Mildew-proof and bacteriostatic liquid biological mildew removal agent as well as preparation method and application thereof

Abstract

The invention provides a mildew-proof and bacteriostatic liquid biological mildew removal agent as well as a preparation method and application thereof, and belongs to the technical field of biological mildew removers for feeding. The preservation number of the bifidobacterium G-1 provided by the invention is CCTCC (China Center For Type Culture Collection) NO: M2024350. The bifidobacterium G-1 provided by the invention can inhibit the growth of mould, has the effect of protecting gastrointestinal tracts of livestock and poultry, and can be applied to the preparation of a biological mildew preventive for feed. The microbial preparation provided by the invention can secrete a plurality of probiotics, supplement intestinal beneficial flora, protect intestinal flora balance and maintain an excellent intestinal microenvironment. The composite biological mildew removing agent provided by the invention has the effects of inhibiting mildew and degrading mycotoxin, reduces the burden of small intestines, and reduces the damage and adverse influence of the mildew and the mycotoxin on livestock and poultry. The liquid preparation of the composite biological mildew removing agent provided by the invention can improve the slaughtering average weight of livestock and poultry and reduce the diarrhea rate and the feed conversion ratio.

Description

Technical Field

[0001] The invention belongs to the technical field of biological mildew inhibitors for feed, and in particular relates to a liquid biological mildew-removing agent for mildew prevention and bacteriostasis, and a preparation method and application thereof. Background Art

[0002] At present, the safety of livestock and poultry products has gradually attracted public attention. In today's environment where antibiotics are banned and restricted, feed, drinking water, environment, utensils and other aspects will affect the production performance of livestock and poultry. Pathogenic bacteria not only cause livestock and poultry to be infected with bacterial diseases, but also produce toxins in the body of livestock and poultry, seriously affecting the growth of livestock and poultry and even death. Feed raw materials and products are susceptible to mold contamination. Molds reproduce and produce mycotoxins under suitable conditions, which not only reduces the nutritional value of feed, changes the palatability of feed, and reduces the amount of animal feed intake, but also the mycotoxins produced will endanger the health of animals. In mild cases, it will cause animal growth stunting, reduced production performance and reduced disease resistance, and in severe cases, it will cause animal poisoning and death. From a deeper level, mycotoxins are hidden and accumulative. Once ingested by livestock and poultry, they will accumulate in different parts of the body, causing adverse symptoms. In severe cases, their by-products will affect human health. Therefore, it is imperative to seek green, environmentally friendly, reasonably priced and easy-to-use feed additives.

[0003] Organic acid is a kind of pollution-free and residue-free preparation. At present, organic acids mainly include citric acid, fumaric acid, malic acid, formic acid, acetic acid, propionic acid, lactic acid and their derivatives, which are basically a mixture of two or more on the market. Although they are effective, they will also inhibit some probiotics in the intestine and affect intestinal health. Most of the existing feed acidifiers in this field need to be used in the form of mixing. The mixing process is complicated and arduous. If it is not operated properly, it may cause discomfort to the operator; if it is not added properly, it will also destroy the effective ingredients in the feed and harm the digestive system of livestock and poultry. Therefore, it is necessary to find a suitable biological acidifier that can achieve the effect of adding an acidifier, have anti-mildew and antibacterial effects, and can also protect the gastrointestinal tract of livestock and poultry and improve production performance. This is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide a Bifidobacterium G-1 capable of inhibiting the growth of mold.

[0005] The second purpose of the present invention is to provide a microbial preparation that can secrete a variety of prebiotics, supplement beneficial intestinal flora, protect the balance of intestinal flora, and maintain an excellent intestinal microenvironment.

[0006] The third object of the present invention is to provide a composite biological mold removal agent, which has the functions of preventing and removing mold, resisting bacteria and protecting the gastrointestinal tract of livestock and poultry.

[0007] The fourth object of the present invention is to provide a composite biological mold removal agent liquid preparation that can be directly drunk. By using it in liquid form and drinking water, it can avoid the problem of easy corrosion of equipment and volatilization when using it in a mixing way, and better achieve the application effect of the preparation; supplying the liquid preparation of the present invention through drinking water can more effectively increase the feed intake during heat stress than through feed, while reducing the pH of drinking water and the buffering capacity of feed, which is beneficial to the crop and forestomach.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides a bifidobacterium G-1. The bifidobacterium G-1 is classified and named as Bifidobacterium sp. G-1, and is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M2024350.

[0010] The present invention also provides a microbial preparation, which comprises the above-mentioned Bifidobacterium G-1 and Bacillus Velez J-1, and the preservation number of the Bacillus Velez J-1 is CCTCC NO: M2021287.

[0011] Preferably, the viable count of Bifidobacterium G-1 is 1000×10 8 CFU / g or more, the viable count of the Velez Bacillus J-1 is 200×10 8 CFU / g or more, the weight ratio of Bifidobacterium G-1 powder to Bacillus Velez J-1 powder is 2-5:6-10.

[0012] The present invention also provides the use of the Bifidobacterium G-1 or the microbial preparation in the preparation of a biological mold removal agent.

[0013] The present invention also provides a composite biological mold removal agent, which comprises a mold inhibitor and the above-mentioned microbial preparation; the mold inhibitor comprises the following raw materials in parts by weight: 0.1 to 1 part of organic acid, 0.5 part of calcium formate, 0.05 to 0.2 part of taurine, and 10 to 30 parts of glucose oxidase.

[0014] Preferably, the organic acid comprises at least two of formic acid, acetic acid, lactic acid and propionic acid.

[0015] The present invention also provides a composite biological mold removal agent liquid preparation, the raw materials of the liquid preparation include the composite biological mold removal agent and water.

[0016] Preferably, the weight proportions of the raw materials in the liquid preparation are: 0.5 to 1 part of the mildew inhibitor, 800 to 1200 parts of water, and 0.9 to 2.5 parts of the microbial preparation.

[0017] Preferably, the pH of the liquid preparation is 3.5 to 5.0.

[0018] The present invention also provides a method for preparing the composite biological mold removal agent liquid preparation, comprising the following steps: mixing the mold inhibitor with water to obtain a mixture, and mixing the mixture with a microbial preparation to obtain the composite biological mold removal agent liquid preparation.

[0019] Beneficial effects of the present invention:

[0020] The Bifidobacterium G-1 provided by the present invention can inhibit the growth of mold, has the function of protecting the gastrointestinal tract of livestock and poultry, and can be applied to the preparation of a biological mold removal agent.

[0021] The microbial preparation provided by the present invention can secrete a variety of prebiotics, supplement beneficial intestinal flora, protect the balance of intestinal flora, and maintain an excellent intestinal microenvironment.

[0022] The composite biological mold removal agent and composite biological mold removal agent liquid preparation provided by the present invention can stimulate the secretion of taste buds and digestive enzymes in the digestive tract of animals, can indirectly inhibit gastric peristalsis, reduce gastric emptying speed, prolong the digestion time of protein in the stomach, and reduce the burden on the small intestine. It also has the function of inhibiting mold and degrading mycotoxins, reducing the damage and adverse effects of mold and its toxins on livestock and poultry.

[0023] The composite biological mold removal agent liquid preparation provided by the present invention has the effect of reducing the pH value of the gastrointestinal tract of livestock and poultry and increasing the activity of digestive enzymes; it can promote the reproduction of microorganisms in the gastrointestinal tract and enhance the body's anti-stress ability. The composite biological mold removal agent liquid preparation provided by the present invention can increase the average weight of livestock and poultry, reduce the diarrhea rate and feed-to-meat ratio, and promote the growth and marketing of livestock and poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the phylogenetic tree;

[0025] Figure 2 The left picture is the result of the control group, and the right picture is the result of the Example 12 group.

[0026] Collection Instructions

[0027] The Bifidobacterium G-1 of the present invention is preserved in the China Type Culture Collection Center, classified and named Bifidobacterium sp. G-1, preserved on February 29, 2024, at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M2024350.

[0028] The Bacillus velezensis J-1 of the present invention is deposited in the China Center for Type Culture Collection, classified and named Bacillus velezensis.J-1, the deposit time is March 29, 2021, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCCNO: M2021287 DETAILED DESCRIPTION

[0029] The invention provides a bifidobacterium G-1. The bifidobacterium G-1 is classified and named as Bifidobacterium sp. G-1, and is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M2024350.

[0030] The bifidobacterium G-1 provided by the present invention is isolated and screened from the roadside soil on the Jinlongsigou Mountain in Yingchengzi Street, Ganjingzi District, Dalian City, Liaoning Province. After DNA extraction, PCR amplification, connection and transformation, the BLAST program is used for comparison, a phylogenetic tree is constructed, and strain identification is performed. The results show that the bacteria isolated and screened by the present invention are bifidobacteria.

[0031] The present invention also provides a microbial preparation, which comprises the above-mentioned Bifidobacterium G-1 and Bacillus Velez J-1, and the preservation number of the Bacillus Velez J-1 is CCTCC NO: M2021287.

[0032] In the present invention, the viable count of Bifidobacterium G-1 is preferably 1000×10 8 CFU / g or more, the viable count of the Velez subtilis J-1 is preferably 200×10 8 CFU / g or more, the weight ratio of the Bifidobacterium G-1 powder to the Velez Bacillus J-1 powder is preferably 2-5:6-10, more preferably 5:10. In the present invention, the process of making Bifidobacterium G-1 into a powder is preferably to ferment and culture Bifidobacterium G-1 and centrifuge the bacterial mud to obtain a highly concentrated bacterial liquid, and then use soluble starch or glucose as a carrier to form a powder through microencapsulation. The process of making Velez Bacillus J-1 into a powder is the same as the process of making Bifidobacterium G-1 into a powder, which will not be repeated here.

[0033] The present invention also provides the use of the above-mentioned Bifidobacterium G-1 or the above-mentioned microbial preparation in the preparation of a biological mold removal agent. The Bifidobacterium G-1 and Bacillus Velez J-1 provided by the present invention can secrete a variety of prebiotics, supplement beneficial intestinal flora, protect the balance of intestinal flora, and maintain an excellent intestinal microenvironment.

[0034] The present invention also provides a composite biological mold removal agent, which comprises a mold inhibitor and the above-mentioned microbial preparation; the mold inhibitor comprises the following raw materials in parts by weight: 0.1 to 1 part of organic acid, 0.5 part of calcium formate, 0.05 to 0.2 part of taurine, and 10 to 30 parts of glucose oxidase.

[0035] In the composite biological mold remover product of the present invention, the mold inhibitor and the microbial preparation can be placed separately and then compounded when used. For example, the raw materials of the mold inhibitor can be mixed and recorded as a mold inhibitor package. In the present invention, the composite biological mold remover can be composed of a liquid package and a microbial preparation powder package, or it can be a compounded concentrated package, or it can be a separate diluted liquid package. In the present invention, the organic acid preferably includes at least two of formic acid, acetic acid, lactic acid and propionic acid. The present invention does not specifically limit the specific source of each raw material in the composite biological mold remover. In the present invention, the organic acid is a liquid, and calcium formate, taurine, and glucose oxidase are solid powders. In the biological mold remover, the liquid and the solid powder can be placed separately, or the liquid and the solid powder can be mixed into a mold inhibitor and placed. The enzyme activity of the glucose oxidase is preferably 10000U / g. In the present invention, the weight portion of the organic acid is preferably 0.2 to 0.8 parts, more preferably 0.3 to 0.6 parts; the weight portion of the taurine is preferably 0.1 to 0.18 parts; the weight portion of the glucose oxidase is preferably 15 to 25 parts, more preferably 18 to 20 parts. In the present invention, glucose oxidase can improve feed digestibility, promote digestion and absorption of nutrients, supplement the deficiency of endogenous enzymes nutritionally, improve feed palatability, fully release feed nutrients, and ensure their absorption and utilization rate.

[0036] The present invention also provides a composite biological mold removal agent liquid preparation, the raw materials of the liquid preparation include the composite biological mold removal agent and water.

[0037] The composite biological mold removal agent liquid preparation provided by the present invention can be drunk directly. In the present invention, the weight proportions of each raw material in the liquid preparation are preferably: 0.5 to 1 part of mold inhibitor, 800 to 1200 parts of water, and 0.9 to 2.5 parts of microbial preparation. The weight proportion of the mold inhibitor is more preferably 0.6 to 0.8 parts; the weight proportion of water is more preferably 900 to 1100 parts; and the weight proportion of the microbial preparation is more preferably 1.1 to 2.2 parts. In the present invention, the pH of the liquid preparation is preferably 3.5 to 5.0, and more preferably 4.0 to 4.5.

[0038] The present invention also provides a method for preparing the composite biological mold removal agent liquid preparation, comprising the following steps: mixing the mold inhibitor with water to obtain a mixture, and mixing the mixture with a microbial preparation to obtain the composite biological mold removal agent liquid preparation.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Example 1

[0043] Enrichment and Screening of Bifidobacterium G-1

[0044] From the roadside on the Jinlongsigou Mountain in Yingchengzi Street, Ganjingzi District, Dalian City, Liaoning Province, the upper, middle and lower layers of soil were taken and mixed, and immediately brought back to the laboratory. Take 10g of the mixed soil and add it to 100mL of sterilized saline. Mix evenly for 30 minutes under anaerobic conditions, take 200μL of the mixture after gradual dilution and spread it on the MRS solid plate, culture anaerobically at 37℃, wait for enriched colonies to appear, pick a single colony and inoculate it on the MRS solid plate, gradually purify and select fast-growing strains to carry out the following antibacterial experiment screening.

[0045] The Oxford cup method was used to prepare the fungal pathogen indicator solution with a concentration of 1×10 8 CFU / mL order of magnitude, take 100 μL and spread it evenly on the NA solid plate, place an Oxford cup, take 200 μL of sample (the samples are respectively bifidobacterium fermentation broth stock solution (bifidobacterium fermentation broth stock solution prepared by the fermentation method described in Example 2), the supernatant after centrifugation of bifidobacterium fermentation broth (the supernatant obtained by centrifuging the bifidobacterium fermentation broth stock solution at 10000 r / min for 10 min) or bifidobacterium fermentation broth 0.45 μm filtrate (the liquid obtained by filtering the supernatant after centrifugation of the bifidobacterium fermentation broth through a sterile 0.45 μm filter)) and put it into an Oxford cup, place it in an incubator at 37°C for anaerobically cultured for 24 h, observe and record, and each treatment group of the strain fermentation broth marked as G-1 has an antibacterial effect on mold, and the antibacterial results of G-1 are shown in Table 1.

[0046] Table 1 Oxford cup test results

[0047] Pathogens Bifidobacterium fermentation liquid Supernatant after centrifugation of Bifidobacterium fermentation broth Bifidobacterium fermentation broth 0.45μm filtrate Fusarium <![CDATA[10.69±0.01 Bb ]]> <![CDATA[10.13±0.01 Cc ]]> <![CDATA[9.57±0.04 Cc ]]> Fusarium moniliforme <![CDATA[10.70±0.01 Bb ]]> <![CDATA[10.44±0.02 Bb ]]> <![CDATA[10.43±0.02 Bb ]]> Fusarium nivalis <![CDATA[16.87±0.02 Aa ]]> <![CDATA[12.96±0.02 Aa ]]> <![CDATA[10.88±0.06 Aa ]]>

[0048] Note: The letters on the shoulders in the table indicate: For longitudinal comparison, the same letters or no letters on the shoulders indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate significant difference (P<0.01).

[0049] The results were judged based on the inhibition zone diameter of <10 for low sensitivity, 10≤inhibition zone diameter≤15 for medium sensitivity, and inhibition zone diameter>15 for high sensitivity. As shown in Table 1, the G-1 fermentation broth stock solution and supernatant had medium and above inhibitory effects on Fusarium, Fusarium verticillata and Fusarium nivalis, and the inhibitory effect on Fusarium nivalis was significantly higher than that on the other two molds. The fermentation broth filtrate had medium inhibitory effects on Fusarium verticillata and Fusarium nivalis, and low inhibitory effects on Fusarium.

[0050] DNA extraction, PCR amplification, and ligation and transformation were performed on the G-1 strain. The BLAST program was used for comparison and phylogenetic tree construction to identify the strain. The BLAST program comparison results are shown in Table 2, and the phylogenetic tree results are shown in Table 3. Figure 1 shown.

[0051] Table 2 BLAST program comparison results

[0052]

[0053] Depend on Figure 1 As shown in Table 2, the 16S rDNA sequence of the G-1 strain isolated and obtained by the present invention is completely consistent with that of the animal Bifidobacterium strain Probio-M8, so the G-1 strain is determined to be Bifidobacterium G-1.

[0054] At present, there are few studies on the effect of bifidobacterium on inhibiting mold. The bifidobacterium G-1 obtained by screening and separation in the present invention can inhibit mold while producing rich beneficial biomass, and has multiple effects.

[0055] Example 2

[0056] The Bifidobacterium G-1 stored in the strain storage tube was streaked and subcultured on an MRS solid plate under sterile conditions, and cultured anaerobically at 37°C for 24-36 hours. A single colony was picked and subcultured on another MRS solid plate. After the colony grew, it was subcultured again according to the above method to complete the strain activation. The activated Bifidobacterium G-1 was inoculated into an MRS liquid culture medium, and cultured anaerobically at 37°C for 20 hours to obtain the Bifidobacterium G-1 seed liquid;

[0057] The Bifidobacterium G-1 seed liquid was inoculated into the final seed liquid MRS medium, and cultured at 37°C for 20 hours under anaerobic static fermentation to obtain the Bifidobacterium G-1 final seed liquid;

[0058] The final seed liquid of Bifidobacterium G-1 was inoculated into a fermentation tank and cultured anaerobically at 37°C for 24 hours to obtain Bifidobacterium G-1 fermentation liquid; the fermentation medium in the fermentation tank was (‰): 5 g of tryptone, 10 g of yeast powder, 3 g of dipotassium hydrogen phosphate, 0.3 g of magnesium sulfate, 0.08 g of manganese sulfate, and the pH was 6.8.

[0059] The Bifidobacterium G-1 fermentation liquid was centrifuged at 8000 r / min for 25 min, and the supernatant was discarded to obtain bacterial sludge;

[0060] The bacterial mud and soluble starch were mixed in a weight ratio of 1:5 and spray-dried by a spray dryer to obtain Bifidobacterium G-1 bacterial powder. The number of viable bacteria of Bifidobacterium G-1 bacterial powder was 1000×10 8 CFU / g.

[0061] The preparation process of Bacillus Velez J-1 powder is the same as that of Bifidobacterium G-1 powder. The viable cell count of Bacillus Velez J-1 powder is 200×10 8 CFU / g.

[0062] Example 3

[0063] A microbial preparation is prepared by mixing the Bifidobacterium G-1 powder and the Bacillus Velez subtilis J-1 powder prepared in Example 2, wherein the weight ratio of the Bifidobacterium G-1 powder to the Bacillus Velez subtilis J-1 powder is 5:10.

[0064] Example 4

[0065] A composite biological mold removal agent, comprising a mold inhibitor package and the microbial preparation obtained in Example 3; the mold inhibitor package comprises 0.1 kg of organic acid, 0.5 kg of calcium formate, 0.05 kg of taurine and 10 kg of glucose oxidase (the enzyme activity of the glucose oxidase is 10000 U / g); the organic acid comprises 0.05 kg of formic acid and 0.05 kg of acetic acid.

[0066] Example 5

[0067] A composite biological mold removal agent, composed of a mold inhibitor and the microbial preparation obtained in Example 3; the mold inhibitor consists of 1 kg of organic acid, 0.5 kg of calcium formate, 0.2 kg of taurine and 30 kg of glucose oxidase (the enzyme activity of the glucose oxidase is 10000 U / g); the organic acid consists of 0.5 kg of acetic acid and 0.5 kg of propionic acid.

[0068] Example 6

[0069] A composite biological mold removal agent, composed of a mold inhibitor and the microbial preparation obtained in Example 3; the mold inhibitor consists of 0.305 kg of organic acid, 0.5 kg of calcium formate, 0.1 kg of taurine and 20 kg of glucose oxidase (the enzyme activity of the glucose oxidase is 10000 U / g); the organic acid consists of 0.2 kg of formic acid, 0.1 kg of acetic acid and 0.005 kg of propionic acid.

[0070] Example 7

[0071] A composite biological mold removal agent, composed of a mold inhibitor and the microbial preparation obtained in Example 3; the mold inhibitor consists of 0.405 kg of organic acid, 0.5 kg of calcium formate, 0.2 kg of taurine and 30 kg of glucose oxidase (the enzyme activity of the glucose oxidase is 10000 U / g); the organic acid consists of 0.2 kg of formic acid, 0.2 kg of lactic acid and 0.005 kg of propionic acid.

[0072] Example 8

[0073] A composite biological mold removal agent, composed of a mold inhibitor and the microbial preparation obtained in Example 3; the mold inhibitor consists of 0.205 kg of organic acid, 0.5 kg of calcium formate, 0.05 kg of taurine and 10 kg of glucose oxidase (the enzyme activity of the glucose oxidase is 10000 U / g); the organic acid consists of 0.1 kg of formic acid, 0.1 kg of acetic acid and 0.005 kg of propionic acid.

[0074] Example 9

[0075] A composite biological mold remover liquid preparation is composed of the composite biological mold remover obtained in Example 4 and water. 0.5 kg of mold inhibitor is mixed with 800 kg of water to obtain a mixture, the mixture is mixed with 0.9 kg of microbial preparation, and the pH is adjusted to 3.5 to obtain the composite biological mold remover liquid preparation.

[0076] Example 10

[0077] A composite biological mold remover liquid preparation is composed of the composite biological mold remover obtained in Example 5 and water. 1 kg of the mold inhibitor is mixed with 1200 kg of water to obtain a mixture, the mixture is mixed with 2.5 kg of the microbial preparation, and the pH is adjusted to 5.0 to obtain the composite biological mold remover liquid preparation.

[0078] Embodiment 11

[0079] A composite biological mold remover liquid preparation is composed of the composite biological mold remover obtained in Example 6 and water. 20.905 kg of mold inhibitor is mixed with 20905 kg of water to obtain a mixture, the mixture is mixed with 20.905 kg of microbial preparation, and the pH is adjusted to 4.38 to obtain a composite biological mold remover liquid preparation.

[0080] Example 12

[0081] A composite biological mold remover liquid preparation is composed of the composite biological mold remover obtained in Example 7 and water. 31.105 kg of mold inhibitor is mixed with 31105 kg of water to obtain a mixture, and the mixture is mixed with 31.105 kg of microbial preparation, and the pH is adjusted to 3.79 to obtain a composite biological mold remover liquid preparation.

[0082] Embodiment 13

[0083] A composite biological mold remover liquid preparation is composed of the composite biological mold remover obtained in Example 8 and water. 10.755 kg of mold inhibitor is mixed with 10755 kg of water to obtain a mixture, the mixture is mixed with 10.755 kg of microbial preparation, and the pH is adjusted to 4.98 to obtain a composite biological mold remover liquid preparation.

[0084] Comparative Example 1

[0085] Take 0.2 kg of formic acid, 0.1 kg of acetic acid, 0.005 kg of propionic acid and 0.1 kg of taurine, mix them, dilute them 2000 times, add 810 kg of water, adjust the pH to 4.41, and obtain the antifungal liquid preparation 1.

[0086] Comparative Example 2

[0087] Take 0.1 kg of formic acid, 0.1 kg of acetic acid, 0.005 kg of propionic acid, 0.5 kg of calcium formate and 0.1 kg of taurine, mix them, dilute them 2000 times, add 1610 kg of water, adjust the pH to 4.40, and obtain the antifungal liquid preparation 2.

[0088] Comparative Example 3

[0089] Take 0.2 kg of formic acid, 0.1 kg of acetic acid, 0.005 kg of propionic acid, 0.5 kg of calcium formate, 0.1 kg of taurine and 20 kg of glucose oxidase (the enzyme activity of the glucose oxidase is 10000 U / g), mix them, dilute them 2000 times, add 41810 kg of water, adjust the pH to 4.40, and obtain the liquid mildew inhibitor 3.

[0090] Comparative Example 4 Bifidobacterium G-1 + Bacillus Velez J-1

[0091] 5 kg of Bifidobacterium G-1 powder and 10 kg of Bacillus Velezii J-1 powder obtained in Example 2 were added to 1000 kg of water and mixed to obtain a bacterial suspension with a pH of 6.56, which was recorded as liquid preparation 4.

[0092] Embodiment 14

[0093] The different liquid preparations obtained in Examples 11 to 13 and Comparative Examples 1 to 4 were placed at room temperature for 0 h, 3 h, 6 h, 9 h and 12 h, respectively, and the pH value was measured to monitor the stability within 12 h. The results are shown in Table 3. It shows that the pH value of the composite biological mold removal agent liquid preparation of the present invention is relatively stable and can be continuously used in actual production.

[0094] Table 3 pH values ​​of different liquid preparations at different time periods

[0095]

[0096] Embodiment 15

[0097] Inhibition of pathogenic bacteria

[0098] After activating each serotype of Escherichia coli and Salmonella, a single colony was inoculated into a liquid culture bottle under sterile conditions, and the live bacteria were counted after liquid shaking culture at 150r / min and 37°C for 24 hours.

[0099] The Oxford cup method was used to measure the inhibition of Escherichia coli by different liquid preparations obtained in Examples 11 to 13 and Comparative Examples 1 to 4:

[0100] After the above-mentioned Escherichia coli and Salmonella were diluted 10 times, 100 μL was taken and coated on the nutrient agar solid plate, and sterile Oxford cups were placed according to the labels to facilitate the statistics of the experimental results. 200 μL of each liquid preparation sample was taken out and dripped into the Oxford cup according to the corresponding label. After all samples were added, the solid plate was placed in a 37°C constant temperature incubator for 24 hours. After the incubation was completed, the Oxford cup was carefully taken out with tweezers, and the diameter of the inhibition zone was measured with an electronic vernier caliper. The results are shown in Table 4. The results are judged according to the inhibition zone diameter <10 for low sensitivity, 10≤inhibition zone diameter≤15 for medium sensitivity, and inhibition zone diameter>15 for high sensitivity. It shows that the composite biological de-mold agent liquid preparation in the present invention has an inhibitory effect on Escherichia coli and Salmonella, among which Example 12 has a high sensitivity in inhibiting the inhibitory effect on each pathogenic bacteria, followed by Example 11 and Example 13, but they are all better than the comparative test group. It can be seen that the composite biological mold removal agent liquid preparation of the present invention has a good inhibitory effect on Escherichia coli and Salmonella, and can be used in actual production applications.

[0101] Table 4 Inhibition of Escherichia coli and Salmonella by different liquid preparations (mm)

[0102] Pathogens Embodiment 11 Example 12 Embodiment 13 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 <![CDATA[Escherichia coli O1]]> 16.48 18.58 15.20 10.02 10.00 11.86 9.56 <![CDATA[Escherichia coli O2]]> 15.98 18.86 15.06 10.05 10.01 11.72 9.48 <![CDATA[Escherichia coli O 78 > 16.30 18.94 15.01 10.12 10.06 11.65 9.01 <![CDATA[Escherichia coli O8]]> 16.68 22.13 15.09 10.25 10.08 12.65 9.03 <![CDATA[Escherichia coli O 139 > 15.91 18.58 15.25 10.28 10.12 11.78 9.59 salmonella 16.02 17.06 15.55 10.02 10.00 11.16 9.06

[0103] Example 16

[0104] Inhibit mold

[0105] After activating various molds, single colonies were inoculated into liquid culture bottles under sterile conditions, and cultured in liquid shaking at 120r / min and 28℃ for 48h before use.

[0106] The Oxford cup method was used to measure the fungus inhibition of different liquid preparations obtained in Examples 11 to 13 and Comparative Examples 1 to 4:

[0107] Aspergillus flavus, Fusarium frutescens, Fusarium moniliforme, Fusarium spp., Penicillium, and Aspergillus niger were added to sterile glass balls and shaken to ensure that the bacteria were uniform as much as possible. After diluting 10 times, 100 μL was applied to a solid plate of a special culture medium for molds, and sterile Oxford cups were placed according to the labels to facilitate the statistics of experimental results. 200 μL of each liquid preparation sample was taken out and dropped into the Oxford cup according to the corresponding label. After all samples were added, the solid plate was placed in a 28°C constant temperature incubator for 24-72 hours, and the growth of the bacteria was observed at any time. The plate was taken out in time. After the culture was completed, the Oxford cup was carefully taken out with tweezers, and the diameter of the inhibition zone was measured with an electronic vernier caliper. The results are shown in Table 5. It shows that the composite biological mold removal agent liquid preparation of the present invention has a good inhibitory effect on a variety of molds and can be used for actual production applications.

[0108] Table 5 Inhibition of mold by different liquid preparations (mm)

[0109]

[0110]

[0111] Embodiment 17

[0112] Safety Experiment

[0113] Twelve Kunming mice weighing 18±2g were selected. After the mice were free to move and adapt for 3 days, they were randomly divided into 2 groups, 6 in each group, recorded as Example 12 group and control group. The Example 12 group was gavaged with 500μL / mouse of the liquid preparation obtained in Example 12, and the control group was gavaged with 500μL / mouse of normal saline. The mice were gavaged for 7 days, and the state and feces of the mice were observed during the experiment. After the experiment, the small intestine was removed, rinsed with 1×PBS solution, wiped dry with filter paper, and placed in 10% formaldehyde fixative for testing.

[0114] The results showed that during the experiment, the mice had normal appearance, smooth fur, free movement, normal feces, and no abnormal phenomena. The results of small intestinal villus height and crypt depth are shown in Table 6, and the results of small intestinal sections are shown in Table 6. Figure 2 shown.

[0115] Table 6 Small intestinal villus height (μm)

[0116] Group Fluff height Crypt depth Control group 344.67±1.19 63.71±0.35 Example 12 Group 371.59±10.93 64.34±0.24

[0117] It can be seen that the height of the villi and the height of the crypts of the mice in the Example 12 group are better than those in the control group, so it can be obtained that the composite biological mold removal agent liquid preparation of the present invention has a positive effect on the intestine. The structure of each layer of the small intestinal tissue in the Example 12 group is clear, the mucosal layer can be seen to have abundant intestinal villi, the mucosal epithelium is intact, the columnar epithelial cells are arranged neatly, no obvious necrosis or shedding is observed, the intestinal glands in the lamina propria are arranged neatly and tightly, the connective tissue of the submucosal layer is dense, no obvious edema is observed, and the muscle fibers in the muscular layer are uniformly colored and arranged neatly, no obvious ulcers are observed, and no obvious inflammatory cell infiltration is observed. It is consistent with the control group.

[0118] Embodiment 18

[0119] The Bacillus Velez J-1 powder prepared in Example 2 and the Bifidobacterium G-1 powder prepared in Example 2 were selected respectively, and the two powders were compounded according to different weight ratios to prepare liquid preparations respectively. The method for preparing the liquid preparation was the same as that in Comparative Example 4. The clinical trial effects of liquid preparations of different groups of powders were compared. The group design is shown in Table 7.

[0120] Table 7 Group design

[0121]

[0122]

[0123] In vivo test comparison of the effects of 6 groups, group 1 to group 6, in clinical broiler and duck tests:

[0124] The white-feathered broilers were divided into 7 groups according to different buildings, with a total of 9,500 in group 1, 9,000 in group 2, 9,500 in group 3, 9,600 in group 4, 8,500 in group 5, 9,800 in group 6, and 9,000 in the control group. Different groups were allowed to freely drink the liquid preparations of the corresponding groups, and the control group was allowed to drink water freely. The white-feathered broilers were selected for use three times throughout the breeding cycle, and were used continuously for 3 days a week at 1-7 days of age, 14-21 days of age, and 28-35 days of age (drinking the liquid preparation for 2 hours every day). Each group was fed the same feed during the experiment, and all groups of white-feathered broilers were routinely immunized.

[0125] During the test, the death and escape of animals in each group and the feed delivery (record the weight of the delivered feed and the weight of the remaining feed before the next delivery) were recorded. The initial fasting body weight was measured after fasting for 12 hours and recorded. The feed consumption was recorded by group. During the entire test, the body condition of the chickens was carefully observed and the death was recorded in detail, including the date of death, number of deaths, group, etc. After the end, the average weight, survival rate, diarrhea rate and feed-to-meat ratio of each group were calculated. The results are shown in Table 8.

[0126] Table 8 Experimental results of broiler chickens with different bacterial powder design groups

[0127] Inspection indicators Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Control group Survival rate% 83 82 85 88 92 89 85 Diarrhea rate% 18 17.5 17 16.5 15 16 20 Feed to meat ratio 1.9 1.88 1.86 1.85 1.83 1.86 1.92 Average weight at market price (kg) 2.3 2.3 2.31 2.33 2.37 2.33 2.27

[0128] Cherry Valley ducks were divided into 7 groups according to different buildings, with 7500 ducks in group 1, 6800 ducks in group 2, 7000 ducks in group 3, 7200 ducks in group 4, 7000 ducks in group 5, 6800 ducks in group 6, and 7000 ducks in the control group. Different groups were allowed to freely drink the liquid preparations of the corresponding groups, and the control group was allowed to drink water freely. Cherry Valley ducks were selected for use three times throughout the breeding cycle, and were used continuously for 3 days per week at 1-7 days of age, 14-21 days of age, and 28-35 days of age (drinking liquid preparations for 2 hours every day). Each group was fed the same feed during the test, and all groups of white-feathered broilers were routinely immunized. The remaining test methods were the same as those of white-feathered broilers, and the results are shown in Table 9.

[0129] Table 9 Effects of meat duck experiment with different bacterial powder design groups

[0130] It can be seen from Tables 8 and 9 that the survival rate, diarrhea rate, feed-to-meat ratio and average weight of broiler chickens and ducks were investigated by groups 1 to 6 of liquid bacterial powder preparations, and it was found that the test results of group 5, namely comparative example 4, were better than those of other groups, that is, the best effect was achieved when the weight ratio of Bacillus velezensis J-1 powder to Bifidobacterium G-1 powder was 5:10.

[0131] Embodiment 19

[0132] In vivo experiments comparing the effects of different liquid preparations obtained in Examples 11 to 13 and Comparative Examples 1 to 4

[0133] White-feathered broilers are designed into 8 groups according to different buildings, corresponding to embodiment 11 group, embodiment 12 group, embodiment 13 group, comparative example 1 group, comparative example 2 group, comparative example 3 group, comparative example 4 group and control group respectively, wherein embodiment 11 group 10000, embodiment 12 group 10000, embodiment 13 group 10000, comparative example 1 group 8800, comparative example 2 group 9200, comparative example 3 group 9000, comparative example 4 group 8000, control group 9000. Different groups are free to drink the liquid preparation of corresponding groups respectively, and the control group is free to drink water. White-feathered broilers are selected to use three times throughout the breeding cycle, and are used continuously for 3 days per week (drinking liquid preparation 2h every day) at 1-7 days of age, 14-21 days of age, and 28-35 days of age. Each group is fed the same feed during the test, and the white-feathered broilers of all groups are routinely immunized. The remaining methods are the same as in Example 18.

[0134] Determination of mycotoxin content in feces: During the entire test period, about 500g of fresh feces excreted by experimental animals were collected from different locations in the shed on the day of release in each group. After the samples were taken out, they were frozen and stored. The test samples were dried at 40-50℃ in batches, crushed by a pulverizer and placed in a sealed bag for testing. The sample test used the mycotoxin ELISA kit provided by Beijing Yisenbao Technology Co., Ltd. to detect the content of aflatoxin (AFB1), vomitoxin (DON) and zearalenone (ZEN) in fecal samples to determine the excretion of mycotoxins after metabolism.

[0135] The results are shown in Table 10. The survival rate and average weight of the Example 12 group were significantly better than those of the other Example groups and the Comparative Example group. The diarrhea rate, the amount of mycotoxins detected at the end of the test, and the feed-to-meat ratio were significantly lower than those of the other Example groups and the Comparative Example group, and the Example 11 and Example 13 were inferior to the Example 12.

[0136] Table 10 Clinical trial results of different liquid preparations

[0137] Inspection indicators Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Control group Survival rate% 87.4 87 88 89 92 86.2 86 Diarrhea rate% 18.4 17.5 16 15.5 12.5 19.5 20.5 Feed to meat ratio 1.88 1.87 1.85 1.84 1.81 1.83 1.91 Average weight at market 2.62 2.63 2.65 2.69 2.89 2.72 2.58

[0138]

[0139] After the overall experiment was completed, the aflatoxin content of the broiler liver and breast muscle was randomly sampled and tested (using the kit of Beijing Yisenbao Biotechnology Co., Ltd.), specifically: the liver and breast muscle of the test chicken were collected after dissection and placed in a culture dish, cleaned with pre-cooled physiological saline, and the excess liquid was wiped off with filter paper and placed in a -20°C refrigerator for testing. The results are shown in Table 11. It shows that the composite biological de-mold agent liquid preparation of the present invention has the effect of degrading aflatoxin in the body.

[0140] Table 11 Results of ELISA determination of aflatoxin in liver and breast muscle

[0141]

[0142] Embodiment 20

[0143] In vivo experiments comparing the effects of different liquid preparations obtained in Examples 11 to 13 and Comparative Examples 1 to 4

[0144] Cherry Valley ducks are divided into 8 groups according to different buildings, corresponding to Example 11 group, Example 12 group, Example 13 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group and control group, wherein 6800 in Example 11 group, 6500 in Example 12 group, 6700 in Example 13 group, 6500 in Comparative Example 1 group, 6900 in Comparative Example 2 group, 7000 in Comparative Example 3 group, 6800 in Comparative Example 4 group and 7000 in control group. Different groups freely drink the liquid preparation of corresponding groups respectively, and the control group freely drinks water. Cherry Valley ducks are selected to be used three times throughout the breeding cycle, and are used continuously for 3 days per week (drinking liquid preparation 2h every day) at 1-7 days of age, 14-21 days of age and 28-35 days of age. Each group is fed the same feed during the test, and all groups of Cherry Valley ducks are routinely immunized. The remaining test methods are the same as in Example 19. The results are shown in Table 12. The survival rate and average weight of the Example 12 group were significantly better than those of the other Example groups and the Comparative Example group. The diarrhea rate, mycotoxin detection amount at the end of the test and feed-to-meat ratio were significantly lower than those of the other Example groups and the Comparative Example group, and the Example 11 and Example 13 were inferior to the Example 12.

[0145] Table 12 Clinical trial results of different liquid preparations

[0146]

[0147] The above results show that the liquid composite biological mold remover of the present invention can effectively improve the growth and production performance of animal organisms, and the liquid composite biological mold remover has the advantages of convenient application and stable effect. The microbial preparation provided by the present invention can be used simultaneously with the mold inhibitor to achieve multiple beneficial effects on the animal organism.

[0148] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Bifidobacterium sp. G-1, characterized in that The Bifidobacterium G-1 is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M2024350.

2. A microbial preparation, characterized in that: The microbial preparation comprises the Bifidobacterium G-1 and Bacillus Velez J-1 according to claim 1, and the deposit number of the Bacillus Velez J-1 is CCTCC NO: M2021287.

3. The microbial preparation according to claim 2, characterized in that The viable count of Bifidobacterium G-1 is 1000×10 8 CFU / g or more, the viable count of the Velez Bacillus J-1 is 200×10 8 CFU / g or more, the weight ratio of Bifidobacterium G-1 powder to Bacillus Velez J-1 powder is 2-5:6-10.

4. Use of the Bifidobacterium G-1 according to claim 1 or the microbial preparation according to any one of claims 2 to 3 in the preparation of a biological mold removal agent.

5. A composite biological mold removal agent, characterized in that: The composite biological mold removal agent comprises a mold inhibitor and the microbial preparation according to claim 3; the mold inhibitor comprises the following raw materials in parts by weight: 0.1 to 1 part of organic acid, 0.5 part of calcium formate, 0.05 to 0.2 part of taurine, and 10 to 30 parts of glucose oxidase.

6. The composite biological mold removal agent according to claim 5, characterized in that: The organic acid includes at least two of formic acid, acetic acid, lactic acid and propionic acid.

7. A composite biological mold removal agent liquid preparation, characterized in that: The raw materials of the liquid preparation include the composite biological mold removal agent according to claim 5 or 6 and water.

8. The liquid preparation of the composite biological mold removal agent according to claim 7, characterized in that: The weight proportions of the raw materials in the liquid preparation are: 0.5 to 1 part of antifungal agent, 800 to 1200 parts of water, and 0.9 to 2.5 parts of microbial preparation.

9. The composite biological mold removal agent liquid preparation according to claim 8, characterized in that: The pH of the liquid preparation is 3.5-5.

0.

10. The method for preparing the composite biological mold removal agent liquid preparation according to claim 8, characterized in that: The method comprises the following steps: mixing a mildew preventer with water to obtain a mixture, and mixing the mixture with a microbial preparation to obtain a composite biological mildew remover liquid preparation.

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