Method for producing microbial cell fragments, microbial cell fragments and their formulations
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- 杣源一郎
- Filing Date
- 2024-05-29
- Publication Date
- 2026-05-26
Smart Images
Figure 0007865709000001 
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Abstract
Description
Technical Field
[0001] The present invention relates to a method for producing a disrupted cell mass, the disrupted cell mass, and a formulation thereof. In particular, the present invention relates to a method for producing a disrupted cell mass, the disrupted cell mass, and a formulation thereof, which can produce a disrupted cell mass having a higher natural immune activation effect while maintaining the advantages of being inexpensive and easily producible.
Background Art
[0002] All organisms have innate immunity that recognizes and eliminates all foreign substances (including invading microorganisms, dead cells, denatured biomolecules, cancer cells, aging cells, etc.) from birth. The proper functioning of innate immunity is important for maintaining health, and diseases associated with the malfunction of innate immunity due to some cause, such as infectious diseases, metabolic diseases, accelerated aging, cancer, infertility, dementia, allergic diseases, etc. are known.
[0003] The biggest cause of the abnormal functioning of innate immunity is stress. Physical or mental stress suppresses macrophages, which are phagocytes that recognize and remove foreign substances that play a central role in innate immunity (Non-Patent Document 1). Although stress is considered to be the cause of many diseases, it is difficult to reduce stress in modern society, so a measure to avoid this is required. As a method for avoiding the suppression of innate immunity by stress, it has been clarified that the suppression of innate immunity by stress can be avoided by controlling the activation of innate immunity using the activation ability of macrophages as an index (Non-Patent Document 2). In particular, natural immune activating substances in food materials have high advantages in that they are easy to ingest, have a food experience and high safety, and are relatively inexpensive.
[0004] The most well-known and commonly used food ingredients for activating innate immunity are probiotics (live bacteria) and biogenics (dead bacteria and their components). Substances derived from bacterial cells that activate innate immunity, including macrophages, include lipopolysaccharide (LPS), lipoteichoic acid, lipoarabinomannan, peptidoglycan, flagenin, lipoprotein, muramyl dipeptide, proteoglycan, genes containing unmethylated cytosine-guanine sequences, and β-glucan. Among these bacterial components, LPS is the substance that activates macrophages at the lowest possible levels. LPS is present in the extracellular membrane of Gram-negative bacteria and has a structure in which a lipid called lipid A is bound to a sugar chain consisting of multiple types of sugars. The sugar chain portion consists of a part called core polysaccharide and a part called O antigen. The lipid A portion of LPS is embedded in the lipid layer of the extracellular membrane. The biological activity of LPS is due to the activation of immune cells via Toll-like receptor 4 (TLR4), MD-2, and CD14 on the cell surface (Non-patent documents 3, 4).
[0005] Regarding the effects of LPS, even oral intake of 10 μg per kg of body weight enhances the ability of macrophages to recognize and remove foreign substances (phagocytic activity), but even greater effects are obtained with intakes of 100 μg to 1000 μg per kg of body weight (Non-Patent Literature 5). Furthermore, in the treatment of arteriosclerosis caused by high-fat diets and dementia due to aging, it has been reported that intake of 1000 μg per kg of body weight yields greater effects than intake of 300 μg (Non-Patent Literature 6, 7). These findings indicate that ingesting more LPS enhances the ability of macrophages to eliminate foreign substances. [Prior art documents] [Patent Documents]
[0006] [Patent Document 1] Patent No. 5511112 [Non-patent literature]
[0007] [Non-Patent Document 1] S. Chen et al., "Macrophages in immunoregulation and therapeutics", Signal Transduction and Targeted Therapy, 2023, Vol.8, No.207
Non-Patent Document 2
Non-Patent Document 3
Non-Patent Document 4
Non-Patent Document 5
Non-Patent Document 6
[0008] Previously, a method for preparing LPS-containing bacterial lysates inexpensively and simply by treating cultured Gram-negative bacteria at 100°C for 10 minutes and then crushing them has been reported (Patent Document 1). This bacterial lysate contains LPS, which activates innate immunity, and other bacterial components. If there is a method to increase the LPS content in this bacterial lysate, the amount of LPS that can be ingested by ingesting the same amount of bacterial lysate can be increased.
[0009] Therefore, we have made every effort to improve the manufacturing method so that we can produce bacterial cell lysates that enhance the natural immune activation effect of the bacteria while maintaining the advantages of low cost and ease of manufacturing. [Means for solving the problem]
[0010] The present invention provides a method for producing microbial cell lysates comprising the steps of mechanically crushing the cells of Gram-negative bacteria to obtain crushed cells, and the crushed cells The amount of LPS in the cell lysate is concentrated by performing ultrafiltration with a fractionation molecular weight of 300,000. The method is characterized by comprising the step of obtaining a cell lysate with a larger molecular weight.
[0011] Also The microbial cell fragments of the present invention are characterized by being produced by the method for producing the microbial cell fragments described above. Furthermore, the formulation of the microbial cell fragments of the present invention is characterized by containing the microbial cell fragments. Furthermore, the formulation of the microbial cell crushed material is preferably a food product, cosmetic product, skincare product, supplement, quasi-drug, or pharmaceutical product. [Effects of the Invention]
[0012] According to the present invention, by adding a single purification step, it is possible to produce cell lysates of Gram-negative bacteria in which the LPS content of the cell lysates and freeze-dried products is increased (for example, by 1.4 times and 4.7 times), and the biological activity is enhanced (for example, by 4.7 times), and a formulation of such cell lysates can be provided. [Modes for carrying out the invention]
[0013] The embodiments for carrying out the present invention will be described in detail below. As a result of diligent research, the inventors have discovered that by adding one inexpensive and simple process to the conventional production of microbial cell lysates, the conventional product exhibits a stronger immune activation-inducing ability, and furthermore, unexpectedly, the activity of LPS is increased, thus completing the present invention.
[0014] Gram-negative bacteria may be cultured with ordinary nutrients and then the bacterial cells may be separated from the culture. As long as it is a Gram-negative bacterium containing LPS, the type of bacteria is not particularly limited. For example, it may be Escherichia coli, Salmonella, Aeromonas, Acinetobacter, Proteus, Serratia, Bordetella pertussis, Yersinia, Neisseria, etc. In particular, acetic acid bacteria, Xanthomonas, Zymomonas, Pantoea, Enterobacter, etc., which are Gram-negative bacteria used in food, are desirable in terms of having food experience. Also, the main molecular weight of the LPS of these Gram-negative bacteria with food experience is 20,000 or less.
[0015] After heat sterilization, the bacterial cells are mechanically disrupted by methods such as stirring, freezing, ultrasonic waves, glass beads, a blender, etc., but the method is not limited. It is desirable to perform the disruption treatment non-enzymatically at low temperature. The disruption of cells can be confirmed under a microscope, and it is preferable that 90% or more of the bacteria are disrupted, and almost 100% is more preferable.
[0016] As a method for evaluating natural immune activation, it can be evaluated by the induction enhancement effect of cytokines, etc. of macrophages. For example, tumor necrosis factor (TNF) induction, interleukin (IL)-1α induction, IL-1β induction, nitric oxide (NO) induction, reactive oxygen induction, cancer cell cytotoxic effect, etc.
[0017] Macrophages are cells that are distributed throughout the body. Macrophages have the function of identifying and eliminating foreign substances, and because they have the ability to adapt to the environment and change their characteristics, the characteristics of macrophages vary depending on the tissue in which they exist. However, since all macrophages have the function of eliminating foreign substances, tissue-isolated cells and cell lines such as microglia in the brain, alveolar macrophages in the lungs, Kupffer cells in the liver, Langerhans cells in the skin, peritoneal macrophages, monocytes derived from blood, and bone marrow cells can be used. For example, natural immune activators are added to macrophage cell lines (RAW264.7, J774.1, THP-1, NR8383), peritoneal macrophages, macrophages induced from peripheral blood monocytes, and bone marrow cells, and TNF, IL-1β, reactive oxygen species, nitric oxide (the metabolite nitrite ion can also be used as the measurement product) induced in the culture supernatant, etc. can be measured to evaluate macrophage activation. Macrophage activity can be evaluated by substances such as LPS, lipoteichoic acid, lipoarabinomannan, peptidoglycan, flagellin, lipoprotein, muramyl dipeptide, proteoglycan, and genes containing unmethylated cytosine-guanine sequences, which are components of Gram-positive and Gram-negative bacteria, even if they are mixed substances.
Example
[0018] [Culture of Pantoea bacteria] A part of the colony of Pantoea agglomerans IG1 strain was scraped off and seeded on Luria-Bertani (LB) agar medium, and cultured overnight in a constant temperature bath at 35°C. One colony was added to 10 mL of sterilized LB medium and pre-cultured at 35°C under shaking conditions of 170 rpm. 0.05 mL of the pre-culture solution was added to each of 8 250 mL baffled flasks containing 80 mL, and cultured with shaking overnight at 35°C to obtain 560 mL of bacterial cell culture solution.
[0019] To evaluate the usefulness of the bacterial cell treatment method, the innate immune activation per unit dry weight of the sample was assessed. Although the culture medium components do not exhibit innate immune activation, the bacterial cell culture solution itself contains substances derived from the culture medium components (approximately 2 g per 100 ml), exceeding the amount of bacterial cells obtained. Therefore, the culture medium components significantly affect the dry weight. Because this affects the weight of the bacterial cell treatment product, it becomes difficult to evaluate the effectiveness of the treatment method. Therefore, we decided to prepare wet bacterial cells with minimal importation from the culture medium by centrifugation and use them for biological activity evaluation. Half of the bacterial cell culture solution (280 ml) was transferred to four 50 mL centrifuge tubes and centrifuged at 3500 rpm for 30 minutes (KUBOTA Model 5220 benchtop centrifuge). The wet bacterial cell weight of the collected bacteria was measured using an electronic balance. A total of 2.032 g of wet bacterial cells of Pantoea strain IG1 was obtained from 280 mL of culture solution.
[0020] [Preparation of conventionally processed microbial cell fragments] 1.0 g of the above wet bacterial cells were transferred to four centrifuge tubes, and phosphate-buffered saline (PBS(-)) (Fujifilm Wako Pure Chemical Industries) was added to prepare 10 mL of each 100 mg / mL suspension. As a treatment for disrupting the wet bacterial cells, each suspension was heated in an autoclave at 100°C for 10 minutes. When the temperature had dropped to 80°C, the suspensions were stirred five times for 30 seconds each using a vortex mixer, and 10 mL of conventional bacterial cell disruption by vortex disruption was prepared from two of the tubes. The remaining two tubes were subjected to vortex disruption in addition to disruption using a Polytron homogenizer at 15000 rpm for 5 minutes. After that, centrifugation was performed at 3500 rpm for 20 minutes to prepare 10 mL of conventional bacterial cell disruption by Polytron disruption.
[0021] [Preparation of the microbial cell fragments of the present invention] To enhance the innate immune activation ability of bacterial cell lysates, it would be desirable to remove components ineffective in activating innate immunity from the lysates inexpensively and easily. As for purification methods, ethanol precipitation requires large amounts of expensive alcohol and time-consuming centrifugation, while gel filtration and ion exchange methods are costly and time-consuming, making them neither inexpensive nor easy. In this study, we attempted ultrafiltration, an industrially simple method for separating components that is used in seawater desalination, drinking water filtration, and sake production.
[0022] Two of the four conventional cell lysates ("Vortex Conventional Method" and "Polytron Conventional Method") were placed in a VIVASPIN 20 (#VS2051, Sartorius) equipped with a filter with a molecular weight cutoff of 300,000. Separation and ultrafiltration were performed by centrifugation at 3000 rpm until the internal liquid was reduced to 0.75 mL. The liquid from the first ultrafiltration (the liquid with a low molecular weight (first ultrafiltration)) was collected in a separate container.
[0023] The remaining 0.75 mL of liquid contains only 7.5% (=0.75 mL / 10 mL * 100) of the substances with a molecular weight less than 300,000 that were present in the original 10 mL of conventionally processed bacterial cell lysate. To further remove the substances with a molecular weight less than 300,000 remaining in this liquid, a second ultrafiltration was performed as follows.
[0024] PBS(-) (9.25 mL) was added to the remaining internal solution (0.75 mL) and suspended. Ultrafiltration was then performed at 3000 rpm until the internal solution was reduced to 0.75 mL, and the external solution from the second ultrafiltration (second external solution) was collected in a separate container. As a result, the residual rate of substances with a molecular weight less than 300,000 in the second 0.75 mL of internal solution was calculated to be 0.56% (=7.5% * 7.5%) compared to the amount contained in the original 10 mL of conventionally processed bacterial cell lysate, which is less than 1 / 100th of the amount before ultrafiltration, and it was judged that sufficient removal had occurred. Furthermore, in order to recover any remaining internal solution adhering to VIVASPIN20, distilled water (9.75 mL) was added to the internal solution to wash the filter, and a total of 10 mL of recovered solution (with a high molecular weight) was obtained.
[0025] LPS typically has a molecular weight of 5,000 to 50,000, but it forms micelles and creates apparent macromolecules. Therefore, LPS remains in the liquid after two ultrafiltrations, and this property can be used to easily improve the purity of LPS. In this study, the recovered liquid obtained by ultrafiltration was used as the bacterial cell lysate of the present invention ("Vortex Invention" and "Polytron Invention").
[0026] [Measurement of the innate immune activation ability of microbial cell fragments] To measure the biological activity per unit weight of each cell lysate obtained by the conventional method and the present invention, 10 mL of each cell lysate (four samples in total, from both the conventional method and the present invention) were centrifuged at 3500 rpm for 20 minutes, and all of the supernatant was collected in a separate centrifuge tube (0.5 mL to 0.67 mL of supernatant from each lysate). Each supernatant was freeze-dried to prepare freeze-dried products. The weight of the freeze-dried products was weighed using an electronic balance (Table 1), and solutions were prepared by adding sterile water for injection to each product to the same concentration of 20 mg / mL (keeping the concentration the same at this stage makes it easier to determine the dry weight of the cell lysates that can induce 1 μM of NO using the conventional method and the present invention when conducting the NO production test, and to compare them). Biological activity measurements based on NO production were performed on these solutions.
[0027] [LPS content measurement using Limulus measurement] The Limulus activity of each cell lysate was measured. Evaluations were performed using a standardized starting amount for each cell lysate, as well as evaluations based on dry weight. Each cell lysate was heated at 37°C for 5 minutes, then sonicated (10 minutes) and mixed using a vortex mixer. The solution was then diluted with sterile water for injection, Limulus reagent was added, and the reaction was measured using a toxicometer (Fujifilm Wako Pure Chemical Corporation) to obtain the amount equivalent to standard E. coli LPS (Fujifilm Wako Pure Chemical Corporation).
[0028] [Measurement results] To compare conventionally prepared bacterial cell lysates with those prepared using the present invention, the initial amount of bacterial cells was kept the same (10 mL), so they should contain the same amount of LPS. However, the LPS content per 10 mL of conventionally prepared bacterial cell lysates prepared by vortex lysate was 9.8 mg, while the LPS content per 10 mL of the present invention's bacterial cell lysates was 13.7 mg (Table 1). Surprisingly, the LPS content of the present invention's bacterial cell lysates increased by 1.4 times compared to that of the conventionally prepared bacterial cell lysates. This result was unexpected.
[0029] The substances in the external liquid (first and second filtration) removed by ultrafiltration (lyophilized weights of 0.24g and 0.12g (Table 1)) were substances with a molecular weight of 300,000 or less, including PBS(-). The LPS content in the external liquid (first and second filtration) was 0.0 mg each (Table 1). Therefore, it is considered that LPS contained in the conventionally processed bacterial cell lysate remained almost entirely in the internal liquid (the lysate of the present invention) even after ultrafiltration, and furthermore, many of the substances that inhibit LPS were filtered into the external liquid. In other words, the bacterial cell lysate contains bacterial-derived components such as proteins, peptides, lipids, nucleic acids, and organic acids, as well as bacterial metabolites, and it is presumed that among these, there are substances that inhibit LPS with a molecular weight of 300,000 or less. Possible LPS-inhibiting substances include polypeptides, surfactants (such as lactonin), polyunsaturated fatty acids (such as docosahexaenoic acid), and metal ions such as calcium and iron.
[0030] Next, the LPS content per gram of freeze-dried product of each cell lysate was calculated. The conventional cell lysate contained 29.6 mg, while the present invention cell lysate contained 139.5 mg (Table 1). By removing 70% of the weight of substances with an apparent molecular weight of 300,000 or less (=(0.33-0.10) / 0.33*100), the LPS content increased 4.7 times compared to the conventional cell lysate. This result does not affect the amount of LPS contained in the conventional cell lysate, but the weight of the present invention cell lysate decreased because 70% of the weight of the substance was removed. Furthermore, the amount of LPS lost by the ultrafiltration method used to prepare the present invention cell lysate was very small, less than 1 / 100.
[0031] Furthermore, the LPS content per gram of dry weight of the cell crushed products prepared by the conventional method and the present invention, both prepared by Polytron crushing, was measured. The conventional method cell crushed product contained 33.7 mg of LPS, while the present invention cell crushed product contained 122.3 mg, resulting in an LPS content 3.6 times higher than the conventional method. (Table 2)
[0032] From the above, it has become clear that the present invention's method for treating microbial cell fragments by ultrafiltration is an excellent preparation method that substantially increases the LPS content regardless of the fragmentation method and produces almost no loss.
[0033] [Table 1]
[0034] [Table 2]
[0035] [Measurement of immunostimulatory effects based on NO production from RAW264.7 cells of conventionally crushed materials and the bacterial cell crushed materials of the present invention] NO production from RAW264.7, a mouse macrophage cell line, after the addition of LPS, was measured using the concentration of nitrite, an NO metabolite, in the culture medium as an indicator. RAW264.7 was purchased from ATCC (No. TIB-71). RAW264.7 cells were collected from the culture flask by pipetting, and the cell concentration was increased to 1.6 × 10⁶ in RPMI1640 medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin). 6 Adjusted to cells / mL. 100 μL of cell suspension (1.6 × 10⁶) 5The cells (100 μL / 100 μL) were transferred to each well of a 96-well flat-bottom plate and used for testing after 3 hours. The lyophilized lysates of each cell were prepared by adding culture medium to a concentration of 200 ng / mL, and serially diluted 10-fold in three steps. 100 μL of each solution was added to the cells. The cells were incubated at 37°C in a 5% carbon dioxide incubator for 24 hours. After incubation, 100 μL of the supernatant was collected in another 96-well plate. The amount of nitrite, an NO metabolite, in the culture medium was measured using Gries' reagent according to standard procedures.
[0036] [result] Using the method described above, the amount of NO produced at each concentration (dilution stage) was measured, and the concentration of the freeze-dried product of each cell lysate that could induce 1 μM of NO was calculated and compared. The conventional method produced 5.6 ng / mL, while the present invention produced 1.2 ng / mL. From this, it was revealed that the cell lysate of the present invention has 4.7 times higher biological activity per unit weight of freeze-dried product compared to the cell lysate of the conventional method. [Examples]
[0037] [Culture of Escherichia coli (NBRC strain 3301)] A portion of a colony of Escherichia coli (NBRC strain 3301) was scraped and seeded onto 802 agar medium, and incubated overnight in a 30°C incubator. One colony was added to 80 mL of sterile 802 medium and pre-cultured at 30°C with shaking at 170 rpm. 0.8 mL of the pre-culture solution was added to eight 250 mL baffled flasks, each containing 80 mL of medium, and incubated overnight at 30°C with shaking to obtain 560 mL of bacterial culture solution. Similar to Example 1, since the culture medium components significantly affect the dry weight and thus the weight of the microbial treatment product, we decided to prepare wet microbial cells with minimal contamination from the culture medium by centrifugation and use them for biological activity evaluation. The microbial culture solution was transferred to a 50 mL centrifuge tube and centrifuged at 3500 rpm for 30 minutes. The wet microbial weight of the collected bacteria was measured using an electronic balance. A total of 4.49 g of wet microbial cells was obtained from 560 mL of culture solution.
[0038] [Preparation of conventionally processed microbial cell fragments] 10 mL of a 100 mg / mL suspension was prepared by adding PBS(-) to 1.0 g of the above wet bacterial cells. As a treatment for disrupting the wet bacterial cells, each suspension was heated in an autoclave at 100°C for 10 minutes. When the temperature had decreased to 80°C, the mixture was stirred five times for 30 seconds each using a vortex mixer to prepare conventionally method-disrupted bacterial cells.
[0039] [Preparation of the microbial cell fragments of the present invention] Ultrafiltration was performed in the same manner as in Example 1. 9.0 mL of conventionally prepared bacterial cell lysate was placed in VIVASPIN 20 with a molecular weight cutoff of 300,000, and separated by ultrafiltration by centrifugation at 3000 rpm until the internal liquid was reduced to 0.25 mL. The external liquid from the first ultrafiltration (external liquid (1st)) was collected in a separate container. An equal amount of PBS(-) as the external liquid (1st) was added to the internal liquid in the container, and after suspension, a second ultrafiltration was performed at 3000 rpm until the internal liquid was reduced to 0.25 mL. The external liquid from the second ultrafiltration (external liquid (2nd)) was collected in a separate container. Furthermore, the remaining internal liquid adhering to the VIVASPIN 20 was collected using distilled water, and a total of 3.0 mL of recovered liquid was obtained by combining it with the internal liquid. This was used as the bacterial cell lysate of the present invention.
[0040] [Measurement of the innate immune activation ability of microbial cell fragments] To measure the biological activity per unit weight of each cell lysate obtained by the conventional method and the present invention, each cell lysate was centrifuged at 3500 rpm for 20 minutes, and the supernatant was collected in a separate centrifuge tube (supernatant of each lysate). 0.7 mL of each supernatant was freeze-dried to prepare a freeze-dried product. The weight of the freeze-dried product was weighed using an electronic balance, and a solution was prepared by adding sterile water for injection to a concentration of 50 mg / mL. The biological activity of this solution was measured based on NO production.
[0041] [LPS content measurement using Limulus measurement] The Limulus activity of each cell lysate was measured. Evaluations were performed using a standardized starting volume for each cell lysate, as well as evaluations based on dry weight. Each cell lysate was heated at 37°C for 5 minutes, then sonicated (10 minutes) and mixed using a vortex mixer. The solution was then diluted with sterile water for injection, and the amount equivalent to standard E. coli LPS was measured using a toxicometer.
[0042] [Measurement results] Similar to Example 1, the initial amount of bacterial cells in each cell lysate was kept the same (10 mL) for comparison. The LPS content per 10 mL of the conventional cell lysate was 4.5 mg, while the LPS content per 10 mL of the present invention cell lysate was 5.3 mg, meaning the LPS content of the present invention cell lysate increased to 1.2 times that of the conventional cell lysate (Table 3). Next, the LPS content per 1 g of the freeze-dried product of each cell lysate was calculated. The conventional cell lysate contained 31.7 mg, while the present invention cell lysate contained 263.9 mg (Table 3).
[0043] Based on the above, the LPS content of the cell lysate produced by the present invention increased 8.3 times compared to the conventional method. In Escherichia coli (NBRC 3301 strain), similar to Example 1, the cell lysate treatment method of the present invention effectively increased the LPS content, demonstrating that it is an excellent preparation method.
[0044] [Table 3]
[0045] [Measurement of immunostimulatory effects based on NO production from RAW264.7 cells of conventionally crushed materials and the bacterial cell crushed materials of the present invention] NO production from cells was measured using the concentration of nitrite, an NO metabolite, in the culture medium after adding LPS to RAW264.7, a mouse macrophage cell line. RAW264.7 cells were cultured in the same manner as in Example 1. The lyophilized lysates of each cell were adjusted by adding culture medium to a concentration of 200 μg / mL, and serially diluted 10-fold in 6-7 steps. The cells were cultured for 24 hours at 37°C in a 5% carbon dioxide incubator. After the culture was complete, 100 μL of the supernatant was collected in a separate 96-well plate. The amount of nitrite, an NO metabolite, in the culture medium was measured using Gries' reagent according to a standard procedure.
[0046] [result] Using the method described above, the amount of NO produced at each concentration (dilution stage) was measured, and the concentration of the freeze-dried product of each cell lysate that could induce 10 μM of NO was calculated and compared. The conventional method produced 19.6 ng / mL, while the present invention produced 2.1 ng / mL. From this, it was revealed that the cell lysate of the present invention has 9.3 times higher biological activity per unit weight of freeze-dried product compared to the cell lysate of the conventional method. [Examples]
[0047] [Culture of acetic acid bacteria Gluconobacter oxydans] A portion of a Gluconobacter oxydans colony was scraped and seeded onto a modified agar medium from the University of Agriculture, and incubated for two nights in a 30°C incubator. One colony was added to 80 mL of sterile modified agar medium from the University of Agriculture and pre-cultured at 30°C with shaking at 170 rpm. 8 mL of the pre-culture solution was placed in eight 250 mL baffled flasks, each containing 70 mL of the pre-culture solution, and incubated at 30°C with shaking for two nights to obtain 560 mL of bacterial culture solution. Similar to Example 1, since the culture medium components significantly affect the dry weight and thus the weight of the bacterial cell treatment product, we decided to prepare moist bacterial cells with minimal contamination from the culture medium by centrifugation and use them for biological activity evaluation. The bacterial cell culture solution was transferred to a 50 mL centrifuge tube and centrifuged at 3500 rpm for 20 minutes. The moist bacterial cell weight of the collected bacteria was measured using an electronic balance. A total of 1.59 g of moist bacterial cells was obtained from 560 mL of culture solution.
[0048] [Preparation of conventionally processed microbial cell fragments] 0.50 g of the above wet bacterial cells were added to PBS(-) to prepare 10 mL of a 100 mg / mL suspension. 5 mole / L of sodium hydroxide aqueous solution was added, and the pH was checked with pH test paper to neutralize it. Subsequently, as a lysation treatment of the wet bacterial cells, each suspension was heated in an autoclave at 100°C for 10 minutes. Once the temperature had decreased to 80°C, the mixture was stirred five times for 30 seconds each using a vortex mixer to prepare conventionally lysed bacterial cells.
[0049] [Preparation of the microbial cell fragments of the present invention] Ultrafiltration was performed in the same manner as in Example 1. 3.8 mL of conventionally prepared bacterial cell lysate was placed in VIVASPIN 20 with a molecular weight cutoff of 300,000, and separated by ultrafiltration by centrifugation at 3000 rpm until the internal liquid was reduced to 0.25 mL. The external liquid from the first ultrafiltration (external liquid (1st)) was collected in a separate container. An equal amount of PBS(-) as the external liquid (1st) was added to the internal liquid in the container, and after suspension, a second ultrafiltration was performed at 3000 rpm until the internal liquid was reduced to 0.25 mL. The external liquid from the second ultrafiltration (external liquid (2nd)) was collected in a separate container. Furthermore, the remaining internal liquid adhering to the VIVASPIN 20 was collected using distilled water, and a total of 1.0 mL of recovered liquid was obtained by combining it with the internal liquid. This was used as the bacterial cell lysate of the present invention.
[0050] [Measurement of the innate immune activation ability of microbial cell fragments] To measure the biological activity per unit weight of each cell lysate obtained by the conventional method and the present invention, each cell lysate was centrifuged at 3500 rpm for 20 minutes, and the supernatant was collected in a separate centrifuge tube (supernatant of each lysate). 0.7 mL to 1.0 mL of each supernatant was freeze-dried to prepare freeze-dried products. The weight of the freeze-dried products was weighed using an electronic balance, and a solution was prepared by adding sterile water for injection to a concentration of 50 mg / mL. The biological activity of this solution was measured based on NO production.
[0051] [LPS content measurement using Limulus measurement] The Limulus activity of each cell lysate was measured. Evaluations were performed using a standardized starting volume for each cell lysate, as well as evaluations based on dry weight. Each cell lysate was heated at 37°C for 5 minutes, then sonicated (10 minutes) and mixed using a vortex mixer. The solution was then diluted with sterile water for injection, and the amount equivalent to standard E. coli LPS was measured using a toxicometer.
[0052] [Measurement results] Similar to Example 1, the initial amount of bacterial cells in each cell lysate was kept the same (10 mL) for comparison. The LPS content per 10 mL of the conventional cell lysate was 11.8 μg, while the LPS content per 10 mL of the cell lysate of the present invention was 14.3 μg, meaning the LPS content of the cell lysate of the present invention increased 2.4 times compared to the conventional cell lysate (Table 4). Next, the LPS content per 1 g of the freeze-dried product of each cell lysate was calculated. The conventional cell lysate contained 62.1 μg, while the cell lysate of the present invention contained 476.7 μg (Table 4).
[0053] Based on the above, the LPS content of the cell lysate of the present invention increased 7.7 times compared to the conventional method (Table 4), and in the case of the acetic acid bacterium Gluconobacter oxydans, similar to Example 1, the cell lysate treatment method of the present invention effectively increased the amount of LPS, demonstrating that it is an excellent preparation method.
[0054] [Table 4]
[0055] [Measurement of immunostimulatory effects based on NO production from RAW264.7 cells of conventionally crushed materials and the bacterial cell crushed materials of the present invention] NO production from cells was measured using the concentration of nitrite, an NO metabolite, in the culture medium after adding LPS to RAW264.7, a mouse macrophage cell line. RAW264.7 cells were cultured in the same manner as in Example 1. Lyophilized lysates of each cell type were prepared by adding culture medium to a total concentration of 2000 μg / mL. Five 10-fold serial dilutions were performed, and the cells were cultured for 24 hours at 37°C in a 5% carbon dioxide incubator. After the culture period, 100 μL of the supernatant was collected in a separate 96-well plate. The amount of nitrite, an NO metabolite, in the culture medium was measured using Gries' reagent according to a standard procedure.
[0056] [result] Using the method described above, the amount of NO produced at each concentration (dilution stage) was measured, and the concentration of the freeze-dried product of each cell lysate that could induce 1 μM of NO was calculated and compared. The conventional method produced 26.2 μg / mL, while the present invention produced 3.4 μg / mL. From this, it was revealed that the cell lysate of the present invention has 7.7 times higher biological activity per unit weight of freeze-dried product compared to the cell lysate of the conventional method.
[0057] All publications, patents, and patent applications cited herein are incorporated herein by reference as such.
Claims
1. A method for producing a lysate of microbial cells, comprising the steps of: mechanically crushing the cells of Gram-negative bacteria to obtain crushed cells; and concentrating the amount of LPS in the lysate by performing ultrafiltration on the crushed cells with a fractional molecular weight of 300,000 to obtain a lysate of microbial cells with a higher molecular weight.
2. A microbial cell fragment characterized by being produced by the method for producing a microbial cell fragment described in claim 1.
3. A compound of crushed microbial cells, characterized in that it contains the crushed microbial cells described in Claim 2.
4. The composition of the microbial cell lysate according to claim 3, characterized in that the composition is a food, cosmetic, skincare product, supplement, quasi-drug, or pharmaceutical product.