CN112369500A - Lactobacillus casei and application thereof in weaned pig feed - Google Patents
Lactobacillus casei and application thereof in weaned pig feed Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/245—Lactobacillus casei
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Abstract
The invention is suitable for the technical field of microbial breeding, and provides a lactobacillus casei strain, wherein the lactobacillus casei strain is lactobacillus casei PLBS004, is preserved in China general microbiological culture Collection center (CGMCC) for 12-17 th in 2018, and has a preservation number of CGMCC NO. 16924; the invention also provides application of the lactobacillus casei in feed for weaned pigs. Therefore, the lactobacillus casei PLBS004 can improve the activity of antioxidant enzymes of animal organisms, reduce the oxidative stress of weaning, and reduce the diarrhea rate and the death rate of weaned piglets, thereby improving the production performance and the economic benefit of the weaned piglets.
Description
Technical Field
The invention relates to the technical field of microbial breeding, in particular to lactobacillus casei and application thereof in weaned pig feed.
Background
After weaning, because the piglets are influenced by various factors such as diet, environment, psychology and the like, oxidative stress is easy to occur, so that the piglets are stagnated in growth, easy to cause diarrhea and seriously harm the health of the piglets. Although antibiotics can effectively relieve diarrhea caused by piglet weaning stress and improve the growth performance of piglets, because the problems of antibiotic resistance and antibiotic residue are more and more paid attention by the society in recent years, the search for a proper antibiotic substitute is an urgent problem to be solved in the development of animal husbandry.
The lactobacillus fermented feed can not only produce a large amount of lactobacillus and metabolic products such as organic acid, enzyme and the like, but also degrade anti-nutritional factors in the feed, improve the palatability of the feed, and improve the digestion and utilization of nutrient substances in the feed, thereby improving the growth performance of animals.
The lactobacillus casei is one of important groups in lactobacillus, has the characteristics of strong acid production, acid resistance, bacteriostasis and the like, can prevent the growth and the propagation of pathogenic bacteria in intestinal tracts, can generate natural antibacterial substances, and has obvious inhibiting and killing effects on the intestinal pathogenic bacteria. The lactobacillus casei not only has antagonistic action on pathogenic bacteria, but also can adhere to epithelial cells of the intestinal tract to play a role in space occupying protection. Meanwhile, the lactobacillus casei can also produce nonspecific immunoregulation factors, stimulate local immune reaction of intestinal tracts and improve the nonspecific immune function of animals.
In view of the above, the prior art is obviously inconvenient and disadvantageous in practical use, and needs to be improved.
Disclosure of Invention
Aiming at the defects, the invention aims to provide lactobacillus casei and application thereof in feed for weaned pigs, and the lactobacillus casei PLBS004 with a proper level is added into daily ration of the weaned pigs, so that the contents of glutathione peroxidase, superoxide dismutase and catalase in the serum of the weaned pigs can be improved, the content of malondialdehyde is reduced, the activity of antioxidant enzymes of animal organisms is improved, the weaned oxidative stress is reduced, and the diarrhea rate and the death rate of the weaned pigs are reduced, thereby improving the production performance and the economic benefit of the weaned pigs.
In order to achieve the aim, the lactobacillus casei is lactobacillus casei PLBS004, is preserved in China general microbiological culture Collection center (CGMCC) in 2018 at 12 and 17 months, and has a preservation number of CGMCC NO. 16924.
The lactobacillus casei according to the invention is obtained from cow milk.
According to the lactobacillus casei, the addition amount of the lactobacillus casei in the basic ration of the weaned piglets is 200-1000 mg/kg.
According to the lactobacillus casei, the addition amount of the lactobacillus casei in the basic ration of the weaned piglets is 507-755 mg/kg.
The invention also provides a method for screening the lactobacillus casei, which comprises the following steps:
step one dilution of the Mixed strains
Putting cow milk into an MRS solid culture medium, culturing for 24 hours at 37 ℃, and performing gradient dilution on strains to obtain diluents with different gradients;
step two preliminary screening of the strains
Respectively taking the diluent, coating the diluent on an MRS solid culture medium added with 1.5-3% of calcium carbonate, and carrying out anaerobic culture in an incubator at 37 ℃ for 36-48 h; selecting a single bacterial colony with a calcium soluble ring as a primary screening bacterial strain;
re-screening of the three strains
And respectively inoculating the primary screened strains into an MRS liquid culture medium, and continuously culturing for 24h to obtain secondary screened strains.
According to the method for screening lactobacillus casei, the MRS solid culture medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef extract, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of triammonium citrate, 5.0g/L of sodium acetate, 0.58g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate tetrahydrate, 801mlg/L of tween and 1L of distilled water.
According to the method for screening lactobacillus casei, the culture conditions of the colonies in the third step are 37 ℃ and 200 rpm.
The lactobacillus casei provided by the invention is applied to weaned pig feed.
The lactobacillus casei PLBS004 provided by the invention has stronger acid resistance, cholate resistance and artificial gastrointestinal fluid resistance, and the lactobacillus casei PLBS004 with a proper level is added into the daily ration of the weaned piglets, so that the contents of glutathione peroxidase, superoxide dismutase and catalase in the serum of the weaned piglets can be improved, the content of malondialdehyde is reduced, the antioxidase activity of animal organisms is improved, the weaning oxidative stress is reduced, and the diarrhea rate and the death rate of the weaned piglets are reduced, thereby improving the production performance and the economic benefit of the weaned piglets.
Drawings
FIG. 1 is a colony morphology of Lactobacillus casei PLBS004 of the present invention;
FIG. 2 is a graph of the growth of Lactobacillus casei PLBS004 according to the invention;
FIG. 3 is a diagram showing an acid resistance test of Lactobacillus casei PLBS004 according to the present invention;
FIG. 4 is a graph of a bile salt resistance test of Lactobacillus casei PLBS004 according to the invention;
FIG. 5 is a diagram of an artificial gastrointestinal fluid tolerance test of Lactobacillus casei PLBS004 according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides lactobacillus casei, which has the following academic name: lactobacillus casei; the lactobacillus casei is lactobacillus casei PLBS004, which is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 of Xilu No.1 of Beijing Kogyo-Yang district, Beijing) in 2018 at 12 and 17 months, and the preservation number is CGMCC NO. 169944.
The invention provides a method for screening and identifying lactobacillus casei PLBS004, which is prepared from cow milk and comprises the following steps:
screening of one Strain
Step one dilution of the Mixed strains
Putting cow milk into MRS solid culture medium, culturing for 24 hours at 37 ℃, and carrying out gradient dilution on the strains to obtain diluents with different gradients.
The MRS solid culture medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef extract, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of triammonium citrate, 5.0g/L of sodium acetate, 0.58g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate tetrahydrate, 801mlg/L of Tween and 1L of distilled water, and sterilizing at 115 ℃ for 20 minutes.
Step two preliminary screening of the strains
Respectively taking the diluent, coating the diluent on an MRS solid culture medium plate added with 1.5-3% calcium carbonate, and carrying out anaerobic culture in an incubator at 37 ℃ for 36-48 h; and selecting a single bacterial colony which grows well and has obvious calcium lysosphere as a primary screening bacterial strain.
Re-screening of the three strains
And respectively inoculating the primary screened strains into an MRS liquid culture medium, and continuously culturing for 24h at 37 ℃ under 200rpm to obtain secondary screened strains.
Identification of two strains
Morphological identification
According to the classification and experimental method of lactobacillus bacteria and Bojie's manual of systematic bacteriology, the morphological identification of colony is carried out on the re-screened strain. (see FIG. 1)
As can be seen from FIG. 1, the colonies are oval or round-like, the edges are regular, the size of the colonies is generally 2-3 mm, and the colonies are milky white.
Identification of Gene sequences
The rescreened strain is sent to the company Limited in the biological engineering (Shanghai) to be sequenced, the obtained 16S rRNA sequence information is compared with the 16S database of NCBI, and the parameter identification is set to be more than 95. And then selecting the first 30 RNA sequences with the highest identification (if the sequence is insufficient, the RNA sequences are completely taken), performing sequence multiple alignment by using muscle software, and constructing a phylogenetic tree by using FastTree software. The results show that the re-screened strain is closest to the published Lactobacillus casei sp.fmnp02, and the evolutionary tree based on the 16S r RNA sequence component also shows that the strain is closest to the evolutionary relationship with Lactobacillus casei sp.fmnp02. Therefore, the rescreened strain was classified as lactobacillus casei, named lactobacillus casei PLBS 004.
In order to verify the strain properties of the lactobacillus casei PLBS004 strain of the present invention, strain property tests including a growth curve of the strain, an acid resistance test, a bile salt resistance test and an artificial gastrointestinal fluid tolerance test were performed.
Growth curve of the Strain
Inoculating activated Lactobacillus casei PLBS004 into MRS liquid culture medium according to the inoculation amount of 1% (v/v), standing and culturing at 37 deg.C for 48h, and measuring the bacterial concentration of the culture solution at 600nm every 4 h. The growth curve of the strain was obtained. (see FIG. 2)
As can be seen from FIG. 2, Lactobacillus casei PLBS004 grows rapidly in MRS medium, and grows exponentially within 4-28 h; with the prolonging of the culture time, the bacterial strains grow to produce acid, the pH value is continuously reduced, and the number of the bacterial strains enters a stable period after about 28 hours.
Acid resistance test
Centrifuging the bacterial suspension of lactobacillus casei PLBS004 for 5min at 4 ℃, then washing bacterial sludge for 3 times by PBS, inoculating the bacterial sludge into a liquid MRS culture medium with the pH value of 3.0 according to the inoculation amount of 3%, culturing at 37 ℃, respectively diluting and coating bacterial liquids at 0, 1, 2 and 3h, and calculating the survival rate by taking a fresh MRS culture medium as a blank control. (see FIG. 3)
As can be seen from FIG. 3, after 3h of growth in MRS at pH 3.0, the survival rate of Lactobacillus casei PLBS004 still reaches above 40%, which indicates that Lactobacillus casei PLBS004 has strong tolerance to acidic environment.
Bile salt resistance test
Inoculating the bacterial suspension of lactobacillus casei PLBS004 into an MRS liquid culture medium with cholate concentration of 0.3% according to the inoculation amount of 3%, respectively sampling samples for gradient dilution by 10 times when culturing for 0, 1, 2, 3 and 4 hours at 37 ℃, taking the diluent with proper gradient for coating and counting, and calculating the survival rate by taking a fresh MRS culture medium as a blank control. (see FIG. 4)
As can be seen from FIG. 4, Lactobacillus casei PLBS004 has high tolerance to bile salt, and the survival rate of the strain is higher than 30% at 3 h.
Artificial gastrointestinal fluid tolerance test
Centrifuging a bacterial suspension of lactobacillus casei PLBS004, washing the bacterial suspension for 3 times by PBS, suspending the bacterial sludge in artificial gastric juice (0.35% pepsin and 0.3% NaCl, and adjusting the pH to 3.0) which is subjected to filter sterilization by a filter membrane, respectively culturing for 0, 1, 2 and 3 hours at the temperature of 37 ℃, taking bacterial liquid for dilution and coating, calculating the number of viable bacteria by a flat plate bacterial colony counting method, and determining the tolerance of the bacterial strain under the condition of acid gastric juice.
After the thalli are treated in the artificial gastric juice for 3 hours, the artificial gastric juice containing the thalli is sucked, added into the artificial intestinal juice (1.1 percent of sodium bicarbonate, 0.3 percent of NaCl, 0.1 percent of trypsin and 0.6 percent of bile salt which are filtered and sterilized by a filter membrane and adjusted to the pH value of 8.0), cultured at 37 ℃, sucked bacterial liquid is respectively diluted and coated at 0 hour, 3 hour, 5 hour, 7 hour and 8 hour, the viable count is calculated by a plate counting method, and the tolerance of the bacterial strain in the intestine is measured.
The survival rate was calculated using fresh MRS medium as blank control. (see fig. 5) when food is digested in the stomach and enters the intestine, the flora growth in the intestine is influenced by various components, and the probiotics need to be able to tolerate trypsin and alkaline environment in the intestine to exert their probiotic effect.
As can be seen from FIG. 5, the survival rate of the Lactobacillus casei PLBS004 can be maintained to be above 40% after the Lactobacillus casei PLBS004 is treated in the simulated artificial intestinal juice for 8 hours, which indicates that the Lactobacillus casei PLBS004 has good tolerance capability of the artificial intestinal juice.
The lactobacillus casei has stronger acid resistance, cholate resistance and artificial gastrointestinal fluid resistance, and can prevent the growth and reproduction of pathogenic bacteria in intestinal tracts, so the lactobacillus casei PLBS004 is applied to the feed for weaned pigs, and in order to verify the application value of the lactobacillus casei PLBS004 in the feed for weaned pigs, the invention also provides an application method of the lactobacillus casei in the feed for weaned pigs.
First, experimental animal and raising management
40 weaned piglets with 21 days age (half of male and half of female) with similar genetic background, gestation frequency and weight are selected and randomly divided into 4 treatment groups (half of male and half of female in each treatment group), including a control group and three test groups. After 7 days of pre-feeding, the control group was fed a basal diet and the test group was supplemented with lactobacillus casei PLBS004 on the basal diet. The feeding period is 32 days.
The test pigs were raised on a nursery bed at a house temperature of about 25 ℃ and all weaned piglets had free access to food and water during the test period. The basic daily ration is prepared according to the formula of a weaned piglet farm.
Second, feed formula
The lactobacillus casei PLBS004 is added into basic daily ration of weaned piglets, and the addition amount is 200-1000 mg/kg.
Third, measuring the index
Measurement of growth Performance
And (4) respectively weighing the pigs on an empty stomach at 0d, 14d and 32d of the test, accurately recording the daily feed intake during the test, calculating the average daily feed intake, the average daily gain and the feed-weight ratio, and counting the diarrhea rate.
Determination of serum antioxidant enzyme Activity
At the 14 th and end of the middle test period (32 th), the blood of the pig anterior vena cava was collected, and the serum was separated and stored frozen at-80 ℃. Detecting the activity of antioxidant enzyme in serum, including glutathione peroxidase, superoxide dismutase, catalase and malondialdehyde.
In order to further verify the application value of the lactobacillus casei PLBS004 in the feed for the weaned pigs, the weaned pigs are fed by the application method, the following examples are provided, and relevant indexes of the weaned pigs in the examples are measured. The feeding management method of the weaned piglets in each example is the same, and only the addition amount of the lactobacillus casei PLBS004 in each example is changed, so the same contents in each example are not listed.
The addition amount of lactobacillus casei PLBS004 in each example is shown in Table I; the related index results of the weaned piglets in each example are shown in the second table and the third table.
Comparative example
After the completion of the culture, the average daily gain of the weaned piglets is 381.12g, the average daily feed intake is 713.24g, the feed-weight ratio is 1.87, and the diarrhea rate is 0.56%; it was found that the content of glutathione peroxidase in the serum of weaned piglets was 1071.4mol/L, the content of superoxide dismutase was 110.8U/ml, the content of catalase was 11.8U/g Hb, and the content of malondialdehyde was 2.9 nmol/ml.
Example 1
The lactobacillus casei PLBS004 of the invention is added into basic daily ration of weaned pigs, and the addition amount is 550 mg/kg.
After the completion of the culture, the average daily gain of the weaned piglets is 452.36g, the average daily feed intake is 760.68g, the feed-weight ratio is 1.68, and the diarrhea rate is 0.21%; the glutathione peroxidase content in the serum of the weaned piglet was 1278.1mol/L, the superoxide dismutase content was 143.1U/ml, the catalase content was 15.6U/g Hb, and the malondialdehyde content was 1.5 nmol/ml.
Example 2
The lactobacillus casei PLBS004 of the invention is added into basic daily ration of weaned pigs, and the addition amount is 640 mg/kg.
After the completion of the culture, the average daily gain of the weaned piglets is 448.94g, the average daily feed intake is 762.70g, the feed-weight ratio is 1.7, and the diarrhea rate is 0.17%; it was found that the content of glutathione peroxidase in the serum of weaned piglets was 1296.8mol/L, the content of superoxide dismutase was 154.0U/ml, the content of catalase was 16.9U/g Hb, and the content of malondialdehyde was 1.6 nmol/ml.
Example 3
The lactobacillus casei PLBS004 of the invention is added into basic daily ration of weaned pigs, and the addition amount is 780 mg/kg.
After the completion of the culture, the average daily gain of the weaned piglets is 450.83g, the average daily feed intake is 771.58g, the feed-weight ratio is 1.71, and the diarrhea rate is 0.19%; it was found that the content of glutathione peroxidase in the serum of weaned piglets was 1308.1mol/L, the content of superoxide dismutase was 148.5U/ml, the content of catalase was 16.2U/g Hb, and the content of malondialdehyde was 1.5 nmol/ml.
Because the implementation processes of the embodiments are the same, the specific processes of other embodiments are not listed, and only the three embodiments with better effects are listed.
TABLE-addition of Lactobacillus casei PLBS004 in the examples in mg/kg
Growth performance of epi-weaned piglets
The content of antioxidant in the serum of the weaned pig with the third layer of the table is%
The embodiment shows that the lactobacillus casei PLBS004 added into the basic ration of the weaned pig can improve the average daily gain and the average daily feed intake of the weaned pig, reduce the feed-weight ratio of the weaned pig, and show that the lactobacillus casei PLBS004 enhances the intestinal tract digestive enzyme activity of the weaned pig and improves the utilization rate of nutrient substances of the weaned pig. The lactobacillus casei PLBS004 is used as a feed additive and added into weaned pig feed, can adjust intestinal flora, enhance the digestion and absorption functions of gastrointestinal tracts, maintain the dominance of probiotics, promote the digestion and absorption of intestines and stomach, and further improve the growth performance and the feed conversion rate of animals.
The lactobacillus casei PLBS004 is added into basic daily ration of the weaned pig, so that the diarrhea rate of the weaned pig can be reduced, and the lactobacillus casei PLBS004 enhances the disease resistance of the weaned pig. The lactobacillus casei PLBS004 forms a biological barrier by combining with intestinal mucosa, promotes cellular immunity and humoral immunity, maintains intestinal microecological balance and normal physiological function, improves the immune function of organisms, reduces the occurrence of diseases and reduces the diarrhea rate of weaned piglets.
The embodiments show that the addition of the lactobacillus casei PLBS004 of the invention into the basic ration of the weaned pig can improve the contents of glutathione peroxidase, superoxide dismutase and catalase in the serum of the weaned pig and reduce the content of malondialdehyde, which shows that the lactobacillus casei PLBS004 can improve the activity of antioxidant enzymes of animal organisms, reduce the oxidative stress of the weaned pig, maintain the intestinal health of the weaned pig, improve the digestion utilization rate of the weaned pig on feed and promote the growth of the weaned pig.
In addition, the above examples show that when the addition amount of the lactobacillus casei PLBS004 in the basic ration of the weaned pig is 507-755 mg/kg, the production performance and nutrient digestibility of the weaned pig are the best, and the diarrhea rate and the death rate are the lowest, so that the optimal addition amount of the lactobacillus casei PLBS004 in the basic ration of the weaned pig is 507-755 mg/kg.
In conclusion, the lactobacillus casei PLBS004 provided by the invention has stronger acid resistance, cholate resistance and artificial gastrointestinal fluid resistance, and the lactobacillus casei PLBS004 with a proper level is added into the daily ration of the weaned pig, so that the contents of glutathione peroxidase, superoxide dismutase and catalase in the blood serum of the weaned pig can be improved, the content of malondialdehyde is reduced, the activity of animal organism antioxidase is improved, the weaning oxidative stress is reduced, and the diarrhea rate and the death rate of the weaned pig are reduced, thereby improving the production performance and the economic benefit of the weaned pig.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. The lactobacillus casei is characterized in that the lactobacillus casei is lactobacillus casei PLBS004, is preserved in China general microbiological culture collection center in 2018, 12 and 17 months, and has the preservation number of CGMCC NO. 16924.
2. The lactobacillus casei according to claim 1, wherein the lactobacillus casei is obtained from bovine milk.
3. The lactobacillus casei according to claim 1, wherein the lactobacillus casei is added in a basal diet of weaned piglets in an amount of 200-1000 mg/kg.
4. The lactobacillus casei according to claim 3, wherein the lactobacillus casei is added in a basal ration for weaned piglets in an amount of 507-755 mg/kg.
5. A method of screening for lactobacillus casei as claimed in claim 1, comprising the steps of:
step one dilution of the Mixed strains
Putting cow milk into an MRS solid culture medium, culturing for 24 hours at 37 ℃, and performing gradient dilution on strains to obtain diluents with different gradients;
step two preliminary screening of the strains
Respectively taking the diluent, coating the diluent on an MRS solid culture medium added with 1.5-3% of calcium carbonate, and carrying out anaerobic culture in an incubator at 37 ℃ for 36-48 h; selecting a single bacterial colony with a calcium soluble ring as a primary screening bacterial strain;
re-screening of the three strains
And respectively inoculating the primary screened strains into an MRS liquid culture medium, and continuously culturing for 24h to obtain secondary screened strains.
6. The method for screening lactobacillus casei according to claim 5, wherein the MRS solid medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef extract, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of triammonium citrate, 5.0g/L of sodium acetate, 0.58g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate tetrahydrate, 801mlg/L of tween and 1L of distilled water.
7. The method for screening Lactobacillus casei according to claim 5, wherein the culture conditions of the colonies in step three are 37 ℃ and 200 rpm.
8. Use of lactobacillus casei according to claim 1 in the feed of weaned piglets.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112625983A (en) * | 2021-03-05 | 2021-04-09 | 山东中科嘉亿生物工程有限公司 | Lactobacillus casei L.Casei21 with treatment effect on diarrhea and application thereof |
| CN113088468A (en) * | 2021-04-09 | 2021-07-09 | 中国农业大学 | Lactobacillus casei Ma. GLRGJ1 and application thereof |
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| CN101333505A (en) * | 2007-06-26 | 2008-12-31 | 内蒙古农业大学 | Applications of lactobacillus casei for antioxidation |
| CN106497828A (en) * | 2016-10-14 | 2017-03-15 | 北京市农林科学院 | A kind of application of lactobacillus casei and its lyophilized formulations in pig feed |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101333505A (en) * | 2007-06-26 | 2008-12-31 | 内蒙古农业大学 | Applications of lactobacillus casei for antioxidation |
| CN106497828A (en) * | 2016-10-14 | 2017-03-15 | 北京市农林科学院 | A kind of application of lactobacillus casei and its lyophilized formulations in pig feed |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112625983A (en) * | 2021-03-05 | 2021-04-09 | 山东中科嘉亿生物工程有限公司 | Lactobacillus casei L.Casei21 with treatment effect on diarrhea and application thereof |
| CN113088468A (en) * | 2021-04-09 | 2021-07-09 | 中国农业大学 | Lactobacillus casei Ma. GLRGJ1 and application thereof |
| CN113088468B (en) * | 2021-04-09 | 2022-06-03 | 中国农业大学 | Lactobacillus casei Ma. GLRGJ1 and application thereof |
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