JP2011030530A - Lactobacillus producing 2-phenylethanol and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new technique for producing 2-phenylethanol originated from a natural product. <P>SOLUTION: There is provided a lactobacillus producing 2-phenylethanol. The method for producing the 2-phenylethanol comprises a mutation treatment process for introducing a mutation to a lactobacillus and a process for screening a phenylalanine analogue-resistant strain from the mutation-introduced lactobacilli. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a composition for producing 2-phenylethanol and use thereof, and more particularly to a composition, kit and method for producing 2-phenylethanol.

  2-Phenylethanol (hereinafter also referred to as 2-PE) is a main component of the scent of roses and has attracted attention as a flavor component. Moreover, 2-PE has an antibacterial action. Furthermore, 2-PE is also used as a preservative for soap because it is difficult to decompose in an alkaline environment.

2-PE is also called phenethyl alcohol, and is a kind of alcohol represented by C ₆ H ₅ CH ₂ CH ₂ OH. It is a colorless liquid that exists widely in nature, and is contained in various essential oils such as carnation, hyacinth, aleppo pine, ylang ylang, geranium, neroli, and kinkweed. It is well miscible with ethanol or ether, but slightly soluble in water (2 mL / 100 mL · H ₂ O).

  Industrially, synthetic 2-PE is produced as a by-product of the catalytic reduction method of styrene oxide or the petrochemical industry. As a further production method of synthesis 2-PE, a method of reacting benzyl chloride and methanol by light irradiation in the presence of a europium compound is also disclosed (see Patent Document 1).

  On the other hand, natural 2-PE can be obtained, for example, by distilling rose petals. Moreover, natural 2-PE can be obtained by fermentation with yeast. As a technique for obtaining more 2-PE in a system using yeast, a method of extracting 2-PE from a distillation residue produced during the production of alcohol (see Patent Document 2), or yeast originally possessed A technology for improving the productivity of 2-PE by improving the 2-PE production ability (see Patent Documents 3 to 5) is disclosed.

JP 6-87769 A (published March 29, 1994) Japanese Patent Laid-Open No. 11-157 (released on January 6, 1999) Japanese Patent Laid-Open No. 3-98577 (published on April 24, 1991) Japanese Patent Laid-Open No. 3-94670 (published on April 19, 1991) Japanese Patent Laid-Open No. 9-224650 (published on September 2, 1997) Japanese Patent Laid-Open No. 6-284884 (published on October 11, 1994) JP 7-67669 A (published March 14, 1995)

  2-PE, which provides the scent of roses, is produced in large quantities around the world and is widely used in detergents and cosmetics. However, since synthetic 2-PE is a chemically synthesized product, when it is used in the human living environment, a high degree of purification is required, and as a result, the purification is very complicated. In recent years, demand for products derived from nature has increased, and development of technology for producing naturally-derived 2-PE is desired from the viewpoint of not only natural flavors but also natural foods.

  This invention is made | formed in view of said problem, The objective is to provide the new technique which manufactures naturally-derived 2-PE.

  The method for producing a lactic acid bacterium producing 2-phenylethanol according to the present invention includes a mutation treatment step for introducing a mutation into the lactic acid bacterium, and a selection step for selecting a phenylalanine analog resistant strain from the lactic acid bacterium having the mutation introduced. It is characterized by that. In the production method according to the present invention, the mutation treatment step is preferably performed by chemical treatment or ultraviolet treatment.

  In the production method according to the present invention, the lactic acid bacterium to be subjected to the mutation treatment step is preferably selected from the group consisting of accession numbers NITE BP-5-8 and NITE P-9-11.

  The lactic acid bacterium according to the present invention is characterized by producing 2-phenylethanol. The lactic acid bacterium according to the present invention is a phenylalanine analog resistant strain, and is preferably a mutant selected from lactic acid bacteria into which an arbitrary mutation has been introduced.

  Moreover, the lactic acid bacteria concerning this invention may be acquired by the said manufacturing method.

  The first composition according to the present invention is characterized in that it contains viable bacteria of lactic acid bacteria in order to produce lactic acid bacteria that produce 2-phenylethanol. The first composition according to the present invention may be in the form of a kit, and in the case of a kit, it is sufficient that a living lactic acid bacterium is viable, but a reagent for treating the lactic acid bacterium with a drug is further provided. It is preferable that it is provided.

  The 2nd composition concerning this invention is characterized by containing the lactic acid bacteria which produce | generate 2-phenylethanol so that a fragrance of rose may be provided. Moreover, the composition concerning this invention may be a composition to which the fragrance of the rose was provided, Preferably, it is a detergent, cosmetics, or a foodstuff.

  The production method according to the present invention includes a step of viably containing a lactic acid bacterium that produces 2-phenylethanol in a target food to produce a food with a rose scent. . In the production method according to the present invention, the lactic acid bacterium is preferably a phenylalanine analog resistant strain. In this case, the production method includes a mutation treatment step for introducing a mutation into the lactic acid bacterium, and phenylalanine from the lactic acid bacterium into which the mutation has been introduced. It is preferable to further include a selection step of selecting an analog resistant strain.

  The production method according to the present invention includes a step of culturing a phenylalanine analog-resistant strain of lactic acid bacteria in a phenylalanine analog-containing medium and a step of extracting 2-phenylethanol from the culture supernatant in order to produce 2-phenylethanol. It is characterized by that. In the production method according to the present invention, the phenylalanine analog-resistant strain of the lactic acid bacterium is preferably obtained by the above-described method for producing a lactic acid bacterium that produces 2-phenylethanol. In this case, a mutation treatment for introducing a mutation into the lactic acid bacterium Preferably, the method further includes a selection step of selecting a phenylalanine analog resistant strain from among the lactic acid bacteria having the mutation introduced therein.

  The present invention can provide natural 2-phenylethanol. Since 2-phenylethanol is the main component of the rose scent, the present invention can provide a product with a rose scent.

It is a figure which shows the 2-phenylethanol biosynthetic pathway in a plant and yeast. It is a figure which shows the result of GC-MS about the preparation (a) of 2-phenylethanol, and the chloroform extract (b) of the culture supernatant of a lactic acid bacteria mutant.

[1: Lactic acid bacteria producing 2-PE and method for producing the same]
The present invention provides a method for producing a lactic acid bacterium that produces 2-phenylethanol (hereinafter also referred to as 2-PE). The production method according to the present invention includes a mutation treatment step for introducing a mutation into lactic acid bacteria, and a selection step for selecting a phenylalanine analog-resistant strain from lactic acid bacteria into which the mutation has been introduced.

  The present invention also provides a lactic acid bacterium that produces 2-PE. The lactic acid bacteria concerning this invention can be manufactured with the manufacturing method concerning this invention.

  2-PE has the following structural formula.

  The 2-PE biosynthetic pathway in plants and yeast is shown in FIG. In plants (for example, roses), an enzyme that synthesizes phenylacetaldehyde from phenylalanine exists and controls biosynthesis to 2-PE in plants. On the other hand, there is no enzyme that synthesizes phenylacetaldehyde from phenylalanine in yeast, but the presence of an enzyme that synthesizes phenylacetaldehyde from phenylpyruvic acid controls the biosynthesis of 2-PE in yeast.

  Although the biosynthetic pathways are partially different, it is known that similar systems for producing 2-PE exist in higher plants and yeast, which are the same eukaryotes. This system is clearly different from the synthetic 2-PE system described above (reduction of styrene oxide).

  Patent Documents 6 and 7 disclose methods for producing 2-PE using prokaryotes. Specifically, the technique described in Patent Document 6 or the like is to convert styrene oxide into 2-PE by contacting with styrene-utilizing corynebacteria. However, although this technique uses a reaction by microorganisms, in principle, it is only a synthetic 2-PE system. Furthermore, Patent Documents 6 and 7 strongly indicate that the presence of styrene oxide reductase is very important for the production of 2-PE using prokaryotes.

  These findings fully suggest that eukaryotic systems such as plants and yeast do not exist in prokaryotes for the biosynthesis of 2-PE. Furthermore, even if a system for biosynthesis of 2-PE exists in prokaryotes, it is well suggested that it is a system involving styrene oxide reductase. Of course, it is no wonder that prokaryotes do not have the same biosynthetic pathway as eukaryotes such as plants and yeast. Indeed, an enzyme that synthesizes phenylacetaldehyde from phenylalanine, which is involved in the biosynthesis of 2-PE in yeast, has not been found so far in lactic acid bacteria. In addition, the present inventors have confirmed that lactic acid bacteria do not produce 2-PE (unpublished).

  Based on these findings, it is highly possible that one skilled in the art would expect a system that produces 2-PE from phenylalanine to exist in prokaryotes or be motivated to confirm the existence of such a system. Have difficulty.

  Patent Document 3 or the like obtains a mutant strain in which the efficiency of biosynthesis from phenylalanine to 2-PE is improved by selecting a p-fluorophenylalanine (PFP) resistant strain from the mutant-treated yeast. Is disclosed. However, as described above, this technique is a technique for improving functions originally possessed by yeast, and is not a technique for imparting new functions to yeast.

  Thus, there is no known technique that can reasonably expect completion of the present invention. Therefore, the present invention cannot be easily conceived by those skilled in the art. Needless to say, the fact that 2-PE can be produced using lactic acid bacteria is beyond the scope of those skilled in the art.

  In the production method according to the present invention, the mutation treatment step may be any method capable of randomly introducing mutations into the gene of lactic acid bacteria, and various known methods (for example, drug treatment (ethyl methanesulfonic acid, nitroso Application of guanidine, etc.), ultraviolet irradiation, etc.) can be utilized. In the present invention, a selection step after the mutation treatment step is essential in order to obtain lactic acid bacteria having a desired character. The selection step may be any method that can select a strain resistant to phenylalanine analogs of lactic acid bacteria, and various known methods (for example, culturing lactic acid bacteria in a medium to which an analog of phenylalanine is added) can be used. That is, the selection step can be a step of adding an analog of phenylalanine to the medium. In this case, the analog of phenylalanine to be added is preferably a purified sample. Examples of the phenylalanine analog applied to the present invention include p-fluorophenylalanine (PFP), which is widely used, but is not limited thereto. In addition, those skilled in the art who read this specification will easily understand that lactic acid bacteria producing 2-PE can be easily obtained without any trial and error based on the description of this specification.

  Further, by repeating the production method according to the present invention, the ability of lactic acid bacteria to produce 2-PE (2-PE production ability) can be enhanced. That is, a mutant strain with enhanced 2-PE production ability can be produced by further performing the mutation treatment step and the selection step on the obtained mutant strain (lactic acid bacterium producing -PE). .

  That is, such additional mutants are also within the scope of the present invention, and repeating the production method according to the present invention is also within the scope of the present invention. Furthermore, the production method according to the present invention may be a method for enhancing the ability to produce 2-PE by lactic acid bacteria.

  Of course, based on the description herein, a person skilled in the art can identify a gene into which a mutation has been introduced. If such a target gene is identified, direct mutation introduction into the target gene may be performed by using genetic engineering techniques (base substitution, insertion and / or addition by recombination or the like). Further, the function of the target gene may be inhibited using a conventionally known genetic engineering technique (for example, antisense method, sense (cosuppression) method, RNAi method, etc.).

  As described above, in the selection step in the present invention, in order to obtain a phenylalanine analog resistant strain, the lactic acid bacterium subjected to the mutation treatment is cultured in a medium containing a phenylalanine analog (for example, p-fluorophenylalanine). It can be a process.

  Lactic acid bacteria have been inoculated into Japanese traditional fermented foods such as cheese, yogurt, fermented butter, miso and pickles, as well as fruit juice and vegetable juice since ancient times. It plays an important role in improving the flavor, structure, nutritional value of foods, and imparting preservability. In recent years, as a physiological effect of lactic acid bacteria, an improvement effect of intestinal flora by inoculation with live lactic acid bacteria or an intestinal regulating action has been clarified, and lactic acid bacteria preparations have been developed as pharmaceuticals.

  As used herein, “lactic acid bacteria” are intended to be bacteria that produce large amounts of lactic acid from sugars by fermentation. The lactic acid bacteria applicable to the present invention may be those that ultimately produce only lactic acid, those that produce other than lactic acid (for example, alcohol or acetic acid), and those that are cocci or bacilli. May be.

  Lactic acid bacteria applicable to the present invention are so-called animal lactic acid bacteria isolated from mammalian milk, intestinal tract, etc., or lactic acid bacteria isolated from meat products (eg, fermented sausages such as nam), grains, It may be a lactic acid bacterium isolated from plant origin such as vegetables and fruits, or fermented foods containing these as raw materials. Examples of animal lactic acid bacteria include Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus juguiri, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus delbrueckii, Lactobacillus salvariilus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus rhamnosus, Lactobacillus casei var. alactosus, Lactobacillus fermenti, Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus cellobio (Separated from flowers in our laboratory), Lactobacillus brevis, Leuconostoc mesenteroides, Teragenococcus halophilus, Lactobacillus curvatus, Lactobacillus fermentum, Pedeococcus pentosaceus, Pedeococcus acidilactici, Bacillus coagulans, Enterococcus mun Examples include dtii.

  In addition, as a specific strain among the lactic acid bacteria mentioned above, Lactobacillus plantarum SN13T strain (separation source: Nam of fermented sausage made in Thailand: pork mince wrapped in banana leaves and fermented), Lactobacillus plantarum SN26T Strain (source: sausage), Lactobacillus plantarum SN35N strain (source: pear), Lactobacillus plantarum JCM1149 strain (source: salted cabbage), Lactobacillus plantarum JCM8348 strain (source: rice), Lactobacillus plantarum IFO3070 strain (JCM1057) Strain), Lactobacillus casei K-1 strain (source: Kameda plant lactic acid bacteria yogurt), Lactobacillus casei JCM 8136 strain (separation source: unknown), Pediococcus pentosaceus (isolation) Source: Otsuka Vegetable Warrior), Pediococcus JCM5885 strain (source: Sake Moromi), Lactobacillus plantarum SN35M strain (source: Melon), Lactococcus lactis subspecies Lactis SN26N strain (source: Nashi), Enterococcus spice SN21I Strains (separation source: fig), Enterococcus munti SN29N strain (separation source: pear), Lactobacillus hilgardi NBRC15886 (separation source: wine) and the like.

  As a plant lactic acid bacterium, the present inventors have so far made Lactobacillus plantarum SN13T strain, Lactobacillus plantarum SN26T strain, Lactobacillus plantarum SN35N strain, Lactobacillus plantarum SN35M strain, Lactococcus lactis subspecies lactis SN26N strain, Enterococcus spp. SN29N strain etc. are isolated.

  The Lactobacillus plantarum SN35M strain is a strain isolated by the present inventors, and is incorporated by the National Institute of Technology and Evaluation (NITE) Patent Microorganism Deposit Center (292-0818, Kazusa Kamashizu, Kisarazu City, Chiba Prefecture 2-5-8). And the deposit number NITE BP-5 (deposit date: July 6, 2004, transfer date: February 16, 2009) and has the following mycological properties: isolates from melon, Gram-positive lactobacilli, homozygous fermentation, catalase-negative, no spore-forming ability, can be cultured even under aerobic conditions, taxonomic position is Lactobacillus plantarum.

  The Lactobacillus plantarum SN35N strain is a strain isolated by the present inventors, and is incorporated by the National Institute of Technology and Evaluation (NITE) Patent Microorganism Deposit Center (292-0818, Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 2-5-8). And the deposit number NITE BP-6 (deposit date: July 6, 2004, transfer date: February 16, 2009) and has the following mycological properties: an isolate from pear, Gram-positive lactobacilli, homozygous fermentation, catalase negative, no spore-forming ability, can be cultured even under aerobic conditions, taxonomic position is Lactobacillus plantarum.

  The Lactobacillus plantarum SN13T strain is a strain isolated by the present inventors, and is incorporated by the National Institute of Technology and Evaluation (NITE) Patent Microorganism Deposit Center (292-0818, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 2-5-8). Is deposited under the accession number NITE BP-7 (consignment date: July 6, 2004, transfer date: January 28, 2009) and has the following mycological characteristics: sausage called Nam from Thailand Isolate, Gram-positive lactobacilli, homozygous fermentation, catalase negative, no spore-forming ability, can be cultured under aerobic conditions, taxonomic position is Lactobacillus plantarum.

  The Lactobacillus plantarum SN26T strain is a strain isolated by the present inventors, and is incorporated by the National Institute of Technology and Evaluation (NITE) Patent Microorganism Deposit Center (292-0818, Kazusa Kamashizu, Kisarazu City, Chiba Prefecture 2-5-8). Is deposited under the accession number NITE BP-8 (date of trust: July 6, 2004, date of transfer: February 16, 2009) and has the following mycological properties: sausage named Nam from Thailand Isolate, Gram-positive lactobacilli, homozygous fermentation, catalase negative, no spore-forming ability, can be cultured even under aerobic conditions, taxonomic position is Lactobacillus plantarum.

  Lactococcus lactis subspecies lactis strain SN26N is a strain isolated by the present inventors, and is incorporated by the National Institute of Technology and Evaluation (NITE) Patent Microorganism Deposit Center (292-0818, Kazusa Kamashizu, Kisarazu City, Chiba Prefecture). 5-8), with the deposit number NITE P-9 (date of deposit: July 6, 2004) and having the following bacteriological properties: isolates from pear, gram-positive lactic acid cocci, homo Type fermentation, catalase negative, no spore-forming ability, can be cultured under aerobic conditions, taxonomic position is Lactococcus lactis subsp. lactis.

  Enterococcus munzti strain SN29N is a strain isolated by the present inventors, and is a patent evaluation microorganism deposit center of the National Institute of Technology and Evaluation (NITE) (292-0818, Kazusa Kamashizu, Kisarazu City, Chiba Prefecture 2-5-8). And has the following bacteriological properties: isolate from pear, gram-positive lactic acid cocci, homo-type fermentation, catalase Negative, no spore-forming ability, culturable under aerobic conditions, taxonomic position is Enterococcus munditi.

  Enterococcus spices SN21I strain is a strain isolated by the present inventors, and is an independent administrative agency, National Institute of Technology and Evaluation (NITE) Patent Microorganism Deposit Center (292-0818, Kazusa Kamashizu, Kisarazu City, Chiba Prefecture 2-5-8). And has the following bacteriological properties: isolate from fig, gram-positive lactic acid cocci, homozygous fermentation, catalase Negative, no spore-forming ability, can be cultured under aerobic conditions, taxonomic position is Enterococcus sp. .

  In the present invention, the lactic acid bacteria described above may be used alone or in combination of two or more.

  The present invention provides a composition (first composition) that contains viable lactic acid bacteria. The first composition is a composition for producing lactic acid bacteria that produce 2-PE. That is, if the 1st composition concerning this invention is used, the lactic acid bacteria which produce | generate 2-PE can be manufactured. Thereby, the product (for example, detergent, cosmetics, foodstuff, etc.) to which the scent of rose by 2-PE was given can be manufactured.

  The composition concerning this invention may further contain the fermentation raw material fermented with lactic acid bacteria. In addition, the composition according to the present invention may further contain sake lees or shochu distillation residues or extracts thereof. In addition, about the sake lees or shochu distillation residue, or these extracts, it is in the patent document (Unexamined-Japanese-Patent No. 2006-42796 (published February 16, 2006)) used as reference in this specification. The details are described and can be easily understood by those skilled in the art.

  As used herein, the term “composition” may be an embodiment in which the components are contained in a single composition or an embodiment in which the components are separately provided in a single kit. That is, the composition according to the present invention may further include a culture medium, but is in the form of a kit comprising the composition containing the above live bacteria of lactic acid bacteria viable and the fermentation raw material separately. Also good. In addition, the composition according to the present invention may further contain a rice fermented product obtained from koji mold, a sake lees or shochu distillation residue, or an extract thereof. However, the composition according to the present invention and the sake lees or shochu distillation residue or these It may be in the form of a kit provided with the extract separately. Further, when the first composition is provided in the form of a kit, the kit may be provided with viable bacteria of lactic acid bacteria so as to survive, but a reagent for treating the lactic acid bacteria with a drug or phenylalanine of lactic acid bacteria It is preferable that a reagent for selecting an analog resistant strain is further provided.

  The fermentation raw material used in the present invention is preferably selected from the group consisting of milk, fruit juice and vegetable juice. Examples of the milk include animal milk (for example, cow's milk, goat milk, and sheep milk), and milk is particularly preferable. The milk may be either non-sterilized milk or pasteurized milk, and concentrated milk or condensed milk prepared from these milks, skim milk, partially skimmed milk or powdered milk obtained by drying these powders, etc. There may be. Examples of fruit juice include, but are not limited to, peach, pear, apple, strawberry, pomegranate, grape, mango, mandarin orange, orange, pineapple and the like, and may be fruit puree. Examples of vegetable juice include, but are not limited to, carrots, tomatoes, cabbage and other juices, and vegetable purees may be used. Fruit juice and vegetable juice are obtained by grinding fruit or vegetable with a mixer or the like, and further squeezing if necessary. Such fruit juices and vegetable juices may be concentrated as appropriate, and the concentrated solution may be used as it is or after being diluted to an appropriate concentration with distilled water or the like. The milk, fruit juice and vegetable juice described above may be used alone or in combination depending on the purpose. Note that fruit or vegetable puree is obtained by performing an enzyme treatment under an ultra-high pressure (for example, 100 MPa), and is rich in dietary fiber. The enzyme used for this enzyme treatment can be appropriately selected according to the type of fruit or vegetable.

  In the present specification, the term “containing viable bacteria viable” is not particularly limited as long as the target strain is maintained in the composition without dying. That is, in the composition, the target viable bacteria may be growing or may be dormant.

  In one embodiment, the first composition preferably contains Lactobacillus plantarum SN13T strain viably, and further contains a fermentation raw material fermented by Lactobacillus plantarum SN13T strain. It is preferable to further contain these extracts. If it is such a form, since the enzyme activity of the living microbe of a Lactobacillus plantarum SN13T strain | stump | stock can be kept high, this living microbe can be contained so that it can survive in a composition. In other embodiments, the first composition comprises live bacteria of Lactobacillus plantarum SN13T strain with suitable excipients. Excipients that do not affect the survival of lactic acid bacteria are well known in the art and include, for example, starch, dry yeast, lactose, sucrose, and the like for processing the composition into tablets (eg, corn Peptide, maltose, etc.). Thus, the composition according to the present invention is intended to be used as a so-called “fermented species”.

[2: Utilization of lactic acid bacteria producing 2-PE]
The present invention also provides a composition (second composition) containing a viable lactic acid bacterium that produces 2-PE. The second composition is a composition for producing a product containing 2-PE. That is, if the 2nd composition concerning this invention is used, the product (For example, detergent, cosmetics, foodstuffs, etc.) to which the fragrance of the rose by 2-PE was provided can be manufactured.

  The 2nd composition may further contain the fermentation raw material fermented by lactic acid bacteria similarly to the 1st composition. In addition, the composition according to the present invention may further contain sake lees or shochu distillation residues or extracts thereof. The fermentation raw material, sake lees, and shochu distillation residue applicable to the second composition may be the same as those of the first composition. The second composition may also be in kit form. Of course, the second composition may also be used as a so-called “fermented seed” in the same manner as the first composition.

  In one embodiment, the second composition viably contains a phenylalanine analog resistant strain derived from Lactobacillus plantarum SN13T strain, and further contains a fermentation raw material fermented by the phenylalanine analog resistant strain. Preferably, it further contains a sake lees or shochu distillation residue or an extract thereof. In such a form, since the enzyme activity of the viable bacteria of the phenylalanine analog resistant strain can be kept high, the viable bacteria can be contained viable in the composition. In other embodiments, the second composition comprises viable bacteria of a phenylalanine analog resistant strain derived from a Lactobacillus plantarum SN13T strain, with suitable excipients. As an excipient | filler which does not affect the survival of the said phenylalanine analog resistant strain, what was mentioned above about the 1st composition should just be employ | adopted.

  If the composition concerning this invention is used, the product (For example, detergent, cosmetics, foodstuffs, etc.) to which the fragrance of the rose by 2-PE was provided can be manufactured.

  In the case of food, the second composition itself may be the target product (edible composition). The edible composition produced using the present invention is an edible composition (ie, food, beverage, etc.) imparted with a rose scent by 2-PE. From the viewpoint of enhancing palatability, health food (function It is extremely useful when provided as a food. The production method of the edible composition according to the present invention is not particularly limited, and examples thereof include cooking, processing, and production by a generally used food or beverage production method. 2-PE obtained by the present invention is It may be contained in the food or beverage. That is, this invention also provides the method of manufacturing the foodstuff to which the fragrance of the rose was provided. The production method according to the present invention only needs to include a step of viably containing a lactic acid bacterium that produces 2-PE in a target food in order to produce a food with a rose scent. Examples of such foods include dairy products (eg, yogurt), health foods (eg, capsules, tablets, powders), beverages (eg, milk beverages, vegetable beverages), and drinks. It is not limited.

  In the case where the above-described second composition itself is an intended edible composition, the composition according to the present invention includes gelatin, agar, saccharides, flavors, pulp, etc. in addition to the desired lactic acid bacteria and fermentation raw materials. Moreover, the raw material normally used for manufacture of fermented food or fermented drinks can also be added. For example, sugars such as sucrose, glucose, fructose, palatinose, trehalose, sugar alcohols such as sorbitol, xylitol, erythritol, reduced starch syrup, high sweetness sweeteners such as aspartame, acesulfame potassium, sucrose fatty acid ester, glycerin fatty acid ester, Emulsifiers such as lecithin, thickeners such as carrageenan, xanthan gum and guar gum, acidulants such as citric acid, lactic acid and malic acid, juices such as lemon juice and orange juice, vitamins, calcium, iron, manganese and zinc Furthermore, it is possible to add herbal medicines such as licorice, katsushi, ginger, or food additives such as herbs, sodium glutamate, gardenia pigments, silicone, and phosphates.

  The present invention further provides a method for producing 2-PE. The production method according to the present invention includes a culture step of culturing a phenylalanine analog-resistant strain of lactic acid bacteria in a phenylalanine analog-containing medium and an extraction step of extracting 2-PE from the culture supernatant in order to produce 2-PE. It is characterized by that. In the production method according to the present invention, the phenylalanine analog resistant strain of the lactic acid bacterium is preferably obtained by the above-described method for producing a lactic acid bacterium that produces 2-PE.

  In addition, the said culture | cultivation process may be a fermentation process which ferments a fermentation raw material with the living microbe of the phenylalanine analog resistant strain of the said lactic acid bacteria. In the fermentation step, it is preferable to ferment the fermentation raw material until the acidity falls within the range of 0.6 to 1.5%. Fermentation should just be performed using a well-known method in the said field | area. You may use what was cooled after completion | finish of fermentation as a fermented drink. Such fermented beverages may be further diluted or sterilized.

[1. Acquisition of mutants
Lactobacillus plantarum SN13T strain was cultured in 10 mL of MRS medium (MERCK) at 37 ° C. for 24 hours. Centrifugation of 200 μL of the culture broth collected the cells, and the cells were washed with 1 mL of a completely synthetic medium not containing phenylalanine (hereinafter, medium A). After washing, the cells were suspended in 1 mL of medium A (containing 1% ethyl methanesulfonic acid) and incubated at 37 ° C. for 30 minutes. The culture was centrifuged to collect the cells, resuspended in 1 mL of medium A, and 100 μL each of the cells were applied to medium A containing 200 μg / mL or 300 μg / mL of p-fluorophenylalanine (PFP). When plated on a medium containing 200 μg / mL PFP, 10 strains of growing clones were obtained after culturing at 37 ° C. for 2 days, and each was obtained from Lactobacillus plantarum SN13T-200-1 to Lactobacillus plantarum SN13T-200-10 Named. When inoculated on a medium containing 300 μg / mL PFP, after culturing at 37 ° C. for 6 days, 5 growing clones were obtained, each of which was obtained as Lactobacillus plantarum SN13T-300-1 to Lactobacillus plantarum SN13T-300-5 Named.

  The obtained 15 mutant strains are maintained under the management of the University of Hiroshima University Industry-Academia Collaboration Center (1-2-2 Kagamiyama, Higashihiroshima City, Hiroshima Prefecture) and should be sold as needed. Is possible.

[2. Production of 2-phenylethanol (2-PE) in mutants]
The obtained 15 mutant strains were cultured in 10 mL of MRS medium at 37 ° C. for 24 hours. 500 μL of each culture solution was inoculated into 50 mL of fresh MRS medium, and cultured at 37 ° C. for 48 hours. The culture solution after culturing was centrifuged to remove the cells, and a culture supernatant was obtained. The obtained supernatant was extracted with 50 mL of chloroform. The extraction operation was performed 3 times. The obtained chloroform layers were combined, and the solvent was distilled off under reduced pressure using an evaporator. The residue was dissolved in an appropriate amount of chloroform and analyzed with a gas chromatography / mass spectrometry (GC-MS) apparatus. 2-PE analysis was also performed as a sample.

  As a result, 3 strains (Lactobacillus plantarum SN13T-300-1, Lactobacillus plantarum SN13T-300-2, and Lactobacillus plantarum SN13T-300-4) were confirmed among the mutants obtained from the medium containing 300 μg / mL PFP. However, all produced 2-PE. This suggests that all 15 mutant strains obtained can produce 2-PE. FIG. 2 shows the results of GC-MS of the sample 2-PE and the results of GC-MS in the chloroform extract of the culture supernatant of the lactic acid bacteria mutant (FIG. 2 (a) and ( b)). Further, it was confirmed by GC-MS that the parent strain did not produce 2-PE (data not shown).

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  The present invention can be easily applied to various products (detergents, foods, cosmetics, etc.) that are desired to have a rose scent.

Claims (12)

A method for producing a lactic acid bacterium that produces 2-phenylethanol,
A production method comprising a mutation treatment step of introducing a mutation into a lactic acid bacterium, and a selection step of selecting a phenylalanine analog-resistant strain from lactic acid bacteria into which the mutation has been introduced.   The manufacturing method according to claim 1, wherein the mutation treatment step is performed by chemical treatment or ultraviolet treatment.   The production method according to claim 1 or 2, wherein the lactic acid bacterium to be subjected to the mutation treatment step is selected from the group consisting of accession numbers NITE BP-5-8 and NITE P-9-11.   Lactic acid bacteria acquired by the manufacturing method of any one of Claims 1-3.   Lactic acid bacteria that produce 2-phenylethanol.   A composition for producing a lactic acid bacterium that produces viable 2-phenylethanol, containing viable lactic acid bacteria.   A composition for imparting the scent of roses, which contains viable lactic acid bacteria that produce 2-phenylethanol.   A food containing viable lactic acid bacteria that produce 2-phenylethanol. A method for producing a food with a rose scent,
The manufacturing method including the process of containing the lactic acid bacteria which produce | generate 2-phenylethanol so that it may survive in the target foodstuff. The lactic acid bacterium is a phenylalanine analog resistant strain,
The production method according to claim 9, further comprising a mutation treatment step of introducing a mutation into the lactic acid bacterium, and a selection step of selecting a phenylalanine analog resistant strain from the lactic acid bacterium into which the mutation has been introduced. A method for producing 2-phenylethanol, comprising:
A method comprising: culturing a phenylalanine analog-resistant strain of lactic acid bacteria in a medium containing phenylalanine analog; and extracting 2-phenylethanol from the culture supernatant. The production method according to claim 11, further comprising a mutation treatment step of introducing a mutation into the lactic acid bacterium, and a selection step of selecting a phenylalanine analog resistant strain from the lactic acid bacterium into which the mutation has been introduced.

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