CN108041299B - Antibiotic-free piglet compound feed - Google Patents
Antibiotic-free piglet compound feed Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- A—HUMAN NECESSITIES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
Abstract
The invention mainly belongs to the technical field of biology, and mainly relates to application of glycine and glucose in combination in preparation of antibiotic-free piglet compound feed for improving sensitivity of bacteria to terramycin and doxycycline, so that the problem of bacterial drug resistance is solved. The invention combines the small molecular substance glycine, glucose and exopolysaccharide, and then prepares the product with other functional feed additives and feed raw materials, improves the body immunity and body functions of piglets through the combined action of the glycine, the glucose and the exopolysaccharide, and improves the sensitivity of bacteria to antibiotics for prevention and treatment when diseases occur in the piglet stage and need to be prevented and treated, thereby achieving the purpose of preventing and treating the harm of bacteria including drug-resistant bacteria.
Description
Technical Field
The invention mainly belongs to the technical field of biology, and relates to a combined animal feed product comprehensively applied in the multidisciplinary field, in particular to application of glycine and glucose in combination in preparation of antibiotic-free piglet compound feed for improving sensitivity of bacteria to terramycin and doxycycline.
Background
The conventional piglet compound feed uses antibiotics, and simultaneously uses inorganic copper sulfate pentahydrate, ferrous sulfate monohydrate, zinc sulfate monohydrate, manganese sulfate monohydrate and other metal trace element feed additives and inorganic phosphorus, so that the compound feed for the conventional piglets for long-term feeding of bred animals causes potential harm of antibiotic resistance and environmental pollution caused by excessive antibiotics which are not digested and absorbed along with feces. In addition, a large amount of inorganic metal trace element feed additives such as copper, iron, zinc, manganese and the like and inorganic phosphorus are used in the feed, the digestibility of animal organisms on the trace elements is not high, part of the trace elements and inorganic phosphorus which are not digested and absorbed are discharged out of bodies through an excretion system, and the environment pollution of soil, underground water and the like is caused by fertilizers or treatment substances formed by excrement.
Although the use of antibiotics plays an essential role in the protection of human health and life and the intensive cultivation of animals, the abuse of antibiotics and the misuse thereof also become key factors threatening human health, livestock and poultry cultivation, aquaculture and ecological environment. Therefore, it is important to control bacterial antibiotic resistance.
Antibiotics are currently used in large quantities in the livestock farming industry. On the one hand, some antibiotics are essential as veterinary drugs for the control of bacterial infections. On the other hand, some antibiotics can promote animal growth. The use of a large amount of antibiotics can lead to the death of a large amount of sensitive bacteria, lead to the mass propagation of drug-resistant bacteria and promote and enhance the drug resistance of bacteria. The use of different antibiotics promotes the generation of multi-drug resistant bacteria, i.e. strains which can resist more than 3 antibiotics are generated. Controlling infection by these multi-drug resistant bacteria often requires replacement of new antibiotics and increased antibiotic doses. However, such a control method tends to make the resistance spectrum of the remaining multiple drug-resistant bacteria wider and the resistance ability stronger. Therefore, the invention of new technical products with little or no antibiotics is of great significance.
In the 50 s of the 20 th century, due to the discovery of remarkable immunocompetence of polysaccharides, various scholars gradually discover that the polysaccharides have unique biological activity from organisms such as fungi, seaweed, higher plants and the like, wherein the functions of promoting and recovering the immune function of organisms by the polysaccharides are particularly prominent. The polysaccharide is used as an immunity promoting and regulating agent, and has antibacterial, antiviral, antiparasitic, antitumor, radioprotective, and antiaging effects. The active polysaccharide is widely valued and researched by people because of wide sources, low price, exact effect and pure nature. The application range of the method is increasingly expanded.
Currently, glycine is mainly used as a nutritional additive and attractant for increasing amino acid in feed for livestock, particularly pets and the like. In the feed for the piglets, glycine and copper, iron, zinc and manganese form an organic chelate in a chelation mode, so that the digestibility of the bred animals on the copper, iron, zinc and manganese is improved, and the using amount of the copper, iron, zinc and manganese is reduced. So far, there are no reports that glycine promotes antibiotics to inhibit the growth of drug-resistant bacteria and that glycine and glucose are combined to promote the action of antibiotics, and there are no reports that glycine, glucose and extracellular polysaccharide are combined in piglet compound feed.
Disclosure of Invention
The invention aims to provide a piglet compound feed which can improve the sensitivity of bacteria to antibiotics for prevention and treatment and prevent the harm of bacteria including drug-resistant bacteria.
In order to achieve the technical purpose, the product application solution of the invention is as follows:
an antibiotic-free piglet compound feed comprises 0.001-5.0 wt% of glycine, 0.01-5.0 wt% of glucose and 0.01-5.0 wt% of exopolysaccharide.
Further, the antibiotic-free piglet compound feed comprises the following components in percentage by weight: 0.001% -5.0% of glycine; 0.05 to 5.0 percent of glucose; 0.01 to 5.0 percent of extracellular polysaccharide; 0.1 to 1.5 percent of calcium dihydrogen phosphate; 0.1 to 1.2 percent of stone powder; 0.1 to 0.6 percent of salt; 0.01% -0.2% of organic copper preparation; 0.01 to 0.2 percent of organic iron preparation; 0.02% -0.2% of organic zinc preparation; 0.02% -0.2% of organic manganese preparation; 0.01-3.0% of organic trace element pre-preparation; 0.1 to 0.6 percent of lysine; 0.02% -0.5% of methionine; 0.02% -0.6% of threonine; 0.01 to 0.05 percent of multi-dimension; betaine 0.01-0.2%; 0.01-0.03% of sweetening agent; 0.01 to 0.3 percent of antioxidant; 0.01 to 0.3 percent of mildew preventive; 0.01% -0.2% of enzyme preparation; 30 to 70.0 percent of high-quality corn; 43 percent of soybean meal 2 to 30.0 percent; 46% of soybean meal 2% -30.0%; 1 to 4.0 percent of soybean oil.
Further, the antibiotic-free piglet compound feed also comprises the following components in percentage by weight: 0 to 2.0 percent of calcium hydrophosphate; 0 to 1.5 percent of choline; 0-0.5% of tryptophan; phytase 0-2.0%; 0-2% of an acidifying agent; high-quality wheat 0-20.0%; 0-20.0% of high-quality barley; 0-15.0% of fermented soybean meal; 0-10.0% of puffed corn; 0-20.0% of puffed soybean; whey powder 0-8.0%; 0 to 5.0 percent of imported fish meal; 0-3.0% of white sugar; 0.01 to 4.0 percent of soybean oil; 0-5.0% of Chinese herbal medicine carrier.
Further, the antibiotic-free piglet compound feed is characterized in that: the Chinese herbal medicine carrier is a plant deep processing mixture with antibacterial and food calling effects, such as pericarpium Citri Tangerinae, fructus crataegi, flos Caryophylli, and Oregano oil.
Further, the antibiotic-free piglet compound feed is characterized in that: the purity of the glycine is more than 99%.
Further, the antibiotic-free piglet compound feed is characterized in that: the glucose is monohydrate glucose, and the purity of the glucose is more than 99.8 percent.
Further, the antibiotic-free piglet compound feed is characterized in that: the exopolysaccharide is a saccharide with an immune enhancement effect, and comprises one or more of microbial exopolysaccharide or plant exopolysaccharide.
Further, the antibiotic-free piglet compound feed is characterized in that: the feed is directly fed to piglets in the piglet stage.
When two micromolecular substances, namely glycine and glucose, are cooperatively used, antibiotics can be promoted to enter a bacterial body, the content of the antibiotics in the bacterial body is increased more obviously, and the sensitivity of clinical drug-resistant bacteria of various bacteria and bacteria to antibiotics such as kanamycin, oxytetracycline, doxycycline, amoxicillin and the like can be improved.
In conclusion, the invention has the pioneering technology that glycine, glucose and extracellular polysaccharide are used as core functional additive raw materials of the antibiotic-free piglet compound feed to be combined with other nutrients and feed raw materials, the antibiotic-free piglet compound feed is prepared by reasonable collocation and scientific combination according to a modern animal nutrition model, small molecular substance glycine and glucose are compounded in the antibiotic-free piglet compound feed for use, and the combined action of the glycine, the glucose and the extracellular polysaccharide improves the piglet body immunity and body functions, improves the sensitivity of bacteria to antibiotics, and achieves the purpose of preventing and treating the harm of bacteria including drug-resistant bacteria. Compared with the application of using veterinary drug powder (antibiotics) as an antibacterial drug-resistant drug in the piglet compound feed for a long time, the compound feed has higher sanitation and safety. Meanwhile, the use amount of the feed additive containing the metal trace elements such as copper, iron, zinc, manganese and the like and inorganic phosphorus is greatly reduced through the combined application of the organic metal elements and the balance of nutrition of piglets, so that the pollution of the environment caused by undigested absorption of the metal trace elements such as copper, iron, zinc, manganese and the like and the inorganic phosphorus in the feed is reduced.
Drawings
FIG. 1 shows the results of a study of the content of antibiotics that can be promoted into bacteria by the addition of glycine and glucose.
FIG. 2 shows the results of a study of glycine and/or glucose to increase kanamycin sensitivity of Staphylococcus aureus.
FIG. 3 shows the results of a study of glycine and/or glucose to increase kanamycin sensitivity in P.aeruginosa.
FIG. 4 shows the results of the study of glycine and/or glucose for increasing the kanamycin sensitivity of the clinical drug-resistant Escherichia coli bacteria.
FIG. 5 shows the results of a study of glycine and/or glucose to increase the sensitivity of Vibrio alginolyticus to kanamycin.
FIG. 6 shows the results of increasing the sensitivity of E.coli to oxytetracycline by the addition of glycine and/or glucose.
FIG. 7 shows the results of the determination of the resistance of Escherichia coli to clinical bacteria.
FIG. 8 shows the results of the synergistic enhancement of oxytetracycline sensitivity by the addition of glycine and/or glucose.
FIG. 9 shows the results of studies on the sensitivity of Edwardsiella tarda to doxycycline, which was improved by the addition of glycine and/or glucose.
FIG. 10 shows the results of a study of the enhancement of the sensitivity of E.coli to doxycycline by the addition of glycine and/or glucose.
FIG. 11 shows the results of studies on the improvement of the sensitivity of Escherichia coli clinical bacteria to doxycycline by the addition of glycine and/or glucose.
FIG. 12 is a result that the addition of glycine and/or glucose can improve the sensitivity of Escherichia coli to amoxicillin.
FIG. 13 shows the results of the determination of the drug resistance of Escherichia coli
FIG. 14 is a result of the synergistic improvement of the sensitivity of clinical bacteria of Escherichia coli to amoxicillin by the addition of glycine and/or glucose.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
Glycine and glucose can increase the amount of antibiotics that enter the body of bacteria
Bacterial death is related to the amount of antibiotic that enters the interior of the bacteria. In order to research the effect of glycine and glucose on promoting antibiotics to enter the bacteria, a single colony of Edwardsiella tarda EIB202 is picked from an LB plate and inoculated into a 5mLLB culture medium, and the medium is subjected to shaking culture at 30 ℃ and 200rpm for 24 hours to reach a saturation state. The bacterial liquid is collected by centrifugation, centrifuged for 5min at 8000rpm, the supernatant is removed, the bacterial cells are washed by 0.85% physiological saline, finally the bacterial cells are suspended by 1 XM 9 (containing 10mM acetate), the OD value of the bacterial liquid is adjusted to 0.2, and then 5mL of the bacterial liquid is respectively dispensed into test tubes to be used as test samples for standby. Dividing the experiment into 5 groups, wherein 2 groups are control groups and are respectively added with no substance and antibiotics; the other 3 groups are experimental groups, and glycine, glucose, glycine and glucose are added under the condition of adding antibiotics. After incubation for 6h at 30 ℃ on a shaker at 200 rpm. The cells were washed by centrifugation, disrupted by ultrasonication, and the kanamycin content was determined using a kanamycin ELISA detection kit (Clevel Technology Group Inc., Tokyo, Navkon). The results are shown in FIG. 1. After glycine is added, the content of antibiotics entering the bacteria is increased by 6.57 times compared with the content of antibiotics entering the bacteria when only antibiotics are added, the content of antibiotics in the bacteria is increased by 4.74 times after glucose is added, and the content of antibiotics entering the bacteria is greatly increased by 13.21 times after glucose and glycine are added. The synergistic effect of glycine and glucose can obviously improve the content of antibiotics entering the bacteria body.
Example 2
Glycine and glucose can improve the sensitivity of various bacteria to kanamycin antibiotic
Various bacteria were picked: staphylococcus aureus (S.aureus), Pseudomonas aeruginosa (P.aeruginosa), Escherichia coli clinical drug-resistant bacterium (Y15), Vibrio alginolyticus (V.algyrinolyticus) were monoclonally cultured in 100ml of LB liquid medium at 37 ℃ or 30 ℃ for 16 hours at 200rpm to reach saturation. 20mL of the bacterial solution was collected, centrifuged at 8000rpm for 5min, the supernatant was removed and the cells were washed with an equal volume of 0.85% physiological saline, and finally suspended with 1 XM 9 (containing 10mM acetate), the OD of the bacterial solution was adjusted to 0.2, and then separately dispensed into 5mL tubes, and after adding kanamycin as a control group and further adding 20mM glycine, 10mM glucose, 20mM glycine and 10mM glucose as test groups, and incubating at 37 ℃ or 30 ℃ and 200rpm for 6 hours in a shaker, 100. mu.L of the bacterial solution was taken for colony counting, and the results are shown in FIGS. 2-5. From these results, it can be seen that, for staphylococcus aureus (fig. 2), the bactericidal efficiency was improved by 16.38 times and 32.75 times by adding 20mM glycine and 10mM glucose, respectively, while the bactericidal efficiency was improved by 327.5 times by adding 20mM glycine and 10mM glucose simultaneously; for pseudomonas aeruginosa (fig. 3), the sterilization efficiency is respectively improved by 1.97 times and 1.71 times after 20mM glycine and 10mM glucose are respectively added, and the sterilization efficiency is improved by 20.99 times after 20mM glycine and 10mM glucose are simultaneously added; for clinical drug-resistant bacteria of escherichia coli (fig. 4), the sterilization efficiency is respectively improved by 1.05 times and 34.86 times after 20mM glycine and 10mM glucose are respectively added, and the sterilization efficiency is improved by 305 times after 20mM glycine and 10mM glucose are simultaneously added; for Vibrio alginolyticus (FIG. 5), the bactericidal efficiency was increased by 1.3 times and 72.75 times by adding 20mM glycine and 10mM glucose, respectively, and by 646.67 times by adding 20mM glycine and 10mM glucose, respectively. The results show that the sterilization efficiency of bacteria including drug-resistant bacteria is improved after glycine and glucose are respectively added, and the sterilization efficiency is remarkably improved after the glycine and the glucose are simultaneously added, so that the sensitivity of various bacteria to kanamycin can be improved by the combined use of the glycine and the glucose.
Example 3
Glycine and/or glucose for improving sensitivity of escherichia coli and clinical drug-resistant bacteria thereof to terramycin
(I) Glycine and/or glucose increase the sensitivity of E.coli to oxytetracycline
Preparation of E.coli test samples: single colonies of E.coli were picked from LB plates and inoculated into 5ml of LB medium, followed by shaking culture at 37 ℃ and 200rpm for 16 hours to reach saturation. The bacterial liquid is collected by centrifugation, centrifuged for 5min at 8000rpm, the supernatant is removed, the bacterial cells are washed by 0.85% physiological saline, and finally suspended by 1 XM 9 (containing 10mM acetate), the OD value of the bacterial liquid is adjusted to 0.2, and then 5mL of the bacterial liquid is respectively dispensed into test tubes for later use.
Dividing the prepared samples into 5 groups, wherein 2 groups are control groups, and no substance is added and terramycin is added respectively; the other 3 groups are experimental groups, and glycine, glucose, glycine and glucose are added under the condition of adding oxytetracycline respectively. After incubation for 6h at 37 ℃ on a shaker at 200rpm, 100. mu.L of the bacterial suspension was counted and the results are shown in FIG. 6. As can be seen from the results, compared with the case of adding oxytetracycline only, the sterilization efficiency is respectively improved by 3.78 times (the survival rate is reduced from 16.38% of that of the oxytetracycline only to 4.33% of that of the oxytetracycline and the glycine) and 4.85 times (the survival rate is reduced to 3.38% of that of the oxytetracycline and the glucose) after adding 20mM glycine and 10mM glucose, the sterilization efficiency is improved by 11.18 times (the survival rate is reduced to 1.47% of that of the oxytetracycline and the glucose and the glycine) after adding the glycine and/or the glucose, the survival rate of the escherichia coli is obviously reduced when being treated with the oxytetracycline, and the two substances can improve the sensitivity of the escherichia coli to the oxytetracycline and have a synergistic effect.
(II) glycine and/or glucose improve sensitivity of clinical drug-resistant bacteria of escherichia coli to terramycin
And (3) determining the drug resistance of clinical drug-resistant bacteria of escherichia coli: escherichia coli is the most predominant and abundant bacterium in animal intestinal tract, and most of the bacteria isolated clinically at present are multi-drug resistant bacteria. A strain of Escherichia coli is obtained by isolation from a pig farm, and the drug resistance of the strain is determined. The results (figure 7) show that the strain has the minimum inhibitory concentration to roxithromycin of 625 micrograms/ml, tetracycline of 6250 micrograms/ml, gentamicin of 2500 micrograms/ml, clindamycin of 25000 micrograms/ml, ceftazidime of 0.488 micrograms/ml, balofloxacin of 62.5 micrograms/ml, ampicillin of 6250 micrograms/ml, and amikacin of 2500 micrograms/ml, which indicates that the escherichia coli clinical bacterium is a multi-drug-resistant bacterium.
The sensitivity research of glycine and/or glucose for improving clinical drug-resistant bacteria of escherichia coli on terramycin: dividing the prepared samples (experimental samples prepared by the method of the escherichia coli) into 5 groups, wherein 2 groups are control groups and are respectively added with no substance and oxytetracycline; the other 3 groups are experimental groups, and glycine, glucose, glycine and glucose are added under the condition of adding oxytetracycline respectively. After incubation for 6h at 37 ℃ on a shaker at 200rpm, 100. mu.L of the broth was counted for colonies, and the results are shown in FIG. 8. As can be seen from the results, compared with the case of adding only terramycin, the sterilization efficiency is respectively improved by 1.32 times (the survival rate is reduced from 72.95% of that of adding only terramycin to 55.22% of that of adding terramycin and glycine) and 1.6 times (the survival rate is reduced to 45.68% of that of adding terramycin and glucose) after adding 20mM glycine and 10mM glucose, while the sterilization efficiency is improved by 2.73 times (the survival rate is reduced to 26.58% of that of adding terramycin and glycine and glucose) after adding 20mM glycine and 10mM glucose, and the survival rate of the clinical drug-resistant bacteria of escherichia coli is obviously reduced after adding glycine and/or glucose, which shows that the two substances can improve the sensitivity of the clinical drug-resistant bacteria of escherichia coli to terramycin and have a synergistic effect.
Example 4
Glycine and/or glucose increase the susceptibility of bacteria to doxycycline
Glycine and/or glucose can improve sensitivity of Edwardsiella tarda to doxycycline
Dividing the prepared samples into 5 groups, wherein 2 groups are control groups, and no substance is added and doxycycline is added respectively; the other 3 groups were experimental groups, and glycine, glucose, glycine and glucose were added in the case of doxycycline addition. After incubation for 6h at 30 ℃ on a shaker at 200rpm, 100. mu.L of the bacterial suspension was counted and the results are shown in FIG. 9. From the results, it was found that the bactericidal efficiency was improved by 5.97 times (the survival rate was reduced from 96.61% by adding doxycycline alone to 16.19% by adding doxycycline and glycine) and 7.08 times (the survival rate was reduced to 13.64% by adding doxycycline and glucose) respectively, compared with the case of adding doxycycline alone, and the bactericidal efficiency was improved by 11.18 times (the survival rate was reduced to 8.64% by adding doxycycline and glucose and glycine) by adding 20mM glycine and 10mM glucose simultaneously.
Glycine and/or glucose can improve sensitivity of Escherichia coli to doxycycline
Dividing the prepared samples into 5 groups, wherein 2 groups are control groups, and no substance is added and doxycycline is added respectively; the other 3 groups were experimental groups, and glycine, glucose, glycine and glucose were added in the case of doxycycline addition. After incubation for 6h at 37 ℃ on a shaker at 200rpm, 100. mu.L of the broth was counted for colonies, and the results are shown in FIG. 10. From the results, it was found that the bactericidal efficiency was improved by 1.49 times (survival rate was reduced from 99.71% by adding doxycycline to 67.24% by adding doxycycline and glycine) and 2.96 times (survival rate was reduced to 33.62% by adding doxycycline and glucose) respectively, compared with the case of adding doxycycline alone, and the bactericidal efficiency was improved by 4.09 times (survival rate was reduced to 24.42% by adding doxycycline and glycine and glucose) by adding 20mM glycine and 10mM glucose simultaneously.
Glycine and/or glucose can improve the sensitivity of Escherichia coli clinical bacteria to doxycycline
Dividing the prepared samples into 5 groups, wherein 2 groups are control groups, and no substance is added and doxycycline is added respectively; the other 3 groups were experimental groups, and glycine, glucose, glycine and glucose were added in the case of doxycycline addition. After incubation for 6h at 37 ℃ on a shaker at 200rpm, 100. mu.L of the broth was counted for colonies, and the results are shown in FIG. 11. From the results, it was found that the bactericidal efficiency was improved by 1.35 times (the survival rate was decreased from 96.59% in the case of doxycycline addition to 71.59% in the case of doxycycline addition and glycine addition) and 1.41 times (the survival rate was decreased to 68.4% in the case of doxycycline addition and glucose addition) respectively by adding 20mM glycine and 10mM glucose, and the bactericidal efficiency was improved by 2.33 times (the survival rate was decreased to 41.4% in the case of doxycycline addition and glycine addition and glucose addition) compared to the case of doxycycline addition alone.
After glycine and/or glucose are/is added, the survival rates of various bacteria including Edwardsiella tarda, Escherichia coli and clinical drug-resistant Escherichia coli bacteria are obviously reduced when the bacteria are treated by doxycycline, and the two substances can improve the sensitivity of the bacteria to the doxycycline and have synergistic effect.
Example 5
Glycine and/or glucose can improve the sensitivity of escherichia coli and escherichia coli clinical bacteria to amoxicillin
Glycine and/or glucose can improve the sensitivity of Escherichia coli to amoxicillin
Preparation of test specimens: single colonies of E.coli were picked from LB plates and inoculated into 5ml of LB medium, followed by shaking culture at 37 ℃ and 200rpm for 16 hours to reach saturation. The bacterial liquid is collected by centrifugation, centrifuged for 5min at 8000rpm, the supernatant is removed, the bacterial cells are washed by 0.85% physiological saline, and finally suspended by 1 XM 9 (containing 10mM acetate), the OD value of the bacterial liquid is adjusted to 0.2, and then 5mL of the bacterial liquid is respectively dispensed into test tubes for later use.
Dividing the prepared samples into 5 groups, wherein 2 groups are control groups, and no substance is added and amoxicillin is added respectively; and the other 3 groups are experimental groups, and glycine, glucose, glycine and glucose are respectively added under the condition of adding amoxicillin. After incubation for 6h at 37 ℃ on a shaker at 200rpm, 100. mu.L of the broth was counted for colonies, and the results are shown in FIG. 12. As can be seen from the results, compared with the case of adding only amoxicillin, the bactericidal efficiency was improved by 9.36 times (the survival rate was reduced from 25.29% of amoxicillin added only to 2.7% of amoxicillin added and glycine added) and 9.56 times (the survival rate was reduced to 2.64% of amoxicillin added and glucose added) respectively, while the bactericidal efficiency was improved by 18.55 times (the survival rate was reduced to 1.36% of amoxicillin added and glycine added and glucose added) simultaneously with 20mM glycine and 10mM glucose.
Glycine and/or glucose can improve the sensitivity of Escherichia coli clinical bacteria to amoxicillin
And (3) determining the drug resistance of clinical drug-resistant bacteria of escherichia coli: escherichia coli is the most predominant and abundant bacterium in animal intestinal tract, and most of the bacteria isolated clinically at present are multi-drug resistant bacteria. A strain of Escherichia coli is obtained by isolation from a pig farm, and the drug resistance of the strain is determined. The results (fig. 13) show that the strain has a minimum inhibitory concentration to roxithromycin of 625 micrograms/ml, a minimum inhibitory concentration to tetracycline of 6250 micrograms/ml, a minimum inhibitory concentration to gentamicin of 2500 micrograms/ml, a minimum inhibitory concentration to clindamycin of 25000 micrograms/ml, a minimum inhibitory concentration to ceftazidime of 0.488 micrograms/ml, a minimum inhibitory concentration to balofloxacin of 62.5 micrograms/ml, a minimum inhibitory concentration to ampicillin of 6250 micrograms/ml, and a minimum inhibitory concentration to amikacin of 2500 micrograms/ml, which indicates that the escherichia coli clinical bacterium is a multi-drug-resistant bacterium.
The sensitivity research of the clinical multiple drug-resistant bacteria of escherichia coli on amoxicillin can be improved by glycine and glucose: dividing the prepared samples (by the same sample preparation method of Escherichia coli) into 5 groups, wherein 2 groups are control groups, and no substance is added and amoxicillin is added; and the other 3 groups are experimental groups, and glycine, glucose, glycine and glucose are respectively added under the condition of adding amoxicillin. After incubation for 6h at 37 ℃ on a shaker at 200rpm, 100. mu.L of the broth was counted for colonies, and the results are shown in FIG. 14. As can be seen from the results, compared with the case of adding only amoxicillin, the bactericidal efficiency was improved by 5.06 times (the survival rate was reduced from 89.32% by adding only amoxicillin to 17.64% by adding amoxicillin and glycine) and 7.55 times (the survival rate was reduced to 11.82% by adding amoxicillin and glucose) by adding 20mM glycine and 10mM glucose, respectively, and the bactericidal efficiency was improved by 13.64 times (the survival rate was reduced to 6.55% by adding amoxicillin and glucose and glycine) by adding 20mM glycine and 10mM glucose simultaneously.
After glycine and/or glucose are added, the survival rate of escherichia coli treated by amoxicillin is remarkably reduced, which shows that glycine and/or glucose can improve the sensitivity of escherichia coli to amoxicillin and have synergistic effect.
Example 6
Use test of antibiotic-free piglet compound feed in piglet nursing stage
Purpose of the experiment
Aiming at the situation and possibility of respiratory tract mixed infection in a pig farm in a low-temperature season and a large temperature difference in a piglet nursing stage, and generally aiming at preventing diseases of piglets by antibiotic combination in the process of carrying out respiratory tract diseases in a staged manner in a pig raising process, the experiment verifies that the antibiotic-free piglet compound feed only contains glycine, glucose and extracellular polysaccharide but does not contain any antibiotic has the effects of preventing diseases and improving the piglet production performance by combining the piglet compound feed with veterinary drug powder (antibiotic).
Test method
1. Animal selection and grouping: preliminary preparation of the experiment was performed starting from the 25-day-old weaning of the piglets at birth. Selecting 30 piglets born by a self-reproduction third-birth fourth-birth similar-period estrus mating sow in a test pig farm, transferring from a birth bed to a nursery pig house according to the conventional method of the pig farm, randomly dividing the weaned piglets into 16 piglets, each 18-20 piglets, selecting 10 piglets with similar body weight and health condition from 35 days old piglets in birth, dividing into 5 groups, and adding 36-38 piglets in 1 group and 2 piglets.
2. Test treatment and daily ration composition: experimental piglets were divided into 5 treatment groups: control group, test group 1, test group 2, test group 3, and test group 4. The feed ration used in 5 groups was as follows:
the feed ration for the control group comprises the following components in parts by weight: the label of the product labels indicates that each 1kg of the product contains 20% of oxytetracycline calcium 500mg and 50% of kitasamycin powder 100mg of commercial additive premix feed for piglets 10%, high-quality corn 47.0%, puffed corn 10.0%, puffed soybean 8.0%, 46% of soybean meal 11.0%, fermented soybean meal 6%, whey powder 3.0%, imported fish meal 2.0%, white sugar 2.0%, soybean oil 1.0%, and the total amount is 100%. 1000mg of amoxicillin powder 10% of veterinary drug for prevention and treatment and 500mg of doxycycline powder 20% of veterinary drug for prevention and treatment are added into each 1kg of the daily ration, and the mixture is uniformly stirred with feed and then continuously used for 7 days.
The experimental group 2 adopts the antibiotic-free piglet compound feed of the patent, and the antibiotic-free piglet compound feed comprises the following components in parts by weight: glycine 0.5%, glucose 2.0%, exopolysaccharide 0.2%, calcium dihydrogen phosphate 0.4%, calcium hydrogen phosphate 0.6%, stone powder 0.6%, salt 0.1%, organic copper preparation 0.15%, organic iron preparation 0.13%, organic zinc preparation 0.13%, organic manganese preparation 0.1%, organic trace element pre-preparation 3.0%, lysine 0.15%, methionine 0.05%, threonine 0.12%, tryptophan 0.07%, multi-vitamin 0.01%, choline 0.08%, sweetener 0.015%, antioxidant 0.2%, mildew preventive 0.1%, enzyme preparation 0.02%, acidulant 0.1%, high-quality corn 48.0%, puffed corn 10.0%, puffed soybean 8.0%, 46% soybean meal 11.0%, fermented soybean meal 5.0%, whey powder 2.0%, imported fish meal 3.0%, white sugar 1.0%, soybean oil 2.0%, dried orange peel powder 1.195%, and total 100%.
Test group 2 feed ration: 1000kg of the antibiotic-free piglet compound feed in the test 2 group is added with the veterinary drug powder for prevention of 20 percent of terramycin calcium powder of 500g and the kitasamycin powder of 50 percent of 100g, and the mixture is stirred uniformly with the feed and then is continuously used for 7 days.
Test group 3 adopts the antibiotic-free piglet compound feed of the patent, and the antibiotic-free piglet compound feed comprises the following components in parts by weight: glycine 0.05%, glucose 5.0%, exopolysaccharide 1.0%, monocalcium phosphate 1.2%, stone powder 0.4%, salt 0.2%, organic copper preparation 0.14%, organic iron preparation 0.14%, organic zinc preparation 0.13%, organic manganese preparation 0.11%, organic trace element pre-preparation 0.02%, lysine 0.35%, methionine 0.02%, threonine 0.03%, multi-vitamin 0.035%, betaine 0.06%, sweetener 0.02%, antioxidant 0.1%, fungicide 0.1%, enzyme preparation 0.02%, acidulant 0.2%, high-quality corn 48.0%, high-quality wheat 10.0%, 46% soybean meal 10.0%, 43% soybean meal 15.0%, whey powder 3.0%, imported fish meal 2.0%, soybean oil 2.0%, dried orange peel powder 0.675%, and 100%.
Test group 3 feed ration: 1000kg of the antibiotic-free piglet compound feed in the test 3 groups is added with 1000g of amoxicillin powder with 10 percent of veterinary drug for prevention and treatment and 500g of doxycycline powder with 20 percent, and the mixture is stirred uniformly with the feed and then is continuously used for 7 days.
Test group 4 adopts the antibiotic-free piglet compound feed of the patent, and the antibiotic-free piglet compound feed comprises the following components in parts by weight: 0.001% of glycine, 0.01% of glucose, 5.0% of exopolysaccharide, 0.7% of monocalcium phosphate, 0.2% of calcium hydrophosphate, 0.9% of stone powder, 0.5% of salt, 0.13% of organic copper preparation, 0.16% of organic iron preparation, 0.1% of organic zinc preparation, 0.09% of organic manganese preparation, 1.2% of organic trace element pre-preparation, 0.45% of lysine, 0.3% of methionine, 0.45% of threonine and 0.12% of tryptophan; 0.05% of multivitamin, 0.07% of betaine, 0.03% of sweetener, 0.2% of antioxidant, 0.1% of mildew preventive, 0.02% of enzyme preparation, 0.219% of acidulant, 50.0% of high-quality corn, 10.0% of puffed soybean, 11.0% of 46% soybean meal, 4.0% of soybean protein concentrate, 2.0% of white sugar and 2.0% of soybean oil, wherein the total amount is 100%.
Test group 4 feed ration: 1000kg of the antibiotic-free piglet compound feed in the test 4 groups is added with 1000g of amoxicillin powder with 10 percent of veterinary drug for prevention and treatment and 500g of doxycycline powder with 20 percent, and the mixture is stirred uniformly with the feed and then is continuously used for 7 days.
The control group, test 1 group, test 2 group, test 3 group and test 4 group were supplemented with the above-mentioned antibiotics and veterinary drugs for prevention and treatment at the time of mixed production of piglet feed starting on day 5 of the official start of the test and continued for 7 days.
3. Feeding management: 5 groups of 10 pigsties are bred in adjacent pigsties of the same pigsty, the ground except the leaky floor is a heat-preservation floor, so that the pigsties can be freely eaten, and the pigsties can be freely drunk by drinking water tanks, so that the ventilation is good. The piglets of each group are fed freely in the same way.
4. And (3) observation and recording: the number of early heads and early weights of piglets, the number of end heads and end weights of piglets are recorded before and after the experiment, the condition of pigs is observed and recorded in the experiment period, and abnormal pigs are treated in time.
Results and discussion
After 30 days of official testing, the results are shown in table 1:
TABLE 1 statistics of test results
From table 1 it can be seen that:
the mortality rates of the test 1 group, the test 2 group, the test 3 group and the test 4 group during the test period are respectively 5.6 percent, 2.6 percent, 2.7 percent, 2.8 percent and 10.8 percent (%) lower than that of the control group;
the daily average weight gain of test 1, test 2, test 3 and test 4 was 354.5, 395.7, 365.0, 388.2 and 266.4 (g/day/head) higher than that of the control group during the test period.
The effect verification tests prove that the glycine, glucose and exopolysaccharide are combined with 20% of oxytetracycline calcium powder and 50% of kitasamycin powder in the piglet stage test 1 group and the piglet stage test 2 group; experiments 3 and 4 show that glycine, glucose and exopolysaccharide are combined with 10% of amoxicillin powder and 20% of doxycycline powder, so that the combination of 20% of oxytetracycline calcium, 50% of kitasamycin powder, 10% of amoxicillin powder and 20% of doxycycline powder which are superior to a control group in the aspects of preventing and treating piglet death and ensuring the production performance is combined. The antibiotic-free piglet compound feed only containing glycine, glucose and exopolysaccharide but no any antibiotic can achieve the purposes of improving the sensitivity of bacteria to the antibiotic, improving the body immunity and body function of piglets and preventing and treating the harm of bacteria including drug-resistant bacteria by combining the antibiotic-free piglet compound feed with proper veterinary drug powder in a piglet stage in a staged manner.
Claims (6)
1. The application of the combination of glycine and glucose in the preparation of the antibiotic-free piglet compound feed for improving the sensitivity of bacteria to terramycin or doxycycline is provided, wherein the bacteria are escherichia coli and escherichia coli clinical drug-resistant bacteria; the antibiotic-free piglet compound feed contains 0.001-5.0 wt% of glycine, 0.01-5.0 wt% of glucose and 0.01-5.0 wt% of extracellular polysaccharide, wherein the extracellular polysaccharide is a saccharide with an immune enhancement effect and is one or more of microbial extracellular polysaccharides.
2. The use of the combination of glycine and glucose according to claim 1 for the preparation of a non-antibiotic piglet formula feed for increasing the sensitivity of bacteria to terramycin or doxycycline, said non-antibiotic piglet formula feed comprising the following components in percentage by weight: 0.001% -5.0% of glycine; 0.05 to 5.0 percent of glucose; 0.01 to 5.0 percent of extracellular polysaccharide; 0.1 to 1.5 percent of calcium dihydrogen phosphate; 0.1 to 1.2 percent of stone powder; 0.1 to 0.6 percent of salt; 0.01% -0.2% of organic copper preparation; 0.01 to 0.2 percent of organic iron preparation; 0.02% -0.2% of organic zinc preparation; 0.02% -0.2% of organic manganese preparation; 0.01-3.0% of organic trace element pre-preparation; 0.1 to 0.6 percent of lysine; 0.02% -0.5% of methionine; 0.02% -0.6% of threonine; 0.01 to 0.05 percent of multi-dimension; betaine 0.01-0.2%; 0.01-0.03% of sweetening agent; 0.01 to 0.3 percent of antioxidant; 0.01 to 0.3 percent of mildew preventive; 0.01% -0.2% of enzyme preparation; 30 to 70.0 percent of high-quality corn; 43 percent of soybean meal 2 to 30.0 percent; 46% of soybean meal 2% -30.0%; 1 to 4.0 percent of soybean oil.
3. The use of a combination of glycine and glucose in the preparation of a non-antibiotic piglet feed formulation for increasing the sensitivity of bacteria to oxytetracycline or doxycycline according to claim 1, said non-antibiotic piglet feed formulation further comprising the following components in weight percent: 0 to 2.0 percent of calcium hydrophosphate; 0 to 1.5 percent of choline; 0-0.5% of tryptophan; phytase 0-2.0%; 0-2% of an acidifying agent; high-quality wheat 0-20.0%; 0-20.0% of high-quality barley; 0-15.0% of fermented soybean meal; 0-10.0% of puffed corn; 0-20.0% of puffed soybean; whey powder 0-8.0%; 0 to 5.0 percent of imported fish meal; 0-3.0% of white sugar; 0.01 to 4.0 percent of soybean oil; 0-5.0% of Chinese herbal medicine carrier.
4. The use of a combination of glycine and glucose as claimed in claim 1 for the preparation of a nonreactive piglet compound feed for increasing the sensitivity of bacteria to oxytetracycline or doxycycline, characterized in that: the purity of the glycine is more than 99%.
5. The use of a combination of glycine and glucose as claimed in claim 1 for the preparation of a nonreactive piglet compound feed for increasing the sensitivity of bacteria to oxytetracycline or doxycycline, characterized in that: the glucose is monohydrate glucose, and the purity of the glucose is more than 99.8 percent.
6. The use of a combination of glycine and glucose as claimed in claim 1 for the preparation of a nonreactive piglet compound feed for increasing the sensitivity of bacteria to oxytetracycline or doxycycline, characterized in that: the feed is directly fed to piglets in the piglet stage.
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Denomination of invention: A compound feed for piglets without resistance Effective date of registration: 20210304 Granted publication date: 20201229 Pledgee: Postal Savings Bank of China Limited Changtai County sub branch Pledgor: FUJIAN NISERVE BIOTECHNOLOGY Co.,Ltd. Registration number: Y2021350000031 |