Brevibacillus laterosporus-based pig intestinal health regulating agent and preparation method thereof

Abstract

The invention relates to the technical field of breeding, and discloses a brevibacillus laterosporus-based pig intestinal health regulating agent and a preparation method thereof, and the regulating agent comprises the following components in parts by mass: 5-10 parts of brevibacillus laterosporus BL1 thallus powder, 80-90 parts of a feed carrier, and 5-10 parts of a protective agent. The brevibacillus laterosporus BL1 strain is directly added into a feed carrier, the brevibacillus laterosporus is used for controlling reproduction of intestinal pathogens, the number of harmful bacteria in intestinal tracts can be remarkably reduced, meanwhile, the brevibacillus laterosporus BL1 can increase the relative abundance of probiotics such as lactobacillus in the intestinal tracts, flora balance is recovered, and the breeding efficiency of the intestinal tracts is improved. According to the present invention, the enterococcus faecium has characteristics of good intestinal tract barrier activity, intestinal tract physical barrier function enhancing by improving the expression level of the intestinal tract barrier related gene, good heat resistance and broad-spectrum antibacterial activity, and solves the problems of antibiotic drug resistance, drug residue, flora imbalance and the like by using the enterococcus faecium to replace antibiotics.

Description

Technical Field

[0001] The invention relates to the technical field of breeding, and in particular to a pig intestinal health regulator based on Brevibacillus laterosporus and a preparation method thereof. Background Art

[0002] With the development of intensive farming, the intestinal health of pigs has received increasing attention. The intestine is the main organ for animal digestion and absorption, and its health directly affects the growth performance and immunity of pigs. However, in modern farming practices, adding antibiotics to feed is a common solution to control the reproduction of intestinal pathogens, but long-term use of antibiotics brings the following problems:

[0003] Antibiotic resistance: Pathogens have become significantly more resistant to antibiotics, resulting in reduced treatment effectiveness and an increased risk of disease outbreaks.

[0004] Drug residues: The residues of antibiotics in animals and the environment have raised concerns about food safety and environmental pollution.

[0005] Imbalance of intestinal flora: The broad-spectrum antibacterial effect of antibiotics may disrupt the balance of intestinal flora, inhibit the growth of beneficial bacteria, and reduce the overall health level of animals. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a pig intestinal health regulator based on Brevibacillus laterosporus and a preparation method thereof, which solves the problem in the prior art that in the pig breeding process, antibiotics need to be used for a long time to control the reproduction of intestinal pathogens, thereby causing antibiotic resistance, drug residues and flora imbalance.

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: a pig intestinal health regulator based on Brevibacillus laterosporus, the regulator comprising the following components in the following mass ratios:

[0008] Brevibacillus laterosporus BL1 bacterial powder: 5-10 parts

[0009] Feed carrier: 80-90 servings

[0010] Protective agent: 5 to 10 parts.

[0011] Preferably, the feed carrier comprises:

[0012] Cereals: corn flour, wheat flour, wheat bran, rice husk flour and sorghum flour;

[0013] Pulses and by-products: soybean meal, pea meal and soybean hull meal;

[0014] Tubers and their by-products: potato flour, cassava flour and sweet potato flour;

[0015] Fiber: oat bran, wheat bran and beet pulp powder;

[0016] Energy supplements: molasses powder and rice bran rapeseed cake powder;

[0017] Mineral carrier: stone powder and calcium hydrogen phosphate;

[0018] Binders and fillers: corn starch, tapioca starch and microcrystalline cellulose.

[0019] Preferably, the protective agent includes: lactose, maltose, sucrose, glucose, seaweed, glycerol, sorbitol, glycerol, gelatin, whey protein, polyethylene glycol, polyvinyl pyrrolidone, vitamin C, starch, dextran and pectin.

[0020] Preferably, the regulator is 5×10 10 The ratio of CFU / kg diet was added to the basal diet.

[0021] Preferably, the Brevibacillus laterosporus BL1 bacterial cell powder includes live bacterial cells and heat-killed bacterial cells.

[0022] The preparation method of a pig intestinal health regulator based on Brevibacillus laterosporus comprises the following steps:

[0023] (1) preparing Brevibacillus laterosporus BL1 bacterial powder;

[0024] (2) mixing bacterial powder with a feed carrier;

[0025] (3) Add protective agent and stir evenly;

[0026] (4) After mixing, the mixture is prepared into a powder or granular product.

[0027] Preferably, in step (1), when preparing the bacterial powder of Brevibacillus laterosporus BL1, live strain powder and inactivated strain powder are included:

[0028] Preparation of live strain powder:

[0029] A. Inoculate the Brevibacillus laterosporus BL1 strain into LB medium for cultivation to obtain bacterial liquid;

[0030] B. Centrifuge the bacterial solution to collect the bacteria;

[0031] C. freeze-drying the collected bacteria and crushing them into live bacteria powder;

[0032] Preparation of inactivated strain powder:

[0033] D. Prepare live strains according to steps A and B;

[0034] Heat the prepared bacterial solution to 100°C and maintain it for 14 to 15 minutes to inactivate the bacterial activity;

[0035] The inactivated bacteria were collected by centrifugation;

[0036] The collected inactivated bacteria are freeze-dried and crushed to produce inactivated bacteria powder.

[0037] Preferably, in step A, the culture medium is cultured under shaking conditions of 35-40°C and 170-190 r / min for 22-24 hours;

[0038] In step B and step D, centrifuges are used for centrifugation, and the centrifugation conditions are 5900-6200 r / min, the temperature is 2-4° C., and the centrifugation time is 9-11 min.

[0039] Preferably, when the bacterial powder is mixed with the feed carrier in step (2), a horizontal mixer is used for the mixing operation, and the stirring speed is set to 50-100 r / min and the stirring time is 10-15 min;

[0040] When the protective agent is added and stirred in step (3), the stirring device is maintained at 80 to 120 r / min and the stirring time is 8 to 12 minutes.

[0041] Preferably, in step (4), when preparing a powdery or granular product:

[0042] Preparation of powdered products:

[0043] The stirred mixture is dried by freeze drying equipment or spraying:

[0044] Freeze drying: Place the mixture in a freeze dryer, set the temperature to -50°C to -80°C, control the vacuum degree to 10 to 50 Pa, and dry for 12 to 24 hours;

[0045] Spray drying: Use a spray dryer with the air inlet temperature set at 120°C to 150°C, the air outlet temperature controlled at 70°C to 90°C, and the feed flow rate at 10 to 30 mL / min until the mixture is dried into powder;

[0046] Granular product preparation:

[0047] The homogenized mixture is passed through an extrusion granulator or a disc granulator to prepare granules:

[0048] Extrusion granulator: The mixture is extruded into granules with a diameter of 1 to 3 mm;

[0049] Disc granulator: adjust the disc angle to 35°~45°, make the mixture roll in the disc to granulate, and the particle diameter is controlled at 1~5mm;

[0050] After granulation is completed, the granules are placed in a drying oven with the temperature set at 50°C to 60°C and the drying time set at 6 to 12 hours to ensure that the moisture content of the granules is less than 10%.

[0051] The invention provides a pig intestinal health regulator based on Brevibacillus laterosporus and a preparation method thereof.

[0052] It has the following beneficial effects:

[0053] 1. The present invention directly adds the Brevibacillus laterosporus BL1 strain into a feed carrier and uses Brevibacillus laterosporus to control the reproduction of intestinal pathogens, thereby significantly reducing the number of harmful bacteria in the intestine. At the same time, Brevibacillus laterosporus BL1 can increase the relative abundance of probiotics such as lactobacillus in the intestine, restore the balance of the flora, and enhance the intestinal physical barrier function by increasing the expression level of intestinal barrier-related genes. In addition, the strain has good heat resistance and broad-spectrum antibacterial properties. By using it instead of antibiotics, the problems of antibiotic resistance, drug residues and flora imbalance are solved.

[0054] 2. The present invention uses live or heat-killed bacteria of Brevibacillus laterosporus BL1, which can not only regulate the metabolic environment in the intestine, but also significantly increase the content of short-chain fatty acids in the intestine, which helps to optimize the level of intestinal metabolites. At the same time, the regulator of the present invention can reduce the concentration of toxic metabolites indole in the intestine, reduce the risk of inflammatory response in the intestine, and further improve the overall health of pigs. In addition, while maintaining its functionality, the heat-killed bacteria improve the storage stability and adaptability of the regulator to processing technology, thereby enhancing its practical application value. DETAILED DESCRIPTION

[0055] The following will be clearly and completely described in conjunction with the technical solutions in the embodiments of the present invention specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] In order to better understand the present invention, the above contents are described in detail below in conjunction with specific embodiments.

[0057] The embodiment of the present invention provides a pig intestinal health regulator based on Brevibacillus laterosporus, wherein the regulator comprises the following components in the following mass ratios:

[0058] Brevibacillus laterosporus BL1 bacterial powder: 5-10 parts

[0059] Feed carrier: 80-90 servings

[0060] Protective agent: 5 to 10 parts.

[0061] Preferably, the feed carrier comprises:

[0062] Cereals: corn flour, wheat flour, wheat bran, rice husk flour and sorghum flour;

[0063] Pulses and by-products: soybean meal, pea meal and soybean hull meal;

[0064] Tubers and their by-products: potato flour, cassava flour and sweet potato flour;

[0065] Fiber: oat bran, wheat bran and beet pulp powder;

[0066] Energy supplements: molasses powder and rice bran rapeseed cake powder;

[0067] Mineral carrier: stone powder and calcium hydrogen phosphate;

[0068] Binders and fillers: corn starch, tapioca starch and microcrystalline cellulose.

[0069] Preservatives include: lactose, maltose, sucrose, glucose, seaweed, glycerol, sorbitol, glycerol, gelatin, whey protein, polyethylene glycol, polyvinyl pyrrolidone, vitamin C, starch, dextran and pectin.

[0070] The regulator was 5×10 10 The ratio of CFU / kg diet was added to the basal diet.

[0071] The bacterial powder of Brevibacillus laterosporus BL1 includes live bacterial cells and heat-killed bacterial cells.

[0072] The present invention provides a method for preparing the above-mentioned regulating agent, comprising the following steps:

[0073] (1) preparing Brevibacillus laterosporus BL1 bacterial powder;

[0074] (2) mixing bacterial powder with a feed carrier;

[0075] (3) Add protective agent and stir evenly;

[0076] (4) After mixing, the mixture is prepared into a powder or granular product.

[0077] In step (1), when preparing the bacterial powder of Brevibacillus laterosporus BL1, the powder includes live strain powder and inactivated strain powder:

[0078] Preparation of live strain powder:

[0079] A. Inoculate the Brevibacillus laterosporus BL1 strain into LB medium for cultivation to obtain bacterial liquid;

[0080] B. Centrifuge the bacterial solution to collect the bacteria;

[0081] C. freeze-drying the collected bacteria and crushing them into live bacteria powder;

[0082] Preparation of inactivated strain powder:

[0083] D. Prepare live strains according to steps A and B;

[0084] Heat the prepared bacterial solution to 100°C and maintain it for 14 to 15 minutes to inactivate the bacterial activity;

[0085] The inactivated bacteria were collected by centrifugation;

[0086] The collected inactivated bacteria are freeze-dried and crushed to produce inactivated bacteria powder.

[0087] In step A, the culture medium is cultured under shaking conditions of 35-40° C. and 170-190 rpm for 22-24 hours;

[0088] In step B and step D, centrifuges are used for centrifugation, and the centrifugation conditions are 5900-6200 r / min, the temperature is 2-4° C., and the centrifugation time is 9-11 min.

[0089] When mixing the bacterial powder with the feed carrier in step (2), a horizontal mixer is used for mixing, and the stirring speed is set to 50-100 r / min and the stirring time is 10-15 min;

[0090] When the protective agent is added and stirred in step (3), the stirring device is maintained at 80 to 120 r / min and the stirring time is 8 to 12 minutes.

[0091] In step (4), when preparing a powder or granular product:

[0092] Preparation of powdered products:

[0093] The stirred mixture is dried by freeze drying equipment or spraying:

[0094] Freeze drying: Place the mixture in a freeze dryer, set the temperature to -50°C to -80°C, control the vacuum degree to 10 to 50 Pa, and dry for 12 to 24 hours;

[0095] Spray drying: Use a spray dryer with the air inlet temperature set at 120°C to 150°C, the air outlet temperature controlled at 70°C to 90°C, and the feed flow rate at 10 to 30 mL / min until the mixture is dried into powder;

[0096] Granular product preparation:

[0097] The homogenized mixture is passed through an extrusion granulator or a disc granulator to prepare granules:

[0098] Extrusion granulator: The mixture is extruded into granules with a diameter of 1 to 3 mm;

[0099] Disc granulator: adjust the disc angle to 35°~45°, make the mixture roll in the disc to granulate, and the particle diameter is controlled at 1~5mm;

[0100] After granulation is completed, the granules are placed in a drying oven with the temperature set at 50°C to 60°C and the drying time set at 6 to 12 hours to ensure that the moisture content of the granules is less than 10%.

[0101] Example 1: Preparation and experimental verification of a regulator based on Brevibacillus laterosporus BL1

[0102] 1. Purpose of the experiment

[0103] This experiment compared the regulators with different bacterial states (active or inactivated) and bacterial ratios to verify their inhibitory effects on four harmful bacteria, namely Streptococcus, Escherichia coli, enterotoxigenic Escherichia coli, and Salmonella, as well as the residual amount of strains in pig feces, so as to screen out the best usage ratio and bacterial state, providing a reference for practical application.

[0104] 2. Experimental Design

[0105] Subjects:

[0106] Thirty-six healthy, uniformly-weight growing pigs (weight 25 ± 1 kg) were selected. The initial abundance of four undesirable bacteria in their feces (Streptococcus, Escherichia coli, enterotoxigenic Escherichia coli, and Salmonella) was similar and recorded. Then, the 36 pigs were randomly divided into 6 groups, 6 pigs in each group.

[0107] 3. Regulator preparation steps (1) Select Brevibacillus laterosporus and mix them into experimental groups 1 to 3

[0108] Set up low dose (3 servings), medium dose (8 servings) and high dose (12 servings)

[0109] Low dose (3 servings): Test whether a small amount of strains can significantly improve the intestinal environment.

[0110] Medium dose (8 copies): Verify whether the moderate ratio of strains is the optimal dose, balancing effect and cost.

[0111] High dose (12 copies): observe whether the high dose can further improve the antibacterial effect, and evaluate whether there are adverse effects of excessive strains;

[0112] Among them, experimental groups one to three, each group is set up with two samples, using active bacteria and inactivated bacteria respectively, so there are experimental groups one to six in total.

[0113] The specific differences are shown in Table 1:

[0114] Table 1:

[0115]

[0116] (2) Experimental groups 1 to 6 were prepared separately, and the preparation steps were as follows:

[0117] The Brevibacillus laterosporus BL1 strain was inoculated into LB medium and cultured at 37°C and 180 rpm for 24 hours.

[0118] The cells were collected using a centrifuge (6000 r / min, 4 °C, 10 min);

[0119] The bacteria were placed in a freeze-drying device and dried at -50 °C for 12 hours to make live bacteria powder (live bacteria count ≥ 1 × 10 10 CFU / g).

[0120] Preparation of inactivated bacterial powder:

[0121] Keep the strain inoculation and centrifugation steps consistent with the preparation of active bacteria;

[0122] Heat the bacterial solution to 100°C for 15 minutes to inactivate the bacterial activity;

[0123] The inactivated bacteria were placed in a freeze-drying device and dried at -50°C for 12 hours to prepare inactivated bacteria powder.

[0124] Regulator mixing and preparation

[0125] Mix bacterial powder (active or inactivated), carrier (corn flour) and protective agent (lactose) according to the experimental group ratio;

[0126] Use a horizontal mixer, set the speed to 50r / min, and stir for 15 minutes to ensure uniform mixing;

[0127] The feed was prepared into pellets with a diameter of 1 to 3 mm and placed in a drying oven at 50° C. for 6 hours.

[0128] 4. Data Collection Methods

[0129] Fecal sampling time:

[0130] Day 0 (before feeding), day 3, day 7, day 15 and day 30.

[0131] Detection indicators:

[0132] the relative abundance of four undesirable bacterial groups (Streptococci, Escherichia coli, enterotoxigenic Escherichia coli, and Salmonella);

[0133] Residue of Brevibacillus laterosporus (CFU / g);

[0134] The experimental data records are shown in Table 2

[0135] Table 2:

[0136]

[0137]

[0138] Data analysis

[0139] By analyzing the data in the experimental table, the following is a detailed analysis of the residual amounts of four undesirable bacteria and Brevibacillus laterosporus:

[0140] 1. Analysis of relative abundance of Streptococcus

[0141] Experimental group 5 (active bacteria, 8 copies):

[0142] The decrease was significant from 25±2% on the 0th day of the experiment to 8±1% on the 30th day, and the antibacterial effect was the best.

[0143] Experimental group 6 (inactivated bacteria, 8 copies):

[0144] The effect was suboptimal, but very close to that of experimental group 5, decreasing from 25±2% on day 0 to 9±1% on day 30.

[0145] Compared with other groups:

[0146] Low-dose groups (Group 1 and Group 3, 3 copies): The relative abundance of Streptococcus decreased to 15±2% and 17±2% on the 30th day, respectively, and the antibacterial effect was significantly weaker.

[0147] High-dose groups (Groups 2 and 4, 12 copies): Although the relative abundance of Streptococcus decreased to 12±1% and 14±2% on the 30th day, there was no significant advantage in the decrease compared with experimental groups 5 and 6, and the high dose increased the cost.

[0148] 2. Analysis of relative abundance of Escherichia coli

[0149] Experimental Group 5:

[0150] It dropped from 22±2% on day 0 to 6±1% on day 30, showing an excellent antibacterial effect.

[0151] Experimental Group 6:

[0152] It dropped from 22±2% on day 0 to 7±1% on day 30, which was close to that of experimental group 5, and the inactivated bacteria avoided the risk of residual live bacteria.

[0153] Compared with other groups:

[0154] The relative abundances of the low-dose groups (Group 1 and Group 3) on the 30th day were 14±2% and 16±2%, respectively, with no significant decrease.

[0155] The relative abundance of E. coli in the high-dose groups (Groups 2 and 4) dropped to 9±1% and 10±1% on the 30th day, close to experimental groups 5 and 6, but the additional cost of the high dose reduced its cost-effectiveness.

[0156] 3. Analysis of relative abundance of enterotoxigenic Escherichia coli

[0157] Experimental Group 5:

[0158] The antibacterial effect was the best, decreasing from 18±2% on the 0th day to 5±1% on the 30th day, and the decrease was stable.

[0159] Experimental Group 6:

[0160] It dropped from 18±2% on day 0 to 6±1% on day 30, an effect second only to that of experimental group 5.

[0161] Compared with other groups:

[0162] The relative abundance of the low-dose groups (Group 1 and Group 3) on the 30th day was 12±1% and 14±2%, respectively, indicating a poor antibacterial effect.

[0163] The relative abundances of the high-dose groups (Groups 2 and 4) on the 30th day were 7±1% and 9±1%, respectively, and the effects were close to those of experimental groups 5 and 6, but the costs were higher.

[0164] 4. Analysis of relative abundance of Salmonella

[0165] Experimental Group 5:

[0166] It dropped from 20±2% on day 0 to 5±1% on day 30, showing the most significant antibacterial effect.

[0167] Experimental Group 6:

[0168] This dropped from 20 ± 2% on day 0 to 6 ± 1% on day 30, which is suboptimal but still an excellent performance.

[0169] Compared with other groups:

[0170] The relative abundance of the low-dose groups (Group 1 and Group 3) on the 30th day was 14±2% and 16±2%, respectively, and the effect was not significant.

[0171] The relative abundances of the high-dose groups (Groups 2 and 4) on the 30th day were 8±1% and 10±1%, respectively, which were slightly inferior to those of experimental groups 5 and 6.

[0172] 5. Analysis of Brevibacillus laterosporus Residues

[0173] Experimental group 5 (active bacteria):

[0174] The residual amount of bacteria detected in feces was 4×10 4 CFU / g is lower than the safety upper limit, indicating that it is safe and controllable.

[0175] Experimental group 6 (inactivated bacteria):

[0176] No bacterial residue was detected in the inactivated bacteria group, which completely avoids the risk of strain spread and better meets high food safety requirements.

[0177] Compared with other groups:

[0178] Residual bacteria were also detected in the low-dose and high-dose active bacteria groups (Group 1 and Group 2), but the number was significantly lower than that in Group 5.

[0179] Summary of the advantages of experimental groups 5 and 6

[0180] Advantages of experimental group 5 (active bacteria, 8 copies):

[0181] Significant antibacterial effect:

[0182] The inhibitory effect on four undesirable bacteria (Streptococcus, Escherichia coli, enterotoxigenic Escherichia coli, and Salmonella) was the best, and the relative abundance of Streptococcus and Salmonella dropped to 8±1% and 5±1%, respectively, on the 30th day.

[0183] High economic efficiency:

[0184] Using a medium dose of 8 parts of bacteria balances the antibacterial effect and cost.

[0185] Bacteria residues can be controlled:

[0186] The residual number of bacteria on the 30th day was 4×10 4 CFU / g, within the safe range.

[0187] Advantages of experimental group 6 (inactivated bacteria, 8 copies):

[0188] Excellent antibacterial effect:

[0189] The antibacterial effect was second only to that of experimental group 5, and the abundance of the four undesirable bacteria decreased to a level close to that of experimental group 5 on the 30th day (9±1% for Streptococcus and 6±1% for Salmonella).

[0190] Sterile residue:

[0191] Inactivated bacteria completely avoid the risk of residual active bacteria and are more suitable for scenarios with high food safety requirements.

[0192] Strong applicability:

[0193] The inactivated bacteria are highly stable in high-temperature feed processing and are suitable for industrial production conditions.

[0194] Comprehensive analysis and conclusion

[0195] Experimental group 5 (active bacteria, 8 copies) and experimental group 6 (inactivated bacteria, 8 copies) are the best solutions for this experiment:

[0196] Experimental group 5 is more suitable for scenarios with extremely high requirements for antibacterial effects and suitable for low-temperature processing;

[0197] Experimental Group 6 is suitable for scenarios with stricter food safety requirements and high-temperature processing technology.

[0198] The recommended dose is 8 servings, ensuring a balance between cost and safety while achieving optimal results.

[0199] Example 2: Comparative experiment based on Brevibacillus laterosporus BL1 and commonly used antibiotics

[0200] 1. Purpose of the experiment

[0201] The purpose of this experiment is to verify the advantage of Brevibacillus laterosporus in controlling undesirable bacteria such as Streptococcus, Escherichia coli, enterotoxigenic Escherichia coli and Salmonella by comparing the antibacterial effects and residues of Brevibacillus laterosporus BL1 (experimental groups 5 and 6) with four commonly used antibiotics, so as to provide a scientific basis for it as an alternative to antibiotics.

[0202] 2. Experimental Design

[0203] Subjects:

[0204] Thirty healthy, uniformly weighted growing pigs (weight 25 ± 1 kg) were selected, with similar initial abundances of four undesirable bacterial groups (Streptococci, Escherichia coli, Enterotoxigenic Escherichia coli, and Salmonella) in their feces.

[0205] Thirty pigs were randomly divided into 6 groups, 5 pigs in each group, including 2 experimental groups (Bacillus laterosporus) and 4 control groups (commonly used antibiotics).

[0206] 3. The experimental group and control group settings are shown in Table 3

[0207] Table 3:

[0208]

[0209] 4. How to use regulators and antibiotics

[0210] Experimental groups (Group 5 and Group 6):

[0211] Preparation method of regulator:

[0212] The preparation method of active bacteria and inactivated bacteria is consistent with that in Example 1.

[0213] The regulators were mixed evenly according to the proportion (8 parts of bacteria, 80 parts of carriers, and 10 parts of protective agents) to prepare pellet feed.

[0214] Control group (group 1 to group 4):

[0215] Antibiotics should be evenly mixed into the feed according to the recommended veterinary dosage.

[0216] 5. Data Collection Methods

[0217] Fecal sampling time:

[0218] Day 0 (before feeding), day 3, day 7, day 15 and day 30.

[0219] Blood sampling time:

[0220] Day 3, Day 7, Day 15 and Day 30.

[0221] Detection indicators:

[0222] Antibacterial effect: relative abundance of Streptococcus, Escherichia coli, enterotoxigenic Escherichia coli, and Salmonella.

[0223] Probiotic balance: changes in relative abundance of Lactobacillus and Bifidobacterium.

[0224] Residue test: Residue of Brevibacillus laterosporus or antibiotics in the blood.

[0225] 6. Experimental data recording and analysis

[0226] The results of the comparison of the inhibitory effects on undesirable flora are shown in Table 4

[0227] Table 4:

[0228]

[0229]

[0230] Experimental summary:

[0231] (1) Analysis of relative abundance of Streptococcus

[0232] Experimental group 5 (active bacteria):

[0233] The abundance of Streptococcus decreased from 25±2% on day 0 to 8±1% on day 30, a decrease of 68%, the largest decrease among all experimental groups.

[0234] Experimental group 6 (inactivated bacteria):

[0235] The abundance of Streptococcus decreased from 25±2% on day 0 to 9±1% on day 30, a decrease of 64%, second only to experimental group 5.

[0236] Control group (antibiotics):

[0237] The abundance of streptococci in the optimal antibiotic group (amoxicillin) on the 30th day was 10±1%, a decrease of 60%, which was inferior to that of experimental groups 5 and 6.

[0238] The abundance of Streptococcus in the other antibiotic groups (enrofloxacin, tetracycline, and polymyxin B) dropped to 11% to 12%, and the antibacterial effect was significantly inferior to that of Brevibacillus laterosporus.

[0239] Advantages analysis:

[0240] The streptococcal inhibition effects of experimental groups 5 and 6 were better than those of the four antibiotics, especially the inhibition effect of experimental group 5 was the most significant.

[0241] The antibacterial effect of Brevibacillus laterosporus is exerted through competitive inhibition and metabolites (such as antimicrobial peptides and extracellular enzymes), avoiding the problem of antibiotic resistance.

[0242] (2) Analysis of relative abundance of Escherichia coli

[0243] Experimental group 5 (active bacteria):

[0244] The best effect was achieved when the abundance of Escherichia coli decreased from 22±2% on day 0 to 6±1% on day 30, a decrease of 73%.

[0245] Experimental group 6 (inactivated bacteria):

[0246] E. coli abundance decreased from 22 ± 2% on day 0 to 7 ± 1% on day 30, a 68% decrease, which is suboptimal.

[0247] Control group (antibiotics):

[0248] The abundance of Escherichia coli in the optimal antibiotic group (enrofloxacin) on the 30th day was 8±1%, a decrease of 64%, which was lower than that in experimental groups 5 and 6.

[0249] The abundance of E. coli in the remaining antibiotic groups (amoxicillin, tetracycline, and polymyxin B) dropped to 9% to 11%.

[0250] Advantages analysis:

[0251] Experimental groups 5 and 6 were superior to all antibiotic groups in terms of the inhibitory effect on Escherichia coli.

[0252] Escherichia coli is a stubborn pathogen, and antibiotic inhibition can easily lead to drug resistance. However, Brevibacillus laterosporus significantly inhibits its growth by regulating the intestinal environment, competing for adhesion sites, and other mechanisms, thus avoiding the risk of drug resistance.

[0253] (3) Analysis of relative abundance of enterotoxigenic Escherichia coli

[0254] Experimental group 5 (active bacteria):

[0255] The abundance decreased from 18 ± 2% on day 0 to 5 ± 1% on day 30, a decrease of 72%, showing the best effect.

[0256] Experimental group 6 (inactivated bacteria):

[0257] The abundance decreased from 18 ± 2% on day 0 to 6 ± 1% on day 30, a decrease of 67%, which is suboptimal.

[0258] Control group (antibiotics):

[0259] The abundance of the optimal antibiotic group (enrofloxacin) was 6±1% on the 30th day, which was comparable to that of experimental group 6 but lower than that of experimental group 5.

[0260] The abundance of the remaining antibiotic groups dropped to 7% to 9%.

[0261] Advantages analysis:

[0262] The inhibitory effect of experimental group 5 was significantly better than that of all antibiotics, and the effect of experimental group 6 was equivalent to that of the best antibiotic group (enrofloxacin), but no antibiotic residue was produced.

[0263] Brevibacillus laterosporus not only directly inhibits enterotoxigenic Escherichia coli, but also indirectly inhibits the reproduction of intestinal probiotics (Lactobacillus and Bifidobacterium) by promoting the growth of probiotics (Lactobacillus and Bifidobacterium).

[0264] (4) Analysis of relative abundance of Salmonella

[0265] Experimental group 5 (active bacteria):

[0266] The best effect was achieved when abundance decreased from 20 ± 2% on day 0 to 5 ± 1% on day 30, a 75% decrease.

[0267] Experimental group 6 (inactivated bacteria):

[0268] Abundance decreased from 20 ± 2% on day 0 to 6 ± 1% on day 30, a 70% decrease, which is suboptimal.

[0269] Control group (antibiotics):

[0270] The abundance of the best antibiotic group (amoxicillin) was 8±1% on day 30, a decrease of 60%.

[0271] The abundance of the remaining antibiotic groups dropped to 9% to 10%.

[0272] Advantages analysis:

[0273] Experimental groups 5 and 6 performed well in inhibiting Salmonella, especially experimental group 5, whose antibacterial effect far exceeded that of all antibiotics.

[0274] Salmonella is an important pathogen that causes intestinal inflammation and foodborne diseases in animals. Brevibacillus laterosporus significantly reduces its abundance by competing for nutrients and releasing antibacterial substances.

[0275] (5) Analysis of changes in probiotics (Lactobacillus and Bifidobacterium)

[0276] Experimental Group 5 and Experimental Group 6:

[0277] The abundance of probiotics increased by 15% to 20%, showing a significant probiotic growth-promoting effect.

[0278] Control group (antibiotics):

[0279] The abundance of probiotics decreased by 10% to 15%, indicating that antibiotics not only inhibited the unhealthy flora, but also had a killing effect on intestinal probiotics, disrupting the intestinal microecological balance.

[0280] Advantages analysis:

[0281] Experimental groups 5 and 6 were able to significantly promote the growth of probiotics, in sharp contrast to the antibiotic group, demonstrating their protective effect on the intestinal microecological environment.

[0282] Brevibacillus laterosporus not only inhibits pathogens but also significantly improves intestinal health by maintaining the balance of intestinal flora.

[0283] Comprehensive analysis and advantages of Brevibacillus laterosporus

[0284] Significant antibacterial effect:

[0285] Experimental group 5 (active bacteria) and experimental group 6 (inactivated bacteria) were better than all antibiotic groups in inhibiting streptococci, Escherichia coli, enterotoxigenic Escherichia coli and Salmonella, especially experimental group 5. Although the effect of experimental group 6 was slightly inferior to that of experimental group 5, it was still better than all antibiotic groups.

[0286] Probiotic protection and promotion: Experimental groups 5 and 6 significantly increased the abundance of probiotics (Lactobacillus and Bifidobacterium) (15% to 20%), while the antibiotic group significantly reduced the abundance of probiotics (10% to 15%), disrupting the balance of intestinal flora. Brevibacillus laterosporus has both antibacterial and probiotic effects through microecological regulation.

[0287] Avoid drug resistance issues: The use of antibiotics can easily induce the production of drug-resistant strains, while Brevibacillus laterosporus exerts its effects through nonspecific competition, nutrient depletion and antibacterial metabolites, without the risk of drug resistance.

[0288] High safety: Brevibacillus laterosporus has no blood residue and does not damage other systems of the body. It is safer and more reliable than antibiotics.

[0289] Conclusion: Brevibacillus laterosporus (experimental groups 5 and 6) is superior to the antibiotic group in terms of antibacterial effect and safety by significantly inhibiting pathogens, protecting probiotics and avoiding drug residues. It is an ideal alternative to antibiotics and is suitable for promotion and application in modern animal husbandry.

[0290] The comparison of residual amount in blood is shown in Table 5

[0291] Table 5:

[0292]

[0293] Summarize:

[0294] 1. Analysis of the experimental group (Bacillus laterosporus)

[0295] Experimental Group 5 (active bacteria) and Experimental Group 6 (inactivated bacteria): During the entire experimental period (day 3, day 7, day 15 and day 30), no residual amount of Brevibacillus laterosporus was detected in the blood (all were 0).

[0296] This is because Bacillus laterosporus mainly acts on the intestinal environment, is not absorbed into the blood, and does not participate in systemic circulation, so it will not cause any form of tissue or blood residue.

[0297] Security advantages:

[0298] No residue: Brevibacillus laterosporus completely avoids accumulation in the blood and has no adverse effects on pig meat quality, viscera or food safety.

[0299] Healthy and friendly: Its action is limited to the intestines, which not only ensures the antibacterial effect but also avoids systemic toxicity problems.

[0300] 2. Analysis of the control group (antibiotics)

[0301] (1) Amoxicillin group (control group 1):

[0302] The residual amount in the blood was detected to be 12±2μg / mL on the 3rd day. Although it gradually decreased over time, it was still 2±1μg / mL on the 30th day.

[0303] Potential issues:

[0304] Even 30 days after stopping the medication, trace amounts of amoxicillin still remain, which may be passed to consumers through pork or offal, posing a food safety risk.

[0305] (2) Enrofloxacin group (control group 2):

[0306] The blood residual level on the 3rd day was 15±3μg / mL, and it was still 3±1μg / mL on the 30th day.

[0307] Potential issues:

[0308] Enrofloxacin is a broad-spectrum antibiotic with a higher residual amount in the blood than amoxicillin and a slower elimination rate. Such residues may cause drug allergies in consumers or the formation of drug-resistant strains.

[0309] (3) Tetracycline group (control group 3):

[0310] The blood residual level on the 3rd day was 18±3μg / mL, and even on the 30th day it was still 3±1μg / mL.

[0311] Potential issues:

[0312] The residue level of tetracycline is higher than that of amoxicillin and enrofloxacin. Its chronic accumulation may lead to excessive drug residues in pork and by-products, posing a threat to consumers' health.

[0313] (4) Polymyxin B group (control group 4):

[0314] The blood residual level on the 3rd day was 20±4μg / mL and remained at 4±2μg / mL on the 30th day.

[0315] Potential issues:

[0316] Polymyxin B has the highest blood residue level, and its long-term use may lead to serious drug resistance problems and have a significant impact on food safety and export standards.

[0317] 3. Comparative analysis:

[0318] The comparison between Brevibacillus laterosporus and antibiotics is shown in Table 6

[0319] Table 6:

[0320]

[0321] 4. Advantages of Brevibacillus laterosporus

[0322] (1) No blood residue

[0323] No blood residues were detected in the experimental group (Bacillus laterosporus) during the entire experimental period, indicating that its effect was limited to the intestine, without systemic spread and not involved in blood circulation.

[0324] Compared with the antibiotic group (antibiotics still had 2-4 μg / mL blood residues even on the 30th day), Brevibacillus laterosporus completely avoided the risk of drug residues.

[0325] (2) Eliminate food safety risks

[0326] Antibiotic residues can be passed to consumers through pork and offal, possibly leading to drug allergies, toxic reactions or the spread of drug-resistant bacteria. However, as a feed additive, Brevibacillus laterosporus does not pose any threat to food safety.

[0327] (3) Avoiding drug resistance issues

[0328] Long-term use of antibiotics can easily induce drug resistance in pathogens, resulting in reduced treatment efficacy. However, Brevibacillus laterosporus inhibits pathogens through metabolites (such as antimicrobial peptides and extracellular enzymes) and has no risk of drug resistance.

[0329] (4) Wide range of applications

[0330] Brevibacillus laterosporus is not only suitable for high-temperature processed feed (inactivated bacteria), but also for low-temperature processed feed (active bacteria), and the selection of antibiotics usually requires strict restrictions on its process conditions.

[0331] in conclusion

[0332] Blood residue:

[0333] Brevibacillus laterosporus does not have any blood residue, completely avoiding food safety risks and drug resistance issues.

[0334] There were still significant residuals in the antibiotic group on day 30, indicating a potential safety risk of antibiotics.

[0335] Comprehensive Security:

[0336] As a green and safe feed additive, Brevibacillus laterosporus has the characteristics of no residue, high antibacterial efficiency and no risk of drug resistance, making it an ideal alternative to antibiotics.

[0337] Recommended application scenarios:

[0338] Experimental Group 5 (active bacteria): suitable for scenarios with high requirements for antibacterial effects and suitable for low-temperature processing.

[0339] Experimental Group 6 (inactivated bacteria): suitable for industrial production conditions with strict food safety requirements and high temperature processing.

[0340] Through blood residue comparison, the significant safety advantages of Brevibacillus laterosporus were verified, providing a scientific basis for its promotion and application as an antibiotic alternative.

[0341] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pig intestinal health regulator based on Brevibacillus laterosporus, characterized in that: The control agent comprises the following components in parts by mass: Brevibacillus laterosporus BL1 bacterial powder: 5-10 parts Feed carrier: 80-90 servings Protective agent: 5 to 10 parts.

2. The pig intestinal health regulator based on Brevibacillus laterosporus according to claim 1, characterized in that: The feed carrier includes: Cereals: corn flour, wheat flour, wheat bran, rice husk flour and sorghum flour; Pulses and by-products: soybean meal, pea meal and soybean hull meal; Tubers and their by-products: potato flour, cassava flour and sweet potato flour; Fiber: oat bran, wheat bran and beet pulp powder; Energy supplements: molasses powder and rice bran rapeseed cake powder; Mineral carrier: stone powder and calcium hydrogen phosphate; Binders and fillers: corn starch, tapioca starch and microcrystalline cellulose.

3. The pig intestinal health regulator based on Brevibacillus laterosporus according to claim 1, characterized in that: The protective agent includes: lactose, maltose, sucrose, glucose, seaweed, glycerol, sorbitol, glycerol, gelatin, whey protein, polyethylene glycol, polyvinyl pyrrolidone, vitamin C, starch, dextran and pectin.

4. The pig intestinal health regulator based on Brevibacillus laterosporus according to claim 1, characterized in that: The regulator was 5×10 10 The ratio of CFU / kg diet was added to the basal diet.

5. The pig intestinal health regulator based on Brevibacillus laterosporus according to claim 1, characterized in that: The Brevibacillus laterosporus BL1 bacterial cell powder includes live bacterial cells and heat-inactivated bacterial cells.

6. A method for preparing a pig intestinal health regulator based on Brevibacillus laterosporus, which is used to prepare the pig intestinal health regulator based on Brevibacillus laterosporus according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) preparing Brevibacillus laterosporus BL1 bacterial powder; (2) mixing bacterial powder with a feed carrier; (3) Add protective agent and stir evenly; (4) After mixing, the mixture is prepared into a powder or granular product.

7. The method for preparing the pig intestinal health regulator based on Brevibacillus laterosporus according to claim 6, characterized in that: In step (1), when preparing the bacterial powder of Brevibacillus laterosporus BL1, the powder includes live strain powder and inactivated strain powder: Preparation of live strain powder: A. Inoculate the Brevibacillus laterosporus BL1 strain into LB medium for cultivation to obtain bacterial liquid; B. Centrifuge the bacterial solution to collect the bacteria; C. freeze-drying the collected bacteria and crushing them into live bacteria powder; Preparation of inactivated strain powder: D. Prepare live strains according to steps A and B; Heat the prepared bacterial solution to 100°C and maintain it for 14 to 15 minutes to inactivate the bacterial activity; The inactivated bacteria were collected by centrifugation; The collected inactivated bacteria are freeze-dried and crushed to produce inactivated bacteria powder.

8. The method for preparing the pig intestinal health regulator based on Brevibacillus laterosporus according to claim 7, characterized in that: In step A, the culture medium is cultured under the conditions of 35-40° C. and 170-190 r / min with shaking for 22-24 hours; In step B and step D, centrifuges are used for centrifugation, and the centrifugation conditions are 5900-6200 r / min, the temperature is 2-4° C., and the centrifugation time is 9-11 min.

9. The method for preparing the pig intestinal health regulator based on Brevibacillus laterosporus according to claim 6, characterized in that: When mixing the bacterial powder with the feed carrier in step (2), a horizontal mixer is used for mixing, and the stirring speed is set to 50-100 r / min and the stirring time is 10-15 min; When the protective agent is added and stirred in step (3), the stirring device is maintained at 80 to 120 r / min and the stirring time is 8 to 12 minutes.

10. The method for preparing the pig intestinal health regulator based on Brevibacillus laterosporus according to claim 6, characterized in that: In step (4), when preparing a powder or granular product: Preparation of powdered products: The stirred mixture is dried by freeze drying equipment or spraying: Freeze drying: Place the mixture in a freeze dryer, set the temperature to -50°C to -80°C, control the vacuum degree to 10 to 50 Pa, and dry for 12 to 24 hours; Spray drying: Use a spray dryer with the air inlet temperature set at 120°C to 150°C, the air outlet temperature controlled at 70°C to 90°C, and the feed flow rate at 10 to 30 mL / min until the mixture is dried into powder; Granular product preparation: The uniformly stirred mixture is passed through an extrusion granulator or a disc granulator to prepare granules: Extrusion granulator: The mixture is extruded into granules with a diameter of 1 to 3 mm; Disc granulator: adjust the disc angle to 35°~45°, make the mixture roll in the disc to granulate, and the particle diameter is controlled at 1~5mm; After granulation is completed, the granules are placed in a drying oven with the temperature set at 50°C to 60°C and the drying time set at 6 to 12 hours to ensure that the moisture content of the granules is less than 10%.

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