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CN113308397B - Probiotics for inhibiting clostridium perfringens and screening method and application thereof - Google Patents

Probiotics for inhibiting clostridium perfringens and screening method and application thereof Download PDF

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CN113308397B
CN113308397B CN202110548346.5A CN202110548346A CN113308397B CN 113308397 B CN113308397 B CN 113308397B CN 202110548346 A CN202110548346 A CN 202110548346A CN 113308397 B CN113308397 B CN 113308397B
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clostridium perfringens
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汪以真
姜子鹏
李文涛
文超越
苏维发
路则庆
靳明亮
王凤芹
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Zhejiang University ZJU
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Abstract

The invention discloses probiotics for inhibiting clostridium perfringens and a screening method and application thereof. The probiotic bacteria are Bacillus amyloliquefaciens BA40, which are preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021535. Wherein the probiotic screening source is pig source intestinal content and feces sample. The probiotic strains are preliminarily screened by a double-layer agar method and an agar diffusion experiment, and then two probiotics with obvious inhibition capacity on clostridium perfringens are obtained by acid resistance, cholate resistance, pancreatin resistance and other experiments: bacillus amyloliquefaciens BA 40. The strain is used for animal experiments for preventing the infection of the clostridium perfringens of mice, so that the problems of weight loss, intestinal form damage, intestinal inflammatory reaction and the like caused by the clostridium perfringens infection can be obviously improved, meanwhile, the probiotic treatment group improves the IgA and IgG content in the serum of the mice, obviously improves the intestinal flora structure of the mice, reduces the diarrhea rate of the mice and provides a new way for replacing antibiotics.

Description

Probiotics for inhibiting clostridium perfringens and screening method and application thereof
Technical Field
The invention belongs to the technical field of biology, particularly relates to the field of development and application of functional bacillus, and particularly relates to probiotics for inhibiting clostridium perfringens and a screening method and application thereof.
Background
Clostridium perfringens causes a variety of diseases in humans and other animals, including human food poisoning, lamb dysentery, piglet diarrhea, chicken necrotizing enteritis, and the like. If the sick animals can not be treated correctly and timely, shock and even death of the sick animals can be easily caused, and even if the young livestock infected with clostridium perfringens can survive after treatment, the growth and development of the sick animals can be influenced. Clostridium perfringens is a main pathogenic bacterium of zoonosis, and once pathogenic conditions are triggered, serious economic losses are brought to human health, animal husbandry and other industries.
Probiotic bacteria refer to living microorganisms that, after a certain amount of addition, are capable of producing a health benefit to the host. The bacillus is a microorganism which has good enzyme production capability, and can promote the health of a host, maintain the balance of intestinal microorganisms of the host, improve the immunity of the host, reduce infectious diseases and the like; meanwhile, the probiotics can also generate bioactive substances such as antibiotics and the like to inhibit the colonization of pathogenic microorganisms. In addition, the probiotics has the characteristics of safety, no pollution and no residue, and is an ideal feed additive. The research shows that the bacillus screened by animal source has better antagonistic capability to clostridium perfringens, thereby effectively preventing ileum lesions of animals, necrotizing colitis and other diseases caused by clostridium perfringens. Therefore, probiotics are also likely to be ideal substitutes for antibiotics.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a probiotic for inhibiting clostridium perfringens and a screening method and application thereof.
A probiotic for inhibiting clostridium perfringens is Bacillus amyloliquefaciens (BA 40) with capability of inhibiting clostridium perfringens, which is preserved in China center for type culture collection, No. 299 of eight branches of Wuchang district, Wuhan city, Hubei, with the preservation date of 2021 year, 5 month and 17 days and the preservation number of M2021535.
The application of the probiotics is used for preventing clostridium perfringens infection of animals.
The application process is as follows:
1) preparation of probiotic bacteria liquid
In LB liquidInoculating Bacillus amyloliquefaciens BA40 in the culture medium, and shake culturing at 37 deg.C for 12 hr to make the concentration of the bacterial liquid reach 1.0 × 108~1.0×109CFU/mL, preparing bacillus amyloliquefaciens BA40 bacterial liquid;
2) application of probiotic bacteria liquid
Selecting 24 mice with the age of 6 weeks C57B/L, and carrying out intragastric administration on 1mL of probiotic bacillus amyloliquefaciens BA40 by a BA40+ clostridium perfringens infected group for 10 days continuously, wherein the disease resistance of the mice is obviously improved after the mice are treated by the probiotic bacillus amyloliquefaciens BA40, the weight, the intestinal form and the intestinal barrier of the mice are obviously changed, the serum IgA and IgG expression of the mice is increased, and the expression of proinflammatory factors IL-1 beta, IL-6 and TNF-alpha of the serum of the mice is reduced.
A method for screening probiotics for inhibiting clostridium perfringens comprises the steps of separating and purifying swine excrement to obtain a plurality of bacillus strains, and carrying out primary screening by a double-layer agar method and an agar diffusion method and secondary screening by bile salt, pancreatin and acidity tolerance experiments to obtain the bacillus strains with bacteriostatic activity; the screening steps are as follows:
1) separation and purification: separating bacillus from swine excrement and purifying to obtain multiple bacillus strains;
2) primary screening: detecting the bacteriostatic ability of the bacillus strain obtained in the step 1) on clostridium perfringens to obtain a strain with bacteriostatic ability;
3) re-screening: detecting the pancreatin tolerance, acidity tolerance and cholate tolerance of the bacillus strain with higher pathogenic bacterium inhibition capability obtained in the step 2), and re-screening to obtain the bacillus strain with clostridium perfringens inhibition capability.
The pig source intestinal contents and the excrement are jejunum, ileum contents and excrement samples obtained after the local Jinhua pig is slaughtered at the age of 180 days.
The separation and purification: diluting the porcine intestinal content and the fecal sample by multiple times, and taking 10 times of the diluted porcine intestinal content and the fecal sample-3、10-4、10-5、10-6Four dilution gradients, spread on LB plate, each dilution gradient has at least 3 plates, single colony is obtained in 37 deg.C incubator for 24h, inoculating loop is used to pickRemoving single colony, inoculating in LB liquid culture medium, after 12h, selecting bacterial liquid with inoculating loop, streaking on LB plate, after 24h, obtaining pure separated bacillus strain.
The primary screening: screening strains with a high inhibition effect on clostridium perfringens by detecting the inhibition condition of the strains;
the specific procedure is to add 100. mu.L of 10 to the lower LB solid plate-3、10-4、10-5、10-6Uniformly coating the intestinal contents and the diluent of the fecal sample with four dilution gradients, cooling an upper layer culture medium to about 50 ℃, adding clostridium perfringens according to the proportion of 5%, mixing, immediately pouring into an upper layer flat plate for paving, placing in a constant temperature incubator at 37 ℃ for standing culture for 12h, making at least 3 flat plates for each dilution gradient, and screening the bacillus with the capability of inhibiting clostridium perfringens;
and then extracting the supernatant of the separated bacillus liquid, adding lower-layer agar into an RCM culture medium of clostridium perfringens according to the addition of 5% by using an Oxford cup method, uniformly placing Oxford cups after the lower-layer agar is solidified, adding 100 mu L of bacillus supernatant into the Oxford cups, placing the oxford cups in an anaerobic incubator at 37 ℃ for 24h, repeating three times on each plate, measuring the diameter of a bacteria inhibiting area, and screening the bacillus strain with strong capability of inhibiting clostridium perfringens.
The secondary screening: the bacillus with stronger bacteriostatic ability is obtained after the primary screening and purification, the bacillus strain with high tolerance ability is obtained by taking the commercially available bacillus subtilis PB6 as a reference strain and taking the acid resistance, the cholate resistance and the pancreatin resistance of the strain as the secondary screening indexes.
The invention can have the following beneficial effects:
the strain is used for animal experiments for preventing the infection of the clostridium perfringens of mice, so that the problems of weight loss, intestinal form damage, intestinal inflammatory reaction and the like caused by the clostridium perfringens infection can be obviously improved, meanwhile, the probiotic treatment group improves the IgA and IgG content in the serum of the mice, obviously improves the intestinal flora structure of the mice, reduces the diarrhea rate of the mice and provides a new way for replacing antibiotics.
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FIG. 1 shows the results of experiments on inhibition of Clostridium perfringens by Bacillus.
Fig. 2 is a probiotic growth curve.
FIG. 3 shows the results of body weight changes in mice.
FIG. 4 shows the results of mouse intestinal tract speculum and electron microscope.
Detailed Description
The invention is further illustrated below with reference to the figures and examples.
Example 1 strain screening:
(1) primary screening of bacillus with bacteriostatic ability from porcine intestinal contents and excrement
Taking the jejunum, ileum intestinal contents and excrement sample 1g of 180-day-old Jinhua pigs, and mixing the materials according to the weight ratio of 1: 10, diluting with sterilized normal saline, water bathing at 75 deg.C for 15min, then diluting with 10 times of sterile water in gradient, selecting 10-3、10-4、10-5、10-6And (2) respectively sucking 100 mu L of bacterial suspension to coat on LB plates, wherein each dilution gradient comprises at least 3 plates, cooling the upper layer LB semisolid culture medium to about 50 ℃, adding activated clostridium perfringens (ATCC 13124) according to the proportion of 5%, mixing, immediately pouring into the upper layer plate to pave, placing in a constant temperature incubator at 37 ℃ for standing and culturing for 12h, and screening the bacillus with the capability of inhibiting clostridium perfringens by making at least 3 plates for each dilution gradient and each pathogenic bacterium.
(2) Purification of bacillus and screening of bacillus for inhibiting clostridium perfringens
And (3) selecting a single colony of the primarily screened bacillus by using an inoculating loop, inoculating the single colony in an LB liquid culture medium, after 12 hours, selecting a bacterial liquid by using the inoculating loop, scribing on an LB plate, and after 24 hours, obtaining the pure separated bacillus strain by using the single colony on the LB plate.
And then extracting the supernatant of the separated bacillus liquid, adding lower-layer agar into an RCM culture medium of clostridium perfringens according to the addition of 5% by using an Oxford cup method, uniformly placing Oxford cups after the lower-layer agar is solidified, adding 100 mu L of the supernatant of the bacillus into the Oxford cups, placing the oxford cups in an anaerobic incubator at 37 ℃ for 24h, repeating three times for each plate, measuring the diameter of a bacteriostatic circle, and screening the bacillus strain with strong capability of inhibiting clostridium perfringens. The results of the experiment are shown in FIG. 1.
TABLE 1 determination of the ability of strains to inhibit Clostridium perfringens
Bacterial strains DBJ TSN JH-5 BA40 PB6
Bacteriostatic diameter (Clostridium perfringens)/cm 1.57 1.81 1.81 1.92 1.92
(3) Re-screening: the bacillus strain with high tolerance is obtained by taking the acid resistance, the cholate resistance and the pancreatin resistance of the strain as the double-screening indexes, and the specific process is as follows:
bacillus subtilis PB6 is a probiotic strain with excellent function of inhibiting clostridium perfringens, and is taken as a reference for carrying out tolerance strain screening.
Several purified strains were inoculated in LB liquid medium, cultured at 37 ℃ for 24 hours, and the OD as absorbance was measured every 2 hours600The growth curve of bacillus was measured at the lower optical density values, and the results are shown in fig. 2.
Preparing a bacillus sterile physiological saline bacterial suspension: inoculating the bacillus colony obtained by primary screening and purification in an LB liquid culture medium, culturing for 12h at 37 ℃, taking the culture solution as an inoculation solution, inoculating the culture solution in 50mL of LB liquid culture medium according to the inoculation amount of 2%, and standing and culturing for 12h to obtain a culture solution of the strain. Centrifuging at 4 deg.C at 10000 rpm, collecting thallus for 10 min, washing twice with sterile normal saline, resuspending thallus with sterile normal saline, and adjusting thallus concentration to 1.0 × 108 CFU/mL。
The acidity tolerance of the bacillus is determined by the following specific operations:
adding appropriate amount of 5 mol/L HCL solution into LB liquid culture medium to make pH value of the culture medium reach 3.5, 4.5, 5.5, respectively, and inoculating at 1 × 10 concentration according to 2% inoculation amount8And (3) placing the bacillus sterile physiological saline resuspension of CFU/mL into a shaker at 37 ℃ for culturing, carrying out plate coating and bacterium counting according to a gradient dilution method after 12 hours, repeating three steps in each gradient, and detecting the proliferation capacity of the strain under different pH conditions. The survival rate is calculated according to the formula: wherein, the A control is the number of bacteria in the control group, and the A sample is the number of bacteria in the sample group.
Survival% = a sample/a control × 100
TABLE 2 determination of acid resistance of the strains
Bacterial strains pH = 3.5. survival (%) pH = 4.5. survival (%) pH = 5.5. survival (%)
DBJ 0.10 9.58 31.35
TSN 0.12 6.10 33.11
JH-5 0.08 2.34 30.45
BA40 0.30 9.57 44.53
PB6 0.16 7.69 41.19
The method for measuring the bile salt resistance of the bacillus specifically comprises the following steps:
sterilizing LB solid culture medium under high pressure, cooling to about 50 deg.C, adding pig bile salt, setting three gradient concentrations of 0.2%, 0.3% and 0.4%, pouring into flat plate, and collecting 100 μ L of 1 × 108And (3) coating a CFU/mL bacillus sterile physiological saline heavy suspension, placing in a constant-temperature incubator at 37 ℃ for 12h, and then recording the bacterial count. Survival rateAnd calculating according to a formula.
TABLE 3 determination of the bile salt resistance of the strains
Bacterial strains 0.2% bile salt survival (%) 0.3% bile salt survival rate (%) 0.4% bile salt survival (%)
DBJ 38.24 11.43 0.07
TSN 59.15 12.12 0.13
JH-5 59.43 7.57 0.07
BA40 67.65 15.24 0.59
PB6 62.80 9.88 0.24
The method for measuring the pancreatic enzyme resistance of the bacillus comprises the following specific operations:
sterilizing LB solid culture medium under high pressure, cooling to about 50 deg.C, adding pancreatin, setting three gradient concentrations of 0.2%, 0.5% and 08%, pouring into flat plate, and collecting 100 μ L of 1 × 108And (4) coating a plate with CFU/mL bacillus sterile normal saline resuspension solution, placing the plate in a constant-temperature incubator at 37 ℃ for 12 hours, and then counting the number of bacteria. The survival rate is calculated according to a formula.
TABLE 4 determination of the pancreatic enzyme resistance of the strains
Bacterial strains 0.2% pancreatic enzyme survival (%) 0.5% pancreatic enzyme survival (%) 0.8% pancreatic enzyme survival (%)
DBJ 45.13 19.67 0.89
TSN 74.13 43.36 0.52
JH-5 39.39 22.36 0.77
BA40 86.49 37.70 1.33
PB6 77.38 28.21 1.22
The result shows that the bacillus has certain tolerance to acid, cholate, pancreatin and the like, and BA40 is selected as a clostridium perfringens inhibiting strain according to the conditions of the three indexes, and then the application capability of the clostridium perfringens inhibiting strain is measured.
Example 2: identification of Strain BA40
The 16S 16S rDNA of the strain BA40 is sequenced, and the comparison of Blast with an NCBI database shows that the homology of BA40 with a model strain Bacillus amyloliquefaciens AB301002.1 is 99.72 percent, and the homology with a model strain Bacillus amyloliquefaciens KX137853.1 is 99.38 percent, so the strain is judged to be the Bacillus amyloliquefaciens. The strain BA40 has been preserved in China center for type culture Collection, No. 299 in the eighth place in Wuchang district, Wuhan city, Hubei province, 5.17.2021, with the preservation number of CCTCC NO: M2021535.
Example 3 prevention of Clostridium perfringens infection with Bacillus amyloliquefaciens BA40
1) Preparation of probiotic bacteria liquid
Inoculating the Bacillus amyloliquefaciens BA40 of claim 6 in LB liquid culture medium, and shake culturing at 37 deg.C for 12 hr to reach a bacterial liquid concentration of 1.0 × 108~1.0×109CFU/mL, preparing bacillus amyloliquefaciens BA40 bacterial liquid; according to the same manner, a bacterial solution was prepared from commercially available Bacillus subtilis PB 6.
2) Application of probiotic bacteria liquid
24 mice of C57B/L at 6 weeks old are selected and randomly divided into four groups, namely a control group, a clostridium perfringens infected group, a BA40+ clostridium perfringens infected group and a PB6+ clostridium perfringens infected group. The group infected by BA40+ clostridium perfringens is intragastrically infused with 1mL of probiotics bacillus amyloliquefaciens BA40 and PB6+ clostridium perfringens for 10 consecutive days, and the group infected by the clostridium perfringens is intragastrically infused with probiotics bacillus subtilis PB6 for 10 consecutive days, and the control group is intragastrically infused with PBS with the same dose; on the eleventh day of the experiment, gavages of 1.0X 10 were performed on three groups except the control group8Clostridium perfringens, and slaughter samples within 72 h.
The content of IL-1 beta in serum is determined, and the specific detection method is carried out according to the kit instruction.
The content of IL-6 in serum is determined, and the specific detection method is carried out according to the kit instruction.
The content of serum TNF-alpha is measured, and the specific detection method is carried out according to the kit instruction.
And (3) measuring the serum IgA content, wherein the specific detection method is carried out according to the kit instruction.
The content of IgG in serum is measured, and the specific detection method is carried out according to the kit instruction.
And (3) measuring the sIgA content of the colon, wherein the specific detection method is carried out according to the kit instruction.
The content of DAO in serum is determined according to the instruction of the kit.
And (3) measuring the content of the serum DLA, wherein the specific detection method is carried out according to the kit instruction.
TABLE 5 Effect of example 3 of the invention on growth Performance in mice
Item Control group Infectious group PB6+ infection group BA40+ infected group
Final weight, g 21.65a 18.25c 19.50b 21.20a
Daily average feed intake, g/day 2.99 a 1.09 c 2.07 b 2.85 a
Length of colon in cm 6.61 ab 5.97 c 6.08 bc 6.83 a
Liver index 4.40 c 6.30 a 5.04 b 4.84 bc
Spleen index 0.36 c 1.10 a 0.56 b 0.45 b
Note: the difference of the shoulder marks of the same person is obvious (P <0.05)
As can be seen from table 5 and the corresponding weight change figure 3, probiotic BA40 prevented post-challenge clostridium perfringens, and none of the mouse body weight, daily average food intake, colon length, liver index, spleen index significantly changed (P >0.05) compared to the control group; compared with the clostridium perfringens infected group, the probiotic BA40 has significant changes in the body weight, daily average feed intake, colon length, liver index and spleen index of post-challenge clostridium perfringens mice (P is less than 0.05).
TABLE 6 Effect of example 3 of the invention on proinflammatory factors and immunoglobulins in mouse serum
Item Control group Infectious group PB6+ infected group BA40+ infected group
IL-1β，μg/mL 144.34 ab 153.08a 123.60bc 113.22c
IL-6，μg/mL 138.99b 167.99a 136.07b 132.73b
TNF-α，μg/mL 666.79ab 731.97a 590.29b 564.45b
IgA，μg/mL 422.85a 504.46b 452.28a 434.47a
IgG，μg/mL 59.90ab 78.64b 64.71ab 55.17b
sIgA，μg/mL 17.09b 21.99a 15.99bc 13.98c
DAO，μg/mL 32.85ab 37.93a 38.28a 26.07b
DLA，μg/mL 103.01b 119.49a 111.57ab 103.36b
As can be seen from Table 6, compared with the control group, the probiotic BA40 has no significant change in the contents of IL-1 beta, IL-6, TNF-alpha, IgA, IgG, sIgA, DAO and DLA in mice of Clostridium perfringens after preventing post-challenge (P > 0.05); compared with a clostridium perfringens infected group, the probiotic BA40 prevents the IL-1 beta, IL-6, TNF-alpha, IgA, IgG, sIgA, DAO and DLA contents of post-challenge clostridium perfringens mice from being obviously changed (P is less than 0.05).
TABLE 7. Effect of example 3 of the invention on the height of villi and depth of crypts in the mouse intestinal tract
Item Control group Infectious group PB6+ infected group Feeling of BA40+Dyeing group
Height of fluff, μm 410.25 210.3 397.85 415.36
Depth of crypt, μm 80.3 120.33 79.68 75.44
Hiding ratio of velvet 5.11 1.75 4.99 5.51
As can be seen from table 7 and the corresponding intestinal HE staining, scanning electron microscopy and transmission electron microscopy images of fig. 4, compared to the control group, probiotic BA40 prevented post-challenge clostridium perfringens, and the height of villi, crypt depth and hiding ratio of villi in mice were all not significantly changed (P > 0.05); compared with the clostridium perfringens infected group, probiotic BA40 prevented significant changes in post-challenge clostridium perfringens mice in villus height, crypt depth and crypt ratio (P < 0.05).
From the above, we can know that: the bacillus amyloliquefaciens BA40 provided by the invention has the capability of inhibiting clostridium perfringens, and meanwhile, the probiotic preparation can effectively prevent mice from infecting clostridium perfringens, improve the disease resistance of organisms of the mice, and simultaneously maintain the intestinal immunity and the intestinal shape capability.

Claims (2)

1. The probiotic bacteria for inhibiting clostridium perfringens are characterized in that Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BA40 with clostridium perfringens inhibiting capability is preserved in China center for type culture collection, No. 299, the preservation date is 2021, 5 and 17 days, and the preservation number is M2021535.
2. Use of probiotic bacteria according to claim 1 for the preparation of a medicament for the prevention of clostridium perfringens infection in an animal.
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Application publication date: 20210827

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Denomination of invention: Probiotics inhibiting Clostridium perfringens and its screening method and application

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