Cell disruption suspension of thraustochytrids microorganism with antioxidant activity

Abstract

To provide antioxidant compositions containing cell debris of Thraustochytrids microorganism as an active ingredient, and methods for producing the cell disruption suspension.SOLUTION: The present inventors have found that the cell disruption suspension of Thraustochytrids microorganisms has excellent antioxidant activity, and completed the present invention.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an antioxidant composition containing, as an active ingredient, cell lysates of thraustochytrid microorganisms, and a method for producing said cell lysates suspension. [Background technology]

[0002] Reactive oxygen species (ROS), generated during various metabolic processes in the body, are known to be involved in the onset and progression of various conditions and diseases (e.g., inflammation, allergic reactions, joint damage, rheumatoid arthritis, osteoporosis, cardiovascular disease, and cancer). For example, when lipid molecules are oxidized by ROS, peroxidation of low-density lipoproteins can occur, leading to the formation of plaque in arteries, which can lead to cardiovascular disease and atherosclerosis. Furthermore, oxidative modification of protein molecules by ROS can cause changes in tissue structure and immune abnormalities, leading to rheumatoid arthritis and connective tissue damage, and altering the appearance and function of the skin.

[0003] In particular, the gastrointestinal tract is known to be the largest ROS-generating tissue in the body. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely used to prevent infarction, induce ROS generation in the gastrointestinal tract as a side effect. The gut microbiota, particularly in the intestine, is disrupted by ROS, facilitating the proliferation of harmful bacteria. ROS are generated by the metabolism of harmful bacteria, and mitochondria in the gastrointestinal tract damaged by toxins produced by harmful bacteria also generate ROS. ROS generated in the gastrointestinal tract not only deteriorates the health of the gastrointestinal tract itself, but also inhibits the elimination of ROS generated by other autoimmune systems and various organs by mobilizing ROS-eliminating mechanisms in the gastrointestinal tract, resulting in the development of various symptoms and diseases.

[0004] Superoxide dismutase (SOD), a representative ROS-scavenging enzyme, catalyzes the conversion of superoxide, a reactive oxygen species, into oxygen and hydrogen peroxide. The resulting hydrogen peroxide, which is also harmful, is then eliminated by catalase and glutathione peroxidase (GPx).

[0005] Various small molecules, such as various vitamins, polyphenols, catechins, glutathione, ascorbate, tocopherol, ubiquinone, bilirubin, and uric acid, can function as natural antioxidants, either in cooperation with or independently of the body's antioxidant mechanisms. For example, vitamin B2 acts as a coenzyme for GPx, and polyphenols have SOD-like activity. Carotenoids also function as antioxidants and may exert protective effects against oxidative stress and related chronic diseases. For example, Canfield et al. (1992) Proc. Soc. Exp. Biol. Med. 200:260 summarizes reports on the relationship between carotenoids and various chronic diseases, such as coronary heart disease, cataracts, and cancer.

[0006] Therefore, pharmaceuticals containing such antioxidant substances as active ingredients can be advantageously used for the treatment or prevention of diseases or symptoms associated with the action of ROS. In fact, it has been reported that administration of antioxidants can prevent gastrointestinal damage (Sha S. et al. (2013), Biomaterials, Nov; Vol. 34 (33), pp. 8393-8400) and improve the intestinal flora (Long BV. et al., (2014), J. Gastroenterol, May; Vol. 49 (5), pp. 806-813).

[0007] Substances with antioxidant activity are also found in various foods, and it is recommended to actively consume them in your daily diet to prevent the harmful effects of ROS and related diseases.

[0008] Furthermore, biological materials containing substances with such antioxidant activity, or compositions with high antioxidant activity obtained by processing them, can be used as antioxidants in foods, as food additives for enriching foods with antioxidant components, or as nutritional supplements, and there is a high demand for them in the food industry.

[0009] For example, biological materials whose extracts have high antioxidant activity include Brassica plants (Brassica spp., Brassicaceae) (JP 2003-81848 A), Kalanchoe plants (Crassulaceae) (JP 2005-29483 A), seeds of the Bignoniaceae family (JP 2006-321730 A), and Water hyacinth (Myrtaceae) (JP 2007-8902 A). Curcumin, contained in turmeric, has been shown to have antioxidant properties. In a study in which bovine aortic endothelial cells were exposed to curcumin, a strong induction of heme oxygenase-1 in the endothelial cells was observed (Motterlinin R. et al., (2000) Free Radic. Biol. Med., April 15; Vol. 28 (8) pp. 1303-1312). It has been confirmed that hydrolysates of ground flounder and peptides isolated from them have ROS-scavenging effects and protective effects against oxidative stress, inhibiting ROS generation, lipid peroxidation, and cell death. The use of peptides isolated from fractions of ground flounder hydrolysates as antioxidant food compositions is being investigated (Patent Publication No. 2020-518572). Extracts of blackcurrant leaves, a member of the Ribes family, have been shown to contain several novel compounds with antioxidant activity, and antioxidants containing these compounds as active ingredients and methods for isolating these novel compounds have been developed (Patent Publication No. 2014-084322).

[0010] Microbial cultures with antioxidant activity are also known, including liquid koji (JP 2013-252125 A), yeast extract (JP 2012-214393 A), and the culture supernatant of the yeast Candida tropicalis pK233 (Nishihara H. et al., (2009) Nippon Shokuhin Kagaku Kogaku Kaishi, Vol. 56 (5) pp255-260).

[0011] Thraustochytrid microorganisms have been widely used for the industrial-scale mass production of useful substances because they have high substance production efficiency, extremely fast growth rates, and the scale of their culture can be easily expanded. In particular, large-scale microbial culture techniques for producing substances with high industrial utility, such as hydrocarbons, oils such as triacylglycerols, or polysaccharides produced by microorganisms, have been highly developed (Patent Publication No. 2764572, G. Chen et al., New Biotechnology 27, 382-289 (2010)), and microorganisms can be used as low-cost, highly efficient biological resources.

[0012] As a prior art for obtaining biologically active biological materials from such microorganisms, Japanese Patent Publication No. 2017-137265 discloses that an ethanol extract of a microorganism belonging to the genus Aurantiochytrium has anti-inflammatory activity. However, in this publication, the microorganisms are suspended in a solvent such as ethanol, allowed to stand for a long period of time, and the supernatant is recovered. Therefore, this extract contains only dissolved components that can be eluted from the cells by the solvent. This extract is clearly distinguishable as a substance from the cell lysate suspension of the present invention, which contains the contents of cells obtained by homogenizing the cells in water, as described below. This substantial difference as a substance is manifested in the difference in physiological activity between the extract of this publication and the cell lysate suspension of the present invention, namely, that the extract of this publication has anti-inflammatory activity, while the cell lysate suspension of the present invention has antioxidant activity. [Prior art documents] [Patent documents]

[0013] [License 1] Special Announcement No. 2003-81848 [License 2] Special Announcement No. 2005-29483 [License 3] Special Announcement No. 2006-321730 [License 4] Special Announcement No. 2007-8902 [Patent Document 5] Special Announcement No. 2020-518572 [License 6] Special Announcement No. 2014-084322 [License 7] : 2013-252125 Announcement [License 8] Special Announcement No. 2012-214393 [License 9] Special Announcement No. 2017-137265 [Non-licensed literature]

[0014] [Non-licensed Document 1] :Canfield, et al., (1992) Proc. Soc. Exp. Biol. Med. 200:260 [Non-licensed Document 2] :Sha S. et al.(2013),Biomaterials, Nov; Vol34 (33), pp8393-8400) [Non-licensed Document 3] :Long BV. et al., (2014), J. Gastroenterol, May; Vol49(5), pp806-813) [Non-licensed Document 4] :Motterlinin R. et al., (2000) Free Radic. Biol. Med.,April 15; Vol. 28 (8) pp1303-1312 [Non-licensed Document 5] :G. Chen. et al. New Biotechnology 27, 382-289 (2010) [Non-patent document 6] :Nishihara H. et al., (2009) Nippon Shokuhin Kagaku Kogaku Kaishi, Vol. 56 (5) pp255-260 Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention relates to an antioxidant composition containing, as an active ingredient, cell lysates of thraustochytrid microorganisms, and a method for producing said cell lysates suspension. [Means for solving the problem]

[0016] The inventors have found that a suspension of disrupted cells of the heterotrophic microorganism Aurantiochytrium exhibits excellent antioxidant activity. Upon further investigation of this antioxidant activity, the suspension was found to have high scavenging activity against all reactive oxygen species, including superoxide, hydroxyl radical, and singlet oxygen, making it an excellent antioxidant biological material.

[0017] In addition to Aurantiochytrium, it was also confirmed that microorganisms from several other genera of Thraustochytrids, to which Aurantiochytrium belongs, also possess high antioxidant capacity, suggesting that this antioxidant capacity is possessed by many Thraustochytrids.

[0018] ROS are known to be involved in various diseases or symptoms in the body, and administration of antioxidants is useful for preventing or suppressing such diseases or symptoms. As shown in the Examples below, a homogenized suspension of Aurantiochytrium cells suppressed cell damage associated with increased ROS production induced by exposure to NSAIDs in normal rat gastric mucosal cells.

[0019] Furthermore, in the following examples, oral ingestion of a suspension of lysed Aurantiochytrium cells was shown to increase the proportion of Bifidobacteriaceae in the intestinal flora. As described above, ROS generated in the digestive tract disrupt the intestinal flora and promote the proliferation of harmful bacteria, resulting in the breakdown of the body's immune system and the development of various diseases. The improvement of the intestinal flora shown in the examples indicates that the antioxidant capacity of the suspension of lysed Aurantiochytrium cells extends to the improvement of ROS-related health conditions through oral ingestion.

[0020] As described above, microalgae are suitable for the mass production of useful substances on an industrial scale, and have a track record of being used in the production of pharmaceuticals, nutritional supplements, feed, and food additives. Therefore, the microalgae cell lysate with high antioxidant activity discovered in the present invention is a novel antioxidant biological material that is inexpensive and suitable for mass production, and will largely meet the high demand in the pharmaceutical, food, feed, and health care markets.

[0021] Therefore, the present application provides the following inventions.

[0022] 1. An antioxidant composition having antioxidant activity, which contains cell lysates of Thraustochytrid microorganisms as an active ingredient. 2. The antioxidant composition according to item 1 for the treatment and prevention and / or alleviation of diseases or symptoms or cosmetic problems associated with reactive oxygen species (ROS) in the body. 3. The antioxidant composition according to item 1, which is a nutritional supplement composition or a feed composition for supporting, improving or enhancing antioxidant function in the body. 4. The antioxidant composition according to any one of items 1 to 3, wherein the thraustochytrid microorganism belongs to any one of the genus Aurantiochytrium, Schizochytrium, Pariechytrium, Botryochytrium, Thraustochytrium, and Sicyoidochytrium. 5. The antioxidant composition according to any one of items 1 to 4, wherein the thraustochytrid microorganism belongs to either the genus Aurantiochytrium or the genus Schizochytrium. 6. A method for producing a cell lysate of a thraustochytrid microorganism to be incorporated as an active ingredient in the antioxidant composition according to any one of items 1 to 5, comprising the following steps: i) disrupting thraustochytrid microbial cells; and ii) suspending the disrupted cells in an aqueous medium; , a production method. 7. A method for producing a cell lysate of a thraustochytrid microorganism to be incorporated as an active ingredient in the antioxidant composition according to any one of items 1 to 5, comprising the following steps: i) suspending thraustochytrid microorganisms in an aqueous medium; and ii) disrupting the suspended cells; , a production method. 8. The production method according to either item 6 or 7, wherein the cell disruption is selected from the group consisting of mechanical disruption, high-pressure disruption, homogenization, ultrasonic disruption, and freeze-thawing. 9. The production method according to any one of items 6 to 8, wherein the aqueous medium is selected from the group consisting of pure water, salt water, culture medium, and buffer solution. 10. The production method according to any one of Items 6 to 9, further comprising: iii) a step of evaluating the antioxidant activity of the produced cell lysate suspension; and iv) a step of modifying step i) or ii) in reference to the evaluation results. 11. The production method according to Item 10, wherein the antioxidant activity is evaluated by measuring the activity of scavenging reactive oxygen species, measuring the activity of scavenging DDPH (2,2-diphenyl-1-picrylhydrazyl) radicals, or evaluating ORAC (oxygen radical absorbance capacity). 12. The antioxidant composition according to any one of items 1 to 5, which is in the form of a lozenge, pill, capsule, liquid medicine or tablet for oral ingestion. [Brief explanation of the drawings]

[0023] [Figure 1]This shows a phylogenetic tree of thraustochytrid microorganisms. The phylogenetic tree shows the lineage of the entire Labyrinthula family, with the group enclosed by the dotted rectangle representing the thraustochytrid lineage. The strain names listed are those evaluated for antioxidant capacity.

[0024] [Figure 2] The results of an evaluation of the antioxidant activity of the microbial cell lysate suspensions according to the present invention by electron spin resonance (ESR) are shown. The horizontal axis of each graph represents the magnetic field, and the vertical axis represents the signal intensity. MilliQ, Aurantiochytrium stored at room temperature for five years, and Spirulina served as negative controls. It was shown that the cell lysate suspensions of all four tested Aurantiochytrium strains possessed significant scavenging activity for the reactive oxygen species hydroxyl radical (OH-).

[0025] [Figure 3] The results of an evaluation of the antioxidant activity of the microbial cell lysate suspension of the present invention by electron spin resonance are shown below. The microbial samples used were Hondaea (AR-4a), Schizochytrium (SSK10-5), Parietichytrium (6F-10b), and Ulkenia (Ota1-10), and the reactive oxygen species used were singlet oxygen (O2) and hydroxyl radical (OH-). Hondaea, Schizochytrium, and Ulkenia exhibited potent reactive oxygen scavenging ability against singlet oxygen and hydroxyl radical. On the other hand, Parietichytrium was evaluated for its effectiveness against singlet oxygen, but not against hydroxyl radical.

[0026] [Figure 4]This study demonstrates the relationship between the preservation method of microbial cells and the antioxidant activity of microbial cell lysate suspensions. Microbial samples used included Aurantiochytrium one month after harvest (old), Aurantiochytrium immediately after harvest (new), refrigerated and frozen versions of each, and dried Schizochytrium after harvest. Singlet oxygen (1O2), superoxide (O2-), and hydroxyl radical (OH-) were used as reactive oxygen species. All forms of Aurantiochytrium and Schizochytrium, regardless of postharvest preservation method, exhibited potent scavenging activity against all reactive oxygen species.

[0027] [Figure 5] This shows the protective effect of microbial cell lysate suspension against cell damage caused by indomethacin (IND), an NSAID. Exposure to 250 μM IND reduced cell viability, but the viability was significantly restored in a concentration-dependent manner by the addition of microbial cell lysate suspension.

[0028] [Figure 6] 1 shows the results of metagenomic analysis of feces collected from male ICR mice orally administered the microbial cell lysate suspension according to the present invention. The proportion of Bifidobacteriaceae significantly increased in mice administered a high dose of the suspension. DETAILED DESCRIPTION OF THE INVENTION

[0029] In the present invention, microorganisms that can be used as a material for the cell lysate suspension are microorganisms belonging to the genus Thraustochytrium. Examples of such microorganisms include, but are not limited to, microbial species of the genera Aurantiochytrium, Schizochytrium, Pariechytrium, Botryochytrium, Thraustochytrium, and Sicyoidochytrium, as well as mutants or microbial strains originating from these. Preferably, the microorganisms that can be used as a material for the cell lysate suspension in the present invention are microorganisms of the genus Aurantiochytrium or Schizochytrium.

[0030] The microorganism used in the present invention is preferably a strain with excellent growth ability. Such a strain may be one that has been collected and isolated from nature, one that has been cloned through mutagenesis and screening, or one that has been established using genetic recombination technology. The characteristics that can be improved in the microbial strain are any characteristics that are advantageous for preparing a product with the desired antioxidant activity in the present invention, such as growth efficiency, tolerance to suboptimal culture conditions (high-density culture, sunlight, nutrients, temperature, pH, ingredient composition, etc.), or antioxidant activity of a cell suspension.

[0031] The cultivation of the microorganisms is based on methods established in the art, that is, normal maintenance cultivation is carried out by inoculating algae into a medium with appropriately prepared ingredients according to a standard method.

[0032] The medium for culturing the microorganism essentially contains salt, a carbon source, and a nitrogen source. Generally, a so-called GTY medium (artificial sea salt 10-40 g / L, D(+) glucose 20-200 g / L, tryptone 10-60 g / L, yeast extract 5-40 g / L) is used for culturing thraustochytrid microorganisms. The medium of the present invention is also basically composed of a combination of these three elements.

[0033] Carbon sources include sugars such as glucose, fructose, sucrose, etc., and alcohols such as glycerol, etc. These carbon sources are added at a concentration of, for example, 20 to 200 g per liter of medium.

[0034] Various nitrogen sources may be added to the culture medium for the microorganism, such as organic nitrogen sources such as sodium glutamate and urea, inorganic nitrogen sources such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate and ammonium nitrate, or biological digests such as yeast extract, corn steep liquor, polypeptone, peptone and tryptone.

[0035] Microorganisms are cultured at a culture temperature of 5 to 40°C, preferably 10 to 35°C, and more preferably 15 to 30°C. Subculture is usually performed every 1 to 10 days, preferably every 3 to 7 days, depending on the growth rate of the microbial strain. Cultivation can be performed by aeration culture, shaking culture, or static culture, with aeration culture or shaking culture being preferred. For long-term storage of microbial strains, an agar medium may be used, which is prepared by adding agar at a concentration of 1.0 to 3.0% to a liquid medium and solidifying it. For longer-term storage, microbial strains may be stored frozen according to standard methods.

[0036] The disrupted cell suspension of the present invention is prepared from the microorganism obtained by culturing. The microbial cells to be disrupted are used in a form suitable for the cell disruption treatment, such as a filtration concentrate, a centrifugation concentrate, a slurry, a paste, a wet or dry pellet, a frozen or spray-dried product, etc. The cell material before disruption may be stored refrigerated or frozen.

[0037] Any appropriate method can be used to disrupt the microbial cell material, including, for example, mechanical disruption using a bead mill or the like, high-pressure disruption, homogenization in an aqueous medium, ultrasonic disruption, freeze-thawing, freeze-pulverization, etc. The method to be used to disrupt the cellular material can be appropriately selected by those skilled in the art depending on the production scale, the quality required for the product, the configuration of the production equipment, etc. Preferably, ultrasonic disruption or high-pressure disruption can be used as the disruption method.

[0038] The disruption product of the microbial cell material can be suspended in a suitable aqueous medium. Alternatively, if the disruption is carried out in an aqueous medium, the aqueous medium may be the same as or different from the suspension medium of the final disrupted suspension. The aqueous medium may be pure water, or may be a salt water such as seawater or saline, a culture medium, a buffer solution, or the like, which can be selected by those skilled in the art depending on the intended use.

[0039] The cell lysate suspension has a high level of antioxidant activity. In the present invention, "antioxidant activity" refers to the ability to remove reactive oxygen species (ROS), such as singlet oxygen (1O2), hydroxyl radicals (OH-), and superoxide anions (O2-), or the ability to prevent or suppress cell or tissue damage caused by ROS in vivo, ROS-related diseases or symptoms, or cosmetic problems.

[0040] The high antioxidant activity of the cell lysate suspension of the present invention is believed to be achieved by disrupting the microbial cells and exposing or dissolving the cell contents in the aqueous medium. In fact, ethanol extraction of Aurantiochytrium microorganisms without disruption, as described in JP 2017-137265 A, does not exhibit such antioxidant activity. It is unclear which component of the intracellular contents of the thraustochytrid microorganisms provides the desired antioxidant activity of the present invention. However, as shown in the Examples below, activity varies depending on the microbial species, and activity decreases in older samples, suggesting that a group of bioactive substances that can be denatured and are specific to the microbial species is involved. Therefore, to maximize the antioxidant activity of the cell suspension, various considerations can be made, such as the selection and culture conditions of a microbial species whose intracellular contents have the desired antioxidant activity, disruption conditions that highly expose the intracellular contents, and an aqueous medium in which the intracellular contents can exert their antioxidant activity. These can be achieved without excessive trial and error by evaluating the antioxidant activity of the produced cell lysate suspension.

[0041] Preferably, the antioxidant activity may be evaluated using any appropriate method, and available evaluation methods include, for example, measurement of reactive oxygen species scavenging activity used in the Examples, measurement of DDPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, ORAC (Oxygen Radical Absorbance Capacity) evaluation, etc. The evaluation results can be used to optimize the manufacturing process of the disrupted cell suspension, such as the preparation conditions of the microbial material, the disruption treatment conditions, the composition of the aqueous medium, and the suspension concentration, or to determine the dosage of the suspension required to produce the desired antioxidant activity.

[0042] In addition, various additives may be added to the disrupted cell suspension to prevent denaturation during the preparation process, enhance antioxidant activity, improve storage stability, change physicochemical properties, or impart other nutritional properties. Such additives include lipids, carbohydrates, organic acids, vitamins, minerals, antibiotics, flavorings, colorings, preservatives, excipients, bulking agents, thickeners, adhesives, hydrating agents, disintegrants, emulsifiers, pH adjusters, and any other additives acceptable in the field to which the final product belongs.

[0043] The cell lysate suspension can be incorporated as an active ingredient into antioxidant pharmaceutical compositions or antioxidant cosmetic compositions for the treatment, prevention, and / or alleviation of various diseases or symptoms associated with ROS in the body. Alternatively, the cell lysate suspension can be incorporated as an active ingredient into nutritional supplement compositions or feed compositions with antioxidant activity. The form, composition, physicochemical properties, dosage, method of administration, and other conditions of the composition for each application are design factors that can be appropriately selected by those skilled in the art in each field depending on the specific application.

[0044] In a further aspect of the invention, the cell lysate suspension may be used alone or in combination with other substances that also have antioxidant or other physiological activities.

[0045] Preferably, the cell lysate suspension can be prepared as an oral preparation, which can further contain one or more optional excipients that are generally accepted as pharmaceuticals.

[0046] Preferably, the cell lysate suspension is formulated as a troche, pill, capsule, liquid medicine, tablet, or the like.

[0047] The oral formulation may preferably be taken as a nutritional supplement for the treatment or prevention of diseases or physical disorders associated with oxidative stress.

[0048] The oral formulation may be taken in combination with one or more additional substances, compounds, drugs or compositions useful in achieving the desired effect. [Example]

[0049] Example 1: Preparation of cell lysate suspension 1. Cell preparation Thraustochytrid cultivation Agar cultures of Aurantiochytrium limacinum strains 4W-ib and NYH2, Aurantiochytrium mangrovei strains 18W-13a and NYH1, Hondaea strain AR-4a, Schizochytrium strain SSK-10-5, Parietichytrium strain 6F-10b, and Ulkenia strain Ota1-10 were inoculated into GTY medium in 500-ml Erlenmeyer flasks and cultured at 100 rpm and 25°C for 7 days. During the culture period, 10 ml of 50% glucose solution was added to each culture on days 4 and 6. These cultures were collected by centrifugation at 3200 rpm for 20 minutes, washed twice with 1.7% saline, frozen at -80°C, and lyophilized under reduced pressure while still frozen.

[0050] 2. Cell disruption treatment The cells obtained in step 1 were adjusted to a concentration of 0.1 g / mL with MilliQ water. After adjustment, the suspension was disrupted using an ultrasonic homogenizer for 5 minutes.

[0051] Example 2: Antioxidant capacity test The antioxidant capacity of the cell lysate suspension obtained in Example 1 was tested using the following three reactive oxygen species measurement methods.

[0052] Singlet oxygen (1O2) measurement method: Acid red and TEMPO were dissolved in MilliQ water to final concentrations of 2 mM and 100 mM, respectively. 100 μL of Acid red, 100 μL of TEMPO, 30 μL of sample, and 70 μL of MilliQ water were mixed and irradiated with 540 nm light for 30 seconds. After irradiation, the sample solution was loaded into a Pasteur pipette and electron spin resonance (ESR) was measured using an electron spin resonance spectrometer (JEOdL-TE, JEOL Ltd., Tokyo). The measurement conditions were as follows: sweep width: 7.5 mT, gain: 50-500, modulation width: 0.2 mT, time contrast: 0.1 s, center field: 335.5 mT, sweep time: 0.5 min.

[0053] Hydroxy radical (OH-) measurement method: The spin trap CYPMPO was dissolved in MilliQ water to a final concentration of 100 mM. 16 μL hydrogen peroxide, 20 μL CYPMPO, 20 μL sample, and 144 μL MilliQ water were mixed and irradiated with 405 nm light for 10 seconds. The ESR of the sample solution after irradiation was measured in the same manner as for singlet oxygen measurement.

[0054] Superoxide anion (O2-) measurement method: The spin trap CYPMPO was dissolved in MilliQ water to a final concentration of 100 mM. Hypoxanthine was dissolved in NaOH to a final concentration of 20 mM. A mixture of 20 μL hypoxanthine and 10 μL CYPMPO was designated Solution A. B mixture of 1.6 μL xanthine oxidase (25 U / mL) and 10 μL CYPMPO was designated Solution B. C mixture of 20 μL sample and 138.4 μL MilliQ water was designated Solution C. The ESR of the sample solutions A, B, and C was measured in the same manner as for singlet oxygen measurement.

[0055] Antioxidant Ability Test 1: Evaluation of Antioxidant Activity of Aurantiochytrium Cell Lysate Suspension The antioxidant activity of cell lysate suspensions of Aurantiochytrium strains 4W-1b, NYH2, 18W-13a, and NYH1 prepared as described in Example 1 above was tested by the hydroxyl radical measurement described above. The results of the test are shown in Figure 2. It was shown that the cell lysate suspensions of all four tested Aurantiochytrium strains had significant scavenging activity for the reactive oxygen species hydroxyl radical (OH-).

[0056] Antioxidant capacity test 2: Evaluation of antioxidant activity of thraustochytrid microbial cell lysate suspension The antioxidant activity of cell lysate suspensions of Hondaea (AR-4a), Schizochytrium (SSK10-5), Parietichytrium (6F-10b), and Ulkenia (Ota1-10), prepared as described in Example 1, was tested using the hydroxyl radical and singlet oxygen assays described above. The results are shown in Figure 3. Hondaea, Schizochytrium, and Ulkenia exhibited potent reactive oxygen scavenging ability against singlet oxygen and hydroxyl radicals. On the other hand, Parietichytrium was evaluated for its effectiveness against singlet oxygen, but not against hydroxyl radicals.

[0057] Antioxidant Ability Test 3: Evaluation of the Antioxidant Activity of the Microbial Cell Lysate Suspension of the Present Invention under Different Storage Conditions Aurantiochytrium cells immediately after harvest (new), refrigerated or frozen samples one month after harvest (old), and dried Schizochytrium cells after harvest were used as microbial cell materials. Disrupted suspensions were prepared as described in Example 1 above, and their antioxidant activities were tested by the above-mentioned hydroxyl radical, superoxide anion, and singlet oxygen measurements. The test results are shown in Figure 4. All forms of Aurantiochytrium and Schizochytrium exhibited potent scavenging activity against all reactive oxygen species, regardless of the post-harvest storage method.

[0058] Example 3: Protective effect of cell lysate suspension against IND-induced cytotoxicity The Aurantiochytrium cell lysate suspension obtained in Example 1 was used to evaluate the protective effect of the cell lysate suspension against IND-induced cell injury. NSAIDs, including IND, are known to induce gastric mucosal injury, and it has also been reported that gastrointestinal injury caused by NSAID administration leads to deterioration of the intestinal bacterial flora. Normal rat gastric mucosal cells were cultured in a 96-well plate at 2 x 10 3 Cells were seeded at 1000 x g / well and cultured for 2 days. After culture, filter-sterilized cell lysate suspensions at 0, 1, or 10 μg / mL were added and cultured for an additional hour. After culture, the supernatant was removed and the cells were exposed to 250 μM IND for 24 hours. After exposure, cell viability was measured using CCK8.

[0059] The test results are shown in Figure 5. Exposure to IND reduced cell viability by 48%, but pretreatment with the cell lysate suspension significantly reduced the reduction in cell viability (p<0.01). This demonstrates that pretreatment with the cell lysate suspension according to the present invention can suppress IND-induced cell damage.

[0060] Example 4: Intestinal flora evaluation test The Aurantiochytrium cell lysate suspension obtained in Example 1 was used to examine its effects on the intestinal microbiota of mice. The gastrointestinal tract is the organ that produces the largest amount of ROS in the body, and increased ROS is associated with the deterioration of the intestinal microbiota and the subsequent onset of various diseases. Male ICR mice were orally administered 0, 1, 10, or 100 mg / mL Aurantiochytrium cell lysate suspension at a dose of 0.1 mL per 10 g of mouse body weight daily for 30 days. On the 31st day, the mice were individually housed in cages, and feces from each mouse were collected after 24 hours and stored at -20°C. Metagenomic analysis was performed using the feces.

[0061] The results of this test are shown in Figure 6. As a result of the metagenomic analysis, the proportion of Bifidobacteriaceae was significantly increased in the 100 mg / mL dose group compared to the other groups. This test demonstrated that administration of the cell lysate suspension of the present invention significantly increases the proportion of Bifidobacteria, a type of beneficial bacteria.

Claims

1. An antioxidant composition having antioxidant activity, which contains cell lysates of Thraustochytrid microorganisms as an active ingredient.

2. 10. The antioxidant composition according to claim 1 for the treatment and prevention and / or alleviation of diseases or symptoms or cosmetic problems associated with reactive oxygen species (ROS) in the body.

3. 2. The antioxidant composition according to claim 1, which is a nutritional supplement or a feed composition for supporting, improving or enhancing antioxidant function in the body.

4. 4. The antioxidant composition according to claim 1, wherein the Thraustochytrid microorganism belongs to any one of the genus Aurantiochytrium, Schizochytrium, Pariechytrium, Botryochytrium, Thraustochytrium, and Sicyoidochytrium.

5. The antioxidant composition according to any one of claims 1 to 4, wherein the thraustochytrid microorganism belongs to either the genus Aurantiochytrium or the genus Schizochytrium.

6. A method for producing a cell lysate of a thraustochytrid microorganism to be incorporated as an active ingredient in the antioxidant composition according to any one of claims 1 to 5, comprising the following steps: i) disrupting thraustochytrid microbial cells; and ii) suspending the disrupted cells in an aqueous medium; , a production method.

7. A method for producing a cell lysate of a thraustochytrid microorganism to be incorporated as an active ingredient in the antioxidant composition according to any one of claims 1 to 5, comprising the following steps: i) suspending thraustochytrid microorganisms in an aqueous medium; and ii) disrupting the suspended cells; , a production method.

8. 8. The method according to claim 6, wherein the disruption of the cells is selected from the group consisting of mechanical disruption, high-pressure disruption, homogenization, ultrasonic disruption, and freeze-thawing.

9. The method according to any one of claims 6 to 8, wherein the aqueous medium is selected from the group consisting of pure water, salt water, culture medium, and buffer solution.

10. The production method according to any one of claims 6 to 9, further comprising: iii) a step of evaluating the antioxidant activity of the produced cell lysate suspension; and iv) a step of modifying step i) or ii) in reference to the evaluation results.

11. The production method according to claim 10, wherein the evaluation of antioxidant activity is carried out by measuring the activity of scavenging reactive oxygen species, measuring the activity of scavenging DDPH (2,2-diphenyl-1-picrylhydrazyl) radicals, or evaluating ORAC (oxygen radical absorbance capacity).

12. The antioxidant composition according to any one of claims 1 to 5, which is in the form of a troche, pill, capsule, liquid medicine or tablet for oral ingestion.

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