JP2018199632A - Spore germination inhibitor - Google Patents

Abstract

To provide a substance that can inhibit germination of spore-forming bacterium spore.SOLUTION: Provided is a germination inhibitor for Paenibacillus bacterium spore containing aspartic acid as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a substance that suppresses germination of bacterial spores.

  Gram-positive spore-forming bacteria are highly resistant to the environment and difficult to control growth. Spore-forming bacteria have long been known as a cause of food spoilage. In recent years, it has been reported that spores are formed in biofilms, and that properties such as heat resistance are different between floating cell-derived spores and biofilm-derived spores. Since most of the bacteria in the environment are considered to exist in a biofilm state, control of spore in the biofilm as well as in the floating cell is important for the suppression of bacterial growth.

  Techniques have been proposed for controlling the spore growth of bacteria that are not easily sterilized by heating or bactericidal agents due to the presence of the spore. For example, a method of sprouting all spores quickly to sterilize them in the state of vegetative cells, or a method of suppressing germination and suppressing proliferation over a long period of time has been attempted.

  Conventionally, amino acids are often used for promoting germination and inhibiting germination. For example, L-aspartic acid is used as a germination promoter for Bacillus (Non-Patent Document 1), while it is used as a germination inhibitor for Clostridium ( Non-patent document 2). Glycine is used as a germination inhibitor for Bacillus (Non-Patent Document 3), while it is used as a germination promoter for Clostridium (Non-Patent Document 4).

  Paenibacillus is a genus of bacteria reclassified from the genus Bacillus, and is distributed in soil, plant rhizosphere, water, insects, foods and the like. Paenibacillus genus bacteria are spore-forming bacteria and have recently attracted attention as food spoilage harmful bacteria. The spores of the genus Paenibacillus have higher resistance to peracetic acid, which is a powerful fungicide, and can grow at low temperatures as compared to other types of spore-forming bacteria (Non-patent Document 5). In relation to various beverage foods, the problem of resistant bacteria such as Paenibacillus spp. Furthermore, the spoilage fungi Paenibacillus sp. And Paenibacillus odorifer were isolated from cooked foods, that these fungi were able to grow at 4 ° C, and that Paenibacillus spp. (Non-patent Document 6).

  On the other hand, in the fields of horticulture and agriculture, Paenibacillus bacteria are used as organic biological fertilizers or microbial preparations for the purpose of promoting plant growth and controlling organisms. In these organic biological fertilizers or microbial preparations, spore formation of Paenibacillus bacteria is considered to be effective for improving the survival rate of the bacteria in the fertilizers and preparations, promoting the growth of the plants, and maintaining the biological control effect. (See Non-Patent Document 7).

Applied and Environmental Microbiology, 2016, doi: 10.1128 / AEM-00594-16. Journal of Bacteriology, 2010, 192 (2): 418-425 Microbiology and Immunology, 1985, 29 (3): 229-241 Journal of Bacteriology, 2016, 198 (20): 2767-2775 Abstracts of Annual Meeting 2016, Presentation Number 2F043 Biocontrol science, 2006, 11 (1): 43-47 Bioresource Technology, 2012, 108: 190-195

  The present invention provides a substance capable of suppressing germination of spores of spore-forming bacteria.

  The present inventors have found that aspartic acid has an action of suppressing germination of spores of the genus Paenibacillus.

Therefore, in one embodiment, the present invention provides a germination inhibitor for Paenibacillus spp. Containing aspartic acid as an active ingredient.
In another embodiment, the present invention provides a growth inhibitor of Paenibacillus bacteria comprising aspartic acid as an active ingredient.
In another embodiment, the present invention provides an agent for preventing microbial contamination by Paenibacillus bacteria, comprising aspartic acid as an active ingredient.
In another embodiment, the present invention provides an agent for improving the stability of a fertilizer or a microbial preparation containing Paenibacillus bacteria, comprising aspartic acid as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, germination of the spore of Paenibacillus genus bacteria can be suppressed effectively irrespective of the presence state of microbes, such as a floating cell and a biofilm. Moreover, according to this invention, the proliferation of the said microbe can be suppressed by suppressing the germination of the spore of Paenibacillus genus bacteria. The present invention is applicable to suppression of germination of Paenibacillus spp. In foods, fertilizers, microbial preparations, and the like. ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to prevent the decay and alteration of foodstuffs by the Paenibacillus spore which contaminated in the manufacturing process or remained after a manufacturing process, without performing the heat sterilization and chemical processing which impair the food flavor. . Further, according to the present invention, the Paenibacillus genus bacteria contained in the fertilizer or microbial preparation can be maintained as spores to improve the survival rate, and the effect of the fertilizer or microbial preparation can be maintained.

Phase contrast microscope observation images of Paenibacillus polymyxa ATCC39564 spore derived from biofilm (BF) cultured in the presence (30 mM) and absence (0 mM) of L-Asp. After 30 minutes of culture. Phase contrast microscope observation images of Paenibacillus polymyxa ATCC39564 spore derived from biofilm (BF) cultured in the presence (30 mM) and absence (0 mM) of L-Asp. After 28 hours of culture. Phase contrast microscopic observation images of Paenibacillus polymyxa ATCC39564 spores cultured in the presence (3 to 30 mM) and absence (0 mM) of L-Asp. After 30 minutes of culture. Phase contrast microscopic observation images of Paenibacillus polymyxa ATCC39564 spores cultured in the presence (30 mM) and absence (0 mM) of L-Asp. After 28 hours of culture. Phase-contrast microscopy images of suspension culture-derived Paenibacillus polymyxa ATCC39564 spores cultured in the presence (30 mM) and absence (0 mM) of D-Asp. After 30 minutes of culture. Phase-contrast microscopy images of suspension culture-derived Paenibacillus sp. KMC150 spores cultured in the presence (30 mM) and absence (0 mM) of L-Asp. After 24 hours of culture. Phase-contrast microscope images of suspension culture-derived Paenibacillus sp. KMC150 spores cultured in the presence (30 mM) and absence (0 mM) of D-Asp. After 16 hours of culture. Phase-contrast microscopy images of Paenibacillus glucanolyticus KMC129 spores cultured in the presence (30 mM) and absence (0 mM) of L-Asp. After 24 hours of culture. Phase-contrast microscopy images of Paenibacillus glucanolyticus KMC129 spores cultured in the presence (30 mM) and absence (0 mM) of D-Asp. After 16 hours of culture.

  In the present invention, aspartic acid is used as an active ingredient for inhibiting germination of Paenibacillus bacteria.

  Furthermore, in the present invention, aspartic acid suppresses germination of Paenibacillus spore bacteria, so that the growth of the bacterium can be suppressed, and further, microbial contamination by the bacterium can be prevented. Therefore, in the present invention, aspartic acid can also be used as an active ingredient for inhibiting the growth of Paenibacillus bacteria or for preventing microbial contamination by Paenibacillus bacteria. These aspects of the present invention are useful in fields such as food production.

  Furthermore, in the present invention, aspartic acid can be used as an active ingredient for improving the stability of fertilizers or microbial preparations containing Paenibacillus bacteria. In the fertilizer or microbial preparation containing the Paenibacillus bacterium, the germination of the spore of the bacterium is suppressed by aspartic acid, thereby improving the survival rate of the bacterium in the fertilizer or the preparation, and Plant growth promotion or biocontrol effect can be maintained. Preferably, the improvement in the stability of the fertilizer or microbial preparation in the present invention means the survival rate of Paenibacillus bacteria in the fertilizer or preparation or the quality of the fertilizer or preparation (as compared to the case where no aspartic acid is added). For example, it refers to improvement or prevention of plant growth promotion or biological control effect.

  In the present invention, the species of the genus Paenibacillus is not particularly limited, and preferred examples include Paenibacillus polymyxa, Paenibacillus chibensis, Paenibacillus glucanolyticus, Paenibacillus glucanolyticus, Paenibacillus massiliensis, Paenibacillus popilliae, Paenibacillus alvei, Paenibacillus sp. And the like, more preferred examples are Paenibacillus sp. Paenibacillus sp., Paenibacillus polymyxa ATCC39564, Paenibacillus sp. KMC150, Paenibacillus glucanolyticus KMC129.

  The spores of the genus Paenibacillus may be in the state of floating cells or may be present in a biofilm. In any case, aspartic acid exhibits an excellent germination inhibitory effect on the spores of Paenibacillus bacteria.

  The aspartic acid used in the present invention may be either L-aspartic acid or D-aspartic acid. Further, the aspartic acid may be a free form or a salt form. Examples of the salt include acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, and the like. Examples of the acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, and phosphate, and acetate, maleate, fumarate, citrate, malate, lactate, α-ketoglutar Organic acid salts such as acid salts, gluconates and caprylates can be mentioned. Examples of the metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt. Examples of the ammonium salt include salts such as ammonium and tetramethylammonium. Examples of the organic amine addition salt include salts of morpholine, piperidine and the like. Examples of the amino acid addition salt include salts with arginine, lysine and the like. Aspartic acid used in the present invention may be at least one selected from the group consisting of L-aspartic acid, D-aspartic acid, and salts thereof.

  Aspartic acid or a salt thereof used in the present invention can be produced by a conventional method. For example, a free form of aspartic acid or a salt thereof can be obtained by a method of isolation and purification from animals or plants containing them, chemical synthesis, fermentation production, or the like. Or you may purchase a commercial item.

  In the present invention, the subject to which the aspartic acid is applied includes the aforementioned spores of the genus Paenibacillus, or substances containing or possibly having the spores of the genus Paenibacillus. Preferably, the aspartic acid in the present invention is a substance containing or possibly containing Paenibacillus spore, a substance for suppressing the germination of Paenibacillus spore, a substance for suppressing the growth of Paenibacillus bacterium, Paenibacillus genus, It can be applied to substances that want to prevent microbial contamination by bacteria. Examples of such applicable substances include foods, packaging or storage containers thereof, fertilizers or microbial preparations containing Paenibacillus bacteria, liquids or biofilms containing or possibly containing Paenibacillus bacteria, and the like. It is done.

  As used herein, “food” includes solid, semi-solid or liquid food, beverage, feed, pet food, and the like, and raw materials thereof. Examples of the food include confectionery such as breads, noodles, rice, and cookies, tablets, jelly, dairy products, soups, frozen foods, instant foods, processed starch products, processed fish products, and other processed foods. , Seasonings, nutritional supplements, and beverages such as tea beverages, coffee beverages, milk beverages, fruit beverages, near water, carbonated beverages, and jelly-like beverages.

  As used herein, “fertilizer” includes fertilizer in a form known in the art, for example, solid, liquid or powder. Preferably, the fertilizer is an organic biological fertilizer. The fertilizer includes organic fertilizer materials, micronutrient fertilizer materials, insecticides, herbicides, plant growth improvers, fungicides, shellicides, algicides, bacterial inoculums, fungal inoculations in addition to the above-mentioned Paenibacillus bacteria It may further include materials, or combinations thereof. The fertilizer may be applied to a plant growth medium (eg, soil, solution, etc.) or may be applied directly to the plant or seed.

  In the present specification, the “microbe preparation” includes a microbial preparation generally used in the field of horticulture or agriculture for the purpose of promoting plant growth, controlling the organism, and the like. The microbial preparation is in the same form as a normal horticultural or agricultural microbial preparation (eg, powder, wettable powder, granule, emulsion, liquid, suspension, flowable, coating agent, etc.). Good. The microorganism preparation may contain a culture solution, a high concentration product or a dried product of the above-mentioned Paenibacillus bacterium alone, or, in addition to the Paenibacillus bacterium, other microorganisms, solid carriers, adjuvants, and other plants. It may further contain other optional components that are permitted to be applied. The microbial preparation may be applied to a plant growth medium (eg, soil, solution, etc.), may be applied to a fertilizer or water applied to the plant, or may be applied directly to the plant or seed.

  As the Paenibacillus bacterium used in the fertilizer and microbial preparation for the purpose of promoting plant growth or controlling the organism, preferably, the above-mentioned Paenibacillus polymixer, Paenibacilis tibensis, Paenibacillus glucanoliticus, Paenibacillus maciliensis, Paenibacillus Examples include Popilie and Paenibacillus albei.

  When suppressing germination of Paenibacillus spp. Contained in the applicable substance, add aspartic acid to the substance, or apply or spray a liquid containing aspartic acid on the surface of the substance, or aspartic acid What is necessary is just to immerse this substance in the liquid containing this. In order to suppress germination of Paenibacillus genus bacterial spores present in foods, fertilizers or microbial preparations, it is preferable to add aspartic acid to the foods, fertilizers or microbial preparations. In order to suppress germination of Paenibacillus spp. Existing in a food package or storage container, it is preferable to apply or spray a liquid containing aspartic acid on the surface of the package or storage container. In order to suppress germination of Paenibacillus spp. Existing in a biofilm, the biofilm is applied or sprayed with a liquid containing aspartic acid, or the biofilm is immersed in a liquid containing aspartic acid, It is preferable to penetrate aspartic acid into the biofilm.

  In the present invention, the application concentration of aspartic acid is not particularly limited as long as it is an effective amount that can suppress germination of Paenibacillus spp. In the application target, but is preferably 2 mM or more, more preferably 3 mM or more, and Preferably, it is 5 mM or more. On the other hand, from the viewpoint of economy, it is preferably 300 mM or less, more preferably 30 mM or less (aspartic acid free form equivalent, the same applies hereinafter). Examples of the range of application concentration of aspartic acid in the present invention include 2 to 300 mM, 2 to 30 mM, 3 to 300 mM, 3 to 30 mM, 5 to 300 mM, and 5 to 30 mM.

  Therefore, according to a preferred embodiment of the present invention, for example, the concentration of aspartic acid in the substance is 0.027 to 4.0% by mass relative to the application target substance such as food, fertilizer, and microbial preparation. 027 to 0.40 mass%, 0.040 to 4.0 mass%, 0.040 to 0.40 mass%, 0.067 to 4.0 mass%, or 0.067 to 0.40 mass% Thus, aspartic acid may be added. Alternatively, when the substance to be applied is a food package or a storage container, 0.027 to 4.0% by mass, 0.027 to 0.40% by mass, 0.040 to 4.0% by mass, 0.040 A liquid containing ~ 0.40 mass%, 0.067-4.0 mass%, or 0.067-0.40 mass% aspartic acid is applied or sprayed onto the substance, or the substance is applied to the liquid. The liquid may be attached to the surface of the substance by dipping. Alternatively, when the substance to be applied is a biofilm, a liquid containing aspartic acid is applied or sprayed on the substance, or the substance is immersed in the liquid so that the concentration of aspartic acid in the substance is 0. 027 to 4.0 mass%, 0.027 to 0.40 mass%, 0.040 to 4.0 mass%, 0.040 to 0.40 mass%, 0.067 to 4.0 mass%, or 0 What is necessary is just to make it become 0.067-0.40 mass%.

  In a preferred embodiment, the concentration of aspartic acid in the germination inhibitor of Paenibacillus spore, the growth inhibitor of Paenibacillus bacterium, and the microbial contamination inhibitor by Paenibacillus of the present invention is preferably 2 to 300 mM, 2 to 30 mM, 3 to 300 mM, 3 to 30 mM, 5 to 300 mM, or 5 to 30 mM.

  In the present invention, the aspartic acid may be used in combination with another substance having an action of suppressing germination of Paenibacillus spore. Examples of such other materials include indole and its analogs. Furthermore, the aspartic acid of the present invention may be used in combination with other substances as long as the effect of suppressing the germination of the Paenibacillus spore is not impaired. For example, it can be used in combination with existing bactericides and preservatives.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

(Method)
(1) Preparation of spore culture medium Paenibacillus spp. KMC150 is a Paenibacillus polymyxa ATCC39564, which was distributed from the American Type Culture Collection (ATCC), and was isolated from the food filling line environment by a conventional method and identified by the sequence of 16S ribosomal RNA. And Paenibacillus glucanolyticus KMC129 isolated from the raw material by a conventional method and identified by the sequence of 16S ribosomal RNA. The Paenibacillus bacterium was cultured overnight in TSB medium (BACTO Tryptic Soy broth: Becton, Dickinson and Company, catalog # 21825). Thereafter, the culture solution of Paenibacillus polymyxa ATCC39564 and Paenibacillus sp. KMC150 was added to 2 × SGG (Schaffer's glucose supplemented with 1% [wt / vol] glycerol) medium so that OD ₆₀₀ = 0.01 (the composition is Journal of bacteriology, 2007, 189 (13): 4920-4931). JASCO V-530 was used for the measurement of OD ₆₀₀ . In addition, the culture solution of Paenibacillus glucanolyticus KMC129 is in accordance with DS (Difco stimulation) medium (composition is Molecular biological methods for Bacillus, CR Harwood, and SM Cutting (eds.), Wiley, 1990, so that OD ₆₀₀ = 0.01. ) Suspended in 100 mL. By culturing the suspension of Paenibacillus polymyxa ATCC39564 in a 500 mL beaker at 30 ° C. for 200 hours, a culture solution containing a biofilm containing spores was obtained. In addition, the suspension of each strain was shake-cultured in a 500 mL bladed Erlenmeyer flask at 30 ° C. and 150 rpm (60 hours for Paenibacillus polymyxa ATCC39564, 50 hours for Paenibacillus glucanolyticus KMC129, 96 hours for Paenibacillus sp. KMC150), A culture solution containing floating cells containing Paenibacillus spore was obtained.

(2) Preparation of Spore Purified Solution The culture solution of (1) was observed with a phase contrast microscope to confirm the formation of spores. Spores were purified from the culture solution of each fungus. Paenibacillus polymyxa spores are collected by centrifuging the culture solution at 4 ° C., 9000 G for 15 minutes, then suspending the collected bacteria in sterile water, and centrifuging at 4 ° C., 9000 G for 15 minutes. Purification by precipitation. Paenibacillus sp. Or Paenibacillus glucanolyticus spores were collected by centrifuging the culture solution at 4 ° C. and 3000 G for 15 minutes, and then the collected bacteria were suspended in sterile water and centrifuged at 4 ° C. and 7000 G for 15 minutes. Purification was performed by separating and precipitating spores. The procedure of spore purification was repeated until cell debris and vegetative cells were removed and 90% or more of the cells became spores when the purified product was observed with a phase contrast microscope. The obtained spore pellet was suspended in cold sterilized water and used as a purified spore solution. The purified solution was stored at 4 ° C. until use.

(3) Culture and spore observation in the presence of aspartic acid In the spore purified solution of Paenibacillus polymyxa derived from biofilm (BF) or suspension culture obtained in (2), L-aspartic acid (L-Asp) or D -Add Nutrient Broth containing aspartic acid (D-Asp) (L-Asp final concentration is 0, 1, 2, 3, 4, 5 or 30 mM, D-Asp final concentration is 0 or 30 mM) and test The cells were cultured at 30 ° C. in a tube. In addition, the spore purified solution of Paenibacillus sp. Or Paenibacillus glucanolyticus derived from suspension culture obtained in (2) was heated at 80 ° C. for 15 minutes, and then AGFK solution (100 mM L-Asp containing 0 or 30 mM L-Asp). -Inoculate a 1/2 AGFK solution containing asparagine, 100 mM D-glucose, 10 mM D-fructose, 100 mM KCl, 10 mM Tris-HCl; pH 8.4), or 0 or 30 mM D-Asp and in vitro Cultured at 30 ° C. Germinated spores are observed as dark spores by phase contrast microscopy. Therefore, the spore culture solution was observed with a phase contrast microscope after 30 minutes of culture and after 16, 24 and 28 hours. In addition, when the spore germinates, it absorbs moisture, and the turbidity decreases. Therefore, the turbidity OD ₆₆₀ of the spore culture solution was measured after 0 to 120 minutes of culture, and the rate of change in turbidity was calculated based on the initial turbidity (0 minute of culture), which was used as an index of germination efficiency. For the measurement of OD ₆₆₀ , a miniphoto518R manufactured by TAITEC was used.

(Example 1) Inhibition of Paenibacillus polymyxa spores by L-Asp Phase-contrast microscopy images of BF-derived Paenibacillus polymyxa ATCC39564 spores cultured for 30 minutes and 28 hours in the presence (30 mM) and absence (0 mM) of L-Asp Are shown in FIGS. 1 and 2, respectively. At both 30 minutes and 28 hours after culturing, the ratio of Dark spores (germinated spores) in the total spores was lower in the presence of L-Asp than in the absence.

  Table 1 shows the results of turbidity measurement of the BF-derived Paenibacillus polymyxa ATCC39564 spore culture solution cultured in the presence or absence of L-Asp. In the presence of 2 mM or more of L-Asp, turbidity reduction due to culture was suppressed, indicating that spore germination was inhibited.

  Phase-contrast microscopy images of suspension culture-derived Paenibacillus polymyxa ATCC39564 spores cultured for 30 minutes and 28 hours in the presence (3 to 30 mM) or absence (0 mM) of L-Asp are shown in FIGS. 3 and 4, respectively. At both 30 minutes and 28 hours after culturing, the ratio of Dark spores in the whole spores decreased in the presence of L-Asp compared to the absence. Furthermore, after 30 minutes of culture, the ratio of Dark spores in the whole spores decreased depending on the concentration of L-Asp.

(Example 2) Inhibition of Paenibacillus polymyxa spore germination by D-Asp Phase contrast microscopic observation image of suspension cell-derived Paenibacillus polymyxa ATCC39564 spore cultured in the presence (30 mM) and absence (0 mM) of D-Asp (30 minutes in culture) The latter is shown in FIG. Spore germination was suppressed in the presence of D-Asp.

(Example 3) Inhibition of Paenibacillus sp. Spore germination by L-Asp Phase contrast microscopic observation image of suspension cell-derived Paenibacillus sp. KMC150 spore cultured in the presence (30 mM) and absence (0 mM) of L-Asp (culture) 24 hours later) is shown in FIG. Spore germination was suppressed in the presence of L-Asp.

(Example 4) Inhibition of germination of Paenibacillus sp. Spore by D-Asp Phase contrast microscopic observation image of cultured cell-derived Paenibacillus sp. KMC150 spore cultured in the presence (30 mM) and absence (0 mM) of D-Asp (culture) 16 hours later) is shown in FIG. Spore germination was suppressed in the presence of D-Asp.

(Example 5) Inhibition of Paenibacillus glucanolyticus spore germination by L-Asp Phase contrast microscopic observation image of floating cell-derived Paenibacillus glucanolyticus KMC129 spore cultured in the presence (30 mM) and absence (0 mM) of L-Asp (cultured for 24 hours) The latter is shown in FIG. Spore germination was suppressed in the presence of L-Asp.

(Example 6) Inhibition of Paenibacillus glucanolyticus spore germination by D-Asp Phase contrast microscopic observation image of suspension-derived Paenibacillus glucanolyticus KMC129 spore cultured in the presence (30 mM) and absence (0 mM) of D-Asp (cultured for 16 hours) The latter is shown in FIG. Spore germination was suppressed in the presence of D-Asp.

Claims (10)

  A germination inhibitor for Paenibacillus spp., Which contains aspartic acid as an active ingredient.   A growth inhibitor of Paenibacillus bacteria comprising aspartic acid as an active ingredient.   An antimicrobial agent for microbial contamination by Paenibacillus bacteria, comprising aspartic acid as an active ingredient.   A stability improver for fertilizers or microbial preparations containing Paenibacillus bacteria, comprising aspartic acid as an active ingredient.   The agent of any one of Claims 1-4 whose said Paenibacillus genus bacteria are Paenibacillus polymixer, Paenibacillus sp, or Paenibacillus glucanolyticus.   A method for inhibiting germination of Paenibacillus spore, comprising applying aspartic acid to Paenibacillus spore.   A method for inhibiting the growth of Paenibacillus bacteria, comprising applying aspartic acid to Paenibacillus bacteria spores.   A method for preventing contamination of a substance by a bacterium belonging to the genus Paenibacillus, comprising applying aspartic acid to the substance containing or possibly the genus Paenibacillus.   A method for improving the stability of a fertilizer or microbial preparation containing Paenibacillus spore, comprising applying aspartic acid to a fertilizer or microbial preparation containing Paenibacillus spore.   The method according to any one of claims 6 to 9, wherein the Paenibacillus bacterium is Paenibacillus polymixer, Paenibacillus sp, or Paenibacillus glucanolyticus.