Bacillus amyloliquefaciens and application thereof

Abstract

The invention provides a bacillus amyloliquefaciens and application thereof. The preservation number of the bacillus amyloliquefaciens is CGMCC (China General Microbiological Culture Collection Center) NO.28723. The bacillus amyloliquefaciens can be used for The bacillus amyloliquefaciens disclosed by the invention can be used for preparing animal feed, and has the advantages of being low in addition amount, capable of relieving serum oxidative injury and intestinal injury of animals, protecting livers of the animals and the like.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to Bacillus amyloliquefaciens and application thereof. Background Art

[0002] Long-term use and abuse of antibiotics not only leave antibiotic residues in livestock and poultry products and manure, damaging the ecological environment and affecting animal production, but also spread through the food chain, causing human resistance to antibiotics and threatening human health. Therefore, finding suitable antibiotic substitutes, vigorously promoting green feed additives, and implementing healthy livestock and poultry breeding are inevitable requirements and trends for the development of my country's animal husbandry industry.

[0003] Substitutes for antibiotics can be traditional Chinese medicine or beneficial microorganisms. Traditional Chinese medicine is not as effective as antibiotics, has no residue, and there are problems such as competition between humans and animals for medicine, and the price is high. Beneficial microorganisms such as Bacillus amyloliquefaciens and Bacillus subtilis are second only to antibiotics in effect, have no residue, and are low in price. Bacillus subtilis is the main one on the market. Bacillus amyloliquefaciens is a close relative of subtilis and has better effects than Bacillus subtilis. However, the performance of different strains of Bacillus amyloliquefaciens can vary greatly. It is of great significance to screen and obtain Bacillus amyloliquefaciens suitable for preparing animal feed. Summary of the invention

[0004] The invention provides a Bacillus amyloliquefaciens and an application thereof, which can be used for preparing animal feed, has the advantages of low addition amount, reducing serum oxidative damage and intestinal damage of animals, protecting the liver of animals, and the like.

[0005] The technical solution of the present invention is achieved in this way: a bacillus amyloliquefaciens, whose preservation number is CGMCCNO.28723.

[0006] Furthermore, the Bacillus amyloliquefaciens is isolated and obtained from pepper fruits.

[0007] The application of the Bacillus amyloliquefaciens in preparing feed.

[0008] Furthermore, the feed contains 50 million / Kg-100 million / Kg of Bacillus amyloliquefaciens.

[0009] Beneficial effects of the present invention:

[0010] The Bacillus amyloliquefaciens of the present invention can be added to feed in low doses (cfu=50 million / kg) to reduce the content of malondialdehyde, improve the oxidative damage of laying hen serum, reduce the content of alanine aminotransferase, protect the liver, maintain the intestinal environment, and reduce intestinal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 The transparent circles are proteases of starch-dissolving No. 17, starch-dissolving No. 7 and starch-dissolving No. 24;

[0013] Figure 2 It is the transparent circle of cellulase No. 17 for degrading starch;

[0014] Figure 3 It is the transparent circle of cellulase No. 17 for degrading starch;

[0015] Figure 4 These are the dilution curves for the control group, low-dose group, and high-dose group;

[0016] Figure 5 Ace index graphs of the control group, low-dose group, and high-dose group;

[0017] Figure 6 Shannon index graphs of the control group, low-dose group, and high-dose group;

[0018] Figure 7 Figure 2 is the Simpson index graph of the control group, low-dose group and high-dose group. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.

[0020] Example 1

[0021] The isolation and acquisition of Bacillus amyloliquefaciens comprises the following steps:

[0022] 1. Isolation medium: PDA (potato dextrose agar) medium

[0023] The preparation steps of PDA culture medium are as follows: (1) Peel the potatoes, weigh 200 g and cut into pieces; (2) Add water and boil for 0.5 h; (3) Filter with culture medium filter cloth; (4) Add glucose and agar in an amount of 2 g / 100 mL, and make up to 1000 mL with water; (5) Autoclave (0.5 h); then pour into a culture dish to form a starch plate.

[0024] 2. Sample processing

[0025] The sample is pepper fruit purchased from the vegetable market in Jun County, Hebi, Henan Province. The surface soil of the pepper fruit is removed with clean water and placed on a sterile operating table; the plant is taken out and cut into small pieces of 0.5 cm × 0.5 cm × 0.5 cm with a scalpel through the tissue cutting method; under sterile conditions, the block material is first disinfected and sterilized in medical ethanol for 3 minutes, then washed with sterile water 3 times, then placed in 0.1% mercuric chloride solution for 5 minutes, and then washed with sterile water 3 times; the edge of the material after surface disinfection is cut off and placed on a sterilized filter paper sheet, so that the surface water is absorbed, and then 4 pieces of culture are placed on each plate, and sterile forceps are used to pick them up and put them in PDA culture medium in turn, and cultured at 28°C for 3-7 days; the strain is purified and preserved on a slant to obtain Bacillus amyloliquefaciens (abbreviated as: No. 17 amyloliquefaciens).

[0026] 3. Statistics and Identification

[0027] Observe the colony morphology of endophytes, observe and identify the morphology of isolated endophytic bacteria through Gram staining; and conduct sequencing identification through a third-party company.

[0028] The Bacillus amyloliquefaciens is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 23, 2023, with a deposit number of CGMCC NO.28723 and a classification name of Bacillus amyloliquefaciens.

[0029] Comparative Example 1

[0030] This embodiment is basically the same as the embodiment 1, except that: the sample is the bio-fermented feed of corn and soybean meal of laying hens in Junxian County, and the amylolytic Bacillus (abbreviated as: amylolytic No. 7) was screened from the bio-fermented feed of corn and soybean meal of laying hens in Junxian County.

[0031] Comparative Example 2

[0032] This embodiment is basically the same as embodiment 1, except that: the sample is the root soil of plants in Yuanling Village, Yichuan County, Luoyang City, and Bacillus amyloliquefaciens (abbreviated as: Amyloliquefaciens No. 24) was screened from the root soil of plants in Yuanling Village, Yichuan County, Luoyang City.

[0033] The starch-dissolving No. 17, starch-dissolving No. 7 and starch-dissolving No. 24 were sequenced and identified by Sangon Biotech (Shanghai) Co., Ltd. The sequencing result of starch-dissolving No. 17 is shown in SEQ ID NO: 1; the sequencing result of starch-dissolving No. 7 is shown in SEQ ID NO: 2; and the sequencing result of starch-dissolving No. 24 is shown in SEQ ID NO: 3.

[0034] Protease transparent circle formula: 10g casein, 3g beef extract powder, 5g sodium chloride, 2g dipotassium hydrogen phosphate, 20g agar, add 1L water.

[0035] Preparation method of protease plate: Mix the various components in the formula, sterilize at 121°C for 30 minutes, and then pour into a culture dish to form a protease plate.

[0036] The actual pictures of the transparent circles of proteases for starch dissolving No. 17, starch dissolving No. 7 and starch dissolving No. 24 are as follows Figure 1 The diameters of the protease transparent zones are shown in Table 1.

[0037]

[0038] Note: Data in the same row without letters or with the same letters indicate no significant difference (P>0.05), while data with different letters indicate significant difference (P<0.05); the same applies to the following tables.

[0039] As shown in Table 1, the transparent zone diameter of the protease of the starch-soluble No. 17 in Example 1 is the largest, and the ability of the starch-soluble No. 17 to produce protease is better than that of the starch-soluble No. 7 and the starch-soluble No. 24.

[0040] Cellulose transparent circle formula: 2g sodium carboxymethyl cellulose, 2g ammonium sulfate, 0.5g sodium chloride, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.4g Congo red, 20g agar, add 1L water. Preparation method of cellulose plate: mix the various components in the formula, then sterilize at 121℃ for 30min, and then pour into the culture dish to form a cellulose plate. The transparent circle of the cellulase No. 17 is as follows Figure 2 As shown by Figure 2 It can be seen that starch solution No. 17 has a good ability to produce cellulase.

[0041] Amylase transparent circle formula: 30g soluble starch, 10g peptone, 5g sodium chloride, 3g beef extract powder, 20g agar, add 1L water. Preparation method of amylase plate: Mix the various components in the formula, then sterilize at 121℃ for 30min, and then pour into the culture dish to form a starch plate. The transparent circle of cellulose enzyme No. 17 is as follows Figure 3 As shown by Figure 3 It can be seen that starch solution No. 17 has a good ability to produce amylase.

[0042] The seed fermentation method of Bacillus amyloliquefaciens is as follows:

[0043] 1. Inoculate 1 mL of the starch-dissolving No. 17 bacterial solution of Example 1 into the seed culture medium for 24 hours. The components of the seed culture medium (g / L) are: 10.0 g of tryptone, 5.0 g of yeast extract powder, and 5.0 g of sodium chloride; culture conditions are: natural pH, temperature 30° C., aerobic culture, and obtain a culture solution;

[0044] 2. After mixing 30 parts of culture medium, 20 parts of water, 95 parts of bran and 5 parts of soybean meal, fermenting for 48 hours, the viable bacteria count CFU = 324 million / g was measured. After natural air drying, the viable bacteria count CFU = 128 million / g was measured.

[0045] The fermented Bacillus amyloliquefaciens is added to the basic feed as animal feed.

[0046] 270 healthy and uniformly weighted 42-week-old Beijing-red laying hens were selected and randomly divided into 3 groups (respectively: control group (C-0), low-dose group (W-1) and high-dose group (W-2)), with 3 replicates in each group and 30 hens in each replicate. The pre-test lasted 7 days and the main test lasted 28 days. During the pre-test period, all laying hens in each group were fed with a basic diet. During the main test period: the control group was fed with a basic diet, the low-dose group was fed with 50 million Bacillus amyloliquefaciens per kilogram on the basis of the basic diet, and the high-dose group was fed with 100 million Bacillus amyloliquefaciens per kilogram on the basis of the basic diet. The basic diet was prepared according to NRC (1994) and "Chicken Feeding Standards" (NY / T33-2004). The composition and nutritional level of the basic diet are shown in Table 2.

[0047] Table 2 Basic feed composition and nutritional level (air-dried basis)

[0048] raw material content Nutritional Levels content corn 63.0 Crude protein 16.82 Soybean meal 24.0 Metabolizable Energy 11.36 Stone powder 8.0 calcium 3.54 Premix 5.0 Available Phosphorus 0.43 total 100.0 Lysine 0.78 Methionine 0.39 Cysteine 0.28

[0049] Note: The premix provides VA8 500IU, VD3 500IU, VE 35mg, VK31mg, VB11.5mg, VB2 4mg, VB6 2mg, VB 12 0.02mg, niacin 30mg, folic acid 0.55mg, pantothenic acid 10mg, biotin 0.16mg, choline chloride 420mg, Cu 10mg, Fe10 mg, Mn 100mg, Zn 100mg.

[0050] After 28 days of the trial period, the effects of Bacillus amyloliquefaciens on serum biochemistry and liver function of laying hens are shown in the following table:

[0051] Table 3 Effect of Bacillus amyloliquefaciens on serum biochemistry

[0052]

[0053] Table 4 Effects of Bacillus amyloliquefaciens on liver function

[0054] project Control group Low dose group High dose group ALT <![CDATA[6.11±0.94 A ]]> <![CDATA[2.58±0.89 B ]]> <![CDATA[3.01±1.21 B ]]> Aspartate aminotransferase 55.49±5.76 60.29±12.14 66.71±8.95

[0055] It can be seen from Tables 2 and 3 that adding low doses (cfu=50 million / Kg) of Bacillus amyloliquefaciens to feed can improve serum oxidative damage in laying hens (reduced malondialdehyde content indicates reduced oxidative damage); and protect the liver (the lower the alanine aminotransferase content, the healthier the liver).

[0056] Figure 4 These are the dilution curves of the control group (C-0), low-dose group (W-1), and high-dose group (W-2). The dilution curves of the tested samples all tend to be flat, indicating that the amount of sequencing data is basically reasonable; the larger the value, the richer the intestinal species and the better the stability. The test group with added Bacillus amyloliquefaciens is better than the control group.

[0057] Figure 5 Figure 2 shows the Ace index graph of the control group (C-0), low-dose group (W-1) and high-dose group (W-2). The Ace index (Abundance-based Coverage Estimator) is an indicator to measure sample coverage. It can calculate the number of unobserved species in the sample based on information such as the number of individuals and species richness of the species in the sample. The Ace index is often used to evaluate the diversity and stability of species in an ecosystem. The larger the value, the better. The values ​​of the low-dose group (W-1) and the high-dose group (W-2) are both greater than those of the control group.

[0058] Figure 6 The Shannon index diagrams for the control group (C-0), low-dose group (W-1) and high-dose group (W-2). The Shannon index is an indicator to measure the species richness and uniformity in an ecosystem. It takes into account the relative abundance of each species in the sample. The more species, the more evenly distributed the relative abundance, and the higher the Shannon index.

[0059] Figure 7 The Simpson index graphs for the control group (C-0), the low-dose group (W-1), and the high-dose group (W-2). The Simpson index is another indicator for measuring species diversity in an ecosystem. It takes into account both the abundance and number of species and is sometimes called the dominance index because it focuses more on the species with the largest number of individuals in the sample. In other words, the more dominant the flora, the lower the richness.

[0060] Depend on Figure 4-7 It can be seen that adding Bacillus amyloliquefaciens is beneficial to maintaining the intestinal environment and reducing intestinal damage.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus amyloliquefaciens, characterized in that: Its deposit number is CGMCC NO.28723.

2. A Bacillus amyloliquefaciens according to claim 1, characterized in that: The amyloliquefaciens bacillus is separated and obtained from pepper fruits.

3. Use of the Bacillus amyloliquefaciens according to claim 1 or 2 in preparing feed.

4. The use according to claim 3, characterized in that: The feed contains 50 million / Kg to 100 million / Kg of Bacillus amyloliquefaciens.

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