WO2017020785A1 - Application of bacteroides fragilis in animal breeding - Google Patents

Abstract

An application of bacteroides fragilis in animal breeding, an application of the bacteroides fragilis in preventing and/or treating gastrointestinal diseases of animals, and an application thereof in pharmaceutical compositions and feed additives. The bacteroides fragilis is ZY-312, and the preservation number is CGMCC No. 10685.

Description

Application of Bacteroides fragilis in animal breeding Technical field

The invention relates to the technical field of microorganisms, medicines, health care products, foods and daily chemical products, in particular to the application of Bacteroides fragilis in animal breeding.

Background technique

China is a big country in animal husbandry. According to statistics from relevant departments, China's production of pig, poultry, meat and eggs ranks first in the world. However, there are many problems in such developed animal husbandry, such as high quality poultry and livestock, and animal resistance. Poor disease ability. According to the recent national animal husbandry epidemic survey, there are hundreds of various types of livestock and poultry diseases in China. Since the 1970s, there have been 37 new diseases of livestock and poultry, and livestock and poultry diseases have become a major obstacle to the development of animal husbandry in China. Therefore, in the process of parenting, farmers will illegally use feed additives such as growth-promoting drugs, sleeping pills, contraceptives, and abuse of antibiotics to increase their yield and disease resistance, resulting in increased resistance of pathogenic microorganisms and residues of drugs in the body. Increased, product quality is seriously damaged, such products have great damage to human health, making China face many barriers in the export of animal products. The development and application of green environmentally-friendly drugs and feed additives that can replace traditional antibiotic drugs and feed additives have become an urgent problem for the livestock industry. At present, microecological preparations have also become the focus of research and development.

Since the 1940s, microbial preparations have been studied abroad for use in feed. In 1947, Miollgaard first discovered that piglets fed with lactic acid bacteria developed better than traditionally fed piglets. In 1967, Letchnikoff used sour milk to treat diarrhea in young animals with remarkable results. In the 1970s, the United States began to use large-scale probiotic feed instead of antibiotics, which can reduce the antibiotic residue of livestock meat, promote growth and improve feed utilization.

At present, the probiotic preparations widely used in feeds can be mainly divided into live bacteria preparations of Lactobacillus, live bacteria preparations of Bacillus, and live bacteria preparations of yeast. Many studies have confirmed that microbial preparations can improve the intestinal micro-ecological environment of animals, improve the body's immunity, enhance the disease resistance of animals, and promote animal growth and development. Research by Fan Guoge et al. showed that the composite probiotic preparation can inhibit the growth of E. coli and can treat piglet diarrhea while improving lipase activity in the intestine. Studies by Bai Yunfei and other scholars have shown that the combination of probiotics can improve the organ index of the green-legged chicken, increase the number of beneficial bacteria in the intestine, and promote its growth. Giangh has also obtained similar results. Studies have shown that the addition of probiotics to the diet can reduce the incidence of diarrhea in weaned piglets, increase the amount of lactic acid bacteria and organic acids in the intestine, and reduce the colonization of E. coli. Bontempo V added yeast to the diet of weaned piglets. The analysis showed that the macrophages of the intestinal mucosa increased significantly and enhanced the resistance to bacterial infection. U.S. Patent Application Serial No. US 595 1977 discloses a variety of probiotic combinations which inhibit the colonization of Salmonella in pigs.

At present, microecological preparations are recognized as environmentally friendly products for non-toxic and non-polluting alternative antibiotic feeds, which are increasingly used in animal husbandry and aquaculture. Some scholars pointed out that the era after antibiotics will be the era of microecological preparations. However, there are few types of excellent strains on the market, and new strains need to be developed.

Bacteroides fragilis is an obligate anaerobic bacterium that is negative for Gram stain, rod-shaped, blunt and densely stained at both ends, and has capsules, no spores, and no motility. It is divided into enterotoxin-producing and non-productive intestines. Toxin type. Bacteroides fragilis is part of the normal flora of human and animal gut, mainly in the colon. In addition, the respiratory tract and genitourinary tract can also grow. Currently, research Most of them are Bacteroides fragilis as a conditional pathogen. When the host mucosa is damaged, it can invade the submucosa, cause infection, and can also flow through the blood, causing the body's organs such as the intestines, abdominal cavity, liver and lungs. Brain tissue purulent infection with abscess and acute and chronic diarrhea; in addition, Bacteroides fragilis also promotes the occurrence of colon and rectal cancer.

A large number of studies have been conducted in the field on Bacteroides fragilis. For example, a Bacteroides strain BF839 is isolated from the well-developed infant or the intestine of a younger animal. It can be used as a live bacterial preparation to increase the growth and development of children, to prevent acute and chronic enteritis, dysbacteriosis, upper respiratory tract infection and nerve. It has good curative effect (see application number "90102847.9", the name is "a beneficial strain and its application" Chinese invention patent application; Zhang Jijie, et al. Clinical application of Bacteroides fragilis (BF839) bacterial liquid. Chinese Journal of Biological Products, 1995, Vol. 8, No. 2, pp. 63-65).

For another example, the application number is “201310095126.7”, the name is “Bacteroides fragilis with probiotic characteristics”; the application number is “201310085744.3”, the name is “Application of Bacteroides fragilis in the preparation of acute radiation-induced enteritis composition”; No. 201310085716.1, entitled "Bacteroides fragilis" disclosed in the Chinese Patent Application entitled "Application of Bacteroides fragilis in the preparation of a composition for treating inflammatory bowel disease", which was isolated from infant feces in 2012. The probiotic Bacteroides strain (deposited by CGMCC No. 7280) can be used for the treatment of inflammatory bowel disease, diarrhea and the like. In addition, through further identification of the strain, it was found that the bacterial strain, culture characteristics, physiological and biochemical reaction results were similar to Bacteroides fragilis, and the BLASTN sequence was compared with the Bacteroides fragilis standard strain ATCC25285. The source is 99%, and the drug sensitivity test suggests that the strain Bd312 is not sensitive to cefradine, amoxicillin, gentamicin, sulfamethoxazole, and trimethoprim. The acute and chronic toxicity test indicates no toxicity (Liu Yangyang, et al. Isolation and identification of non-toxic fragile Bacteroides in infants. Chinese Journal of Medicine, 2014, Vol. 94, No. 30, pp. 2372-2374).

At present, there is a lack of microecological preparations containing Bacteroides fragilis on the market, and the application field of the bacteria needs to be expanded.

Summary of the invention

The technical problem to be solved by the present invention is to provide a use of Bacteroides fragilis ZY-312 in animal breeding, in particular, a pharmaceutical composition and a feed additive for preventing and/or treating gastrointestinal diseases in animals.

In order to achieve the above object, the present invention provides the use of Bacteroides fragilis in animal breeding.

The above-mentioned Bacteroides fragilis application, wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312, and the accession number is CGMCC No. 10685.

In order to better achieve the above object, the present invention also provides the use of Bacteroides fragilis in the prevention and/or treatment of gastrointestinal diseases of animals, the Bacteroides fragilis is Bacteroides fragilis ZY-312, and the accession number is CGMCC No. 10685.

In order to better achieve the above object, the present invention also provides a pharmaceutical composition for preventing and/or treating gastrointestinal diseases of an animal, wherein the pharmaceutical composition comprises a pharmaceutically effective dose of Bacteroides fragilis ZY-312 .

In order to better achieve the above object, the present invention also provides a feed additive for preventing and/or treating gastrointestinal diseases of an animal, wherein the feed additive comprises an effective amount of Bacteroides fragilis ZY-312.

The feed additive described above, wherein the effective amount is a dose effective to maintain a healthy intestinal microflora, and the amount of the Bacteroides fragilis ZY-312 added in the feed is ≥1×10 ⁶ CFU/g.

The feed additive described above, wherein the effective amount is a dose effective to reduce the growth of pathogenic bacteria and viruses in the animal, and the amount of the Bacteroides fragilis ZY-312 added in the feed is ≥1×10 ⁶ CFU/g.

The above-mentioned feed additive, wherein the effective amount is a dose effective to increase animal feed utilization efficiency or animal survival rate, and the added amount of the Bacteroides fragilis ZY-312 in the feed is ≥1×10 ⁶ CFU/g.

In the above feed additive, wherein the effective amount is a dose effective to increase the weight of the animal, the amount of the Bacteroides fragilis ZY-312 added in the feed is ≥1×10 ⁶ CFU/g.

The above feed additive, wherein the animal is a pig or a poultry.

The feed additive described above, wherein the pig is a piglet or a sow, and the poultry is a chicken.

The technical effects of the present invention are:

The invention is isolated and purified from the well-developed baby feces, and the Bacteroides fragilis ZY-312 selected from a large number of Bacteroides fragilis strains is proved to be free of the enterotoxin gene bft, and is an avirulent strain having The characteristics of probiotics, after fermentation culture, staining microscopy, physiological and biochemical characteristics analysis and animal experiments, found that compared with other existing Bacteroides fragilis strains, Bacteroides fragilis ZY-312 has outstanding antibiotics, stomach acid and other probiotics. The characteristics can effectively overcome the shortcomings of the existing vulnerable Bacteroides fragilis in the digestive tract, and is a preferred strain of a new generation of microecological preparations, and has broad application prospects. The use of the Bacteroides fragilis ZY-312 of the present invention for preventing and/or treating gastrointestinal diseases in animals can be used for controlling intestinal diseases of animals. The bacteria is safe and reliable, and has a good development prospect in the field of animal husbandry.

The invention is described in detail below with reference to the accompanying drawings and specific embodiments.

DRAWINGS

Figure 1 is a view showing the colony morphology of Bacteroides fragilis ZY-312 after anaerobic culture;

Figure 2 is a Gram stained microscopic examination map of Bacteroides fragilis ZY-312 of the present invention (1000 ×);

Figure 3 is a scanning electron microscope observation view of Bacteroides fragilis ZY-312 of the present invention (30000 ×);

Figure 4 is a comparison diagram of gel electrophoresis results of the PCR product of the present invention;

Figure 5 is a comparison diagram of gel electrophoresis results of PCR products of the present invention;

Figure 6 is a phylogenetic tree established based on genome-wide sequence comparison;

7A-7D are diagrams showing the physiological condition of the piglets in the present invention.

The Bacteroides fragilis ZY-312 of the present invention was deposited with the China General Microorganisms Collection and Management Center (CGMCC) on April 2, 2015, and its deposit number is CGMCC No. 10685, and the deposit address is Chaoyang District, Beijing. No. 3, No. 1 Courtyard, Beichen West Road.

detailed description

The structural principle and working principle of the present invention will be specifically described below with reference to the accompanying drawings:

Embodiments of the present invention include: the present invention screens a large number of Bacteroides fragilis strains by screening a large amount of feces from healthy infants, and identifies a new Bacteroides fragilis, named ZY, by physical and chemical experiments. -312, compared with the existing Bacteroides fragilis strains, it has probiotic properties such as bile-tolerant salt and gastric acid resistance, which can make up for some defects of the original beneficial bacteria, and still maintain the bile salts and acidic conditions in the digestive tract. Highly biologically active, it is a preferred strain of probiotic products. The Bacteroides fragilis ZY-312 was preserved in China's common microbial strains on April 2, 2015. The Deposit Management Center (CGMCC) has a deposit number of CGMCC No.10685 and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

Bacteroides fragilis ZY-312 can be used in animal breeding, or in the prevention and/or treatment of gastrointestinal diseases in animals, and can also be prepared into a pharmaceutical composition. The pharmaceutical composition contains a pharmaceutically effective amount of Bacteroides fragilis ZY-312. Furthermore, the pharmaceutical composition may also contain a suitable pharmaceutically acceptable pharmaceutical carrier and an animal culture acceptable carrier. The Bacteroides fragilis ZY-312 of the present invention can also be formulated as a feed additive for preventing and/or treating gastrointestinal diseases in animals. The feed additive contains an effective amount of Bacteroides fragilis ZY-312. Wherein the animal is, for example, a pig, and may include piglets and sows, and may also be poultry such as chickens, ducks or geese. The effective amount of Bacteroides fragilis ZY-312 can effectively maintain healthy intestinal microflora, effectively reduce the growth of pathogenic bacteria and viruses in animals, effectively improve animal feed utilization efficiency, effectively improve animal survival rate and effectively Increase the weight of the animal.

The invention will now be further described in conjunction with specific embodiments. It should be noted that the Bacteroides fragilis or the pharmaceutical composition containing the Bacteroides fragilis of the present invention or the feed additive for use in the prevention and/or treatment of gastrointestinal diseases of the present invention can be administered to a subject. Applied to the indications described above and exhibiting the functions described above, all dosage forms within the scope of the invention have been tested, hereinafter only for purposes of illustration, only a few of which are described in the examples. However, it should not be construed as limiting the invention.

Unless otherwise stated, the reagents used in the present invention are commercially available.

Example 1

Isolation and purification of Bacteroides fragilis ZY-312

Reagents and instruments

(1) Medium A: Bacteroides-Bile-escin (BBE) agar (Qingdao Haibo Biotechnology Co., Ltd., Item No.: HB7028) was added with a modified formula. The specific ingredients are as follows:

Table I

(2) Medium B: Bacteroides-bile-escin (BBE) agar (Qingdao Haibo Biotechnology Co., Ltd., item number: HB7028) was added with a modified formula. The specific ingredients are as follows:

Table II

(3) Medium C: Buffalo broth (Qingdao Haibo Biotechnology Co., Ltd., article number: HB0241) was added with fetal bovine serum in an amount of 5% (v/v) (Sijiqing, article number: HB0205).

(4) Experimental equipment

2.5L sealed culture tank (Mitsubishi Gas Chemical Co., Ltd., C-31)

Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd., model: DHP-9082)

Microscope (Nikon Instruments (Shanghai) Co., Ltd., model: E100)

PCR instrument (App Applied Biosystems, Model: Applied

PCR system 9700)

Electrophoresis instrument (Beijing Liuyi Instrument Factory, model: DYCP-32B)

(5) Reagents

Anaerobic gas bag (Mitsubishi Gas Chemical Co., Ltd., article number: C-1)

Bacterial DNA Extraction Kit (Bacterial DNA Kit) OMEGA, USA, article number: D3350-01)

Taq Enzyme (Bao Bioengineering (Dalian) Co., Ltd., Item No.: DR100A)

Agarose (Brand: Biowest, Cat. No. 91622)

Sulfamethoxazole (sigma, article number: S7507-10G)

Trimethoprim (sigma, article number: T7883-5G)

Vitamin K1 (Qingdao Rishui Biotechnology Co., Ltd., Item No.: 21005)

DL1000DNA Marker (Bao Bioengineering (Dalian) Co., Ltd., Item No.: D526A)

Enterotoxin-producing strain of Bacteroides fragilis (provided by Sun Yong, Department of Gastroenterology, Southern Hospital, isolated from patients with clinical diarrhea)

Bacteroides fragilis standard strain ATCC25285 (purchased from Guangdong Institute of Microbiology)

Bacteroides fragilis strain Bd312 (the deposit number is CGMCC No.7280, provided by Guangzhou Zhiguang Biotechnology Co., Ltd.)

BF839 strain (isolated from totem probiotics).

(6) Medium configuration

Medium A configuration: 61.5 g of BBE medium was weighed, dissolved in 1000 mL of distilled water by heating, autoclaved at 121 ° C for 15 minutes, and cooled to about 50 ° C. Addition of filter-sterilized sulfamethoxazole 1 g, trimethoprim 4g and 50mL of sterile defibrated sheep blood, mix well, pour into the sterile plate, and set aside.

Medium B configuration: 61.5 g of BBE medium was weighed, dissolved in 1000 mL of distilled water, autoclaved at 121 ° C for 15 minutes, and cooled to about 50 ° C. Addition of filter-sterilized sulfamethoxazole 1 g, trimethoprim 4g, mix well, pour into a sterile plate and set aside.

Medium C configuration: Weighed 28.1 g of Brucella broth medium, dissolved in 1000 mL of distilled water with heating and stirring, and filled in a triangular flask, and autoclaved at 121 ° C for 15 minutes, and set aside. Add 5% fetal calf serum before use.

Brinell broth configuration: Weigh 24.1g of Brucella broth culture medium, stir it in 1000mL distilled water with heating and stirring, dispense triangle flask, autoclave at 121 °C for 15 minutes, and set aside.

method:

1, separation and purification

Fresh baby feces 0.5 g were taken and placed in a triangular flask containing 4.5 mL of Brucella broth and shaken for 1 minute. 0.1 mL was dripped on the medium, streaked, placed in an anaerobic tank, and cultured at 37 ° C for 48 hours. A typical colony was picked up in liquid medium for 24 h for Gram staining. The morphology was observed under a microscope, and the bacterial solution of Gram-negative bacteria was selected, streaked into a blood plate, and cultured for 48 hours under anaerobic conditions. According to the morphological characteristics of the colony on the plate and the microscopic observation of the staining characteristics, size, bulb shape and distribution of the cells, it was judged whether or not to purify. If the bacteria are impure, continue the above steps and repeat the passages several times until a purified strain is obtained.

2, colony characteristics

Bacteroides fragilis ZY-312 was cultured on a blood plate for 48 hours, showing a round micro-convex, translucent, white, smooth surface, no hemolysis, colony diameter of 1-3mm, see Figure 1.

3, the shape under the microscope

B. fragilis ZY-312 was subjected to Gram staining microscopy. It was a Gram-negative bacterium with a typical rod shape. Both ends were blunt and densely stained. The uncolored part of the cells was shaped like a vacuole. See Figure 2.

4, the shape of the electron microscope

The fixative was fixed and observed by scanning electron microscopy. Microscopically, the Bacteroides fragilis ZY-312 is 0.5 to 0.8×1 to 4.5 μm in size, without flagella, and without spores. See Figure 3.

5, biochemical identification

Table 3 shows the results of biochemical identification (in the table, + indicates positive, - indicates negative)

Table 3

Determination of reaction substrate	result
Tryptophan	-
Urea	-

glucose	+
Mannitol	-
lactose	+
sucrose	+
maltose	+
Salicyl alcohol	-
Xylose	+
Arabic candy	-
gelatin	-
Qiyeling	+
glycerin	-
Cellobiose	-/+
Mannose	+
Pine syrup	-
Raffinose	+
Sorbitol	-
D	-
Trehalose	-

API20A (Biochemical Reaction Identification Plate, French BioMerieux Co., Ltd.) physiological and biochemical reaction results show that Bacteroides fragilis ZY-312 can ferment glucose, lactose, sucrose, maltose, xylose, escin, mannose, raffinose , in line with the characteristics of Bacteroides fragilis.

Example 2

Identification of Bacteroides fragilis ZY-312

The polymerase chain reaction (PCR) primers (synthesized by Infineon (Shanghai) Trading Co., Ltd.) have the following sequence:

Primer pair 1:

Forward primer: 5'-ACGCTTGCACCCTCCGTATTA-3’

Reverse primer: 5'-GCTTAGAGTTTGATCCTGGCTCAG-3'

Primer pair 2:

Forward primer: 5'-TGGGTGGTTGCTGCCTGGACACA-3'

Reverse primer: 5'-CATCCGGGTATGGATATGAA-3'

Primer pair 3:

Forward primer: 5'-GATGCTCCAGTTACAGCTTCCATTG-3'

Reverse primer: 5'-CGCCCAGTATATGACCTAGTTCGTG-3'

Bft gene primer pair:

Forward primer: 5'-GACGGTGTATGTGATTTGTCTGAGAGA-3’

Reverse primer: 5'-ATCCCTAAGATTTATTATCCCAAGTA-3'

1. PCR identification (PCR, polymerase chain reaction, is a commonly used method for rapidly amplifying genes)

(1) 16S rRNA sequence determination

The above strain was inoculated on medium A, and cultured at 37 ° C for 48 hours under anaerobic conditions. A single strain was inoculated into a liquid medium, and cultured at 37 ° C for 48 hours. The DNA extraction kit extracts bacterial DNA (Tiangen Biochemical Technology (Beijing) Co., Ltd., article number: DP302-02) as a PCR template DNA.

Amplification of 16S rRNA gene sequence: primer pair 1 amplified fragment size is about 531 bp; primer pair 2 amplified fragment size is 518 bp; primer pair 3 amplified fragment size is about 970 bp.

A 20 μL PCR reaction system was used: 10 μL of Taq enzyme, 2 μL of template DNA, 1 μL of each of the forward and reverse primers, and 6 μL of sterile deionized water.

The PCR reaction conditions were: pre-denaturation at 95 ° C for 5 min, denaturation at 95 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 72 ° C for 45 s, 30 cycles, and extension at 72 ° C for 10 min.

The PCR product was electrophoresed on a 2% agarose gel under electrophoresis conditions of 100 V for 15 min.

The results of gel electrophoresis of PCR products are shown in Figure 4. Among them, lanes 1 and 2 are primer pair 1 and primer pair 2 amplification products respectively; lanes 4 and 5 are primer pair 1 and primer pair 2 amplification products respectively (repetitive PCR Results); lanes 3 and 6 are primer pair 3 amplification products; lane 7 is DNA molecular weight standard (DL1000 DNA marker). The isolated strain DNA was amplified by PCR with primer pair 1 and the product size was 531 bp. After PCR amplification with primer pair 2, the product size was 518 bp. After PCR amplification with primer pair 3, the product size was 970 bp, which was in line with expectations. The strain is Bacteroides fragilis.

The PCR product was subjected to nucleotide sequence determination (Shenzhen Huada Gene Technology Co., Ltd.) for a total of 2 samples. The sequencing results were performed on a BLAST alignment (http://www.ncbi.nlm.nih.gov/BLAST/) on Genbank (a database of DNA sequences established by the National Center for Biotechnology Information), see Table 3.

The results showed that it was isolated as a strain of Bacteroides fragilis.

Table 4 shows the results of BLAST alignment of 16S rRNA sequences (partial)

Table 4

(2) PCR detection of bft gene

The strains screened by the sequencing were inoculated into the medium C, and cultured at 37 ° C for 48 hours under anaerobic conditions. 2 mL of the culture solution was taken, and DNA was extracted using a bacterial DNA extraction kit as a PCR template DNA. The bft gene is amplified using the bft gene primer, and the amplified fragment size should be 294 bp.

Using 20 μL PCR reaction system: 10 μL of Taq enzyme, 2 μL of template DNA, 1 μL of each of the upper and lower primers, and sterile separation 6 μL of water.

The PCR reaction conditions were: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s for 30 cycles and extension at 72 °C for 10 min. The PCR product was subjected to 2% agarose gel electrophoresis, and the electrophoresis conditions were 100 V for 15 min.

The results are shown in Fig. 5. The lanes 1, 2, 3 and 4 are the electrophoresis results of the isolated strain ZY-312; the 5, 6, and 7 are the electrophoresis results of the enterotoxin-producing strain Bacteroides; the lane 8 is the DL1000 DNA marker. Lanes 4 and 5 are amplification products of bft gene primer pair; lanes 1 and 7 are primer pair 2 amplification products; 2, 6 are primer pair 1 amplification products; and 3 lanes are primer pair 3 amplification products.

The results showed that ZY-312 was a strain of Bacteroides fragilis and did not contain the enterotoxin bft gene, which was a new avirulent strain.

2. Whole genome sequencing analysis and identification

Whole genome sequencing of Bacteroides fragilis ZY-312 (Shenzhen Huada Gene Technology Co., Ltd.), the sequencing results are compared with the published strain sequences, using the treebest software to construct the NJ-tree using the adjacency method or using the software PhyML to adopt the maximum However, the maximum likelihood tree is constructed. The phylogenetic tree shows (see Figure 6) that Bacteroides fragilis ZY-312 is on the same branch as Bacteroides fragilis standard strain ATCC25285 (NCTC9343), indicating that Bacteroides fragilis ZY-312 is a new Bacteroides fragilis, and ATCC25285 is homologous.

Virulence gene analysis was performed on the whole genome sequencing results to verify whether it contains the toxin-producing bft gene. The results showed that the whole genome of Bacteroides fragilis ZY-312 does not contain the bft gene, which is a new Bacteroides fragilis that does not produce enterotoxin.

Example 3

Tolerance of Bacteroides fragilis ZY-312 to gastric acid

1. Preparation of artificial gastric juice (according to the preparation method of artificial gastric juice in Chinese Pharmacopoeia in 2010)

23.4 mL of concentrated HCl was dissolved in 100 mL of purified water to obtain dilute hydrochloric acid. Take 8.2 mL of dilute hydrochloric acid, add 400 mL of purified water and 5 g of pepsin (

Pig source, 1:15000), to a volume of 500mL. Artificial gastric juice was obtained by magnetic stirring at 37 ° C overnight.

2, bacterial preparation

The bacterial liquid was collected, centrifuged, the supernatant was discarded, and the physiological saline was resuspended, centrifuged again, and the supernatant was discarded, and the cells were reserved.

3, adding artificial gastric juice to determine the number of viable bacteria

Artificial gastric juice was added to the cells, and resuspended, and the number of viable cells at 0, 1, 2, and 3 hours was measured.

The viable count was performed by a 10-fold serial dilution method: 100 μL of the bacterial solution was added to 900 μL of the medium, and the gradient was gradually diluted to a suitable concentration. Four concentration gradients were applied to each plate point, and each gradient was repeated for 3 times, each time 20 μL was spotted. 37 ° C, anaerobic culture for 48 h, the number of colonies (counting the number of colonies from 3 to 30 concentration gradient).

Viable count (CFU/mL) = sum of three spotted colonies / 3 × 50 × dilution

Table 5 shows the results of gastric acid tolerance test of different strains (data is the log value of live bacteria concentration, h is hour)

Table 5

Probiotics must enter the gastrointestinal tract of the human body and reach a certain concentration in order to function. Probiotics must first pass through the stomach in the state of live bacteria to enter the intestines from the oral cavity to the intestinal tract. The time for food (especially fluid) to pass through the stomach is typically 1-2 hours. According to the different diet structure, the pH value of human gastric juice fluctuates greatly, usually at pH 3.0, which can reach pH 1.5 when fasting or eating acidic food, and up to pH 4-5 when eating alkaline food. The acidic environment activates pepsinogen, which kills bacteria that enter the stomach with food. If probiotics are to have a probiotic effect in the human body, they must have certain acid resistance and pepsin resistance.

The results showed that compared with other strains of Bacteroides fragilis, the viable bacterial concentration of Bacteroides fragilis ZY-312 was still higher after 3h, while the viable concentration of other strains decreased rapidly with time, indicating the tolerance of ZY-312 to gastric acid. Good, with good probiotic potential and application prospects.

Example 4

Bacterial tolerance test of Bacteroides fragilis ZY-312

1. Experimental materials

Tryptone soy broth (TSB, OXOID, item number: CM0129B)

Tryptone soy agar (referred to as TSA, OXOID, article number: CM0131B)

Beef bile powder (Shanghai Shenggong, article number: ON1210)

Fetal bovine serum (MP Biomedicals, article number: 2916754)

2. Preparation of strains and reagents

Bile powder solution: Add bovine bile powder to TSB and set three final concentrations of 10g/L (1% bovine powder), 20g/L (2% bovine powder) and 40g/L (4% bovine). powder). After sterilization, serum (final concentration 50 mL/L) was added for use. At the same time, the TSB without bile powder was used as a control.

Strain culture and collection: strains (ZY-312, Bd312, BF839, ATCC25285) were statically cultured at 37 °C, anaerobic liquid static culture to late logarithmic growth (about 14-16 hours), dispensed into centrifuge tubes, and each tube was dispensed. 3 ml of the bacterial solution was centrifuged at 4000 rpm for 5 minutes at room temperature. The bacteria were washed once with 0.01 M PBS (centrifugation at room temperature, 4000 rpm for 5 minutes), the supernatant was discarded, and the precipitate was used.

3. Culture in artificial bile powder medium

The washed bacteria were resuspended with the above bile powder solution, and the initial bacterial solution concentration was adjusted to 1 × 10 ⁸ CFU/mL with the bile powder-containing solution. At 37 ° C, anaerobic culture for 1, 2, 4 hours, the number of viable bacteria was counted by plating, and the number of bacteria at 0 hours was used as a control. The experiment was done 3 times in parallel.

4. Calculate bacterial tolerance to gallbladder

Comparing the results of the above three time points with the corresponding 0-hour results, the results of the strains tolerant to the bile powder after different time in the artificial bile powder solution were obtained, with mean ± standard deviation and statistical results. Description.

Table 6 shows the results of the anti-biliary powder test of SK08 strain (n=3)

Table 6

The results are shown in Table 4. When observed at 0-4h, ZY-312 can grow normally in 1%, 2%, and 4% bile powder. The higher the viable powder concentration, the higher the viable count, ZY-312 The number of viable bacteria was significantly higher than that of other strain groups. The results indicate that ZY-312 is tolerant to bile salts and is significantly superior to other strains.

Bile salts are sodium or potassium salts formed by the binding of bile acids secreted by hepatocytes to glycine or taurine, which are the main components of bile involved in digestion and absorption. After the bile salt is discharged into the small intestine, most of it is absorbed into the blood by the small intestinal mucosa, and then enters the liver to form bile. The mass concentration of bile salts in the human small intestine fluctuates in the range of 0.03 to 0.3 g/100 mL.

For living cells, bile salts can destroy cell membranes, so tolerance to bile salts is one of the important indicators for evaluating probiotics. Probiotics produce bile salt hydrolase, which catalyzes the hydrolysis of glycine and taurine-bound bile salts to amino acid residues and free bile salts. Strains with bile salt dissociation ability can lower serum cholesterol levels in high cholesterol populations and prevent hypercholesterolemia in normal people. The concentration of bile salts in the digestive tract is not fixed. At the beginning of the digestion and digestion, the mass concentration is 15-20 g/L, and then the mass concentration is reduced to about 3 g/L. Probiotics must survive normal biliary concentrations during passage through the gastrointestinal tract. To colonize the small intestine, they must be resistant to bile salts. Therefore, ZY-312 has a better application prospect than other strains of Bacteroides fragilis.

Example 5

Effect of Bacteroides fragilis ZY-312 on epidemic diarrhea in piglets

Primary reagent

DMEM (Dulbecco's modified eagle medium) was purchased from Shanghai Yingjie Jieji Biological Co., Ltd.; RNA extraction The kit was purchased from OMEGA Bio-Tek;

dNTPs, rTaq enzyme, reverse transcriptase, Marker DL2000, etc. were all purchased from Bao Bio (Dalian) Co., Ltd.

Main instrument

Thermo 3111 CO ₂ incubator, produced by Thermo Electron Corporation;

XWJ3-1 inverted microscope, produced by Chongqing Optical Instrument Factory;

96-well cell culture plate, brand: Nunclon, produced by Thermo Fisher Scientific GmbH, Germany;

2720 PCR Amplifier, produced by Applied Biosystems, USA;

Tanon 4120 gel imaging system, purchased from Guangdong Yuwei Biological Co., Ltd.

20 piglets of 2 days old were randomly divided into 4 groups of 5 heads each. The first group was the first-inset post-dose group. The piglets were artificially challenged with porcine epidemic diarrhea at 2 days of age, and the oral test bacteria were started on the second day after the challenge, 2 mL/time/day, continuous administration for 7 days; The second group was the first dose group, and the oral test bacteria were started on the second day after the birth of the piglets, 2mL/time, 2 times/day (1 time in the morning and evening), and the epidemic diarrhea was artificially challenged after 3 days of continuous feeding; The third group was the disease control group. Piglets were challenged with porcine epidemic diarrhea at 2 days of age as a positive control for the disease; the fourth group was a blank control group, and no treatment was performed. Artificial breast feeding was performed every day as a normal group control.

After the start of the experiment, the incidence and mortality of piglets were observed and collected. Piglet feces and vomit were collected for PCR detection of porcine epidemic diarrhea virus. The dead piglets were dissected and the intestinal lesions were observed. The small intestines were collected and fixed with tissue fixative. After the end, the live piglets were sacrificed, the intestinal lesions were observed, and the small intestines were collected and fixed with tissue fixative.

Table 7 shows the anti-porcine epidemic diarrhea of Bacteroides fragilis ZY-312

Table 7

As shown in Table 7, the incidence: the mortality of the first group and the second group was 0, and there were 2, 1 vomiting and diarrhea, respectively.

At the end of the observation period, the recovery was normal and the virus test was negative. The third group had a mortality rate of 60%, five had vomiting and diarrhea, and the virus was positive. The fourth group is normal.

The changes of tissue were observed: the stomach wall and small intestine wall of the first and second groups were normal, no congestion was observed, mesenteric capillaries were normal; the third group was mesenteric congestion, mesenteric lymph node hyperemia, intestinal wall thinning, stomach wall bleeding The fourth group of stomach wall, mesenteric lymph node and intestinal wall were normal, see Figure 7A-7D, Figure 7A is the first group, the stomach wall and small intestine wall are normal, no congestion, mesenteric capillaries are normal; Figure 7B is the first In the second group, there was no congestion in the mesentery and the stomach wall was normal. The small intestine wall is thin; Figure 7C is the third group, mesenteric congestion, mesenteric lymph node hyperemia, intestinal wall thinning, stomach wall bleeding embolism; Figure 7D is the fourth group, the stomach wall, mesenteric lymph nodes and intestinal wall are normal.

The experimental results confirmed that Bacteroides fragilis ZY-312 can resist porcine epidemic diarrhea virus, prevent and/or treat epidemic diarrhea in piglets, repair intestinal wall of piglets, and improve the survival rate of piglets.

Example 6

Application of Bacteroides fragilis ZY-312 in piglet breeding

Fermentation of Bacteroides fragilis ZY-312: Activated ZY-312 was inoculated into tryptic soy broth culture medium and anaerobic cultured at 37 ° C for 12 h. The method of using Bacteroides fragilis ZY-312 in feed: the lyophilized and pulverized powder of the fermenting bacteria are thoroughly stirred and used until the basal diet is added, and the amount of Bacteroides fragilis ZY-312 added in the feed is ≥1× 10 ⁶ CFU/g.

50 weaned piglets aged around 24 days were divided into 5 groups according to body weight, litter size and gender. They were control group, antibiotic control group, low dose group, middle dose group and high dose group. head. The antibiotic control group was supplemented with colistin sulfate for the basal diet, and the other three treatment groups were supplemented with Bacteroides fragilis ZY-312 probiotic powder, which were low dose group (B. fragilis ZY-312 was 1×10 ⁷ CFU/g), middle dose group (1×10 ⁸ CFU/g of Bacteroides fragilis ZY-312), high dose group (1×10 ⁹ CFU/g of Bacteroides fragilis ZY-312).

During the test, the piglets were fed separately according to the group, and their eating, drinking, activities, etc. were observed, diarrhea and the like were recorded, and the food intake of each group was recorded in detail. Each litter was weighed on an empty stomach 0 and 30 days after the start of the test.

Calculation method: daily gain = (average weight at the end of the test - initial average body weight) / test days; average daily feed intake = total consumption / total feeding day; feed to meat ratio = total consumption / total weight gain; diarrhea Rate = number of diarrhea / total feeding days × 100%.

Table 8 shows the effect of Bacteroides fragilis ZY-312 on the growth of piglets

Table eight

Table 9 shows the effect of Bacteroides fragilis ZY-312 on diarrhea rate in piglets

Table 9

The above results indicate that (see Tables 8 and 9), Bacteroides fragilis ZY-312 can increase the growth rate of piglets, reduce the ratio of feed to meat, and reduce the rate of diarrhea in piglets.

Example 7

Application of Bacteroides fragilis ZY-312 in sow breeding

Fermentation of Bacteroides fragilis ZY-312: Activated ZY-312 was inoculated into tryptic soy broth culture medium and anaerobic cultured at 37 ° C for 12 h. The method of using Bacteroides fragilis ZY-312 in feed: the lyophilized and pulverized powder of the fermenting bacteria are thoroughly stirred and used until the basal diet is added, and the amount of Bacteroides fragilis ZY-312 added in the feed is ≥1× 10 ⁶ CFU/g.

Twenty sigma female sows aged 55-60 days with age, weight, body condition and parity were selected and randomly divided into 2 groups, control group and test group, 10 rats in each group. The control group was fed with basal diet. The experimental group was fed the basal diet + Bacteroides fragilis ZY-312 powder, and the Bacteroides fragilis ZY-312 ≥ 1 × 10 ⁶ CFU / g. Indicator determination: Count the number of litters per litter, the number of live pigs, the birth weight and the birth weight; use the remaining material of the clear bottom tank to calculate the actual amount of feed.

Test animal management: The two groups have the same management conditions, according to the routine operation of daily management, one round and one sow, all pigs in the same pig house, managed by the same person, fed 3 times a day, free drinking water, all sows An average of 2.5 kg/day of feeding was restricted.

(1) Effect of Bacteroides fragilis ZY-312 on sow performance

Table 10 shows the breeding and breeding of sows

Table ten

According to the test results, the average litter size of the test group was 7.6% higher than that of the control group, the average birth weight was 4.7% higher than that of the control group, the average newborn litter weight was increased by 5.5%, and the average litter size and litter size of the litter were lower. They were reduced by 67% and 91%, respectively, with significant differences.

(2) Improvement of feed utilization rate of sows by Bacteroides fragilis ZY-312

The test time was 60 days, and the feed consumption of the sow during the test period is shown in the following table.

Table 11 shows the feed consumption of sows during the test period.

Table XI

The test results showed that (see Table 10 and Table XI), during the whole test period, the test group consumed 150Kg of head and the control group consumed 158kg. The test group reduced the consumption of the head by 5% compared with the control group, and the feeding cost decreased. .

Example 8

Application of Bacteroides fragilis ZY-312 in chicken breeding

Fermentation of Bacteroides fragilis ZY-312: Activated ZY-312 was inoculated into tryptic soy broth culture medium and anaerobic cultured at 37 ° C for 12 h. The method of using Bacteroides fragilis ZY-312 in the feed: the freeze-dried and pulverized bacterial powder of the fermenting bacteria are thoroughly stirred and used until the base diet is used.

500 chicks were randomly divided into 4 groups, 125 in each group, 1 group was the control group, 2 groups, 3 groups, and 4 groups were the experimental group. 2, 3, 4 are low dose group (B. fragilis ZY-312 is 1 × 10 ⁷ CFU / g), medium dose group (B. fragilis ZY-312 is 1 × 10 ⁸ CFU / g), high dose group (Bacteroides fragilis ZY-312 is 1 × 10 ⁹ CFU / g). The basic composition of the test group diet and the control diet were the same, the only difference was that the test group diet was supplemented with Bacteroides fragilis ZY-312 powder. From the beginning to the end of the test, the test group and the control group should be strictly separated. The management of the control group was carried out in accordance with the normal management method. All daily drugs and antibiotics were carried out according to the standards, and the disinfecting reagents used were the same as before; the test group did not use all antibiotic drugs. In the feed, the original breeding material was used 1 week before the brooding (1-7 days old), that is, the feed was consistent with the control group, and the B. fragilis ZY-312 powder was added to the feed in the second week.

The temperature and humidity control temperature in the house should not rise more than 5 °C per week, and the fluctuation should not exceed 2 °C every day. The effects of Bacteroides fragilis ZY-312 powder on the survival rate, weight of the basket, ratio of meat to meat and the number of rows of chicks are shown in Table 12.

Table 12 shows the application of Bacteroides fragilis ZY-312 in raising chickens

Table 12

The results in Table 12 showed that compared with the control group, the experimental composition, the weight of the slaughter, and the ratio of the meat to the meat were significantly different, and the survival rate was significantly higher than that of the control group.

The application of the Bacteroides fragilis ZY-312 of the present invention in preventing and/or treating gastrointestinal diseases of animals can be used for controlling intestinal diseases of animals, improving immunity and disease resistance of animals, reducing the prevalence of animals, and improving animals. Feed conversion rate, increase animal body weight, promote its growth and development performance, improve the quality of meat, eggs, milk and other products, can also eliminate antibiotic drug residues, purify the environment and protect the ecological environment. The bacteria is safe and reliable, and has a good development prospect in the field of animal husbandry.

The invention may, of course, be embodied in a variety of other embodiments without departing from the spirit and scope of the invention. Changes and modifications are intended to be included within the scope of the appended claims.

Claims (11)

1. Application of Bacteroides fragilis in animal breeding.
2. The Bacteroides fragilis application according to claim 1, wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312, and the accession number is CGMCC No. 10685.
3. The use of Bacteroides fragilis in the prevention and/or treatment of gastrointestinal diseases of animals, the Bacteroides fragilis is Bacteroides fragilis ZY-312, and the accession number is CGMCC No. 10685.
4. A pharmaceutical composition for preventing and/or treating gastrointestinal diseases in an animal, characterized in that the pharmaceutical composition comprises a pharmaceutically effective amount of Bacteroides fragilis ZY-312.
5. A feed additive for preventing and/or treating gastrointestinal diseases in an animal, characterized in that the feed additive comprises an effective amount of Bacteroides fragilis ZY-312.
6. The feed additive according to claim 4, wherein the effective amount is a dose effective to maintain a healthy intestinal microflora, and the amount of the Bacteroides fragilis ZY-312 added to the feed is ≥ 1 × 10 ⁶ CFU/g.
7. The feed additive according to claim 4, wherein the effective amount is a dose effective to reduce the growth of pathogenic bacteria and viruses in the animal, and the amount of the Bacteroides fragilis ZY-312 added to the feed is ≥1 ×10 ⁶ CFU/g.
8. The feed additive according to claim 4, wherein the effective amount is a dose effective to increase animal feed utilization efficiency or animal survival rate, and the amount of the Bacteroides fragilis ZY-312 added to the feed is ≥1× 10 ⁶ CFU/g.
9. The feed additive according to claim 4, wherein the effective amount is a dose effective to increase the weight of the animal, and the amount of the Bacteroides fragilis ZY-312 added to the feed is ≥ 1 × 10 ⁶ CFU/g.
10. A feed additive according to any one of claims 4-8, wherein the animal is a pig or a poultry.
11. The feed additive according to claim 9, wherein the pig is a piglet or a sow, and the poultry is a chicken.

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