Lactic acid bacteria extract, its preparation and its antioxidant and radiation-resistant applications

Abstract

The present invention relates to a lactic acid bacteria extract, a preparation method and an application thereof. 60 The intracellular contents of lactic acid bacteria after Co-γ ray irradiation. The lactic acid bacteria extract of the present invention has readily available raw materials, a simple preparation process, high resistance to oxidative damage and radiation damage, and strong application value.

Description

Technical Field

[0001] The present invention relates to a lactic acid bacteria extract, a preparation method and application thereof. Background Art

[0002] With the advancement of science and technology and the widespread application of nuclear technology, ionizing radiation has permeated every aspect of life. X-rays and nuclear waste are common man-made radiation sources; uncharged neutrons and charged alpha particles are natural radiation. While ionizing radiation brings convenience to people, it also has certain health impacts. Ionizing radiation can produce reactive oxygen species (ROS) free radicals in tissues and cells, interfering with macromolecules such as DNA and proteins, inducing cell damage and abnormal cell function, leading to damage to the human and animal body, such as the hematopoietic system, immune system, and digestive system, ultimately causing functional disorders in multiple organs, pathology, and even death.

[0003] With a deeper understanding of radiation damage and protection, many radioprotectants have been discovered. Radioprotectants fall into two categories: one is primarily synthetic drugs, such as tryptophan compounds and sulfur-containing compounds, but these suffer from significant side effects, short duration of action, and high cost. The other is natural radioprotectants, such as polysaccharides and polyphenols, which offer advantages such as long duration of action, low cost, and no significant toxic side effects. Natural radioprotectants are primarily classified into plant polyphenols, plant polysaccharides, alkaloids, saponins, anthocyanins, and vitamins. Plant polyphenols and plant polysaccharides are the most numerous, and plant polysaccharides from various sources have been extensively studied. However, research on radioprotectants derived from microorganisms is relatively limited. The search for new, highly effective, non-toxic, and long-term natural radioprotectants has become a current research priority.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The primary object of the present invention is to provide a lactic acid bacteria extract.

[0006] The second object of the present invention is to provide a method for preparing the lactic acid bacteria extract.

[0007] The third object of the present invention is to provide applications of the lactic acid bacteria extract.

[0008] In order to achieve the purpose of the present invention, the technical solution adopted is:

[0009] The present invention provides a lactic acid bacteria extract, which contains 60 Intracellular contents of lactic acid bacteria after Co-γ ray irradiation.

[0010] Optionally, the lactic acid bacteria extract is a freeze-dried powder of the intracellular matter.

[0011] The present invention also provides a method for preparing the lactic acid bacteria extract, which comprises at least the following steps:

[0012] S1, activate the lactic acid bacteria twice, culture the second generation to the logarithmic phase, and use 60 Co-γ irradiation treatment;

[0013] S2, collect the cells, break them, and collect the supernatant by centrifugation;

[0014] S3. Freeze drying.

[0015] Optionally, the culture temperature is 30-37°C, preferably 35°C.

[0016] Optionally, the 60 The Co-γ irradiation treatment is performed with a total dose of 50 to 750 Gy, preferably 250 to 500 Gy; the irradiation time is 60 to 180 min, preferably 120 min.

[0017] Optionally, in S2, the fermentation liquid after irradiation is centrifuged to collect the bacterial cells and disrupt them;

[0018] The conditions for the disruption are: ultrasonic disruption of cells at 200-500W, working for 2-4s, resting for 4-6s, and the disruption treatment time is 10-20min;

[0019] Preferably, the cells are disrupted by ultrasound at 300 W, with a working period of 3 seconds and a rest period of 5 seconds, and the disruption treatment time is 15 minutes.

[0020] Optionally, the centrifugation condition is 0-6°C, 8000-10000 rpm, and the centrifugation time is 8-12 min.

[0021] Optionally, in S2, the crushing step further includes a washing step;

[0022] The washing step is to wash the bacteria 2 to 4 times with 75 mM phosphate buffer solution (PBS).

[0023] Optionally, in S3, the supernatant is filtered and sterilized and then freeze-dried, and the freeze-drying conditions are:

[0024] Precool it at -80 to -70°C overnight, and freeze-dry it in a vacuum freeze dryer with a cold trap temperature of -60 to -55°C, a vacuum degree of 3 to 5 Pa, and a freezing time of 36 to 48 hours.

[0025] The present invention also relates to the use of the lactic acid bacteria extract in preparing products for resisting and preventing radiation damage.

[0026] The present invention also relates to the use of the lactic acid bacteria extract in preparing products for resisting and preventing oxidative damage.

[0027] The present invention has at least the following beneficial effects:

[0028] The raw materials of the lactic acid bacteria extract of the invention are easily available, the preparation process is simple, the extract has high effects of resisting oxidation damage and radiation damage, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The effect of different radiation doses on the survival of lactic acid bacteria;

[0030] Figure 2 The protective effect of radiation-induced intracellular substances of lactic acid bacteria on H2O2 damage to lactic acid bacteria;

[0031] Figure 3 The radiation protection effect of normal lactic acid bacteria intracellular matter (CFE) on AML-12 cells;

[0032] Figure 4 The radiation protection effect of the lactic acid bacteria intracellular substance (IR-CFE) on AML-12 cells in Example 1;

[0033] Figure 5 The results are a comparison of the radiation protection effects of the normal lactic acid bacteria intracellular matter irradiation group (CFE+IR) and the lactic acid bacteria intracellular matter irradiation group (IR-CFE+IR) of Example 1 on AML-12 cells;

[0034] Figure 6 The effect of lactic acid bacteria intracellular substances on peripheral blood leukocytes of irradiated mice;

[0035] Figure 7 The effect of lactic acid bacteria intracellular substances on peripheral blood red blood cells of irradiated mice;

[0036] Figure 8 The effect of lactic acid bacteria intracellular substances on SOD in the serum of irradiated mice;

[0037] Figure 9 The effect of lactic acid bacteria intracellular substances on MDA in the serum of irradiated mice;

[0038] Figure 10 The effect of lactic acid bacteria intracellular substances on GSH-PX in the serum of irradiated mice;

[0039] Figure 11 The effect of lactic acid bacteria intracellular substances on GSH in the serum of irradiated mice. DETAILED DESCRIPTION

[0040] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0041] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular also includes the plural. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0042] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] Lactic acid bacteria is a general term for bacteria that can produce large amounts of lactic acid using fermentable carbohydrates. 60 Certain components in Co-γ-induced lactic acid bacteria have radiation protection and antioxidant effects, thus completing the present invention. Although radiation stimulation causes certain damage to lactic acid bacteria, it can induce a series of responses in the lactic acid bacteria, allowing them to return to a normal physiological state. Some substances produced during this process can reduce the damage caused by radiation to the body. This type of radiation protectant is directly derived from the lactic acid bacteria cells after radiation induction and can be directly obtained through ultrasonic disruption. The raw materials are readily available, the preparation method is simple, the cost is low, and it can be used for industrial implementation.

[0044] The present invention provides a lactic acid bacteria extract containing 60 The intracellular contents of lactic acid bacteria after Co-γ ray irradiation can be used as a radiation protectant and antioxidant.

[0045] Preferably, the lactic acid bacteria extract is a freeze-dried powder of intracellular matter.

[0046] The present invention also relates to a method for preparing the lactic acid bacteria extract, which comprises at least the following steps:

[0047] S1, activate the lactic acid bacteria twice, culture the second generation to the logarithmic phase, and use 60 Co-γ irradiation treatment;

[0048] S2, collect the cells, break them, and collect the supernatant by centrifugation;

[0049] S3. Freeze drying.

[0050] Optionally, the culture temperature is a constant temperature culture of 30-37°C, preferably 35°C.

[0051] Optional, 60 Co-γ treatment is performed with a total dose of 50 to 750 Gy, preferably 250 to 500 Gy, and the irradiation time is 120 minutes. Studies in the present invention have shown that when lactic acid bacteria are irradiated at low doses and slowly, they activate multiple stress mechanisms to resist the adverse environment, thereby increasing the content of active ingredients in the intracellular matter and improving antioxidant and radiation resistance.

[0052] Optionally, in S2, the fermentation liquid after irradiation is centrifuged to collect the bacterial cells and crush them.

[0053] The disruption conditions of the embodiment of the present invention are: ultrasonic disruption of cells at 200-500W, working for 2-4s, resting for 4-6s, and the disruption treatment time is 10-20min.

[0054] The preferred disruption conditions of the present invention are to disrupt the cells by ultrasound at 300W, with an operating time of 3s and a rest time of 5s, for a disruption time of 15min. Disrupting the cells under these conditions can increase the content of the active ingredient in the extract.

[0055] The centrifugation conditions of the embodiment of the present invention are 0-6° C., 8000-10000 rpm, and a centrifugation time of 8-12 min.

[0056] Optionally, in S2, a washing step is further included before disruption, the purpose of which is to remove the culture medium in the bacterial cells. The washing conditions are: washing the bacterial cells 2 to 4 times with 75 mM phosphate buffered saline (PBS).

[0057] Optionally, in S3, the supernatant is filtered and sterilized and then freeze-dried. The freeze-drying conditions are:

[0058] Precool it at -80 to -70°C overnight, and freeze-dry it in a vacuum freeze dryer with a cold trap temperature of -60 to -55°C, a vacuum degree of 3 to 5 Pa, and a freezing time of 36 to 48 hours.

[0059] In vivo experiments have shown that the lactic acid bacteria extract of the present invention can increase the number of white blood cells and red blood cells in mice after radiation, and can reduce the level of oxidative stress in the serum of mice after radiation. Superoxide dismutase (SOD) is an important enzyme in the body's antioxidant system that can catalyze the dismutation reaction of superoxide anion free radicals and alleviate oxidative damage to cells. The body produces oxygen free radicals through enzyme systems and non-enzymatic systems. The latter can attack polyunsaturated fatty acids in biological membranes, trigger lipid peroxidation, and thus form lipid peroxides, such as malondialdehyde (MDA), etc. Therefore, the MDA content can reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cell damage. Glutathione peroxidase (GSH-PX) is an important peroxide decomposition enzyme that can specifically catalyze the reduction reaction of reduced glutathione (GSH) to lipid peroxides, removing harmful peroxide metabolites in cells. GSH is a substrate necessary for GSH-PX to catalyze the decomposition of peroxides and is one of the most important antioxidants in cells, playing an important role in maintaining cell biological functions. Radiation significantly reduced SOD and GSH-PX enzyme activities and GSH levels in mouse serum, while increasing MDA levels, indicating that radiation can induce oxidative stress in mice. Intragastric administration of the freeze-dried lactic acid bacteria powder of the present invention alleviated oxidative stress levels in the serum of irradiated mice, increased serum antioxidant enzyme activity, and reduced MDA levels, a lipid peroxidation product.

[0060] The present invention also found that the intracellular substances of lactic acid bacteria can significantly improve the survival rate of lactic acid bacteria under H2O2 oxidative stress. This indicates that when lactic acid bacteria are irradiated with low doses and slowly, they activate multiple stress mechanisms to resist the adverse environment. This improves the lactic acid bacteria's antioxidant capacity, and their intracellular substances can scavenge H2O2, reducing the damage caused by H2O2 to the lactic acid bacteria.

[0061] Example 1

[0062] 1. Activate the lactic acid bacteria twice and culture the second generation to the logarithmic phase: Use MRS medium to activate Lactococcus lactis subsp. Lactis IL1403 frozen in a glycerol tube twice, maintain the incubator temperature at around 35°C, and culture the second generation to the logarithmic phase (16 hours);

[0063] 2. Conduct 60 Co-γ ray radiation treatment: 60 The radiation treatment was performed with Co-γ rays, with a total radiation dose of 500 Gy and a radiation treatment time of 120 min.

[0064] 3. Collect the cells by centrifugation and wash: centrifuge at 4000 rpm for 10 min to collect the cells, and wash them twice with 75 mM phosphate buffer;

[0065] 4. Ultrasonic disruption: Suspend 200 mL of the fermentation broth in 50 mL of phosphate buffer and disrupt the cells using ultrasonication. Ultrasonic disruption conditions: 300 W power, 3 seconds on, 5 seconds off, for a total of 15 minutes.

[0066] 5. Centrifuge and collect the supernatant; after crushing, centrifuge at 10000 rpm for 10 minutes to obtain the supernatant;

[0067] 6. Filter the supernatant with a filter membrane and freeze-dry it: Filter the supernatant with a 0.22 μm filter membrane, place it in an ultra-low temperature refrigerator (-80°C) for pre-cooling overnight, and finally freeze-dry it in a vacuum freeze dryer with a cold trap temperature of -60°C, a vacuum degree of 5 Pa, and a freeze-drying time of 36 to 48 hours to obtain freeze-dried powder of lactic acid bacteria.

[0068] The culture medium used above is MRS culture medium, and the components per liter of culture medium are shown in Table 1:

[0069] Table 1

[0070]

[0071] Experimental Example 1 Effect of different radiation doses on the survival of lactic acid bacteria

[0072] Operation steps: Activate the lactic acid bacteria twice, culture the second generation to the logarithmic phase and then irradiate. The radiation doses are:

[0073] Group 1: total dose of 250 Gy and irradiation time of 60 min;

[0074] Group 2: total dose of 500 Gy and irradiation time of 120 min;

[0075] Group 3: The total dose was 750 Gy and the irradiation time was 180 min.

[0076] After treatment, the number of surviving lactic acid bacteria is determined by the following method:

[0077] After treatment, 0.5 mL of fermentation liquid was taken from each sample, 4.5 mL of sterile deionized water was added, and gradient dilution was performed in sequence, with a dilution factor of 10. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , take appropriate dilution gradient (10-4 , 10 -5 and 10 -6 ) 200 μL of sample solution was taken and plated, and each gradient was repeated three times. After culturing in an incubator at 37°C for 48 hours, the number of viable bacteria was counted and compared with the control group to investigate the effect of different radiation doses on the survival of lactic acid bacteria. The experimental results are shown in Figure 2. Figure 1 (The dilution gradient is 10 -5 and 10 -6 ) (*P<0.05 and ***P<0.001 compared with the Control group).

[0078] Experimental Example 2

[0079] The preparation steps of normal lactic acid bacteria intracellular matter (CFE) freeze-dried powder are as follows:

[0080] 1. Activate the lactic acid bacteria twice and culture the second generation to the logarithmic phase:

[0081] 2. Collect the cells and wash them;

[0082] 3. Ultrasonic fragmentation;

[0083] 4. Centrifuge and collect the supernatant:

[0084] 5. Filter the supernatant with a membrane and freeze-dry it.

[0085] The specific conditions are the same as in Example 1.

[0086] The anti-oxidative damage experiment was carried out. The specific experimental method is as follows:

[0087] 1. Cultivate lactic acid bacteria Lactococcus lactis subsp. Lactis IL1403 to the logarithmic phase to obtain a lactic acid bacteria suspension;

[0088] 2. Dissolve the lyophilized powder of Example 1 and CFE in water to prepare solutions with a concentration of 10 mg / mL respectively;

[0089] 3. Incubate the following mixtures:

[0090] PBS group: a mixture containing 3 mL of lactic acid bacteria suspension, 1 mL of PBS, and 1 mL of 5 mmol / L H2O2 solution was incubated at 30°C for 1 h;

[0091] CFE group: a mixture containing 3 mL of lactic acid bacteria suspension, 1 mL of 10 mg / mL (final concentration 2 mg / mL) control drug solution, and 1 mL of 5 mmol / L H2O2 solution was incubated at 30°C for 1 h;

[0092] IR-CFE group: A mixture containing 3 mL of lactic acid bacteria suspension, 1 mL of 10 mg / mL (final concentration 2 mg / mL) drug solution of Example 1, and 1 mL of 5 mmol / L H2O2 solution was incubated at 30°C for 1 h;

[0093] 4. After incubation, take 0.5 mL of fermentation liquid and add 4.5 mL of sterile deionized water. Then dilute the mixture in a gradient of 10. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , take appropriate dilution gradient (10 -4 , 10 -5 and 10 -6 200 μL of the sample solution was aspirated and plated, with three replicates for each gradient. After 48 hours of incubation at 37°C, the viable cells were counted and compared with the control group to investigate the protective effect of the irradiated lactic acid bacteria intracellular matter against H2O2 damage. Figure 2 (The dilution gradient is 10 -4 ) as shown ( ** P < 0.01 and *** P < 0.001 compared with the PBS group; ## P < 0.01 compared with the CFE group).

[0094] like Figure 2 As shown, intracellular substances of lactic acid bacteria can significantly improve their survival rate under 5mmol / L H2O2 oxidative stress, and irradiated intracellular substances have a better protective effect than normal intracellular substances. When lactic acid bacteria are given low-dose, slow irradiation, they activate multiple stress mechanisms to resist the adverse environment. During this process, lactic acid bacteria enhance their antioxidant capacity, and their intracellular substances can scavenge H2O2, reducing its damage to lactic acid bacteria.

[0095] Experimental Example 3

[0096] Normal lactic acid bacteria intracellular matter (CFE) freeze-dried powder was prepared according to the method in Experimental Example 2, and radiation-induced lactic acid bacteria intracellular matter (IR-CFE) freeze-dried powder was prepared according to the method in Example 1.

[0097] The MTT assay was used to detect the radioprotective effects of CFE and IR-CFE on AML-12 cells. The specific steps are as follows:

[0098] 1. The lactic acid bacteria extracts of Example 1 and the comparative drug were dissolved in water to prepare solutions with concentrations of 0 μg / mL, 1000 μg / mL, 2000 μg / mL, 4000 μg / mL, 6000 μg / mL, and 8000 μg / mL, respectively;

[0099] 2. Collect normal mouse liver cells AML-12 cells in the logarithmic growth phase and prepare a single cell suspension. Inoculate 4000 cells per well in a 96-well plate. Add 90 μL of culture medium to each well. After culturing in an incubator for 12 hours, add 10 μL of intracellular substances at different concentrations. The specific grouping is as follows:

[0100] Normal lactic acid bacteria intracellular matter non-irradiated group (CFE) and normal lactic acid bacteria intracellular matter irradiated group (CFE+IR): 10 μL of CFE intracellular matter at different concentrations was added to make the final concentrations of CFE intracellular matter 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL, respectively. At the same time, 10 μL of culture medium was added as a control well, and 10 μL of epigallocatechin gallate (EGCG) was added as a positive control well, and the final concentration of EGCG was 10 μg / mL.

[0101] Example 1 Reagent Non-Irradiated Group (IR-CFE) and Example 1 Reagent Irradiated Group (IR-CFE+IR): 10 μL of CFE intracellular matter at different concentrations was added to make the final concentrations of IR-CFE intracellular matter 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL, respectively. At the same time, 10 μL of culture medium was added as a control well, and 10 μL of epigallocatechin gallate (EGCG) was added as a positive control well, and the final concentration of EGCG was 10 μg / mL.

[0102] 3. The cells of the control reagent irradiation group (CFE+IR) and the Example 1 reagent irradiation group (IR-CFE+IR) were cultured for 12 hours and then 60 Co-γ ray radiation treatment, with a radiation dose of 6 Gy;

[0103] The control reagent non-irradiated group (CFE) and the Example 1 reagent non-irradiated group (IR-CFE) were not irradiated and cultured for 24 hours.

[0104] 4. After the incubation is completed, the culture medium was aspirated, and 10 μL of serum, 10 μL of MTT solution (5 mg / mL), and 90 μL of culture medium were added to each well. After continuing to incubate for 4 hours, the culture medium was aspirated, and 100 μL of DMSO solution was added to each well. After shaking for 10 minutes, the absorbance at 492 nm was measured using a microplate reader.

[0105] The experimental results obtained are as follows Figures 3 to 5 As shown. Among them, Figure 3 The radiation protection effect of normal lactic acid bacteria intracellular matter (CFE) on AML-12 cells. Figure 4 The radiation protection effect of the lactic acid bacteria intracellular substance (IR-CFE) on AML-12 cells in Example 1 is shown in FIG. Figure 5 The results are a comparison of the radiation protection effects of the normal lactic acid bacteria intracellular matter irradiation group (CFE+IR) and the reagent irradiation group (IR-CFE+IR) of Example 1 on AML-12 cells. ** P < 0.01 and *** P < 0.001 compared with the 0 μg / mL CFE group; ## P < 0.01 compared with the 0 μg / mL IR-CFE group; ^^^ P < 0.001 compared with the CFE+IR group at the same concentration).

[0106] like Figures 3 to 5 As shown, irradiation significantly reduced cell count at a sample concentration of 0 μg / mL (i.e., without sample addition). However, the addition of 100 to 800 μg / mL of CFE and IR-CFE significantly increased post-irradiation cell count, demonstrating that intracellular substances protect cells from radiation damage. At a concentration of 600 μg / mL, the sample's proliferative and radioprotective effects reached their maximum. At the same concentration, IR-CFE exhibited higher absorbance than CFE, indicating that the irradiation-induced intracellular substances provided greater protection.

[0107] Experimental Example 4

[0108] 1. Drugs and experimental equipment

[0109] Positive drugs: Liquejun tablets, lyophilized powder of Example 1;

[0110] Experimental equipment: 60 Co-γ-ray radiation source.

[0111] 2. Experimental Animals and Methods

[0112] Experimental subjects: Kunming mice, provided by Henan Animal Experiment Center.

[0113] Experimental methods: 105 mice were adaptively cultured for one week and randomly divided into 7 groups, with 15 mice in each group. They were divided into control group (NC group), model group (IR group), positive control group (PC group), high-dose lactic acid bacteria intracellular substances group (CFE-H group), low-dose radiation-induced lactic acid bacteria intracellular substances group (IR-CFE-L group), medium-dose radiation-induced lactic acid bacteria intracellular substances group (IR-CFE-M group), and high-dose radiation-induced lactic acid bacteria intracellular substances group (IR-CFE-H group).

[0114] After 30 days of continuous gavage, all mice in each group except the control group were given 60 The mice were irradiated uniformly with Co-γ radiation at a single dose of 6 Gy at a dose rate of 2 Gy / min. Following irradiation, the mice were deprived of food but not water, and subsequent indicators were measured.

[0115] Control group (NC group): 10mL / kg normal saline, gavage for 30 consecutive days, no 60 Co-γ irradiation treatment.

[0116] Model group (IR group): 10mL / kg normal saline, continuous gavage for 30 days 60 Co-γ irradiation treatment.

[0117] Positive control group (PC group): 12 mg / kg of Licodone tablets aqueous solution, continuously gavaged for 30 days 60 Co-γ irradiation treatment.

[0118] Lactobacillus intracellular high-dose group (CFE-H group): 200 mg / kg of Lactobacillus intracellular lyophilized powder (Example 1) aqueous solution, continuously gavaged for 30 days and then 60 Co-γ irradiation treatment.

[0119] The low-dose group of radiation-induced lactic acid bacteria intracellular matter (IR-CFE-L group) was given 50 mg / kg of lyophilized powder of radiation-induced lactic acid bacteria intracellular matter (Example 1) in aqueous solution, and then given intragastric administration for 30 consecutive days. 60 Co-γ irradiation treatment.

[0120] The radiation-induced lactic acid bacteria intracellular matter medium dose group (IR-CFE-M group): 100 mg / kg radiation-induced lactic acid bacteria intracellular matter freeze-dried powder (Example 1) aqueous solution, continuous gavage for 30 days after 60 Co-γ irradiation treatment.

[0121] Radiation-induced lactic acid bacteria intracellular high-dose group (IR-CFE-H group): 200 mg / kg radiation-induced lactic acid bacteria intracellular freeze-dried powder (Example 1) aqueous solution, continuous gavage for 30 days after 60 Co-γ irradiation treatment.

[0122] 3. Evaluation method

[0123] 3.1 Peripheral blood assay

[0124] 20 μL of blood was collected from the mouse eyeball and placed in a buffer solution. The number of peripheral blood white blood cells and red blood cells was detected using a blood cell analyzer.

[0125] The experimental results are as follows Figure 6 、 Figure 7 As shown. Among them, Figure 6 The effect of lactic acid bacteria intracellular substances on peripheral blood leukocytes of irradiated mice. Figure 7 The effect of lactic acid bacteria intracellular substances on peripheral blood red blood cells of irradiated mice. # P < 0.05 and ### P < 0.001 compared with the NC group; *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the IR group; ^^ P < 0.01 compared with the IR-CFE-H group (n = 7).

[0126] Depend on Figure 6 、 Figure 7 It can be seen that radiation can significantly reduce the number of white blood cells and red blood cells in the blood of mice. Oral administration of radiation-induced lactic acid bacteria intracellular substances can increase the number of white blood cells and red blood cells in irradiated mice, and the effect is better when administered with a high dose.

[0127] 3.2 Antioxidant enzyme assay

[0128] (1) Determination of antioxidant enzymes in serum: Blood was collected from the mouse eyeballs and centrifuged at 4000 rpm, 4°C, and 10 min to prepare serum. The SOD, GSH-PX, GSH, and MDA enzyme activities, as well as the GSH content and MDA content in serum were determined using the Nanjing Jiancheng SOD kit, MDA kit, GSH-PX kit, and GSH kit.

[0129] The experimental results are as follows Figures 8 to 11 shown. Figure 8 The effect of lactic acid bacteria intracellular substances on SOD in the serum of mice after irradiation. Figure 9 The effect of lactic acid bacteria intracellular substances on MDA in the serum of irradiated mice. Figure 10 The effect of lactic acid bacteria intracellular substances on GSH-PX in the serum of mice after irradiation. Figure 11 Effects of lactic acid bacteria intracellular substances on serum GSH in irradiated mice ( ## P < 0.01 and ### P < 0.001 compared with the NC group; * P<0.05, ** P < 0.01 and *** P<0.001 compared with IR group; ^P<0.05, ^^ P < 0.01 and ^^^ P < 0.001 compared with the CFE-H group (n = 7).

[0130] Depend on Figures 8 to 11It can be seen that radiation can significantly reduce the SOD enzyme activity, GSH-PX enzyme activity and GSH content in mouse serum, while increasing the MDA content, indicating that radiation can lead to increased oxidative stress levels in mice. Oral administration of the freeze-dried lactic acid bacteria powder of the present invention can reduce the oxidative stress level in the blood of mice, increase the activity of antioxidant enzymes in the blood, and reduce the content of lipid peroxidation product MDA in the blood. The freeze-dried lactic acid bacteria powder of the present invention has a better effect than normal freeze-dried lactic acid bacteria powder.

[0131] In summary, the results of animal experiments show that radiation-induced lactic acid bacteria intracellular substances (Example 1) can increase the number of white blood cells and red blood cells in irradiated mice, increase the activity of antioxidant enzymes in the serum of irradiated mice, and reduce the MDA content, indicating that radiation-induced lactic acid bacteria intracellular substances exert radiation protection effects through the hematopoietic system and antioxidant system.

[0132] Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the claims. Any person skilled in the art may make several possible changes and modifications without departing from the concept of the present application. Therefore, the scope of protection of the present application shall be based on the scope defined by the claims of the present application.

Claims

1. Lactic acid bacteria extract, the lactic acid bacteria extract contains 60 The intracellular matter of Lactococcus lactis induced by Co-γ ray irradiation is derived from Lactococcus lactis subspecies lactis IL1403. 60 The Co-γ irradiation treatment uses a total dose of 50 to 750 Gy to treat the bacteria, and the irradiation time is 60 to 180 minutes.

2. The lactic acid bacteria extract according to claim 1, wherein The intracellular material obtained was freeze-dried powder.

3. The lactic acid bacteria extract according to claim 1 or 2, characterized in that Its preparation comprises the following steps: S1. Activate the lactic acid bacteria twice, culture the second generation to the logarithmic phase or culture for 16 hours, and use 60 Co-γ irradiation treatment; S2, collect the cells, break them, and collect the supernatant by centrifugation; S3. Freeze drying.

4. The lactic acid bacteria extract according to claim 3, wherein The culture temperature is 30°C to 37°C.

5. The lactic acid bacteria extract according to claim 3, wherein The culture temperature was 35°C.

6. The lactic acid bacteria extract according to claim 3, wherein The bacteria were treated with a total dose of 250 to 500 Gy.

7. The lactic acid bacteria extract according to claim 3, wherein The irradiation time is 120 minutes.

8. The lactic acid bacteria extract according to claim 3, wherein In S2, the fermentation liquid after irradiation is centrifuged to collect the bacterial cells and then the cells are disrupted.

9. The lactic acid bacteria extract according to claim 8, wherein The conditions for the disruption are: ultrasonically disrupting cells at 200-500W, working for 2-4 seconds, resting for 4-6 seconds, and the disruption treatment time is 10-20 minutes.

10. The lactic acid bacteria extract according to claim 9, wherein The cells were disrupted by ultrasound at 300 W, with an operation time of 3 s and a rest time of 5 s, and the disruption treatment time was 15 min.

11. The lactic acid bacteria extract according to claim 3, wherein The supernatant was collected under the centrifugal conditions of 0-6°C, 8000-10000 rpm, and a centrifugal time of 8-12 min.

12. The lactic acid bacteria extract according to claim 3, wherein In S2, a washing step is further included before the crushing; The washing step is to wash the bacteria 2 to 4 times with 75 mM phosphate buffer solution (PBS).

13. The lactic acid bacteria extract according to claim 3, wherein In S3, the supernatant is filtered and sterilized and then freeze-dried. The freeze-drying conditions are: Precool it at -80 to -70°C overnight, and freeze-dry it in a vacuum freeze dryer with a cold trap temperature of -60 to -55°C, a vacuum degree of 3 to 5 Pa, and a freezing time of 36 to 48 hours.

14. Use of the lactic acid bacteria extract according to any one of claims 1 to 13 in the preparation of a protective agent, wherein the protective agent is used to resist and prevent radiation damage.

15. The use according to claim 14, characterized in that: Applications of the protectant include anti-oxidative damage and prevention of oxidative damage.

16. Use of the lactic acid bacteria extract according to any one of claims 1 to 13 in the preparation of a protective agent, wherein the protective agent is used for resisting oxidative damage and preventing oxidative damage.

Images