Method for producing bacterial body crushed product, bacterial body crushed product, and formulation thereof

Abstract

The present invention pertains to an easy and low-cost method for producing a bacterial cell crushed product, the method configured so that it is possible to produce a bacterial cell crushed product having a higher natural immunity activation effect. The method comprises: a step for mechanically crushing gram negative bacterium cells to obtain crushed bacterial cells; and a step for purifying the crushed bacterial cells by molecular weight fractionation to obtain a bacterial cell crushed product of larger molecular weights. Thus, the LPS content of the bacterial cell crushed product and the lyophilized product can be increased (e.g., 1.4 times and 4.7 times), and the bioactivity performance can be enhanced (e.g. 4.7 times).

Description

Method for producing crushed bacterial cell material, crushed bacterial cell material and blend thereof

[0001] The present invention relates to a method for producing disrupted bacterial cell material, disrupted bacterial cell material, and blends thereof, and in particular to a method for producing disrupted bacterial cell material that can produce disrupted bacterial cell material with a higher natural immune activation effect while maintaining the advantage of being inexpensive and easy to produce, disrupted bacterial cell material, and blends thereof.

[0002] All living organisms are born with an innate immune system that recognizes and eliminates all foreign substances (including invading microorganisms, dead cells, denatured biomolecules, cancer cells, senescent cells, etc.) Healthy functioning of the innate immune system is important for maintaining good health, and diseases that are known to be associated with impaired functioning of the innate immune system for some reason include infectious diseases, metabolic diseases, accelerated aging, cancer, infertility, dementia, and allergic diseases.

[0003] Stress is the major cause of innate immune dysfunction. Physical or mental stress suppresses macrophages, phagocytes that recognize and eliminate foreign substances and play a central role in innate immunity (Non-Patent Document 1). Stress is thought to be the cause of many diseases, but reducing stress is difficult in modern society, so strategies to avoid it are needed. As a method for avoiding stress-induced suppression of innate immunity, it has been shown that controlling innate immune activation using macrophage activation as an indicator can avoid stress-induced innate immune suppression (Non-Patent Document 2). In particular, innate immune activators in the form of food ingredients have the advantages of being easy to ingest, being commonly eaten, being highly safe, and being relatively inexpensive.

[0004] The most well-known commonly used food ingredients that activate innate immunity include probiotics (live bacteria) and biogenics (killed bacteria and their components). Bacterial components that activate innate immunity, including macrophages, include lipopolysaccharide (LPS), lipoteichoic acid, lipoarabinomannan, peptidoglycan, flagenin, lipoproteins, muramyl dipeptide, proteoglycans, genes containing unmethylated cytosine-guanine sequences, and β-glucans. Of these bacterial components, LPS is the one that activates macrophages in the smallest amounts. LPS is present in the outer membrane of Gram-negative bacteria and consists of a lipid called lipid A bound to a glycan consisting of multiple sugars. The glycan portion consists of a core polysaccharide and an O-antigen. The lipid A portion of LPS is embedded in the lipid layer of the outer membrane. The biological activity of LPS activates immune cells via Toll-like receptor 4 (TLR4), MD-2, and CD14 on the cell surface (Non-patent Documents 3 and 4).

[0005] Oral ingestion of LPS at a dose of 10 μg per kg of body weight enhances the ability of macrophages to recognize and eliminate foreign substances (phagocytic activity), but even greater effects are observed at doses of 100 μg to 1,000 μg per kg of body weight (Non-Patent Document 5). It has also been reported that a dose of 1,000 μg per kg of body weight is more effective than a dose of 300 μg in treating arteriosclerosis caused by a high-fat diet and dementia due to aging (Non-Patent Documents 6 and 7). These findings suggest that ingesting larger amounts of LPS enhances the ability of macrophages to eliminate foreign substances.

[0006] Patent No. 5511112

[0007] S. Chen et al., "Macrophages in immunoregulation and therapeutics," Signal Transduction and Targeted Therapy, 2023, Vol.8, No.207; T. Nakamoto et al., "Treatments for the Activating Macrophages that Reduces Surgical Stress and Postoperative Mortalities from Bacterial Infections and Tumor Metastases," In Vivo, 2007, Vol.21, No.2, pp.357-364; Wikipedia "Lipopolysaccharide," [online], [Retrieved May 10, 2024], Internet <URL: http: / / ja.wikipedia.org / wiki / Lipopolysaccharide>; Wikipedia "Lipid A," [online], [Retrieved May 10, 2024], Internet <URL: http: / / ja.wikipedia.org / wiki / LipidA>; H. Inagawa et al., "Primed Activation of Macrophages by Oral Administration of Lipopolysaccharide Derived from Pantoea agglomerans", In Vivo, 2016, Vol.30, No.3, p.205-212Y. Kobayashi et al., "Oral administration of Pantoea agglomerans derived lipopolysaccharide prevents development of atherosclerosis in high-fat diet-fed apoE-deficient mice via ameliorating hyperlipidemia, "pro-inflammatory mediators and oxidative responses", PLOS ONE, 2018, Vol.13, No.3Y. Kobayashi et al., "Oral administration of Pantoea agglomerans derived lipopolysaccharide prevents metabolic dysfunction and Alzheimer's disease-related memory loss in senescence accelerated prone 8 (SAMP8) mice fed a high-fat diet", PLOS ONE, 2018, Vol.13, No.6.

[0008] A method for preparing a bacterial cell lysate containing LPS at low cost and easily has been reported (Patent Document 1). This bacterial cell lysate contains LPS, which activates innate immunity, as well as other bacterial components. If a method could be developed to increase the LPS content in this bacterial cell lysate, the amount of LPS ingested from the same amount of bacterial cell lysate could be increased.

[0009] Therefore, we made extensive efforts to improve the production method so that we could produce a bacterial cell lysate that enhances the innate immune activation effect of the bacterial cell while maintaining the advantages of low cost and ease of production.

[0010] The method for producing disrupted bacterial cell material of the present invention is characterized by comprising the steps of mechanically disrupting Gram-negative bacterial cells to obtain disrupted bacterial cells, and purifying the disrupted bacterial cells by molecular weight fractionation to obtain disrupted bacterial cell material with a higher molecular weight.

[0011] The purification is preferably carried out by ultrafiltration. The molecular weight cutoff of the ultrafiltration is preferably 300,000. The disrupted bacterial cell lysate of the present invention is characterized by being produced by the method for producing disrupted bacterial cell lysate. A blend of disrupted bacterial cell lysate of the present invention is characterized by containing the disrupted bacterial cell lysate. The blend of disrupted bacterial cell lysate is preferably a food, cosmetic, skin care product, supplement, quasi-drug, or pharmaceutical product.

[0012] According to the present invention, by adding a single purification step, it is possible to prepare gram-negative bacterial cell lysates that have an increased LPS content (e.g., 1.4-fold and 4.7-fold) and increased biological activity (e.g., 4.7-fold) in the cell lysates and lyophilized products, and it is also possible to provide formulations of such cell lysates.

[0013] The present inventors have conducted extensive research and discovered that adding an inexpensive and simple step to the conventional production of bacterial cell lysate results in a stronger immune activation-inducing ability than the conventional product and, moreover, unexpectedly enhances the activity of LPS, leading to the completion of the present invention.

[0014] Gram-negative bacteria may be cultured with standard nutrients and then isolated from the culture. The type of Gram-negative bacteria is not particularly limited as long as they contain LPS. Examples include Escherichia coli, Salmonella, Aeromonas, Acinetobacter, Proteus, Serratia marcescens, Bordetella pertussis, Yersinia, and Neisseria. Gram-negative bacteria used in food, such as Acetobacter, Xanthomonas, Zymomonas, Pantoea, and Enterobacter, are particularly desirable because they have a history of consumption. Furthermore, the molecular weight of the LPS from these Gram-negative bacteria is primarily 20,000 or less.

[0015] After heat sterilization, the bacterial cells are mechanically disrupted by stirring, freezing, ultrasonication, glass beads, a blender, or other methods, but the method is not limited thereto. The disruption is preferably carried out non-enzymatically at low temperature. The disruption of the cells can be confirmed under a microscope, and the disrupted bacteria should be 90% or more, preferably nearly 100%.

[0016] Innate immune activation can be evaluated by measuring the induction and enhancement of macrophage cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-1α, IL-1β, nitric oxide (NO), reactive oxygen species, and cancer cell cytotoxicity.

[0017] Macrophages are cells distributed throughout the body. They have the ability to identify and eliminate foreign substances, but because they can adapt to their environment, their characteristics vary depending on the tissue in which they reside. However, all macrophages possess this function, and tissue-isolated cells and established cell lines, such as brain microglia, lung alveolar macrophages, liver Kupffer cells, skin Langerhans cells, peritoneal macrophages, blood-derived monocytes, and bone marrow cells, can be used. For example, macrophage cell lines (RAW264.7, J774.1, THP-1, NR8383), peritoneal macrophages, peripheral blood monocytes, and macrophages differentiated from bone marrow cells, can be treated with innate immune activators. Macrophage activation can be assessed by measuring the induced levels of TNF, IL-1β, reactive oxygen species, and nitric oxide (although the metabolic product nitrite can also be used as a measurement product) in the culture supernatant. Macrophage activity can be evaluated for its ability to activate innate immunity even when the substance contains a mixture of LPS, lipoteichoic acid, lipoarabinomannan, peptidoglycan, flagenin, lipoprotein, muramyl dipeptide, proteoglycan, and genes containing unmethylated cytosine and guanine sequences, which are components of gram-positive and gram-negative bacteria.

[0018] [Cultivation of Pantoea] A portion of a colony of Pantoea agglomerans strain IG1 was scraped and plated on Luria-Bertani (LB) agar medium and cultured overnight in an incubator at 35°C. One colony was added to 10 mL of sterilized LB medium and precultured at 35°C with shaking at 170 rpm. 0.05 mL of the preculture was added to each of eight 250 mL baffled flasks containing 80 mL of LB medium, and cultured overnight with shaking at 35°C to obtain 560 mL of bacterial culture medium.

[0019] To evaluate the usefulness of the bacterial cell treatment method, we evaluated the innate immune activation per dry weight of the sample. While medium components do not activate the innate immune system, the bacterial cell culture itself contains substances derived from the medium components (approximately 2 g per 100 ml), exceeding the yield of the bacterial cell mass. Therefore, the medium components significantly affect the dry weight. This influence on the weight of the bacterial cell treatment product makes it difficult to evaluate the effectiveness of the treatment method. Therefore, we prepared wet bacterial cells with minimal carryover from the culture medium by centrifugation and used them for bioactivity evaluation. Half of the bacterial cell culture (280 ml) was transferred to four 50 mL centrifuge tubes and centrifuged at 3,500 rpm for 30 minutes (KUBOTA Model 5220 tabletop centrifuge). The wet bacterial weight of the harvested bacteria was measured using an electronic balance. A total of 2.032 g of Pantoea IG1 wet bacterial cells was obtained from 280 mL of culture.

[0020] [Preparation of Conventional Bacterial Cell Lysate] 1.0 g of the above wet bacterial cells was transferred to four centrifuge tubes, and phosphate-buffered saline (PBS(-)) (Fujifilm Wako Pure Chemical Industries) was added to prepare 10 mL of a 100 mg / mL suspension. To disrupt the wet bacterial cells, each suspension was heated in an autoclave at 100°C for 10 minutes. Once the temperature had cooled to 80°C, the tubes were vortexed five times for 30 seconds. Two tubes were vortex-disrupted to prepare 10 mL of conventional bacterial cell lysate ("Vortex Conventional Method"). The remaining two tubes were vortex-disrupted and then homogenized using a Polytron homogenizer at 15,000 rpm for 5 minutes. The tubes were then centrifuged at 3,500 rpm for 20 minutes to prepare 10 mL of conventional bacterial cell lysate ("Polytron Conventional Method").

[0021] [Preparation of the bacterial cell lysate of the present invention] To enhance the innate immune activation ability of the bacterial cell lysate, it is necessary to inexpensively and easily remove components ineffective in innate immune activation from the bacterial cell lysate. Regarding purification methods, ethanol precipitation requires a large amount of expensive alcohol and requires laborious centrifugation procedures, while gel filtration and ion exchange methods are not inexpensive or simple because they can only separate a small amount of substance and are laborious, resulting in high costs. In this study, ultrafiltration, an industrially simple component separation method used in seawater desalination, drinking water filtration, sake production, etc., was attempted.

[0022] Ten mL of each of the conventional bacterial cell lysates from two of the four tubes (the "conventional vortex method" and the "conventional Polytron method") was placed in a VIVASPIN 20 (#VS2051, Sartorius) equipped with a filter with a molecular weight cutoff of 300,000, and subjected to ultrafiltration by centrifugation at 3,000 rpm until the internal solution became 0.75 mL. The external solution from the first ultrafiltration (external solution with a small molecular weight (first)) was collected in a separate container.

[0023] The remaining 0.75 mL of the internal solution contained only 7.5 (= 0.75 mL / 10 mL * 100)% of the substances with molecular weights less than 300,000 contained in the original 10 mL of conventional bacterial cell lysate. In order to further remove the substances with molecular weights less than 300,000 remaining in this internal solution, a second ultrafiltration was performed as follows.

[0024] The remaining internal solution (0.75 mL) was added to PBS(-) (9.25 mL) and suspended. Ultrafiltration was then performed at 3000 rpm until the internal solution reached 0.75 mL. The external solution from the second ultrafiltration (external solution (second)) was collected in a separate container. The residual percentage of substances with molecular weights less than 300,000 in the second 0.75 mL internal solution was calculated to be 0.56% (= 7.5% * 7.5%) compared to the original 10 mL of conventionally disrupted bacterial cell material. This was less than 1 / 100th of the amount before ultrafiltration, indicating that sufficient removal had been achieved. Furthermore, to recover any residual internal solution adhering to the 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 (high molecular weight) was obtained.

[0025] Although LPS normally has a molecular weight of 5,000 to 50,000, it forms micelles, apparently forming large molecules. Therefore, LPS remains in the internal solution after two rounds of ultrafiltration, and by utilizing this property, the degree of LPS purification can be easily increased. The solution recovered by ultrafiltration was used as the bacterial cell lysate of the present invention ("Vortex present invention" and "Polytron present invention").

[0026] [Measurement of the innate immune activation ability of bacterial cell lysates] To measure the bioactivity per weight of each bacterial cell lysate obtained by the conventional method and the present invention, 10 mL of each of the four bacterial cell lysates (combined), obtained by the conventional method and the present invention, was centrifuged at 3500 rpm for 20 minutes, and the entire supernatant was collected in a separate centrifuge tube (0.5 mL to 0.67 mL of each lysate supernatant). The supernatant of each lysate was lyophilized to prepare a lyophilized product. The lyophilized product was weighed using an electronic balance (Table 1), and water for injection was added to prepare a solution with the same concentration of 20 mg / mL. (Maintaining the same concentration at this stage makes it easier to determine and compare the dry weights of the conventional and present bacterial cell lysates capable of inducing 1 μM NO during NO production testing.) The bioactivity of these solutions was measured based on NO production.

[0027] [LPS content measurement by Limulus assay] Limulus activity was measured for each bacterial cell lysate. Evaluations were performed using the same starting amount of each bacterial cell lysate and per dry weight. Each bacterial cell lysate was heated at 37°C for 5 minutes, sonicated for 10 minutes, and vortexed. The solution was then diluted with water for injection, and Limulus reagent was added. The reaction was monitored using a toxinometer (Fujifilm Wako Pure Chemical Corporation) to measure the amount of standard E. coli LPS (Fujifilm Wako Pure Chemical Corporation).

[0028] [Measurement Results] In order to compare the bacterial cell lysate prepared by the conventional method and the bacterial cell lysate prepared by the present invention, the starting bacterial volume was the same (10 mL), so they should contain the same amount of LPS. However, the LPS content per 10 mL of the bacterial cell lysate prepared by the conventional method using vortex disruption was 9.8 mg, while the LPS content per 10 mL of the bacterial cell lysate prepared by the present invention was 13.7 mg (Table 1). Surprisingly, the LPS content of the bacterial cell lysate prepared by the present invention was 1.4-fold higher than that of the bacterial cell lysate prepared by the conventional method. This result was unexpected.

[0029] The substances removed by ultrafiltration in the external solution (first and second runs) (lyophilized weights: 0.24 g and 0.12 g, respectively (Table 1)) were substances with molecular weights of 300,000 or less, including PBS(-). The LPS content in the external solution (first and second runs) was 0.0 mg (Table 1). Therefore, it is likely that the LPS contained in the conventional bacterial cell lysate remained in the internal solution (the present invention's lysate) even after ultrafiltration, and that most of the LPS-inhibiting substances were filtered into the external solution. This suggests that the bacterial cell lysate contains bacterial components such as proteins, peptides, lipids, nucleic acids, and organic acids, as well as bacterial metabolic products, among which are substances that inhibit LPS with molecular weights of 300,000 or less. Potential LPS-inhibiting substances include polypeptides, surfactants (e.g., lactonin), polyunsaturated fatty acids (e.g., docosahexaenoic acid), and metal ions such as calcium and iron.

[0030] Next, the LPS content per gram of each lyophilized bacterial cell lysate was calculated to be 29.6 mg for the conventional bacterial cell lysate and 139.5 mg for the present bacterial cell lysate (Table 1). By removing 70% (= (0.33 - 0.10) / 0.33 * 100) of the weight of substances with apparent molecular weights of less than 300,000, the LPS content increased by 4.7-fold compared with the conventional bacterial cell lysate. This result does not affect the amount of LPS contained in the conventional bacterial cell lysate, but is due to the removal of 70% of the weight of the substances, resulting in a decrease in the weight of the present bacterial cell lysate. Furthermore, the loss of LPS by the ultrafiltration method used to prepare the present bacterial cell lysate was extremely small, less than 1 / 100 of the original amount.

[0031] Furthermore, the LPS content per gram of dry weight of the conventionally disrupted bacterial cell lysates and the present invention, prepared by Polytron disruption, was measured. The LPS content per gram of dry weight was 33.7 mg for the conventionally disrupted bacterial cell lysate and 122.3 mg for the present invention, which is 3.6 times higher than that of the conventional method (Table 2).

[0032] From the above, it has become clear that the ultrafiltration method of the present invention for treating disrupted bacterial cells is an excellent preparation method in which the LPS content is substantially increased with almost no loss, regardless of the disruption method.

[0033]

[0034]

[0035] [Measurement of immunopotentiating activity based on NO production from RAW264.7 cells using conventional cell lysates and the present invention's cell lysates] After adding LPS to RAW264.7, a mouse macrophage cell line, NO production from the cells 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 harvested from a culture flask by pipetting and adjusted to a cell concentration of 1.6 x 10 in culture medium (RPMI1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin). 6 The cell suspension was adjusted to 100 μL (1.6 × 10 5 A total of 100 μL of each lyophilized bacterial cell lysate (cells / 100 μL) was transferred to each well of a 96-well flat-bottom plate and used in the test after 3 hours. Culture medium was added to each lyophilized bacterial cell lysate to adjust the concentration to 200 ng / mL, and three 10-fold serial dilutions were made, each of which was added in 100 μL to the cells. The cells were cultured for 24 hours at 37°C in a 5% CO2 incubator. After the culture was completed, 100 μL of the supernatant was collected and transferred to another 96-well plate. The amount of nitrite, a product of NO metabolism, in the culture medium was measured using Griess reagent according to standard methods.

[0036] [Results] Using the above method, the amount of NO produced at each concentration (dilution step) was measured, and the concentration of each freeze-dried cell lysate that could induce 1 μM of NO was calculated and compared. The conventional method yielded 5.6 ng / mL, while the present invention yielded 1.2 ng / mL. This demonstrates that the bioactivity per freeze-dried product weight of the bacterial cell lysate of the present invention is 4.7 times higher than that of the bacterial cell lysate obtained using the conventional method.

[0037] [Cultivation of Escherichia coli (NBRC 3301 strain)] A portion of an Escherichia coli (NBRC 3301 strain) colony was scraped and plated on 802 agar medium and cultured overnight in a constant temperature bath at 30°C. One colony was added to 80 mL of sterilized 802 medium and precultured at 30°C with shaking at 170 rpm. Eight 250 mL baffled flasks containing 80 mL of medium were each filled with 0.8 mL of preculture medium and cultured overnight with shaking at 30°C to obtain 560 mL of bacterial cell culture. As in Example 1, since medium components significantly affect the dry weight and thus the weight of the bacterial cell product, wet bacterial cells with minimal carryover from the culture medium were prepared by centrifugation and used for bioactivity evaluation. The bacterial cell culture was transferred to a 50 mL centrifuge tube and centrifuged at 3500 rpm for 30 minutes. The wet bacterial weight of the harvested bacteria was measured using an electronic balance. A total of 4.49 g of wet cells was obtained from 560 mL of the culture medium.

[0038] [Preparation of conventional bacterial cell disruption product] PBS(-) was added to 1.0 g of the above wet bacterial cells to prepare 10 mL of a 100 mg / mL suspension. To disrupt the wet bacterial cells, each suspension was heated in an autoclave at 100°C for 10 minutes. Once the temperature had dropped to 80°C, the suspension was stirred five times for 30 seconds using a vortex mixer to prepare conventional bacterial cell disruption products.

[0039] [Preparation of the bacterial cell lysate of the present invention] Ultrafiltration was performed in the same manner as in Example 1. 9.0 mL of the conventional bacterial cell lysate was placed in a VIVASPIN 20 tube with a molecular weight cutoff of 300,000, and centrifuged at 3,000 rpm until the internal solution reached 0.25 mL. The external solution from the first ultrafiltration (external solution (1st)) was collected in a separate container. The internal solution in the container was suspended in an equal volume of PBS(-) as the external solution (1st), and then a second ultrafiltration was performed at 3,000 rpm until the internal solution reached 0.25 mL. The external solution from the second ultrafiltration (external solution (2nd)) was collected in a separate container. Furthermore, the remaining internal solution adhering to the VIVASPIN 20 tube was collected using distilled water, and combined with the internal solution to obtain 3.0 mL of recovered solution. This was designated the bacterial cell lysate of the present invention.

[0040] [Measurement of the innate immune activation ability of bacterial cell lysates] To measure the bioactivity per weight of each bacterial cell lysate obtained by the conventional method and the present invention, each bacterial cell lysate was centrifuged at 3500 rpm for 20 minutes, and the supernatant was collected in a separate centrifuge tube (each lysate supernatant). 0.7 mL of each lysate supernatant was freeze-dried to prepare a freeze-dried product. The freeze-dried product was weighed using an electronic balance, and water for injection was added to prepare a solution at 50 mg / mL. The bioactivity of this solution was measured based on NO production.

[0041] [LPS content measurement by Limulus assay] Limulus activity of each bacterial cell lysate was measured. Evaluation was performed using the same starting amount of each bacterial cell lysate and per dry weight. Each bacterial cell lysate was heated at 37°C for 5 minutes, sonicated (10 minutes), and stirred using a vortex mixer. The resulting solution was then diluted with water for injection, and the amount of standard E. coli LPS was measured using a toxinometer.

[0042] [Measurement Results] As in Example 1, the starting bacterial volume of each bacterial cell lysate was the same (10 mL) for comparison, but the LPS content per 10 mL of bacterial cell lysate prepared by the conventional method was 4.5 mg, while the LPS content per 10 mL of bacterial cell lysate prepared by the present invention was 5.3 mg, meaning that the LPS content in the bacterial cell lysate prepared by the conventional method was 1.2-fold higher than that in the bacterial cell lysate prepared by the present invention (Table 3). Next, the LPS content per 1 g of each lyophilized bacterial cell lysate was calculated to be 31.7 mg for the bacterial cell lysate prepared by the conventional method, while it was 263.9 mg for the bacterial cell lysate prepared by the present invention (Table 3).

[0043] From the above, the LPS content in the bacterial cell lysate of the present invention was increased 8.3 times compared to that obtained by the conventional method. As in Example 1, the bacterial cell lysate treatment method of the present invention substantially increased the LPS content in Escherichia coli (NBRC 3301 strain), demonstrating that it is an excellent preparation method.

[0044]

[0045] [Measurement of Immunostimulatory Activity Based on NO Production from RAW264.7 Cells Using Conventional Cell Lysates and Cell Lysates of the Present Invention] After adding LPS to RAW264.7, a mouse macrophage cell line, NO production from the cells was measured using the concentration of the NO metabolite, nitrite, in the culture medium as an indicator. RAW264.7 cells were cultured as in Example 1. Lyophilized samples of each cell lysate were adjusted to 200 μg / mL with culture medium, diluted 10-fold in 6-7 steps, and cultured for 24 hours at 37°C in a 5% CO2 incubator. After incubation, 100 μL of supernatant was collected in a separate 96-well plate. The amount of nitrite, an NO metabolite, in the culture medium was measured using Griess reagent according to standard methods.

[0046] [Results] Using the above method, the amount of NO produced at each concentration (dilution step) was measured, and the concentration of each freeze-dried cell lysate that could induce 10 μM of NO was calculated and compared. The results were 19.6 ng / mL for the conventional method and 2.1 ng / mL for the present invention. This demonstrates that the biological activity per freeze-dried product weight of the bacterial cell lysate of the present invention is 9.3 times higher than that of the bacterial cell lysate obtained using the conventional method.

[0047] [Cultivation of the Acetic Acid Bacterium Gluconobacter oxydans] A portion of a Gluconobacter oxydans colony was scraped and plated on modified agar medium (Nippon University of Agriculture and Technology) and cultured for two nights in a 30°C incubator. One colony was added to 80 mL of sterilized modified medium (Nippon University of Agriculture and Technology) and precultured at 30°C with shaking at 170 rpm. Eight 250 mL baffled flasks containing 70 mL of medium were each filled with 8 mL of preculture medium and cultured with shaking at 30°C for two nights to obtain 560 mL of bacterial culture. As in Example 1, because medium components significantly affect the dry weight and thus the weight of the bacterial cell product, wet bacterial cells with minimal carryover from the culture medium were prepared by centrifugation and used for bioactivity evaluation. The bacterial culture was transferred to a 50 mL centrifuge tube and centrifuged at 3500 rpm for 20 minutes. The wet bacterial weight of the harvested bacteria was measured using an electronic balance. A total of 1.59 g of wet bacterial cells was obtained from the 560 mL culture medium.

[0048] [Preparation of Conventional Bacterial Cell Lysate] PBS(-) was added to 0.50 g of the above wet bacterial cells to prepare 10 mL of a 100 mg / mL suspension. 5 moles / L of aqueous sodium hydroxide solution was added, and the pH was confirmed with pH test paper and neutralized. Each suspension was then heated in an autoclave at 100°C for 10 minutes to disrupt the wet bacterial cells. Once the temperature had dropped to 80°C, the suspension was stirred five times for 30 seconds using a vortex mixer to prepare a conventional bacterial cell lysate.

[0049] [Preparation of the bacterial cell lysate of the present invention] Ultrafiltration was performed in the same manner as in Example 1. 3.8 mL of the conventional bacterial cell lysate was placed in a VIVASPIN 20 tube with a molecular weight cutoff of 300,000, and centrifuged at 3,000 rpm until the internal solution reached 0.25 mL. The external solution from the first ultrafiltration (external solution (1st)) was collected in a separate container. The internal solution in the container was suspended in an equal volume of PBS(-) as the external solution (1st), and then a second ultrafiltration was performed at 3,000 rpm until the internal solution reached 0.25 mL. The external solution from the second ultrafiltration (external solution (2nd)) was collected in a separate container. Furthermore, the remaining internal solution adhering to the VIVASPIN 20 tube was collected using distilled water, and combined with the internal solution to obtain 1.0 mL of recovered solution. This was designated the bacterial cell lysate of the present invention.

[0050] [Measurement of the innate immune activation ability of bacterial cell lysates] To measure the bioactivity per weight of each bacterial cell lysate obtained by the conventional method and the present invention, each bacterial cell lysate was centrifuged at 3500 rpm for 20 minutes, and the supernatant was collected in a separate centrifuge tube (each lysate supernatant). 0.7 mL to 1.0 mL of each lysate supernatant was freeze-dried to prepare a freeze-dried product. The freeze-dried product was weighed using an electronic balance, and water for injection was added to prepare a solution with a concentration of 50 mg / mL. The bioactivity of this solution was measured based on NO production.

[0051] [LPS content measurement by Limulus assay] Limulus activity of each bacterial cell lysate was measured. Evaluation was performed using the same starting amount of each bacterial cell lysate and per dry weight. Each bacterial cell lysate was heated at 37°C for 5 minutes, sonicated (10 minutes), and stirred using a vortex mixer. The resulting solution was then diluted with water for injection, and the amount of standard E. coli LPS was measured using a toxinometer.

[0052] [Measurement Results] As in Example 1, the starting bacterial volume (10 mL) of each bacterial cell lysate was the same for comparison, but the LPS content per 10 mL of the bacterial cell lysate prepared by the conventional method was 11.8 μg, while the LPS content per 10 mL of the bacterial cell lysate prepared by the present invention was 14.3 μg, meaning that the LPS content in the bacterial cell lysate prepared by the conventional method was 2.4-fold higher than that in the bacterial cell lysate prepared by the present invention (Table 4). Next, the LPS content per gram of each lyophilized bacterial cell lysate was calculated to be 62.1 μg for the bacterial cell lysate prepared by the conventional method and 476.7 μg for the bacterial cell lysate prepared by the present invention (Table 4).

[0053] From the above, it was found that the LPS content in the cell lysate of the present invention was 7.7 times higher than that in the conventional method (Table 4). As in Example 1, the cell lysate treatment method of the present invention substantially increased the LPS content in the acetic acid bacterium Gluconobacter oxydans, demonstrating that it is an excellent preparation method.

[0054]

[0055] [Measurement of Immunostimulatory Activity Based on NO Production from RAW264.7 Cells Using Conventional Cell Lysates and Cell Lysates of the Present Invention] After adding LPS to RAW264.7, a mouse macrophage cell line, NO production from the cells was measured using the concentration of the NO metabolite nitrite in the culture medium as an indicator. RAW264.7 cells were cultured as in Example 1. Lyophilized samples of each cell lysate were adjusted to 2000 μg / mL with culture medium, diluted 10-fold in five steps, and cultured for 24 hours at 37°C in a 5% CO incubator. After incubation, 100 μL of supernatant was collected in a separate 96-well plate. The amount of nitrite, an NO metabolite, in the culture medium was measured using Griess reagent according to standard methods.

[0056] [Results] Using the above method, the amount of NO produced at each concentration (dilution step) was measured, and the concentration of each freeze-dried cell lysate that could induce 1 μM of NO was calculated and compared. The results were 26.2 μg / mL for the conventional method and 3.4 μg / mL for the present invention. This demonstrates that the biological activity per freeze-dried product weight of the freeze-dried cell lysate of the present invention is 7.7 times higher than that of the conventional method.

[0057] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for producing disrupted bacterial cells, comprising the steps of: mechanically disrupting Gram-negative bacterial cells to obtain disrupted bacterial cells; and purifying the disrupted bacterial cells by molecular weight fractionation to obtain disrupted bacterial cells with a higher molecular weight.

2. The method for producing disrupted bacterial cell material according to claim 1, wherein the purification is carried out by ultrafiltration.

3. The method for producing disrupted bacterial cell material according to claim 2, characterized in that the molecular weight cutoff in the ultrafiltration method is 300,000.

4. A disrupted bacterial cell product produced by the method for producing disrupted bacterial cell product according to claim 1.

5. A blend of disrupted bacterial cells, characterized in that the disrupted bacterial cell material according to claim 4 is blended therein.

6. The composition of disrupted bacterial cells according to claim 5, characterized in that the composition is a food, cosmetic, skin care product, supplement, quasi-drug or pharmaceutical product.

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