TW201934017A - A feed additive for decreasing brachyspira hyodysentriae infections in pigs and its manufacturing method - Google Patents

Abstract

The present invention relates to a feed additive, especially containing the wheat bran and soybean solid fermentation and fermentative products obtained by Bacillus subtilis, and apply to decreasing Brachyspira hyodysentriae bacteria population in pigs with feed additives.

Description

Feed additive for reducing infection of porcine erythra and its manufacturing method

The present invention relates to a pig feed additive; in particular, it comprises a Bacillus subtilis fermentation product obtained by solid-state fermentation of bran and soybean meal, and a pig feed additive capable of reducing the infection of pig intestinal erythra.

Swine hoarfrost is a bacterial disease that can cause severe chancretion in pigs. It is a disease caused by Gram-negative bacteria such as Brachyspira hyodysenteriae. Infected pigs can produce blood samples when they are severe, and Treponema pallidum can secrete proteases and lipopolysaccharides to destroy intestinal epidermal cells and produce hemorrhagic ulcers. As a result, pigs cannot properly absorb nutrients and defecation, and even die. Treponema pallidum can survive in feces for up to two weeks. , Still has the ability to infect, and can be carried by vectors such as mice, cats, dogs, cockroaches and infect healthy pigs. This disease is more common in growing and finishing pigs, so it has a huge loss to the pig farm (Alvarez-Ordó ez et al., 2013; Paster and Dewhirst, 2000). Compared with the common intestinal pathogenic microorganisms such as Escherichia coli and Salmonella in the taxonomy of Treponema pallidum, the former is a distinct difference between the proteophytes and the latter two proteobacteria, as shown in the table below. The prior art points out that the surface bactericidal function is aimed at the conventional bacteria, and does not include Borrelia burgdorferi.

In the past, the method of prevention and treatment was to use carbadox antibiotics. However, because of the high cytotoxicity of carbadox, it is easy to cause pig poisoning. Therefore, the Agricultural Committee announced on July 18, 105, the agricultural defense word No. 1051471970 Several antibiotics, such as Cabad, are banned, so there is currently no good response to porcine redhead. Only antibiotics can be relied on, but the addition of antibiotics in the feed can easily lead to the problem of pork drug residues.

The research and development of green feed additives produced by solid-state fermentation of probiotics allows livestock and poultry farmers to have more non-drug alternatives to reduce the drug residues of livestock products and improve the safety of livestock products. The present invention uses pigs as experimental animals and uses bran The husk and soybean meal are used as solid metabolites to produce surface hormones as index metabolites. The obtained fermentation product of Bacillus subtilis is dried and pulverized, and then added to the feed to control the use of Borrelia burgdorferi.

Figure 1 shows the analysis of surface primes by Bacillus subtilis fermentation.

Figure 2 shows the results of the surface hormone analysis by Bacillus subtilis on the bran and soybean meal cereal ferment on the 4th to 6th.

Figure 3 shows the results of the reduction of the surface tension of pure water by the surface hormone produced by the bran and soybean meal culture medium.

Fig. 4 shows that the surface hormone inhibits the occurrence of hemolytic circles in Borrelia burgdorferi.

Fig. 5 is an electron micrograph of surface perforation on Borrelia burgdorferi by surface perforation.

Figure 6 shows the effect of pigs on intestinal flora after being fed for four weeks with different concentrations of surface ferment.

In the present invention, Bacillus subtilis BCRC14718 strain is used for different grains including solid state fermentation culture. Configure a 250g space pack with a moisture content of 80% in the substrate. Sterilize at 121 ° C for 30 minutes. Allow the solid medium to cool. Connect the Bacillus subtilis bacterial solution with 4% of the total fermentation substrate at about 2 × 107 CFU / mL. After culturing at -35 ° C for 4-6 days, drying and milling are performed.

The fermentation product of Bacillus subtilis was analyzed by HPLC using HPLC. The conditions of HPLC analysis were: Lichrocart 250-4 Lichrospher 100 RP-18 column and Acetontril: 3.8mM Trifluoroacetic acid = 4: 1 (v / v) as the eluent. The flow rate was 1 mL / min, and the HPLC analysis was performed under the condition of 210 wavelengths. The analysis results show that the surface hormone retention time is about 23 minutes, as shown in FIG. 1. Bran and soybean meal mixed fermentation on the 4th to 6th produced the surface hormone analysis results shown in Figure 2, the results of solid state fermentation can produce 975ppm.

The fermented product produced by Bacillus was acid-precipitated and freeze-dried to extract the surface hormones, and the concentrations of 50, 100, 200, 400, and 800 ppm were serially diluted to determine the surface tension of the liquid using a surface tension meter. The results showed 800 ppm The surface tension can be reduced from 72 dyne / cm of pure water to 50 dyne / cm at 800 ppm. As shown in Figure 3.

Using Brachyspira hyodysentriae as the inhibitory zone test, apply 200ul of pig bacillus liquid to 1.5% agarose in BHI medium, which will contain 50ppm And a 100 ppm surface peptide patch were infiltrated into the sample solution to completely absorb it to saturation, and then placed in an anaerobic tank at 42 ° C for 2 days to observe the inhibition zone and observe the inhibition zone size, as shown in FIG. 4.

Fermented surface hormones against P. suis, using a scanning electron microscope to observe the surface damage of P. suis, the surface hormones 0,100 ppm, 250 ppm were cultured with Treponema pallidum for 1 hour, and the cell mass was collected by centrifugation at 2000 xg. Replace with 2.5% glutaraldehyde solution for 24 hours, add osmium tetroxide for dyeing for 1.5 hours, and then gradually dehydrate with different concentrations of ethanol. Finally, use 100% ethanol for dehydration, and then use a critical point dryer and a metal coating machine for gold plating. , And observe on the machine. The results showed that a large number of holes were formed on the surface of the bacterial cells at 100 ppm, and 250 ppm showed that all the bacterial cells were destroyed. The results are shown in FIG. 5. Animal test of Bacillus subtilis ferment, the test is divided into three groups: control group (feed without Bacillus subtilis ferment, feed with Bacillus subtilis ferment each containing 50ppm and 100ppm surface hormone. Each treatment is repeated three times, each repeat 4 weaned piglets, the test period was 28 days, and the number of faeces was collected on the day of the end of the test. The results of Figure 6 show that the intestinal flora was 100 ppm in the surface hormone group, which can significantly reduce the number of Treponema pallidum bacteria and the large intestine compared with the control group. Bacillus and increase the number of Lactobacillus.

Claims (8)

A method for manufacturing a feed additive for reducing infection of S. suis, including the following steps: A. Take 5-10 mL of Bacillus subtilis activated bacterial solution, the concentration of the bacterial solution is 2 × 10 ⁷ CFU / mL, and inoculate it at 200- 250 grams of bran and soybean meal solid-state fermentation substrate; B. solid-state fermentation at 30-35 ℃ for 2-6 days; C. drying and pulverizing the fermentation product; D. surface metabolites Analysis; E. Added to pig feed. For example, the manufacturing method of item 1 of the patent scope is pigs. For example, the manufacturing method of the third item of the patent application, wherein the surface hormone content of the fermentation product on the fourth day of fermentation is 975 ppm at the highest. A feed additive for reducing S. suis infection is obtained by a manufacturing method such as the scope of application for patents Nos. 1-3, and is characterized by adding 0, 50, and 100 ppm of the surface hormone content in the solid fermentation product of Bacillus subtilis. index. For example, the pig feed supplement for item 4 of the patent application scope, in which the surface hormone content of the solid fermentation product of Bacillus subtilis is 0, 50, and 100 ppm, and the pig's final spirulina is 100 ppm in the control pig's feces. At the same time, it decreased by 62.5% and 34.4%, respectively. For example, the pig feed supplement for item 4 of the patent application scope, in which the reduction of porcine intestinal pathogenic bacteria infection means that when the control group is 100 and the supplement containing 100 ppm surface hormone is administered, the intestinal E. coli is 47.4%, and the number of bacteria is Decreased 52.6%, the number of lactic acid bacteria was 134.2%, and the number of bacteria increased by 34%. For example, the pig feed supplement for patent application No. 4 is based on Bacillus subtilis. The surface hormone content in the fermentation product was 100 ppm. A feed additive as claimed in the scope of patent application No. 4 is used to reduce the infection of swine intestinal bacillus.