Biological preparation for inhibiting growth of clostridium perfringens and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to a biological preparation for inhibiting growth of clostridium perfringens and application of the biological preparation. It is found that glycyrrhizic acid can inhibit growth of clostridium perfringens and has no obvious inhibiting effect on growth of pediococcus acidilactici and enterococcus faecalis. By compounding glycyrrhizic acid, pediococcus acidilactici and enterococcus faecalis, the bacteriostasis rate on clostridium perfringens can be remarkably improved, and when the concentration of glycyrrhizic acid in fermentation liquor is 150 mg / kg, the addition amount of pediococcus acidilactici is 0.1 mL 1 * 10 < 5 > CFU / mL, and the addition amount of enterococcus faecalis is 0.1 mL 1 * 10 < 7 > CFU / mL, the comprehensive bacteriostasis rate on clostridium perfringens can reach 99.36%; through compounding, the additive amount of glycyrrhizic acid is reduced, the cost is reduced, the antibacterial efficacy is improved, popularization and application are facilitated, and the feed additive is of great significance to development of natural and efficient feed additives.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology and relates to a biological agent for inhibiting the growth of Clostridium perfringens. Background Art

[0002] In broiler production, the incidence of necrotic enteritis is extremely high, mainly showing two symptoms: clinical and subclinical. The clinical symptoms are mainly manifested as a sudden increase in the mortality rate of the chicken flock, a short duration of clinical symptoms, and a mortality rate that can be as high as 50%. Subclinical necrotic enteritis is more common, manifested as decreased appetite, listlessness, huddling motionless, growth retardation, weight loss, bloody diarrhea, intestinal mucosal damage, etc. The hidden nature of this disease has caused huge economic losses to the poultry industry. It is estimated that the economic losses caused by broiler necrotic enteritis globally are as high as $2 billion, and with the expansion of the breeding scale, the losses will be even greater. Chicken necrotic enteritis is mainly caused by Clostridium perfringens, which is a Gram-positive anaerobic spore-forming bacterium, rod-shaped, and the formed spores can survive in various environments, commonly found in wastewater, dust, air, and the intestines of healthy animals. Its size changes with the environment, and it can competitively grow with other microorganisms in the intestine. It is a conditional pathogen, divided into seven subtypes: A, B, C, D, E, F, and G types, and can produce toxins such as α, β, ε, ι, cpe, and NetB. Currently, type A is the main prevalent type globally, but types C, F, and G have also been detected. Among them, types A and G have the most serious impact on poultry, mainly causing avian necrotic enteritis. Inhibiting the growth of Clostridium perfringens is currently the most effective strategy for preventing and treating avian necrotic enteritis.

[0003] Antibiotics are the most effective way to kill Clostridium perfringens. However, with the release of regulations banning the addition of antibiotics to feed, there is an urgent need to develop new biological agents to replace the use of antibiotics. Commonly used biological agents mainly include plant extracts, probiotics, prebiotics, acidifiers, and enzyme preparations, etc. Patent 202310235441.9 discloses a compound probiotic for treating Clostridium perfringens infection and its application, including Bacillus subtilis, Lactobacillus sakei, and Enterococcus faecalis. The formula of this application can effectively improve intestinal damage and intestinal inflammatory response caused by Clostridium perfringens infection; and has the same efficacy as antibiotics, and can be used as a substitute for antibiotics in actual production to prevent and treat Clostridium perfringens. Chen Keyu compounded glycyrrhiza extract and scutellaria extract at different concentrations and found that the Chinese medicine compound could inhibit the growth of Clostridium perfringens. Adding a certain dose of Chinese medicine compound (1000mg / kg) to the feed could relieve the clinical symptoms of chicken necrotic enteritis caused by the colonization of Clostridium perfringens, improve the reduced growth performance caused by the infection; relieve the damage of chicken intestinal structure caused by Clostridium perfringens, and improve the inflammatory response (Chen Keyu. Research on the Therapeutic Effect of Chinese Medicine Compound on Chicken Necrotic Enteritis Caused by Clostridium perfringens [D]. Sichuan Agricultural University, 2023.).

[0004] Therefore, developing biological agents that can inhibit the growth and adhesion of pathogenic bacteria and improve the production performance of animals is one of the currently ideal measures against chicken necrotic enteritis, and it is of great significance for developing anti-chicken necrotic enteritis products without toxic side effects and drug resistance. Summary of the Invention

[0005] To solve the above problems, the present invention proposes a biological agent for inhibiting the growth of Clostridium perfringens and its application.

[0006] The technical solution of the present invention is realized as follows:

[0007] On the one hand, the present application proposes a biological agent for inhibiting the growth of Clostridium perfringens, and the above biological agent contains glycyrrhizic acid, Pediococcus acidilactici, and Enterococcus faecalis.

[0008] Preferably, the concentration of the above glycyrrhizic acid is 50 - 200mg / kg.

[0009] Preferably, the preservation number of the above Pediococcus acidilactici is GDMCC 1.263, preserved in the Guangdong Provincial Microbial Culture Collection Center, and the bacterial liquid concentration is 1×10 5 -1×10 7 CFU / mL.

[0010] Preferably, the preservation number of the above Enterococcus faecalis is CGMCC 1.2135, preserved in the China General Microbiological Culture Collection Center, and the bacterial liquid concentration is 1×10 5 -1×107 CFU / mL.

[0011] On the other hand, the present application also provides a method for inhibiting the growth of Clostridium perfringens. The above method is an anaerobic co-culture method, and the steps are as follows: inoculate the activated glycyrrhizic acid, Pediococcus acidilactici, and Enterococcus faecalis into a liquid medium for fermentation, and co-culture the fermentation broth with Clostridium perfringens.

[0012] Preferably, the above liquid medium is MRS liquid medium or RCM liquid medium.

[0013] Preferably, the concentration of glycyrrhizic acid in the above fermentation broth is 50 - 200 mg / kg, and the bacterial solution concentrations of Pediococcus acidilactici and Enterococcus faecalis are both 1×10 5 -1×10 7 CFU / mL.

[0014] In the third aspect, the above biological agent is used for inhibiting the growth of Clostridium perfringens.

[0015] In the fourth aspect, the above biological agent is used for preparing a product for preventing and treating Clostridium perfringens infection.

[0016] In the fifth aspect, the above biological agent is used for preventing and treating avian necrotic enteritis.

[0017] Preferably, the above avians are broiler chickens.

[0018] The present invention has the following beneficial effects:

[0019] The present application provides a biological agent for inhibiting the growth of Clostridium perfringens and its application, and it is found that glycyrrhizic acid can inhibit the growth of Clostridium perfringens, and has no obvious inhibitory effect on the growth of Pediococcus acidilactici and Enterococcus faecalis. When glycyrrhizic acid, Pediococcus acidilactici, or Enterococcus faecalis acts alone, the bacteriostatic rate also shows an increasing trend with the increase of concentration and addition amount; in order to reduce the dosage of glycyrrhizic acid and lower the cost, the present application combines glycyrrhizic acid, Pediococcus acidilactici, and Enterococcus faecalis, and it is found that the bacteriostatic rate against Clostridium perfringens is significantly improved. When the concentration of glycyrrhizic acid in the co-fermentation broth is 150 mg / kg, the addition amount of Pediococcus acidilactici is 0.1 mL 1×10 5 CFU / mL, and the addition amount of Enterococcus faecalis is 0.1 mL 1×10 7 CFU / mL, the comprehensive bacteriostatic rate against Clostridium perfringens can reach 99.36%; inhibiting the growth of Clostridium perfringens is currently the most effective strategy for preventing and treating avian necrotic enteritis. By combining, the addition amount of glycyrrhizic acid is reduced, the cost is lowered, and the bacteriostatic effect is improved, which is beneficial to popularization and application, and is of great significance for developing natural and highly efficient feed additives. Description of the Drawings

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 The inhibitory effects of glycyrrhizic acid at different concentrations on the growth of Clostridium perfringens.

[0022] Figure 2 The inhibitory effects of different addition amounts of Pediococcus acidilactici on the growth of Clostridium perfringens.

[0023] Figure 3 The inhibitory effects of different addition amounts of Enterococcus faecalis on the growth of Clostridium perfringens.

[0024] Figure 4 The effects of the combination of glycyrrhizic acid, Pediococcus acidilactici and Enterococcus faecalis on the gas production of Clostridium perfringens. Detailed implementation manners

[0025] The following will clearly and completely describe the technical solutions of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0027] The materials used in this application are as follows:

[0028] 1. Strains and plant extracts:

[0029] Clostridium perfringens (ATCC - 13124) and Pediococcus acidilactici (GDMCC 1.263) were both purchased from the Guangdong Provincial Microbial Culture Collection Center; Enterococcus faecalis (CGMCC 1.2135) was purchased from the China General Microbiological Culture Collection Center; glycyrrhiza extract, ≥24%: was purchased from Gansu Fanzhi Pharmaceutical Co., Ltd.

[0030] 2. Main reagents and materials:

[0031] The RCM medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0032] The TSC medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0033] Tryptone was purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0034] Yeast extract powder was purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0035] Anhydrous glucose was purchased from Tianjin Damao Chemical Reagent Factory.

[0036] Anhydrous sodium acetate was purchased from Tianjin Damao Chemical Reagent Factory.

[0037] Ammonium citrate was purchased from Zhengzhou Paini Chemical Reagent Factory.

[0038] Dipotassium hydrogen phosphate was purchased from Tianjin Damao Chemical Reagent Factory.

[0039] Magnesium sulfate was purchased from Tianjin Huadong Reagent Factory.

[0040] Manganese sulfate was purchased from Tianjin Damao Chemical Reagent Factory.

[0041] Tween 80 was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.

[0042] Sodium chloride was purchased from Tianjin Damao Chemical Reagent Factory.

[0043] Agar powder was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0044] 3. Medium:

[0045] MRS liquid medium: Accurately weigh 15 g of tryptone, 10 g of yeast extract powder, 20 g of anhydrous glucose, 5 g of anhydrous sodium acetate, 2 g of ammonium citrate, 2 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate, 0.02 g of manganese sulfate, and 1 mL of Tween, dissolve them in 1000 mL of distilled water, stir until fully dissolved, and sterilize at 121 °C under high-pressure steam for 20 min.

[0046] MRS solid medium: Accurately weigh 15 g of tryptone, 10 g of yeast extract powder, 20 g of anhydrous glucose, 5 g of anhydrous sodium acetate, 2 g of ammonium citrate, 2 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate, 0.02 g of manganese sulfate, 1 mL of Tween, and 20 g of agar powder, dissolve them in 1000 mL of distilled water, stir until fully dissolved, and sterilize at 121 °C under high-pressure steam for 20 min.

[0047] RCM liquid medium (Clostridium enrichment medium): Accurately weigh 3.8 g of RCM medium powder, dissolve it in 100 mL of distilled water, stir until fully dissolved, and sterilize at 121 °C under high-pressure steam for 20 min.

[0048] TSC medium (Tryptone - Sulfite - Cycloserine Agar Base): Accurately weigh 4.7 g of TSC medium powder and dissolve it in 100 mL of distilled water. Stir until fully dissolved, and sterilize it at 121 °C under high - pressure steam for 20 min.

[0049] 4. Activation and cultivation of strains:

[0050] Activation and cultivation of Pediococcus acidilactici: Take out the freeze - dried bacterial powder from the refrigerator (4 °C), pick a small amount of the bacterial powder and activate it in MRS liquid medium. Use the plate coating method to pick a single colony and inoculate it into MRS liquid medium, and incubate it statically in a constant - temperature incubator at 37 °C for 24 h for subsequent experiments.

[0051] Activation and cultivation of Enterococcus faecalis: Take out the freeze - dried bacterial powder from the refrigerator (4 °C), pick a small amount of the bacterial powder and activate it in MRS liquid medium. Use the plate coating method to pick a single colony and inoculate it into MRS liquid medium, and incubate it statically in a constant - temperature incubator at 37 °C for 24 h for subsequent experiments.

[0052] Activation and cultivation of Clostridium perfringens strains: Take out the freeze - dried bacterial powder from the refrigerator (4 °C), pick a small amount of the bacterial powder and activate it in RCM liquid medium. Use the plate pouring method to pick a single colony and inoculate it into RCM liquid medium, and incubate it statically in an anaerobic incubator at 37 °C for 24 h for subsequent experiments.

[0053] Example

[0054] In vitro antibacterial experiment: The antibacterial tests of glycyrrhizic acid, Pediococcus acidilactici, Enterococcus faecalis, and the combined antibacterial tests of glycyrrhizic acid with Pediococcus acidilactici and Enterococcus faecalis all adopt the anaerobic co - culture method.

[0055] 1. Study on the effect of glycyrrhizic acid with different concentrations on inhibiting Clostridium perfringens

[0056] Co - culture different concentrations of glycyrrhizic acid with Clostridium perfringens, and determine the effect of different concentrations of glycyrrhizic acid on inhibiting Clostridium perfringens.

[0057] Co - culture glycyrrhizic acid with final concentrations of 50 mg / kg, 100 mg / kg, 150 mg / kg, 200 mg / kg with Clostridium perfringens. The addition amount of Clostridium perfringens is 0.1 mL of 1×10 8 CFU / mL bacterial solution. Add Clostridium perfringens alone (without adding glycyrrhizic acid) as the control group. Incubate for 24 h, determine the viable count of Clostridium perfringens in the fermentation broth, and calculate the inhibition rate. There are 5 groups in total for the experiment, including 4 experimental groups and 1 control group, with 3 replicates in each group. The experiment uses 20 mL anaerobic tubes, and the reaction system is 15 mL in total.

[0058] Effect of glycyrrhizic acid with different concentrations on inhibiting Clostridium perfringens

[0059] As Figure 1 shown, when co - culturing glycyrrhizic acid at different concentrations with Clostridium perfringens, there were significant differences in the inhibition rates after 24 h. Moreover, the higher the concentration of glycyrrhizic acid added, the higher the antibacterial rate, indicating that glycyrrhizic acid has an obvious inhibitory ability on the growth of Clostridium perfringens. When the concentration of glycyrrhizic acid in the fermentation broth reached 200 mg / kg, the antibacterial rate against Clostridium perfringens reached 98.37%. Considering actual production, the addition amount of 200 mg / kg of glycyrrhizic acid is relatively large. At present, the cost is a bit high, which is not conducive to popularization and application. Therefore, in subsequent research, the concentration of glycyrrhizic acid was reduced.

[0060] 2. Study on the effect of different concentrations of Pediococcus acidilactici in inhibiting Clostridium perfringens

[0061] Co - culture different concentrations of Pediococcus acidilactici with Clostridium perfringens and determine the effect of different concentrations of Pediococcus acidilactici in inhibiting Clostridium perfringens.

[0062] Co - culture Pediococcus acidilactici with an addition amount of 0.1 mL of 1×10 5 CFU / mL, 0.1 mL of 1×10 6 CFU / mL, 0.1 mL of 1×10 7 CFU / mL with Clostridium perfringens. The addition amount of Clostridium perfringens is 0.1 mL of 1×10 8 CFU / mL bacterial liquid. Add only Clostridium perfringens (without adding Pediococcus acidilactici) as the control group. Culture for 24 h, determine the viable count of Clostridium perfringens in the fermentation broth, and calculate the inhibition rate. There are 5 groups in total for the experiment, including 4 experimental groups and 1 control group, with 3 replicates in each group. The experiment uses 20 - mL anaerobic tubes, and the reaction system is 15 mL in total.

[0063] Effect of different concentrations of Pediococcus acidilactici in inhibiting Clostridium perfringens

[0064] As Figure 2 shown, co - culture different addition amounts of Pediococcus acidilactici with Clostridium perfringens for 24 h. The results show that as the addition amount of Pediococcus acidilactici increases, the antibacterial rate against Clostridium perfringens shows an increasing trend, reaching a maximum of 54.55%.

[0065] 3. Study on the effect of different concentrations of Enterococcus faecalis in inhibiting Clostridium perfringens

[0066] Co - culture different concentrations of Enterococcus faecalis with Clostridium perfringens and determine the effect of different concentrations of Enterococcus faecalis in inhibiting Clostridium perfringens.

[0067] Co - culture with an addition amount of 0.1 mL of 1×10 5 CFU / mL, 0.1 mL of 1×10 6CFU / mL, 0.1 mL of 1×10 7 CFU / mL of Enterococcus faecalis and Clostridium perfringens were co - cultured. The addition amount of Clostridium perfringens was 0.1 mL of 1×10 8 CFU / mL bacterial solution. Clostridium perfringens was added alone (Enterococcus faecalis not added) as the control group. After culturing for 24 h, the viable count of Clostridium perfringens in the fermentation broth was measured, and the inhibition rate was calculated. There were 5 groups in total for the experiment, including 4 experimental groups and 1 control group, with 3 replicates in each group. 20 - mL anaerobic tubes were used for the experiment, and the reaction system was 15 mL in total.

[0068] Effect of different concentrations of Enterococcus faecalis on inhibiting Clostridium perfringens

[0069] As Figure 3 shown, Enterococcus faecalis and Clostridium perfringens were co - cultured for 24 h with different addition amounts. As the addition amount of Enterococcus faecalis increased, the inhibition rate of Clostridium perfringens gradually increased, reaching 64.85%.

[0070] 4. Effect of different concentrations of glycyrrhizic acid on the growth of Pediococcus acidilactici in different media

[0071] Different concentrations of glycyrrhizic acid and Pediococcus acidilactici were co - cultured in MRS liquid medium and RCM medium respectively to measure the effect of different concentrations of glycyrrhizic acid on the growth of Pediococcus acidilactici.

[0072] Glycyrrhizic acid with final concentrations of 50 mg / kg, 100 mg / kg, and 150 mg / kg was co - cultured with Pediococcus acidilactici. The addition amount of Pediococcus acidilactici was 0.1 mL of 1×10 6 CFU / mL bacterial solution. Pediococcus acidilactici was added alone (glycyrrhizic acid not added) as the control group. After culturing for 24 h, the pH and viable count of the fermentation broth were measured, and the effect of glycyrrhizic acid on the growth of Pediococcus acidilactici was analyzed by comparing with the control group. There were 8 groups in total for the experiment, including 6 experimental groups and 2 control groups, with 3 replicates in each group. 20 - mL anaerobic tubes were used for the experiment, and the reaction system was 15 mL in total.

[0073] Effect of different concentrations of glycyrrhizic acid on the growth of Pediococcus acidilactici in different media

[0074] As shown in Table 1 and Table 2, after different concentrations of glycyrrhizic acid were co - cultured with Pediococcus acidilactici in RCM and MRS media for 24 h, there were significant differences in pH, but the numerical differences were small; there were significant differences in the viable count in the RCM medium, and the viable count of Pediococcus acidilactici with added glycyrrhizic acid was significantly higher than that of the control group (P < 0.05), while in the MRS medium, the differences were not significant (P > 0.05). In general, whether in the RCM or MRS medium, glycyrrhizic acid does not inhibit the growth of Pediococcus acidilactici, and subsequent orthogonal compounding experiments can be carried out.

[0075] Table 1 pH and viable cell count of Pediococcus acidilactici co-cultured with glycyrrhizic acid at different concentrations in RCM medium

[0076]

[0077] Note: “—” indicates no inhibition rate. Different lowercase letters in the same column of the table represent significant differences (P<0.05), and the same lowercase letters represent no significant differences (P>0.05).

[0078] Table 2 pH and viable cell count of Pediococcus acidilactici co-cultured with glycyrrhizic acid at different concentrations in MRS medium

[0079]

[0080] Note: “—” indicates no inhibition rate. Different lowercase letters in the same column of the table represent significant differences (P<0.05), and the same lowercase letters represent no significant differences (P>0.05).

[0081] 5. Effects of glycyrrhizic acid at different concentrations on the growth of Enterococcus faecalis in different media

[0082] Glycyrrhizic acid at different concentrations was co-cultured with Enterococcus faecalis in MRS liquid medium and RCM medium respectively to determine the effects of glycyrrhizic acid at different concentrations on the growth of Enterococcus faecalis.

[0083] Glycyrrhizic acid with final concentrations of 50mg / kg, 100mg / kg, and 150mg / kg was co-cultured with Enterococcus faecalis. The addition amount of Enterococcus faecalis was 0.1mL of 1×10 6 CFU / mL bacterial solution. Single addition of Enterococcus faecalis (without adding glycyrrhizic acid) was used as the control group. After culturing for 24h, the pH and viable cell count of the fermentation broth were measured, and the effects of glycyrrhizic acid on the growth of Enterococcus faecalis were analyzed by comparing with the control group. There were 8 groups in total in the experiment, including 6 experimental groups and 2 control groups, with 3 replicates in each group. 20mL anaerobic tubes were used in the experiment, and the reaction system was 15mL in total.

[0084] Effects of glycyrrhizic acid at different concentrations on the growth of Enterococcus faecalis in different media

[0085] As shown in Table 3 and Table 4, after co-culturing with Enterococcus faecalis in RCM and MRS media at different concentrations of glycyrrhizic acid for 24 h, there were significant differences in pH, but the numerical differences were small; there were no significant differences in the viable counts of Enterococcus faecalis. Comparing Table 3 and Table 4, the pH of co-culturing different concentrations of glycyrrhizic acid with Enterococcus faecalis in RCM medium was higher than that in MRS medium, and the viable counts of Enterococcus faecalis were lower than those in MRS medium. In conclusion, whether in RCM or MRS medium, glycyrrhizic acid does not inhibit the growth of Enterococcus faecalis, and subsequent orthogonal compounding experiments can be carried out.

[0086] Table 3 pH and viable counts of co-culturing different concentrations of glycyrrhizic acid with Enterococcus faecalis in RCM medium

[0087]

[0088] Note: “—” indicates no inhibition rate. Different lowercase letters in the same column of the table indicate significant differences (P < 0.05), and the same lowercase letters indicate no significant differences (P > 0.05).

[0089] Table 4 pH and viable counts of co-culturing different concentrations of glycyrrhizic acid with Enterococcus faecalis in MRS medium

[0090]

[0091] Note: “—” indicates no inhibition rate. Different lowercase letters in the same column of the table indicate significant differences (P < 0.05), and the same lowercase letters indicate no significant differences (P > 0.05).

[0092] 6. Effects of compounding of glycyrrhizic acid, Pediococcus acidilactici and Enterococcus faecalis on the inhibitory effect against Clostridium perfringens

[0093] Compound different concentrations of glycyrrhizic acid, Pediococcus acidilactici and Enterococcus faecalis according to the orthogonal scheme, co-culture with Clostridium perfringens, and determine the effects of the compounding of glycyrrhizic acid, Pediococcus acidilactici and Enterococcus faecalis on the inhibitory effect against Clostridium perfringens.

[0094] Compound glycyrrhizic acid with final concentrations of 50 mg / L, 100 mg / L, 150 mg / L and Pediococcus acidilactici and Enterococcus faecalis with an addition amount of 0.1 mL of 1×10 5 CFU / mL, 0.1 mL of 1×10 6 CFU / mL, 0.1 mL of 1×10 7 CFU / mL according to the orthogonal scheme, and co-culture with an addition amount of 0.1 mL of 1×10 8Co-culture with Clostridium perfringens at CFU / mL, with single addition of Clostridium perfringens as the control group. Incubate for 24 h, determine the viable count of Clostridium perfringens in the fermentation broth, and calculate the inhibition rate. There are 10 groups in total for the experiment, including 9 experimental groups and 1 control group, with 3 replicates in each group. The experiment uses 20 mL anaerobic tubes, and the reaction system is 15 mL in total.

[0095] Effect of the combination of glycyrrhizic acid, Pediococcus acidilactici, and Enterococcus faecalis on the inhibitory effect against Clostridium perfringens

[0096] Figure 4 As shown, in experimental groups 5, 7, 8, and 9, compared with the control group and other groups, there are the fewest interface bubbles, indicating that the growth of Clostridium perfringens is inhibited.

[0097] As shown in Table 5, experimental groups 5, 6, 7, 8, and 9 are significantly better than other groups. Among them, in experimental group 7, the content of glycyrrhizic acid is 150 mg / kg, the addition amount of Pediococcus acidilactici is 0.1 mL of 1×10 5 CFU / mL, and the addition amount of Enterococcus faecalis is 0.1 mL of 1×10 7 CFU / mL has the strongest inhibitory effect on Clostridium perfringens, and the inhibition rate reaches 99.07%. The optimized combination obtained by orthogonal optimization is A3B2C3, that is, the content of glycyrrhizic acid is 150 mg / kg, the addition amount of Pediococcus acidilactici is 0.1 mL of 1×10 6 CFU / mL, and the addition amount of Enterococcus faecalis is 0.1 mL of 1×10 7 CFU / mL; the primary and secondary relationship of the influence of each factor on the inhibition rate of Clostridium perfringens is: A > B > C, that is, glycyrrhizic acid > Pediococcus acidilactici > Enterococcus faecalis.

[0098] Table 5 Inhibition rate of different concentrations of glycyrrhizic acid, Enterococcus faecalis, and Pediococcus acidilactici against Clostridium perfringens in RCM medium

[0099]

[0100] Table 6 Analysis of variance table for orthogonal experiment

[0101]

[0102] As shown in Table 6, the corrected model P < 0.01, and the difference is extremely significant, indicating that glycyrrhizic acid, Pediococcus acidilactici, and Enterococcus faecalis all have inhibitory effects on the growth of Clostridium perfringens; both the R-square and the adjusted R-square are greater than 0.8, indicating that the orthogonal experiment has a good fitting degree.

[0103] As shown in Table 7, the orthogonal optimal group and Test Group 7 were significantly superior to Test Group 5. The orthogonal optimal group had the highest antibacterial rate, but there was no significant difference from Test Group 7. Considering the addition amount, it was considered that Test Group 7 was the optimal test group. At this time, the concentration of glycyrrhizic acid in the fermentation broth was 150 mg / kg, the addition amount of Pediococcus acidilactici was 0.1 mL 1×10 5 CFU / mL, and the addition amount of Enterococcus faecalis was 0.1 mL 1×10 7 CFU / mL.

[0104] Table 7 Verification Table of Orthogonal Test

[0105]

[0106] The present invention discovers that glycyrrhizic acid can inhibit the growth of Clostridium perfringens, and has no obvious inhibitory effect on the growth of Pediococcus acidilactici and Enterococcus faecalis. The compounding of glycyrrhizic acid, Pediococcus acidilactici and Enterococcus faecalis can significantly improve the antibacterial rate against Clostridium perfringens. When the concentration of glycyrrhizic acid in the fermentation broth is 150 mg / kg, the addition amount of Pediococcus acidilactici is 0.1 mL 1×10 5 CFU / mL, and the addition amount of Enterococcus faecalis is 0.1 mL 1×10 7 CFU / mL, the comprehensive antibacterial rate against Clostridium perfringens is the highest.

[0107] The above are only the preferred embodiments of the present invention, and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A biological agent for inhibiting the growth of Clostridium perfringens, characterized in that: The biological preparation contains glycyrrhizic acid, Pediococcus acidilactici and Enterococcus faecalis.

2. The biological agent according to claim 1, characterized in that: The concentration of the glycyrrhizic acid is 50-200 mg / kg.

3. The biological agent according to claim 1, characterized in that: The preservation number of the lactic acid Pediococcus is GDMCC1.263, and the concentration of the bacterial solution is 1×10 5 -1×10 7 CFU / mL.

4. The biological agent according to claim 1, characterized in that: The deposit number of the Enterococcus faecalis is CGMCC1.2135, and the concentration of the bacterial solution is 1×10 5 -1×10 7 CFU / mL.

5. A method for inhibiting the growth of Clostridium perfringens, characterized in that: The method comprises the following steps: inoculating glycyrrhizic acid and activated Pediococcus acidilactici and Enterococcus faecalis into a liquid culture medium for fermentation, and co-culturing the fermentation liquid with Clostridium perfringens.

6. The method according to claim 5, characterized in that: The liquid culture medium is MRS liquid culture medium or RCM liquid culture medium.

7. The method according to claim 6, characterized in that: The concentration of glycyrrhizic acid in the fermentation broth is 50-200 mg / kg, and the concentration of the bacterial broth of Pediococcus acidilactici and Enterococcus faecalis is 1×10 5 -1×10 7 CFU / mL.

8. Use of the biological agent according to any one of claims 1 to 4 in inhibiting the growth of Clostridium perfringens.

9. Use of the biological agent according to any one of claims 1 to 4 in the preparation of a product for resisting Clostridium perfringens infection.

10. Use of the biological preparation according to any one of claims 1 to 4 in preventing and treating necrotic enteritis in poultry.

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