A heat-inactivated lactobacillus rod-shaped bacteria agent, a preparation method and application thereof

Abstract

The application discloses a kind of heat inactivated Lactobacillus coryniformis bacterial agent and its preparation method and application.The active ingredient of the heat inactivated Lactobacillus coryniformis bacterial agent is heat inactivated Lactobacillus coryniformis, and the heat inactivated Lactobacillus coryniformis is the bacteria obtained by treating Lactobacillus coryniformis at 80 DEG C for 10 min, and the Lactobacillus coryniformis is Lactobacillus coryniformis NA-3, and its registration number in China General Microbiological Culture Collection Center is CGMCC No.18480.By experiment, it is proved that the heat inactivated Lactobacillus coryniformis NA-3 bacterial agent has antioxidant activity, can effectively scavenge various free radicals, and can be used for preparing antioxidant, to scavenge harmful free radicals of animal or human body;The heat inactivated Lactobacillus coryniformis NA-3 bacterial agent has immunoregulatory activity, and can enhance cellular immunity.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, specifically relating to a heat-inactivated Lactobacillus rod-shaped bacterial agent, its preparation method, and its application. Background Technology

[0002] Lactobacillus has unique and excellent effects in animal husbandry, such as maintaining the balance of intestinal microbiota, preventing animal diseases, and improving the production performance of farmed animals. It can also reduce the use of antibiotics in feed and lower the resistance of related pathogens. Using lactic acid bacteria preparations in the breeding process is one of the effective ways to reduce antibiotic residues in animal feed and reduce the development of drug resistance in farmed animals. However, live lactobacillus has drawbacks such as efficacy degradation and mutation during application, insensitivity to high temperatures and humidity, high viable cell count decay rate and short survival time at room temperature, requiring transportation and storage under low-temperature conditions, and the risk of transferring resistance genes with long-term use, and even causing live bacteria allergies in the host. These drawbacks limit its application in livestock and aquaculture.

[0003] *Lactobacillus corynebacterium* is a common lactobacillus found in fermented vegetables. In naturally fermented sauerkraut from Northeast my country, it accounts for up to 17.29% of the lactic acid bacteria, indicating its abundant source. *Lactobacillus corynebacterium* can secrete bacteriocins, which can control the growth of other microorganisms; it can also secrete lactic acid, effectively inhibiting the proliferation of harmful bacteria. Research on *Lactobacillus corynebacterium* is still limited both domestically and internationally, and its market application is also rare, with even fewer reports on its use in the livestock and aquaculture industries. Research on heat-inactivated lactobacilli focuses primarily on *Lactobacillus plantarum*, with others including *Lactobacillus casei*, *Lactobacillus rhamnosus*, and *Lactobacillus acidophilus*. Currently, no research reports have been found on inactivated *Lactobacillus corynebacterium*. As a lactobacillus with excellent characteristics and abundant sources, *Lactobacillus corynebacterium* is a dominant microorganism in many fermented foods, possessing potential application advantages. The main reason for the lack of practical application of *Lactobacillus corynebacterium* is the weak research on its beneficial life properties, which needs to be strengthened. Summary of the Invention

[0004] The technical problem to be solved by this invention is that the efficacy of active lactobacilli deteriorates and mutates during application, the product is not resistant to high temperature and high humidity, and the number of live bacteria decreases rapidly and the survival period is short at room temperature, and it must be transported and stored under low temperature conditions.

[0005] To address the aforementioned technical problems, the present invention first provides a microbial agent.

[0006] The active ingredient of the bacterial agent provided by the present invention may be heat-inactivated Lactobacillus coryniformis. The heat-inactivated Lactobacillus coryniformis is a bacterium obtained by treating Lactobacillus coryniformis at 80°C for 10 min. The Lactobacillus coryniformis is Lactobacillus coryniformis NA-3, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 18480.

[0007] The above-mentioned microbial agent has at least one of the following properties:

[0008] 1) Antioxidant or free radical scavenger;

[0009] 2) Regulate immune function.

[0010] The free radical may be at least one of ABTS free radical, DPPH free radical, nitric oxide free radical, hydroxyl free radical and superoxide free radical, and the regulation of immune function may be to stimulate macrophages to produce NO, IL-6, TNF-α and / or reactive oxygen species.

[0011] This invention also provides the application of heat-inactivated Lactobacillus rod-shaped bacteria in the preparation of bacterial agents.

[0012] The application of heat-inactivated Lactobacillus coryniformis provided by this invention in the preparation of bacterial agents, wherein the heat-inactivated Lactobacillus coryniformis is a bacterium obtained by treating Lactobacillus coryniformis at 80°C for 10 min, and the Lactobacillus coryniformis is Lactobacillus coryniformis NA-3, whose registration number at the China General Microbiological Culture Collection Center is CGMCC No. 18480.

[0013] In the above applications, the free radical may be at least one of ABTS free radical, DPPH free radical, hydroxyl free radical and superoxide free radical.

[0014] In the above applications, the bacterial agent may have the property of enhancing the ABTS free radical scavenging ability of the Lactobacillus rod-shaped.

[0015] The aforementioned microbial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0016] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0017] Products containing the aforementioned microbial agents are also within the scope of protection of this invention. These products include, but are not limited to, pharmaceuticals, food, health products, animal feed, or animal feed additives.

[0018] The aforementioned drugs may include pharmaceutically acceptable carriers. The carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Various dosage forms can be formulated using these materials, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, microspheres, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. Various carriers known in the art can be widely used to formulate unit dosage forms into suppositories. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. Furthermore, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparation if necessary.

[0019] The above-mentioned drugs may be used for human or animal (veterinary) purposes. The dosage forms of the drugs include, but are not limited to, tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, microspheres, transdermal preparations, lozenges, suppositories, or lyophilized powder injections.

[0020] The method for preparing the above-mentioned bacterial agent also falls within the scope of protection of this invention.

[0021] The above-mentioned method for preparing the bacterial agent may include using heat-inactivated Lactobacillus coryniformis as the active ingredient to obtain the bacterial agent. The heat-inactivated Lactobacillus coryniformis is a bacterium obtained by treating Lactobacillus coryniformis at 80°C for 10 min. The Lactobacillus coryniformis is Lactobacillus coryniformis NA-3, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 18480.

[0022] This invention provides a heat-inactivated Lactobacillus coryniformis NA-3 bacterial agent and its potential applications in antioxidation and immunomodulation. Experiments have demonstrated that the heat-inactivated Lactobacillus coryniformis NA-3 bacterial agent possesses antioxidant activity, effectively scavenging various free radicals, and can be used to prepare antioxidants to eliminate harmful free radicals in animals or humans. Furthermore, the heat-inactivated Lactobacillus coryniformis NA-3 bacterial agent possesses immunomodulatory activity, enhancing intestinal immunity in animals, and can be used in the preparation of immune enhancers in feed.

[0023] Preservation Instructions

[0024] Strain name: Lactobacillus corynebacterium

[0025] Latin name: Lactobacillus coryniformis

[0026] Strain number: NA-3

[0027] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0028] Collection institution abbreviation: CGMCC

[0029] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0030] Date of preservation: September 10, 2019

[0031] CGMCC Registration Number: CGMCC No. 18480 Attached Figure Description

[0032] Figure 1The results are shown by scanning electron microscopy before (A) and after (B) inactivation of Lactobacillus NA-3.

[0033] Figure 2 Results of Lactobacillus NA-3 free radical scavenging (L: active Lactobacillus NA-3; HK: heat-inactivated Lactobacillus NA-3).

[0034] Figure 3 Results of NO production in RAW264.7 macrophages after treatment with live and heat-killed Lactobacillus NA-3.

[0035] Figure 4 Results of RAW264.7 macrophage cell viability after treatment with live and heat-killed Lactobacillus NA-3.

[0036] Figure 5 Results of IL-6(A) and TNF-α(B) production in RAW264.7 macrophages treated with live and heat-killed Lactobacillus NA-3.

[0037] Figure 6 Results of reactive oxygen species (ROS) production in RAW264.7 macrophages treated with live and heat-killed Lactobacillus NA-3.

[0038] Figure 7 The results are shown by scanning electron microscopy before (A) and after (B) inactivation of Lactobacillus casei NA-2.

[0039] Figure 8 Results of scavenging hydroxyl radicals in Lactobacillus casei NA-2 (Live: active Lactobacillus casei NA-2; Heat-killed: heat-inactivated Lactobacillus casei NA-2).

[0040] Figure 9 Results of NO production in RAW264.7 macrophages after treatment with live and heat-killed Lactobacillus casei NA-2.

[0041] Figure 10 Results of RAW264.7 macrophage cell viability after treatment with live and heat-killed Lactobacillus casei NA-2.

[0042] Figure 11Results of IL-6(A) and TNF-α(B) production in RAW264.7 macrophages treated with live and heat-killed Lactobacillus casei NA-2. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0044] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate. IBM SPSS Statistics 26 statistical software was used to process the data. Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and P < 0.05 indicated a significant difference compared to the control.

[0045] The *Lactobacillus coryniformis* NA-3 described in the following examples has been granted Chinese invention patent number CN 110982736B. It was deposited on September 10, 2019, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 18480, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. *Lactobacillus coryniformis* NA-3 is also known as *Lactobacillus coryniformis* CGMCC No. 18480.

[0046] Example 1: Preparation of microbial agent

[0047] This embodiment provides two bacterial agents: a heat-inactivated Lactobacillus coryniformis agent and a live Lactobacillus coryniformis agent. The active ingredient in the heat-inactivated Lactobacillus coryniformis agent is heat-inactivated Lactobacillus coryniformis, obtained by treating Lactobacillus coryniformis NA-3 at 80°C for 10 minutes. The active ingredient in the live Lactobacillus coryniformis agent is Lactobacillus coryniformis NA-3. The specific preparation method is as follows:

[0048] 1. Activation of the strain

[0049] Lactobacillus coryniformis NA-3, frozen at -80℃, was streaked onto MRS solid medium and cultured anaerobically at 37℃ for 48 h. Single colonies from the solid medium were picked and cultured anaerobically at 37℃ for 24 h in 8 mL of MRS liquid medium. The culture was then transferred to 100 mL of MRS liquid medium at a 1% inoculum and cultured anaerobically at 37℃ for 16-18 h to obtain Lactobacillus coryniformis in the logarithmic growth phase. This yielded the Lactobacillus coryniformis NA-3 culture medium.

[0050] 2. Preparation of microbial agents

[0051] 2.1 Preparation of heat-inactivated Lactobacillus rod-shaped bacterial agent

[0052] After centrifuging the fresh *Lactobacillus coryniformis* NA-3 culture medium from step 1 at 10,000 rpm for 5 min, the culture medium was removed, and the bacterial cells were collected. The bacterial cells were washed twice with sterile PBS buffer (phosphate buffer), and the concentration of *Lactobacillus coryniformis* NA-3 was adjusted to 10⁻⁶ using sterile PBS buffer. 8 CFU / mL, take 10 mL of the above bacterial suspension into a 50 mL sterile centrifuge tube, and treat at 80℃ for 5 min, 10 min, 15 min, 20 min and 30 min respectively. After the bacterial suspension returns to room temperature, take 100 μL of the bacterial suspension and spread it on MRS solid medium. Incubate anaerobically at 37℃ for 48 h. No colonies appeared.

[0053] Lactobacillus casei NA-2 was treated with the same heat treatment method as Lactobacillus corynebacterium. Lactobacillus casei NA-2 is described in "Antibacterial potential of a novel Lactobacillus casei strain isolated from Chinese northeast sauerkraut and the antibiofilm activity of its exopolysaccharides." Food & Function 2020, 11(5):4697-4706., and is available to the public from our laboratory. It was also plated on MRS solid medium and anaerobically cultured at 37°C for 48 h. The results showed that some viable bacteria (100 cfu / mL < viable bacteria count < 300 cfu / mL) of Lactobacillus casei NA-2 were still present after treatment at 80°C for 5 min, while no viable bacteria grew in the bacterial suspensions treated at 80°C for 10 min, 15 min, 20 min, and 30 min. To ensure that all live bacteria are inactivated, this invention uses a heat treatment at 80°C for 10 minutes as the treatment condition for obtaining heat-inactivated Lactobacillus rod-shaped bacterial agent.

[0054] 2.2 Preparation of Active Rod-shaped Lactobacillus Inoculant

[0055] The only difference from 2.1 is that the 80℃ heating treatment for 10 minutes was not performed; all other operations are the same. The specific method is as follows: After centrifuging the fresh *Lactobacillus coryniformis* NA-3 culture medium from step 1 at 10000 rpm for 5 minutes, the culture medium was removed, and the bacterial cells were collected. The bacterial cells were washed twice with sterile PBS buffer (phosphate buffer solution), and the concentration of *Lactobacillus coryniformis* NA-3 was adjusted to 10% using sterile PBS buffer. 8 The concentration of cfu / mL was used to obtain an active rod-shaped Lactobacillus inoculum.

[0056] 3. Microstructure of the bacterial agent

[0057] The microstructures of the heat-inactivated *Lactobacillus rod-shaped* bacterial agent (2.1) and the live *Lactobacillus rod-shaped* bacterial agent (2.2) were observed using scanning electron microscopy (SEM). The results are as follows: Figure 1 As shown. Active rod-shaped Lactobacillus inoculant ( Figure 1 (A) and heat-inactivated Lactobacillus rod-shaped bacterial agent ( Figure 1The overall morphology of B) is significantly different. The surface of active rod-shaped lactobacilli cells has many irregular linear attachments, while the surface attachments of heat-inactivated rod-shaped lactobacilli are significantly reduced, and even completely disappeared on some cells. These cell attachments may be extracellular polysaccharides or other attachment components produced by lactobacilli.

[0058] Example 2: Evaluation of the in vitro antioxidant activity of the bacterial agent

[0059] Using the heat-inactivated Lactobacillus rod-shaped bacterial agent from Example 1 (2.1) and the active Lactobacillus rod-shaped bacterial agent from Example 1 (2.2) as samples, their antioxidant activity was evaluated by an in vitro free radical scavenging experiment. The specific operation is as follows:

[0060] 1. ABTS free radical scavenging experiment

[0061] First, mix 5 mL of 7 mM ABTS stock solution with 88 μL of 140 mM potassium persulfate solution, and let stand overnight in the dark. Then, dilute with deionized water until the wavelength at 734 nm is adjusted to 0.700 ± 0.02, thus obtaining ABTS. + Working solution, ready for use.

[0062] Set up an experimental group and a control group, with three replicates for each group, as follows: For the experimental group, administer 400 μL of ABTS... + After mixing the working solution and 100 μL of sample, incubate in the dark for 6 min, centrifuge at 12000 rpm for 5 min, and transfer 200 μL of supernatant to a 96-well plate. OD values ​​are then measured using a microplate reader. 734nm The OD 734nm Named OD Ai The control group received 400 μL of ABTS. + After mixing the working solution with 100 μL of 10 mM PBS (phosphate buffered saline) at pH 7.4, incubate in the dark for 6 min, centrifuge at 12000 rpm for 5 min, and transfer 200 μL of the supernatant to a 96-well plate. OD values ​​are then measured using a microplate reader. 734nm The OD 734nm Named OD Ao The ABTS radical scavenging rate is calculated using the following formula: ABTS radical scavenging rate = (OD Ao -OD Ai ) / OD Ao ×100%.

[0063] 2. DPPH free radical scavenging experiment

[0064] Take 150 μL of sample and mix thoroughly with 150 μL of 0.1 mM DPPH solution (prepared with DPPH as solute and methanol as solvent) to form the sample group; mix thoroughly with 150 μL of 10 mM PBS (phosphate buffered saline) at pH 7.4 to form the blank control group. Perform three replicates for each group. After standing at room temperature in the dark for 30 min, centrifuge at 12000 rpm for 5 min, and then transfer 200 μL of the supernatant to a 96-well plate for OD measurement using a microplate reader. 517 The scavenging rate of free radical DPPH = (OD) 空白 –OD 样品 ) / (OD 空白 )×100%.

[0065] 3. NO free radical scavenging experiment

[0066] 100 μL of sample and 50 μL of sodium nitroprusside solution (100 mM) were mixed to form the sample group; 100 μL of PBS buffer (10 mM, pH 7.4) and 50 μL of sodium nitroprusside solution (100 mM) were mixed to form the blank control group. Each group was performed in triplicate. After standing at room temperature in the dark for 2 h, centrifuged at 12000 rpm for 5 min, 100 μL of the supernatant was transferred to each well of a 96-well plate. 100 μL of Grise's reagent was added to each well, and after standing in the dark for 15 min, the OD was measured using a microplate reader. 550 NO free radical scavenging rate = (OD 空白 –OD 样品 ) / (OD 空白 (×100%). Griess reagent is a reagent formed by mixing aminobenzenesulfonic acid and α-naphthylamine in acetic acid solution. p-Aminobenzenesulfonic acid reagent (Reagent A): Mix 30 mL of glacial acetic acid and 100 mL of deionized water. Weigh 0.5 g of p-aminobenzenesulfonic acid and dissolve it in the above mixture. Store at 4°C protected from light. α-Naphthylamine reagent (Reagent B): Weigh 0.1 g of α-naphthylamine and dissolve it in 100 mL of hot distilled water. After the solution cools to room temperature, add 30 mL of glacial acetic acid. Store at 4°C protected from light. Mix equal volumes of reagent A and reagent B to obtain Griess reagent. It should be prepared fresh before use.

[0067] 4. Hydroxyl radical (·OH) scavenging experiment

[0068] First, 50 μL of PBS (10 mM, pH 7.4), 25 μL of 1,10-phenanthroline (2.5 mM), 25 μL of FeSO4 (2.5 mM), and 25 μL of H2O2 (20 mM) were sequentially added to a 96-well plate. After thorough mixing, 100 μL of the heat-inactivated Lactobacillus rod-shaped bacterial agent from Example 1 (2.1) and the active Lactobacillus rod-shaped bacterial agent from Example 1 (2.2) were added, mixed, and incubated at 37°C for 1 h to form the sample group (As). Then, 50 μL of PBS (10 mM, pH 7.4), 25 μL of 1,10-phenanthroline (2.5 mM), 25 μL of FeSO4 (2.5 mM), and 25 μL of H2O2 were sequentially added to a 96-well plate. After thorough mixing, 100 μL of H2O2 was added... PBS (10 mM, pH 7.4) was mixed and incubated at 37°C for 1 h as the negative control (Ao). 50 μL of PBS (10 mM, pH 7.4), 25 μL of 1,10-phenanthroline (2.5 mM), 25 μL of FeSO4 (2.5 mM), and 25 μL of H2O2 (20 mM) were sequentially added to a 96-well plate, mixed thoroughly, and then 100 μL of PBS (10 mM, pH 7.4) was added. After mixing, the plate was incubated at 37°C for 1 h as the blank control (Ac). All treatments were performed in triplicate. The absorbance of the reaction solution at 536 nm was measured. The scavenging activity of hydroxyl radicals was calculated using the following formula: Scavenging activity (%) = (As-Ac) / (Ao-Ac) × 100%.

[0069] 5. Superoxide radicals (·O) 2 -)Clearance Experiment

[0070] 50 μL of Tris-HCl buffer (pH 8.0, 150 mM) and 25 μL of pyrogallol (1.50 mM, dissolved in 10 mM HCl) were mixed thoroughly, and then 100 μL of the heat-inactivated Lactobacillus caudatus agent from Example 1 (2.1) and the active Lactobacillus caudatus agent from Example 1 (2.2) were added, and the mixture was thoroughly mixed again to form the sample group (A11). 50 μL of Tris-HCl buffer (pH 8.0, 150 mM) and 25 μL of 10 mM HCl were mixed thoroughly, and then 100 μL of the heat-inactivated Lactobacillus caudatus agent from Example 1 (2.1) and the active Lactobacillus caudatus agent from Example 1 (2.2) were added, and the mixture was thoroughly mixed again to form the sample control group (A10). 50 μL of Tris-HCl buffer (pH 8.0, 150 mM) and 25 μL of pyrogallol (1.50 mM, dissolved in 10 mM HCl) were mixed thoroughly to form the sample control group (A10). After mixing with HCl, add 100 μL of PBS (10 mM, pH 7.4) and mix thoroughly again, serving as the negative control group (A01); mix 50 μL of Tris-HCl buffer (pH 8.0, 150 mM) and 25 μL of 10 mM HCl, add 100 μL of PBS (10 mM, pH 7.4) and mix thoroughly again, serving as the blank control group (A00). Incubate at room temperature for 30 min, and measure the absorbance of the reaction solution at 325 nm. Perform three replicates for each treatment group. The ability to scavenge superoxide radicals generated by the autoxidation of pyrogallol is the superoxide radical scavenging activity of the two samples, calculated as follows: Scavenging activity (%) = [1 - (OD) / (10 mM HCl)] / (10 mM HCl) ...] / (10 mM HCl) / (10 mM HCl)] / (10 mM HCl) / (10 mM HCl)] / (10 mM HCl) / (10 mM HCl)] / (10 mM HCl)] / (10 mM HCl)] / (10 mM HCl)] / (10 mM HCl)] / (10 mM HCl)] / (10 mM HCl)] / (10 mM HCl)] / (10 mM HCl)] / (10 A11 -OD A10 ) / (OD A01 -OD A00 )]×100%.

[0071] The above experimental results are as follows Figure 2 As shown in the figure, both active and heat-inactivated Lactobacillus rod-shaped agents have varying degrees of free radical scavenging ability. The two different forms of bacteria have the strongest scavenging ability against ABTS free radicals, followed by DPPH. The scavenging abilities against hydroxyl radicals and superoxide radicals are not significantly different, while the activity against NO free radicals is the weakest.

[0072] Comparing the free radical scavenging activities of active and heat-inactivated Lactobacillus, it can be seen that the heat-inactivated Lactobacillus rod-shaped bacteria can scavenge ABTS with an activity of up to 58%, which is about 12% higher than that of the active strain. For DPPH, hydroxyl radicals and superoxide radicals, although the scavenging activity of the heat-inactivated strain is not significantly better than that of the active strain, they all have different degrees of scavenging efficacy, with scavenging rates of 32%, 20% and 27%, respectively.

[0073] Example 3: Evaluation of the in vitro immunomodulatory activity of heat-inactivated Lactobacillus coryniformis NA-3.

[0074] Macrophages were stimulated with different amounts of live and heat-inactivated Lactobacillus bacteria, and their nitric oxide (NO) levels and cell activity were measured. Their immunomodulatory activity was evaluated by measuring IL-6 and TNF-α cytokines. The specific procedures are as follows.

[0075] 1. Nitric oxide (NO) determination

[0076] RAW264.7 macrophages were seeded in 10cm cell culture dishes, and 7mL of DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (penicillin 10000U / mL, streptomycin 10000μg / mL, solvent 0.01M PBS) was added. The cells were cultured at 5% CO2 and 37°C under constant humidity. Cells were seeded into 96-well plates at a density of 100,000 cells per well, with 200μL of medium per well, and cultured overnight. The heat-inactivated *Lactobacillus* inoculum from Example 1 (2.1) and the live *Lactobacillus* inoculum from Example 2.2 were centrifuged at 12000rpm for 5min, and then resuspended in DMEM medium containing 10% FBS to obtain cells with a concentration of 10... 8 The sample bacterial suspension had a cfu / mL concentration and a bacterial cell count of 10. 7 The sample bacterial suspension and bacterial cell content were 10 cfu / mL. 6 CFU / mL bacterial suspensions were prepared. The overnight cell culture medium in 96-well plates was removed, and different bacterial suspensions were added, with four replicates for each concentration. DMEM medium (10% FBS, 1% penicillin-streptomycin solution) was used as a negative control, and 1 μg / mL lipopolysaccharide (LPS) as a positive control. The plates were incubated at 5% CO2 and 37°C for 24 h. After overnight macrophage culture, different bacterial suspensions were added, and the plates were incubated at 5% CO2 and 37°C for 24 h. The cell culture medium was then collected, centrifuged at 12000 rpm for 5 min, and the supernatant was analyzed for nitric oxide (NO). The Gliese assay was used to determine the NO content in the supernatant.

[0077] Sodium nitrite was used as the standard in this experiment, and the NO content was expressed as nitrite nitrogen. The specific procedure was as follows: 100 μL of supernatant and 100 μL of Griess reagent were added to each well of a 96-well plate. Simultaneously, 100 μL of 0, 6.25, 12.5, 25, 50, and 100 μM sodium nitrite solutions were added as standards, and then added to each well with 100 μL of Griess reagent. Each group was performed in triplicate. The plates were incubated at room temperature for 5 min, and the absorbance of the reaction solution at 550 nm was measured. A standard curve was constructed with the absorbance after the reaction of Griess reagent with sodium nitrite solution as the ordinate (y) and the concentration of sodium nitrite as the abscissa (x). Based on the linear relationship between absorbance and sodium nitrite concentration, the NO content in the cell culture medium, expressed as nitrite nitrogen, was calculated. Griess reagent is a reagent formed by mixing aminobenzenesulfonic acid and α-naphthylamine in acetic acid solution. p-Aminobenzenesulfonic acid reagent (Reagent A): Mix 30 mL of glacial acetic acid and 100 mL of deionized water. Weigh 0.5 g of p-aminobenzenesulfonic acid and dissolve it in the mixture. Store at 4°C protected from light. α-Naphthylamine reagent (Reagent B): Weigh 0.1 g of α-naphthylamine and dissolve it in 100 mL of hot distilled water. After cooling the solution to room temperature, add 30 mL of glacial acetic acid. Store at 4°C protected from light. Mix equal volumes of Reagent A and Reagent B to obtain Griess' reagent, which should be prepared fresh before use.

[0078] The results are as follows Figure 3 As shown, with the increase of the number of active and heat-inactivated Lactobacillus rod-shaped bacteria, the amount of NO produced by the stimulated cells increased. 10 6 The activity of cfu / mL and the heat-inactivated strains did not stimulate the cells to produce NO, mainly because the number of Lactobacillus bacteria was low; when the number of bacteria increased to 10... 7 -10 8 At cfu / mL, live bacterial strains were more effective at stimulating NO production than heat-inactivated strains. However, heat-inactivated strains also have the ability to stimulate NO production in cells and have the potential to replace live bacterial strains, even at concentrations of 10. 8 CFU / mL bacterial density has similar functions to LPS.

[0079] 2. Detection of cell viability

[0080] Cell viability was determined using the MTS method (Promega, Madison, WI, USA).

[0081] RAW264.7 macrophages were seeded in 10cm cell culture dishes, and 7mL of DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (penicillin 10000U / mL, streptomycin 10000μg / mL, solvent 0.01M PBS) was added. The cells were cultured at 5% CO2 and 37°C under constant humidity. Cells were seeded into 96-well plates at a density of 100,000 cells per well, with 200μL of medium per well, and cultured overnight. The heat-inactivated *Lactobacillus* inoculum from Example 1 (2.1) and the live *Lactobacillus* inoculum from Example 2.2 were centrifuged at 12000rpm for 5min, and then resuspended in DMEM medium containing 10% FBS to obtain cells with a concentration of 10... 8 The sample bacterial suspension had a cfu / mL concentration and a bacterial cell count of 10. 7 The sample bacterial suspension and bacterial cell content were 10 cfu / mL. 6 The sample bacterial suspension was prepared at cfu / mL. Macrophages were treated with the above sample bacterial suspension for 24 h, with four replicates for each concentration. DMEM medium (10% FBS, 1% penicillin-streptomycin solution) was used as a negative control, and 1 μg / mL lipopolysaccharide (LPS) was used as a positive control.

[0082] After collecting the cell culture medium, 2 mg / mL MTS solution (100 μL / well) was added to each well of a 96-well plate, and the plates were incubated at 37°C for 3 hours. The absorbance at 490 nm was then measured using a microplate reader. Cell viability (%) = OD 样品 / OD 阴性对照 ×100%. The cell viability of macrophages after treatment with active and heat-inactivated *Lactobacillus* is as follows: Figure 4 As shown, 10 6 The activity of CFU / mL and the heat-inactivated strains do not affect cell growth; however, with increasing bacterial concentration, 10 7 Both forms of *Lactobacillus* present at cfu / mL affected cell viability, with viability 20% lower compared to the negative control group; when the bacterial concentration reached 10... 8 At cfu / mL, cell viability was below 50%, and only 20% of cells treated with live bacteria were viable, significantly lower than that of heat-inactivated Lactobacillus. This indicates that cell viability decreases with increasing bacterial count, suggesting that high bacterial concentrations have a certain impact on cell growth. Compared to live bacteria, heat-inactivated bacteria have a smaller impact on cell viability.

[0083] 3. Measurement of cytokines TNF-α and IL-6

[0084] RAW264.7 macrophages were seeded in 10cm cell culture dishes, and 7mL of DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (penicillin 10000U / mL, streptomycin 10000μg / mL, solvent 0.01M PBS) was added. The cells were cultured at 5% CO2 and 37°C under constant humidity. Cells were seeded into 96-well plates at a density of 100,000 cells per well, with 200μL of medium per well, and cultured overnight. The heat-inactivated *Lactobacillus* inoculum from Example 1 (2.1) and the live *Lactobacillus* inoculum from Example 2.2 were centrifuged at 12000rpm for 5min, and then resuspended in DMEM medium containing 10% FBS to obtain cells with a concentration of 10... 8 The sample bacterial suspension had a cfu / mL concentration and a bacterial cell count of 10. 7 The sample bacterial suspension and bacterial cell content were 10 cfu / mL. 6 The sample bacterial suspension was prepared at cfu / mL. Macrophages were treated with the above sample bacterial suspension for 24 h, with four replicates for each concentration. DMEM medium (10% FBS, 1% penicillin-streptomycin solution) was used as a negative control, and 1 μg / mL lipopolysaccharide (LPS) was used as a positive control.

[0085] Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IL-6 and TNF-α in cell culture medium, employing ELISA Max. TM The Deluxe kit (BioLegend, San Diego, CA, USA) can be used for testing according to the instructions. Results are as follows: Figure 5 As shown. By Figure 5 China A and Figure 5 From B, we know that 10 7 Both active and heat-inactivated lactobacilli produced small amounts of IL-6 and TNF-α, with the active strains significantly stimulating higher cell production; 10 8 CFU / mL live Lactobacillus does not stimulate cells to produce IL-6 and TNF-α, mainly because the cell viability is too low. However, heat-inactivated Lactobacillus can significantly stimulate cells to produce IL-6 and TNF-α and has the potential to be a positive control (LPS).

[0086] 4. Detection of Reactive Oxygen Species (ROS)

[0087] This experiment used 2',7'-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe to measure the intracellular reactive oxygen species (ROS) content. The specific procedure is as follows.

[0088] RAW264.7 macrophages were seeded in 10cm cell culture dishes, and 7mL of DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (penicillin 10000U / mL, streptomycin 10000μg / mL, solvent: 0.01M PBS) was added. The cells were cultured at 5% CO2 and 37°C under constant humidity. Cells were seeded into black 96-well plates at a density of 50,000 cells per well, with 200μL of medium per well, and cultured overnight. The heat-inactivated *Lactobacillus* inoculum from Example 1 (2.1) and the active *Lactobacillus* inoculum from Example 2.2 were centrifuged at 12000rpm for 5min, and then resuspended in DMEM medium containing 10% FBS to obtain cells with a concentration of 10... 8 The sample bacterial suspension had a cfu / mL concentration and a bacterial cell count of 10. 7 The sample bacterial suspension and bacterial cell content were 10 cfu / mL. 6 A sample bacterial suspension with cfu / mL.

[0089] Macrophages were treated with the above-mentioned bacterial suspensions for 24 h, with four replicates for each concentration. DMEM medium (10% FBS, 1% penicillin-streptomycin solution) was used as a negative control, and 1 μg / mL lipopolysaccharide (LPS) as a positive control. After 24 h of treatment, the cell supernatant was removed, and adherent cells were washed with cold PBS buffer (10 mM, pH 7.4) under ice bath conditions. 200 μL of PBS buffer containing 10 μM DCFH-DA was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 30 min. The fluorescence intensity (excitation 485 nm, emission 528 nm) of each well was measured using a Synergy H1 microplate reader. Higher intensity indicated higher reactive oxygen species (ROS) content. The results are shown below. Figure 6 As shown.

[0090] As shown in the figure, 10 6 -10 7 Active *Lactobacillus rod-shaped* at cfu / mL can stimulate the production of intracellular reactive oxygen species. 8 The bacterial concentration (CFU / mL) has a significant impact on cell viability, with lower reactive oxygen species (ROS) production. Compared to live *Lactobacillus*, heat-inactivated *Lactobacillus* exhibits the same efficacy; however, ROS production gradually increases with increasing bacterial concentration, reaching a higher level at 10⁻⁶ CFU / mL. 7 -10 8 The heat-inactivated strains with cfu / mL have the same efficacy as LPS, exhibiting immunomodulatory effects.

[0091] Example 4: In vitro antioxidant evaluation of inactivated Lactobacillus casei NA-2

[0092] 1. The preparation process of Lactobacillus casei NA-2 inoculant and the inactivated Lactobacillus casei NA-2 inoculant is the same as in Example 1. The inactivation conditions are 80℃ for 10 min. The microstructure of Lactobacillus casei NA-2 before and after inactivation is as follows: Figure 7 As shown, there are significant differences between live Lactobacillus casei NA-2 and inactivated Lactobacillus casei strain NA-2; the former has a smooth cell wall, while the latter has wrinkled cell wall surfaces.

[0093] 2. In vitro hydroxyl radical (·OH) scavenging experiment of active and inactivated Lactobacillus casei NA-2 inoculum.

[0094] The operation steps are the same as in Example 2, and the results are as follows: Figure 8 As shown in the results, the ability of *Lactobacillus casei* NA-2 to scavenge hydroxyl radicals showed a consistent trend with that of *Lactobacillus corynebacterium* NA-3, with the active bacterial agent showing higher activity than the heat-inactivated agent. There was no significant difference in the scavenging ability between active *Lactobacillus casei* NA-2 and *Lactobacillus corynebacterium* NA-3, but the scavenging activity of heat-inactivated *Lactobacillus corynebacterium* NA-3 was significantly higher than that of heat-inactivated *Lactobacillus casei*, by approximately 10%.

[0095] Comparison of the hydroxyl radical scavenging results of heat-inactivated Lactobacillus casei NA-2 and Lactobacillus corynebacterium NA-3 shows that the heat-inactivated Lactobacillus corynebacterium NA-3 has a significantly better ability to scavenge hydroxyl radicals than the heat-inactivated Lactobacillus casei NA-2.

[0096] Example 5: Evaluation of the in vitro immunomodulatory activity of heat-inactivated Lactobacillus casei NA-2.

[0097] 1. Cell culture and processing

[0098] The experiment was still conducted using mouse mononuclear macrophages RAW264.7, and the operation steps were the same as in Example 3.

[0099] 2. Nitric oxide (NO) determination

[0100] The operation steps are the same as in Example 3, and the results are as follows: Figure 9 As shown, the results exhibit a similar trend to those for Lactobacillus NA-3. 10 6 The activity of cfu / mL and the heat-inactivated strains did not stimulate the cells to produce NO, mainly because the number of Lactobacillus bacteria was low; when the number of bacteria increased to 10... 7 At cfu / mL, active Lactobacillus casei NA-2 stimulates macrophages to produce large amounts of NO, with yields approaching those of the positive control group (LPS-treated group); while at 10 8 NO was not detected in cells treated with live Lactobacillus casei NA-2 at cfu / mL, a significant difference compared to the results for Lactobacillus rod-shaped NA-3. For heat-inactivated Lactobacillus casei NA-2, 10 7 A bacterial agent at CFU / mL can only stimulate macrophages to produce a very small amount of NO, while 108 A bacterial agent at cfu / mL can stimulate macrophages to produce large amounts of NO, comparable to LPS production.

[0101] 3. Detection of cell viability

[0102] The operation steps are the same as in Example 3, and the results are as follows: Figure 10 As shown. Similar to the effect of *Lactobacillus corynebacterium* NA-3, its macrophage activity decreases with increasing colony count. For heat-inactivated agents, 10... 7 The effects of CFU / mL heat-inactivated Lactobacillus casei NA-2 and heat-inactivated Lactobacillus rod-shaped bacteria NA-3 on macrophage growth were relatively similar. Macrophage activity after treatment with live Lactobacillus casei NA-2 was significantly lower than that after treatment with live Lactobacillus rod-shaped bacteria NA-3, and 10 8 Macrophages treated with cfu / mL live Lactobacillus casei NA-2 showed no detectable cell viability, indicating a significant impact on cell growth. In contrast, live Lactobacillus corynebacterium NA-3 caused significantly less damage to cells than live Lactobacillus casei NA-2.

[0103] 4. Measurement of cytokines TNF-α and IL-6

[0104] The operation steps are the same as in Example 3, and the results are as follows: Figure 11 As shown. Due to 10 8 CFU / mL active Lactobacillus casei NA-2 has a significant impact on cell viability, therefore it will not stimulate cells to produce TNF-α and IL-6. 10 8 CFU / mL heat-inactivated Lactobacillus casei NA-2 stimulates macrophages to produce TNF-α and IL-6. TNF-α production is even comparable to the positive control (LPS), which may lead to overimmunity and inflammation. 10 7 CFU / mL activity and heat-inactivated Lactobacillus casei NA-2 produce relatively small amounts of TNF-α and IL-6. 10 7 There was no significant difference in TNF-α production between cfu / mL live Lactobacillus casei NA-2 and heat-inactivated Lactobacillus casei NA-2-stimulated cells (123 pg / mL - 133 pg / mL). Compared with Lactobacillus casei NA-2, 10 7The effects of live Lactobacillus casei NA-3 (cfu / mL) and heat-inactivated Lactobacillus casei NA-3 on stimulating TNF-α production in cells showed a significant difference. The former produced 138 pg / mL, while the latter produced 61 pg / mL. This indicates that there was almost no difference in TNF-α production stimulated by live Lactobacillus casei NA-2 and live Lactobacillus casei NA-3. However, heat-inactivated Lactobacillus casei NA-2 produced approximately twice as much TNF-α as heat-inactivated Lactobacillus casei NA-3. Since high levels of TNF-α can lead to inflammation, heat-inactivated Lactobacillus casei NA-3 is considered safer. The IL-6 results also showed that 10... 7 CFU / mL live *Lactobacillus casei* NA-2 stimulates cells to produce 66 pg / mL of IL-6, approximately 25 pg / mL lower than live *Lactobacillus cordata* NA-3. However, there is no significant difference in IL-6 production between heat-inactivated *Lactobacillus casei* NA-2 and heat-inactivated *Lactobacillus cordata* NA-3. A comparison shows that 10 7 Both heat-inactivated Lactobacillus casei NA-2 and heat-inactivated Lactobacillus corynebacterium NA-3 (cfu / mL) can stimulate cells to produce TNF-α and IL-6, activating cellular immunity. However, heat-inactivated Lactobacillus corynebacterium NA-3 stimulates cells to produce relatively less TNF-α, has higher safety, and is more suitable for research on immunotherapeutic agents.

[0105] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A microbial preparation, characterized in that, The active ingredient of the microbial preparation is heat-inactivated *Lactobacillus corynebacterium*, which is obtained by treating *Lactobacillus corynebacterium* at 80°C for 10 minutes. The *Lactobacillus corynebacterium* is *Lactobacillus corynebacterium* (…). Lactobacillus coryniformis NA-3, its registration number at the China General Microbiological Culture Collection Center is CGMCC No.18480.

2. The microbial preparation according to claim 1, characterized in that, The microbial preparation has at least one of the following properties: 1) Scavenging free radicals; 2) Regulate immune function.

3. The microbial preparation according to claim 2, characterized in that, The free radical is at least one of ABTS free radical, DPPH free radical, hydroxyl free radical and superoxide free radical. The regulation of immune function involves stimulating macrophages to produce NO, IL-6, TNF-α, and / or reactive oxygen species.

4. Application of heat-inactivated *Lactobacillus* in the preparation of microbial preparations, wherein the heat-inactivated *Lactobacillus* is a bacterium obtained by treating *Lactobacillus* at 80°C for 10 min, and the *Lactobacillus* is *Lactobacillus* (…). Lactobacillus coryniformis NA-3, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 18480; The microbial preparation has at least one of the following properties: 1) Scavenging free radicals; 2) Regulate immune function.

5. The application according to claim 4, characterized in that, The free radical is at least one of ABTS free radical, DPPH free radical, hydroxyl free radical and superoxide free radical.

6. The application according to claim 4 or 5, characterized in that, The microbial preparation has the property of enhancing the ABTS free radical scavenging ability of the Lactobacillus rod-shaped.

7. A product containing any of the microbial preparations described in claims 1-3.

8. The product according to claim 7, characterized in that, The product is a drug, health product, animal feed, or animal feed additive.

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