CN112226389A - Planting culture method of intestinal probiotic groups of Sanhuang young chickens and application of intestinal probiotic groups - Google Patents
Planting culture method of intestinal probiotic groups of Sanhuang young chickens and application of intestinal probiotic groups Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N1/20—Bacteria; Culture media therefor
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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
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
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Abstract
The invention discloses a method for cultivating probiotics colony of chicken intestinal canal of Sanhuang broiler and application thereof, separating probiotics bacterial strains from different intestinal sections of duodenum, jejunum, ileum, caecum and chicken feces of chicken intestinal canal, comparing bacterial strain tolerance, enzyme production and acid production capacity, cultivating and screening bacterial strains with high viable count and rich enzyme production, screening bacterial strains in a compounding way, cultivating and optimizing the compounding proportion in a planting way to obtain a compound microbial agent consisting of Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici, Bacillus safensis (Bacillus safensis), Propionibacterium freudenreichii (Propionibacterium freudenreichii) and Clostridium butyricum (Cloidium butyricum), applying the microbial agent to the feeding of the Sanhuang broiler, wherein the probiotics colony grows and breeds rapidly, the viable count is high, the microbial agent is beneficial to the digestion of the chick, the immunity of the chick is improved, the survival rate of the chick is improved obviously, and the survival rate of the chick is increased, has wide application value for the microbial inoculum in the chick breeding.
Description
Technical Field
The invention belongs to the field of culture methods of microbial floras, and particularly relates to a field planting culture method of intestinal probiotic floras of sanhuang young chickens and application thereof.
Background
A Microbial ecological agent (Microbial ecological agent) is a biological agent or a viable bacteria agent prepared by selecting beneficial microorganisms and special nutrient substances thereof in an animal body and performing special processing processes such as culture, fermentation and drying under the guidance of a Microbial ecological balance theory, a Microbial ecological prevention theory, a Microbial ecological nutrition theory and the like, and can supplement normal microorganisms lacking or reduced in intestinal tracts in quantity or variety and maintain or adjust the balance of the Microbial ecological environment in the intestinal tracts. Has the functions of preventing and treating diseases and health care, has no toxic or residual side effect, and is popular with people in recent years. The selection of strains is a key factor influencing the action effect of the microecological preparation, and the following principles are followed when the strains are selected: firstly, the bacterial strain belongs to normal flora of animals, can ensure that probiotics can smoothly enter intestinal tract for field planting and can rapidly grow and reproduce, and better exert the probiotic function. Secondly, the probiotic effect of the strain is good, it can tolerate low pH and bile in the stomach and it can be rapidly colonized on the intestinal mucosa. Thirdly, the strain is safe and has no toxic and side effects. Fourth, it has good storage stability, is easy to produce and maintain its viable state.
The three-yellow-meat chick refers to a chick which is just hatched for 0-50 days. The digestive function of the chicks is very weak, the feed with soft texture needs to be prepared, the chicks which just come out of the shell have enough egg yolks in the bodies, and partial nutrient substances can be supplied to the chicks within 3 to 5 days. The chick is eaten after 12-24h after hatching, the death rate of the chick is lowest during the period of eating, but a large amount of microorganisms are introduced into the intestinal tract of the chick at the same time, which is the largest influence factor on the survival rate of the chick. The poultry intensive culture is all supporting from fodder to aquaculture device now, and the microorganism live bacteria only two kinds of using-way can intervene, and one kind is that solid powder can use with chicken fodder mixing together, and the second kind is that liquid microbial inoculum uses through adding to drinking water line, and the former does not have special requirement, and the latter requires that the bacterial liquid dispersibility is good, can not block up the waterline. Because the intestinal tract of the poultry is short, the solid bacterial powder is in a dormant state, and the solid bacterial powder is usually discharged out of the body after entering the intestinal tract and not being activated when being directly fed, so that the exertion of the action effect is limited. The liquid microbial inoculum is always in a high-activity state in the culture stage, and can produce a large amount of active metabolites (enzymes, organic acids, active small peptides, amino acids, antibiotics and the like) during culture, and the advantages enable the liquid microbial inoculum to enter the intestinal tract without activation, and the metabolites are not affected by gastric acid, bile salts and the like, so that the liquid microbial inoculum can act faster and has more remarkable effect. Therefore, the compounding of the multiple non-antagonistic microbial agents can further improve the utilization level and utilization rate of the feed of animals compared with a single strain, thereby reducing the feed meat/egg ratio. However, in the actual application process, different strains are compounded, and how to effectively control the viable count and the metabolic product stability of each strain is a difficult problem in the development process and application of the microbial agent at present.
Disclosure of Invention
Aiming at the technical problems of antibiotic abuse, high nonreactive breeding mortality of the chicks, the stability of the viable count of the compound microbial inoculant, the types and the yield of metabolites and the like in the process of the desuperheating breeding of the three-yellow-meat chicks. The invention aims to provide a planting culture method and application of intestinal probiotic groups of Sanhuang young chickens. Through separating and screening high-quality strains from duodenum, jejunum, ileum, cecum and chicken feces of different intestinal sections of chicken intestinal tracts, comparing the resistance, enzyme production and acid production capacity of the strains, obtaining the high-quality strains which have high viable count and can produce rich protease, lipase, amylase, lactic acid, acetic acid, propionic acid, formic acid and citric acid, carrying out compound planting culture and proportion optimization on the screened strains, finally obtaining the composite microbial agent consisting of Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici, Bacillus safensis (Bacillus safensis), Propionibacterium freudenreichii and Clostridium butyricum (Clostridium butyricum), applying the composite microbial agent to the feeding of the three-yellow broiler chickens, wherein the probiotic group grows and breeds rapidly in the chicken intestinal tracts, the number is high, the digestion of the chickens is facilitated, the immunity of the chickens is improved obviously, and the survival rate of the chicks is increased, has wide application value for the microbial inoculum in the chick breeding.
In order to achieve the technical purpose, the invention is realized by the following technical scheme:
the invention provides a planting culture method of intestinal probiotic groups of Sanhuang young broiler chickens, which specifically comprises the following steps:
(1) screening and separating different intestinal sections of duodenum, jejunum, ileum and cecum of the intestinal tract of the free-range local chicken and chicken manure, directionally separating bacillus, lactobacillus, bifidobacterium and clostridium butyricum, obtaining a representative strain through morphology and comparison of staining and purification, and identifying the strain.
(2) Culturing the screened bacillus, lactobacillus, bifidobacterium and clostridium butyricum serving as probiotics in a corresponding culture medium: culturing the bacillus in an LB liquid culture medium at 37 ℃ and 180rpm/min for 48h, performing static culture and activation on the lactobacillus in an MRS liquid culture medium at 37 ℃ for 48h, performing static culture on clostridium butyricum in an RCM culture medium at 37 ℃ for 48h, and performing anaerobic static culture on the bifidobacterium in the MRS liquid culture medium at 37 ℃ for 48 h; and measuring the inhibition zone of each bacterium, selecting the strain with the most obvious inhibition zone effect, and primarily screening high-quality strains.
(3) And (3) carrying out tolerance screening and enzyme production and acid production capability comparison on the screened probiotics, and screening out the bacterial strains with strong acid resistance, cholate resistance, gastrointestinal fluid resistance, acid production capability and rich enzyme production.
(4) And (3) combining the Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici (Pediococcus acidilactici), Bacillus safensis (Bacillus safensis), Propionibacterium freudenreichii (Propionibacterium freudenreichii) and Clostridium butyricum (Clostridium butyricum) obtained by screening in the step (3) into a composite microbial agent suitable for colonization culture of intestinal probiotics of the sanhuang broiler chickens, and mixing the 5 strains according to a volume ratio of 10: 10: 50: 50: 50, compounding, and finally determining the intestinal probiotic groups suitable for the planting cultured chicks of the three-yellow broiler chicken.
(5) And (3) feeding 0-day-old chicks with the probiotic groups prepared in the step (4) and compounded by Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici (Pediococcus acidilactici), Bacillus safensis (Bacillus safensis), propionibacterium freudenreichii and Clostridium butyricum (Clostridium butyricum), and planting and culturing in the intestinal tracts of the chicks to quickly establish the probiotic groups with strong vigor.
In the invention, the composite microbial agent obtained by the method for colonizing and culturing intestinal probiotics of the three-yellow broiler chicken is prepared by mixing Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici (Pediococcus acidilactici), Bacillus safensis (Bacillus safensis), Propionibacterium freudenreichii (Propionibacterium freudenreichii) and Clostridium butyricum (Clostridium butyricum) according to the volume ratio of 10: 10: 50: 50: 50 are compounded to obtain.
In the invention, the compound microbial agent is applied to the intestinal probiotic flora field planting of the three-yellow young chickens.
In the invention, the compound microbial agent is applied to the feeding of the three-yellow broiler chicken.
After the technical scheme is adopted, the invention has the following beneficial effects:
(1) compared with the conventional microbial agent for feeding, the compound microbial agent prepared by the method for planting and culturing the intestinal probiotics of the three-yellow young chickens has the improvement effects on the production performance (feed-meat ratio, survival rate and average weight), the number of intestinal escherichia coli (pathogenic bacteria), lactobacillus and bifidobacterium (beneficial bacteria), the concentration of immune globulin A and G and the like.
(2) The intestinal probiotic colony planting culture method for the Sanhuang young chickens, provided by the invention, can not only ensure the growth of strains without antagonism, but also effectively control the metabolites of microbial colonies and generate abundant protease, lipase and amylase, thereby obviously improving the digestion function of the Sanhuang young chickens.
(3) The composite microbial agent prepared by the method for planting and culturing the intestinal probiotic groups of the three-yellow young chickens has high viable count, can quickly establish the advantageous bacteria groups of the caecum in the intestinal tracts of the three-yellow young chickens, stimulates and improves the immunity and helps digestion.
Description of the drawings:
is free of
Detailed Description
The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.
The invention has the following measurement indexes: the growth performance indexes such as weight, feed conversion ratio, survival rate and the like are measured, 1 healthy three-yellow-meat chick is randomly extracted every repetition after the broiler chicken is fed for 28 days and weighed on an empty stomach in the morning, and the biochemical indexes of blood are measured by a full-automatic biochemical analyzer (Olypus AU600 type).
Example 1: intestinal probiotic group planting culture method for Sanhuang young chicken
A field planting culture method of intestinal probiotic groups of Sanhuang young chickens specifically comprises the following steps:
(1) screening and separating different intestinal sections of duodenum, jejunum, ileum and cecum of the intestinal tract of the free-range local chicken and chicken manure, directionally separating bacillus, lactobacillus, bifidobacterium and clostridium butyricum, obtaining a representative strain through morphology and comparison of staining and purification, and identifying the strain.
(2) Culturing the screened bacillus, lactobacillus, bifidobacterium and clostridium butyricum serving as probiotics in a corresponding culture medium: culturing the bacillus in an LB liquid culture medium at 37 ℃ and 180rpm/min for 48h, performing static culture and activation on the lactobacillus in an MRS liquid culture medium at 37 ℃ for 48h, performing static culture on clostridium butyricum in an RCM culture medium at 37 ℃ for 48h, and performing anaerobic static culture on the bifidobacterium in the MRS liquid culture medium at 37 ℃ for 48 h; and measuring the inhibition zone of each bacterium, selecting the strain with the most obvious inhibition zone effect, and primarily screening high-quality strains.
(3) Comparing the tolerance, enzyme production and acid production of the screened probiotics, and screening out the bacterial strains with strong acid resistance, cholate resistance, gastrointestinal fluid resistance, acid production capability and rich enzyme production.
(4) The above steps (A) and (B)3) The screened and obtained Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici (Pediococcus acidilactici), Bacillus safensis (Bacillus safensis), Propionibacterium freudenreichii (Propionibacterium freudenreichii) and Clostridium butyricum (Clostridium butyricum) form a compound microbial agent suitable for colonization culture of intestinal flora of the yellow broiler chickens, and the 5 strains are mixed according to the volume ratio of 10: 10: 50: 50: 50, compounding, and finally determining the intestinal probiotic groups suitable for the planting cultured chicks of the three-yellow broiler chicken.
(5) And (3) the probiotic groups prepared in the step (4) and compounded by Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici (Pediococcus acidilactici), Bacillus safensis (Bacillus safensis), propionibacterium freudenreichii and Clostridium butyricum (Clostridium butyricum) are planted and cultured in the intestinal tract of the chicks by feeding 0 day old chicks, and the strong probiotic groups are quickly established.
Example 2: intestinal probiotic group planting culture method for Sanhuang young chicken
(1) Isolation, purification and characterization of the strains
And (3) buying free-ranging local chickens of farmers and herdsmen in south northern Xinjiang, and fasting the local chickens for 3 days without water prohibition to completely empty the food in the intestinal tracts of the local chickens. After the live chicken is slaughtered, selecting different intestinal sections of duodenum, jejunum, ileum, cecum and chicken manure of each chicken intestinal tract, separating, binding each intestinal section by using a sterile cotton rope, marking each intestinal section, shearing off the different intestinal sections by using sterile scissors, placing on sterile gauze, shearing off the intestinal tract by using sterile scissors in a sterile environment, hanging adhesive on the surface of the intestinal wall by using a sterile spoon, and placing on the intestinal wallAdding 50ml sterile physiological saline into 50ml triangular flask with glass beads at a volume of 50ml/150ml, shaking in shaking table at room temperature for 30min, and performing gradient dilution under sterile operation for 10 min-4、10-5And 10-6Culturing on various culture media at constant temperature of 37 ℃, and culturing anaerobic microorganisms at constant temperature of 37 ℃ in an anaerobic culture tank (introducing carbon dioxide or nitrogen into the tank to discharge oxygen in the tank to form an anaerobic environment). Directionally separating bacillus (bacillus licheniformis, bacillus subtilis and bacillus coagulans), lactic acid bacteria (enterococcus faecium, enterococcus lactis, lactobacillus acidophilus, lactobacillus bulgaricus, lactobacillus casei, lactobacillus plantarum, lactobacillus lactis and pediococcus pentosaceus), bifidobacterium (bifidobacterium bifidum) and clostridium butyricum, and simultaneously separating chicken intestinal pathogenic bacteria, wherein the culture medium used for separation and identification is shown in the attached table 1.
Table 1: chicken intestinal flora separation culture medium
| Culture medium | Name (R) |
| Lactic acid bacteria isolation medium | MRS agar (0.75% CaCO added)3) |
| Culture medium for spore bacteria | LB agar |
| Clostridium anaerobe selective culture medium | TSN agar |
| Clostridium butyricum culture medium | RCM medium |
| Escherichia coli identification culture medium | EMB agar |
| Salmonella and shigella identification medium | SS agar |
A total of 30 representative strains were purified by morphological and staining comparison, and then 16S rRNA gene sequencing was performed on the strains to construct phylogenetic trees by sequence alignment with GenBank database, and the results are shown in Table 2. The 30 strains are respectively classified into lactobacillus: pediococcus, enterococcus, Lactobacillus; bifidobacteria: bifidobacterium, bacillus, clostridium butyricum; pathogenic bacteria: escherichia, Salmonella, Shigella.
Table 2: isolation and identification of intestinal microorganisms
(2) Probiotic activity comparative test
According to the species and characteristics of probiotics in each intestinal segment, the inhibition of intestinal pathogenic bacteria is a screening criterion, and according to the characteristic that the oxygen content of the intestinal segment is gradually reduced from duodenum, jejunum, ileum to cecum, the duodenum and the jejunum mainly take aerobic microorganisms as main bacteria, the ileum micro-anaerobe mainly takes lactic acid bacteria as main bacteria, and the cecum anaerobe mainly takes anaerobic bacteria as main bacteria.
Culturing the screened probiotics in LB liquid culture medium (spore bacteria) at 37 ℃ and 180rpm/min for 48h, performing static culture and activation on lactobacillus in MRS liquid culture medium at 37 ℃ for 48h, and performing static culture on clostridium butyricum in RCM culture medium at 37 ℃ for 48 h; shaking-culturing pathogenic bacteria in a common liquid culture medium (NB) for 16h, cooling sterilized Nutrient Agar (NA) culture medium to about 46 ℃, inoculating 1% of the pathogenic bacteria, mixing uniformly, pouring a flat plate under an aseptic ultra-clean workbench, after the flat plate is cooled and solidified, punching by using a puncher, adding 100 mu L of lactobacillus bacterial liquid into each hole, standing for 3h at normal temperature, placing in a 37 ℃ incubator, and culturing overnight. The inhibition zone of each lactic acid bacterium was measured, and the strain with the most significant inhibition zone effect was selected for the subsequent co-culture experiment, and the specific results are shown in table 3.
Table 3: inhibition effect of chicken-origin intestinal probiotics on chicken-origin pathogenic bacteria (in vitro plate bacteriostasis ring method)
According to the data in Table 3, 7 strains of Bacillus have stronger bacteriostatic activity against three pathogenic bacteria, including Bacillus velezensis (DJ-H3), Bacillus subtilis (TS-S6), Bacillus safensis (HJ-M15), Bacillus haynesii (TS-S7), Bacillus methylotrophicus (BC-K1), Bacillus pusilus (BJ-K3) and Bacillus tequilensis (HJ-K5). Screening 3 strains of lactic acid bacteria: lactobacillus plantarum (HJ-M11), Enterococcus Feacium (EM-S1), Pediococcus acidilactici (HJ-S5), anaerobic strain 2: clostridium butyricum (HJ-M1), Bifidobacterium bifidum (HJ-M12), Propionibacterium strain 1: propionibacterium freudenreichii (BC-K2).
(3) Probiotic tolerance comparative test
The results of this experiment, in which the tolerance of 13 selected strains was compared and the survival rate of the strains at different pH values and bile salt concentrations and the tolerance of each strain to human gastrointestinal fluids were examined, are shown in tables 4 to 7.
Table 4: comparison of acid resistance Properties of Probiotics
Table 5: comparison of the bile salt resistance of probiotic bacteria
Table 6: comparison of Artificial gastrointestinal fluid resistance
Table 7: comparison of Artificial intestinal fluid resistance
From the results in tables 4-7, it can be seen that the spore formation rate of each spore bacterium is 99.21% after the culture is finished, and the spore formed by each spore bacterium has strong resistance to acid, bile salt, intestinal fluid and gastric juice, wherein the influence of the pH value on the spore bacterium is the largest, when the pH value is 1.5, the viable bacteria of the spore bacterium are placed for 0.5h, and the viable bacteria number is not more than 61% at most; the viable count increases to about 80% at a pH of 2.5 and to about 90% at a pH of 3.5. The influence difference of bile salt, intestinal juice and gastric juice on various probiotics is not obvious, the survival rate is at least kept above 90% under the condition of the highest concentration, and except that propionic acid bacteria are sensitive to the performance of bile salt and intestinal juice, the viable count is at least above 80%, which shows that the bacteria have potential probiotic application effect.
(4) Comparison test of enzyme and acid production capability of probiotics
The extracellular enzyme activities generated by the strains after 48h of culture are compared by utilizing a micro-method enzyme reagent detection kit, the spores use LB liquid culture medium as a basic culture medium, the spores with the same concentration are inoculated, then 1ml of bacterial liquid is absorbed at 12000rpm/min for centrifugation after 48h of culture, then 0.1ml of supernatant is absorbed and added into an enzyme activity kit for constant temperature culture at 37 ℃ to measure the enzyme activities, and the results are shown in tables 8-9.
Table 8: comparison of enzyme production by spore bacteria
Table 9: acid production comparison by probiotics
As can be seen from the results shown in the attached tables 8 to 9, both the Bacillus and Clostridium butyricum produce abundant proteases, lipases and amylases, and a small amount of cellulases. The enzyme producing capability of each spore bacterium is relatively equivalent, wherein the capability of clostridium butyricum for producing various enzymes is strongest, and the enzyme producing capability of other spore bacteria is not greatly different. Compared with probiotics in acid production capacity, the homolactic acid bacteria Lactobacillus plantarum HJ-M11 has the strongest acid production capacity, the pediococcus acidilactici has the second highest acid production capacity, the two bacteria do not produce other types of acids, and the heterolactic acid bacteria EM-S1 and HJ-M12 can produce lactic acid, acetic acid and formic acid. Clostridium butyricum is capable of producing acetic acid and butyric acid, whereas Propionibacterium freudenreichii BC-K2 is only capable of detecting propionic acid.
(5) Compound planting culture test of probiotic strains
According to the characteristic that fermentation metabolites of each strain are relatively constant under the condition of a constant culture medium, and the requirement of the three-yellow-meat chick in the breeding stage, different strains and metabolites thereof are compounded. Setting up an orthogonal analysis experiment, and selecting Bacillus safensis HJ-M15, Bacillus haynesii TS-S7, Bacillus tequilensis HJ-K5, Lactobacillus plantarum HJ-M11, Enterococcus Feacium EM-S1, Pediococcus acidicicus HJ-S5, Clostridium butyricum HJ-M1, Bifidobacterium bifidum HJ-M12, Propionibacterium freudenreichii BC-K2. The orthogonal experimental table is shown in table 10,5 factors, 4 levels 1,5,10,50 ml. Feeding tests are carried out on the 16 groups of compound microbial inoculum, yellow-feathered broilers of 0 day age are taken as research objects, 20 chickens in each group are added into drinking water after the microbial inoculum is diluted according to a ratio of 1:1000, chick feed is feed for opening of Tiankang finished chick, the chick freely eats food and drinks water, and each group of chick is isolated from each other by using an independent water fountain and an independent net cage frame (2m multiplied by 1m is 2 m)2) And recording the survival rate, the weekly weight gain and the feed dosage of the chickens within 30 days of an experimental period, carrying out PCA analysis on the values by using SPSS, extracting representative values of the values and carrying out orthogonal result analysis.
Table 10: weekly weight gain of yellow-feathered broilers
As can be seen from the results of the attached Table 10, when the strains were tested in a volume ratio of Bacillus haynesii TS-S7: pediococcus acidilactici HJ-S5: bacillus safensis HJ-M15: propionibacterium freudenreichii BC-K2: clostridium butyricum HJ-M1 ═ 10: 10: 50: 50: 50 of the compound microbial inoculum has the greatest influence on each index of the chicks. The intestinal canal of the young chicken of 0 day old is in an aseptic state, and the intestinal canal has short hair holding capacity and the like at this stage, and the intestinal canal is digested and has weak absorption capacity, so that the strong probiotic flora is required to be quickly established by feeding probiotics. In addition, the chicks in this stage have weak immunity, and have few and single cecal section microbial flora, so that a cecal dominant group needs to be quickly established to stimulate and improve immunity and help digestion. The feed digestion and utilization are promoted by utilizing spore bacteria with high enzyme production and bacteriostatic activity, the compound lactobacillus reduces the pH value of the intestinal tract and elutes the adhesion of pathogenic bacteria such as escherichia coli, the clostridium butyricum and the bifidobacterium increase the cecal immunostimulation function and promote the immunity of chicks.
The number of viable bacteria of each strain was further determined under the conditions of a fixed medium, and the specific results are shown in table 11.
Table 11: viable count and acid yield of strain on specific culture medium
The microbial strains are prepared from Bacillus haynesii TS-S7 (Bacillus marinus) according to the compound proportion: pediococcus acidilactici HJ-S5 (Pediococcus acidilactici): bacillus safensis HJ-M15 (Bacillus safensis): propionibacterium freudenreichii BC-K2 (Propionibacterium freudenreichii): clostridium butyricum HJ-M1 (Clostridium butyricum) ═ 10: 10: 50: 50: 50 when the compound microbial agent is prepared, the growth of various strains is normal, no mutual inhibition is generated, the number of viable bacteria is high, and the acid production types are various, thereby providing a basis for the application of the compound microbial agent in chicken feeding.
Example 3: application of compound microbial agent prepared by intestinal probiotic group planting culture method of Sanhuang young broiler chickens
In the embodiment, the application effect of the compound microbial agent prepared by the intestinal probiotic colony colonization culture method of the three-yellow-meat chicks in the feeding of the three-yellow-meat chicks is examined, six colony houses (average 1 million feathers/one colony) of a certain three-yellow-meat chicken farm in Xinjiang rock river valley are selected, the growth performance of the microbial agent on the meat-yellow chicken under different dilution concentration gradients and the influence of the material-meat ratio are compared, three dilution gradients are set, the ratio of 1:500(T1), 1:1000(T2), 1:1500(T3), 1:2000(T4) and 1:3000(T5) are set, the commercial compound microbial agent is fed according to the instruction ratio as a control group 1 (CK1), the normal feeding group without adding the microbial agent is used as a control (CK2), the growth performance of the three-yellow-meat chicks in a period of 7-42 days is monitored, the influence of the immune organ index of the three-yellow-meat chicks caused by using different microbial agents is evaluated, the feeding start date of, comparing the difference of each organ index of the three-yellow-meat chicken, further examining the influence of different microbial agents on the cecal microorganisms and serum immune globulin of the three-yellow-meat chicken, and the specific result is 12-15.
Table 12: growth performance of 7-42-day-old three-yellow-meat yellow chicks
Table 13: influence of different treatment groups on immune index of Sanhuang meat yellow chicks
Table 14: influence of compound microbial agent on cecal microorganisms of three-yellow-meat chicks
| Group of | Lactobacillus strain | Bifidobacterium | Escherichia coli |
| T1 | 5.95±0.29a | 4.42±0.85a | 2.89±0.28a |
| T2 | 6.06±0.22a | 4.69±0.36a | 1.96±0.44a |
| T3 | 5.98±0.32a | 4.46±0.25a | 2.34±0.35a |
| T4 | 5.90±0.31a | 4.39±0.34a | 2.64±0.28a |
| T5 | 6.02±0.20a | 4.53±0.31a | 2.26±0.25a |
| CK1 | 4.75±0.35a | 3.09±0.34a | 4.86±0.35a |
| CK2 | 3.92±0.44b | 1.61±0.47b | 7.68±0.22c |
Note: number unit of flora: CFU (1X 10)8) Per gram of intestinal contents;
② different superscript letters in the same column represent significant difference (P <0.05), and the same superscript letters represent insignificant difference (P > 0.05).
Table 15: effect of different treatments on immunoglobulin levels in Sanhuang broiler chicks
| Group of | Immunoglobulin A | Immunoglobulin G |
| T1 | 4762.03±362.13b | 17989.35±967.35a |
| T2 | 4882.59±453.63b | 18150.36±657.27a |
| T3 | 4851.32±244.53a | 17645.31±856.38a |
| T4 | 4805.15±325.25b | 18089.45±811.65a |
| T5 | 4825.51±296.31a | 17105.34±832.16a |
| CK1 | 4533.18±130.66a | 16855.67±980.8ab |
| CK2 | 3154.06±342.48c | 11487.19±239.12c |
Note: the letters in the same column indicate no significant difference (P >0.05), and the letters in the adjacent columns indicate significant difference (P <0.05) and the letters in the other columns indicate very significant difference (P < 0.01).
As can be seen from the data in the attached tables 12-15, compared with the situation that the compound microbial agent for the three-yellow-meat chicks prepared by the invention is fed without a microbial agent and a commercially available microbial agent, the production performance of the compound microbial agent for the three-yellow-meat chicks prepared by the invention is obviously superior to that of other groups, wherein the average weight of the broilers is 3.13kg, the weight of the broilers is increased by 3.07kg in the whole period, and the survival rate of the broilers is more than 97.73 percent; according to the invention, the cecal microorganisms of the three-yellow-meat chicks fed by different compound microbial agents are further investigated, the lactobacillus and bifidobacterium (probiotics) content of the three-yellow-meat chicks fed by the prepared compound microbial agent for the three-yellow-meat chicks are improved, and intestinal tract large intestine bacillus (pathogenic bacteria) is inhibited, so that the digestive function of the three-yellow-meat chicks is obviously enhanced; the serum immunoglobulin of the three-yellow-meat chick fed by different microbial agents is measured, and the result shows that the compound microbial agent provided by the invention is obviously superior to a commercial microbial agent control group without being added, the concentrations of serum immunoglobulin A and immunoglobulin G of the three-yellow-meat chick are further increased, the effect of an organism for transferring nutrient substances is enhanced, the deposition of muscle protein is promoted, and the humoral immune response of the organism is enhanced simultaneously, so that the average body weight of the three-yellow-meat chick is obviously increased.
The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art based on the foregoing description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (4)
1. A field planting culture method for intestinal probiotic groups of Sanhuang young chickens is characterized by comprising the following steps:
(1) screening and separating chicken manure and different intestinal sections of duodenum, jejunum, ileum and caecum of intestinal tracts in free-range local chickens raised by farmers and herdsmen, directionally separating bacillus, lactobacillus, bifidobacterium and clostridium butyricum, obtaining representative strains through morphology and comparison of staining and purification, and identifying the strains;
(2) culturing the screened bacillus, lactobacillus, bifidobacterium and clostridium butyricum serving as probiotics in a corresponding culture medium: culturing the bacillus in an LB liquid culture medium at 37 ℃ and 180rpm/min for 48h, statically culturing and activating the lactobacillus in an MRS liquid culture medium at 37 ℃ for 48h, statically culturing the clostridium butyricum in an RCM culture medium at 37 ℃ for 48h, and culturing the bifidobacterium in the LB liquid culture medium at 37 ℃ and 180rpm/min for 48 h; measuring the inhibition zone of each bacterium, selecting the strain with the most obvious inhibition zone effect, and primarily screening high-quality strains;
(3) comparing the tolerance, enzyme production and acid production capabilities of the screened probiotics, and screening out bacterial strains with strong acid resistance, cholate resistance, gastrointestinal fluid resistance, acid production capability and rich enzyme production;
(4) the above steps (A) and (B)3) Screening to obtain a composite microbial agent suitable for colonization culture of intestinal flora of the sanhuang broiler chicken, wherein the composite microbial agent comprises Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici (Pediococcus acidilactici), Bacillus safensis (Bacillus safensis), Propionibacterium freudenreichii (Propionibacterium freudenreichii) and Clostridium butyricum (Clostridium butyricum), and the 5 kinds of the microbial agents are mixed according to the volume ratio of 10: 10: 50: 50: 50, compounding, and finally determining the intestinal probiotic groups of the fixedly planted and cultured chicks suitable for the three-yellow-meat chicks;
(5) and (3) feeding the 0-day-old chicks to colonize and culture the probiotics groups compounded by the Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici (Pediococcus acidilactici), Bacillus safensis (Bacillus safensis), propionibacterium freudenreichii and Clostridium butyricum (Clostridium butyricum) prepared in the step (4) in the intestinal tracts of the chicks, and quickly establishing the strong probiotics groups.
2. The method for permanent planting and culturing intestinal probiotics of the sanhuang broiler chicken as claimed in claim 1, wherein the complex microbial inoculant is prepared from 5 species of Bacillus marinus (Bacillus haynesii), Pediococcus acidilactici (Pediococcus acidilactici), Bacillus safensis (Bacillus safensis), Propionibacterium freudenreichii (Propionibacterium freudenreichii), Clostridium butyricum (Clostridium butyricum) according to the volume ratio of 10: 10: 50: 50: 50 are compounded to obtain.
3. The use of the compound microbial inoculant of claim 2 in intestinal probiotic flora colonization of sanhuang broiler chickens.
4. The use of the complex microbial inoculant of claim 2 in the feeding of sanhuang broiler chickens.
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