CN113180159A - Lysozyme feed additive prepared by fermentation process - Google Patents
Lysozyme feed additive prepared by fermentation process Download PDFInfo
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- CN113180159A CN113180159A CN202110611966.9A CN202110611966A CN113180159A CN 113180159 A CN113180159 A CN 113180159A CN 202110611966 A CN202110611966 A CN 202110611966A CN 113180159 A CN113180159 A CN 113180159A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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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
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry
Abstract
The invention relates to a feed additive containing lysozyme, wherein the lysozyme is prepared by a fermentation process, and the feed additive comprises the following specific steps: placing the liquid culture medium in a triangular flask, inoculating strain seeds of streptomyces griseus according to the inoculum size of 4% v/v, performing shake flask fermentation under the fermentation conditions of 20-40 ℃ and 300r/min at 150-. The lysozyme feed additive obtained by the fermentation process has good prevention and treatment effects on piglet diarrhea, and has better using effect by being matched with the traditional Chinese medicine extract.
Description
Technical Field
The invention relates to the field of feeds, and particularly relates to a feed additive containing lysozyme.
Background
In recent years, with the highly intensive production of the livestock industry, the occurrence of livestock diseases is increasing day by day, and the death rate caused by the diseases is increasing year by year, so that great economic loss is caused to the livestock industry. Diarrhea is one of the main factors affecting the survival rate and growth and development of piglets, and the traditional prevention and treatment measures are the use of antibiotics. Although the use of antibiotics in livestock and poultry feed plays an important role in effectively controlling diseases, improving the feed conversion rate and ensuring the stable supply of livestock products, the problem of bacterial drug resistance is particularly prominent due to the overrange, high-dose or multi-variety and low-dose long-term use of antibiotics in the feed production and the abuse and indiscriminate use of antibiotics in the breeding process. With the general ban on antibiotics in feed in 2006 of the european union and the general ban on antibiotics in feed in 1 month 1 of 2017 of U.S. bulletin, antibiotic replacement development has been a hot spot of animal nutrition research.
Lysozyme (also called cell wall hydrolase) can specifically act on bacterial cell wall peptidoglycan molecules, and can break beta-1, 4 glycosidic bonds between N-acetylmuramic acid and acetyl glucosamine to loosen the bacterial cell wall to achieve the effect of dissolving bacteria. Lysozyme is widely distributed in higher animal and plant tissues and secretions thereof, protozoa, insects, plants and various microorganisms.
The lysozyme is used as one of nonspecific immune factors in normal body fluid and tissues of a human body, has various pharmacological effects, participates in various immune reactions of the body, has antibacterial and antiviral effects, and has an important effect of keeping physiological balance of the body in a normal defense function and nonspecific immunity of the body. It can improve and enhance macrophage phagocytic and digestive functions, activate leukocyte phagocytic function, and improve leukopenia caused by cytostatics, thereby enhancing body resistance. The lysozyme can directly hydrolyze gram-positive bacteria, and can also hydrolyze gram-negative bacteria such as escherichia coli and the like under the participation of secretory immunoglobulin A and complement. It also has the function of dissolving drug-resistant bacteria, and has the characteristics of obvious curative effect and small side effect on human body, so that it is an ideal medicinal enzyme. Lysozyme can directly act with virus protein with negative charge, and forms double salt with DNA, RNA and apoprotein to inactivate virus. The lysozyme as an enzyme antibacterial drug can participate in mucopolysaccharide metabolism, can play a role of strong inflammation diminishing when being matched with oral administration and external application, can be combined with various acidic substances inducing inflammation to inactivate the acidic substances, can enhance the curative effects of antibiotics and other drugs, and improves the mucopolysaccharide metabolism of tissue matrixes, thereby achieving the purposes of diminishing inflammation and repairing tissues.
The lysozyme is an essential tool enzyme in genetic engineering, cell engineering and fermentation engineering. In foreign countries, lysozyme is often used for the extraction of substances in the body of bacteria. The method is characterized in that sensitive thallus suspension is treated by lysozyme in a proper buffer solution, then ultrasonic waves, refrigerated centrifugation and other means are combined to obtain cell-free extract, and required thallus substances such as protein, nucleic acid, enzyme, active polypeptide and the like can be obtained by further refining, so that the demand of the development of the biological industry on lysozyme preparations is increasing day by day.
At present, the lysozyme which is most seen in the market is egg white lysozyme, but the lysozyme only has an effect on gram-positive bacteria, and the application range of the lysozyme is limited. In recent years, various sources of lysozyme are widely researched at home and abroad, but research reports on preparation of microbial lysozyme by a fermentation method are few, so that the application of the non-egg white lysozyme in actual industrial mass production is limited.
The traditional Chinese medicine has the characteristics of improving and regulating the body state while resisting bacteria, so that the development of a preparation for controlling piglet diarrhea by utilizing the special traditional Chinese medicine theory and rich botanical medicine resources in China becomes an important subject for maintaining and developing the pig industry.
Pulsatillae radix is perennial Ranunculaceae plant, and has a length of 50cm, dense white and long hair, thick and conical main root, and sometimes distorted brown outer skin. Pulsatilla chinensis, being cold in nature and entering large intestine meridian, has the effects of clearing heat and removing toxicity, cooling blood and checking diarrhea, and is a good medicine for treating heat-toxicity and bloody dysentery, and is commonly used for removing damp-heat in intestines and stomach. In addition, the traditional Chinese medicine composition can be used for treating malaria, or can be used for treating pruritus vulvae and swelling vulvae by being decocted for external washing, and has good effect on treating bacillary dysentery and amoebic dysentery in recent years. Pulsatillae radix has wide antibacterial effect, and its extract has good tranquilizing and analgesic effects, wherein the commonly used effective components are saponin and anemonin.
The fresh Chinese pulsatilla root juice, decoction and ethanol extract have obvious antibacterial effect in vitro, are sensitive to staphylococcus aureus, pseudomonas aeruginosa and shigella dysenteriae and also have obvious inhibiting effect on shigella dysenteriae, bacillus subtilis, salmonella and the like. In addition, studies have shown that the antibacterial active ingredients of pulsatilla chinensis are anemonin and protoanemonin, both of which have strong antibacterial effects.
Disclosure of Invention
Aiming at the defects of the prior art, the invention obtains the feed additive with good effect on preventing and treating the diarrhea of the piglets by screening the fermentation source and the fermentation process of the lysozyme and optimizing the compatibility of the lysozyme and the traditional Chinese medicine extract.
The technical scheme of the invention is as follows: a feed additive containing lysozyme is prepared by a fermentation process, and comprises the following specific steps: placing the liquid culture medium in a triangular flask, inoculating strain seeds of streptomyces griseus according to the inoculum size of 4% v/v, performing shake flask fermentation under the fermentation conditions of 20-40 ℃ and 300r/min at 150-.
Preferably, the liquid medium comprises glucose and peptone.
More preferably, the concentration of the glucose is 10g/L, and the concentration of the peptone is 5 g/L.
Preferably, the Streptomyces griseus is selected from Streptomyces griseus S-35.
Preferably, the fermentation condition is 30 ℃ and 200 r/min.
Preferably, the gel column chromatography uses a Sephadex G-100 gel column, and the cation exchange column chromatography uses a CM Sepharose Fast Flow column.
Preferably, the ammonium sulfate salting-out method comprises the following steps: adding ammonium sulfate into the supernatant to 20% saturation, stirring for 1 hr, standing for 1 hr, centrifuging at 10000r/min, dissolving the precipitate with distilled water, packaging into dialysis bag with molecular weight of 10000, and dialyzing in 10mmol/L PBS; adding ammonium sulfate into the supernatant in the dialysis bag to 70% saturation, stirring for 1h, standing for 1h, centrifuging at 10000r/min, collecting precipitate, and dialyzing to obtain dialysate.
Preferably, the feed additive also comprises traditional Chinese medicine extracts.
More preferably, the traditional Chinese medicine extract is a pulsatilla chinensis extract.
Preferably, the extraction method of the pulsatilla chinensis bunge extract comprises the following steps: heating radix Pulsatillae with 5-20 times of water, boiling, decocting with slow fire for 0.5-2 hr, pouring out medicinal liquid, adding 2-5 times of water, decocting, repeating for 1-3 times, each for 0.5-2 hr; mixing the medicinal liquids, concentrating, standing overnight, and centrifuging at 2500 r/min for 10 min to obtain supernatant; collecting supernatant, and spray drying to obtain dry powder.
More preferably, the extraction method of the pulsatilla chinensis extract is as follows: heating radix Pulsatillae with 10 times of water, boiling, decocting with slow fire for 1 hr, pouring out medicinal liquid, adding 3 times of water, decocting, repeating for 2 times, each for 1 hr; mixing the medicinal liquids, concentrating, standing overnight, and centrifuging at 2500 r/min for 10 min to obtain supernatant; collecting supernatant, and spray drying to obtain dry powder.
Most preferably, the weight ratio of the pulsatilla chinensis extract to the lysozyme is 1: 1.
The invention also provides the application of the feed additive containing lysozyme in the preparation of the medicines for preventing and treating piglet diarrhea.
The invention has the beneficial effects that:
the inventor obtains the lysozyme with good effect on preventing and treating the diarrhea of the piglets by screening the fermentation source of the lysozyme and optimizing the fermentation process parameters, further finds that the prevention and treatment effect is better by using the lysozyme and the Chinese medicinal extract together, and proves that the feed additive has certain guiding significance on the research and development of the antibiotic-free feed additive and the prevention and treatment of the diseases such as the diarrhea of the piglets.
Detailed Description
Preparation of lysozyme by fermentation process
Example 1:
a liquid medium was prepared in which glucose was 10g/L and peptone was 5 g/L. Placing 25mL liquid culture medium in a triangular flask, inoculating 4% (v/v) thallus seed of Streptomyces griseus S-35, performing shake flask fermentation under 30 deg.C and 200r/min, performing fermentation culture for 24h, centrifuging the fermentation liquid to remove thallus, collecting supernatant, sequentially salting out with ammonium sulfate (adding ammonium sulfate into the supernatant to 20% saturation, stirring for 1h, standing for 1h, centrifuging at 10000r/min, dissolving precipitate with distilled water, packaging into a dialysis bag with molecular weight of 10000, dialyzing in 10mmol/L PBS, adding ammonium sulfate into the supernatant to 70% saturation, stirring for 1h, standing for 1h, centrifuging at 10000r/min, collecting precipitate, dialyzing to obtain dialysate, Sephadex G-100 gel column chromatography and Sepharose Fast Flow cation exchange column chromatography, freeze drying to obtain lysozyme L1.
Example 2:
a liquid medium was prepared in which glucose was 10g/L and peptone was 5 g/L. Placing 25mL liquid culture medium in a triangular flask, inoculating 4% (v/v) thallus seed of Streptomyces griseus S-35, performing shake flask fermentation under 20 deg.C and 150r/min, performing fermentation culture for 24h, centrifuging the fermentation liquid to remove thallus, collecting supernatant, sequentially salting out with ammonium sulfate (adding ammonium sulfate into the supernatant to 20% saturation, stirring for 1h, standing for 1h, centrifuging at 10000r/min, dissolving precipitate with distilled water, packaging into a dialysis bag with molecular weight of 10000, dialyzing in 10mmol/L PBS, adding ammonium sulfate into the supernatant to 70% saturation, stirring for 1h, standing for 1h, centrifuging at 10000r/min, collecting precipitate, dialyzing to obtain dialysate, Sephadex G-100 gel column chromatography and Sepharose Fast Flow cation exchange column chromatography, freeze drying to obtain lysozyme L2.
Example 3:
a liquid medium was prepared in which glucose was 10g/L and peptone was 5 g/L. Placing 25mL liquid culture medium in a triangular flask, inoculating 4% (v/v) thallus seed of Streptomyces griseus S-35, performing shake flask fermentation under 40 deg.C and 300r/min, performing fermentation culture for 24h, centrifuging the fermentation liquid to remove thallus, collecting supernatant, sequentially salting out with ammonium sulfate (adding ammonium sulfate into the supernatant to 20% saturation, stirring for 1h, standing for 1h, centrifuging at 10000r/min, dissolving precipitate with distilled water, packaging into a dialysis bag with molecular weight of 10000, dialyzing in 10mmol/L PBS, adding ammonium sulfate into the supernatant to 70% saturation, stirring for 1h, standing for 1h, centrifuging at 10000r/min, collecting precipitate, dialyzing to obtain dialysate, Sephadex G-100 gel column chromatography and Sepharose Fast Flow cation exchange column chromatography, freeze drying to obtain lysozyme L3.
Second, preparation of Chinese medicine extract
Example 4:
heating radix Pulsatillae with 10 times of water, boiling, decocting with slow fire for 1 hr, pouring out medicinal liquid, adding 3 times of water, decocting, repeating for 2 times, each for 1 hr; mixing the medicinal liquids, concentrating, standing overnight, and centrifuging at 2500 r/min for 10 min to obtain supernatant; collecting supernatant, and spray drying to obtain Pulsatilla chinensis extract P1.
Example 5:
heating radix Pulsatillae with 20 times of water, boiling, decocting with slow fire for 2 hr, pouring out medicinal liquid, adding 5 times of water, decocting, repeating for 3 times, each for 2 hr; mixing the medicinal liquids, concentrating, standing overnight, and centrifuging at 2500 r/min for 10 min to obtain supernatant; collecting supernatant, and spray drying to obtain Pulsatilla chinensis extract P2.
Example 6:
heating radix Pulsatillae with 5 times of water, boiling, decocting with slow fire for 0.5 hr, pouring out medicinal liquid, adding 2 times of water, decocting, repeating for 1 time, each for 0.5 hr; mixing the medicinal liquids, concentrating, standing overnight, and centrifuging at 2500 r/min for 10 min to obtain supernatant; collecting supernatant, and spray drying to obtain Pulsatilla chinensis extract P3.
Thirdly, the feed additive of the invention has the influence on the prevention and treatment of piglet diarrhea
The egg white lysozyme used in the following experiments was purchased from Shanghai Hui Shi pharmaceutical science and technology Co., Ltd, the fermented lysozyme was lysozyme L1 prepared in example 1, and the Pulsatilla chinensis extract was Pulsatilla chinensis extract P1 prepared in example 4, unless otherwise specified.
1. Test protocol
The test was conducted in the Hainin scientific and technological pasture of the animal husbandry and veterinarian institute of the agricultural academy of Zhejiang province. According to the principle that the weights are similar and the male and female halves are in each group, 100 weaned piglets are randomly selected and are randomly divided into 5 groups (blank group, egg white lysozyme group, lysozyme L1 group, Chinese pulsatilla root extract P1 group and mixed group), and each group has 20 piglets. The basal diet formula and nutritional level fed during the test are shown in table 1.
TABLE 1 basic ration formula and Nutrition horizon
Note: the premix contains minerals (calcium, phosphorus, salt, copper, iron, manganese, zinc, selenium, iodine, and cobalt), vitamins (vitamin A, vitamin D, vitamin B1, vitamin B2, vitamin B12, biotin, folic acid, pantothenic acid, nicotinic acid, vitamin K, and vitamin E), and part of amino acids (lysine, methionine, threonine, and tryptophan), and no antibiotics.
The scheme of adding the feed additive into the basic ration for each test group is as follows:
(1) blank group: basal ration;
(2) egg white lysozyme group: 1.5 kg/ton of basic ration and egg white lysozyme;
(3) lysozyme L1 group: basic ration and lysozyme L11.5kg/ton;
(4) pulsatillae radix extract P1 group: basic ration and the pulsatilla extract P11.5kg/ton;
(5) a mixed group (lysozyme L1 and the pulsatilla chinensis extract P1 are mixed according to the weight ratio of 1: 1): basic ration, lysozyme L10.75kg/ton and Chinese pulsatilla root extract P10.75kg/ton.
2. Raising management and determination index
The test herd had free access to food and water, was fed on a separate meal 4 times a day (time 7:00, 11:00, 15:00, 19:00 respectively), the immunization program was performed according to the pig farm feeding management program, and diarrhea was immediately treated with neomycin sulfate and multivitamins in combination with oral rehydration salts. The whole test period is 40 days (30 days old-70 days old).
And observing and recording the diarrhea occurrence condition of each group of piglets in detail every day, and calculating the diarrhea rate.
The diarrhea rate (%). percent (total number of piglets in the test period/(test days × number of piglets) × 100% of diarrhea in piglets).
After the test is finished, collecting the feces samples of all groups of piglets, extracting the genomic DNA of fecal bacteria, sequencing amplified fragments by using a primer which is a specific primer of a bacteria 16S rDNA V4 area, then carrying out bioinformatics analysis on high-quality sequencing data, and mainly investigating the content of Cyanobacteria (cyanophyta) representing harmful bacteria in feces.
3. Test results
TABLE 2 Effect of the feed additive of the present invention on the prevention and treatment of diarrhea in piglets
| Test group | Diarrhea Rate (%) |
| Blank group | 8.8％ |
| Egg white lysozyme group | 4.6％ |
| Lysozyme L1 group | 2.1％ |
| Pulsatilla chinensis extract P1 group | 3.0％ |
| Mixing group | 1.1％ |
The test results show that (see table 2), compared with commercial egg white lysozyme, the lysozyme produced by the fermentation process has better effect of preventing and treating piglet diarrhea, and the fermentation process provided by the invention is proved to have a promoting effect on the production of lysozyme with high pharmacological activity. In addition, the effect of preventing and treating piglet diarrhea of the mixed group combined with the pulsatilla chinensis extract is more obvious, and an unexpected excellent effect is generated.
In addition, the sequencing result of the piglet manure-like 16S rDNA shows that compared with the egg white lysozyme group, the content of the harmful bacteria of the Cyanobacterium (Cyanophyta) in the lysozyme L1 group and the mixed group is respectively reduced by 53.28% and 68.29%, which indicates that the lysozyme is more beneficial to the establishment of beneficial flora in piglet intestinal tracts than the commercially available lysozyme.
In conclusion, the feed additive, in particular the antibiotic-free piglet breeding integrated technology by using the composition of the lysozyme and the traditional Chinese medicine extract prepared by the invention to replace antibiotics is feasible, and the effect is obviously superior to that of a commercial lysozyme preparation.
The above-mentioned embodiments are only preferred embodiments of the present invention, and it should be noted that, for those skilled in the art, several modifications can be made without departing from the present invention, and these should also be considered as within the scope of the present invention.
Claims (10)
1. The feed additive containing lysozyme is characterized in that the lysozyme is prepared by a fermentation process, and the specific steps are as follows: placing the liquid culture medium in a triangular flask, inoculating strain seeds of streptomyces griseus according to the inoculum size of 4% v/v, performing shake flask fermentation under the fermentation conditions of 20-40 ℃ and 300r/min at 150-.
2. The feed additive of claim 1 wherein the liquid medium comprises glucose and peptone.
3. The feed additive according to claim 2, wherein the concentration of glucose is 10g/L and the concentration of peptone is 5 g/L.
4. The feed additive of claim 1 wherein the streptomyces griseus is selected from the group consisting of streptomyces griseus S-35.
5. The feed additive according to claim 1, wherein the fermentation conditions are 30 ℃ and 200 r/min.
6. The feed additive according to any one of claims 1 to 5, wherein the feed additive further comprises a herbal extract.
7. The feed additive of claim 6, wherein the Chinese medicinal extract is Pulsatilla chinensis extract.
8. The feed additive as claimed in claim 7, wherein the extraction method of the pulsatilla chinensis extract is as follows: heating radix Pulsatillae with 5-20 times of water, boiling, decocting with slow fire for 0.5-2 hr, pouring out medicinal liquid, adding 2-5 times of water, decocting, repeating for 1-3 times, each for 0.5-2 hr; mixing the medicinal liquids, concentrating, standing overnight, and centrifuging at 2500 r/min for 10 min to obtain supernatant; collecting supernatant, and spray drying to obtain dry powder.
9. The feed additive of claim 8, wherein the weight ratio of lysozyme to pulsatillae radix extract is 1: 1.
10. Use of a feed additive according to any one of claims 1 to 9 for the manufacture of a medicament for the prevention and treatment of diarrhoea in piglets.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5374545A (en) * | 1989-04-24 | 1994-12-20 | Novo Nordisk A/S | Cell wall lytic enzymes from bacillus pabuli |
| JP2005080605A (en) * | 2003-09-10 | 2005-03-31 | Yaizu Suisankagaku Industry Co Ltd | Method for producing sugar composition containing n-acetylglucosamine and food or drink containing the sugar composition |
| CN1687397A (en) * | 2004-12-21 | 2005-10-26 | 中国水产科学研究院黄海水产研究所 | New type marine microorganism of lysozyme and bacillus 5-12 of producing lysozyme in high yield |
| CN102102095A (en) * | 2010-11-30 | 2011-06-22 | 浙江大学 | Method for preparing lysozyme by fermenting marine streptomyces |
| CN102847017A (en) * | 2012-08-26 | 2013-01-02 | 裔群英 | Oral solution for prevention and treatment of piglet diarrhea, and preparation method thereof |
| US20160051599A1 (en) * | 2013-04-09 | 2016-02-25 | Novozymes A/S | Compositions and methods for improving the health of aquatic animals |
| CN105454695A (en) * | 2016-01-25 | 2016-04-06 | 浙江艾杰斯生物科技有限公司 | Feed additive for preventing and curing porcine diarrhea |
| CN106421176A (en) * | 2016-11-30 | 2017-02-22 | 李生涛 | Traditional Chinese medicine powder for preventing and curing weaned piglet diarrhea |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101912048B (en) * | 2010-08-13 | 2013-06-05 | 中牧实业股份有限公司 | T4 lysozyme premix for animals and preparation method thereof |
| CN103333937B (en) * | 2013-06-06 | 2015-01-21 | 徐州工程学院 | Technique for preparing antimicrobial peptide by using Bacillus subtilis |
| CN104920897A (en) * | 2015-06-30 | 2015-09-23 | 江西瑞普康恩农牧发展有限公司 | Feed used for piglets to increase daily weight gain and reduce diarrhea |
-
2017
- 2017-04-14 CN CN202110611966.9A patent/CN113180159B/en active Active
- 2017-04-14 CN CN201710243612.7A patent/CN108719607B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5374545A (en) * | 1989-04-24 | 1994-12-20 | Novo Nordisk A/S | Cell wall lytic enzymes from bacillus pabuli |
| JP2005080605A (en) * | 2003-09-10 | 2005-03-31 | Yaizu Suisankagaku Industry Co Ltd | Method for producing sugar composition containing n-acetylglucosamine and food or drink containing the sugar composition |
| CN1687397A (en) * | 2004-12-21 | 2005-10-26 | 中国水产科学研究院黄海水产研究所 | New type marine microorganism of lysozyme and bacillus 5-12 of producing lysozyme in high yield |
| CN102102095A (en) * | 2010-11-30 | 2011-06-22 | 浙江大学 | Method for preparing lysozyme by fermenting marine streptomyces |
| CN102847017A (en) * | 2012-08-26 | 2013-01-02 | 裔群英 | Oral solution for prevention and treatment of piglet diarrhea, and preparation method thereof |
| US20160051599A1 (en) * | 2013-04-09 | 2016-02-25 | Novozymes A/S | Compositions and methods for improving the health of aquatic animals |
| CN105454695A (en) * | 2016-01-25 | 2016-04-06 | 浙江艾杰斯生物科技有限公司 | Feed additive for preventing and curing porcine diarrhea |
| CN106421176A (en) * | 2016-11-30 | 2017-02-22 | 李生涛 | Traditional Chinese medicine powder for preventing and curing weaned piglet diarrhea |
Non-Patent Citations (6)
| Title |
|---|
| T YOSHIMOTO, D TSURU: "Studies on bacteriolytic enzymes. II. Purification and some properties of two types of staphylolytic enzymes from Streptomyces griseus", 《J BIOCHEM》 * |
| 刘晓晔等: "白头翁汤对大鼠跨内皮迁移的中性粒细胞溶菌酶的影响", 《畜牧兽医学报》 * |
| 张琇等: "海洋细菌S-12-86的产溶菌酶条件", 《中国水产科学》 * |
| 盛精: "白头翁、生姜防治仔猪白痢及传染性肠炎的经验", 《畜牧与兽医》 * |
| 船津胜，鹤大典，李光福: "《溶菌酶》", 31 December 1982, 山东科学技术出版社 * |
| 赵昕，等: "灰色链霉菌RX-17溶菌酶R1的纯化及性质研究", 《微生物学报》 * |
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| CN108719607A (en) | 2018-11-02 |
| CN108719607B (en) | 2021-07-06 |
| CN113180159B (en) | 2022-09-27 |
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