CN116536205B - Clostridium butyricum composite microbial inoculum and application thereof - Google Patents
Clostridium butyricum composite microbial inoculum and application thereof Download PDFInfo
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- CN116536205B CN116536205B CN202310516234.0A CN202310516234A CN116536205B CN 116536205 B CN116536205 B CN 116536205B CN 202310516234 A CN202310516234 A CN 202310516234A CN 116536205 B CN116536205 B CN 116536205B
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- clostridium butyricum
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Classifications
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- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
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- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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Abstract
The invention discloses a clostridium butyricum composite microbial inoculum and application thereof in endogenous reduction of odor components in livestock and poultry feces. The compound microbial inoculum provided by the invention can be used as a feed additive, can reduce the production of indole, skatole and ammonia nitrogen in the intestinal tracts of birds endogenously, has the reduction rate of 18.67%, 19.25% and 16.5%, and reduces the contents of ammonia, organic acid and the like in the culture environment. In addition, the microbial compound bacteria agent can also prevent and treat intestinal diseases, maintain the balance of intestinal flora of livestock and poultry, improve the disease resistance of organisms, enhance the immunity, increase the feed conversion rate, reduce the meat-to-feed ratio and improve the growth performance. The cultivation method is suitable for poultry farms with various scales.
Description
Technical Field
The invention relates to the technical field of functional microorganisms, in particular to a clostridium butyricum composite microbial agent and application thereof.
Background
In the livestock and poultry farming industry, harmful gas generated by high-density farming severely restricts the healthy development of the industry. For example, the harmful gas generated by the chicken manure can pollute the air in the house, reduce the production performance of poultry, spread diseases, cause the air quality in the cultivation field and nearby to be reduced, and pose a threat to human health. Researches show that the contribution rate of the breeding environment to the productivity of the livestock and poultry can reach 30-40%. In actual production, the methods of ventilation, removing the excrement and urine, spraying the adsorbent and the like are adopted to reduce the odor gas in the colony house. The method for spraying the adsorbent has high cost, is inconvenient for large-scale cultivation and has unsatisfactory effect; ventilation causes the temperature of the colony house to drop sharply, and the production performance of livestock and poultry is easily affected.
At present, the microbial feed additive is a biological agent prepared by fermenting beneficial microorganisms approved by the agricultural department, and is the best substitute of antibiotics. The microbial product is used for improving the health level of livestock and poultry and the metabolites thereof, can regulate the micro-ecological imbalance in the livestock and poultry, keep micro-ecological balance, and is a microbial product for stimulating the propagation and growth of beneficial bacteria of the animals and resisting the growth of harmful bacteria. The research shows that the concentration of harmful gases in the colony house can be effectively reduced by adding the microbial agent into the daily ration of the poultry.
Clostridium butyricum (Clostridium butyricum), an obligately anaerobic gram-positive bacillus, belongs to the genus Clostridium. The cell morphology is: the diameter is 0.6-1.2 mu m, the length is 0.3-0.7 mu m, the two ends are round, the middle part is slightly expanded, and the flagellum is in a rod shape, single or paired and can move. Clostridium butyricum is used as a feed additive, is not influenced by gastric acid and bile acid, can promote the proliferation of beneficial intestinal flora (bifidobacteria and lactobacillus) of animals, inhibit the growth and proliferation of harmful bacteria and putrefying bacteria in the intestinal tract, and correct the disorder of intestinal flora; the main metabolite butyric acid is a main nutrient substance for regenerating and repairing intestinal epithelial tissue cells, and has important significance in reducing abuse of feed antibiotics, reducing bacterial drug resistance of animals and guaranteeing animal health.
Zhou Anshu and the like in the study of the inhibition effect of clostridium butyricum on harmful bacteria and beneficial bacteria in the intestinal tracts of livestock (feed exposition, 1 st year 2023) show that clostridium butyricum has a certain inhibition effect on main harmful bacteria in the intestinal tracts of livestock, has the feeding value of regulating and controlling the balance of intestinal flora of livestock, and the total quantity of lactobacillus and bifidobacterium in the intestinal tracts of livestock can be obviously improved by singly adding clostridium butyricum in the diet, so that the production performance of livestock is improved; xu Fei discloses that clostridium butyricum has the characteristics of acid resistance, high temperature resistance, high pressure resistance and multiple antibiotics resistance in the aspects of pig breeding production, has the advantages of safety, no toxicity, greenness, no residue, good stability, and multiple excellent biological functions of regulating intestinal microorganism colony balance, improving intestinal mucosa morphology, improving organism growth performance, digestibility, immunity, oxidation resistance and the like, is widely applied to livestock breeding industry as a novel feed additive, and simultaneously provides the difference of the sources of bacterial strains, the bacterial strains obtained by separating from pig intestinal tracts have obvious effects on pigs, are not necessarily effective on chickens, and have different sources of clostridium butyricum preparations and different application objects. Sun Yushuang et al in the "influence of mannooligosaccharide-clostridium butyricum on faecal flora of 14-day-old broiler chickens" indicate that the addition of clostridium butyricum and mannooligosaccharide or the combined addition of clostridium butyricum and mannooligosaccharide in the daily ration of broiler chickens has obvious change of dominant flora on 5 classification levels of phylum, class, order, family, genus and the like, and plays a role in regulating the structure of intestinal flora.
In summary, the main problems of the microbial feed additive are the selection of excellent species and the decrease in activity of the microbial feed additive due to the inactivation of the species. How to screen high-efficiency bacterial strains and to be matched with reasonable formulas, plays an important research significance in the optimal effect of the microbial feed additive.
Disclosure of Invention
The invention aims to: aiming at the defects of the prior art, the invention provides the dominant clostridium butyricum strain and further provides the composite microbial inoculum to reduce the generation of endogenous odor of livestock and poultry and reduce the generation of harmful gases in the colony house, thereby providing references for popularization and application.
Specifically, the invention further provides a clostridium butyricum composite microbial inoculum, which comprises the following components in parts by weight: 55-90 parts of clostridium butyricum bacterial agent, 6-25 parts of polyglutamic acid, 1-10 parts of jerusalem artichoke extract and 3-10 parts of bile acid. The clostridium butyricum agent comprises clostridium butyricum, the clostridium butyricum is classified and named as clostridium butyricum SDS-01 (Clostridium butyricum SDS-01), and is preserved in China center for type culture Collection (China center for type culture Collection) at the 3 rd month and 10 th year, the preservation address is China university of Wuhan, and the preservation number is CCTCC NO: m2023287. Clostridium butyricum is collected in the soil of the autotrophic chicken farm.
Preferably, the composite microbial inoculum comprises the following components in parts by weight: 75-80 parts of clostridium butyricum bacterial agent, 10-15 parts of polyglutamic acid, 3-5 parts of jerusalem artichoke extract and 6-8 parts of bile acid.
In a specific embodiment, the composite microbial inoculant comprises the following components in parts by weight: 78.33 parts of clostridium butyricum bacterial agent, 12.33 parts of polyglutamic acid, 4.00 parts of jerusalem artichoke extract and 7.67 parts of bile acid.
Preferably, the polyglutamic acid has a weight average molecular weight of 10kDa to 1000kDa; more preferably, the polyglutamic acid has a weight average molecular weight of 50-200kDa.
The jerusalem artichoke extract is prepared by placing jerusalem artichoke dry powder into water, wherein the feed-liquid ratio is 1:30-60, ultrasonic assisted extraction, hot water extraction at 50-70deg.C for 60-120min, centrifuging to obtain supernatant extract, and spray drying to obtain Jerusalem artichoke extract.
Further, the invention provides a clostridium butyricum bacterial agent, which comprises clostridium butyricum, wherein the total number of effective viable bacteria in the bacterial agent is 1.0X10 8～2×109 CFU/mL.
The invention further provides application of any one of the composite microbial inoculum, the clostridium butyricum and the microbial inoculum containing the same in endogenous reduction of odor components in livestock and poultry feces.
Specifically, the odor component comprises any one or more of indole, 3-methylindole and ammoniacal nitrogen.
The invention further provides application of any one of the composite microbial inoculum, the clostridium butyricum and the microbial inoculum containing the same in adjusting intestinal flora, improving intestinal microecology and enhancing disease resistance of livestock and poultry, wherein the intestinal flora adjustment is as follows: inhibiting Salmonella colonization, promoting growth of Thick-walled bacteria and actinomycetes, and regulating the ratio of Bacteroides.
The application further discloses application of any one of the composite microbial inoculum, the clostridium butyricum and the microbial inoculum containing the same in improving feed conversion rate and growth performance of livestock and poultry, wherein the growth performance comprises weight, slaughter performance and laying rate, and the slaughter performance comprises half-clean rate, full-clean rate, pectoral muscle rate and leg muscle rate.
When in use, the clostridium butyricum, the microbial inoculum containing the clostridium butyricum or any one of the compound microbial inoculum is added into the feed or drinking water of the cultured animals for use.
Wherein, the feed additive is mixed with basic ration for feeding according to the mass ratio of 0.001 to 0.1 percent, or diluted by clean water according to the proportion of 1:50 to 1:500 and added into drinking water of livestock and poultry
The beneficial effects are that: according to the clostridium butyricum SDS-01 and the composite microbial inoculum thereof, on one hand, the release amount of malodorous gases such as indole, skatole and ammoniacal nitrogen in livestock and poultry manure can be effectively reduced, the generation of malodorous gases is reduced from the source, and the reduction rate is 18.67%, 19.25% and 16.5%, and ammonia and organic acid in the environment. On the other hand, the clostridium butyricum SDS-01 composite microbial inoculum has remarkable enhancement effect on the growth performance of broiler chickens on the basis of the effects, and has certain economic benefit; meanwhile, the infection of salmonella to the broiler chicken can be resisted, and the normal growth and muscle quality of the broiler chicken are ensured. Specifically, the clostridium butyricum SDS-01 composite microbial inoculum can increase the body resistance of broiler chickens and reduce the damage of salmonella to the broiler chickens. In the experiment, 40% of the toxicity attack model group is positive, obvious focus appears on the liver of the broiler chicken, and the focus of the liver of the broiler chicken is obviously reduced by about 50% due to the intervention of clostridium butyricum added in the ration before and clostridium butyricum added in the ration after the toxicity attack; clostridium butyricum is added into daily ration to intervene before and after the toxin is removed, and the liver has no obvious focus. After feeding clostridium butyricum for 1-2 weeks, the bacterial load of the liver is obviously lower than that of a virus attack control group, and the continuous feeding clostridium butyricum has the best effect, and at the moment, the liver has almost no salmonella lesion part, so that the clostridium butyricum composite bacterial agent can effectively inhibit the colonization of salmonella. In addition, the clostridium butyricum SDS-01 composite microbial inoculum can effectively increase the feed conversion rate, reduce the meat-to-feed ratio and improve the growth performance, compared with a control group, the average body weight gain of the clostridium butyricum SDS-01 composite microbial inoculum added with the feed in the whole broiler raising period is greatly improved compared with the control group, the daily average feed intake is increased by 1.7%, and the feed-to-meat ratio is reduced by 4.70%. The clostridium butyricum and the compound bacterial agent thereof can not only carry out deodorization treatment on the excrement alone, but also be compounded with other microorganisms and/or chemical substances with deodorization function, and the deodorization effect is also obvious.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a schematic representation of the colony morphology of Clostridium butyricum;
FIG. 2 is a schematic diagram showing the effect of a microbial inoculum on chicken livers;
FIG. 3 shows the relative abundance of cecal microbiota levels in broilers;
FIG. 4 is the effect of clostridium butyricum addition on indole, skatole and ammoniacal nitrogen content;
fig. 5 is a graph of weight gain of white feather broilers.
Detailed Description
The invention is further illustrated below in connection with specific examples. With respect to the specific methods or materials used in the embodiments, those skilled in the art may perform conventional alternatives based on the technical idea of the present invention and are not limited to the specific descriptions of the embodiments of the present invention.
Example 1 screening and identification of Clostridium butyricum SDS-01.
Collecting microorganism sample from soil of Nanjing Lishui chicken farm, adding into 0.9% physiological saline, shaking, adopting 10-time gradient dilution method, coating soil microorganism bacterial liquid on clostridium value-added culture medium plate, placing into 2.5L anaerobic culture bag after the culture medium is completely solidified, adding anaerobic gas production bag, sealing, and culturing at 37deg.C for 24 hr. And (3) microscopic examination is carried out on single colonies growing on the solid plate of the clostridium multiplication medium, single colonies conforming to the characteristics of clostridium are streaked and inoculated on the solid plate of the fresh clostridium multiplication medium, and anaerobic culture is continued until single colonies with basically consistent forms are obtained. Colonies containing the calcium-dissolving ring were selected, and streaking was repeated on the above plate to obtain a single strain. The colony morphology is shown in the figure. Wherein, the clostridium proliferation culture medium formula (1L): glucose 5.0g, lunar peptone 10.0g, beef powder 10.0g, yeast powder 3.0g, soluble starch 1.0g, sodium chloride 5.0g, sodium acetate 3.0g, L-cysteine hydrochloride 0.5g, agar 0.5g and pH 6.8+ -0.2.
And (3) sending the single strain obtained by screening to China center for strain identification, and comprehensively analyzing experimental data such as cell morphology, physiological and biochemical experiments, 16S rRNA gene sequences and the like of the strain, wherein an identification result is clostridium butyricum (Clostridium butyricum). The result of the 16S rRNA gene sequence determination is shown as SEQ ID NO. 1.
Example 2 culturing and preparation method of Clostridium butyricum SDS-01.
The total effective viable bacteria of the clostridium butyricum SDS-01 microbial preparation is more than or equal to 1.0X10 8 CFU/mL, wherein the main effective viable bacteria clostridium butyricum is 1X 10 8～2×109 CFU/mL. The preparation method comprises the following steps:
(1) Plate culturing, namely using a solid activating culture medium to activate clostridium butyricum, inoculating clostridium butyricum on the plate of the solid activating culture medium by a streak inoculation method, and culturing at 50 ℃ for 36-48 hours to revive the clostridium butyricum to form single colonies. Wherein the solid activation culture medium is prepared according to the following weight percentages: 1% of peptone, 0.5% of yeast powder, 1% of sodium chloride, 2% of agar and the balance of distilled water, sterilizing at 121 ℃ for 20min, and cooling for standby.
(2) Preparing shake flask seeds, namely using a shake flask and a liquid activation culture medium, respectively inoculating the strains with good growth vigor and no pollution in the step (1) into the liquid activation culture medium by using an inoculating loop, performing anaerobic static culture for 36-48 hours at the temperature of 30-35 ℃ in a 500mL triangular flask to obtain shake flask seed liquid, and performing bacterial strain identification by sampling in a sterile operation to ensure that the bacterial strain is correct;
Wherein the liquid activation culture medium is prepared according to the following weight percentages: 1% of peptone, 0.5% of yeast powder, 1% of sodium chloride and the balance of distilled water, sterilizing for 20min at 121 ℃, and cooling for standby;
(3) The method comprises the steps of (1) domesticating and culturing, namely adding a liquid domestication culture medium into a 1L large triangular flask by using a clostridium butyricum single strain in the domestication and culturing step (2), inoculating the single strain in the step (2) for domestication and culturing, wherein the inoculum size is 5%, culturing at 33-37 ℃, standing and culturing for 36-48 h, and carrying out passage for two generations;
Wherein the domestication culture medium is prepared according to the following weight percentages: 3% of molasses, 0.5% of yeast powder, pH of 6.0-6.5 and the balance of distilled water, sterilizing for 15min at 115 ℃, and cooling for standby.
(4) And (3) fermenting and culturing, namely adding 800L of the fermentation medium into 1 ton of stainless steel fermentation tank, inoculating 9% of the liquid strain subjected to domestication and culture in the step (3), fermenting for 72 hours under normal temperature anaerobism, calculating the viable count under a microscope, and when the total number of effective viable bacteria is 1.5X10 9 CFU/ml, wherein the spore rate can reach 95.3%, stopping stirring, and spray-drying the bacterial liquid to obtain the clostridium butyricum preparation, namely bacterial powder.
The invention determines that the most ideal viable bacteria number and spore rate can respectively reach 1.5 multiplied by 10 9 CFU/ml and 95.3 percent when the optimal fermentation culture medium composition and the inoculation amount are 9 percent, and the inorganic salt component has no obvious influence on the growth of thalli.
Example 3 clostridium butyricum SDS-01 complex microbial agent formulation was optimized.
(1) Screening of composite microbial inoculant components
Early experiments show that the clostridium butyricum SDS-01 screened by the method has the endogenous deodorizing effect when being used in poultry cultivation, and in order to enhance the use effect, a clostridium butyricum SDS-01 compound form is adopted to prepare a compound microbial inoculum, and the compound substances to be selected are as follows: gamma polyglutamic acid, polylysine and nattokinase for assisting in promoting feed conversion and absorption; konjak extract, jerusalem artichoke extract and bean dreg extract for assisting in regulating intestinal flora composition; and bile acid, citrus oil and taurine for assisting in enhancing feed digestion and growth are compounded with clostridium butyricum SDS-01 through a single factor experimental design and added into basic ration of broiler chickens, and components with good compounding effect are screened by taking the generation amount of indole, trimethylindole and ammoniacal nitrogen in the cecum content (excrement after excretion) of the broiler chickens as indexes. Wherein, the high performance liquid chromatography is used for measuring the content of indole and skatole (3-methylindole), and the indophenol blue method is used for measuring the content of ammoniacal nitrogen.
Among them, the gamma-polyglutamic acid used in the following examples had a weight average molecular weight of 100kDa.
The Jerusalem artichoke extract is prepared by placing Jerusalem artichoke dry powder in water with a feed-to-liquid ratio of 1:50, ultrasonic assisted extraction, extracting with hot water at 60deg.C for 100min, centrifuging to obtain supernatant extract, and spray drying to obtain Jerusalem artichoke extract.
The konjak extract is prepared by placing konjak dry powder in water with a feed-to-liquid ratio of 1:20, ultrasonic assisted extraction, hot water extraction at 40 ℃ for 100min, centrifugation to obtain supernatant extract, spray drying to obtain konjak extract.
The bean dreg extract is prepared by placing konjak dry powder into water, wherein the feed-liquid ratio is 1:20, ultrasonic assisted extraction, hot water extraction at 90 ℃ for 60min, centrifugation to obtain supernatant extract, spray drying to obtain the bean dreg extract.
TABLE 1
TABLE 2
TABLE 3 Table 3
(2) Optimization of component proportion of composite microbial inoculant
The proportions of clostridium butyricum SDS-01, polyglutamic acid, jerusalem artichoke extract and bile acid components which are obtained through screening are optimized, four-factor four-level orthogonal designs are designed, the compound microbial inoculum is prepared by uniformly mixing according to the proportions designed in the table, 25 compound microbial inoculum with different proportions are added into daily ration of broilers according to 0.1%, and the optimal proportion is optimized by measuring the reduction rate of the excrement of the broilers, indole, trimethyl indole and ammonia nitrogen. As a result, the 11 # composite microbial inoculum is added into daily ration of broiler chickens, the reduction rate of total indole, trimethyl indole and ammoniacal nitrogen is the lowest, the 11 # ratio is judged to be the optimal compounding ratio according to the minimum reduction rate, the 11 # composite microbial inoculum is used for subsequent experiment examples, meanwhile, the polyglutamic acid, the jerusalem artichoke extract and bile acid can cooperate with clostridium butyricum through research judgment, and the generation of odor is reduced by improving amino acid metabolism, adjusting flora composition and the like.
TABLE 4 Table 4
EXAMPLE 4 Clostridium butyricum SDS-01 composite microbial inoculum and preparation method thereof
The clostridium butyricum SDS-01 composite microbial inoculum comprises clostridium butyricum SDS-01, polyglutamic acid, jerusalem artichoke extract and bile acid, and the preparation method comprises the following steps: the clostridium butyricum SDS-01 bacterial powder prepared in the example 2, polyglutamic acid, jerusalem artichoke extract and bile acid are adopted, uniformly mixed and granulated according to the weight ratio of 78:12:4:8, and the clostridium butyricum SDS-01 composite bacterial agent is obtained, wherein the effective viable count of the clostridium butyricum SDS-01 bacterial powder is 1.26X10 9 CFU/g.
The application method of the composite microbial inoculum comprises the following steps: mixing with basic ration for feeding according to the mass ratio of 0.001-0.1%, feeding livestock and poultry animals, diluting with clean water according to the mass ratio of 1:50-1:500, and adding into drinking water of livestock and poultry animals.
Example 5 Effect of feed-added clostridium butyricum SDS-01 Complex microbial inoculant on disease resistance of broiler chickens
The basic ration of corn, bean pulp and oil is uniformly mixed according to the proportion of table 5 to prepare basic ration of broiler chickens, 125 fries of 15-day-old white feather broiler chickens are selected for feeding, the basic ration is divided into a control group, a salmen virus eliminating group and a salmen virus eliminating-microorganism microbial inoculum group, the microorganism microbial inoculum group is based on the basic ration of the broiler chickens, clostridium butyricum SDS-01 composite microbial inoculum prepared in the example 4 is added, the chickens are fed according to the grouping design of table 6, and the chickens are weighed after being fed for 23 days, and the growth state of the chickens is observed. Wherein, premix 1 is commercial broiler premix, and each kilogram of feed provides the following components: vitamin A6132.00 IU, vitamin D3 3579IU, vitamin E11.00IU, vitamin K1.6 mg, vitamin B12.07 mg, vitamin B2.11 mg, vitamin B6.62 mg, vitamin B3.82 mg, calcium pantothenate 12mg, niacin 22.00mg, folic acid 0.4mg, manganese 60.00mg, iron 42mg, copper 2.5mg, zinc 45mg, iodine 0.9mg, se 0.25mg.
Table 5 basic ration composition of broiler chickens
TABLE 6 grouping design
The results are shown in Table 7, the toxicity attack model group shows 40% positive, the liver of the broiler chicken shows obvious focus, the focus of the liver of the broiler chicken is obviously reduced by intervention of clostridium butyricum compound bacteria agent added in daily ration before and after toxicity attack, and the liver has no obvious focus (Table 7). The liver bacterial load is obviously lower than that of a virus-attack control group, and the liver has almost no salmonella lesion part, so that the clostridium butyricum composite bacterial agent can effectively inhibit the colonization of salmonella, and the number of liver lesions, the positive rate and the bacterial load of chickens can be obviously reduced by continuous feeding.
TABLE 7 influence of clostridium butyricum SDS-01 Complex microbial inoculant addition on liver lesions and salmonella load of broiler chickens
When the salmonella challenge experiment is carried out, the clostridium butyricum compound bacterial agent group is added, so that the damage to broiler chickens caused by salmonella enteritidis infection of chickens is effectively reduced. And observing the liver of the broiler chicken subjected to experimental dissection, wherein the broiler chicken fed with the clostridium butyricum composite microbial inoculum has no sign of disease or is cured completely, and the bacterial load is reduced to almost vanish, as shown in table 2 and figure 2. The results of 16s sequencing analysis on the flora structures of the intestinal tracts of the broilers show that the salmonella infection group shows the lowest relative abundance, the lactobacillus is most obviously reduced, the clostridium butyricum compound microbial inoculum is added to feed to promote the growth of the firmicutes and actinomycetes, the proportion of the bacteroides is reduced, flora disorder caused after salmonella infection can be effectively regulated, as shown in figure 3, the oxygen can be rapidly consumed by clostridium butyricum spore germination, a hypoxia environment is produced, the growth of intestinal anaerobic probiotics is facilitated, the stability of the intestinal tracts of the broilers is not changed by adding the clostridium butyricum compound microbial inoculum, the proportion of partial flora is only changed, and the flora is optimally regulated under the condition that the original flora abundance is not destroyed. Meanwhile, the compound of gamma polyglutamic acid, jerusalem artichoke extract, bile acid and the like can also enhance the efficacy of influencing the intestinal microbial structure of broiler chickens, thereby supporting the potential application of the compound as intestinal probiotics.
Example 6 effect of adding clostridium butyricum SDS-01 complex inoculant on broiler manure malodorous compounds.
The clostridium butyricum SDS-01 composite microbial inoculum (the viable count is 1.26X10 9 CFU/g) prepared in the example 4 is uniformly mixed with basic ration of corn, bean pulp and oil according to the proportion shown in the table 5, 400 chicken fries of 1 day old white feather broiler are selected for feeding, the chicken fries are divided into a control group and a composite microbial inoculum group, the control group is fed with basic ration (the basic ration shown in the table 5), 0.01% clostridium butyricum composite microbial inoculum is added into each group on the basis of the basic ration, the broiler is slaughtered after 38 days of feeding, the cecum content (feces after excretion) is taken out, the indole content and the manure content (3-methylindole) are measured by adopting a high performance liquid chromatography, and the ammonia nitrogen content is measured by an indophenol blue method.
The results show that the clostridium butyricum composite microbial agent provided by the invention can obviously reduce the content of indole, skatole and ammoniacal nitrogen in the excrement of the broiler chickens, the reduction rate is 18.67%, 19.25% and 16.5%, and the clostridium butyricum composite microbial agent can obviously reduce the generation of odor compounds in the excrement of the broiler chickens by endogenous addition of the clostridium butyricum composite microbial agent, as shown in fig. 4.
Example 7 Effect of feed-added Clostridium butyricum SDS-01 Complex microbial inoculant on environmental gases in henhouses
In example 5, after 38 white feather broilers were raised, the environmental gases of the chicken house environment of the basic ration group and the clostridium butyricum complex microbial inoculum were collected by using a gas sampling bag, and the gas composition and the change of volatile organic compounds in the chicken house environment were measured by a gas phase method, and the results show that after clostridium butyricum complex microbial inoculum is added, the ammonia content in the chicken house environment is reduced by 11.73% and the organic acid content is reduced by 12.58% compared with the control group, as shown in table 8. The clostridium butyricum composite microbial inoculum is added into the feed, so that the concentration of odor in the environment of the henhouse can be effectively reduced, and the environment of the henhouse can be improved.
TABLE 8 analysis of environmental constituents of henhouse
| Sequence number | Component name | Control group | Composite microbial inoculum group |
| 1 | Carbon dioxide | 0.0828Vo1％ | 0.1336Vo1％ |
| 2 | Oxygen gas | 21.4283Vo1％ | 22.6175Vo1％ |
| 3 | Nitrogen gas | 78.4787Vo1％ | 77.2387Vo1％ |
| 4 | Ammonia gas | 7.76mg/m3 | 6.85mg/m3 |
| 5 | Acetic acid | 2.2205mg/m3 | 2.1375mg/m3 |
| 6 | Isobutyric acid | 5.2443mg/m3 | 5.106mg/m3 |
| 7 | Valeric acid | 5.277mg/m3 | 4.9497mg/m3 |
Example 8 effect of feed additive clostridium butyricum SDS-01 complex microbial inoculum on growth performance of broiler chickens.
The clostridium butyricum SDS-01 composite microbial inoculum (the viable count is 1.26 multiplied by 10 9 CFU/g) prepared in the example 4 is uniformly mixed with basic ration of corn, bean pulp and oil according to the proportion shown in table 5, 288 chicken fries of 1 day old white feather broiler are selected for feeding, the chicken fries are divided into a control group and a microbial inoculum group, the control group is fed with basic ration of broiler (shown in table 5), 0.01% clostridium butyricum composite microbial inoculum is added to each group on the basis of basic ration, and after feeding for 42 days, growth performance indexes such as weighing and the like are tested.
As shown in fig. 5, compared with the control group, the average body weight gain of the group added with clostridium butyricum composite microbial inoculum is greatly improved compared with the control group in the whole broiler raising period, the daily average feed intake is increased by 1.7%, and the feed conversion ratio is reduced by 4.70%. Each chicken feeds 90.6 g daily, the feed intake is 3171.9 g in the whole test period, and the addition amount of clostridium butyricum products is 0.32 g; 1.569 g more food than the control group, 54.915 g more food in the whole test period; the weight gain was 83.72g throughout the test period. In terms of slaughter performance, after clostridium butyricum is added, the half-breech-free rate, the full-breech-free rate, the pectoral muscle rate and the leg muscle rate of the broiler chickens are all obviously improved (Table 9).
Table 9 slaughtering performance of broilers
Note that: a, b are saliency groupings, different groupings are labeled, representing significant differences between groups.
Example 9 application effect of clostridium butyricum SDS-01 composite microbial inoculum in laying hen breeding.
To evaluate the application effect of the clostridium butyricum SDS-01 composite microbial inoculum prepared in the example 4 in the laying hen breeding, 120 sea-blue brown laying hens (380 days old) were selected for the breeding experiment, and the breeding experiment was divided into a control group and a clostridium butyricum SDS-01 composite microbial inoculum group, wherein the control group is fed with basic ration, and the composition is shown in Table 10. Starting on day 8, adding 0.1% clostridium butyricum SDS-01 composite microbial inoculum on the basis of basic diet, and continuously raising for 42 days, wherein the test chickens are kept in cages, and the control group and the microbial inoculum group chicken cages are placed separately and are free to eat and drink water, and are naturally illuminated in the daytime and illuminated in the morning and evening. And observing the weight, feed intake, egg yield and the like of the chicken flock, recording, randomly selecting 30 chickens in each treatment on the 49 th day of the experiment, and testing the content of indole, 3-methylindole and ammoniacal nitrogen in the intestinal contents of the chickens. Wherein, premix 2 is a commercially available premix for laying hens, and each kilogram of premix comprises the following components: vitamin A37500 IU, vitamin D3 10000IU, vitamin E250IU, vitamin K3 mg, vitamin B1.5 mg, vitamin B2 152mg, vitamin B6 80mg, nicotinic acid 500mg, calcium pantothenate 525mg, choline 6g, iron 875mg, copper 10mg, zinc 1000mg, manganese 1000mg, iodine 2.5mg, selenium 2.5mg.
Table 10 basic ration composition for laying hen
| Raw materials | Content (%) |
| Corn | 58.2 |
| Bean pulp | 27.7 |
| Soybean lecithin oil powder | 1.9 |
| Stone powder | 8.3 |
| Premix 2 | 3.9 |
| Totals to | 100 |
Table 11 shows that, compared with the control group, the average only average feed intake and average daily gain are respectively improved by 4.28% and 13.95% by adding 0.1% clostridium butyricum SDS-01 composite microbial inoculum into the feed during the whole broiler raising period, and the feed conversion rate is reduced by 7.8% compared with the control group; the average laying rate is improved by 4.78 percent.
TABLE 11 influence of clostridium butyricum SDS-01 complex inoculant added to diet on growth performance of laying hens
The feed is added with 0.1% clostridium butyricum SDS-01 composite microbial inoculum group, the reduction rate of indole, skatole and ammonia nitrogen in cecum content is 69.45%, 55.99% and 39.52%, and the reduction rate is shown in Table 12, which shows that the clostridium butyricum composite microbial inoculum provided by the invention can be used for remarkably reducing the generation of odor compounds in intestinal tract content of laying hens endogenously.
TABLE 12 determination of indole and 3-methylindole and Ammonia Nitrogen content in the content of the cecum of chickens
The invention provides clostridium butyricum and the thought and method for the livestock and poultry intestinal tract regulator, the method and the way for realizing the technical scheme are a plurality of, the above is only the preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by the person skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (5)
1. The clostridium butyricum composite microbial inoculum is characterized by comprising the following components in parts by weight: 75-80 parts of clostridium butyricum bacterial agent, 10-15 parts of polyglutamic acid, 3-5 parts of jerusalem artichoke extract and 6-8 parts of bile acid, wherein the clostridium butyricum bacterial agent comprises clostridium butyricum, the clostridium butyricum is classified and named clostridium butyricum (Clostridium butyricum) SDS-01, and the clostridium butyricum is preserved in China center for type culture collection (CCTCC NO) at 3-10 of 2023: m2023287, wherein the weight average molecular weight of the polyglutamic acid is 10 kDa-1000kDa; the jerusalem artichoke extract is prepared by placing jerusalem artichoke dry powder into water, wherein the feed-liquid ratio is 1:30-60, ultrasonic assisted extraction, hot water extraction at 50-70deg.C for 60-120min, centrifuging to obtain supernatant extract, and spray drying to obtain Jerusalem artichoke extract;
The clostridium butyricum bacterial agent is prepared by the following method:
(1) Plate culture: using a solid activation culture medium, activating clostridium butyricum by a flat plate, inoculating clostridium butyricum on the flat plate of the solid activation culture medium by a streak inoculation method, and culturing for 36-48 hours at 50 ℃ to revive the clostridium butyricum to form single colonies; wherein the solid activation culture medium is prepared according to the following weight percentages: 1% of peptone, 0.5% of yeast powder, 1% of sodium chloride, 2% of agar and the balance of distilled water, sterilizing at 121 ℃ for 20min, and cooling for later use;
(2) Manufacturing shaking bottle seeds: inoculating the strains with good growth vigor and no pollution in the step (1) into the liquid activation culture medium by using a shake flask and the liquid activation culture medium respectively, culturing for 36-48 hours by using a 500mL triangular flask at 30-35 ℃ in an anaerobic standing way to obtain shake flask seed liquid, and performing strain identification by sampling in a sterile operation way to ensure that the strains are correct;
Wherein the liquid activation culture medium is prepared according to the following weight percentages: 1% of peptone, 0.5% of yeast powder, 1% of sodium chloride and the balance of distilled water, sterilizing for 20min at 121 ℃, and cooling for standby;
(3) The method comprises the steps of (1) domesticating and culturing, namely adding a liquid domestication culture medium into a 1L large triangular flask by using a clostridium butyricum single strain in the domestication and culturing step (2), inoculating the single strain in the step (2) for domestication and culturing, wherein the inoculum size is 5%, culturing at 33-37 ℃, standing and culturing for 36-48 h, and carrying out passage for two generations;
Wherein the domestication culture medium is prepared according to the following weight percentages: 3% of molasses, 0.5% of yeast powder, pH of 6.0-6.5 and the balance of distilled water, sterilizing at 115 ℃ for 15min, and cooling for standby;
(4) Fermenting and culturing, namely adding 800L of the fermentation medium into 1 ton of stainless steel fermentation tank, inoculating the liquid strain subjected to domestication and culture in the step (3), fermenting for 72 hours under normal-temperature anaerobic condition with the inoculation amount of 9%, calculating the viable count under a microscope, stopping stirring when the total number of effective viable bacteria is 1.5X10 9 CFU/ml, and spray-drying the bacterial liquid to obtain the clostridium butyricum preparation.
2. The application of the composite microbial inoculum of claim 1 in endogenous reduction of odor components in livestock and poultry feces, wherein the odor components comprise any one or more of indole, 3-methylindole and ammoniacal nitrogen, and the livestock and poultry are chickens.
3. The application of the composite microbial agent in adjusting intestinal flora, improving intestinal microecology and enhancing disease resistance of livestock and poultry, which is characterized in that the intestinal flora adjustment is as follows: inhibiting Salmonella colonization, promoting growth of Thick-walled bacteria and actinomycetes, and regulating the proportion of Bacteroides, wherein the livestock and poultry are chickens.
4. The use of the composite microbial agent of claim 1 for improving feed conversion rate and growth performance of livestock and poultry, wherein the livestock and poultry are chickens, and the growth performance comprises weight, slaughter performance and laying rate, and wherein the slaughter performance comprises half-clean rate, full-clean rate, pectoral muscle rate and leg muscle rate.
5. Use according to any one of claims 2 to 4, wherein the clostridium butyricum complex microbial inoculum according to claim 1 is added to feed or drinking water for farmed animals.
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