CN110577907A - Bifidobacterium animalis and application thereof - Google Patents
Bifidobacterium animalis and application thereof Download PDFInfo
- Publication number
- CN110577907A CN110577907A CN201910851231.6A CN201910851231A CN110577907A CN 110577907 A CN110577907 A CN 110577907A CN 201910851231 A CN201910851231 A CN 201910851231A CN 110577907 A CN110577907 A CN 110577907A
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- Prior art keywords
- cys
- gly
- bifidobacterium animalis
- ala
- bifidobacterium
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Links
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Abstract
The invention relates to the technical field of zoobiology, and discloses bifidobacterium animalis and application thereof. The invention belongs to Bifidobacterium animalis subsp.lactis B18 with the preservation number of CGMCC No.17535 and the preservation date of 2019, 04 and 09 months and the preservation unit: china general microbiological culture Collection center. The invention has strong tolerance to gastric acid and bile salt, long survival time, high survival rate, better inhibition effect on escherichia coli, and better synergistic growth effect with other types of lactic acid bacteria. The feed is used for feeding dairy cows, and can effectively improve the production performance of the dairy cows, improve the quality of fresh milk and reduce the number of somatic cells.
Description
Technical Field
the invention relates to the technical field of zoobiology, in particular to animal bifidobacterium and application thereof.
Background
Bifidobacterium animalis is closely related to human beings and animals in life, is a lactic acid bacterium commonly found in intestinal tracts of infants and animals, can pass through the stomach and colonize in the intestinal tracts to play a beneficial role, has multiple functions of maintaining the balance of flora in the intestinal tracts, promoting nutrient absorption, relieving lactose intolerance, inhibiting the formation of tumor cells, regulating immunity, delaying senescence and the like, and currently, research reports show that the bifidobacterium animalis has more application fields including food, feed, medical care, cosmetics, beauty treatment, aquaculture, industrial and agricultural production, biological preservatives, probiotic preparations and the like.
The bifidobacterium animalis has various physiological functions and can be used as a source of host nitrogen, the initial growth pH value of the bifidobacterium animalis is 6.7-7.0, and the bifidobacterium animalis does not grow under the environment with the pH value of below 4.5-5.0 or the pH value of above 8.0-8.5; when the acid resistance and the bile salt resistance of the thalli are insufficient, the thalli do not wait for entering the intestinal tract, lose the activity of the thalli, and can not pass through the stomach smoothly and plant in the intestinal tract, so that the flora balance in the intestinal tract is adjusted.
To exert these prebiotic effects, it must first be able to tolerate the digestive processes of the human gastrointestinal tract and resist the killing action of gastric juice and bile. Berrada et al reported that food was emptied in the stomach for 90min, the pH in the stomach was 3.0, and the minimum pH was 1.3, and the concentration of human intestinal bile juice was between 0.03% and 0.3%. Therefore, the screening and culturing of animal bifidobacteria which can tolerate gastric acid and cholate concentration has an extremely important role.
Disclosure of Invention
The invention provides bifidobacterium animalis which has stronger tolerance to gastric acid and bile salt, long survival time and high survival rate and can effectively improve the milk yield of dairy cows and reduce the number of somatic cells and application thereof.
The technical problem to be solved is that:
In order to solve the technical problems, the invention adopts the following technical scheme:
the Bifidobacterium animalis belongs to Bifidobacterium animalis subsp.lactis B18, the preservation number is CGMCC No.17535, the preservation date is 09 months 04 in 2019, and the preservation unit is as follows: china general microbiological culture Collection center.
The bifidobacterium animalis B18 has the tolerance to gastric acid and bile salt of human body or animal.
The bifidobacterium animalis of the invention is further characterized in that after the bifidobacterium animalis B18 is placed at room temperature for 60 days, the total number of live bacteria in the bacterial liquid is not less than 8.0 multiplied by 1011cfu/ml。
the bifidobacterium animalis B18 has an inhibition effect on the escherichia coli flora.
The bifidobacterium animalis of the invention is further compounded with one or more of the following lactic acid bacteria, so that the bifidobacterium animalis B18 has a synergistic growth effect:
Lactobacillus acidophilus (Lactobacillus acidophilus);
Lactobacillus delbrueckii subsp. bulgaricus;
Lactobacillus casei subsp. casei;
Streptococcus thermophilus (Streptococcus thermophilus);
Lactobacillus paracasei (Lactobacillus paracasei);
Streptococcus lactis (Streptococcus lactis);
Enterococcus faecium (Enterococcus faecium);
Lactococcus lactis subsp. lactis;
Lactobacillus buchneri (Lactobacillus buchneri);
Bifidobacterium bifidum (Bifidobacterium bifidum).
A method for culturing animal bifidobacterium, the strain of animal bifidobacterium B18 is inoculated into a fermentation culture medium for fermentation, the fermentation culture medium comprises yeast extract, sucrose and K2HPO4。
A preparation method of freeze-dried animal bifidobacterium powder comprises the steps of uniformly mixing fermented bacterium mud of the animal bifidobacterium B18 in claim 1 with a protective agent according to the mass ratio of 1: 4-6, and freeze-drying at the temperature of-30 to-40 ℃.
The preparation method of the animal bifidobacterium lyophilized powder further comprises the following components in parts by weight: 5-20 g of skim milk, 5-15 g of T809 starch, 5-10 g of lactose, 8-15 g of trehalose, 0.5-1.5 g of fructo-oligosaccharide, 1-3 g of xylo-oligosaccharide, 0.5-1.5 g of isomalto-oligosaccharide, 0.5-1 g of inulin, 0.5-1 g of stachyose, 1-2 g of sodium glutamate and 100-200 ml of distilled water.
Application of bifidobacterium animalis and application of the bifidobacterium animalis B18 in food or animal feed.
An application of Bifidobacterium animalis lyophilized powder in milk cow breeding is provided, which is prepared from Bifidobacterium animalis B18.
Compared with the prior art, the invention has the following beneficial effects:
the bifidobacterium lactis strain is separated from intestinal tracts of healthy cows and is identified as bifidobacterium animalis subsp lactis B18. It can obtain high viable count in improved fermentation medium, and contains 2.0 × 109～8.0×109Viable bacteria above cfu/ml; the freeze-drying protective agent and the process can achieve higher survival rate after optimization, and the Chinese medicine still contains 2.5 multiplied by 1011～9.2×1011Viable bacteria of cfu/g or more. The bifidobacterium animalis B18 can be added into the milk cow feed to reduce the use amount of antibiotics, improve the production performance of the milk cow, improve the quality of fresh milk and reduce the number of somatic cells.
The bifidobacterium animalis B18 has strong tolerance to gastric acid and bile salt, and experiments prove that the bifidobacterium animalis B18 can tolerate an acid environment with the pH value of 2.0 and 1.5 percent of the concentration of the bile salt, so that the bifidobacterium animalis B18 can enter human bodies and animal bodies to play a role.
Bifidobacterium animalis B18 has good inhibitory effect on Escherichia coli, and has viable count of 106The inhibition zone in the cfu/ml nutrient agar plate of the escherichia coli can reach 20 mm.
the Bifidobacterium animalis B18 still remained 8.0X 10 after survival for 60 days at room temperature11The number of viable bacteria of cfu/ml is 3.0X 10 after 360 days11The number of viable bacteria cfu/ml is 33.3 percent; the bifidobacterium animalis B18 can prolong the shelf life of products and effectively reduce the cost.
Bifidobacterium animalis B18 has good synergistic growth effect with other types of lactobacillus, and can be used as food and animal feed additive.
Biological preservation Instructions
and (3) classification and naming: bifidobacterium animalis subsp.lactis B18 with the preservation number of CGMCC No.17535 and the preservation date of 2019, 04/09 days and the preservation unit: the general microbiological center of China Committee for culture Collection of microorganisms, is No. 3 of Xilu No.1 of Beijing, Chaoyang, China academy of sciences.
the bifidobacterium animalis of the invention will be further described with reference to the accompanying drawings.
drawings
FIG. 1 is a morphological diagram (microscopic magnification: 1500 times) of Bifidobacterium animalis B18 of the present invention.
Detailed Description
The bifidobacterium animalis B18 of the invention, CGMCC No.17535, is separated from the intestinal tract of a milk cow and identified as bifidobacterium animalis subspecies lactis, and the specific separation and identification method is as follows.
1. Separation method
Weighing 25g to 225ml of sample in sterile buffer salt solution (adding a proper amount of glass beads) as mother solution, shaking for 10min, fully mixing uniformly, performing gradient dilution by using the sterile buffer salt solution, selecting three to five suitable gradients, sucking 0.1ml of sample in MRS culture medium, performing streak separation, and repeating each dilution gradient for two times. And putting the culture dish into an incubator at 37 ℃ for culturing for 48-72 hours, and then selecting a smooth, convex and neat-edged white or milky colony for microscopic examination. The suspected colony is separated and purified again in MRS culture medium according to the steps until the colony is completely purified.
Morphological identification: gram stain, cell morphology, presence or absence of spores, and cell morphology map is shown in FIG. 1.
physiological and biochemical identification: catalase, oxidase, carbohydrate to produce acid.
Molecular biological identification, 16SrRNA sequence analysis and tuf gene sequence analysis.
2. Test results
2.1 measurement of physiological and biochemical Properties
The results of the specific measurements of cell morphology and physiological and biochemical properties are shown in Table 1.
TABLE 1 measurement results of physiological and biochemical characteristics
2.2, according to the sequence determination results of the 16SrRNA and the tuf gene, B18 is identified as animal bifidobacterium lactis.
3. Identification results
In combination with the above identification results, B18 of the present invention is bifidobacterium animalis subsp.
The method for culturing Bifidobacterium animalis B18 of the present invention is as follows.
Test strains: bifidobacterium animalis B18.
Culture medium and culture conditions:
MRS culture medium is selected for activation and counting culture, and other common culture medium with the same activation effect can be adopted; the MRS culture medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract and KH2PO42g, 2g of citric acid diamine, 2g of sodium acetate, 20g of glucose, 801 ml of Tween and MgSO4·7H2O 0.58g、MnSO4·4H20.25g of O, adding distilled water to a constant volume of 1L, adjusting the pH value to 6.2-6.4, and sterilizing at 121 ℃ for 15 min.
The strain is fermented by using a self-made fermentation culture medium, and the fermentation culture medium comprises the following components: 10-30 g of yeast extract and K2HPO41-5 g, 1-5 g of citric acid diamine, 1-5 g of sodium acetate, 5-30 g of cane sugar, 5-20 ml of tomato juice, 5-20 ml of carrot juice, 1-5 ml of pork liver extract, 1-2 ml of Tween 800.5 and MgSO4·7H2O 0.5～3g、MnSO4·4H20.05-1 g of O and 0.05-1 g of sodium glutamate, using distilled water to fix the volume to 1L, adjusting the pH value to 5.5-7.0, and sterilizing for 5-20 min at 105-125 ℃.
in the independently developed fermentation medium, the use of a carbon source, a nitrogen source and a phosphorus source is optimized and selected, the MRS medium is used as a control group and a test basis, different carbon sources of lactose, sucrose, glucose and fructose, different nitrogen sources of tryptone, soybean peptone, yeast extract and beef extract, and different nitrogen sources of K2HPO4、KH2PO4、Na2HPO4、NaH2PO4Obtaining different culture media as different phosphorus sources, inoculating activated B18 into the culture media, culturing at 37 deg.C for 24h, diluting by 10 times, measuring OD value of bacterial liquid at 600nm wavelength, comparing the influence of different carbon sources, nitrogen sources and phosphorus sources on B18 growth with MRS culture media as control, and determining that the optimum carbon source, nitrogen source and phosphorus source are sucrose, yeast extract, K2HPO4。
Through orthogonal experiments, the growth influence of all factors on B18 is determined in sequence by yeast extract>sucrose>K2HPO4And obtaining the specific composition formula of the fermentation medium.
The bifidobacterium animalis B18 is respectively inoculated into 150ml of the fermentation medium and MRS medium with the inoculum size of 2-5%, static culture is carried out for 24h at 37 ℃, and counting culture is carried out for 48h at 37 ℃ after dilution by 10 times.
The detection proves that the number of the viable bacteria in the fermentation medium is 7.6 multiplied by 109cfu/ml, which is obviously higher than 1.2 multiplied by 10 of MRS culture medium9cfu/ml. Fermentation according to the inventionThe carbon source, the nitrogen source and the phosphorus source selected in the culture medium can better promote the growth of B18.
The culture method of the animal bifidobacterium B18 specifically comprises the following steps:
Step one, activating glycerol seeds: inoculating a glycerol preservation strain into an MRS liquid test tube, and performing static culture at 35-40 ℃ for 10-15 hours;
Step two, seed culture: taking a proper amount of activated bacteria liquid according to the inoculation amount of 2-5%, inoculating the activated bacteria liquid into a 250ml triangular flask filled with 150ml of MRS liquid culture medium, and performing static culture for 10-15 hours at the temperature of 35-40 ℃;
Step three, fermentation culture: taking a proper amount of the culture solution according to the inoculation amount of 2-5%, inoculating the culture solution into a 10L fermentation tank filled with a fermentation culture medium, adjusting the temperature to 35-40 ℃, the rotating speed to 50-100 r/min, and culturing under the environment of initial pH 6.5-7.0;
Step four, supplementing materials and adjusting acid: after fermentation culture is carried out for 11-15 h, the speed is regulated to 1-5 ml/min, 400-500 ml of material is supplemented, the pH is adjusted to be 5.5-6.5, and fermentation is continued;
Step five, terminating fermentation: and (3) cooling the fermentation tank to about 20 ℃ after the bacteria grow for 20-24 hours, and finishing the fermentation and discharging.
The cultured bifidobacterium animalis B18 is usually subjected to freeze-drying treatment to prepare freeze-dried powder for prolonging the storage life and facilitating the application, and the specific preparation method is as follows.
Centrifuging the zymocyte liquid of the animal bifidobacterium B18 at 4 ℃ for 5-10min at the centrifugal rotation speed of 4000-; uniformly mixing the bacterial sludge and the protective agent in a mass ratio of 1: 4-6, putting the mixture into a cold trap according to a certain amount, pre-freezing the mixture to-30 to-40 ℃, then taking out the cold trap, setting freeze drying parameters, and drying the freeze drying parameters to obtain freeze-dried bacterial powder, wherein the specific control parameters of freeze drying are shown in table 2.
Table 2 control of the freeze-drying Condition parameters
| Control temperature (. degree.C.) | Drying time (hours) |
| -25～-35 | 1～4 |
| -15～-25 | 1～4 |
| -5～0 | 4～6 |
| 1～5 | 1～8 |
| 15～25 | 1～4 |
| 25～35 | 1～4 |
Wherein the protective agent mixed with the bacterial sludge comprises the following components: 5-20 g of skim milk, 5-15 g of T809 starch, 5-10 g of lactose, 8-15 g of trehalose, 0.5-1.5 g of fructo-oligosaccharide, 1-3 g of xylo-oligosaccharide, 0.5-1.5 g of isomalto-oligosaccharide, 0.5-1 g of inulin, 0.5-1 g of stachyose and 1-2 g of sodium glutamate are dissolved in 100-200 ml of water. Sterilizing at 105-115 deg.C for 10-20 min.
The freeze-dried bacterial powder is subjected to a dilution pouring plate counting method to determine the viable count, and the specific determination result is shown in table 3.
TABLE 3 shape results of lyophilized powder
| Parameter(s) | Results |
| Colour(s) | White colour |
| traits | Powder of |
| Water content% | 1.27％ |
| Viable count cfu/ml | 9.2×1011 |
Bile salt and acid resistance test of animal bifidobacterium B18
1. Test method
1.1 bile salt tolerance test
Bifidobacterium animalis B18 was strictly anaerobically cultured in sterile modified MRS medium at 37 ℃ for 15 hours. The activated strain culture solution is inoculated into sterile modified MRS liquid culture media containing different cholate concentrations (the cholate concentration is 0.2%, 0.5% 1.0%, 1.5%, 2.0%, 2.5%) by 2% inoculation amount, and the modified MRS culture solution containing no cholate is used as a control. Culturing at 37 deg.C for 0h, 2h, 4h, 6h, and 24h, and sampling to determine viable count.
1.2 acid tolerance test
Bifidobacterium animalis B18 was cultured in sterile modified MRS medium at 37 deg.C under strict anaerobic condition for 15h, and the activated culture medium was inoculated into sterile modified MRS at different pH values (pH values of 2.0, 2.5, and 3.0, respectively) at an inoculum size of 2%, while the culture medium at pH6.5 was used as a control. Anaerobic culture at 37 deg.C, sampling after 0min, 30min, 60min, 90min, and 120min respectively, and determining viable count.
2. Test results
2.1 results of bile salt tolerance test
The tolerance of the bifidobacterium animalis B18 to different concentrations of bile salts is shown in Table 4, when the concentration of the bile salts is less than or equal to 1.5%, the bifidobacterium animalis B18 has no inhibition effect on the bile salts, the number of live bacteria is continuously increased within 6 hours, and the tolerance of the bifidobacterium animalis B18 to the bile salts is very strong.
TABLE 4B 18 tolerance to various concentrations of bile salts
| Concentration of bile salts | 0h(cfu/ml) | 2h | 4h | 6h | 24h |
| CK | 4.1×107 | 1.2×108 | 2.5×108 | 3.5×108 | 3.9×109 |
| 0.2％ | 2.0×107 | 2.2×107 | 3.0×107 | 5.7×107 | 8.2×108 |
| 0.5％ | 2.9×106 | 6.4×106 | 9.5×106 | 1.6×107 | 3.4×108 |
| 1.0％ | 3.3×106 | 5.0×106 | 6.2×106 | 1.8×107 | 1.9×108 |
| 1.5％ | 1.9×106 | 4.5×106 | 6.3×106 | 9.8×106 | 8.9×107 |
| 2.0％ | 2.5×106 | 3.5×106 | 5.4×106 | 8.7×105 | 7.1×105 |
| 2.5％ | 2.3×106 | 3.3×106 | 3.5×106 | 3.7×106 | 3.1×106 |
The concentration of bile salt in the small intestine of a human body is between 0.03 and 0.3 percent, and in the test, the blank control test shows that the number of viable bacteria in the test environment is continuously increased along with the prolonging of the test time; in both the 0.2% and 0.5% group of experiments, which most closely approximate the actual intestinal bile salt concentration, the number of viable bacteria in the experimental environment was also increasing continuously, and the fold increase was similar to that of the CK group, and was not much different. In two groups of tests of 1.0% and 1.5% with obviously higher bile salt concentration, the number of live bacteria in the test environment is also increased continuously, but after 6 hours, the increase of the number of live bacteria is slightly slowed down, but no obvious inhibiting effect is generated. In two groups of experiments with the bile salt concentration higher than 2.0%, the growth and the rise of the number of living bacteria within 6h are obviously slow but still in the growth trend, and the number of the living bacteria is slightly reduced between 6h and 24h, so that the inhibition effect of the bile salt on the animal bifidobacterium B18 is reflected. Therefore, the bifidobacterium animalis B18 has strong tolerance to bile salts, and can smoothly pass through small intestine and enter large intestine under normal physiological conditions to regulate the intestinal flora balance.
2.2 results of acid tolerance test
the change of the viable count of the bifidobacterium animalis B18 in different acidic culture solutions is shown in Table 5, and a large amount of viable bacteria still exist after 2 hours of culture under the condition of pH2.0, which indicates that the bifidobacterium animalis B18 has strong tolerance to acid.
TABLE 5B 18 tolerance to acid
| 0min | 30min | 60min | 90min | 120min | |
| pH6.5 | 2.6×107 | 3.3×107 | 4.4×107 | 5.1×107 | 4.9×107 |
| pH3.0 | 1.3×107 | 2.8×107 | 3.2×107 | 3.3×107 | 3.0×107 |
| pH2.5 | 2.3×107 | 2.2×107 | 2.1×107 | 1.8×107 | 1.9×107 |
| pH2.0 | 2.5×107 | 2.0×107 | 1.5×107 | 1.3×107 | 1.1×107 |
The pH value in human stomach is between 2-3, in this experiment, the known test group with the pH value of 6.5 suitable for the adjacent bifidobacteria can know that the viable count in the test environment is slowly increased along with the extension of the test time and is not obviously reduced after 90 min. In the test group of pH3.0 in acid environment, the number of live bacteria in the test environment also slowly increases, and does not obviously decrease after 90min, and the change trend is similar to that of pH6.5 and is not obviously different. In the test group of pH2.5 in the acid environment, the early inhibition effect is gradually shown, and after the test group adapts to the growth environment, the number of the viable bacteria is slightly increased after 90 min. Under the extremely acidic environment of pH2.0, the number of viable bacteria gradually decreased with the increase of the test time, but within 2h, the number of viable bacteria was 2.5X 107cfu/ml to 1.1X 107cfu/ml, the order of magnitude is not changed, the reduction of the quantity is not obvious, and therefore, the bifidobacterium animalis B18 has stronger tolerance to the acid environment, the time for food (particularly fluid) to pass through the stomach is relatively short, and the food generally enters the small intestine after 1-2 h, so that the acid resistance of the bifidobacterium animalis B18 can ensure that the bifidobacterium animalis B18 can smoothly pass through the stomach to reach the intestinal tract and exert the physiological activity of the bifidobacterium animalis.
BIOLOGICAL AGRINST TEST OF BIOFORGANISM BIFOLIBRATION OF BIOFORGANISM BIFOBACTERIUM B18 TO ETHIC coli AND LACTIC ACIDS
1. Test method
1.1 strains
Bifidobacterium animalis B18, Escherichia coli, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Streptococcus lactis, Lactobacillus delbrueckii subsp.
1.2 culture Medium
MRS liquid and solid media, tomato juice solid and liquid media, nutrient broth solid and liquid media.
1.3 Strain activation
activating animal bifidobacterium: and unfreezing the bifidobacterium animalis placed in a low-temperature freezer in a water bath at the temperature of 40-50 ℃, sucking 0.2ml of the bifidobacterium animalis under the aseptic condition, inoculating the bifidobacterium animalis into an MRS liquid culture medium, culturing at the constant temperature of 37 ℃ for 12-14 h, and activating for 2-3 generations.
Activation of lactic acid bacteria: with Bifidobacterium animalis, lactobacillus is activated by MRS liquid culture medium, and streptococcus lactis is activated by tomato juice culture medium.
Activation of E.coli: inoculating Escherichia coli in refrigerator into liquid nutrient broth culture medium at an inoculum size of 2%, culturing at 37 deg.C for 12h, and activating for two generations.
1.4 spreading nutrient agar plate
The activated Escherichia coli is uniformly coated on nutrient agar plate by coating method, and the viable bacteria amount is required to be 106 cfu/ml.
1.5 cultivation of lactic acid bacteria by decantation
Uniformly spreading activated lactobacillus and streptococcus lactis on MRS plate and tomato juice solid plate respectively by pouring method, wherein viable bacteria amount is required to be 106cfu/ml。
1.6 preparation of Bifidobacterium animalis fermentation broth
Preparing fermentation supernatant from the activated bifidobacterium animalis fermentation liquor by adopting a low-temperature centrifugation method, wherein the centrifugation conditions are as follows: 4 ℃, 5000-8000 rpm, 5-10 min.
1.7 Observation of bacteriostatic effect of Bifidobacterium animalis fermentation broth
Oxford cup method: naturally drying the paved nutrient agar plate, MRS solid plate and tomato juice solid plate of the indicator bacteria for 30min, uniformly placing an oxford cup (with the inner diameter of 6mm) on the plate under an aseptic condition, slightly pressing down to ensure that no gap exists between the oxford cup and the contact surface of the plate and a plate culture medium, then sucking 0.2ml of fermentation supernatant into the cup, culturing at the constant temperature of 37 ℃ for 24h, observing the size of a bacteriostatic zone, and measuring the diameter of the bacteriostatic zone. Three replicates were made for each sample and the results averaged. In the control group, 0.2ml of MRS liquid culture medium was added to the Oxford cup under the same culture conditions.
2. Test results
The results of observing the bacteriostatic effect of the bifidobacterium animalis B18 fermentation broth are specifically shown in table 6.
TABLE 6 bacteriostatic effect of Bifidobacterium animalis B18 fermentation broth
As can be seen from Table 6, most of the bifidobacteria and lactobacilli have inhibitory effects on coliform, but the effect of inhibiting Bifidobacterium animalis B18 is particularly significant.
Experiments show that the bifidobacterium animalis B18 and most of lactic acid bacteria can synergistically grow, so that the application range of the bifidobacterium animalis B18 is expanded, and specific experimental results are shown in Table 7.
TABLE 7 combination of Bifidobacterium animalis with lactic acid bacteria (antagonism test)
| Bacterial species name | Test results | Bacterial species name | Test results |
| YDL200 Lactobacillus acidophilus | － | YDL18 Lactobacillus casei | － |
| YDL124 Lactobacillus acidophilus | － | YDL267 Lactobacillus paracasei | － |
| YDL163 Lactobacillus acidophilus | － | YDS151 Streptococcus thermophilus | － |
| YDL192 Lactobacillus acidophilus | － | YDS179 Streptococcus thermophilus | － |
| YDL137 Lactobacillus acidophilus | － | YDS194 Streptococcus thermophilus | － |
| YDL184 Lactobacillus bulgaricus | － | YDB176 Bifidobacterium bifidum | － |
| YDL162 Baogaria milk rod | － | YDS125 Streptococcus lactis | － |
| YDL197 Lactobacillus casei | － | YDL174 Lactobacillus plantarum | － |
| YDL108 Lactobacillus casei | － | YDS011 lactococcus lactis | － |
| YDL261 Lactobacillus buchneri | － | YDS170 Streptococcus thermophilus | + |
room temperature tolerance test of Bifidobacterium animalis B18
1. Test method
The animal bifidobacterium B18 bacterial powder of the same batch is respectively packed into 7 sterilized aluminum foil bags, each aluminum foil bag sample is vacuumized, and each vacuumized aluminum foil bag sample is placed in a room at normal temperature. One sample was taken every 60 days to determine the viable count of B18.
2. Test results
The relationship between the change in viable cell count and the number of days under the room temperature storage conditions is shown in Table 8.
TABLE 8B 18 tolerance to Room temperature
| Days of storage (d) | Viable count (cfu/ml) |
| 0 | 9.0×1011 |
| 60 | 8.0×1011 |
| 120 | 7.2×1011 |
| 180 | 6.1×1011 |
| 240 | 5.3×1011 |
| 300 | 4.5×1011 |
| 360 | 3.0×1011 |
Generally, the shelf life of the product containing the bifidobacterium animalis is about half a year at room temperature, and in the test, the bifidobacterium animalis B18 still has 3.0 multiplied by 10 after being stored for 360 days at room temperature11The viable count of cfu/ml is 33.3 percent. Therefore, B18 can effectively prolong the shelf life of the product and reduce the cost.
Application of bifidobacterium animalis B18 serving as feed additive in dairy cow feeding
1. Test method
200 cows with similar individual sizes and ages in days are selected and randomly divided into a control group and a test group, and each group carries out feeding experiments by 100 cows.
The control group of cows is fed with basic daily ration, and the test group is fed with basic daily ration added with animal bifidobacterium B18 lyophilized powder, wherein the addition ratio of the animal bifidobacterium B18 lyophilized powder is 1 per mill.
2. Test results
The test cows within each group were observed and recorded daily and the specific test results are shown in table 9.
Table 9B 18 Effect on cows
| Item | Control group | test group |
| Daily milk yield (kg) | 2701 | 2853 |
| Number of somatic cells (ten thousand/ml) | 57.57 | 30.13 |
As can be seen from table 9, compared with the control group of daily-fed cows, the daily average milk yield of the test group of cows added with the bifidobacterium animalis B18 lyophilized powder was increased by 10.75%; the number of the somatic cells is reduced by 52.34 percent compared with that of the control group. Therefore, the addition of the B18 bacterial powder can effectively improve the milk yield of the dairy cows and reduce the number of somatic cells.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
SEQUENCE NO.1
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
Beijing Bojin Yuan Biotechnology Ltd
Wangcong Tong
<120> animal bifidobacterium and application thereof
<130> 2019.06.19
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1428
<212> 16S rRNA Gene
<213> Bifidobacterium lactis
<400> 1
aaggtctacc cttagacggc tccccccaca agggtcgggc caccggcttc gggtgctacc 60
cactttcatg acttgacggg cggtgtgtac aaggcccggg aacgcattca ccgcggcgtt 120
gctgatccgc gattactagc gactccgcct tcacgcagtc gagttgcaga ctgcgatccg 180
aactgagacc ggttttcagc gatccgcccc acgtcaccgt gtcgcaccgc gttgtaccgg 240
ccattgtagc atgcgtgaag ccctggacgt aaggggcatg atgatctgac gtcatcccca 300
ccttcctccg agttgacccc ggcggtccca catgagttcc cggcatcacc cgctggcaac 360
atgcggcgag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 420
gacgaccatg caccacctgt gaaccggccc cgaagggaaa ccgtgtctcc acggcgatcc 480
ggcacatgtc aagcccaggt aaggttcttc gcgttgcatc gaattaatcc gcatgctccg 540
ccgcttgtgc gggcccccgt caatttcttt gagttttagc cttgcggccg tactccccag 600
gcgggatgct taacgcgttg gctccgacac gggacccgtg gaaagggccc cacatccagc 660
atccaccgtt tacggcgtgg actaccaggg tatctaatcc tgttcgctcc ccacgctttc 720
gctcctcagc gtcagtgacg gcccagagac ctgccttcgc cattggtgtt cttcccgata 780
tctacacatt ccaccgttac accgggaatt ccagtctccc ctaccgcact ccagcccgcc 840
cgtacccggc gcagatccac cgttaggcga tggactttca caccggacgc gacgaaccgc 900
ctacgagccc tttacgccca ataaatccgg ataacgctcg caccctacgt attaccgcgg 960
ctgctggcac gtagttagcc ggtgcttatt cgaacaatcc actcaacacg gccgaaaccg 1020
tgccttgccc ttgaacaaaa gcggtttaca acccgaaggc ctccatcccg cacgcggcgt 1080
cgctgcatca ggcttgcgcc cattgtgcaa tattccccac tgctgcctcc cgtaggagtc 1140
tgggccgtat ctcagtccca atgtggccgg tcaccctctc aggccggcta cccgtcaacg 1200
ccttggtggg ccatcacccc gccaacaagc tgataggacg cgaccccatc ccatgccgca 1260
aaagcatttc ccaccccacc atgcgatgga gcggagcatc cggtattacc acccgtttcc 1320
aggagctatt ccggtgcaca gggcaggttg gtcacgcatt actcacccgt tcgccactct 1380
caccccgaca gcaagctgcc agggatcccg ttcgactgca tggtaagc 1428
SEQUENCE NO.2
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
Beijing Bojin Yuan Biotechnology Ltd
Wangcong Tong
<120> Bifidobacterium animalis and use thereof
<130> 2019.06.19
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 873
<212> tuf Gene sequence
<213> Bifidobacterium animalis subsp
<400> 1
Cys Gly Ala Gly Ala Gly Ala Gly Cys Thr Cys Gly Ala Ala Gly Ala
1 5 10 15
Gly Cys Ala Gly Cys Gly Thr Gly Gly Thr Ala Thr Cys Ala Cys Cys
20 25 30
Ala Thr Cys Ala Ala Cys Ala Thr Thr Gly Cys Cys Cys Ala Cys Ala
35 40 45
Thr Cys Gly Ala Gly Thr Ala Cys Cys Ala Gly Ala Cys Gly Gly Cys
50 55 60
Cys Ala Ala Gly Cys Gly Thr Cys Ala Cys Thr Ala Cys Gly Cys Cys
65 70 75 80
Cys Ala Cys Gly Thr Cys Gly Ala Cys Thr Gly Cys Cys Cys Gly Gly
85 90 95
Gly Cys Cys Ala Cys Gly Cys Cys Gly Ala Cys Thr Thr Cys Gly Thr
100 105 110
Gly Ala Ala Gly Ala Ala Cys Ala Thr Gly Ala Thr Cys Ala Cys Cys
115 120 125
Gly Gly Cys Gly Cys Thr Gly Cys Cys Cys Ala Gly Ala Thr Gly Gly
130 135 140
Ala Thr Gly Gly Cys Gly Cys Cys Ala Thr Cys Cys Thr Cys Gly Thr
145 150 155 160
Thr Gly Thr Gly Gly Cys Cys Gly Cys Cys Ala Cys Cys Gly Ala Cys
165 170 175
Gly Gly Cys Cys Cys Gly Ala Thr Gly Gly Cys Cys Cys Ala Gly Ala
180 185 190
Cys Cys Cys Gly Cys Gly Ala Gly Cys Ala Cys Gly Thr Gly Cys Thr
195 200 205
Gly Cys Thr Cys Gly Cys Cys Cys Gly Thr Cys Ala Gly Gly Thr Cys
210 215 220
Gly Gly Cys Gly Thr Cys Cys Cys Gly Ala Ala Gly Ala Thr Cys Cys
225 230 235 240
Thr Cys Gly Thr Cys Gly Cys Thr Cys Thr Gly Ala Ala Cys Ala Ala
245 250 255
Gly Thr Gly Cys Gly Ala Thr Ala Thr Gly Gly Thr Cys Gly Ala Thr
260 265 270
Gly Ala Cys Gly Ala Ala Gly Ala Gly Cys Thr Cys Ala Thr Cys Gly
275 280 285
Ala Gly Cys Thr Cys Gly Thr Cys Gly Ala Ala Gly Ala Ala Gly Ala
290 295 300
Gly Gly Thr Cys Cys Gly Cys Gly Ala Cys Cys Thr Cys Cys Thr Cys
305 310 315 320
Gly Ala Cys Gly Ala Gly Ala Ala Cys Gly Gly Cys Thr Thr Cys Gly
325 330 335
Ala Cys Cys Gly Cys Gly Ala Cys Thr Gly Cys Cys Cys Gly Gly Thr
340 345 350
Cys Gly Thr Gly Cys Ala Cys Ala Cys Cys Thr Cys Cys Gly Cys Thr
355 360 365
Thr Ala Cys Gly Gly Cys Gly Cys Thr Cys Thr Gly Cys Ala Thr Gly
370 375 380
Ala Cys Gly Ala Cys Gly Cys Thr Cys Cys Gly Gly Ala Thr Cys Ala
385 390 395 400
Cys Gly Ala Cys Ala Ala Gly Thr Gly Gly Gly Thr Thr Gly Cys Cys
405 410 415
Ala Cys Cys Ala Thr Cys Ala Ala Gly Gly Ala Gly Cys Thr Cys Ala
420 425 430
Thr Gly Gly Ala Cys Gly Ala Cys Gly Thr Cys Gly Ala Cys Gly Ala
435 440 445
Gly Thr Ala Cys Ala Thr Cys Cys Cys Gly Ala Cys Cys Cys Cys Gly
450 455 460
Gly Thr Cys Cys Ala Cys Gly Ala Cys Cys Thr Cys Gly Ala Cys Ala
465 470 475 480
Ala Gly Cys Cys Gly Thr Thr Cys Cys Thr Gly Ala Thr Gly Cys Cys
485 490 495
Gly Ala Thr Cys Gly Ala Gly Gly Ala Cys Gly Thr Cys Thr Thr Cys
500 505 510
Ala Cys Cys Ala Thr Cys Thr Cys Cys Gly Gly Cys Cys Gly Thr Gly
515 520 525
Gly Cys Ala Cys Cys Gly Thr Cys Gly Thr Cys Ala Cys Cys Gly Gly
530 535 540
Thr Cys Gly Thr Gly Thr Cys Gly Ala Gly Cys Gly Cys Gly Gly Cys
545 550 555 560
Ala Ala Gly Cys Thr Gly Cys Cys Gly Ala Thr Cys Ala Ala Cys Ala
565 570 575
Cys Gly Ala Ala Cys Gly Thr Cys Gly Ala Gly Ala Thr Cys Gly Thr
580 585 590
Cys Gly Gly Cys Ala Thr Cys Cys Gly Cys Cys Cys Gly Ala Cys Cys
595 600 605
Cys Ala Gly Ala Cys Cys Ala Cys Cys Ala Cys Cys Gly Thr Cys Ala
610 615 620
Cys Cys Thr Cys Cys Ala Thr Cys Gly Ala Gly Ala Cys Cys Thr Thr
625 630 635 640
Cys Cys Ala Cys Ala Ala Gly Cys Ala Gly Ala Thr Gly Gly Ala Thr
645 650 655
Gly Ala Gly Thr Gly Cys Gly Ala Gly Gly Cys Cys Gly Gly Cys Gly
660 665 670
Ala Cys Ala Ala Cys Ala Cys Cys Gly Gly Thr Cys Thr Gly Cys Thr
675 680 685
Gly Cys Thr Cys Cys Gly Cys Gly Gly Cys Ala Thr Cys Ala Ala Cys
690 695 700
Cys Gly Cys Ala Cys Cys Gly Ala Cys Gly Thr Cys Gly Ala Gly Cys
705 710 715 720
Gly Thr Gly Gly Cys Cys Ala Gly Gly Thr Cys Gly Thr Gly Thr Cys
725 730 735
Thr Gly Cys Thr Cys Cys Gly Gly Gly Thr Thr Cys Gly Gly Thr Cys
740 745 750
Ala Cys Cys Cys Cys Gly Cys Ala Cys Ala Cys Cys Ala Ala Gly Thr
755 760 765
Thr Cys Gly Ala Ala Gly Gly Cys Gly Ala Ala Gly Thr Cys Thr Ala
770 775 780
Cys Gly Thr Cys Cys Thr Thr Ala Cys Cys Ala Ala Gly Gly Ala Thr
785 790 795 800
Gly Ala Gly Gly Gly Cys Gly Gly Cys Cys Gly Thr Cys Ala Cys Thr
805 810 815
Cys Gly Cys Cys Gly Thr Thr Cys Thr Thr Cys Thr Cys Gly Ala Ala
820 825 830
Cys Thr Ala Cys Cys Gly Thr Cys Cys Gly Cys Ala Gly Thr Thr Cys
835 840 845
Thr Ala Cys Thr Thr Cys Cys Gly Cys Ala Cys Cys Ala Cys Cys Gly
850 855 860
Ala Cys Gly Thr Cys Ala Cys Cys Gly
865 870
Claims (10)
1. A Bifidobacterium animalis characterized in that: belongs to Bifidobacterium animalis subsp.lactis B18 with the preservation number of CGMCC No.17535 and the preservation date of 2019, 04/09 days and the preservation unit: china general microbiological culture Collection center.
2. Bifidobacterium animalis according to claim 1, characterized in that: the animal bifidobacterium B18 has tolerance to gastric acid and bile salt of human body or animals.
3. bifidobacterium animalis according to claim 1, characterized in that: after the bifidobacterium animalis B18 is placed for 360 days at room temperature, the total number of live bacteria in the bacterial liquid is not less than 3.0 multiplied by 1011cfu/ml。
4. bifidobacterium animalis according to claim 1, characterized in that: the animal bifidobacterium B18 has an inhibiting effect on escherichia coli flora.
5. Bifidobacterium animalis according to claim 1, characterized in that: the bifidobacterium animalis B18 is compounded with one or more of the following lactic acid bacteria, and has a synergistic growth effect:
lactobacillus acidophilus (Lactobacillus acidophilus);
Lactobacillus delbrueckii subsp. bulgaricus;
lactobacillus casei subsp. casei;
Streptococcus thermophilus (Streptococcus thermophilus);
Lactobacillus paracasei (Lactobacillus paracasei);
Streptococcus lactis (Streptococcus lactis);
Enterococcus faecium (Enterococcus faecium);
Lactococcus lactis subsp. lactis;
Lactobacillus buchneri (Lactobacillus buchneri);
Bifidobacterium bifidum (Bifidobacterium bifidum).
6. A method for culturing Bifidobacterium animalis as claimed in any of claims 1 to 5, wherein: inoculating the strain of the bifidobacterium animalis B18 into a fermentation culture medium for fermentation, wherein the fermentation culture medium comprises yeast extract, sucrose and K2HPO4。
7. A preparation method of animal bifidobacterium lyophilized powder is characterized in that: uniformly mixing fermented bacterial sludge of the bifidobacterium animalis B18 in the claim 1 with a protective agent according to the mass ratio of 1: 4-6, and then freezing and drying at the temperature of-30 to-40 ℃.
8. The method of claim 7, wherein: the protective agent comprises the following components in parts by weight: 5-20 g of skim milk, 5-15 g of T809 starch, 5-10 g of lactose, 8-15 g of trehalose, 0.5-1.5 g of fructo-oligosaccharide, 1-3 g of xylo-oligosaccharide, 0.5-1.5 g of isomalto-oligosaccharide, 0.5-1 g of inulin, 0.5-1 g of stachyose, 1-2 g of sodium glutamate and 100-200 ml of distilled water.
9. Use of a bifidobacterium animalis as claimed in claim 1 wherein: the bifidobacterium animalis B18 is applied to food or animal feed.
10. The application of freeze-dried animal bifidobacterium powder is characterized in that: application of lyophilized powder prepared from Bifidobacterium animalis B18 in milk cow feeding is provided.
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| CN112094785A (en) * | 2020-10-12 | 2020-12-18 | 中科美大(福建)生物科技有限公司 | Bifidobacterium animalis as well as preparation and application thereof |
| CN113197311A (en) * | 2020-07-03 | 2021-08-03 | 内蒙古蒙牛乳业（集团）股份有限公司 | Lactic acid bacteria composition and preparation method thereof |
| CN114480229A (en) * | 2022-04-15 | 2022-05-13 | 微康益生菌(苏州)股份有限公司 | Bifidobacterium animalis subsp lactis strain WKB148 and product and application thereof |
| CN118109363A (en) * | 2024-03-25 | 2024-05-31 | 内蒙古科拓生物有限公司 | Composite probiotic composition for preventing or treating cancer, preparation method and application thereof |
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| CN113197311A (en) * | 2020-07-03 | 2021-08-03 | 内蒙古蒙牛乳业（集团）股份有限公司 | Lactic acid bacteria composition and preparation method thereof |
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| CN114480229A (en) * | 2022-04-15 | 2022-05-13 | 微康益生菌(苏州)股份有限公司 | Bifidobacterium animalis subsp lactis strain WKB148 and product and application thereof |
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