Bacteriostatic composition, and bacterial agent, veterinary drug and feed containing same

Abstract

The invention relates to the technical field of nano engineering and probiotics, in particular to a bacteriostatic composition and a fungicide, a veterinary drug and a feed containing the bacteriostatic composition. The invention provides a bacteriostatic composition prepared by simultaneously mixing nano iron sulfide and lactobacillus, which can protect the intestinal colonization ability of lactobacillus and other beneficial bacteria, so that the nano iron sulfide promotes bacterial ferroptosis of pathogenic bacteria in the intestinal tract. The nano iron sulfide and the lactobacillus synergistically inhibit the growth of pathogenic bacteria such as escherichia coli, salmonella typhimurium and staphylococcus aureus in vitro and in intestinal tracts, have no adverse effect on beneficial bacteria and host intestinal epithelial cells, and are beneficial to intestinal health. The invention provides a natural and efficient application mode, and has very high conversion value.

Description

Technical Field

[0001] This invention relates to the fields of nanoengineering and probiotics, and more specifically to an antibacterial composition and bacterial agents, veterinary drugs and feeds containing the antibacterial composition. Background Technology

[0002] Intestinal bacterial infections are a significant factor in causing bacterial enteritis and diarrhea in humans and animals. Antibiotics are a commonly used treatment for bacterial infections. However, the bactericidal mechanism of antibiotics indiscriminately kills both pathogenic and beneficial bacteria, leading to intestinal ecological imbalance. Therefore, long-term use of antibiotics can easily induce antibiotic-associated diarrhea. Furthermore, antibiotic overuse can lead to the emergence of "superbugs" and environmental pollution. To date, no effective alternative antibiotics have been found for treating bacterial infections.

[0003] Probiotics play a vital role in protecting intestinal homeostasis. Specifically, once a certain number of beneficial bacteria colonize the gut, they inhibit the growth of pathogenic bacteria, thus maintaining healthy intestinal homeostasis. Probiotics are also a common method for treating bacterial infections and diarrhea. However, due to various limiting factors in probiotic colonization in the gut, the use of probiotics alone has not shown good efficacy in treating bacterial infections. Currently, new probiotic utilization methods are emerging, such as combining exogenous compounds with probiotics, genetically engineered probiotics, and probiotics coated with materials, to better colonize the gut, produce abundant beneficial metabiotics, and promote intestinal health. To date, the optimal combination for using probiotics in combination with other probiotics has not yet been discovered.

[0004] Nano-iron sulfide materials possess certain antibacterial properties, effectively killing Gram-positive and Gram-negative pathogens by inducing lipid peroxidation in bacteria, making them a promising antibiotic alternative. However, the effects of nano-iron sulfide on probiotics remain unclear to date. Summary of the Invention

[0005] The present invention provides an antibacterial composition that has antibacterial effect and strong in vitro and in vivo bactericidal activity against Staphylococcus aureus 25923, Escherichia coli K88 and Salmonella typhimurium SL1344.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides an antibacterial composition, wherein the antibacterial composition is prepared by mixing 10 [components] in a volume ratio of 1:1. 5 -10 9It is prepared by combining CFU / ml of Lactobacillus and 1-100 mg / ml of nano-iron sulfide, which is an enzyme-like nanomaterial synthesized by the Nanoenzyme Engineering Laboratory of the Institute of Biophysics, Chinese Academy of Sciences. The Lactobacillus and nano-iron sulfide involved are biocompatible and promote the activity and growth of Lactobacillus both in vivo and in vitro. When the concentration of Lactobacillus is below 10... 5 At a concentration of CFU / ml, its effect is weaker and its action is slower. The highest concentration of vaginal lactobacillus provided in this invention example is 10. 9 CFU / ml represents the plateau phase of vaginal lactobacillus growth, and this concentration is set as the maximum concentration. When the concentration of nano-iron sulfide is below 1 mg / ml, a good combined antibacterial effect cannot be achieved; when the concentration of nano-iron sulfide is above 100 mg / ml, it cannot jointly inhibit lactobacillus.

[0007] In the antibacterial composition, the lactobacillus may include vaginal lactobacillus ( Lactobacillus vaginalis Lactobacillus reuteri ( Lactobacillus reuteri ), Lactobacillus amyloliquefaciens ( Lactobacillus amylovorus ), Lactobacillus mucosa ( Lactobacillus mucosae LM1, Agile Lactobacillus ( Lactobacillus agilis ) and Lactobacillus casei ( Lacticaseibacillus casei The vaginal lactobacillus described therein can inhibit bacteria, resist oxidation, regulate intestinal flora, and has the advantage of protecting the liver and intestines.

[0008] In the antibacterial composition, the lactobacillus includes live, heat-inactivated, lyophilized powder, and fermented forms, and has the advantages of stability, diverse flavors, production of antibacterial postbiotics, and easy digestibility.

[0009] Secondly, the present invention provides a microbial agent containing the above-mentioned antibacterial composition. The microbial agent of the present invention can be a microbial agent made from Lactobacillus after treatment with nano-iron sulfide, or it can be a Lactobacillus microbial agent containing nano-iron sulfide material. It can be a liquid or solid microbial agent, and can be prepared using conventional techniques and by adding excipients permitted in the field of microbial preparations.

[0010] Thirdly, the present invention provides a veterinary drug containing the above-mentioned antibacterial composition.

[0011] Fourthly, the present invention provides a feed in which lactobacillus and nano-iron sulfide are contained at a ratio of 10 mg / kg of feed. 9This product is made by mixing CFU of Lactobacillus with 100-300 mg of nano-iron sulfide in the form of feed. When the dosage of nano-iron sulfide is below 100 mg / kg, a significant synergistic bactericidal effect with Lactobacillus cannot be achieved; when the dosage of nano-iron sulfide is above 300 mg / kg, it will cause the adverse effect of iron overload. The Lactobacillus may include Lactobacillus vaginalis, Lactobacillus reuteri, Lactobacillus amylophilus, Lactobacillus mucosa LM1, Lactobacillus agilei, and Lactobacillus casei.

[0012] In addition, the present invention also provides fermentation products of Lactobacillus treated with nano-iron sulfide, prebiotics and post-biotics of Lactobacillus treated with nano-iron sulfide, and pretreatment and processing steps of Lactobacillus treated with nano-iron sulfide.

[0013] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: It provides an antibacterial composition that effectively kills harmful bacteria such as *Escherichia coli*, *Salmonella typhimurium*, and *Staphylococcus aureus*, promotes the growth and enhances the vitality of beneficial bacteria, has no adverse effects on humans and animals, and simultaneously possesses antioxidant properties, maintaining intestinal homeostasis and intestinal health. The antibacterial composition provided by this invention has good probiotic potential, conversion potential, and the potential to replace antibiotics in treating bacterial infections. It can be used in animal feed products and probiotic pretreatment processes, benefiting animal intestinal health.

[0014] This invention provides a method for protecting probiotics, which utilizes the enzyme-like activity of nanozymes to endow lactobacilli with antioxidant capacity and enhance the tricarboxylic acid cycle and sugar metabolism of lactobacilli, thereby increasing the monosaccharide substrates for lactobacillus growth and enhancing the vitality of lactobacilli. Attached Figure Description

[0015] Figure 1 This invention presents a preferred embodiment of the Oxford cup test and in vivo feeding test of nano-iron sulfide and Lactobacillus. In the figure, A represents the Oxford cup inhibition zone of nano-iron sulfide alone. B represents the Oxford cup inhibition zone 4 of Lactobacillus supernatant and bacterial cells. C represents the Oxford cup inhibition zone of nano-iron sulfide combined with Lactobacillus. The three indicator bacteria in the Oxford cup test were Staphylococcus aureus 25923, Escherichia coli K88, and Salmonella typhimurium SL1344. The negative control was sterile saline, and the positive control was doxycycline. D represents the effect of 50, 100, 300, and 500 mg / kg nano-iron sulfide on inhibiting Salmonella colonization in piglets and the content of the inflammatory factor interleukin-1β (IL-1β). E represents the synergistic effect of nano-iron sulfide and vaginal Lactobacillus on inhibiting the growth of Salmonella, Escherichia coli, and Staphylococcus aureus in the piglet intestine.

[0016] Figure 2This invention provides a preferred embodiment of how nano-iron sulfide promotes the proliferation of Lactobacillus. In this embodiment, A represents the growth curves of Lactobacillus after treatment with different concentrations of nano-iron sulfide. B represents the relative abundance of intestinal Lactobacillus in piglets fed with nano-iron sulfide. C represents the relative abundance of intestinal Lactobacillus in piglets fed with both nano-iron sulfide and Lactobacillus. D represents the enterotype diagram of Lactobacillus in piglets fed with both nano-iron sulfide and Lactobacillus. E represents the Lactobacillus species included in the enterotype formation promoted by nano-iron sulfide, namely Lactobacillus reuteri, Lactobacillus amyloliquefaciens, Lactobacillus mucosa LM1, Lactobacillus agileis, and Lactobacillus casei.

[0017] Figure 3 The preferred embodiment of this invention utilizes nano-iron sulfide to improve the antioxidant capacity of Lactobacillus. Here, A represents the Lactobacillus tricarboxylic acid cycle signaling pathway enriched with nano-iron sulfide. B represents the succinic acid content of Lactobacillus after treatment with nano-iron sulfide. C represents the monosaccharide compound products of Lactobacillus after treatment with nano-iron sulfide. D represents the transcriptional levels of sugar transport and metabolic genes in Lactobacillus after treatment with nano-iron sulfide.

[0018] Figure 4 The preferred embodiment of this invention uses nano-iron sulfide to improve the antioxidant capacity of Lactobacillus. A represents the improvement in the transcriptional level of the oxidoreductase gene in Lactobacillus by nano-iron sulfide. B represents the malondialdehyde level in Lactobacillus. C represents the amount of reactive oxygen species produced by Lactobacillus.

[0019] Figure 5 In a preferred embodiment of the present invention, nano-iron sulfide combined with Lactobacillus improves intestinal health. A represents the histological morphology and score of the piglet colon. B represents the immunohistochemical staining of claudin-1 tight junction protein in the piglet intestine. Detailed Implementation

[0020] The antibacterial composition provided by this invention comprises 1-100 mg / ml of nano-iron sulfide and 10 5 CFU / ml-10 9 Mix CFU / ml of Lactobacillus liquid, fermented Lactobacillus, or fermentation supernatant at a 1:1 ratio and use the mixture directly, or freeze-dry it into a dry powder before use; or add nano-iron sulfide to 1% Lactobacillus in advance, and when the Lactobacillus reaches the maximum viable count, mix the nano-iron sulfide and Lactobacillus mixture well and use directly, or freeze it into a dry powder before use.

[0021] The microbial agent provided by this invention can be prepared by the following method, but is not limited thereto: First, 10% starch is mixed with 50% microcrystalline cellulose, 12% maltodextrin, and 5-10% disintegrant, then granulated and dried to obtain granules. Then, 30% of the prepared Lactobacillus powder (all Lactobacillus types provided by this invention) is mixed with a mixture of nano-iron sulfide and 1.5% aerosol silica in equal increments, and then mixed with the remaining starch to obtain a mixture. The above granules are then mixed with the mixture and 1% lubricant to obtain an intermediate before tableting. Tablets with a hardness of over 5 kg are obtained under a pressure of 5-20 kN. A dry binder is added to control the moisture content to below 3%. The storage conditions of the Lactobacillus agent are controlled; Lactobacillus is stored under anaerobic or semi-anaerobic conditions, and light, low temperature, low humidity, and a suitable pH for Lactobacillus are controlled.

[0022] The veterinary drug provided by this invention can be prepared by the following methods, but not limited to: when nano-iron sulfide is co-cultured with Lactobacillus to the logarithmic growth phase-plateau phase (maximum viable count) or after fermentation, the mixed culture supernatant or fermentation broth is collected, and high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used for detection, screening, extraction, synthesis, or semi-synthesis of antimicrobial drugs, anti-inflammatory drugs, etc. that conform to the veterinary prescription drug catalog.

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0024] Example 1: Inoculate vaginal lactobacilli at a rate of 1% into MRS medium and incubate at 37°C for 10 days. 9 CFU / ml. Add 10 9 CFU / ml vaginal lactobacilli were centrifuged at 10,000 rpm at room temperature, and the supernatant and precipitate were separated. The vaginal lactobacillus precipitate was resuspended in sterile physiological saline. Staphylococcus aureus 25923, Salmonella typhimurium SL1344, and Escherichia coli K88 were used as indicator bacteria and inoculated at 1% into LB solid medium, which was then perforated using sterile Oxford cups. 10 mg / ml nano-ferric sulfide was added, and the resulting 10 mg / ml solution was centrifuged. 9 CFU / ml vaginal lactobacillus bacterial culture supernatant obtained by centrifugation 10 9CFU / ml vaginal lactobacillus cell pellets were used as test reagents, with 200 μl added to each well. Sterile physiological saline was used as a negative control, and 0.5 mg / ml doxycycline as a positive control. The mixtures were incubated at 37°C for 20-24 hours. The size of the inhibition zone was measured using calipers to evaluate the antibacterial effect. Nano-iron sulfide at concentrations of 30 mg / kg, 100 mg / kg, 300 mg / kg, and 500 mg / kg was set as a relief group for Salmonella infection. Serum and feces from piglets were collected for the detection of inflammatory factors and fecal Salmonella counting to screen for suitable nano-iron sulfide concentrations. Vaginal lactobacillus and nano-iron sulfide were added at a ratio of 10 mg / kg feed. 9 CFU of vaginal lactobacillus and 300 mg of nano-iron sulfide were mixed into the diet of weaned piglets and fed freely for 14 days. Colonic digesta of weaned piglets were collected for 16S rRNA microbial sequencing to detect the abundance of Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli in the colon. Figure 1 Results A showed that the inhibition diameters of nano-iron sulfide against Staphylococcus aureus 25923, Salmonella typhimurium SL1344, and Escherichia coli K88 were 4.01 mm, 5.12 mm, and 2 mm, respectively. Figure 1 Results B showed that the antibacterial effects of vaginal lactobacillus supernatant on Staphylococcus aureus 25923, Salmonella typhimurium SL1344, and Escherichia coli K88 were 15.88 mm, 12.22 mm, and 10.00 mm, respectively. Figure 1 The volume ratio of Lactobacillus and iron sulfide used in C was 1:1. The results showed that the combined inhibition diameters of nano-iron sulfide and vaginal Lactobacillus were 27.22 mm, 26.88 mm, and 26.40 mm. Figure 1 Results D showed that, compared with 100 mg / kg, 300 mg / kg and 500 mg / kg, 30 mg / kg of nano-iron sulfide had a poor bactericidal effect in inhibiting Salmonella colonization. Compared with 100 mg / kg and 300 mg / kg, 50 mg / kg and 500 mg / kg of nano-iron sulfide did not alleviate inflammation. Although 500 mg / kg of nano-iron sulfide reduced the colonization of intestinal Salmonella, the high concentration led to an iron overload effect that promoted intestinal inflammation. Therefore, this concentration is not suitable for actual production. Figure 1 The results showed that 300 mg / kg nano-iron sulfide and 10 9 The combined bactericidal effect of CFU / kg *Lactobacillus vaginalis* in the intestines of weaned piglets. In conclusion, nano-iron sulfide and *Lactobacillus vaginalis* have a good combined antibacterial effect.

[0025] Example 2: Lactobacillus vaginalis seed culture was inoculated into MRS liquid medium at a concentration of 1%, with the addition of 0, 1, 10, and 100 mg / ml of nano-iron sulfide. The treated Lactobacillus vaginalis was then incubated at 37°C for 24 hours, and the growth curves of Lactobacillus vaginalis treated with different concentrations of nano-iron sulfide were recorded during the 24-hour period. Nano-iron sulfide was then mixed separately into the diet of weaned piglets at a concentration of 100 mg / kg feed, or mixed with Lactobacillus vaginalis at a concentration of 10 mg / kg feed. 9 CFU of vaginal lactobacillus and 100 or 300 mg of nano-iron sulfide were mixed into the diet of weaned piglets and fed freely for 14 days. Colonic digesta of weaned piglets were collected for 16S rRNA microbial sequencing to detect the relative abundance of colonic lactobacillus. Figure 2 Results showed that adding different concentrations of nano-iron sulfide to the culture medium during in vitro culture of vaginal lactobacilli promoted the growth and viability of vaginal lactobacilli. Figure 2 B and Figure 2 Results showed that nano-iron sulfide can promote the colonization and relative abundance of vaginal lactobacilli in the gut, either alone or in combination with vaginal lactobacilli, and improve the structure of the gut microbiota. Figure 2 Results D showed the lactobacillus enterotype formed in the gut after treatment with nano-iron sulfide. In conclusion, nano-iron sulfide promotes lactobacillus proliferation. Figure 2 The results showed that the lactobacillus types included in the lactobacillus enterotypes promoted by nano-iron sulfide were Lactobacillus reuteri, Lactobacillus amyloliquefaciens, Lactobacillus mucosa, Lactobacillus agileis, and Lactobacillus casei. The abundance of these lactobacillus types increased under the action of nano-iron sulfide, and they jointly formed the lactobacillus enterotypes of piglets, which synergistically killed bacteria with nano-iron sulfide.

[0026] Example 3: Inoculate the vaginal lactobacillus seed culture at a rate of 1% into MRS liquid medium. Treat the inoculated vaginal lactobacillus that has not yet begun to grow with 1 or 10 mg / ml nano-ferric sulfide. Incubate at 37°C. Take samples during the logarithmic growth phase (12 or 18 hours of culture, at which point the number of lactobacilli is approximately 10). 5 Or 10 7 Approximately 0.1 g of Lactobacillus precipitate (CFU / ml) was washed in sterile PBS buffer and then used for prokaryotic transcriptomics sequencing and metabolomics sequencing. Figure 3 Results showed that treatment with nano-iron sulfide enhanced the signaling pathway of the tricarboxylic acid cycle in Lactobacillus. Figure 3 Results B showed that it increased the relative content of succinic acid, an intermediate metabolite in the tricarboxylic acid cycle. Figure 3Results C and D showed that nano-iron sulfide enhanced the gluconeogenesis ability of Lactobacillus, specifically manifested in an increase in the relative content of monosaccharide compounds in Lactobacillus metabolites, and an increase in the transcriptional levels of genes involved in sugar transport and metabolism after treatment with nano-iron sulfide. In conclusion, nano-iron sulfide increased the metabolic substrates and bacterial activity of Lactobacillus.

[0027] Example 4: Add 10 mg / ml of nano-iron sulfide to MRS medium and inoculate with 1% vaginal lactobacilli. Incubate at 37°C and collect samples during the logarithmic growth phase (18 hours of culture, at which point the number of lactobacilli is approximately 10). 7 Approximately 0.1 g of Lactobacillus precipitate (CFU / ml) was washed in sterile PBS buffer and used for prokaryotic transcriptomics sequencing, detection of malondialdehyde levels, and detection of reactive oxygen species production. Figure 4 Results A show that nano-iron sulfide increased the expression of the oxidoreductase gene in *Lactobacillus*. Results B and C show that nano-iron sulfide did not affect reactive oxygen species production or malondialdehyde levels in *Lactobacillus*. In conclusion, nano-iron sulfide enhanced the antioxidant capacity of *Lactobacillus*.

[0028] Example 5: Nano-iron sulfide and lactobacillus promote gut health Vaginal lactobacillus and nano-iron sulfide are added at a ratio of 10 per kg of feed. 9 CFU of vaginal lactobacillus and 300 mg of nano-iron sulfide were mixed into the diet of weaned piglets and provided to them on an ad libitum basis for 14 days. Diarrhea rate, intestinal histological scores, and immunohistochemical staining of the intestinal barrier protein tight junction protein-1 (Claudin-1) were recorded. The diarrhea rate in the control group was approximately 20%; the diarrhea rate in the Salmonella typhimurium group was approximately 80%; the diarrhea rate in the nano-iron sulfide-relief group was 40%; the diarrhea rate in the lactobacillus-only-relief group was 40%; and the combined nano-iron sulfide and lactobacillus improved the diarrhea rate in weaned piglets by 20%. Figure 5 Results showed that nano-iron sulfide and lactobacillus improved the histological morphology of the piglet colon, including the number and size of goblet cells, inflammatory infiltration and inflammatory cell aggregation, and crypt gland morphology. Figure 5 Results B showed that nano-iron sulfide and Lactobacillus improved the expression of intestinal tight junction protein-1 (Claudin-1) and the integrity of the intestinal barrier. In conclusion, nano-iron sulfide and Lactobacillus synergistically promote intestinal health in animals.

[0029] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A composition for antibacterial use, characterized in that, The antibacterial composition is prepared by mixing 10 at a volume ratio of 1:

1. 5 -10 9 It is made from CFU / ml of lactobacillus and 1-100mg / ml of nano-iron sulfide.

2. The antibacterial composition according to claim 1, characterized in that, The lactobacilli include vaginal lactobacilli, Lactobacillus reuteri, Lactobacillus amyloliquefaciens, Lactobacillus mucosae LM1, Lactobacillus agileis, and Lactobacillus casei.

3. The antibacterial composition according to claim 1, characterized in that, The lactobacillus includes live bacteria, heat-inactivated bacteria, freeze-dried powder, and fermented bacteria.

4. A microbial agent, characterized in that, The bacterial agent contains the antibacterial composition according to any one of claims 1 to 3.

5. A veterinary drug, characterized in that, The veterinary drug contains the antibacterial composition according to any one of claims 1 to 3.

6. A feed, characterized in that, The feed contains lactobacillus and nano-iron sulfide at a ratio of 10 mg / kg. 9 It is made by mixing CFU of lactobacillus and 100-300mg of nano-iron sulfide into the diet.

7. The feed according to claim 6, characterized in that, The lactobacilli include vaginal lactobacilli, Lactobacillus reuteri, Lactobacillus amyloliquefaciens, Lactobacillus mucosae LM1, Lactobacillus agileis, and Lactobacillus casei.

Images