JP5466453B2 - Aliphatic aldehyde-degrading microorganisms - Google Patents

Description

  The present invention relates to a microorganism having an ability to decompose aliphatic aldehydes.

  Aldehydes are widely present in nature as aroma components of various substances. Aromatic aldehydes such as vanillin are added as fragrances to many industrial products including foods and used. Pyridoxal, a kind of vitamin B6 compound, is a useful aromatic aldehyde used for amino acid metabolism and neurotransmission.

  On the other hand, aliphatic aldehydes contain many substances that cause unpleasant odors. For example, n-hexanal is one of the causative substances of soybean-specific green bean odor, and nonenal is a causative substance of aging odor.

  Acetaldehyde, which is an aliphatic aldehyde, has a fruit-like aroma at low concentrations and is naturally contained in foods such as fruits and fruit juices, vegetables, dairy products and bread. It is also naturally contained in beverages such as tea and soft drinks, beer, wine and distilled liquor. However, acetaldehyde also causes food flavor inhibition. For example, in processed soybean powder materials used as foods, acetaldehyde is considered to cause blue odor and raw odor along with hexanal, which is an aliphatic aldehyde (Patent Document 1).

  Acetaldehyde is also a cause of blue odor, off-flavor, and altered odor in brewed liquors such as beer and sake, beer-style alcoholic beverages, and distilled spirits such as shochu, and is called a flavor-inhibiting substance (off-flavor). There is a report that the amount of acetaldehyde in the final product has been reduced by examining fermentation conditions, adding other substances such as tannic acid, or selecting raw materials for the purpose of improving off-flavor etc. due to acetaldehyde (Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5).

  Acetaldehyde in alcohol is also known as a hangover component. Alcohol (ethanol) taken into the body by drinking is oxidized to acetaldehyde by alcohol dehydrogenase, converted to acetic acid by aldehyde dehydrogenase, and finally decomposed into carbon dioxide and water and released outside the body. Half of yellow races, including Japanese, have acetaldehyde dehydrogenase with low activity, so acetaldehyde that has temporarily accumulated in the body due to drinking, so the proportion of people who show hangover symptoms such as headache and nausea is Western Higher than Excess acetaldehyde is secreted into the blood and released by exhalation, accumulates in saliva and causes bad breath.

  As means for removing acetaldehyde in saliva and food afterwards, use of L-cysteine (sulfur-containing amino acid) or barley green juice is considered (Non-patent Document 1). This is a method devised based on the fact that L-cysteine and flavonoids in green juice add to acetaldehyde and inactivate it. However, the complex formation reaction of flavonoids in L-cysteine or green juice and acetaldehyde is a reversible reaction, and acetaldehyde may be regenerated. In addition, L-cysteine and green juice itself are substances having a unique taste and odor, have a great influence on the flavor in foods and beverages, and are difficult to use in practical use. Further, a method of degrading aldehyde by adding an aldehyde-degrading enzyme isolated from animals or plants can be considered. However, since these enzymes generally have low substrate specificity, they have the disadvantage of decomposing both harmful aliphatic aldehydes and useful aromatic aldehydes. Furthermore, many known aldehyde-degrading enzymes require the presence of NAD, which is a coenzyme, but NAD non-requiring aldehyde-degrading enzymes are preferable from the viewpoint of cost.

Aldehyde oxidase and aldehyde dehydrogenase mainly exist as aldehyde degrading enzymes. Aldehyde oxidase uses oxygen to catalyze the oxidation of aldehyde to produce carboxylic acid and hydrogen peroxide. Aldehyde dehydrogenase catalyzes the oxidation of aldehyde using NAD ⁺ or NADP ⁺ as a coenzyme. For example, it has been reported that Bradyrhizobium japonicum, a soybean rhizobia, produces n-hexanal dehydrogenase capable of degrading n-hexanal (Non-patent Document 2).

  In addition, a method for reducing the unpleasant odor of alcoholic beverages, a method for deodorizing cereals, and a method for reducing the unpleasant odor of foods using acetic acid bacteria have been reported (Patent Document 6, Patent Document 7, and Patent Document 8).

JP 2006-129877 A JP 2008-1113587 A Japanese Patent No. 3943122 Japanese Patent No. 3640946 JP 2000-60531 A Japanese Patent Publication No. 8-22220 Japanese Patent No. 3025409 JP 62-294046 A

Salaspuro, V., et al., Int. J. Cancer (2002) 97, 361-364 Soy protein research vol.11. P.67-70, (2008)

  An object of the present invention is to provide a method for reducing a fatty acid aldehyde, which is a causative substance of a flavor-inhibiting substance and an aging odor in foods and beverages, by a safe means. As one of the methods, aliphatic aldehydes can be obtained by microbial fermentation using bacteria that are naturally present in the human body, such as food microorganisms and intestinal bacteria, or bacteria that are harmless to humans in the environment. A method of decomposing and removing can be considered, but for this, i) it does not act on other aldehydes such as vanillin which is a preferred aroma component of foods and beverages, and ii) oxidized coenzyme (for cost reasons) It is desirable to satisfy that no addition of NAD or the like is required.

  The present inventors have extensively searched for an enzyme-producing bacterium having properties that meet the purpose of use from among a large number of microorganisms. As a result, 384 strains of acetaldehyde-degrading bacteria were isolated, and 203 strains of acetaldehyde-degrading bacteria were obtained. 64 strains out of 203 strains were confirmed to degrade vanillin. Bacteria that showed aldehyde-degrading activity under acidic conditions (pH 3.5) were 50 strains out of 203 strains. From these results, 16 strains that completely decomposed acetaldehyde but did not decompose vanillin under acidic conditions were selected. These 16 strains were subjected to 16S rRNA sequence analysis and Gram staining to identify microbial species. In addition, the base sequences of 16 SrRNAs of 16 selected strains were analyzed, and microorganism species were estimated using BLAST. Moreover, it was clarified that the crude enzyme solution extracted from the AL-5, AL-7, and AL-11 strains showed aldehyde oxidase activity even in the absence of NAD. In addition, they found that these bacteria actually have the ability to degrade acetaldehyde in saliva and food, and found that they also have the ability to degrade other aliphatic aldehydes such as n-hexanal and nonenal, The present invention has been completed.

That is, the present invention is a microorganism belonging to the genus Pseudomonas or Pantoea, which has an ability to resolve aliphatic aldehydes and has no ability to resolve vanillin.
In addition, the present invention is a microorganism belonging to the genus Pseudomonas or Pantoea, which has an ability to decompose aliphatic aldehydes under conditions of pH 3.0 or more and 4.0 or less, and has no ability to decompose vanillin. Such microorganisms have an advantage that they exhibit acetaldehyde-degrading activity, for example, in the stomach, which is an acidic environment, and in acidic foods and drinks.

  In addition, the present invention is a microorganism belonging to the genus Pseudomonas or Pantoea, which has an ability to degrade aliphatic aldehydes but not to vanillin, where the ability to resolve aliphatic aldehydes does not require NAD. It is a certain microorganism. Further, the microorganism is a microorganism having an Naldehyde non-requiring aliphatic aldehyde decomposing ability due to acetaldehyde oxidase.

  The present invention is also Pseudomonas sp. FERM ABP-10988, Pantoea sp. FERM ABP-10989, Pantoea sp. FERM ABP-10990, or mutants thereof that degrade aliphatic aldehydes but not vanillin.

  The present invention further includes a composition containing the microorganism of the present invention, or a composition containing a cell disruption solution, a crude enzyme solution or a purified enzyme prepared from the microorganism of the present invention. In particular, the composition is a food or drink, a cosmetic, a laundry treatment agent, or a deodorant.

The present invention is also an oral hygiene agent containing the above-described microorganism of the present invention, or an oral hygiene agent containing a cell disruption solution, a crude enzyme solution or a purified enzyme prepared from the above-described microorganism of the present invention.
The present invention further relates to a method for reducing aliphatic aldehyde in foods or beverages, characterized by adding the microorganism of the present invention.

  The present invention relates to microorganisms that degrade aliphatic aldehydes but do not degrade vanillin, particularly Pseudomonas sp. Strain AL-5 (deposit number FERM ABP-10988), Pantoea sp. Strain AL-7 strain (deposit number FERM ABP-10989) Pantoea sp. Strain AL-11 (deposit number FERM ABP-10990) is provided. Such microorganisms can be used to reduce aliphatic aldehydes that cause off-flavors in foods without affecting the preferred aroma components. In particular, according to the present invention, acetaldehyde in saliva that causes bad breath can be reduced.

FIG. 1 shows the 16S rDNA sequence (full length) of the AL-5 strain. FIG. 2 shows the 16S rDNA sequence (part) of the AL-7 strain. FIG. 3 shows the 16S rDNA sequence (part) of the AL-11 strain. FIG. 4 shows the molecular phylogenetic analysis results of Al-5 strain using 16S rDNA. The numbers near the branch molecules indicate the bootstrap value, and the lower left line indicates the scale bar. FIG. 5 shows changes in activity depending on the presence or absence of NAD ⁺ using the crude enzyme of the AL-5 strain.

Aliphatic Aldehyde Degrading Microorganism The present invention is a microorganism belonging to the genus Pseudomonas or Pantoea, which has an ability to decompose aliphatic aldehyde but does not have the ability to decompose vanillin.

  Strains that meet these requirements can also be obtained by fresh screening. An example of microbial screening is as follows: 1) A soil sample obtained from a park, forest, field, etc. is suspended in physiological saline, and the supernatant is applied to a selective medium containing acetaldehyde. It can be obtained by separating, evaluating acetaldehyde-degrading activity and vanillin-degrading activity, and 3) selecting a bacterial strain that degrades acetaldehyde but does not degrade vanillin. From these, the microorganism of the present invention may be obtained by further selecting a strain that shows activity even under acidic conditions and a strain that does not require NAD for the acetaldehyde degradation reaction.

  As representative strains, Pseudomonas sp. AL-5 strain (deposit number FERM ABP-10988), Pantoea sp. Strain AL-7 strain (deposit number FERM) deposited on July 28, 2008, at the Patent Organism Depositary. ABP-10989), Pantoea genus AL-11 strain (deposit number FERM ABP-10990). The estimation of microbial species and the analysis of mycological characteristics can be performed, for example, by the method of Example 2-6 of the present specification. The identification and bacteriological characterization results of the AL-5, AL-7, or AL-11 strains are shown in Tables 1, 2, and 4-8, and FIGS. 1-4 of this specification. ing.

  The microorganisms of the present invention have the ability to degrade not only acetaldehyde but also other aliphatic aldehydes such as hexanal and nonenal. The microorganism of the present invention is particularly useful in the food field because it can degrade aliphatic aldehydes that cause these unpleasant odors. Furthermore, the microorganism of the present invention may be a wild strain or a mutant strain as long as it is a microorganism belonging to the genus Pseudomonas or Pantoea and decomposes an aliphatic aldehyde but does not decompose vanillin.

  Mutant strains can be obtained without mutation treatment with ethyl methanesulfonic acid, a commonly used mutation agent, treatment with other chemicals such as nitrosoguanidine and methylmethanesulfonic acid, ultraviolet irradiation, or treatment with a mutation agent. It can also be obtained by so-called spontaneous mutation.

  The phrase “has no resolution for vanillin” means that vanillin is not decomposed at all or has a weak decomposition activity but is extremely weak in decomposition activity compared to aliphatic aldehyde decomposition. For example, the resolution of vanillin is about 1/10, preferably about 1/50, compared with the resolution of aliphatic aldehydes.

  As a medium used for culturing the microorganism of the present invention, any medium can be used without particular limitation as long as it is a medium that can grow a microorganism that is capable of degrading an aliphatic aldehyde, which is a genus Pseudomonas or Pantoea. For example, a medium prepared in [peptone, 5 g / l; yeast extract, 2 g / l: NaCl, 8.5 g / l; pH 7.0] can be used, but is not limited thereto. The medium used for the growth of the microorganism of the present invention specifically contains a carbon source that can be assimilated by the microorganism of the present invention, such as glucose, and a nitrogen source that can be assimilated by the microorganism of the present invention. An organic nitrogen source such as peptone, meat extract, yeast extract, corn steeple liquor and the like, and an inorganic nitrogen source such as ammonium sulfate and ammonium chloride can be contained. Further, if desired, a salt composed of a cation such as sodium ion, potassium ion, calcium ion or magnesium ion and an anion such as sulfate ion, chlorine ion or phosphate ion may be contained. Furthermore, trace elements, such as vitamins and each acid, can also be contained. The concentration of the carbon source is, for example, about 0.1% to 10%. The concentration of the nitrogen source is, for example, about 0.01% to 5%, although it varies depending on the type. The concentration of inorganic salts is, for example, about 0.001% to 1%. Preferably, an aliphatic aldehyde such as acetaldehyde is added to the medium for the purpose of removing contamination with microorganisms that cannot decompose the aliphatic aldehyde. The concentration of the aliphatic aldehyde can be appropriately determined, but is preferably 0.1%.

  The microbial cells of the microorganism of the present invention may be used in a state suspended in a culture solution, or may be collected or concentrated from the culture solution by a usual method such as centrifugation. The recovered or concentrated cells can be immobilized on an appropriate carrier and used as immobilized cells. The collected or concentrated bacterial cells may be used as a powder by a usual method such as freeze-drying, or the powder and various excipients may be mixed and used as a dosage form such as a powder, tablet, capsule or the like. Good.

A method for preparing a cell extract from the microorganism of the present invention can be appropriately selected by those skilled in the art from ordinary methods such as ultrasonic disruption and surfactant treatment.
In addition, from the microorganisms of the present invention, those skilled in the art can prepare an enzyme extraction fraction or an aliphatic aldehyde-degrading enzyme preparation using ordinary knowledge. These may be used as they are, or may be immobilized on an appropriate carrier and used as an immobilized enzyme.

The composition containing the microorganism of the present invention The present invention relates to a composition containing the microorganism of the present invention, or a composition containing a cell disruption solution, a crude enzyme solution, or a purified enzyme prepared from the microorganism of the present invention. In particular, the composition is a food or drink, a cosmetic, a laundry treatment agent, or a deodorant.

  The composition of the present invention is added with live cells, dead cells, immobilized cells, cell extract of the microorganism of the present invention, or an aliphatic aldehyde-degrading enzyme preparation purified from these microorganisms. More preferably, catalase is also added in order to immediately decompose hydrogen peroxide that may be a by-product during the decomposition of the aliphatic aldehyde. In addition, additives, preservatives, pigments and the like are appropriately added according to the respective compositions such as foods and drinks, cosmetics, laundry treatment agents and deodorants.

  The food or drink of the present invention can remove the aliphatic aldehyde present or generated in the body by ingesting the microorganism of the present invention. As long as it has the effect of the present invention, a sweetener, a sour agent, a fragrance, an antioxidant and the like may be added as appropriate. Specific product forms include troches, gums, candy, tablets, powders, drinks and the like. Methods for adding additives and processing into foods or beverages can be appropriately selected and carried out by those skilled in the art in consideration of the properties of the product and the like.

  The cosmetic of the present invention is used for the purpose of suppressing unpleasant odors such as body odor and aging odor. Examples of product forms include soaps, emulsions, lotions, creams, shampoos, conditioners, and hair sprays. The deodorant of the present invention is also used for the purpose of suppressing unpleasant odors such as body odors and aging odors. The product form is, for example, spray, liquid or solid.

The laundry treatment agent of the present invention is used for the purpose of removing unpleasant odors such as body odor and aging odor. Examples of product forms include laundry detergent, laundry soap, laundry finish, and softener.
The oral hygiene agent containing the microorganism of the present invention The present invention is an oral hygiene agent containing the microorganism of the present invention, or a cell disruption solution, crude enzyme solution, or purified enzyme prepared from the microorganism of the present invention. .

  The oral hygiene agent of the present invention is added with live cells, dead cells, immobilized cells, cell extract of the microorganism of the present invention, or an aliphatic aldehyde-degrading enzyme preparation purified from these microorganisms. Preferably, catalase is also added in order to immediately decompose hydrogen peroxide that may be by-produced during the decomposition of the aliphatic aldehyde. In addition, additives and the like are added as appropriate. The oral hygiene agent of the present invention can remove an aliphatic aldehyde present or generated in the mouth.

  The mouth sanitary agent of the present invention includes, as a non-limiting example, a mouth refreshing agent, a mouth odor preventing agent, a mouth odor reducing agent, or a mouthwash, and specific product forms include tablets, sprays, and liquids. It is pasty.

  The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

About 153 samples of about 1.0 g each were collected from a selection park of aliphatic aldehyde-degrading and vanillin non-degrading bacteria , forests, fields, etc., and stored at 4 ° C. A soil sample (0.1 g) is suspended in physiological saline (10 ml), and a supernatant (100 μl) is added to an agar medium containing acetaldehyde [acetaldehyde, 1 g / l; peptone, 5 g / l; yeast extract, 2 g / l: NaCl, 8.5 g / l; pH 7.0; agar, 15 g / l]. After stationary culture at 30 ° C., the grown acetaldehyde-resistant colonies (384 strains) were divided into new plates and inoculated one after another, cultured again at 30 ° C., and stored at 4 ° C.

The acetaldehyde decomposition activity was evaluated as follows. The isolated colony was suspended in an acetaldehyde standard reaction solution [acetaldehyde, 2 mM; NAD ⁺ , 2 mM; potassium phosphate buffer (pH 6.0), 10 mM; final volume, 500 μl] and allowed to stand at room temperature for 24 hours. After the reaction, the supernatant was obtained by centrifugation at 15,000 × g for 10 minutes. 50 μl of the supernatant, 100 μl of 200 mM acetate buffer (pH 3.5), and 40 μl of 0.1% 3-methyl-2-benzothiazolone hydrazone (MBTH) solution were mixed, and the solution was incubated at 50 ° C. for 30 minutes. Next, 190 μl of 0.6% ammonium iron sulfate (III) / acetic acid acidic aqueous solution [iron iron (III) sulfate 12 hydrate, 6 g / l; acetic acid, 50 ml / l] was added, and the mixture was further reacted at room temperature for 20 minutes. If the aldehyde remains, the solution is colored blue. After the reaction, it was diluted to 1 ml with water, 200 μl of the solution was transferred to a 96-well microtiter plate, and the absorbance at 620 nm was measured with SpectraMax 340PC (Molecular Devices). Strains with reduced absorbance were designated as acetaldehyde-degrading bacteria. The method for evaluating the vanillin degrading activity of acetaldehyde-degrading bacteria is that the reaction solution is changed to a vanillin reaction solution [vanillin, 4 mM; NAD ⁺ , 4 mM; potassium phosphate buffer (pH 6.0), 10 mM] and the concentration of the MBTH solution. Is the same as the method for evaluating acetaldehyde-degrading activity, except that it is 0.5% and is not diluted with water after colorimetry. The ethanol-degrading activity of acetaldehyde-degrading bacteria was also evaluated. The evaluation method is the same as the acetaldehyde decomposition activity evaluation method except that the reaction solution is changed to an ethanol reaction solution [ethanol, 100 μl / ml; potassium phosphate buffer (pH 6.0), 10 mM]. However, this time, ethanol was decomposed when aldehyde was present in the solution.

In order to investigate the influence of pH on aldehyde decomposition, acetaldehyde decomposition activity under acidic conditions (pH 3.5) was evaluated. The evaluation method is the same as the acetaldehyde decomposition activity evaluation method described above except that the reaction solution is changed to an acetaldehyde acidic reaction solution [acetaldehyde, 2 mM; NAD ⁺ , 2 mM; acetate buffer (pH 3.5), 10 mM].

  Through the above operation, 384 strains of acetaldehyde-degrading bacteria were isolated from the soil sample, and 203 strains of acetaldehyde-degrading bacteria were obtained. 64 strains out of 203 strains were confirmed to degrade vanillin. Bacteria that showed aldehyde-degrading activity under acidic conditions (pH 3.5) were 50 strains out of 203 strains. Sixteen strains that completely decomposed acetaldehyde under the reaction conditions described above but did not decompose vanillin and showed activity even under acidic conditions were selected.

16SrDNA partial sequence analysis of selected strain Using universal primers 520F and 1400R for authentic bacteria, a part of 16SrDNA was amplified by colony PCR for the selected strain [reaction solution composition: template, 10 μl; ExTaq buffer, 1 ×; dNTPs, 0.2 mM; 520F primer (5′-GTGCCCAGCMGCCCGCCG-3 ′; M: AorC), 1.0 μM; 1400R primer (5′-ACGGGGCGTGTGTRC-3 ′ ;, R: AorG), 1.0 μM; ExTaq DNA Polymerase ), 0.5 U; final solution volume, 20 μl; reaction conditions: 96 ° C., 1 minute; (96 ° C., 30 seconds; 55 ° C., 30 seconds; 72 ° C., 1 minute) × 30 cycles]. After the PCR product was subjected to agarose gel electrophoresis, the target PCR product (about 900 bp) was gel collected and purified using GenElute Agarose Spin Columns (SIGMA). The purified PCR product was TA cloned into pCR2.1-TOPOvector using TOPO TA Cloning Kit (Invitrogen), and E. coli TOP10 (One Shot TOP10 Chemically Competent Cells, Invitrogen) was transformed. Transformant is LB agar medium [tryptone, 10 g / l; yeast extract, 5 g / l; NaCl, 10 g / l; agar, 15 g / l; ampicillin, 50 μg / ml; 5-bromo-4-chloro-3-indolyl -β-D-galactopyranoside (X-Gal), 80 μg / ml] and cultured at 37 ° C. for 16 hours. From the grown colonies, colonies having plasmids with inserted inserts were selected by blue / white screening, and 2 × YT medium [tryptone, 16 g / l; yeast extract, 10 g / l; NaCl, 5 g / l; ampicillin, 50 μg / ml] and again cultured for 14 hours. After culturing, the plasmid was recovered, subjected to a sequencing reaction, and the base sequence was analyzed by CEQ 2000XL DNA Analysis System (Beckman Coulter). After the analysis, the microorganism species was estimated from the base sequence obtained using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) (Table 1).

  The 16S rDNA sequences of AL-5, AL-7, and AL-11 are shown in FIGS.

Identification of selected strains by Gram staining Gram staining was performed using Faber G (Nissui) for confirmation of classification by 16S rDNA sequence analysis performed in Example 2. The cells were applied to a slide glass and allowed to dry naturally. After drying, the cells were fixed by flame fixation. After staining with a 0.2% Victoria Blue solution for 1 minute, it was gently washed from the back of the slide glass so that running water was not directly applied to the coated surface, and decolorized using a 2% picric acid ethanol solution. After gently washing with water again, staining was performed with a 0.25% safranin solution for 1 minute. After gently washing with water, removing water and drying, the sample was observed under a microscope at 1000 times. Bacillus cereus (Gram positive) and E. coli (Gram negative) were co-stained and used as controls. The results are shown in Table 2. AL-12 was estimated to be a gram-positive bacterium, Micrococcus genus, by 16S rDNA analysis, but the results of the Gram staining performed this time were negative. The result of Gram staining may vary depending on the culture conditions.

Evaluation of Acetaldehyde Degrading Activity of Crude Enzyme Fraction The crude enzyme fraction was fractionated from the strain selected and identified in Example 1, and their acetaldehyde degrading activity was examined in detail.

  Aldehyde oxidase (ALOD) activity of fractions obtained by preparing cell extracts from AL-2, AL-4, AL-5, AL-8, and AL-11 and subjecting them to anion exchange chromatography I investigated. Further, the fractions showing the main peak of activity were collected to obtain a crude enzyme solution, and the pH dependency and NAD dependency of each ALOD activity were examined. As a result, it was found that the crude enzyme solution prepared from AL-5 and AL-8 showed stable ALOD activity between pH 4-8. It was also found that the crude enzyme solutions prepared from AL-2, AL-4, AL-5, AL-8, and AL-11 all showed activity in the absence of NAD.

Molecular phylogenetic analysis of AL-5 strain by 16SrDNA full-length sequence Molecular phylogenetic analysis by the full length 16SrDNA of AL-5 strain was performed to estimate the microorganism species. The microbial cells used for the analysis were cultivated at 30 ° C. for 24 hours under aerobic conditions using Nutrient agar medium (Oxoid, Hampshire, England). DNA extraction was performed according to the protocol using InstaGene Matrix (BIO RAD, CA, USA). PCR was performed with PrimeSTAR HS DNA Polymerase (Takara Bio, Shiga) using the extracted DNA as a template and the primers shown in Table 3.

  Next, a sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA), and the base sequence was analyzed with ABI PRISM 3100 Genetic Analyzer System (Applied Biosystems, CA, USA). The analysis software used was Auto Assembler (Applied Biosystems, CA, USA), Apollon (Techno Suruga, Shizuoka), CLUSTAL W, MEGA ver 3.1. And the homology search with the base sequence obtained using the Apollon DB bacteria reference strain database (Techno Suruga, Shizuoka) and the international base sequence database (GenBank / DDBJ / EMBL) was performed.

  As a result of homology search against a bacterial reference strain database using BLAST, the AL-5 16S rDNA base sequence showed high homology to Pseudomonas-derived 16S rDNA, and the homology rate was 99.3%. It showed the highest homology to 16S rDNA of the alcaligenes LMG1224 strain. Also in the homology search for GenBank / DDBJ / EMBL, the AL-5 16S rDNA base sequence showed high homology to Pseudomonas-derived 16S rDNA. a homology of 99.3% with respect to 16S rDNA of L. alcaligenes LMG1224 strain It showed a homology of 98.7% with 16S rDNA of otitidis MCC10330 strain. From these facts, it is considered that AL-5 is highly likely to belong to Pseudomonas. Therefore, in this molecular phylogenetic analysis, 16S rDNA derived from a reference strain of a species belonging to Pseudomonas which is considered to be relatively related was obtained, and a molecular phylogenetic tree was prepared.

  As a result of molecular phylogenetic analysis, 16S rDNA of AL-5 A phylogenetic branch was formed with 16 SrDNA of alcaligenes (FIG. 4). Since the bootstrap value indicating the reliability of the phylogenetic branch (branch) of AL-5 is relatively high at 88%, AL-5 is P. amidst known species belonging to Pseudomonas. considered to be most closely related to alcaligenes. However, both 16S rDNAs are not completely identical, and AL-5 and P.I. From the phylogenetic branch of alcaligenes, the possibility that they are different species cannot be denied. Therefore, from the results of this molecular phylogenetic analysis, AL-5 was changed to P.P. Pseudomonas sp., which is closely related to alcaligenes. Estimated.

Morphological observation and physiological / biochemical property test of AL-5 strain Morphological observation and physiological / biochemical property test of AL-5 strain were also conducted. The cells used for morphological observation and physiological / biochemical property tests were cultured at 30 ° C. for 24 hours using Nutrient agar medium (Oxoid, Hampshire, England). The obtained bacterial cells were subjected to Gram staining using Faber G “Nissui” (Nissui Pharmaceutical, Tokyo) and observed for morphology with a photochemical microscope BX50F4 (Olympus, Tokyo). Further, catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F) were also tested based on the method of BARROW et al. Furthermore, a test using API20NE (bioMerieux, Lyon, France) was performed. Further, as an additional test, fluorescent dye production, starch hydrolysis, casein hydrolysis, lipase (Tween 80) activity, and lecithinase activity on King's B agar medium were tested.

  The cells used in the analysis of the fatty acid composition of the cells were cultured at 28 ° C. for 24 hours using Trypticase Soy Broth + Granular Agar medium (Becton Dickinson, MD, USA). Fatty acid extraction and measurement from the obtained bacterial cells were carried out according to a cell fatty acid composition analysis operation manual (Version 6) of Sherlock Microbial Identification System (Version 5.0) (MIDI, DEIUSA). The fatty acid composition obtained was checked against the TSBA40 database of MIS Standard Libraries.

  As a result of fatty acid analysis, C18: 1ω7c (29.87%), C16: 0 (19.36%), Sum In Feature 3 (C15: 0 iso 2OH + C16: 1ω7c) (18.22%) were recognized as the main fatty acids. (Table 4). As a result of collation with the MIDI database, P. aeruginosa has been identified as a bacterial species having a similar fatty acid composition. mendocina (similarity 0.168), P.M. alcaligenes (similarity 0.095) was searched (Table 5). However, the degree of similarity is low, and it is considered that there is no data on the corresponding bacterial species in the MIDI database according to the criteria for similarity of MIDI (SI: Similarity Index). If it has a composition, 1.000 is given, and if it is 0.500 or more as a guide, it is judged that they are similar).

  AL-5 is a gram-negative gonococci having motility, which oxidizes glucose, and both catalase and oxidase reactions were positive (Table 6). In the API test, AL-5 showed arginine dihydrolase activity, did not hydrolyze exrin, but hydrolyzed gelatin (Table 7). AL-5 produced a fluorescent dye on King'sB agar medium, did not hydrolyze casein, showed no lecithinase activity, showed lipase (Tween 80) activity, and hydrolyzed casein (Table 7).

  These properties are similar to those described in P.A., except that they produce fluorescent dyes. Almost similar to the general properties of alcaligenes. However, P.I. alcaligenes is known as a non-fluorescent dye-producing species in the genus Pseudomonas, and in addition, it also assimilates AL-5 glucose. Different from the properties of alcaligenes. In addition, the physiological properties obtained from this test and P.I. fluorescens and P.I. When compared with species producing fluorescent dyes such as putida, no matching species were found. Therefore, AL-5 is judged to be a new species of Pseudomonas from this physiological and biochemical property test, and Pseudomonas sp. Estimated.

  The AL-5 strain, AL-7 strain and AL-11 strain were transferred to the Pseudomonas genus FERM ABP-10988, Pantoea genus FERM ABP-10989, and Pantoea genus FERM on July 28, 2008, respectively. Received as ABP-10990.

Effect of NAD ^{+ on} aldehyde-degrading enzyme activity from AL-5 strain
Preparation of AL-5 crude enzyme solution The AL-5 strain is 50 ml of LB medium containing 1% ethanol [tryptone, 10 g / l; yeast extract, 5 g / l; NaCl, 10 g / l] at 30 ° C. for 24 hours at 180 rpm. Cultured with shaking. After culture, the cells were collected by centrifugation at 6000 × g for 15 minutes. The collected cells were resuspended in 20 ml of 10 mM potassium phosphate buffer (KPB) (pH 7.0), centrifuged again at 6000 × g for 15 minutes, and the supernatant was removed. The bacterial cells were resuspended in 10 mM KPB (pH 7.0) twice the amount collected, and disrupted by ultrasonic waves. After crushing, the mixture was centrifuged at 20000 × g, and the resulting supernatant was dialyzed against 10 mM KPB (pH 7.0). And the thing of the state which removed the low molecule derived from a microbial cell body by dialysis was used as the crude enzyme liquid.
Influence of NAD ⁺ on aldehyde oxidoreductase activity The enzyme reaction was performed at 30 ° C for 24 hours with an enzyme reaction solution (2 mM acetaldehyde; (+2 mM NAD ⁺ ); 50 mM KPB (pH 7.0); crude enzyme solution; final solution volume 250 μl). I let you. After the reaction, 50 ml of the supernatant, 100 ml of 200 mM acetate buffer (pH 3.5) and 40 ml of 0.1% 3-methyl-2-benzothiazolone hydrazone (MBTH) solution were mixed, and the solution was incubated at 50 ° C. for 30 minutes. Next, 190 ml of 0.6% ammonium iron sulfate (III) / acetic acid acidic aqueous solution [iron ammonium sulfate (III) .12 hydrate, 6 g / l; acetic acid, 50 ml / l] was added, and the mixture was further reacted at room temperature for 20 minutes. If the aldehyde remains, the solution is colored blue. After the reaction, it was diluted to 1 ml with water, 200 ml of the solution was transferred to a 96-well microtiter plate, and the absorbance at 620 nm was measured by SpectraMax340PC (Molecular Devices). The activity shown by NAD ⁺ in the absence and NAD ⁺ -independent activity, a value obtained by subtracting the activity from NAD ⁺ -independent activity shown by NAD ⁺ presence was NAD ⁺ dependent activity.
Influence of NAD ⁺ on aldehyde oxidoreductase activity The crude enzyme solution of AL-5 showed aldehyde oxidation activity even in the absence of NAD ⁺ . The addition of NAD ^+, showed higher activity than NAD ⁺ absence (Fig. 5). This is the crude enzyme solution of AL-5 results considered both enzymes enzyme having (acetaldehyde oxidase) and NAD ⁺ dependent activity (acetaldehyde dehydrogenase) with NAD ⁺ -independent activity is present (Fig. 5) .

Evaluation of acetaldehyde-degrading activity in saliva by AL-5 strain Acetaldehyde, 1 ml / l; peptone, 5 g / l; yeast extract, 2 g / l: NaCl, 8.5 g / l in a medium (pH 7.0, 5 liters) The AL-5 strain was cultured at 30 ° C. for 2 days. The cells were collected by centrifugation (6,000 rpm, 15 minutes) and washed with physiological saline. Cells (wet weight, about 0.1 g) were suspended in saliva (0.5 ml) to which acetaldehyde was added to 2 mM, and incubated at 37 ° C. for 5 hours. The cells were removed from the reaction solution by centrifugation (6,000 rpm, 15 minutes), and the concentration of acetaldehyde contained in the supernatant was measured by the method described in Example 1. As a result, acetaldehyde was detected in the supernatant. I couldn't.

Evaluation of acetaldehyde-degrading activity in saliva using AL-5 strain bacterial cell extract The cells of AL-5 obtained by culturing in the same manner as in Example 7 (wet cell weight 3.6 g) were ice-cooled. The obtained suspension was suspended in 0.01 M phosphate buffer (pH 7.0, 10 ml) and crushed by sonication (20 kHz, total 1 minute). The cell disruption solution was centrifuged (10,000 rpm, 15 minutes), and the supernatant was recovered as a cell extract. Catalase (Sigma, USA, 10 mg) was added to the bacterial cell extract, and dialyzed sufficiently at 4 ° C against 0.01 M phosphate buffer (pH 7.0) to obtain a crude enzyme solution. 0.1 ml of the crude enzyme solution was suspended in saliva (0.5 ml) to which acetaldehyde was added to 2 mM, and incubated at 37 ° C. for 5 hours. When the concentration of acetaldehyde contained in the saliva was measured by the method described in Example 1, acetaldehyde could not be detected.

Evaluation of Salivary Acetaldehyde Degradation Activity by AL-7 Strain and AL-11 Strain AL-7 strain or AL-11 strain was cultured at 30 ° C. for 2 days in a medium having the same composition as in Example 7. The cells were collected by centrifugation (6,000 rpm, 15 minutes) and washed with physiological saline. Bacterial cells (wet weight, about 0.2 g each) were suspended in saliva (0.5 ml) to which acetaldehyde was added to 2 mM, and incubated at 37 ° C. for 5 hours. Bacteria were removed from the reaction solution by centrifugation (6,000 rpm, 15 minutes), and the concentration of acetaldehyde contained in the supernatant was measured by the method described in Example 1. As a result, strains AL-7 and AL-11 were measured. Acetaldehyde could not be detected in the supernatant in any of the strains.

Evaluation of Salivary Acetaldehyde Decomposition Activity Evaluation Using AL-7 Strain and AL-11 Strain Bacteria Extracts The cells of AL-11 obtained in the same manner as in Example 9 (wet cell weight, 5.4 g) were added to ice. The suspension was suspended in a cooled 0.01 M phosphate buffer (pH 7.0, 10 ml) and pulverized by sonication (20 kHz, total 1 minute). The cell disruption solution was centrifuged (10,000 rpm, 15 minutes), and the supernatant was recovered as a cell extract. Catalase (Sigma, USA, 10 mg) was added to the bacterial cell extract, and dialyzed sufficiently at 4 ° C against 0.01 M phosphate buffer (pH 7.0) to obtain a crude enzyme solution. 0.1 ml of the crude enzyme solution was suspended in saliva (0.5 ml) to which acetaldehyde was added to 2 mM, and incubated at 37 ° C. for 5 hours. When the concentration of acetaldehyde contained in the saliva was measured by the method described in Example 1, acetaldehyde could not be detected.

Evaluation of acetaldehyde-degrading activity in saliva by AL-5 strain
Acealdehyde; 1 ml / l, peptone; 5 g / l, yeast extract; 2 g / l, NaCl; medium AL-5, 8.5 g / l (pH 7.0, 200 ml), strain AL-5 at 30 ° C. for 3 days Cultured. The cells were collected by centrifugation (5000 rpm, 10 minutes) and washed with physiological saline. Cells (wet weight, about 0.1 g) were suspended in saliva (0.5 ml) to which acetaldehyde was added to 2 mM, and incubated at 37 ° C. for 5 hours. After the reaction, the supernatant was obtained by centrifugation at 15,000 × g for 10 minutes. Using 450 μl of the supernatant as an internal standard, it was put into a glass vial to which 50 μl of 100 ppm isobutanol was added, and the acetaldehyde concentration was measured by a headspace gas chromatograph method. The instrument used was a Varian CP-3800 gas chromatograph equipped with a Tekmar 7000 type headspace autosampler and an INNOWAX19091N-233 capillary column (length 30 m, inner diameter 0.25 mm, film 0.25 μm). As a result, as in the case of measuring by the method described in Example 8, acetaldehyde was not detected in the supernatant by the above measuring method.

Evaluation of acetaldehyde-degrading activity in saliva with AL-5 strain extract
The cells of AL-5 obtained by culturing in the same manner as in Example 7 (cell wet weight, 7.3 g) were ice-cooled 0.1 M phosphate buffer (pH 7.0, 15 ml). And crushed by sonication (20 kHz, total 10 minutes). The cell disruption solution was centrifuged (15000 rpm, 15 minutes), and the supernatant was recovered as a cell extract. This was dialyzed sufficiently at 4 ° C. against 0.01 M phosphate buffer (pH 7.0) to obtain a crude enzyme solution. 0.1 ml of the crude enzyme solution was suspended in saliva (0.5 ml) to which acetaldehyde was added to 2 mM, and incubated at 37 ° C. for 5 hours. When the concentration of acetaldehyde contained in the saliva was measured by the method described in Example 12, as in Example 9, acetaldehyde was not detected in the supernatant.

Evaluation of acetaldehyde-degrading activity in saliva by AL-11 strain
Strain AL-11 was cultured for 3 days at 30 ° C. in a medium having the same composition as in Example 7. The cells were collected by centrifugation (5000 rpm, 10 minutes) and washed with physiological saline. Cells (wet weight, about 0.1 g each) were suspended in saliva (0.5 ml) to which acetaldehyde was added to 2 mM, and incubated at 37 ° C. for 5 hours. The cells were removed from this reaction solution by centrifugation (15000 rpm, 1 minute), and the concentration of acetaldehyde contained in the supernatant was measured by the method described in Example 12. In either case, acetaldehyde was not detected in the supernatant.

Evaluation of Acetaldehyde Degradation Activity in Saliva Using AL-11 Strain Bacteria Extract The cells of AL-11 obtained in the same manner as in Example 14 (wet cell weight, 2.0 g) were cooled with ice to 0.1 M. It was suspended in a phosphate buffer (pH 7.0, 4 ml) and crushed by ultrasonication (20 kHz, total 10 minutes). The cell disruption solution was centrifuged (15000 rpm, 15 minutes), and the supernatant was recovered as a cell extract. This was dialyzed sufficiently at 4 ° C. against 0.01 M phosphate buffer (pH 7.0) to obtain a crude enzyme solution. 0.1 ml of the crude enzyme solution was suspended in saliva (0.5 ml) to which acetaldehyde was added to 2 mM, and incubated at 37 ° C. for 5 hours. When the concentration of acetaldehyde contained in the saliva was measured by the method described in Example 12, as in Example 11, acetaldehyde was not detected in the supernatant.

Evaluation of n-hexanal and 2-nonenal decomposition activity by AL-5 bacterial cell extract The reaction solution obtained by adding n-hexanal or 2-nonenal to 2 ppm to the crude enzyme solution obtained in the same manner as in Example 13 ( 0.6 ml) was suspended in 0.03 ml of the crude enzyme solution and incubated at 37 ° C. for 10 minutes. When the concentrations of n-hexanal and 2-nonenal contained in this reaction solution were measured by the method described in Example 12, n-hexanal and 2-nonenal were not detected.

Evaluation of n-hexanal and 2-nonenal decomposition activity using AL-11 bacterial cell extract The reaction solution obtained by adding n-hexanal or 2-nonenal to 2 ppm to the crude enzyme solution obtained in the same manner as in Example 15 ( 0.6 ml) was suspended in 0.03 ml of the crude enzyme solution and incubated at 37 ° C. for 10 minutes. When the concentrations of n-hexanal and 2-nonenal contained in this reaction solution were measured by the method described in Example 12, n-hexanal and 2-nonenal were not detected.

Claims (14)

Pseudomonas sp. FERM BP-10988 strain, Pantoea sp. FERM BP-10989 strain, Pantoea sp. FERM BP-10990 strain, or a mutant thereof, which is a microorganism having an ability to decompose aliphatic aldehydes but not to vanillin. The microorganism according to claim 1, wherein the aliphatic aldehyde is acetaldehyde, hexanal and / or nonenal. The microorganism according to claim 1, wherein the aliphatic aldehyde is acetaldehyde. The microorganism according to any one of claims 1 to 3, which has an ability to decompose aliphatic aldehyde under conditions of pH 3.0 or more and 4.0 or less. The microorganism according to any one of claims 1 to 4, wherein the aliphatic aldehyde resolution is not required for NAD. The microorganism according to claim 5, wherein the NAD non-requiring aliphatic aldehyde resolution is due to acetaldehyde oxidase. The composition containing the microorganism as described in any one of Claims 1-6. A composition comprising a cell disruption solution, a crude enzyme solution or a purified enzyme prepared from the microorganism according to any one of claims 1 to 6. The composition according to claim 7 or 8, wherein the composition is a food or drink, a cosmetic, a laundry treatment agent, or a deodorant. An oral hygiene agent comprising the microorganism according to any one of claims 1 to 6. An oral sanitary agent comprising a cell disruption solution, a crude enzyme solution, or a purified enzyme prepared from the microorganism according to any one of claims 1 to 6. A method for reducing the concentration of an aliphatic aldehyde in a food or beverage, wherein the microorganism according to any one of claims 1 to 6 is added to a food or drink. 13. The method according to claim 12, wherein the aliphatic aldehyde is acetaldehyde, hexanal and / or nonenal. The method according to claim 12, wherein the aliphatic aldehyde is acetaldehyde.