CN111154677B - Lactobacillus acidophilus and application thereof - Google Patents
Lactobacillus acidophilus and application thereof Download PDFInfo
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- CN111154677B CN111154677B CN202010033981.5A CN202010033981A CN111154677B CN 111154677 B CN111154677 B CN 111154677B CN 202010033981 A CN202010033981 A CN 202010033981A CN 111154677 B CN111154677 B CN 111154677B
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Abstract
The invention provides lactobacillus acidophilus and application thereof. The lactobacillus acidophilus Ma.SSRGJ1 is a gram-positive bacterium, can grow in an acid environment with the pH value of more than 3.0, has strong bile salt resistance and bacteriostatic ability on gram-negative bacteria, has safe and reliable effect when being used for feeding animals after being prepared into a microbial inoculum, and has positive effects on promoting digestion and absorption of nutrient substances, improving feed conversion efficiency and promoting growth.
Description
Technical Field
The invention belongs to the field of microbiology and feeding probiotics, and particularly relates to lactobacillus acidophilus and application thereof.
Background
In the livestock breeding industry, the microecological preparation is gradually replacing feed antibiotics as an environment-friendly feed additive, can improve the intestinal microecological environment, promote the proliferation of beneficial bacteria in the intestinal tracts of livestock and poultry, prevent or inhibit the propagation of harmful bacteria, adjust the balance of intestinal flora, obviously reduce the ammonia odor of animal excrement, reduce mosquito, fly and insect pests, improve and optimize the livestock and poultry breeding ecological environment, reduce environmental pollution, improve the anti-stress capability and immunity of animals, improve the conversion rate of animal feed, reduce the production cost and increase the economic benefit.
The ideal strain which can be directly fed for the microecological preparation is as follows: firstly, people and animals cannot be pathogenic, and hybrid seeds cannot be generated between the pathogenic microorganisms and the pathogenic microorganisms; secondly, the propagation is easy in vitro and in vivo, and the in vitro propagation speed is high; ③ the drug can survive in low pH and bile and can be implanted into intestinal mucosa; fourthly, substances such as lactic acid, hydrogen peroxide and the like can be generated in the fermentation process; can synthesize the inhibitors for enteropathogenic bacteria such as escherichia coli, salmonella, staphylococcus, clostridium and the like without influencing the activity of the inhibitors; sixthly, the survival rate of the live bacteria is high after processing, and the stability at high temperature is good after the live bacteria is mixed into the feed; preferably from the animal's own intestinal tract; is favorable for promoting the growth and development of hosts and improving the disease resistance.
The microecologics have been widely used in the livestock breeding industry due to important probiotic effect, but the study of feeding lactobacillus acidophilus microecologics is rare. The feeding lactobacillus acidophilus microecological preparation has the functions of probiotics and has the characteristics of high activity and excellent stability. With the improvement of the preparation process and the deep research, the microecological preparation is expected to be widely applied.
Lactobacillus acidophilus belongs to bacteria which are published by the FDA (1989) in the United states and the department of agriculture (1999) in China and have no pathogenicity to animals and can be directly used for animal feed. A large number of experiments at home and abroad also prove that the lactobacillus acidophilus is beneficial to establishing normal flora in animals, resisting the invasion of harmful microorganisms, improving the production performance of the animals and preventing and treating diseases. The lactobacillus acidophilus can not only adjust the balance of intestinal flora, but also secrete antibiotics such as lactobacillus acidophilus, acidophilus bacteriocin, lactobacillin and the like, thereby generating antagonism to intestinal pathogenic bacteria and inhibiting the proliferation of intestinal undesirable microorganisms. Currently, the applications of lactobacillus acidophilus are mainly focused on the dairy industry, the pharmaceutical industry and other industrial applications. The application of lactobacillus acidophilus in dairy products is mainly fermented dairy products, and the application in the pharmaceutical industry is mainly due to the special health-care effect of lactobacillus acidophilus.
Disclosure of Invention
The lactobacillus acidophilus provided by the invention is acid-resistant, cholate-resistant and good in bacteriostatic effect.
In order to achieve the purpose, the invention provides Lactobacillus acidophilus (Lactobacillus acidophilus) Ma.ssrgj1, which is obtained by separating pig intestinal tracts of a certain pig raising experimental base in beijing and screening after ultraviolet mutagenesis. The strain ma. ssrgj1 is Lactobacillus acidophilus (Lactobacillus acidophilus) by 16S rRNA gene sequence analysis. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Lu 1 of North Chen of the south facing Yang district in Beijing, the institute of microbiology of China academy of sciences, zip code 100101) in 11.12.2019, and is classified and named as Lactobacillus acidophilus with the preservation number of CGMCC NO. 18939.
The microbiological properties of lactobacillus acidophilus ma.ssrgj1 are: gram-positive bacteria, wherein the cell shape is rod-shaped, the tail end of the rod is round, and no spore exists; the size of a single colony is less than or equal to 1mm, the color is milky white and opaque, the surface of the colony is smooth and wet, and the edge is neat and glossy. The thallus can grow in an acid environment with a pH value of more than 3.0, and has strong bile salt resistance. And has certain bacteriostatic ability, and especially has obvious effect of inhibiting escherichia coli and salmonella.
In a second aspect, the present invention provides a microbial inoculum containing said lactobacillus acidophilus ma.
In a third aspect, the present invention provides a feed additive or animal feed containing the lactobacillus acidophilus or its bacterial agent.
The number of viable bacteria of Lactobacillus acidophilus Ma.SSRgJ1 in the feed additive is 1 × 106CFU/g～1×1010CFU/g; preferably, the feed additive contains Lactobacillus acidophilus Ma.SSRgJ1 with viable count of 1 × 108CFU/g～1×109CFU/g。
The number of viable bacteria of Lactobacillus acidophilus Ma.SSRgJ1 in the animal feed is 1 × 105CFU/kg～1×108CFU/kg, preferably 1X 107CFU/kg～1×108CFU/kg。
The probiotic effect of the lactobacillus acidophilus ma.ssrgj1 is identified by an in vitro method, and the result shows that the lactobacillus acidophilus ma.ssrgj1 can resist acid, acid and bile salt, can resist the internal environment of the gastrointestinal tract, and has the potential of probiotics.
Based on the fact that lactobacillus acidophilus Ma.SSRGJ1 has outstanding bacteriostatic properties, the medicine or the composition containing lactobacillus acidophilus Ma.SSRGJ1 or the microbial inoculum thereof, in particular bacteriostatic medicines, belong to the protection scope of the invention.
In a fourth aspect, the present invention provides a preservative containing said lactobacillus acidophilus or its bacterial agent.
In a fifth aspect, the invention provides the application of the lactobacillus acidophilus or the microbial inoculum thereof in bacteriostasis, including non-treatment purpose application.
The use as described above, said bacterium being a gram-negative bacterium.
Preferably, the bacteria include, but are not limited to, Escherichia (Escherichia) bacteria or Salmonella (Salmonella) bacteria, more preferably Escherichia coli, Salmonella. Such as Escherichia coli O157, Escherichia coli K99, Salmonella pullorum (Salmonella pullorum) CVCC 1791.
In a sixth aspect, the invention provides any one of the following applications of the lactobacillus acidophilus or its bacterial agent:
1) used for preparing feed additive;
2) for increasing feed conversion rate;
3) is used for promoting animal growth, increasing animal weight and disease resistance;
4) used in the fields of food, medicine or health care products;
5) can be used in the field of antiseptic.
In a seventh aspect, the present invention provides a method for preparing a lactobacillus acidophilus agent, the method comprising: fermenting and culturing lactobacillus acidophilus Ma.SSRGJ1, centrifuging the fermentation liquor, taking the precipitate to obtain bacterial sludge, mixing with a freeze-drying protective agent, and freeze-drying.
Preferably, the fermentation medium used is: 30g/L of whey powder, 30g/L of soybean meal, 5g/L of glucose, 4g/L of sodium chloride, 0.1g/L of zinc sulfate, 0.6g/L of manganese sulfate, 1g/L of magnesium sulfate and 0.05% v/v of defoaming agent.
The fermentation conditions were: introducing mixed gas with the volume ratio of nitrogen to hydrogen of 9:1 at the temperature of 35 ℃ and the rotation speed of 200rpm under the stirring condition, and fermenting and culturing for 16 h.
The lyoprotectant may be: 10% skimmed milk powder + 6% lactose. The dosage ratio of the bacterial sludge to the freeze-drying protective agent is 3:1 in terms of g: mL.
The invention provides application of lactobacillus acidophilus Ma.SSRgJ1 or a microbial inoculum thereof in inhibiting gram-negative bacteria. Wherein, the gram-negative bacteria include but are not limited to escherichia coli and salmonella. In one embodiment of the invention, the lactobacillus acidophilus ma.ssrgj1 has strong inhibition capacity on escherichia coli O157 and salmonella CVCC1791, and the diameters of inhibition zones reach 2.80cm and 2.17cm respectively.
The lactobacillus acidophilus Ma.SSRGJ1 provided by the invention has the advantages that the feed utilization rate is improved, and the digestion and absorption of nutrient substances in the feed are promoted; enhancing animal immunity, increasing daily gain, and reducing feed conversion ratio; no pollution, no residue, biological environmental protection and the like, and has obvious effects on promoting the growth of animals and improving the weight of the animals.
Drawings
Fig. 1 is a colony morphology of lactobacillus acidophilus ma.
Fig. 2 is a gram stain of lactobacillus acidophilus ma.
Fig. 3 shows the results of acid resistance measurement of lactobacillus acidophilus ma.
Fig. 4 shows the results of the bile salt resistance test of lactobacillus acidophilus ma.
Fig. 5 is a growth curve of lactobacillus acidophilus ma.
Fig. 6 shows the results of the bacteriostatic test of lactobacillus acidophilus ma. (ii) a
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The MRS medium used in the following examples was formulated as follows: weighing 10g of peptone, 5g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 801.08 g of tween-801.08 g, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate and magnesium sulfate (MgSO)4·7H2O)0.2g, manganese sulfate (MnSO)4·4H2O)0.05g and agar 15g, and adjusting the pH to 6.2 by adding distilled water to a constant volume of 1L. Placing into a sterilizing pot, sterilizing at 121 deg.C for 20 min.
Example 1 isolation and characterization of Lactobacillus acidophilus (Lactobacillus acidophilus) Ma
Isolation of Strain Ma. SSRgJ1
1. Isolation culture of strains
1g of intestinal chyme sample of a pig from a pig raising experimental base of China agriculture university in Haisheu district, Beijing is put into a test tube with 9ml of normal saline, a vortex machine is used for shaking and mixing uniformly to obtain a 1:10 diluent, the diluent is taken for 10 times of incremental dilution, and then 1ml of each of 3 diluents with proper gradients is selected and coated on an MRS culture medium. Culturing at 37 deg.C for 48-72 hr, observing and recording colony morphology, picking single colony with good growth, and streaking for separation and purification. The bacterial suspension was prepared with physiological saline.
2. Ultraviolet mutagenesis and screening of strains
And (3) pouring the sterilized MRS culture medium into culture dishes, after solidification, coating the bacterial suspension obtained in the step (1) on a flat plate, controlling bacterial colonies to be about 50-80 in each culture dish, culturing for 12 hours, and then, carrying out mutagenesis for 30s at a distance of 20cm from an ultraviolet lamp.
Selecting the mutagenized strain, inoculating the strain in an MRS liquid culture medium, culturing for 24 hours, measuring the OD value, selecting the strain with higher growth speed, coating a proper amount of bacterial suspension on an MRS plate, culturing for 24 hours in a constant-temperature incubator at 37 ℃, and carrying out the next step of gram staining.
3. Gram staining of the Strain
Dropping a drop of sterilized distilled water on a glass slide, selecting a single colony (a colony morphology chart is shown in figure 1) which grows fast after mutagenesis, dissolving the single colony in water, scraping the single colony by a scraper, and drying and fixing the single colony on an alcohol lamp. Dripping crystal violet staining solution, staining for 2min, washing with water, and naturally drying; dripping iodine solution for 2min, washing with water, and naturally drying; dropwise adding 50S of alkaline fuchsin ethanol solution, washing with water, and naturally drying; when the purple cells were observed on a common optical microscope, the red cells were negative, and the results are shown in FIG. 2. And selecting gram-positive bacilli to carry out a spore staining experiment in the next step.
4. Spore staining of strains
And (3) taking the strain which grows at a higher speed after mutagenesis in the step (2), dripping a drop of sterilized distilled water on a glass slide, selecting a single strain, dissolving the single strain in the water, scraping the single strain by a scraper, and drying and fixing the single strain on an alcohol lamp. Dripping 3-5 drops of 5% malachite green solution, heating on alcohol lamp for 3-5min, washing with water, and naturally drying; dripping lycopene solution for dyeing for 2min, washing with water, and naturally drying; when observed under a common optical microscope, the spores are green, and the cells are red. Finally obtaining a gram-positive spore-free bacterial strain through the separation and screening in the steps 1-4. The strain was numbered ma.
Identification of Strain Ma
1. Morphological identification
Single colonies of strain ma.ssrgj1 in log growth phase and stable colony size are described as follows: the size of a single colony is less than or equal to 1mm, the single colony is circular, the color is milky white and opaque, the surface of the colony is moist and smooth, and the edge is regular.
Subsequently, the strain ma.ssrgj1 in the logarithmic growth phase was stained, and the form of the cells was observed by an optical microscope. The isolated and screened strain ma. ssrgj1, which is gram positive, rod-shaped in cell morphology and free of spores.
2. 16S rRNA Gene sequence homology analysis
The extraction of the total DNA of the bacteria adopts a bacterial genome DNA extraction kit of Tiangen Biochemical technology Co. The extracted sample is sent to Shanghai Megi biological medicine science and technology Limited for sequencing. BLAST homology comparison is carried out on the determination result in a GenBank database, and the strain type is determined to be Lactobacillus acidophilus (Lactobacillus acidophilus). The sequencing result is shown in SEQ ID NO 1.
16S rRNA gene sequencing is carried out, and the bacterium is identified as Lactobacillus acidophilus according to the sequencing result, the microbiological characteristics and the physicochemical characteristics.
Example 2 stress resistance assay of Lactobacillus acidophilus Ma.SSRgJ1
1. Heat resistance test
Placing the bacterial liquid of Lactobacillus acidophilus Ma.SSRgJ1 in a water bath pot for 20 minutes, respectively treating with the bacterial liquid at 60 ℃, 80 ℃ and 100 ℃, wherein each treatment is repeated for 3 times, and measuring the viable count by adopting a pouring method after the treatment is finished.
After 8lg (cfu/ml) of lactobacillus acidophilus Ma. SSRGJ1 is treated at 60 ℃ for 20 minutes, the viable count is 5.86lg (cfu/ml), and after the lactobacillus acidophilus is treated at 80 ℃ for 20 minutes, the viable count is close to 0, which indicates that the lactobacillus acidophilus cannot tolerate high temperature and needs to be coated in industrial production.
2. Acid resistance detection
Will 108The CFU/ml Lactobacillus acidophilus Ma SSRGJ1 is respectively inoculated into MRS culture media with pH values of 2.0, 3.0, 4.0 and 5.0, and the viable count of the Lactobacillus acidophilus is respectively measured by adopting a plate pouring method at 1h, 2h, 3h and 4 h.
When the pH value is 5.0, 4.0 and 3.0, the lactobacillus acidophilus can normally grow, and when the pH value is 2.0, the growth of the lactobacillus acidophilus is slightly inhibited, but the viable count can still be maintained at 7.48lg (cfu/ml). The strain has strong acid tolerance. The results show that lactobacillus acidophilus ma.
3. Bile salt resistance detection
Activated lactobacillus acidophilus Ma.SSRGJ1 is diluted by sterile normal saline in a multiplying ratio, a proper dilution gradient is selected, 200 microliters of diluent is absorbed and placed in a sterile culture dish for 6 times, then MRS culture medium containing sodium taurocholate (0.1%, 0.2%, 0.3% and 0.4%) with different concentrations is poured on the plate, the plate is cultured at 37 ℃ for 4 hours, colonies are counted every 1 hour, meanwhile, MRS culture medium containing no sodium taurocholate is poured on the plate, the plate is cultured at 37 ℃ for 48 hours, and the colonies are counted to serve as a control group. The results in FIG. 4 show that the viable cell count at different bile salt concentrations does not decrease significantly over time. The effect of 0.1%, 0.2% and 0.3% of bile salts on lactobacillus acidophilus ma. After 4 hours of action of 0.4% bile salts, the viable count decreased slightly, but there was no significant difference. The lactobacillus acidophilus Ma. SSRGJ1 has stronger bile salt resistance.
4. Antibiotic susceptibility testing
Lactobacillus acidophilus ma.ssrgj1 with appropriate concentration was spread on MRS medium, 1 drug sensitive paper sheet was uniformly attached to each dish, cultured for 36 hours, and the size of the zone of inhibition was observed (table 1).
TABLE 1 Lactobacillus acidophilus Ma. SSRgJ1 susceptibility results to different antibiotics
| Name (R) | Diameter of bacteriostatic circle (mm) | Sensitivity of the device |
| Ampicillin | 12 | Mianmin |
| Doxycycline | 15 | Gao Min |
| Penicillin | 23 | |
| Kanamycin | ||
| 8 | Hyposensitivity | |
| Gentamicin | 9 | Hyposensitivity |
| Erythromycin | 22 | Extreme sensitivity |
| Cefalexin | 22 | |
| Ciprofloxacin | ||
| 8 | Hyposensitivity | |
| Chloromycetin | 29 | Extreme sensitivity |
The experimental results show that the lactobacillus acidophilus Ma.SSRGJ1 CGMCC No.18939 does not have good drug resistance, so the lactobacillus acidophilus Ma.SSRGJ1 is safe and reliable to use as a feeding probiotic.
5. Bacteriostatic experiment of lactobacillus acidophilus Ma.SSRGJ1 on gram-negative bacteria
Pouring prepared MRS culture medium into a culture dish, placing an oxford cup after the culture medium is solidified, pouring LB culture medium mixed with 1% of pathogenic bacteria (Escherichia coli O157, Escherichia coli K99 and salmonella CVCC1791) into the upper layer, and solidifying for use. 200 microliters of thalli, bacterial liquid and supernatant are respectively added into the oxford cup, carefully placed into a constant-temperature incubator at 37 ℃ and vertically cultured for 24 hours, and the size of a bacteriostatic zone is checked. The result shows that the lactobacillus acidophilus MA.SSRGJ1 has very obvious bacteriostasis effect on Escherichia coli O157 and salmonella CVCC1791, the size of the bacteriostasis zone is 2.8cm and 2.17cm respectively, and the lactobacillus acidophilus also has certain bacteriostasis effect on Escherichia coli K99, and the bacteriostasis zone reaches 2cm (figure 6).
Example 3 growth curve assay for Lactobacillus acidophilus Ma.SSRgJ1
The growth curve represents the dynamic change of the bacteria in the new and suitable environment until the whole process of aging and death. The viable count was calculated by inoculating Lactobacillus acidophilus Ma.SSRgJ1 in an amount of 10% (v/v) into MRS medium, culturing at 37 ℃ for 20 hours, and measuring OD600 values every 2 hours using MRS medium without added bacteria solution as a blank. The experiment was repeated three times, the results were averaged, the data were recorded and growth curves were plotted. As shown in fig. 5, lactobacillus acidophilus ma. ssrgj1 is in the logarithmic growth phase at 2-16 hours, with a higher propagation rate. The number of lactobacillus acidophilus ma.ssrgj1 tends to be stable at 16-20 hours, at plateau.
EXAMPLE 4 preparation of Lactobacillus acidophilus preparation
1. The fermentation medium formula comprises: whey powder 30g/L, soybean meal 30g/L, glucose 5g/L, sodium chloride 4g/L, zinc sulfate 0.1g/L, manganese sulfate 0.6g/L, magnesium sulfate 1g/L, defoaming agent 0.05% (v/v), adding water and fully dissolving to prepare the fermentation medium.
2. Sterilizing with high temperature steam at 115 deg.C for 30 min.
3. When the temperature of the fermentation medium is reduced to 35 ℃, 5% (v/v) of bacterial liquid with the age of 20 hours is inoculated.
4. Stirring at 35 deg.C and 200rpm, introducing mixed gas of nitrogen and hydrogen at a volume ratio of 9:1, fermenting for 16 hr, and canning to obtain Lactobacillus acidophilus with viable count of 1 × 109cfu/ml fermentation broth. Centrifuging the fermentation liquor at 4 deg.C to obtain precipitate and obtain bacterial sludge.
5. 100mL of freeze-drying protective agent is added into 300g of bacterial sludge, and the mixture is uniformly mixed by using an oscillator to prepare bacterial suspension. Precooling at minus 80 ℃ for 1.5 hours, and quickly transferring the frozen sample to a freeze dryer for freeze-drying for 24 hours to ensure that the water content of the fungus powder reaches about 3 percent. The formula of the freeze-drying protective agent is as follows: 10% skimmed milk powder + 6% lactose.
Example 5 safety evaluation of Lactobacillus acidophilus Ma. SSRgJ1 formulations
In this example, a mouse is used as an experimental animal, and the safety of lactobacillus acidophilus is evaluated by a gavage test method, which specifically comprises the following steps:
1. the freeze-dried powder of lactobacillus acidophilus agent prepared by the method of example 4 has the number of lactobacillus acidophilus Ma.SSRGJ1 bacteria of 1 multiplied by 10 determined by plate counting9cfu/g。
2. Selecting about 8 weeks old mice 72, randomly dividing into 4 groups (group A is control group and is administered with sterile normal saline, group B is high dose group according to 1 × 109The bacterial liquid is filled in cfu/cfu, and the C group is a medium dose group according to 1 multiplied by 108The bacterial liquid is filled in cfu/bacterium, and the group D is a low-dose group according to 1 multiplied by 107Amount of cfu/mouse), 3 replicates per group, 6 mice per replicate.
3. The administration is carried out once every nine morning hours for 21 days.
The laboratory mouse room controls the constant temperature and humidity, the natural illumination, the mouse freely takes food and drinks water, and the mouse cage is cleaned once every 7 days. In the experimental process, the state, survival condition, presence or absence of clinical abnormal symptoms and the like of the mice were observed and recorded every day.
Detection indexes are as follows:
(1) on the day of experiment, blood samples of experimental mice are obtained by adopting a heart blood taking mode, and serum is obtained after static centrifugation and is used for detecting blood biochemical indexes such as albumin, total protein, high-density lipoprotein, low-density lipoprotein, triglyceride, cholesterol, urea, tumor cell necrosis factor and the like in the serum.
(2) The whole heart, liver, spleen and kidney were weighed (bilaterally) and wet-weighed, and the heart index ═ heart wet weight/body weight × 100%, liver index ═ liver wet weight/body weight × 100%, spleen index ═ spleen wet weight/body weight × 100%, and kidney index ═ kidney wet weight/body weight × 100% were calculated, respectively.
TABLE 2 survival of mice in different treatment groups
As can be seen from Table 2, after the mice were gavaged with Lactobacillus acidophilus Ma.SSRGJ1 CGMCC No.18939 for 21 days, the mice of each treatment group survived, indicating that the Lactobacillus acidophilus is safe for animals.
TABLE 3 organ coefficients of mice of different treatment groups
| Group A | Group B | Group C | Group D | |
| Heart and heart | 0.59 | 0.68 | 0.65 | 0.63 |
| Liver disease | 5.64 | 5.58 | 5.53 | 5.61 |
| Spleen | 0.44 | 0.43 | 0.46 | 0.44 |
| Kidney (A) | 1.31 | 1.37 | 1.32 | 1.35 |
As can be seen from Table 3, the organ index of the treated mice was not significantly changed from that of the control group, indicating that the Lactobacillus acidophilus did not cause abnormality in the organs of the mice.
The results of detecting albumin, total protein, high density lipoprotein, low density lipoprotein, triglyceride, cholesterol, urea, tumor cell necrosis factor, etc. in the mouse serum by using a biochemical analyzer all show normal, which indicates that the lactobacillus acidophilus preparation provided in embodiment 4 of the present invention does not affect various physiological indexes of the mouse.
Example 6 application of Lactobacillus acidophilus Ma. SSRgJ1 preparation
In the experiment, 72 weaned piglets of 28-day old Du-grown ternary hybrid are selected, the experimental period is 45 days, the piglets are divided into 2 groups according to the random block group design, each group has 6 repetitions, and each repetition has 6 pigs. Group A was a control group (basal diet group), group B was a treatment group (basal diet supplemented with 260g/t of the Lactobacillus acidophilus preparation prepared in example 4, and the effective viable count was 1X 109cfu/g)。
During the test period, piglets are raised in a totally enclosed nursing pigsty, the temperature is controlled to be 25-27 ℃, and the piglets are fed with free food and water. The basal diet does not contain any antibiotics, and the immunization of the piglets is carried out according to a conventional immunization program.
Measurement indexes are as follows: the production performance of the weaned piglets of each treatment group specifically comprises the following indexes:
1. the piglet feed intake was recorded every day and the average daily feed intake was calculated after the experiment was completed.
2. Piglet body weights were recorded on the day the experiment began and ended and the average daily gain was calculated.
3. The feed-meat ratio was calculated from the test results of A, B in terms of average daily feed intake/average daily gain.
4. During the test period, the fecal condition of the piglets is observed and recorded at 10:00 a day in the morning, and the diarrhea rate of the weaned piglets is calculated, wherein the diarrhea rate (%) is (number of diarrhea heads multiplied by the number of diarrhea days)/(number of pigs multiplied by the number of test days) multiplied by 100%.
TABLE 4 influence of Lactobacillus acidophilus preparation addition to basal ration on weaned pig productivity
As can be seen from Table 4, the average daily feed intake and average daily gain of the piglets in the treated group are both significantly higher than those in the control group (P <0.05), and the feed-meat ratio is lower than that in the control group, which indicates that the feed benefit is better by adding the Lactobacillus acidophilus preparation. The diarrhea rate of the treated group is obviously reduced compared with that of the control group, which shows that the lactobacillus acidophilus has the function of improving the diarrhea of the piglets.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> university of agriculture in China
<120> Lactobacillus acidophilus and uses thereof
<130> KHP201110099.3
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<213> Lactobacillus acidophilus (Lactobacillus acidophilus)
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ctgttgttag agaagaacaa ggatgagagt aactgttcat cccttgacgg tatctaacca 480
gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc 540
cggatttatt gggcgtaaag cgagcgcagg cggtttctta agtctgatgt gaaagccccc 600
ggctcaaccg gggagggtca ttggaaactg ggagacttga gtgcagaaga ggagagtgga 660
attccatgtg tagcggtgaa atgcgtagat atatggagga acaccagtgg cgaaggcggc 720
tctctggtct gtaactgacg ctgaggctcg aaagcgtggg gagcaaacag gattagatac 780
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gctgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa 900
aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga 960
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gcaaagtgac aggtggtgca tggttgtcgt cagctcgggc gtgaaatgtt gggttaagtc 1080
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Claims (11)
1. Lactobacillus acidophilus (Lactobacillus acidophilus) Ma.SSRGJ1 with the preservation number of CGMCC NO. 18939.
2. A microbial preparation comprising the Lactobacillus acidophilus according to claim 1.
3. A feed additive or animal feed containing the Lactobacillus acidophilus of claim 1 or the microbial agent of claim 2.
4. A preservative comprising the Lactobacillus acidophilus strain according to claim 1 or the microbial agent according to claim 2.
5. A pharmaceutical agent comprising the Lactobacillus acidophilus of claim 1 or the microbial agent of claim 2.
6. Use of lactobacillus acidophilus according to claim 1 or the preparation according to claim 2 for bacteriostasis for non-therapeutic purposes.
7. Use according to claim 6, wherein the bacteria are gram-negative bacteria.
8. Use according to claim 7, wherein the gram-negative bacteria are Escherichia (Escherichia) or Salmonella (Salmonella) bacteria.
9. The use according to claim 8, wherein the Escherichia (Escherichia) bacterium is Escherichia coli and the Salmonella (Salmonella) bacterium is Salmonella.
10. Use of any of the following lactobacillus acidophilus according to claim 1 or the bacterial agent according to claim 2:
1) used for preparing feed additive;
2) for increasing feed conversion rate;
3) used for preparing food, medicine or health product;
4) can be used for preparing antiseptic.
11. A method for preparing a lactobacillus acidophilus agent, the method comprising: fermenting and culturing the lactobacillus acidophilus of claim 1, centrifuging the fermentation liquid to obtain a precipitate, mixing the precipitate with a freeze-drying protective agent, and freeze-drying;
the fermentation medium used for the fermentation culture comprises: whey powder 30-35g/L, soybean meal 30-35g/L, glucose 4-6g/L, sodium chloride 3-5g/L, zinc sulfate 0.1-0.2g/L, manganese sulfate 0.6-0.7g/L, magnesium sulfate 0.8-1.2g/L and defoaming agent 0.05-0.08% v/v;
the fermentation conditions were: introducing mixed gas with the volume ratio of nitrogen to hydrogen of 9:1 at the temperature of 35 ℃ and the rotation speed of 200rpm under the stirring condition, and fermenting and culturing for 16 h.
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