Compound microbial inoculant for fermenting chicken compound feed and application thereof

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a compound microbial agent for fermenting chicken compound feed and application thereof.The strains in the compound microbial agent include bacillus subtilis J-4, bacillus subtilis TU-1, bacillus methylotrophicus SD48 and bacillus subtilis J530.The compound microbial agent of the present application can produce various bioactive substances in the process of fermenting chicken compound feed, improve the content of bioactive substances in the feed, enhance the immunity of chickens, and thus can replace antibiotics.Furthermore, the nutritional ingredients and active substances contained in the chicken compound feed fermented by the compound microbial agent can improve the absorption and utilization of the feed by chickens, improve the feed conversion rate, reduce the feed cost, and the obtained chicken compound feed also has good palatability.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a compound microbial agent for fermenting chicken feed and its application. Background Technology

[0002] High-density intensive farming of egg-laying and broiler chickens easily leads to the spread of infectious animal diseases, causing animal deaths and resulting in huge economic losses. Therefore, antibiotics are often widely used in fermented chicken feed for high-density intensive farming of egg-laying and broiler chickens. However, the extensive use of antibiotics can lead to problems such as reduced disease resistance in egg-laying and broiler chickens, increased drug resistance in pathogens, and excessive drug residues in meat and egg products.

[0003] The Ministry of Agriculture and Rural Affairs issued Announcement No. 194, requiring feed production enterprises to cease production of commercial feed containing antibiotic-containing feed additives (excluding traditional Chinese medicine) after July 1, 2020. The ban on antibiotics in feed, the reduction or restriction of antibiotics in livestock farming, and the elimination of antibiotics in products have become an inevitable trend in the development of the livestock industry. However, implementing a ban on antibiotics in feed may lead to a decline in livestock production levels and a significant increase in farming costs.

[0004] Therefore, developing antibiotic alternatives and reasonable alternatives has become the key to the healthy, sustainable, and green development of the aquaculture industry. Summary of the Invention

[0005] To address the above-mentioned technical problems, this invention provides a compound microbial agent for fermented chicken feed and its application. When used in fermented chicken feed, this compound microbial agent produces various bioactive substances during the fermentation process that enhance chicken immunity. It can strengthen the chicken's immune system, regulate intestinal flora, promote intestinal health, and reduce the occurrence of diseases, thus replacing antibiotics.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a compound microbial agent for fermented chicken feed, wherein the strains in the compound microbial agent include Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus subtilis SD48 and Bacillus subtilis J530.

[0008] The compound microbial agent of this invention can produce a variety of bioactive substances during the fermentation of chicken feed, increasing the content of bioactive substances in the feed and enhancing the immunity of chickens, thus replacing antibiotics. Furthermore, the nutrients and bioactive substances contained in the chicken feed fermented by this compound microbial agent can improve the absorption and utilization of feed by chickens, increase feed conversion efficiency, reduce feed costs, and the resulting chicken feed also has good palatability.

[0009] The Bacillus subtilis J-4 strain in this invention was screened from the feces of healthy broiler chickens and preserved in the laboratory of the Department of Pharmaceutical Engineering, College of Life Sciences, Hebei Agricultural University. This strain has been disclosed in "Optimization of Fermentation Conditions for the Production of Antibacterial Substances by Bacillus subtilis J-4 Strain" (Zhai Yujie et al., Henan Agricultural Sciences, 2017, 46(2):131-135), and the applicant promises to release this strain to the public within twenty years from the date of this patent application. Bacillus subtilis J-4 can produce peptide antibacterial substances, which can be precipitated by 60% saturated ammonium sulfate and degraded by pepsin and trypsin, exhibiting excellent anti-Escherichia coli activity. Adding Bacillus subtilis J-4 to feed can prevent the growth of Escherichia coli in the chicken's intestines, reduce the occurrence of diseases, increase the average daily weight gain of chickens, reduce the feed conversion ratio, and improve feed conversion efficiency.

[0010] The Bacillus subtilis TU-1 strain in this invention was screened from the cecum of healthy rabbits and preserved in the laboratory of the Department of Pharmaceutical Engineering, College of Life Sciences, Hebei Agricultural University. This strain has been disclosed in "The Effects of TU-1 Bacterial Agent as an Alternative to Antibiotics on the Activity of Digestive Enzymes, Cecal Microbiota and Growth Performance of Rabbits" (Su Ke et al., Henan Agricultural Sciences, 2018, 47(6):134-138). The applicant promises to release this strain to the public within twenty years from the date of this patent application. After entering the chicken intestine, Bacillus subtilis TU-1 grows and multiplies on its own, promoting the production of various digestive enzymes such as protease, amylase, cellulase and lipase in the intestine, thereby promoting the digestion and absorption of nutrients in the feed by chickens. It also inhibits the growth of pathogens through competitive attachment sites, biological oxygen depletion, nutrient competition, and the production of antibacterial substances, thus maintaining the balance of the intestinal microecology, thereby preventing and reducing the diarrhea rate and improving the production performance of chickens.

[0011] The methyltrophic Bacillus SD48 strain in this invention was obtained by screening using acidic skim milk medium as the limiting medium and is preserved in the laboratory of the Department of Pharmaceutical Engineering, College of Life Sciences, Hebei Agricultural University. This strain has been disclosed in "Optimization of conditions for fermentation of soybean meal to produce soybean peptides by acid-resistant protease-producing Bacillus SD4" (Zhou Xinghua et al., Henan Agricultural Sciences, 2019, 48(8):140-146), and the applicant promises to release this strain to the public within twenty years from the date of this patent application. Methyltrophic Bacillus SD48 has high acid resistance and high acid protease activity. Using this methyltrophic Bacillus SD48 to ferment soybean meal feed can significantly reduce the pH value of the feed, increase the content of acid-soluble protein and antioxidant activity, and can almost completely degrade β-conglycinin and glycinin, increase the content of acid-soluble protein, greatly improve the nutritional value of soybean meal feed, promote the absorption of protein by chickens, and reduce protein residue in feces.

[0012] The Bacillus subtilis J530 strain in this invention was screened from the chicken intestine and is disclosed in patent application number CN110643550A entitled "A Bacillus subtilis J530 strain producing urease inhibitor and its application," which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO:18603. When this Bacillus subtilis J530 is used to ferment chicken compound feed, it can produce short-chain organic acids such as formic acid, acetic acid, propionic acid, and lactic acid, as well as ethanol, and also produces substances that inhibit urease activity. Organic acids and alcohols can lower the pH of fermented chicken feed to below 4.5, while also enhancing its acidity and aroma, thus improving palatability. They can also effectively inhibit mold growth and extend the shelf life of fermented chicken feed. When 15% wt of fermented chicken feed obtained from Bacillus subtilis J530 is added to the chicken diet, the inhibition rate of urease activity in chicken feces can reach 98.37%, which can prevent the conversion of uric acid in chicken feces into ammonia, thereby reducing ammonia emissions from feces and significantly reducing the content of ammonia and other odorous gases in the chicken house.

[0013] Preferably, the ratio of viable bacteria of Bacillus subtilis J-4, Bacillus subtilis TU-1, Bacillus licheniformis SD48 and Bacillus subtilis J530 is (1-2):(1-2):(1-2):(1-2).

[0014] Fermenting chicken feed with the above-mentioned compound microbial agent can produce more peptides, antimicrobial proteins, acidic proteases, amylases, cellulases, organic acids, alcohols, substances that inhibit urease activity, and other bioactive substances, thus enhancing their efficacy.

[0015] Preferably, the compound microbial agent is composed of single-strain agents of Bacillus subtilis J-4, Bacillus subtilis TU-1, Bacillus methyltrophicus SD48, and Bacillus subtilis J530.

[0016] Preferably, the viable counts of Bacillus subtilis J-4, Bacillus subtilis TU-1, Bacillus methyltrophicus SD48, and Bacillus subtilis J530 in the single-strain agent are all (1.0-1.5) × 10⁻¹⁰. 10 CFU / g; the carrier is any one or more of calcium carbonate, starch, or fine wheat bran.

[0017] The carriers of the single-strain agent in this invention, namely calcium carbonate, starch, and fine wheat bran, are feed-grade powdered industrial or agricultural products with strong dispersibility in water. These carriers provide nutrients for the growth and reproduction of Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus subtilis SD48, and Bacillus subtilis J530.

[0018] Secondly, the present invention also provides the application of the above-mentioned compound microbial agent in the preparation of fermented chicken compound feed, including: inoculating the compound microbial agent into chicken compound feed for anaerobic fermentation, thereby obtaining fermented chicken compound feed.

[0019] The fermented chicken feed obtained using the compound microbial agent of this invention can produce bioactive substances such as peptides, antimicrobial proteins, acidic proteases, amylases, cellulases, organic acids and alcohols, and substances that inhibit urease activity. These bioactive substances can promote the absorption of nutrients by chickens, prevent the growth of pathogenic bacteria in the chicken's intestines, maintain the balance of the intestinal microecology, prevent and reduce the rate of diarrhea and reduce the occurrence of diseases, thus replacing antibiotics. At the same time, it can also improve feed conversion efficiency, reduce feed conversion ratio, and improve the production performance of chickens. It can reduce the residual protein in feces, reduce the ammonia emission in feces, and significantly reduce the content of ammonia and other odorous gases in the chicken house, thereby reducing stress, reducing respiratory diseases in chicken flocks, and improving health. It can also improve the palatability of fermented chicken feed.

[0020] Adding the fermented chicken feed at a rate of 5.0wt%-30wt% to the chicken diet and allowing the chickens to consume it freely can significantly increase the levels of immune factors such as IgA, IgG, and IgM, as well as interferon IFN-r in the serum of laying hens, thereby improving the humoral immunity and disease resistance of chickens.

[0021] Preferably, the fermented chicken feed includes any one or more of corn flour, soybean meal, wheat bran, rice bran, or ammonium sulfate.

[0022] Preferably, the fermented chicken feed comprises at least the following components in parts by weight: 50-70 parts corn flour, 15-25 parts soybean meal, 5-15 parts wheat bran, 2-6 parts rice bran, and 0.5-3 parts ammonium sulfate.

[0023] The chicken compound feed using the above components and proportions can provide a solid culture medium for the compound microbial agent of the present invention, which is conducive to its large-scale growth and reproduction, thereby generating more bioactive substances during the fermentation of chicken compound feed.

[0024] Preferably, before carrying out the anaerobic fermentation, water equivalent to 40%-50% of the mass of the fermented chicken feed is added.

[0025] Chicken feed with the above-mentioned amount of water is more suitable as a solid culture medium for compound microbial agents. In practice, the compound microbial agent can be dispersed in water and then added to the chicken feed, completing the addition of water and inoculation of the bacterial strains at the same time, followed by anaerobic fermentation.

[0026] Preferably, the anaerobic fermentation conditions are 20-35℃ for 7-14 days.

[0027] The temperature range and fermentation time described above in this invention are suitable for the growth and reproduction of the compound microbial agent.

[0028] Preferably, the total inoculum amount of the compound microbial agent is (1.0-10)×10⁻⁶. 7 CFU / g. Attached Figure Description

[0029] Figure 1 This experiment uses the plate growth confrontation method to test the four strains of the compound microbial agent of this invention.

[0030] Figure 2 This experiment uses the plate growth confrontation method to test the four strains of the compound microbial agent of this invention.

[0031] Figure 3 This experiment uses the plate growth confrontation method to test the four strains of the compound microbial agent of this invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0033] Fermented feed technology mainly includes microbial fermented feed technology and microbial-enzyme co-fermentation technology.

[0034] Microbial fermentation feed technology refers to using agricultural and industrial by-products with low nutritional value, low price, and poor palatability, such as crop straw, cottonseed meal, rapeseed meal, wheat bran, distiller's grains, and potato residue, as substrates. Under artificially controlled conditions, microorganisms ferment these materials to degrade some macromolecular nutrients such as cellulose, protein, and fat into easily absorbed sugars, soluble peptides, and organic acids, forming bio-feeds that are palatable, nutritious, and contain a large number of beneficial microorganisms.

[0035] In existing technologies, the fermentation effect of using a single strain of microorganisms for microbial fermentation of feed is poor, and the yield of the target product is low.

[0036] This invention utilizes a compound microbial agent containing Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus SD48, and Bacillus subtilis J530 to ferment chicken compound feed. This process enables more complete degradation of nutrients in the feed, improves the conversion and utilization rate of specific substances in the feed, reduces the content of anti-nutritional factors in the feed, increases the content of specific products, improves the flavor and palatability of the feed, and enhances the utilization value of the feed.

[0037] The present invention will be further described below with reference to specific embodiments.

[0038] Example 1

[0039] A compound microbial agent for fermented chicken feed is formulated by blending four single-strain agents—Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus subtilis SD48, and Bacillus subtilis J530—in a 1:1:1:1 live bacteria ratio. Each single-strain agent consists of live bacteria and a carrier, and the live bacteria count of each of the four agents in each single-strain agent is 1.0 × 10⁻⁶. 10 CFU / g; the carrier is fine wheat bran.

[0040] The preparation method of single-strain agent is as follows:

[0041] Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus subtilis SD48, and Bacillus subtilis J530 strains were cultured on NA medium for 24 h. The cultured strains were then inoculated into NB medium and cultured on a shaker at 35–37 °C and 200 rpm for 48 h. The resulting bacterial sludge was obtained by centrifugation and spray-dried to obtain high concentrations of Bacillus subtilis J-4 powder, Bacillus subtilis TU-1 powder, and methyltrophic Bacillus subtilis SD48. Bacillus SD48 and Bacillus subtilis J530 bacterial powders were mixed with fine wheat bran to obtain Bacillus subtilis J-4, Bacillus subtilis TU-1, Bacillus SD48, and Bacillus subtilis J530 bacterial powders. 10 CFU / g).

[0042] Figure 1-3 This is an experiment using the plate growth confrontation method to test the four bacterial strains of the compound bacterial agent of this invention. From... Figure 1-3 It can be seen that there is no mutual inhibition among Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus SD48 and Bacillus subtilis J530.

[0043] Example 2

[0044] A compound microbial agent for fermented chicken feed is formulated by blending four single-strain agents—Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus subtilis SD48, and Bacillus subtilis J530—in a viable cell ratio of 2:1:2:1. Each single-strain agent consists of viable bacteria and a carrier, and the viable cell count of each of the four agents in each single-strain agent is 1.3 × 10⁻⁶. 10CFU / g; the carrier is light calcium carbonate.

[0045] The preparation method of the single-strain agent is the same as in Example 1.

[0046] Example 3

[0047] A compound microbial agent for fermented chicken feed is formulated by combining four single-strain agents—Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus SD48, and Bacillus subtilis J530—in a 2:2:2:2 live bacteria ratio. Each single-strain agent consists of live bacteria and a carrier, and the live bacteria count of Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus SD48, and Bacillus subtilis J530 in each single-strain agent is 1.5 × 10⁻⁶. 10 CFU / g; the carrier is light calcium carbonate.

[0048] The preparation method of the single-strain agent is the same as in Example 1.

[0049] Example 4

[0050] The application of the compound microbial agent of Example 1 in the preparation of fermented chicken compound feed includes at least the following steps:

[0051] Take 58 parts corn flour, 20 parts soybean meal, 15 parts wheat bran, 5 parts rice bran, 2 parts ammonium sulfate, and 40 parts water. Disperse the compound microbial agent from Example 1 into the added water, so that the total inoculum amount of the four types of bacteria is 2.0 × 10⁻⁶. 7 CFU / g. After mixing, pack into a 50L fermentation tank, compact and seal, and ferment for 14 days at 20-35℃.

[0052] The pH of the fermented chicken feed was determined to be 4.21. The fermented chicken feed contained 1.86 g / kg of peptides, exhibited an inhibitory activity against *E. coli* of 50.15 U / g, and had activities of 6.20 U / g, 190.64 U / g, 619.04 U / g, 365.15 U / g, and 350.63 U / g, respectively. The acid-soluble protein content / crude protein content was 38.25 wt%. The degradation rates of glycinin and β-conglycinin in soybean meal were 85.27% and 82.62%, respectively.

[0053] Example 5

[0054] The application of the compound microbial agent of Example 2 in the preparation of fermented chicken compound feed includes at least the following steps:

[0055] Take 64 parts corn flour, 20 parts soybean meal, 10 parts wheat bran, 4 parts rice bran, 2 parts ammonium sulfate, and 45 parts water. Disperse the compound microbial agent from Example 2 into the added water, so that the total inoculum amount of the four types of bacteria is 3.0 × 10⁻⁶. 7 CFU / g. After mixing, pack the mixture into fermented chicken feed bags, press and seal them, 25kg per bag; ferment at 20-25℃ for 10 days.

[0056] The pH of the fermented chicken feed was determined to be 4.17. The fermented chicken feed produced 1.97 g / kg of peptides, inhibited Aspergillus flavus activity by 160.18 U / g, and inhibited Escherichia coli activity by 68.32 U / g. The activities of amylase, protease, cellulase, lipase, and glucosidase in the fermented chicken feed reached 69.52 U / g, 185.14 U / g, 619.65 U / g, 225.89 U / g, and 352.18 U / g, respectively. The acid-soluble protein content / crude protein content was 39.17 wt%. The degradation rates of soy globulin and β-conglycinin in soybean meal were 82.87% and 83.58%, respectively.

[0057] Example 6

[0058] The application of the compound microbial agent of Example 3 in the preparation of fermented chicken compound feed includes at least the following steps:

[0059] Take 70 parts corn flour, 15 parts soybean meal, 5 parts wheat bran, 6 parts rice bran, 2 parts ammonium sulfate, and 50 parts water. Disperse the compound microbial agent from Example 2 into the added water, so that the total inoculum amount of the four types of bacteria is 3.0 × 10⁻⁶. 7 CFU / g. After mixing, pack the mixture into fermented chicken feed bags, press and seal them, 25kg per bag; ferment at 20-25℃ for 10 days.

[0060] The pH of the fermented chicken feed was determined to be 4.13. The fermented chicken feed produced 1.89 g / kg of peptides, inhibited Aspergillus flavus activity by 158.36 U / g, and inhibited Escherichia coli activity by 67.53 U / g. The activities of amylase, protease, cellulase, lipase, and glucosidase in the fermented chicken feed reached 70.35 U / g, 186.83 U / g, 605.17 U / g, 234.21 U / g, and 361.73 U / g, respectively. The acid-soluble protein content / crude protein content was 32.68 wt%. The degradation rates of soy globulin and β-conglycinin in soybean meal were 83.27% and 82.67%, respectively.

[0061] Example of effect 1

[0062] Experimental subjects: 400 laying hens of good health and similar weight at 35 weeks of age were selected and divided into 4 groups of 100 each.

[0063] Experimental group: fed fermented chicken feed and diet as described in Examples 4-6. Control group: fed only normal diet.

[0064] Test method:

[0065] Experimental group: 5.0wt%-30wt% of the fermented chicken compound feed of Examples 4-6 was mixed with the daily diet and allowed to be fed freely by laying hens for 7 consecutive days.

[0066] Control group: The chickens were fed a normal diet and allowed to eat freely for 7 consecutive days.

[0067] Table 1 Results of the feeding experiment

[0068]

[0069] Table 1 shows that the feeding trial of laying hens indicated that replacing 15 wt% of the basal diet with fermented chicken compound feed increased the egg production rate by 6.34% (P<0.05), reduced the average first-time laying rate by 45.35% (P<0.05), improved egg appearance, increased the average Haugh unit by 24.54% (P<0.05), and reduced nitrogen excretion by 20.10% (P<0.05). This improved the laying hen's productivity, extended the laying period, and reduced mortality. It also promoted the absorption of nutrients and improved feed utilization. Furthermore, it reduced ammonia concentration in the chicken house by 61.83% (P<0.01), thereby reducing respiratory diseases, stress, and overall health in the flock.

[0070] Example 2

[0071] Serum immune proteins of laying hens in the experimental and control groups were measured, and the results are shown in Table 2.

[0072] Table 2. Effects of feeding fermented chicken feed on serum immunoglobulins in laying hens

[0073]

[0074] Note: Different lowercase letters in the superscript of data in the same column indicate significant differences (P<0.05), while the same letter indicates no significant difference.

[0075] As shown in Table 2, replacing 15% of the basal diet with fermented chicken feed can significantly increase the levels of immune factors such as IgA, IgG, and IgM, as well as interferon IFN-r in the serum of laying hens (P < 0.05), thereby improving the humoral immunity and antiviral capacity of laying hens, indicating that it can enhance the immunity of laying hens.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound microbial agent for fermenting chicken feed, characterized in that, The compound microbial agent is composed of single-strain agents of Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus SD48 and Bacillus subtilis J530, and the ratio of the number of live bacteria of Bacillus subtilis J-4, Bacillus subtilis TU-1, Bacillus licheniformis SD48 and Bacillus subtilis J530 is (1-2):(1-2):(1-2):(1-2).

2. The compound microbial agent according to claim 1, characterized in that, The viable counts of Bacillus subtilis J-4, Bacillus subtilis TU-1, methyltrophic Bacillus subtilis SD48, and Bacillus subtilis J530 in the single-strain agent were all (1.0-1.5) × 10⁻¹⁰. 10 CFU / g; and / or The carrier of the single-strain agent is any one or more of calcium carbonate, starch, or fine wheat bran.

3. The application of the compound microbial agent according to claim 1 or 2 in fermented chicken feed, characterized in that, The compound microbial agent is inoculated into chicken compound feed for anaerobic fermentation to obtain fermented chicken compound feed, which includes: 50-70 parts corn flour, 15-25 parts soybean meal, 5-15 parts wheat bran, 2-6 parts rice bran, and 0.5-3 parts ammonium sulfate.

4. The application according to claim 3, characterized in that, Before carrying out the anaerobic fermentation, add water equivalent to 40%-50% of the mass of the chicken compound feed.

5. The application according to claim 3, characterized in that, The anaerobic fermentation conditions are 20-35°C for 7-14 days; the total inoculum size of the compound microbial agent is (1.0-10) × 10⁻⁶. 7 CFU / g.

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