A composite microecological preparation and its application in improving duck production performance

Abstract

The present invention relates to a composite probiotic preparation and its use in improving the production performance of ducks. The composite probiotic preparation comprises, by weight, 25-35 parts of Clostridium butyricum powder, 10-15 parts of Bacillus coagulans powder, 10-15 parts of Enterococcus faecalis powder, 5-10 parts of yeast cell walls, 0.05-0.1 parts of antimicrobial peptides, and 5-10 parts of auxiliary materials. The composite probiotic preparation has the advantages of stable properties, can be mixed with feed for feeding, and is easy to use. It can reduce or replace the use of antibiotics in feed, improve the ecological balance of duck intestinal flora, enhance immune function, promote duck growth, and increase feed utilization.

Description

Technical Field

[0001] The present invention relates to the technical field of microecological preparations, and in particular to a composite microecological preparation and its application in improving the production performance of ducks. Background Art

[0002] my country is a major waterfowl farming country. As the farming industry continues to develop towards scale, intensification, and greening, waterfowl farming is becoming increasingly refined and scientific. In recent years, due to various considerations, my country has successively introduced policies to restrict the use of antibiotics in the farming industry. This has created opportunities for the development of microecological green and pollution-free feed additives.

[0003] Probiotics are microorganisms that parasitize humans and livestock, improving the host's microecological balance and exerting beneficial effects. Probiotics regulate the balance of the gastrointestinal flora, prevent gastrointestinal diseases, antagonize pathogens, and enhance the immunity and resistance of livestock and poultry. They also inhibit the growth of various pathogens, demonstrating their beneficial effects. Probiotic metabolism produces organic acids such as lactic acid, acetic acid, and butyric acid. Accumulating these acids significantly lowers the pH of the environment, thereby inhibiting the growth and reproduction of putrefactive and pathogenic bacteria. Probiotics compete with pathogens for adhesion sites and nutrients, and secrete antibacterial substances, inhibiting their colonization, accelerating their excretion, and maintaining a balanced intestinal microbial flora. Furthermore, probiotics can improve protein digestibility, promote the digestion and absorption of lipids, enhance the utilization of calcium and phosphorus, and promote the absorption of vitamin D.

[0004] Therefore, those skilled in the art hope to develop new microecological preparations to reduce the use of antibiotics, improve the growth performance of waterfowl, and increase the economic benefits of waterfowl farming. Summary of the Invention

[0005] The purpose of the present invention is to provide a composite microecological preparation and its application in improving the production performance of ducks. The microbial preparation is beneficial to reducing the mortality rate, lowering the feed-to-meat ratio, and improving the egg production rate, and can replace or reduce the use of antibiotics in feed.

[0006] To this end, the present invention provides a composite microecological preparation, which includes, by weight, 25 to 35 parts of Clostridium butyricum powder, 10 to 15 parts of Bacillus coagulans powder, 10 to 15 parts of Enterococcus faecalis powder, 5 to 10 parts of yeast cell walls, 0.05 to 0.1 parts of antimicrobial peptides, and 5 to 10 parts of auxiliary materials.

[0007] In some embodiments, the effective viable count of Clostridium butyricum in the Clostridium butyricum powder is 1 to 4×10 10 CFU / g, the effective viable bacteria count of Bacillus coagulans in the Bacillus coagulans powder is 1~2×10 10CFU / g, the effective viable bacteria count of Enterococcus faecalis in the Enterococcus faecalis powder is 5~10×10 10 CFU / g.

[0008] In some embodiments, the strain of Clostridium butyricum is CICC No. 23847, the strain of Bacillus coagulans is CICC No. 21736, and the strain of Enterococcus faecalis is CCTCC NO: M 2022020.

[0009] In some embodiments, the yeast cell wall is a baker's yeast cell wall containing 25% to 30% β-glucan.

[0010] In some embodiments, the auxiliary material is porous starch.

[0011] In some embodiments, the amino acid sequence of the antimicrobial peptide is GEKLKKIGQKIKNFFQKLIKIAAKVGSNLL (SEQ ID NO: 1).

[0012] The second aspect of the present invention provides a method for preparing the composite probiotic preparation, which comprises: weighing each raw material by weight, uniformly mixing the Clostridium butyricum powder, the Bacillus coagulans powder, the Enterococcus faecalis powder, the antimicrobial peptide, the yeast cell wall and the auxiliary materials, adding deionized water, and stirring to obtain a mixed slurry; and sequentially subjecting the mixed slurry to low-temperature granulation, spheronization, and low-temperature vacuum drying to obtain the composite probiotic preparation.

[0013] In some embodiments, the moisture content of the mixed slurry is 16% to 20%, for example, about 16%, 17%, 18%, 19%, 20%, etc.

[0014] In some embodiments, the temperature of the low-temperature granulation is 25°C-42°C.

[0015] In some embodiments, the low-temperature vacuum drying conditions are: drying temperature of 25° C.-42° C., working pressure ≤300 mm water column, and drying time of 20 min-50 min.

[0016] The third aspect of the present invention provides the use of the composite probiotic preparation in preparing feed.

[0017] In some embodiments, the feed is waterfowl feed.

[0018] In some embodiments, the addition amount of the composite probiotic preparation in the feed is 1-2‰ (w / w).

[0019] Compared with the prior art, the technical solution of the present invention has at least the following improvements:

[0020] (1) The strains selected for the composite microecological preparation provided by the present invention have been screened through multiple tests, and the three bacteria have a synergistic effect. Among them, Clostridium butyricum can regulate the balance of animal intestinal flora, promote the growth of beneficial bacteria in the animal intestinal tract, antagonize and inhibit the proliferation of harmful bacteria in the intestine, increase the activity of digestive enzymes and lysozymes, and repair intestinal mucosa; Bacillus coagulans has a high degree of adhesion to the intestinal wall, supports good digestive function, and can quickly establish a dominant probiotic community in the intestine; Enterococcus faecalis can promote the establishment of a probiotic community in the intestine, which helps reduce the rate of diarrhea.

[0021] (2) The raw materials used in the present invention are all healthy and beneficial, with no side effects. Yeast cell walls firmly adsorb mycotoxins in feed through intermolecular forces such as hydrogen bonds and van der Waals forces, preventing toxins from being absorbed by the intestines. Antimicrobial peptides have strong antibiotic activity against Gram-positive bacteria, Gram-negative bacteria, and fungi, and have no side effects such as hemolysis.

[0022] (3) The composite microecological preparation of the present invention can be used for waterfowl (such as ducks). It has the advantages of stable properties, can be mixed with feed for feeding, and is easy to use. It can reduce or replace the use of antibiotics in feed, and has the effects of improving the ecological balance of the intestinal flora of ducks, enhancing immune function, promoting duck growth, and improving feed utilization. DETAILED DESCRIPTION

[0023] The exemplary embodiments of the present disclosure will be described in more detail below. It should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. Instead, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0024] Example 1

[0025] This example provides fermentation methods for Clostridium butyricum, Bacillus coagulans, and Enterococcus faecalis, respectively, to prepare fermentation broth and bacterial powder of each bacterial species, which are used in the subsequent preparation of a composite microecological preparation.

[0026] 1. Fermentation of Clostridium butyricum

[0027] (1) Clostridium butyricum (CICC No. 23847) was inoculated into a seed culture medium (2 g yeast powder, 15 g beef extract, 10 g tryptone, 5 g glucose, 1 g soluble starch, 5 g sodium chloride, 2 g sodium acetate trihydrate, 0.5 g L-cysteine ​​hydrochloride, distilled water to 1000 mL, pH 7.0), and cultured under anaerobic conditions at 37°C for 12 h to obtain a first-level seed solution of Clostridium butyricum.

[0028] (2) inoculating the first-level seed liquid of Clostridium butyricum into a seed culture medium (the same as the seed culture medium in step (1)), and culturing under anaerobic conditions at 37°C for 12 hours to obtain the second-level seed liquid of Clostridium butyricum;

[0029] (3) The secondary seed liquid of Clostridium butyricum was inoculated into a fermentation tank. The fermentation medium was as follows: 30 g soluble starch, 15 g yeast powder, 20 g ammonium sulfate, 10 g sodium chloride, 10 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 0.2 g manganese sulfate, 2 g calcium carbonate, and distilled water to 1000 mL. The pH value was 7.0. The fermentation was carried out under anaerobic conditions at 37 °C for 18 h to prepare Clostridium butyricum fermentation liquid. After washing with distilled water, centrifugation, and low-temperature drying, Clostridium butyricum powder was obtained. The effective viable count of Clostridium butyricum was about 2.5 × 10 10 CFU / g.

[0030] 2. Fermentation of Bacillus coagulans

[0031] (1) Inoculate Bacillus coagulans (CICC No. 21736) into a slant culture medium (5 g beef extract, 15 g peptone, 5 g yeast powder, 1 g sodium chloride, 5 g glucose, 15 g agar, distilled water to 1000 mL, pH 7.0) and culture at 37°C under aerobic conditions for 12 h.

[0032] (2) The slant-cultured Bacillus coagulans colony was inoculated into a seed culture medium (15 g of peptone, 2 g of beef extract, 1 g of sodium chloride, and distilled water to 1000 mL, pH 7.0), and cultured at 37°C under aerobic conditions for 12 h to prepare a Bacillus coagulans seed solution;

[0033] (3) The Bacillus coagulans seed liquid was inoculated into a fermentation tank. The fermentation medium was as follows: 5 g corn flour, 5 g glucose, 30 g soybean meal, 0.5 g fish meal, 10 g calcium carbonate, 0.1 g ammonium sulfate, 0.02 g potassium dihydrogen phosphate, 0.02 g magnesium sulfate, 0.02 g manganese sulfate, and distilled water to 1000 mL. The pH value was 7.0. The fermentation was carried out at 37 ° C under aerobic conditions for 18 h to prepare the Bacillus coagulans fermentation liquid. After washing with distilled water, centrifugation, and low-temperature drying, the Bacillus coagulans powder was obtained. The effective viable cell count of Bacillus coagulans was about 1×10 10 CFU / g.

[0034] 3. Enterococcus faecalis

[0035] (1) Enterococcus faecalis (CCTCC NO: M 2022020) was inoculated into MRS solid slant medium (10 g peptone, 10 g beef extract, 5 g yeast powder, 20 g glucose, 1 g Tween-80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g diammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 6 g calcium carbonate, 16 g agar, distilled water to 1000 mL, adjusted to pH 7.0), and cultured at 38°C under aerobic conditions for 12 h;

[0036] (2) The slant cultured E. faecalis was inoculated into a seed culture medium (MRS liquid culture medium) and cultured at 37°C under aerobic conditions for 12 h to prepare an E. faecalis seed solution;

[0037] (3) The Enterococcus faecalis seed liquid obtained in step (2) was inoculated into a fermentation tank, and the fermentation medium was as follows: 15 g of peptone, 10 g of glucose, 5 g of yeast powder, 5 g of sodium acetate, 4 g of diammonium citrate, 1 g of Tween80, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 6 g of calcium carbonate, and distilled water was added to 1000 mL, and the pH value was adjusted to 7.0; the culture was aerobic at 37°C for 18 h to prepare the Enterococcus faecalis fermentation liquid, which was washed with distilled water, centrifuged, and dried at low temperature to obtain the Enterococcus faecalis powder. The effective viable count of Enterococcus faecalis was about 8×10 10 CFU / g.

[0038] Example 2

[0039] This example provides an antimicrobial peptide, the sequence of which is shown in SEQ ID NO: 1. This antimicrobial peptide is derived by fusion recombination of the cathelin-related antimicrobial peptide CRMAP (SEQ ID NO: 2, GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ) with the CPF-ST3 antimicrobial peptide (SEQ ID NO: 3, GLLGPLLKIAAKVGSNLL). During the design of the fusion antimicrobial peptide, the core sequence of CRMAP, representing positions 11-28 of SEQ ID NO: 2, was selected for its antimicrobial activity. For CPF-ST3, the key residues K8 and C-terminal amino acids, representing positions 7-18 of SEQ ID NO: 3, were retained, and the L7I mutation was used to improve steric hindrance after attachment to CRMAP. The resulting antimicrobial peptide possesses an α-helical structure, enhancing its antimicrobial activity without increasing its peptide length. It exhibits strong antibiotic activity against Gram-positive and Gram-negative bacteria, as well as fungi, without side effects such as hemolysis. We commissioned GenScript Biotech Co., Ltd. to prepare an antimicrobial peptide (SEQ ID NO: 1) with a purity of over 95% through existing solid-phase chemical synthesis methods, which was then used in the subsequent preparation of complex microecological preparations.

[0040] Table 1 Minimum inhibitory concentration of antimicrobial peptides

[0041]

[0042] Example 3

[0043] This embodiment provides a composite microecological preparation, which is prepared according to the following method:

[0044] (1) Yeast cell walls (containing 30% β-glucan, purchased from Wuhan Huaxiang Kejie Biotechnology Co., Ltd.) were crushed and passed through an 80-mesh sieve. The following raw materials were weighed and mixed uniformly by weight to obtain the first material: 25 parts of Clostridium butyricum powder prepared in Example 1, 10 parts of Bacillus coagulans powder, 15 parts of Enterococcus faecalis powder, 0.05 parts of the antimicrobial peptide prepared in Example 2, 5 parts of sieved yeast cell walls, and 8 parts of porous starch.

[0045] (2) adding deionized water to the first material obtained in step (1), and stirring to obtain a mixed slurry, wherein the water content of the mixed slurry is about 17%;

[0046] (3) The mixed slurry obtained in step (2) is subjected to low-temperature granulation, spheronization and low-temperature vacuum drying in sequence to prepare a composite microecological preparation; wherein the temperature for low-temperature granulation is 30°C, the temperature for low-temperature vacuum drying is 30°C, the working pressure is ≤300 mm water column, and the drying time is 20 min.

[0047] Example 4

[0048] This example provides a composite probiotic preparation, which was prepared by the same method as in Example 3, except for the amounts of the following raw materials: 30 parts of Clostridium butyricum powder, 12 parts of Bacillus coagulans powder, 10 parts of Enterococcus faecalis powder, 0.06 parts of antimicrobial peptide, 8 parts of yeast cell walls, and 5 parts of porous starch were weighed in parts by weight.

[0049] Example 5

[0050] This example provides a composite probiotic preparation, which was prepared by the same method as in Example 3, except for the amounts of the following raw materials: 35 parts of Clostridium butyricum powder, 15 parts of Bacillus coagulans powder, 10 parts of Enterococcus faecalis powder, 0.08 parts of antimicrobial peptide, 10 parts of yeast cell walls, and 10 parts of porous starch were weighed in parts by weight.

[0051] Comparative Example 1

[0052] This comparative example provides a composite probiotic preparation, which was prepared by the same method as Example 3, except for the amounts of the following raw materials: 35 parts of Clostridium butyricum powder, 20 parts of Enterococcus faecalis powder, 0.05 parts of antimicrobial peptide, 5 parts of yeast cell walls, and 8 parts of porous starch were weighed in parts by weight.

[0053] Comparative Example 2

[0054] This comparative example provides a composite probiotic preparation, which was prepared by the same method as Example 3, except for the amounts of the following raw materials: 25 parts of Clostridium butyricum powder, 10 parts of Bacillus coagulans powder, 15 parts of Enterococcus faecalis powder, 5 parts of yeast cell walls, and 8 parts of porous starch were weighed in parts by weight.

[0055] Experimental Example 1

[0056] The experiment involved 6,000 one-day-old Cherry Valley ducklings. Using a single-factor design, the ducks were randomly divided into six groups of 1,000 ducks each. Each group received a Zhonghui broiler duck feed as the base diet. The control group received no additional compound probiotics, while the remaining five groups received the compound probiotics from Examples 3-5 and Comparative Examples 1-2, respectively, at a dosage of 2‰ (i.e., 2 kg / ton of base diet).

[0057] The experimental ducks were reared on thick ground bedding and managed according to standard procedures, with free access to feed and water. The growth of the ducks was observed for 42 days. Growth performance indicators were measured by recording daily feed consumption, observing growth, morbidity, and mortality, and recording mortality. The ducks were weighed regularly, and their average daily weight gain, average daily feed intake, and average feed-to-weight ratio were calculated. At the end of the 42-day rearing period, the production performance indicators for each group are shown in Table 2.

[0058] Table 2 Production performance indicators of each group

[0059]

[0060] Experimental Example 2

[0061] The experiment selected 3,000 280-day-old Shaoxing white ducks (laying hens) and randomly divided them into 6 groups of 500 ducks each. They used Nanchang Haida laying duck feed as the basic diet. The control group did not add any additional compound probiotics, while the other 5 groups added the compound probiotics of Examples 3 to 5 and Comparative Examples 1 to 2, respectively, at an addition rate of 1‰ (i.e., 1 kg / ton of basic diet). The ducks were managed and raised in a conventional manner, and their growth was observed. The breeding experiment lasted for 42 days. Egg production, egg weight, duck morbidity and mortality, etc. were recorded daily. At the end of the 42-day breeding period, the egg production rate and mortality rate of each group are shown in Table 3.

[0062] Table 3 Egg production rate and mortality rate of each group

[0063]

[0064] The foregoing description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be readily conceived by a person skilled in the art within the technical scope disclosed herein are intended to be encompassed within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A composite microecological preparation, characterized in that: The invention comprises, by weight, 25-35 parts of Clostridium butyricum powder, 10-15 parts of Bacillus coagulans powder, 10-15 parts of Enterococcus faecalis powder, 5-10 parts of yeast cell walls, 0.05-0.1 parts of antimicrobial peptide, and 5-10 parts of auxiliary materials; the amino acid sequence of the antimicrobial peptide is SEQ ID NO: 1; the strain of Clostridium butyricum is CICC No. 23847, the strain of Bacillus coagulans is CICC No. 21736, and the strain of Enterococcus faecalis is CCTCC NO: M2022020.

2. The complex microecological preparation according to claim 1, characterized in that The effective viable bacteria count of the Clostridium butyricum powder is 1 to 4×10 10 CFU / g, the effective viable bacteria count of the Bacillus coagulans powder is 1~2×10 10 CFU / g, the effective viable count of the Enterococcus faecalis powder is 5~10×10 10 CFU / g.

3. The complex probiotic preparation according to claim 1, wherein The yeast cell wall is a baker's yeast cell wall containing 25% to 30% beta-glucan.

4. The complex probiotic preparation according to claim 1, wherein The auxiliary material is porous starch.

5. The method for preparing the composite probiotic preparation according to any one of claims 1 to 4, characterized in that: include: The raw materials are weighed in parts by weight, and the Clostridium butyricum powder, the Bacillus coagulans powder, the Enterococcus faecalis powder, the antimicrobial peptide, the yeast cell wall and the auxiliary materials are uniformly mixed, deionized water is added, and the mixture is stirred to obtain a mixed slurry; the mixed slurry is sequentially subjected to low-temperature granulation, spheronization, and low-temperature vacuum drying to obtain the composite microecological preparation.

6. The preparation method according to claim 5, wherein The temperature of the low-temperature granulation is 25°C-42°C.

7. The preparation method according to claim 5, wherein The conditions for the low-temperature vacuum drying are: drying temperature of 25° C.-42° C., working pressure ≤300 mm water column, and drying time of 20 min-50 min.

8. Use of the composite probiotic preparation according to any one of claims 1 to 4 in preparing feed; the feed is waterfowl feed.

9. The use according to claim 8, characterized in that In the feed, the added amount of the composite microecological preparation is 1-2‰.

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