JP2018033437A - Microbe and enzyme which degrade trimethylamine and method of application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel method to address the problem that the application of conventional methods was difficult in low-temperature conditions of refrigerators and under alkaline conditions, as related to trimethylamine which is a source of unpleasant odors, as the storage conditions of food which produce trimethylamine such a fresh fish is mostly 4°C or less and trimethylamine which has dissolved in water and the like is alkaline.SOLUTION: A method of degrading trimethylamine in low temperature conditions such as in a refrigerator and under conditions exhibiting alkalinity in liquid or food products uses Brevundimonas vesicularis bacterial strain and a degrading enzyme derived from said bacterial strain. The method uses Brevundimonas vesicularis bacterial strain (National Institute of Technology and Evaluation Accession No. NITEAP-02293) which can grow in low temperature, alkaline conditions and has trimethylamine degrading capabilities.SELECTED DRAWING: None

Description

  This invention is a causative substance of an unpleasant odor generated from food, domestic wastewater, domestic animal manure, waste, etc., and has the ability to resolve trimethylamine, which is designated as a regulated substance by the Malodor Control Law, Brevundimonas vesicularis: Independent Administrative Institution Product Evaluation Technology Infrastructure Deposit Number NITE AP-02293) and a trimethylamine-degrading enzyme obtained from this microorganism.

  Trimethylamine is known and commonly recognized as an unpleasant odor arising from foods such as seafood. The cause of the occurrence is that it is decomposed and produced from trimethylamine-N-oxide contained in the body of seafood by the trimethylamine-N-oxide reductase derived from the seafood of the fish or from the microorganisms attached to the body surface. It is a substance known to do (Non-patent Document 1).

  On the other hand, trimethylamine is designated as a specific malodorous substance in Article 2 of the Enforcement Regulations of the Malodor Control Law under Japanese law. The range of the regulation standard concerning the concentration of specific malodorous substances at the site boundary is shown, and the content in the atmosphere is 0.005 / million or more and 0.07 / m or less. For this reason, it is desired to quickly disassemble and remove.

  Trimethylamine may be generated from the causative substance even when stored at 0 ° C. or lower. For this reason, it may be confirmed not only at normal temperature but also in general waste and domestic wastewater at low temperatures. In the ranch as well as in the waste, the occurrence of domestic animals such as manure is observed.

The boiling point of trimethylamine is about 3 ° C. (boiling point 2.87 ° C., 1 atm of 760 mmHg) (Non-patent Document 2). For this reason, it is generally recognized as an unpleasant odor under the domestic temperature. Regarding solubilization in water, it is a substance that is easily soluble at 8.90 × 10 ⁵ mg / L under a temperature condition of 30 ° C. When dissolved in an aqueous solution, trimethylamine is an alkaline substance, and thus has a characteristic of changing the aqueous solution to alkaline.

  As a means for removing the unpleasant odor of trimethylamine, a method of neutralizing the alkaline trimethylamine while mixing the sprayed acidic substance is used. Similarly, as a neutralization technique developed using microorganisms, rice bran is fermented with various microorganisms, and for amines containing trimethylamine, fermentation products such as lactic acid, acetic acid, propionic acid, butyric acid, succinic acid, etc. There has been reported a method of neutralizing and deodorizing by reacting an acidic substance (Patent Document 1 published in 2015).

  In these methods, trimethylamine always maintains a neutral state. However, the substance itself continues to accumulate in the attached part and does not decrease. Depending on the conditions, there is a problem in that neutralized trimethylamine accumulates in a specific place. Furthermore, there is a problem that an unpleasant odor is generated when trimethylamine is liberated again due to some external factor such as pH fluctuation.

  For this reason, in order to deodorize trimethylamine, it is necessary to decompose it directly, and it is desired to develop a safe and safe degrading microorganism or degrading enzyme that is versatile and can be used repeatedly.

As the trimethylamine-degrading microorganism, Lactobacillus paracasei (2011 Published Patent Document 2), culture condition pH 5.0-6.5, culture temperature 25-40 ° C or 30-37 ° C have been reported. Lactobacillus paracasei and Isachenchia orientalis (Issatchenkia orientalis) were used for the culture conditions of pH 3.0-6.0 and culture temperature 25-40 ° C. (2010 published patent document 3), respectively. In addition, Erminia sp (No3937-GTS strain 1993 published patent document 4) is a culture condition of pH 6.0 to 7.5 and a culture temperature of 27 to 37 ° C. It has been reported as a degrading microorganism (Table 1), but trimethylamine is alkaline, but all literatures are used under acidic conditions from near neutral pH. It can be seen that it is used after being modified.

  Trimethylamine is an alkaline substance, and is known to be released into gas and recognized as an unpleasant odor when an organic substance such as meat or fish containing trimethylamine o-N-oxide is decomposed by enzymes or microorganisms. .

  When alkaline trimethylamine decomposition is performed using microorganisms, it is necessary to neutralize trimethylamine first so that it does not volatilize from the object for deodorization purposes. As described above, since the boiling point of trimethylamine is about 3 ° C., it becomes difficult to hold on the object and easily volatilizes under alkaline conditions, and easily volatilizes in the air.

  When trimethylamine generated from food is decomposed using microorganisms, the microorganisms hardly grow under alkaline conditions, and additional conditions such as addition of a pH adjuster are necessary. For this reason, it has been difficult to use unless some artificial method is added.

  When attempting to decompose trimethylamine using a microorganism that grows under alkaline conditions, it is necessary to keep the medium alkaline during the growth of the microorganism. On the other hand, since trimethylamine volatilizes from a culture medium etc. in alkaline conditions, utilization was difficult.

  For this reason, it is desirable that the microorganisms have a function of producing acidic substances such as malic acid themselves and capturing alkaline trimethylamine in the metabolic system by a neutralization reaction. If it cannot be captured, it may volatilize and produce an unpleasant odor due to trimethylamine.

  As another concept, a method for decomposing trimethylamine by utilizing a plurality of microorganisms at the same time has been reported. In this method, it is shown that malodor is suppressed with a raw material composed of 80 or more kinds of microorganisms, mainly lactic acid bacteria, yeasts, filamentous fungi, actinomycetes, and photosynthetic bacteria, using urine from livestock as a raw material ( (Mainly ammonia odor decomposition) (Patent Document 5 published in 2001).

  Other methods using a plurality of microorganisms include Candida Krusei, Candida crusei var. Candida Krusei var transitoria, Isachenchia tericola, Isachenchia occidentalis (patented in 19 years), Isachenchia ocssentula (Iss).

  Other methods that use Lactobacillus formumum or Pichia kluyveri use foods such as beans, cereals, and potatoes as starting points for culture materials, and suppress malodor while growing A method (Patent Document 7 published in 1999) has been reported.

  Similarly, several microorganisms such as Bacillus subtilis, Bacillus natto, Bacillus coagulans, Bacillus macerans, Bacillus macerans, and Enterobacter sac. Enterobacter agglomerans, Streptococcus faecalis (Streptococcus fecalis), Streptococcus cremoris, Streptococcus lactis (us), Streptococcus lactis (us) Rhizopus formosaensis, Aspergillus oryzae, Nitrosomanas agilis and zestomonas cariophile, Pseudomonas cary, Pseudomonas cary A method of suppressing it has been reported (1996 published patent document 8, 1995 published patent documents 9, 10).

  On the other hand, when using multiple microorganisms to deodorize trimethylamine for a target that needs to keep the sanitary environment in good condition, such as food, the target object will be spoiled by the seeded microorganisms. However, it was difficult to introduce a new method.

  From reported cases so far, a temperature condition of 20 ° C. or higher is used as a temperature condition for decomposing trimethylamine. However, trimethylamine generated from foods and the like in daily life is generated in a state of being stored at a low temperature such as a refrigerator of 4 ° C. or lower and the internal temperature of truck transportation. Similarly, foods handled at a store are often temperature controlled at 10 ° C. or less. All of them are stored under low temperature conditions using a refrigerator or the like. For this reason, there has been a demand for the development of microorganisms and enzymes that exert the effect of decomposing trimethylamine at low temperatures.

  In addition, a method has been proposed that uses the masking effect of trehalose and maltitol to suppress the production of trimethylamine (Patent Document 11 published in 1999).

  The masking action is a method characterized by deodorizing by enclosing a malodorous substance, but from the chemical aspect, if the amount of trimethylamine to be deodorized exceeds the deodorizing ability of the masking agent, masking is performed. The effect is saturated and there is a problem that an unpleasant odor is immediately generated. Actually, it is impossible to immediately determine whether the masking effect is saturated, and there is a problem that the amount used is unclear. Furthermore, there has been a problem that the limit of deodorization changes depending on the capacity of the masking agent.

  When the masking agent is used by spraying on an object such as food, there is a problem that the deodorizing effect varies depending on the local concentration of trimethylamine. In order to mask trimethylamine evenly, it is difficult to deodorize with a small amount of masking agent. Therefore, if it is used in a large amount, it causes problems in the flavor, taste, freshness, etc. of the food, making it difficult to use.

JP, 2015-119552, A JP 2011-30737 A JP 2010-117872 A JP-A-5-137567 JP 2001-187126 A JP-A-11-104222 Japanese Patent Laid-Open No. 11-346761 JP-A-8-059418 JP-A-7-222790 Japanese Patent Laid-Open No. 7-222791 Japanese Patent Laid-Open No. 11-308983 Japanese Patent Laid-Open No. 2006-340676

  Enzymatic and non-enzymatic degradation of trimethylamine-N-oxide at temperatures below 0 ° C. Meiko Kimura, Nobuo Seki, Ikuo Kimura, Journal of Japanese Fisheries Society 68 (1) 85-91, 2002 (II) Results of initial environmental risk assessment of chemical substances (14 substances) Item 7, Trimethylamine, 1. Basic matters concerning substances, environmental risk assessment of chemical substances, Vol. 12, published by the Ministry of the Environment, 2011

  An object of the present invention is to provide a method capable of decomposing trimethylamine even under alkaline conditions generated from an organic substance, and capable of decomposing even at a low temperature of 10 ° C. or lower.

  Trimethylamine is widely known as a substance that attracts insects such as flies and cockroaches, and it is also known that when it occurs in a ranch or a food manufacturing factory, these insects are attracted and sanitary conditions are deteriorated. In particular, when a bug such as a cockroach is attracted by trimethylamine in a food manufacturing factory, the risk of causing contamination of the product is increased. For this reason, the present invention aims to be used as an aid to a method for solving these problems.

  The present inventor has sought various strains having excellent biodegradation action alone under alkaline conditions in which trimethylamine is dissolved in water or the like at low temperatures, and repeated various studies. As a result, Brevandimonas vesicularis that can grow under low temperature and alkaline conditions and has trimethylamine-degrading ability (Brevundimonas vesicularis) from the edible part of foods that have been eaten by humans since ancient times The organization deposit number NITE AP-02293) was found and completed.

  An isolated method of Brevundimonas vesicularis strain that degrades trimethylamine is shown below.

  The medium used for the isolation was configured by imitating the meaty surface of fresh fish that is likely to generate trimethylamine, and applying a minimal nutrient medium (MEM). The medium components were used under the conditions shown in Table 2. The medium was sterilized by a sterile filtration membrane having a pore size of 0.2 μm (micrometer) so that other microorganisms were not mixed.

  In the composition of Table 2, the nitrogen source used in the intracellular metabolic system uses only trimethylamine in place of amino acids, and cannot decompose except for microorganisms that decompose trimethylamine and can be used in the intracellular metabolic system. Prepared under conditions.

  It goes without saying that the scope of the invention is not limited to this. A non-selective medium and various types of media can be used as long as the necessary nutrients are contained other than the conditions shown in Table 2.

  The method that led to the discovery of the strain was to wipe off food microorganisms from a plurality of clean fresh fish edible parts with a sterilized cotton swab and apply to a plate medium under the conditions shown in Table 2 to confirm the trimethylamine neutralization resolution. Went. Samples inoculated with microorganisms were selected from 6 samples under the same conditions, and used to count the number of colonies generated on the plate medium. The trimethylamine concentration was 0.0, 0.1, 0.3, 1.0, and 3.0 mM (mmol), and the culture time was 10 ° C. for 10 days (Table 3).

  An average of 5.8 colonies with the highest trimethylamine concentration at 1.0 mM was confirmed, and an average of 1.2 colonies were confirmed even at a concentration of 3.0 mM higher than 1.0 mM. For this reason, all colonies produced at a trimethylamine concentration of 1.0 or 3.0 mM were picked up.

  Although it was in the state of the medium, it was pH 7.7 before adding trimethylamine (Table 2). When 1.0 mM trimethylamine was added, the pH changed to alkaline at pH 8.2, and when trimethylamine was added at 3.0 mM, the pH became 9.9, indicating a further alkaline state. The microorganisms forming the resulting colonies have trimethylamine resolution under these conditions.

  Trimethylamine was added to the composition shown in Table 2 to prepare a 1.0 mM trimethylamine liquid medium. 1 mL of this liquid medium was added to a sterilized 1.5 mL sample tube, and the colonies picked up in the previous item were isolated in 1 / 1.5 mL sample tube and cultured at 10 ° C. for 10 days.

  After culturing for 10 days, each 1.5 mL sample tube was centrifuged at 1000 rpm for 15 minutes. After removing the supernatant, 1 mL of 0 ° C. physiological saline was added to the microbial strain recovered at the bottom of the sample tube by centrifugal force, and the mixture was mixed well, then again centrifuged at 1000 rpm for 15 minutes and washed.

  After repeating washing three times, trimethylamine was added to the composition shown in Table 2 to prepare a 3.0 mM trimethylamine plate medium. 0.1 ml each of the washed strain diluted 10-fold with physiological saline was inoculated on this medium, and the resulting colonies were confirmed again after culturing at 10 ° C. for 10 days. As a result, colonies were confirmed.

  Trimethylamine is added to the composition shown in Table 2 to prepare a 1.0 mM trimethylamine liquid medium, and the liquid medium is subdivided into 1.5 mL portions in a sterilized 2.0 mL sample tube. Isolated in a 0 mL sample tube and used as a stock strain.

(A) Characteristics of microorganisms When cultured on a non-selective plate medium at a temperature of 10 ° C. for 7 days, the following morphological characteristics are observed. 1) Cell shape: Neisseria gonorrhoeae 2) Size 0.8 μm × 2.0 to 3.0 μm 3) Multiformation: No 4) Motility: Yes 5) Spore: No ability to form

(B) Culture properties 1) Color on non-selective plate medium: milky white, 2) Colony transparency: translucent, 3) Colony gloss: Yes, 4) Surface morphology: Smooth, 5) Growth state: Good, 6) Non-selective medium condition: Grows on the surface

(C) Determination of Gram Yin / Positive In the examination of the mycological properties, Gram staining was determined using the Burmy M staining kit manufactured by Muto Chemical Co., Ltd. From the determination results, this strain was a gram-negative bacterium.

(D) Determination of Negative / Positive Oxidase Activity The oxidase activity was determined using a Reagent Stain Dropper and 0.5 mL oxidase (Oxidase), which are products of Japan BD. From the determination results, this strain was positive for oxidase activity.

(E) Negative / positive determination of indole activity The indole activity was determined using a Reagent Stain Dropper and 0.5 mL DMACA indole (Indole), which are products of Japan BD. From the determination results, this strain was negative in indole activity.

(F) Physiological Properties For the bacterial species identification, Becton Dickinson BD BBL Crystal E / NF (for industrial use) that can be confirmed from the physiological aspect was used. As a result, the following characteristics of the trimethylamine-degrading bacterium were shown. 1) Gram staining: negative, 2) oxidase activity: positive, 3) indole activity: negative, 4) arabinose: negative, 5) mannose: negative, 6) sucrose: negative, 7) melibiose: negative, 8) rhamnose: Negative, 9) sorbitol: negative, 10) mannitol: negative, 11) adonitol: negative, 12) galactose: negative, 13) inositol: negative, 14) pnp phosphate: negative, 15) pn- p-α-β-glucoside: negative, 16) pnp-β-galactoside: negative, 17) proline nitroanilide: negative, 18) pnp-bisphosphate: negative, 19) pn -P-xyloside: negative, 20) pnp-α-arabinoside: negative, 21) pnp-phosphorylcholine: negative, 22) pnp-β-glucuronide: negative, 23) pnp-N-acetylglucosaminide: negative, 24) L-γ-glutamine-p-nitroanilide: negative, 25) esculin: positive, 26) phenylalanine: negative, 27) urea: negative, 28) Glycine: negative, 29) citric acid: negative, 30) malonic acid: negative, 31) tetrazolium: negative, 32) arginine: negative, 33) lysine: negative, 34) state on agarose medium: hydrolysis Absent.

  It was shown that the conserved strain that is a trimethylamine-degrading bacterium is a species of the genus Brevundimonas from the physiological aspect. In addition, since esculin positive was shown, it was shown that it is a Brevundimonas vesicularis strain.

  From these results, the microorganism of the present invention is a Brevundimonas vesicularis strain, and the ability of the present strain to degrade trimethylamine was confirmed by the inventors' tests. Therefore, trimethylamine can be efficiently decomposed by adding this strain or a trimethylamine-degrading enzyme derived from this strain to a treatment system containing trimethylamine.

  Here, the obtained conserved strain was deposited as a Brevundimonas vesicularis strain with the deposit number NITE AP-02293 to the National Institute of Technology and Evaluation.

[Gas chromatography analysis conditions]
The analytical column uses Thermon-3000 + KOH (2 + 2)% Sunpak-N60 / 100 mesh (column length: 2.1 meters, column diameter: φ3.2 millimeters Shinwa Chemical Co., Ltd. product), and nitrogen is used as a carrier gas at 50 mL / min. Using. As temperature conditions, the column temperature was 120 ° C., the inlet temperature was 230 ° C., and the detection temperature was 230 ° C. As the detector, a flame ionization detector (FID) can be used to measure the value of measurement and the retention time obtained according to the amount of trimethylamine (FIG. 1).

[Method for quantifying trimethylamine degradation amount]
In order to quantify the trimethylamine concentration by gas chromatography, a trimethylamine concentration was prepared in a plurality of conditions of 0.0, 0.5, 1.0, and 2.0 mM in the liquid medium shown in Table 2, and used as a standard solution. These standard solutions are measured by gas chromatography to produce a calibration curve (FIG. 2), and the amount of trimethylamine degradation can be quantified by comparing with a measurement sample in which trimethylamine degradation has been performed by Brevundimonas vesicularis strain. In order to suppress errors, the sample to be measured was kept in a frozen storage state at −20 ° C. in a tightly-sealed state until immediately before the measurement, and was thawed to 0 ° C. during use.

[Degradation amount of trimethylamine of Brevundimonas vesicularis strain]
A sample for measuring the amount of trimethylamine degradation is prepared in the liquid medium shown in Table 2 under the condition of a trimethylamine concentration of 1 mM, added with Brevundimonas vesicularis strain, cultured at 10 ° C. for 7 days, and errors are suppressed. Therefore, it was kept in a frozen storage state at −20 ° C. in a sealed state until immediately before measurement, and thawed to 0 ° C. when used. Trimethylamine in the medium is decomposed and consumed as a nitrogen source which is a nutrient component of the strain during the culture for 7 days (FIG. 3). The measured amount of trimethylamine obtained was compared by integrating the interval from retention time 3.3 seconds to 4.8 seconds, and as a result, about 45% decomposition of trimethylamine was confirmed (FIG. 4).

Measurement data obtained by analysis by gas chromatography with trimethylamine 0.0, 0.5, 1.0, and 2.0 mM added to the medium shown in Table 2 according to [Gas chromatography analysis conditions] Calibration curve based on the integral of trimethylamine measurement data by [Quantitative determination of trimethylamine degradation amount] Result of measurement of trimethylamine degradation amount using gas chromatography with [Trimethylamine degradation amount of Brevundimonas vesicularis strain] The result of integrating the measurement result of trimethylamine degradation amount obtained by [Trimethylamine degradation amount of Brevundimonas vesicularis strain]

Claims (7)

  A method of degrading trimethylamine using trimethylamine resolution of Brevundimonas vesicularis strain.   A method of use, wherein trimethylamine-degrading enzyme derived from Brevundimonas vesicularis strain is added to a target such as fish to generate trimethylamine, and the concentration of trimethylamine in the target is reduced.   A method comprising decomposing and reducing trimethylamine in a treatment system in addition to a treatment system containing trimethylamine using trimethylamine resolution of Brevundimonas vesicularis strain.   A method of decomposing trimethylamine by mixing trimethylamine-degrading enzyme derived from Brevundimonas vesicularis strain with cosmetics such as cream. The Brevundimonas spp. Bacterium according to claim 1 or 2, wherein the Brevundimonas vesicularis strain has the following mycological properties.
(1) Gram staining negative (2) Bacterial morphology: rod (3) Cell pigment: orange (4) Physiological properties: catalase positive, oxidase positive, gelatin degradability positive, esculin hydrolysis positive (5) glucose, Utilization of maltose and malic acid   The Brevundimonas vesicularis strain (National Institute for Product Evaluation and Technology NITE AP-02293) according to any one of claims 1 to 5, or a mutant thereof is cultured in a medium, A method for collecting trimethylamine-degrading enzyme from cultured metabolites.   A trimethylamine-degrading enzyme characterized by culturing the Brevundimonas vesicularis strain or variant thereof according to any one of claims 1 to 5 in a medium, and collecting the trimethylamine-degrading enzyme from the cells or from a cultured metabolite. Production method.

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