maltose through the absorbance value.
2.3.2 determination of Amylase Activity in crude enzyme solution
Inoculating the activated bacillus liquid into a seed culture medium III, culturing for 12h at 37 ℃, then inoculating into a fermentation culture medium III with the inoculation amount of 5%, performing shake culture for 160r/min, culturing for 48h at 37 ℃, and supplementing calcium carbonate after fermenting for 24 h. Centrifuging after fermentation, and collecting supernatant, namely the crude enzyme solution to be detected. The following operations were performed in this order: 20ml test tubes with a plug scale are taken, sterilized, dried and numbered in advance. Taking 1.00ml of crude enzyme solution, adding 2% of soluble starch lml and 3ml of distilled water, preheating in a water bath at 60 ℃ for 5min, and adding 0.1mol/L of citric acid buffer solution (pH6.0) lml. Preserving heat in water bath at 60 deg.C for 30min, adding 1.5ml DNS solution, boiling in water bath for 5min, rapidly cooling with running water, and adding distilled water to desired volume of 20 ml. The enzyme was inactivated by placing the blank in a boiling water bath for 10min before adding substrate, and the rest of the procedure was as above. Fixing volume, shaking, and measuring OD with spectrophotometer520The value of (c). One unit of enzyme activity (U) was defined as the production of l. mu. mol maltose per ml crude enzyme solution per minute under the above conditions.
According to the results of the enzyme production initial selection test, 19 strains with the diameter of the staining circle/the diameter of the colony being more than or equal to 1.90 are selected in the cellulase production check test, 31 strains with the diameter of the staining circle/the diameter of the colony being more than 1.97 are selected in the protease production check test, and 16 strains with the diameter of the staining circle/the diameter of the colony being more than or equal to 1.23 are selected in the amylase production check test for testing. The results of the enzyme production check test are basically consistent with the results of the enzyme production initial selection test, and further prove that the strain BSWJ2017003 has the capabilities of producing cellulase, protease and amylase, and the strain has higher capabilities of producing cellulase, protease and amylase.
EXAMPLE 3 preparation of Bacillus subtilis powder
Activating the frozen and preserved bacillus subtilis BSWJ2017003 in a nutrient broth culture medium, inoculating the activated bacillus subtilis BSWJ2017003 into a solid culture medium (1.3% of soybean meal, 1.3% of corn flour, 0.5% of bran, 0.3% of peptone, 2% of glucose, 0.1% of beef extract, 0.5% of sodium chloride, 1.2% of agar and the balance of water, wherein the mass percentages of the components are 100%, and the pH value is 6.5-7.5), culturing for 72-96 hours, picking bacterial colonies for microscopic examination, collecting thalli when a large number of spores are formed, adding a proper amount of carriers (corn flour and the like) according to the ratio of 1:100, drying at 65 ℃ to prepare bacillus subtilis powder, and detecting the viable count of the bacillus powder by a dilution method.
Example 4 application of Bacillus subtilis powder in improving growth performance of rex rabbits
1. Animal testing and design of testing
After 120 35-day-old weaned rex rabbits (1000 + -200 g) were acclimatized for one week, they were randomly divided into 6 treatment groups, each of which was 20 rex rabbits. Group 1 was a blank control group fed basal diet without antibiotic addition; the 2 nd to 6 th groups are Bacillus subtilis powder test groups, and the addition amount of the Bacillus subtilis powder in the feed is 1.0 multiplied by 104、1.0×105、1.0×106、1.0×107、1.0×108cfu/g, the test period is 8 weeks, and the test time is 2016, 11 months-2017, 1 month. The basal diet formula is shown in table 4. Reference is made to the Nutrition of the Rabbit (second edition 2010) Rabbit feeding standard.
TABLE 4 basic diet formula and nutritional ingredients
Note: the contents of vitamins and trace elements in each kilogram of feed are as follows: mn90 mg; zn50 mg; fe90 mg; cu10 mg; i0.4mg; se0.2mg; co0.4mg; VA5000 IU; VD3500IU；VE10IU；VK0.5mg；VB11.5mg；VB26.0 mg; 12mg of pantothenic acid; 35mg of nicotinic acid; VB66.0 mg; (ii) a Folic acid 0.8 mg; VB120.01 mg; biotin 0.18mg
2. Animal testing method
The method adopts laminated cage culture, and 1 plant is cultured per cage. Natural ventilation, free intake of food and drinking water.
3. Growth performance index detection
Weighing on empty stomach at seven morning days 1, 8, 15, 22, 29, 36, 43, 50, 57 of the test period, counting feed intake per week, and calculating daily feed intake, daily weight gain and material-to-weight ratio index per week and 1-8 weeks.
4. Intestinal enzyme activity detection
On the 57 th day of the test, 6 rex rabbits were randomly selected from each of the groups 1, 3, 4 and 5, the duodenum, jejunum, ileum and caecum were aseptically separated, the contents thereof were collected, and the protease and amylase activities of the contents of the duodenum, jejunum and ileum and the CMC enzyme and filter paper enzyme activities of the contents of the caecum were determined according to the above enzyme activity determination method. One enzyme activity unit (U) is defined as the amount of enzyme required to produce one μmol substrate per minute per g intestinal content under the conditions described above.
5. Statistical method
Test data One-way Anova analysis was performed on the data using SPSS software, with P <0.05 as the significance level and the test values expressed as "mean ± standard deviation".
6. Analysis of results
6.1 preliminary selection of enzyme produced by Bacillus
TABLE 5 Primary selection of cellulase produced by Bacillus
The results show (table 5), bacillus subtilis BSWJ2017003 produced staining circles by CMC-Na plate culture, staining, observation and measurement, and the ratio of the staining circle diameter/colony diameter was 1.94.
TABLE 6 Primary screening of protease produced by Bacillus
The results show (table 6), that bacillus subtilis BSWJ2017003 produced transparent circles as measured by casein plate culture, observation, and the ratio of the diameter of the stained circle/the diameter of the colony was 2.00.
TABLE 7 Primary screening for amylase produced by Bacillus
The results show (table 7) that bacillus subtilis BSWJ2017003 produced transparent circles as measured by starch plating, staining, observation, and the ratio of the stained circle diameter/colony diameter was 1.32.
6.2 rescreening of Bacillus enzyme production
TABLE 8 rescreening of cellulase, protease and amylase produced by Bacillus
The results show (Table 8) that the CMC enzyme, the filter paper enzyme, the protease and the amylase of the bacillus subtilis BSWJ2017003 have the activities of 15.40U/mL, 2.19U/mL, 6.56U/mL and 3.54U/mL respectively.
6.3 influence of Bacillus subtilis on feed intake of Rex rabbits
TABLE 9 influence of Bacillus subtilis on daily food intake of rex rabbits (g/day)
The results show (Table 9) that there was a difference in the daily food intake between the groups tested, and the daily food intake of the test rex rabbits was lower than that of the blank control group at weeks 1, 3, 5, 6, 7 and 8, and weeks 1-8.
6.4 influence of Bacillus subtilis on daily gain of Rex rabbits
TABLE 10 influence of Bacillus subtilis on the daily gain of Rex rabbits (g/day)
Note: results are expressed as mean ± sd, with significant differences (P <0.05) for data in the same column with different letters, as shown in the tables below.
The results show (table 10) that there are differences in body weight differences between the groups, but it is difficult to find a clear rule between the doses of added bacillus subtilis. The difference between the test groups was not significant compared to the control group throughout the test.
6.5 influence of Bacillus subtilis on Rex Rabbit feed weight ratio
TABLE 11 influence of Bacillus subtilis on Rex Rabbit feed weight ratio
The results show (table 11 and fig. 2) that the material weight ratio of each test group tended to decrease and the material weight ratio of group 4 significantly decreased during the test period, except for the increase in the material weight ratio of group 6 at week 3, group 6 at week 4, and group 3 at week 7, as compared to the control group; the material weight ratio of the 4 th group and the 5 th group is obviously reduced compared with the control group from the whole test, which indicates that 1.0 x 10 is added6cfu/g、1.0×107cfu/g of bacillus subtilis can improve the feed conversion rate, thereby reducing the feed-to-weight ratio.
6.6 influence of Bacillus subtilis on enzyme activity of rex rabbit intestinal tract
TABLE 12 influence of Bacillus subtilis on intestinal enzyme activity of Rex rabbit
Based on the results of the 1-8 week material weight ratios, 3 groups (group 3, group 4, and group 5) with smaller material weight ratios were selected from the group 2-6, and the group 1 (as a control) was added to the selected group to make 4 groups for the subsequent tests.
The results show (Table 12) that the enzyme activities of the intestines of group 3, except for the duodenal amylase, the jejunal protease and the ileal amylase, were significantly increased compared with the control group, and the proteases and amylases of the duodenum, the jejunum and the ileum and the CMC enzyme of the caecum of group 4 and group 5 were significantly increasedAnd filter paper enzyme were both significantly elevated. This indicates the addition of 1.0X 106cfu/g and 1.0X 107The cfu/g bacillus subtilis can obviously improve the intestinal enzyme activity, promote the digestion and absorption of nutrient substances and further improve the production performance of the rex rabbits.
In conclusion, the results of animal experiments show that the bacillus subtilis accounts for 1.0X 106～107The cfu/g dosage is added into basic ration of the weaned rex rabbit, after 8 weeks of feeding, the daily feed-weight ratio of the rex rabbit is obviously increased, the intestinal enzyme activity is obviously increased, and the digestion and absorption of the rex rabbit on nutrient substances are promoted, so that the growth performance of the rex rabbit is improved. The bacillus subtilis disclosed by the invention has a good effect of improving the growth performance of the rex rabbits, and has a great significance for large-scale rex rabbit breeding.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Sichuan university of agriculture Sichuan province grassland scientific research institute
<120> Rex rabbit-derived Bacillus subtilis and application thereof in improving growth performance of Rex rabbits
<130>2017
<160>1
<170>PatentIn version 3.3
<210>1
<211>1073
<212>DNA
<213> Rex rabbit-derived Bacillus subtilis
<400>1
gtgatacgga gctataatgc agtcgagcgg acagatggga gcttgctccc tgatgttagc 60
ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa 120
ccggggctaa taccggatgc ttgtttgaac cgcatggttc aaacataaaa ggtggcttcg 180
gctaccactt acagatggac ccgcggcgca ttagctagtt ggtgaggtaa tggctcacca 240
aggcaacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc 300
ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360
gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac 420
aagtaccgtt cgaatagggc ggtaccttga cggtacctaa ccagaaagcc acggctaact 480
acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta 540
aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg 600
tcattggaaa ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt 660
gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg 720
acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780
taaacgatga gtgctaagtg ttacggggtt tccgcccctt agtgctgcag ctaacgcatt 840
aagcactccg cctggggagt acggtcgcaa gactgaaact caaggaattg acgggggccc 900
gcacaagcgg tggagcatgt ggtttaattc gaagcacgcg aagaacctta ccaggtctga 960
catcctctga catcctagag ataggacgtc cccttcgggg cagagtgaca gtggtgcatg 1020
atgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caa 1073
Claims (5)
1. Bacillus subtilis (B.subtilis)Bacillus subtilis) The application of the bacillus subtilis in improving the growth performance of the rex rabbits is characterized in that a bacillus subtilis preparation is added into basic ration of the rex rabbits, and the content of the bacillus subtilis in the basic ration per gram is 1.0 multiplied by 106~107cfu;
The bacillus subtilis is deposited in the China center for type culture Collection in 2017, 5 and 31 months, and the deposition address is as follows: eight-path No. 299 in Wuchang district, Wuhan university Collection, 430072, preservation number CCTCC NO: m2017290.
2. The use of claim 1, wherein the preparation of bacillus subtilis is a powder of bacillus subtilis.
3. The use of claim 2, wherein the bacillus subtilis powder is prepared by the following method: activating the frozen and preserved bacillus subtilis in a nutrient broth culture medium, inoculating the bacillus subtilis into a solid culture medium, culturing for 72-96 h, picking bacterial colonies for microscopic examination, collecting thalli, adding a carrier, and drying at 65 ℃ to prepare bacillus subtilis powder, namely a bacillus subtilis preparation;
the formula of the solid culture medium is as follows: 1.3 percent of soybean meal, 1.3 percent of corn flour, 0.5 percent of bran, 0.3 percent of peptone, 2 percent of glucose, 0.1 percent of beef extract, 0.5 percent of sodium chloride, 1.2 percent of agar and the balance of water, wherein the mass percentage of the components is 100 percent, and the pH value is 6.5-7.5.
4. The use of claim 1, wherein the preparation of Bacillus subtilis has a viable bacteria content of 2X 1010cfu/g。
5. The use according to any one of claims 1 to 4, wherein the Bacillus subtilis formulation is added to the basal ration of rex rabbits at the time of feed granulation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660534A (en) * | 2012-05-08 | 2012-09-12 | 李德元 | Bacillus subtilis preparation as well as preparation method and application thereof |
| CN102787083A (en) * | 2012-04-27 | 2012-11-21 | 辽宁凯为生物技术有限公司 | Bacillus subtilis and application |
| CN103060222A (en) * | 2012-09-19 | 2013-04-24 | 中国农业科学院饲料研究所 | Bacillus subtilis B27 with probiotic effect and application thereof |
| CN104774781A (en) * | 2014-08-26 | 2015-07-15 | 汕头大学 | Bacillus subtilis DCU and use thereof |
-
2017
- 2017-06-28 CN CN201710509085.XA patent/CN107236691B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787083A (en) * | 2012-04-27 | 2012-11-21 | 辽宁凯为生物技术有限公司 | Bacillus subtilis and application |
| CN102660534A (en) * | 2012-05-08 | 2012-09-12 | 李德元 | Bacillus subtilis preparation as well as preparation method and application thereof |
| CN103060222A (en) * | 2012-09-19 | 2013-04-24 | 中国农业科学院饲料研究所 | Bacillus subtilis B27 with probiotic effect and application thereof |
| CN104774781A (en) * | 2014-08-26 | 2015-07-15 | 汕头大学 | Bacillus subtilis DCU and use thereof |
Non-Patent Citations (5)
| Title |
|---|
| Bacillus subtilis Improves Immunity and Disease Resistance in Rabbits;Guo M 等;《Front Immunol》;20170329;第8卷;第1-13页 * |
| Tu-2制剂对肉兔生产性能及肠道微生物菌群的影响;顾雅君 等;《河南农业科学》;20140515;第43卷(第5期);摘要，第165页左栏第3段，第1.2节，第2.1节和表2 * |
| 一株枯草芽孢杆菌的分离鉴定及抑菌活性分析;许国洋 等;《饲草与饲料》;20151231(第12期);第144-146,315页 * |
| 枯草芽孢杆菌制剂对断奶獭兔生长性能和免疫的影响;裴跃明 等;《饲料研究》;20160323(第6期);第1.1节，第1.3节，第4节 * |
| 枯草芽孢杆菌对普通级实验兔生产性能的初探;胡艳 等;《科技广场》;20121231(第12期);第3节 * |
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