CN114181882B - Method for improving manganese ion content in bacillus subtilis and composite microbial inoculum - Google Patents
Method for improving manganese ion content in bacillus subtilis and composite microbial inoculum Download PDFInfo
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- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 117
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 116
- 239000002131 composite material Substances 0.000 title claims description 6
- 229910001437 manganese ion Inorganic materials 0.000 title abstract description 17
- 238000000034 method Methods 0.000 title abstract description 15
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 title abstract description 6
- 239000002068 microbial inoculum Substances 0.000 title description 6
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- 238000004321 preservation Methods 0.000 claims abstract description 7
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- 229960001231 choline Drugs 0.000 description 1
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- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
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- 235000019319 peptone Nutrition 0.000 description 1
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 1
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
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- 238000003307 slaughter Methods 0.000 description 1
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- 239000001476 sodium potassium tartrate Substances 0.000 description 1
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- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
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Abstract
The invention provides a method for improving manganese ion content in bacillus subtilis, which is to ferment and culture the bacillus subtilis and clostridium butyricum simultaneously in a culture medium rich in manganese ions. The bacillus subtilis strain is a bacillus subtilis NT66 strain, is preserved in No. 3 of No.1 West Lu of Beijing, chaoyang district, china general microbiological culture Collection center of China Committee for microbiological research institute of Chinese academy of sciences, 11.8.2021, and has a preservation number of CGMCC No.23751. According to the invention, the bacillus subtilis strain domesticated by high manganese ions and the clostridium butyricum are jointly cultured according to a certain proportion, so that the quantity of the bacillus subtilis can be increased, the small peptide content in the domesticated bacillus subtilis strain can be effectively increased, the prepared bacillus product can effectively enhance the immunity of animals, promote the growth and development and improve the utilization rate of feed.
Description
Technical Field
The invention belongs to the technical field of livestock and poultry micro-ecological preparation, and particularly relates to a method for improving bacillus subtilis contentManganese ion (Mn) 2+ ) Content method and composite microbial inoculum.
Background
In the field of livestock and poultry breeding, proper manganese ions (Mn) are added into feed 2+ ) Can promote growth and development of livestock and poultry and enhance the quality of eggshells of eggs. The manganese digestion and absorption efficiency is reported to be low, and the poultry only has 2 to 5 percent. Inorganic manganese Mn directly added into feed 2+ Most of the waste water is discharged out of the machine body; particularly in large-scale livestock and poultry breeding, the content of manganese ions discharged into the environment is very high, water eutrophication is easily caused after daily accumulation, and Mn in livestock and poultry products is caused 2+ The content is high; because the livestock and poultry manure contains metal Mn 2+ High Mn content in produced agricultural products 2+ Also high residual levels, thus compromising food safety and the overall ecological environment. Therefore, a method for promoting the growth and development of livestock and poultry, maintaining the intestinal health and improving the eggshell strength of the livestock and poultry and reducing the emission of manganese is researched, and the method has great social value for the nutrition precision and the maintenance of the ecological good cycle of the animal husbandry.
However, bacillus subtilis, which has not been acclimated, has some inertness to metal enrichment, especially Mn 2+ Enrichment is very limited. The bacillus subtilis which is subjected to intensive domestication culture by a certain method can greatly enhance and enrich Mn 2+ Tolerance to high concentrations of Mn 2+ The enrichment of the ions has good adaptability and bearing capacity. The specificity of the domesticated bacteria lays a good foundation for producing the manganese-rich bacillus subtilis feed additive.
Disclosure of Invention
The invention aims to provide a method for improving the content of manganese ions in bacillus subtilis, so that the content of the manganese ions in the bacillus subtilis is effectively improved, and the defects of the prior art are overcome.
The method for improving the manganese ion content in the bacillus subtilis provided by the invention is to simultaneously ferment and culture the bacillus subtilis and clostridium butyricum in a culture medium rich in manganese ions;
the concentration of manganese ions in the culture medium is 15-70.0 mg/L; preferably 40.0mg/L.
As a concrete description of the examples, a concrete composition of the culture medium is as follows: 5-15 g/L of peptone, 1-5 g/L of beef extract powder, 1-10 g/L of NaCl and Mn 2+ The concentration is 15-70.0 mg/L, and the pH value is 5.5-7.9 +/-0.2.
Further, the Bacillus subtilis contains high concentration of Mn 2+ The ionic culture medium is acclimatized.
The Bacillus subtilis strain is a manganese-resistant Bacillus subtilis NT66 strain which is preserved in No. 3 of No.1 Xilu-Xingyi, beijing, and China general microbiological culture Collection center (CGMCC) of the institute of microbiology, china academy of sciences, with the preservation number of CGMCC No.23751, 11.8 days in 2021.
The invention also provides a composite microbial inoculum which comprises domesticated bacillus subtilis and clostridium butyricum;
preferably, the number ratio of the bacillus subtilis NT66 strain to the clostridium butyricum in the composite microbial inoculum is 1: 1-2.
According to the invention, the bacillus subtilis strain domesticated by high manganese ions is co-cultured with clostridium butyricum, so that the quantity of the bacillus subtilis can be increased, and the small peptide content in the domesticated bacillus subtilis strain is effectively increased. The bacillus subtilis NT66 strain and clostridium butyricum combined microbial inoculum has good use effect, can effectively enhance the immunity of animals, inhibit harmful bacteria, maintain the health of intestinal tracts, promote the growth and development and improve the utilization rate of feed.
Drawings
FIG. 1: a graph of the effect of acclimation on the morphology of bacillus subtilis;
FIG. 2: the fermentation graph of the bacillus subtilis manganese in the liquid culture medium containing the clostridium butyricum supernatant is shown, wherein the control group is that the bacillus subtilis manganese is fermented in a common liquid culture medium, and the experimental group is that the bacillus subtilis manganese is fermented in the liquid culture medium containing the clostridium butyricum supernatant.
FIG. 3: a fermentation graph of clostridium butyricum in a liquid culture medium containing a bacillus subtilis manganese supernatant, wherein a control group is that the clostridium butyricum is fermented in a common liquid culture medium; the experimental group is that clostridium butyricum is fermented in a liquid culture medium containing a bacillus subtilis manganese supernatant.
FIG. 4: protein standard curve graph;
FIG. 5 is a schematic view of: determining figures of butyric acid standards with different concentrations by liquid chromatography;
FIG. 6: butyric acid standard curve chart.
Detailed Description
The bacillus subtilis is not a resident bacterium of the intestinal tract, only belongs to a passerby, cannot be planted and grown in the intestinal tract for a long time, enters the gastrointestinal tract along with food, is finally discharged out of the body together with excrement, and plays a role in a culture environment. The bacillus subtilis belongs to aerobic bacteria, has strong gastric acid and bile salt resistance, can keep higher activity, and inhibits the propagation of harmful bacteria in intestinal tracts. The invention jointly cultures the bacillus subtilis and the clostridium butyricum, so that more organic manganese can be enriched in bacillus subtilis.
The present invention will be described in detail with reference to specific embodiments.
Example 1: culturing and domesticating bacillus subtilis
By gradually increasing Mn in the medium 2+ Domestication is carried out in a mode of increasing the concentration and the culture temperature; after the domesticated strain is continuously cultured for a plurality of generations, the strain is compared with the bacillus subtilis before domestication in terms of thallus morphology, enzyme activity, small peptides, amino acids and the like, the property characteristics of the domesticated bacillus subtilis are further determined, and stable passage can be realized. The effect of acclimation on bacillus subtilis morphology is shown in figure 1. As can be seen from FIG. 1, the manganese-rich Bacillus subtilis strain becomes thicker and longer under the same times of strain, and the surface texture is clear and smooth. The appearance of the bacillus subtilis is obviously changed under the domestication effect of the manganese ions.
Wherein a domesticated and cultured Bacillus subtilis NT66 strain capable of tolerating high-concentration manganese sulfate (300 mg/L) is preserved in No. 3 Xilu No.1 Beijing, the sunward area of Beijing, 11.8 days in 2021, and the China general microbiological culture Collection center of the institute of microbiology of China, with the preservation number of CGMCC 23751.
The manganese ion enrichment fermentation is respectively carried out by adopting domesticated copper-rich bacillus subtilis (the preservation number is CGMCC No. 15329), zinc-rich bacillus subtilis (the preservation number is CGMCC No. 15330) and manganese-rich bacillus subtilis (the preservation number is CGMCC No. 23751). Respectively inoculating copper-resistant, zinc-resistant and manganese-resistant bacillus subtilis strains in a culture medium containing 40mg/L manganese ions, culturing at 37 ℃ for 24 hours, centrifuging, eluting and drying bacterial liquid, and measuring the bacterial content and the bacterial organic manganese content.
The result shows that when the addition amount of manganese is 40.0mg/L, the thallus content is 0.46g/L, the organic manganese content of the thallus is 2521.26mg/kg, which is far greater than the manganese content of the copper-rich bacillus subtilis and the zinc-rich bacillus subtilis, and the domesticated strain has a better manganese-rich effect. The manganese-rich effect of the bacterial strain of the invention can not be achieved by the previously domesticated copper-rich bacillus subtilis or zinc-rich bacillus subtilis.
Moreover, since high-temperature tolerance acclimation is also performed in manganese-rich acclimation, the screened acclimated copper-rich bacillus subtilis has higher temperature tolerance than that of the acclimated conventional bacillus subtilis. The temperature of 30-40 ℃ is the proper growth temperature of the bacillus subtilis, and the bacillus subtilis breeds one generation every 20-30 minutes under the temperature condition. And the temperature exceeds 50 ℃, the growth speed of the manganese-rich bacillus subtilis is inhibited, but spores can be formed to resist high-temperature environment. The manganese-rich bacillus subtilis domesticated by the invention can survive at a high temperature of more than 60 ℃, and the survival rate is greatly higher than that of the common bacillus subtilis strain which is not domesticated. The yeast can not survive at the temperature of over 60 ℃, the bifidobacterium can not survive at the temperature of over 70 ℃, and the manganese-rich bacillus subtilis screened and domesticated by the method can also survive for about 10 minutes at the temperature of 100 ℃. The domesticated manganese-rich bacillus subtilis has obvious high temperature resistance, so that the requirement that probiotics added in the livestock and poultry pellet feed have the high temperature resistance can be met.
Example 2: symbiotic effect of bacillus subtilis NT66 strain and clostridium butyricum
1. Growth promoting effect of bacillus subtilis NT66 strain and clostridium butyricum cell-free supernatant
Clostridium butyricum (Clostridium butyricum) used in the test was purchased from Koehania bioengineering, inc., shandong Su.
1) Preparation of two sterile cell-free supernatants: respectively selecting a ring of strains from test tube inclined planes of the two strains to inoculate in a corresponding 250ml culture medium, repeating each strain for 6 times, culturing at 37 ℃ for 24 hours (clostridium butyricum anaerobic culture), centrifuging at 6000/min for 10min, and then carrying out autoclaving to obtain two-strain cell-free supernatant.
Respectively selecting a ring of strains from test tube inclined planes of the two strains, inoculating the strains into corresponding 250ml of culture medium, repeating the steps for 3 strains respectively, culturing the strains at 37 ℃ for 24 hours (clostridium butyricum anaerobic culture) to obtain seed solutions of the two strains, and performing microscopic examination and counting respectively.
(2) The symbiotic effect of the two bacteria has influence on the growth of the bacteria: the fermentation broth was sampled every 4 hours and measured for OD600, and the growth curves of Bacillus subtilis NT66 strain were recorded, as shown in FIGS. 2 and 3, respectively. FIG. 2 shows that the growth promoting effect of the clostridium butyricum fermentation product on the Bacillus subtilis NT66 strain is verified, and an experimental group shows that the cell-free supernatant of clostridium butyricum is added into the culture medium of the acclimatized Bacillus subtilis NT66 strain, and the OD600 value of the final culture solution is 1.49 at most; the control group was Bacillus subtilis NT66 without addition of Clostridium butyricum cell-free supernatant, and the OD600 value of the final culture solution was 1.23 at the maximum. FIG. 3 demonstrates the growth promoting effect of the fermentation product of the acclimated Bacillus subtilis on Clostridium butyricum, and the experimental group shows that the culture medium of Clostridium butyricum contains the acclimated Bacillus subtilis supernatant, and the final culture medium has an OD600 value of 0.88 at most; the control group showed growth of C.butyricum without acclimatized B.subtilis supernatant, and the final culture had an OD600 of 0.58 at the maximum.
The results show that the domesticated bacillus subtilis NT66 strain can form a synergistic effect with clostridium butyricum, so that the growth of the bacillus subtilis NT66 strain is promoted; meanwhile, the bacillus subtilis NT66 strain also has the capability of promoting the growth of clostridium butyricum.
Bacillus subtilis to prepare3% of inoculum size was added to a solution containing 40.0mg/kg Mn 2+ The fermentation was completed after culturing in a shaking incubator at a constant temperature of 150r/min at 37 ℃ for 24 h. And centrifuging the fermentation liquor at 6000r/min and 4 ℃ to obtain wet thalli, washing the wet thalli for 2-3 times by using deionized water, and drying the wet thalli at 55 ℃ to obtain bacillus subtilis powder. Weighing a certain amount of bacillus subtilis powder, and measuring the content of organic manganese in the bacteria. Under the conditions, the cell content was 0.40g/L, and the content of organic manganese in the cells was 1337.55mg/kg. Compared with the bacillus subtilis thallus content which is obtained by the combined culture of the domesticated bacillus subtilis NT66 strain and the clostridium butyricum, the bacillus subtilis thallus content is reduced by 15 percent, and the thallus manganese content is reduced by 46.76 percent.
2. Effect of synergistic Effect on Small peptide content
Centrifuging two bacteria liquid fermentation broth for 10min at the temperature of 20ml and 4 ℃ at 6000r/min, taking 5ml of supernate, adding 0.4mol/L TCA solution with the same volume, standing for 0.5h, centrifuging for 10min at the speed of 4000r/min, and removing insoluble protein and long-chain peptide. And centrifuging for 5min at 4000r/min, and taking a certain amount of supernatant to perform biuret method determination.
(1) The preparation method of the reagent used in the test comprises the following steps: standard protein solution: accurately weighing 0.4g of bovine serum albumin, dissolving with distilled water to a constant volume of 100mL, and storing in a refrigerator at 4 ℃; biuret reagent: weighed 1.50g copper sulfate (CuSO) 4 :5H 2 O) and 6.0g of sodium potassium tartrate (KNaC) 4 H 4 O 6 ·4H 2 O), dissolving in 500mL of water, adding 10% NaOH solution (300mL) while stirring, diluting to 1L with water, and storing in a plastic bottle; 10% trichloroacetic acid (TCA): 1mL of TCA was weighed out accurately and dissolved in distilled water to a constant volume of 10mL.
(2) Calibration of protein standard curve: accurately weighing 1.0g of sample, adding 9mL of water, mixing uniformly, adding 10mL of 10% TCA solution, shaking, mixing uniformly, standing for 30min, and finally centrifuging to obtain supernatant for later use.
Adding the above reagents respectively as shown in Table 1 below, mixing, reacting in dark for 30min, measuring absorbance at 540nm wavelength after reaction, and taking protein concentration as abscissa and absorbance as ordinate as standard curve. The results of the standard curve are shown in FIG. 4.
(3) And (3) measuring the content of the small peptide in the fermentation liquor: accurately transferring 1mL of supernatant into a test tube by using a pipette, adding 4mL of distilled water, uniformly mixing, adding 5mL of biuret reagent, uniformly mixing, reacting for 30min in a dark place, measuring the absorbance value at a wavelength of 540nm after the reaction is finished, and substituting the absorbance value into a standard curve to calculate the small peptide content of the sample. The protein standard curve is plotted in table 1. The small peptide content results are shown in table 2.
Table 1: drawing table of protein standard curve
Table 2: influence table of symbiotic effect of bacillus subtilis manganese and clostridium butyricum on small peptide content
Note: BS is domesticated bacillus subtilis NT66 strain; the CB supernatant is the supernatant obtained after fermentation of clostridium butyricum; CB is clostridium butyricum; the BS supernatant is the supernatant obtained after fermentation of the bacillus subtilis NT66 strain.
The CB supernatant liquid BS fermentation promotion test verifies that the clostridium butyricum fermentation liquor has the effect of promoting the bacillus subtilis NT66 to generate small peptides, the BS group is the small peptide content in the normal bacillus subtilis NT66 fermentation liquor, and the BS + CB supernatant liquid group is the small peptide content in the bacillus subtilis NT66 fermentation liquor based on the clostridium butyricum supernatant liquid. The test result shows that the content of the small peptide produced by the bacillus subtilis NT66 strain is obviously increased under the influence of the clostridium butyricum fermentation liquor (p is less than 0.01).
The BS supernatant CB-promoting fermentation test verifies that the fermentation liquor of the bacillus subtilis NT66 strain has the effect of promoting the clostridium butyricum to generate small peptides, the CB group is the content of the small peptides in the normal clostridium butyricum fermentation liquor, and the CB + BS supernatant group is the content of the small peptides in the clostridium butyricum fermentation liquor based on the bacillus subtilis NT66 strain supernatant; the test result shows that the content of the small peptide produced by the clostridium butyricum is obviously increased (p is less than 0.01) under the influence of the fermentation liquor of the bacillus subtilis NT66 strain.
The results show that the bacillus subtilis NT66 strain and clostridium butyricum have synergistic effect.
Example 3: influence of synergistic fermentation of manganese bacillus subtilis NT66 strain and clostridium butyricum on butyric acid content
(1) Drawing a butyric acid standard curve: the concentrations of prepared butyric acid standard substances are respectively 25ppm, 50ppm, 100ppm, 500ppm, 1000ppm, 2500ppm and 5000ppm, and all the butyric acid standard substances with the concentrations pass through a 0.22 mu m water system film and are processed. The measurement of butyric acid standard and butyric acid standard curve are shown in fig. 5 and fig. 6.
(2) Determination of butyric acid content in sample: the test is divided into two parts, the CB supernatant fluid BS fermentation promotion test is to study the change of butyric acid content of clostridium butyricum fermentation liquor on bacillus subtilis NT66 strains in the growth process, two groups of BS, BS + CB supernatant fluid are set, and the BS is the fermentation of the bacillus subtilis NT66 strains in a basic liquid culture medium; the BS + CB supernatant was the bacillus subtilis NT66 strain fermented in a basal medium supplemented with clostridium butyricum supernatant. The CB supernatant liquid CB fermentation promotion test is to study the change of butyric acid content of clostridium butyricum generated in the growth process of the bacillus subtilis manganese fermentation liquid, two groups of CB, CB + BS supernatant liquid are set, and the CB group is to ferment the clostridium butyricum in a basic liquid culture medium; the group of CB + BS supernatants is the fermentation of Clostridium butyricum in a basal medium supplemented with the supernatant of Bacillus subtilis NT66 strain. The results are detailed in Table 3.
Table 3: table for influence of symbiotic effect of bacillus subtilis NT66 strain and clostridium butyricum on butyric acid content
Note: BS is domesticated bacillus subtilis manganese; the CB supernatant is the supernatant obtained after fermentation of clostridium butyricum; CB is clostridium butyricum; the BS supernatant is the supernatant obtained after the manganese fermentation of the bacillus subtilis, and the contrast groups are respectively the fermentation of the BS and the CB in a common culture medium; the experimental groups were fermented with BS supernatant and CB supernatant added, respectively.
The results in Table 3 show that when the Bacillus subtilis NT66 strain is fermented in a culture medium containing Clostridium butyricum supernatant, the content of butyric acid produced by the strain is obviously improved compared with that of a control group (p is less than 0.01); when clostridium butyricum is fermented in a culture medium containing a supernatant of bacillus subtilis NT66 strain, the content of butyric acid produced by the clostridium butyricum is also remarkably improved compared with that of a control group (p is less than 0.01). This indicates that the Bacillus subtilis NT66 strain can have a synergistic effect with Clostridium butyricum fermentation, and increase the content of butyric acid produced in the fermentation process. The characteristics of the bacteria have important reference value for preparing probiotic preparations.
FIG. 5 shows the measured values of butyric acid standards at different concentrations. A butyric acid standard curve was obtained by plotting a standard curve using a butyric acid concentration gradient as the abscissa and a measured value as the ordinate (see FIG. 6). The result of fig. 5 shows that the peak-off time of the sample to be measured is consistent with that of the standard substance, and the content of the substance measured in the sample to be measured is proved to be the content of butyric acid; the peak area size of the graph represents the measured content of butyric acid, and different peak areas are obtained according to the measured samples. The result shows that the domesticated bacillus subtilis and clostridium butyricum can produce higher content of butyric acid through synergistic fermentation.
Example 4: influence of bacillus subtilis NT66 strain on growth and development of meat ducks
In the experiment, 180 male Beijing cherry valley duck ducklings of 7 days old are selected and fed adaptively for a period of time. The weight was randomly divided into 6 groups of 5 replicates each, with 6 replicates each. This experiment used 5 treatment groups and 1 control group, NP group: feeding basal diet (the ingredients of the basal diet are shown in Table 4) to the blank control group; BS group: adding a bacillus subtilis NT66 strain domesticated by manganese on the basis of basic daily ration; group CB: adding clostridium butyricum on the basis of basic ration; BS + CB (1: adding a mixed probiotic preparation (mixed and added by a manganese domesticated bacillus subtilis NT66 strain and clostridium butyricum in a ratio of 1; BS + CB (1: adding a mixed probiotic preparation (mixed and added by manganese acclimatized bacillus subtilis NT66 strain and clostridium butyricum in a ratio of 1; BS + CB (2: a mixed probiotic preparation (a mixture of 2Co-adding), the added probiotic preparation ensures that the concentration of the bacteria is 1 multiplied by 10 8 CFU/ml. The experimental design is shown in table 5. The feeding period was 6 weeks, and slaughter sampling was performed after the 6 th week.
Table 4: ingredient table of basic feed for meat duck
1 The premix provides the following micronutrients (complete food per kg): VA 12 000IU 3 2 500IU，VE 20mg，VK 3 3mg，VB 1 3mg，VB 2 8mg，VB 6 7mg，VB 12 0.03mg, 20mg of D-pantothenic acid, 50mg of nicotinic acid, 1.5mg of folic acid, 0.1mg of biotin, 500mg of choline, 9mg of copper (as copper sulfate), 110mg of zinc (as zinc sulfate), 100mg of iron (as ferrous sulfate), 100mg of manganese (as manganese sulfate), 0.16mg of selenium (as sodium selenite), and 0.6mg of iodine (as potassium iodide).
2 The contents of nutrient components are calculated values
Table 5: meat duck feeding test design table
Test results show that the weight of the meat duck fed by the manganese NT66 strain of the bacillus subtilis or the synergistic feeding of the NT66 strain of the bacillus subtilis and clostridium butyricum is obviously higher than that of a control group (p is less than 0.01) every day when the meat duck is fed by the bacillus subtilis and the NT66 strain at the age of 35 days; for feed intake, except for the BS + CB (2). In addition, the feed is added with the bacillus subtilis NT66 strain or a mixed preparation of the bacillus subtilis NT66 strain and clostridium butyricum, so that the feed conversion ratio can be remarkably reduced (p is less than 0.01), and the conversion rate of the meat duck feed can be improved by the domesticated manganese ion-rich bacillus subtilis (Table 6).
Table 6: growth performance table for 35-day-old meat ducks
In conclusion, the invention adopts the gradual increase of Mn in the culture medium 2+ The bacillus subtilis NT66 strain obtained by domesticating and screening by a concentration and culture temperature method not only has the advantages of bacillus subtilis, but also has the capability of remarkably improving the capability of converting inorganic manganese into thallus organic manganese, greatly improves the utilization rate of trace elements and the feed conversion rate of livestock and poultry, promotes growth and development, and better exerts the biological function of the organic manganese.
Claims (1)
1. A composite strain is characterized by comprising bacillus subtilis and clostridium butyricum; the preservation number of the bacillus subtilis strain is CGMCC No.23751, and the number ratio of the bacillus subtilis strain to clostridium butyricum is 1: 1-2.
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