JP5244415B2 - Antifungal substance - Google Patents

Description

  The present invention relates to the genus Candida, Saccharomyces, Pichia, Rhodotorula, Clavispora, and Torulaspora. An antifungal substance that inhibits spore germination, a fungicide and a food preservative containing the antifungal substance, fungi using the antifungal substance or the yeast, and Alternatively, the present invention relates to a method for inhibiting the growth of yeast and a novel Candida sp. That produces the antifungal substance.

  Various antibacterial substances are used as food preservatives in order to prevent spoilage and deterioration due to microorganisms and to enhance the preservation of food. By improving the preservability of food, it is possible to efficiently prevent the food from being spoiled in the distribution process, and therefore it is possible to expect an improvement in manufacturing cost, and it is also preferable from the viewpoint of food safety. For this reason, the development of an excellent antibacterial substance is desired. Here, antibacterial substances can be classified mainly into chemically synthesized substances such as propionic acid and sorbic acid, and naturally-derived substances obtained from microorganisms, etc. From the point of view, it is preferably a naturally derived substance.

  In recent years, various types of bacteriocin derived from lactic acid bacteria, such as nisin, have been known as natural antibacterial substances (for example, see Non-patent Document 1). Bacteriocin is usually a peptide or protein, and is easily decomposed by digestive enzymes or the like, so it is very safe. It is also known to have excellent antibacterial activity mainly against gram-positive bacteria.

Other methods for producing food preservatives containing antibacterial substances having excellent antibacterial activity against both gram-positive and gram-negative bacteria include, for example, (1) nutrients, inorganic salts and carbon sources necessary for yeast growth As described above, the yeast is grown in a medium containing glucose until reaching the stationary phase, and then, if necessary, a carbon source is added to the medium from which the yeast has been removed, and repeated cultivation is repeated twice or more until the stationary phase is reached. A method for producing a food preservative containing an antibacterial substance produced by yeast, characterized by being an aqueous solution, is disclosed (for example, see Patent Document 1).
Somoto et al., Food and Development, Vol. 41, No. 3, p73-75, 2006 Japanese Patent No. 2545739

Not only bacteria such as Gram-positive bacteria but also fungi such as mold and yeast cause food rot and the like. In particular, since mold is a major factor in the decay of many foods, it is preferable that a substance having antifungal activity capable of suppressing mold growth can be used as a food preservative or antibacterial agent.
However, bacteriocin such as nisin generally has antibacterial activity only against gram-positive bacteria, and has a problem that it cannot inhibit the growth of fungi such as mold and yeast. Moreover, since the food preservative obtained by the method (1) is an antibacterial substance derived from yeast, it can be used safely for food and has heat resistance. No. 1 describes nothing about antibacterial activity against mold.

On the other hand, although organic acids such as propionic acid have a certain effect on the inhibition of fungal growth, they are chemically synthesized substances, so they are less safe than naturally derived substances and have a unique smell. Therefore, there is a problem that the taste of food is not preferable.
In addition, organic acids have an antibacterial activity mainly under acidity, and have a problem of poor antibacterial properties against neutral foods. The decrease in antibacterial activity in the neutrality of organic acids is because organic acids are almost completely ionized in neutral solutions, but organic acids that are dissociated and negatively charged are difficult to be taken into the cells of microorganisms. It is believed that.

An object of the present invention is to provide an antifungal substance that is safe and can neutrally suppress the growth of fungi such as mold and yeast.
The present invention also provides an antifungal agent and a food preservative containing the antifungal substance, a fungus producing the antifungal substance, and a fungus and / or yeast using the antifungal substance or the fungus. An object is to provide a method for inhibiting proliferation.

  As a result of earnest research to solve the above problems, the present inventors believe that any antifungal substance produced by food-derived microorganisms can be safely used as a food preservative, and various types of Among microorganisms isolated from cheese, a microorganism having antifungal activity capable of suppressing the growth of Aspergillus niger or Rhizopus stroniffer at pH 7.0, and The present invention was completed by finding antifungal substances produced by microorganisms.

That is, the present invention relates to food-derived Candida maltosa O9-NP9 (Accession No. NITE BP-297), Saccharomyces cerevisiae H-1 (Accession No. NITE BP-473), Clavispora sp. P-5 (Clavispora sp. P-5, accession number NITE BP-474), Candida pseudointermedia IAM12510, Pichia membranifaciens JCM1455, Pichia nakase 3 exigua) JCM9554, Rhodotorula acta uta) JCM9494, selected from Filibasidium capsuligenum JCM2273, Candida terabra JCM9566, Saccharomyces cerevisiae Np An antifungal substance that inhibits germination of mold spores and has the following characteristics is produced.
(1) It has antifungal activity at pH 7.0, (2) the culture solution obtained by culturing the yeast in 10% (w / w) skim milk medium has antifungal activity, (3) 10% ( w / w) When the supernatant of the culture medium in which the yeast was cultured in a skim milk medium was ultrafiltered using an ultrafiltration membrane having a MWCO (Molecular Weight Cut Off) of 10 kD, (4) The antifungal activity is deactivated by heat treatment at 55 ° C. for 15 minutes.
The present invention also provides a fungicide containing the antifungal substance described above.
Moreover, this invention provides the food preservative containing the said antifungal substance.
The present invention also provides a method for inhibiting mold growth, characterized by using the antifungal substance described above.
The present invention also provides a method for inhibiting fungal growth, characterized by using the food-derived yeast described above.
The present invention also provides Candida maltosa O9-NP9 (Accession No. NITE BP-297) characterized by producing the antifungal substance described above.
The present invention also provides Saccharomyces cerevisiae H-1 (Accession No. NITE BP-473) characterized by producing any one of the antifungal substances described above.
The present invention also provides Clavispora Spices P-5 (Accession No. NITE BP-474) characterized by producing any one of the antifungal substances described above.

The antifungal substance of the present invention can effectively suppress the growth of fungi such as mold and yeast. Since the antifungal substance of the present invention is a substance produced by yeast isolated from cheese, it can be used not only for foods but also for cosmetics and pharmaceuticals. In particular, since it exhibits antifungal activity in neutrality, it is possible to effectively suppress the growth of fungi and the like even in neutral foods. Since the antifungal substance of the present invention can safely extend the storage period of foods and the like, an improvement in production cost is also expected.
Moreover, the antifungal substance of the present invention can be efficiently produced by the yeast that produces the antifungal substance of the present invention.
Furthermore, by the method for inhibiting the growth of molds and / or yeasts of the present invention, the growth of fungi such as molds and yeasts can be suppressed safely and easily.

  The antifungal substance of the present invention is produced by microorganisms obtained from foods and has antifungal activity at pH 7.0. This is because such an antifungal substance can be safely used for foods and the like, and can also be effective for neutral foods and the like.

  As the microorganism that produces the antifungal substance of the present invention, for example, a strain having antifungal activity capable of suppressing the growth of mold and the like at pH 7.0 is selected from microorganisms isolated from various foods. Can be obtained. This is because a strain having antifungal activity at pH 7.0 is considered to produce the antifungal substance of the present invention. Hereinafter, the method for obtaining the antifungal substance of the present invention and the microorganism producing the antifungal substance will be described in more detail.

1. Acquisition of microorganism group The present inventors focused on cheese, which is one of good fermented foods, in order to acquire microorganisms that produce antifungal substances from food, and separated microorganisms by a conventional method. Specifically, after pouring cheese with a sterilized cheese grater, 0.05 g is added to 5 mL of a sterilized 10% (w / v) skim milk solution dispensed into a test tube with a screw cap. The culture was allowed to stand for a period of time. The precipitate collected by centrifuging the test tube with the screw cap was washed twice with a sterilized physiological phosphate buffer, and then diluted with a physiological phosphate buffer to prepare a sample. The sample was smeared on an MRS agar medium and then cultured at 28 ° C. to isolate microorganisms. Cheese is Camembert cheese (made in Japan), Camembert cheese (made in France), Fromage (made in Japan), Mozzarella cheese (made in Germany), 3 types of gouda cheese (made in the Netherlands), cream cheese (made in Italy), goat milk cheese ( France), cheese food (made in Australia), Hokkaido cheddar cheese (made in Japan), low fat cheddar cheese (made in the Netherlands), white cheddar cheese (made in England), caspian cheese (made in Iran), 14 types in total Used cheese. Microorganisms isolated from one type of cheese were combined into one fungal group.

2. Selection of fungal group containing bacteria with antifungal activity Inoculate 14 types of obtained fungal group into 10% (w / w) skim milk medium, and culture at 28 ° C. for 48 hours to prepare bacterial group culture solution did. The fungal group culture solution was streaked on a 10% (w / w) skim milk flat plate medium, and then a soft agar medium in which spores of Aspergillus niger or Rhizopus stronifer were suspended was overlaid.
As a result of observing the skim milk plate culture medium after culturing at 28 ° C. for 48 hours, growth of Aspergillus niger and the like in the vicinity of some colonies in the skim milk plate medium of the O-9 fungus group derived from Caspian cheese (manufactured by Iran). Was observed to be suppressed. In the other 13 types of fungal groups, growth inhibition of Aspergillus niger or the like was not observed. From this result, it was found that bacteria that produce an antifungal substance exist in the O-9 fungus group.

3. Selection of bacteria group in which bacteria having antifungal activity exist at pH 7.0 (1) Preparation of sample for measuring antifungal activity O-9 bacteria group was inoculated into 10% (w / w) skim milk medium, and 28 Culturing at 48 ° C. for 48 hours to prepare a fungal culture solution. The bacterial cell culture solution was subjected to ultrasonic treatment to uniformly disperse the bacterial cells, and then centrifuged at 3,000 × g for 10 minutes to separate into a supernatant and a precipitate. Since the obtained supernatant was about pH 4, a supernatant sample was prepared by adjusting the pH to 7.0 using a 5% (w / v) sodium carbonate solution. A precipitate sample was prepared by adding an approximately equal amount of a 10% (w / w) skim milk solution adjusted to pH 7.0 to the resulting precipitate. In order to suppress bacterial growth and facilitate measurement of antifungal activity, an antibacterial agent described in Example 1 below was added to each sample in an amount equivalent to 1/10. Further, a sample solution for measurement of antifungal activity was prepared by adding 1/9 equivalent amount of Sabouraud medium (glucose: 40 g / L, peptone S: 10 g / L) to each antibacterial agent-containing sample. .

(2) to each well of the measurement of antifungal activity 96-well microplates, the 3 2-fold serial dilutions ⁽² 0-2 ⁹ times) of the adjusted sample solution (1) were each added in 130 [mu] L. A Sabouraud medium was used for dilution of the sample solution. As a control, a well to which only 130 μL of Sabouraud medium was added was used. Furthermore, 50 μL each of 1.0 × 10 ⁴ sports / mL Aspergillus niger NBRC 9455 spore suspension prepared by the method described in Reference Example 1 described later was added to each well. The 96-well microplate was cultured at 28 ° C. for 48 hours, and then each well was observed with a microscope.

  FIG. 1 shows the result of microscopic observation. Lane A in the figure shows the dilution series of the supernatant sample solution derived from the O-9 fungus group, Lane B shows the dilution series of the precipitate sample solution derived from the O-9 fungal group, and Lane C shows the control. Yes. ○ in the figure represents each well, x in ○ indicates a well in which the growth of Aspergillus niger was observed, ○ in ○ indicates no growth of Aspergillus niger In other words, the wells in which growth inhibition was observed were respectively shown. The growth of Aspergillus niger was determined by germination of Aspergillus niger spores and hyphal elongation. That is, the growth inhibition of Aspergillus niger means a state in which germination and elongation of mycelia are prevented from the spores of Aspergillus niger.

  As shown in FIG. 1, growth of Aspergillus niger was observed in the control wells. On the other hand, in the case of the sample solution derived from the O-9 fungus group, the grown Aspergillus niger was grown in all wells to which the sediment sample solution was added and wells to which the 8- to 512-fold dilution of the supernatant sample solution was added. However, no growth of Aspergillus niger was observed in wells to which a 1 to 4 dilution of the supernatant sample solution was added.

As a result of the observation, the antifungal substance of the present invention is present in the supernatant sample solution derived from the O-9 fungus group, and the growth of Aspergillus niger was suppressed by the antifungal substance. It is clear. Therefore, it was found that the O-9 fungus group contains bacteria having antifungal activity at pH 7.0, that is, bacteria producing the antifungal substance of the present invention.
In addition, since growth inhibition of Aspergillus niger was observed in the wells to which the supernatant sample solution was added, the antifungal substance of the present invention produced by the bacterium was cultured in 10% (w / w) skim milk medium. In some cases, it is clear that it has the property of transferring into the culture medium.

4). Heat resistance of antifungal substance In order to examine the heat resistance of the antifungal substance of the present invention present in the supernatant sample solution derived from the group of O-9 bacteria, the O- adjusted in 3 (1) above. Supernatant samples from 9 bacterial groups were heat-treated at 100 ° C. for 5 minutes and then ice-cooled, adjusted to pH 7.0 as in 3 (1) above, added with antibacterial agent and Sabouraud medium, and heated A treated supernatant sample solution was prepared. When the antifungal activity of the heat-treated supernatant sample solution was measured in the same manner as in 3 (2) above, no inhibition of Aspergillus niger growth was observed. From the results of the observation, it was found that the antifungal substance of the present invention has the property that the antifungal activity is deactivated by heat treatment at 100 ° C. for 5 minutes. In addition, since the antibacterial substance contained in the food preservative described in Patent Document 1 is not inactivated by heat treatment, it is apparent that the substance is different from the antifungal substance of the present invention.

5. Size of antifungal substance In order to examine the size of the antifungal substance of the present invention present in the supernatant sample solution derived from the group of O-9 bacteria, the O- prepared in 3 (1) above was used. Supernatant samples from 9 bacterial groups were subjected to ultrafiltration using an ultrafiltration membrane having a MWCO of 10 kD, and passed through the ultrafiltration membrane and the polymer fraction retained on the ultrafiltration membrane surface. Fractionated into a low molecular fraction. Using the high molecular fraction and the low molecular fraction as samples, adjusting the pH to 7.0 as in 3 (1) above, adding an antibacterial agent and Sabouraud medium, and adding the supernatant high molecular fraction sample solution and the supernatant A low molecular fraction sample solution was prepared. When the antifungal activity of each of the supernatant polymer fraction sample solution and the supernatant low molecular fraction sample solution was measured in the same manner as 3 (2) above, the well containing the supernatant polymer fraction sample solution was added. Then, growth inhibition of Aspergillus niger was observed, but not in the wells to which the supernatant low molecular fraction sample solution was added. From the results of the observation, the antifungal substance of the present invention is fractionated into the polymer fraction, and when ultrafiltration is performed using an ultrafiltration membrane having a MWCO of 10 kD, an ultrafiltration membrane is obtained. It is clear that it has the property of being retained on the surface.

  Caspian cheese is a cheese having a very high salt concentration compared to other cheeses. Therefore, considering the possibility that the fungus producing the antifungal substance of the present invention is halophilic, the O-9 fungus group is added to a 10% (w / w) skim milk medium containing 2.5% NaCl. Except for the inoculation, antifungal activity was measured in the same manner as 3 above. As a result, growth inhibition of Aspergillus niger was observed in the wells to which a 1 to 16-fold dilution of the supernatant sample solution was added. That is, since the antifungal activity was increased about 4 times by culturing in a medium containing 2.5% NaCl, the fungus producing the antifungal substance of the present invention has a sufficient amount even under a high salt concentration. It was found that antifungal substances can be produced.

6). Isolation of Bacteria Producing Antifungal Substance of the Present Invention After applying the O-9 bacterial group culture solution prepared in 3 (1) above to a BCP (bromocresol purple) plate medium, the culture was performed at 37 ° C. Cultured for 48 hours. From the BCP plate medium, a total of 48 colonies were obtained: 6 colonies with yellowing around the colonies and 42 colonies with no yellowing observed. Each well of a 48-well microplate containing BCP agar medium was inoculated with the fungi picked from the obtained 48 colonies, and further cultured at 37 ° C. for 48 hours.

  From 48 cultured strains, 10 strains (P1-P10 strain) out of 42 strains whose medium remained purple, 3 strains (Y1-Y3 strain) among 6 strains whose media had turned yellow, Obtained. The obtained 13 strains were each inoculated into 10% (w / w) skim milk medium and cultured at 37 ° C. for 48 hours to prepare a culture solution. The supernatant obtained by centrifuging the culture solution in the same manner as in 3 (1) above was adjusted to pH 7.0, and then an antibacterial agent and Sabouraud medium were added to obtain a supernatant sample solution derived from each strain. Adjusted. In the same manner as 3 (2) above, a 2-fold serial dilution series of the supernatant sample solution was added to a 96-well microplate, and antifungal activity was measured.

2 (a) and 2 (b) show the results of observing each well with a microscope after culturing the 96-well microplate at 28 ° C. for 48 hours, and (c) and (d) were also cultured for 36 hours. Each of the subsequent observation results is shown. C in the figure represents a control. The circles in the figure represent each well, the circles in the circles indicate wells in which the growth of Aspergillus niger was observed, and the circles in the circles indicate growth inhibition of Aspergillus niger. Each observed well is represented.
From the results of the observation, the antifungal activity was not observed at all in the Y1 to Y3 strains as in the control, but the growth of Aspergillus niger was observed in the P1 to P10 strains. However, all of the P1 to P10 strains are apparently strains capable of producing an antifungal substance having antifungal activity at pH 7.0.

7. Identification of bacteria producing antifungal substance at pH 7.0 Among P1 to P10 strains, identification of P9 strain (hereinafter referred to as O9-NP9 strain) and P5 strain (hereinafter referred to as O9-NP5 strain) was attempted.

7-1. Identification of O9-NP9 strain First, in order to examine the genetic properties, the base sequence of 26S rDNA of the O9-NP9 strain was identified by a conventional method. Specifically, using DNA extracted from the culture solution of O9-NP9 strain and NL1, NL2, NL3 and NL4 primers (O'Donnell, Fusarium and its near relatives, CAB International, p225-233, 1993) The base sequence of the 26S rDNA-D1 / D2 region was identified. For DNA extraction, DNeasy Plant Mini Kit (manufactured by QIAGEN) is used, and for PCR, puReTaq Ready-To-Go PCR beads (manufactured by GE Healthcare Biosciences) and BigDye Terminator v3.1 Cycle Sequen BioPly ABI PRISM 3100 Genetic Analyzer System (Applied Biosystems) was used to identify the base sequence.
When the homology search was performed on the international nucleotide sequence database (GenBank / DDBJ / EMBL), NRRL, which is a reference strain of Candida maltosa, a kind of ascomyceous mycelium anamorph (asexual era) yeast. It showed 100% homology with Y-17777T (accession number: U45745). Therefore, the O9-NP9 strain was estimated to be a microorganism belonging to Candida maltosa.

  The mycological properties of the O9-NP9 strain are shown below. The bacteriological properties were measured by using the O9-NP9 strain cultured on a YM agar medium (Becton Dickinson) at 25 ° C. for 2 days to 1 month. In addition, Yeast: Characteristics and identification, 3rd edition (Barnett et al, Cambridge University), sugar fermentability test, carbon source utilization test, nitrogen source utilization test, vitamin requirement test, temperature tolerance test, drug resistance test Press, 2000) and The Yeast, a taxonomic study, 4th edition (Kurtzman et al, Elsevier, 1998).

Mycological properties (1) Colony observation (at the time of visual observation when cultured on a YM agar plate at 25 ° C. for 4 days)
Peripheral shape: almost all edges, raised state: flat, surface shape: smooth, glossy and properties: weak gloss, wetness, color: white to cream color (2) cultural and morphological properties (with YM agar plate) (At the time of observation with an optical microscope at 25 ° C. for 2 days)
Vegetative cell size and shape: from 3 to 6 μm diameter oval to cylindrical growth format: multipolar budding hyphae, etc .: formation of pseudohyphae (3) formation of sexual reproductive organs (25 ° C. on YM agar plate) (When observed with an optical microscope when cultured for 1 month): None

(7) Growth at 37 ° C .: Positive (8) Growth at 40 ° C .: Negative (9) Growth in 0.01% cycloheximide-containing medium: Positive (10) Medium containing 10% sodium chloride and 5% glucose (11) Growth on vitamin-deficient medium: negative (12) Formation of starch-like substance: negative (13) Decomposition of urea: negative

  FIG. 3 is an electron micrograph of O9-NP9 strain cultured on a YM agar plate at 25 ° C. for 2 days.

  O9-NP9 strains are galactose fermentable, inositol, erythritol, raffinose, cellobiose, melezitose, galactose, lactose, soluble starch, L-rhamnose, DL-lactate, xylitol, and nitrate assimilation, vitamin-deficient medium, 0. It showed bacteriological properties consistent with Candida maltosa in 01% cycloheximide medium at 37 ° C and 40 ° C. On the other hand, Candida maltosa was supposed to ferment trehalose, whereas the O9-NP9 strain did not show trehalose fermentability.

  From these genetic and mycological properties, it was confirmed that the O9-NP9 strain is a Candida maltosa strain. The base sequence of the 26S rDNA-D1 / D2 region was 100% homologous to the Candida maltosa NRRL Y-17777T strain, but because of the difference in trehalose fermentability, the Candida maltosa strain O9-NP9 was It is clear that it has different properties from the maltosa NRRL Y-17777T strain.

  Therefore, the applicant deposited the Candida maltosa O9-NP9 strain at the Patent Microorganism Deposit Center, Product Evaluation Technology Foundation (Deposit date: January 10, 2007). The accession number is NITE BP-297.

7-2. Identification of O9-NP5 strain First, in order to investigate the genetic properties, the base sequence of 26S rDNA of the O9-NP5 strain was identified in the same manner as the O9-NP9 strain. When a homology search was performed on the international nucleotide sequence database (GenBank / DDBJ / EMBL), the nucleotide sequence was 100% homologous to Clavispora lucitanie MTCC1001 (accession number: AF538871), which is a kind of ascomycomycelium. However, it showed 93.8% homology with Clavispora lucitanie NRRL Y-11827T (accession number: U44817), which is a reference strain of Clavispora lucitanie. In addition, there was no known type of reference strain showing 99% or more homology with the base sequence.
The mycological properties of the O9-NP5 strain are shown below. The bacteriological properties were measured in the same manner as for the O9-NP9 strain.

Mycological properties (1) Colony observation (at the time of visual observation when cultured on a YM agar plate at 25 ° C. for 4 days)
Peripheral shape: full edge, raised state: cushion shape, surface shape: smooth, glossy and properties: butter-like, wet, color: white to cream color (2) cultural and morphological properties (25 on YM agar plate) (At the time of observation with an optical microscope when cultivated at ℃ for 2 days)
Size and shape of vegetative cells: Spherical to wide elliptical or oblate circular shape with a diameter of 2 to 6 μm: Multipolar budding hyphae, etc .: Formation of pseudohyphae (3) Formation of sexual reproductive organs (YM agar plate) At 25 ° C. for 1 month and observed with an optical microscope): None

(7) Growth at 37 ° C .: Positive (8) Growth at 40 ° C .: Positive (9) Growth in 10 μg / mL cycloheximide-containing medium: Positive (10) In 10% sodium chloride and 5% glucose-containing medium Growth: Positive (11) Growth in a medium containing 50% glucose: Positive (12) Growth in a vitamin-deficient medium: Negative (13) Formation of starch-like substance: Negative (14) Degradation of urea: Negative (15) Gelatin liquefaction: negative

  The O9-NP5 strain exhibited glucose fermentability, did not exhibit nitrate assimilation, and exhibited bacteriological properties consistent with those of the genus Clavispora. Moreover, in almost all the characteristics, it showed a mycological property similar to that of Clavispora lucitanie, but showed a different mycological property from that of Clavispora lucitanie in the assimilation of raffinose, soluble starch, and hexadecane.

  From these genetic and mycological properties, it was confirmed that the O9-NP5 strain is a clavispora sp. In addition, because of low homology with the reference strain of Clavispora lucitanie and assimilability of raffinose and the like, Clavispora lucitanie O9-NP5 strain is a new strain of Clavispora lucitanie, or Clavispora lucitanie It was estimated to be a new bacterial species closely related to

Therefore, the applicant deposited Clavispora Lucitanie O9-NP5 strain as “Clavispora Spices P-5 strain” at the Patent Microorganism Depositary, National Institute of Technology and Evaluation ( Deposit date: January 16, 2008). Day). The accession number is NITE BP-474 .
In the present specification, “Clavispora lucitane O9-NP5 strain” and “Clavispora species P-5 strain” represent the same strain.

8). Obtaining Yeast Producing Yeast Antifungal Substance of the Present Invention In the same manner as when Candida maltosa strain O9-NP9 was isolated except that YM agar medium was used instead of MRS agar medium, One strain of yeast producing an antifungal substance was obtained from lot Caspian cheese. The one strain was named O9-H1 strain, and the bacteria were identified.

First, in order to examine the genetic properties, the base sequence of 26S rDNA of the O9-H1 strain was identified in the same manner as the O9-NP9 strain. When a homology search was performed on the international nucleotide sequence database (GenBank / DDBJ / EMBL), the base sequence of Saccharomyces cerevisiae, which is a kind of ascomycomycelium, was NRRL Y-12632T (accession number). : AY048154) and 100% homology. Therefore, the O9-H1 strain was estimated to be a microorganism belonging to Saccharomyces cerevisiae.
The mycological properties of the O9-H1 strain are shown below. The bacteriological properties were measured in the same manner as for the O9-NP9 strain.

Mycological properties (1) Colony observation (at the time of visual observation when cultured on a YM agar plate at 25 ° C. for 4 days)
Peripheral shape: almost all edges, raised state: central protrusion shape, surface shape: smooth, glossy and properties: butter-like, wet, color tone: bright cream color (2) cultural and morphological properties (with YM agar plate) (At the time of observation with an optical microscope when cultured at 25 ° C for 3 days)
Size and shape of vegetative cells: wide oval shape with a diameter of 3 to 8 μm to circular growth type: multipolar budding (3) Formation of sexual reproductive organs (optical when cultured on YM agar plate at 25 ° C. for 20 days (When observed under a microscope): Ascomb-like enlarged cells were observed, but no ascospore formation was observed.

(7) Growth at 30 ° C .: Positive (8) Growth at 37 ° C .: Positive (9) Growth at 40 ° C .: Positive (10) Growth at 1 μg / mL cycloheximide-containing medium: Positive (11) Growth in 10% sodium chloride and 5% glucose-containing medium: positive (12) Growth in 50% glucose-containing medium: positive (13) Growth in vitamin-deficient medium: negative (14) Formation of starch-like substance: negative (15) Urea decomposition: negative

  The O9-H1 strain showed rapid sugar fermentability such as glucose, galactose, sucrose, etc., did not show nitrate utilization, and showed bacteriological properties consistent with those of the genus Saccharomyces. Moreover, in almost all the characteristics, it showed mycological properties similar to Saccharomyces cerevisiae. On the other hand, the O9-H1 strain showed bacteriological properties different from Saccharomyces cerevisiae in that it did not ferment raffinose and showed inulin utilization.

  From these genetic and mycological properties, it was confirmed that the O9-H1 strain is a Saccharomyces cerevisiae species. The base sequence of the 26S rDNA-D1 / D2 region was 100% homologous to the Saccharomyces cerevisiae NRRL Y-12632T strain. However, since it differs in raffinose fermentation ability and inulin utilization, the Saccharomyces cerevisiae O9-H1 strain is It is apparent that Saccharomyces cerevisiae NRRL Y-12632T has different properties.

Therefore, the applicant deposited the Saccharomyces cerevisiae O9-H1 strain as “Saccharomyces cerevisiae H-1 strain” at the Patent Microorganism Depositary, National Institute of Technology and Evaluation ( Deposited date: January 16, 2008). Day). The accession number is NITE BP-473 .
In the present specification, “Saccharomyces cerevisiae O9-H1 strain” and “Saccharomyces cerevisiae H-1 strain” represent the same strain.

  The antifungal activity in the present invention means an activity of inhibiting spore germination among the activities of suppressing the growth of fungi such as mold and yeast. By inhibiting spore germination, generation and growth of mold and the like can be effectively prevented.

  The antifungal substance of the present invention is selected from the group consisting of Candida spp., Saccharomyces spp., Pichia spp., Rhodotorula spp., Clavispora spp. It is an antifungal substance that inhibits spore germination produced by one yeast.

  The antifungal substance of the present invention has (1) an antifungal activity at pH 7.0, and (2) a culture solution obtained by culturing the yeast in a 10% (w / w) skim milk medium. And (3) when the supernatant of the culture medium obtained by culturing the yeast in 10% (w / w) skim milk medium is ultrafiltered using an ultrafiltration membrane having a MWCO of 10 kD, It is characterized by being held on the ultrafiltration membrane surface. Further, (4) the antifungal activity may be deactivated by a heat treatment at 55 ° C. for 15 minutes.

  The Candida spp. Producing the antifungal substance of the present invention is preferably Candida maltosa, Candida tropicalis, or Candida pseudointermedia, and particularly preferably Candida maltosa. . Examples of the Candida maltosa include Candida maltosa O9-NP9 strain, Candida maltosa IAM12247, and Candida maltosa IAM12248. Examples of the Candida tropicalis include Candida tropicalis IAM4965. Examples of the Candida pseudo intermedia include Candida pseudo intermedia IAM12510.

  The Pichia spp. Producing the antifungal substance of the present invention is preferably Pichia fermentas, and particularly preferably Pichia fermentas JCM1824. Further, Rhodotorula genus producing the antifungal substance of the present invention is preferably Rhodotorula acta, particularly preferably Rhodotorula acta JCM9494. The Saccharomyces spp. Producing the antifungal substance of the present invention is preferably Saccharomyces cerevisiae, and particularly preferably Saccharomyces cerevisiae H-1. In addition, the clavispora spp. Producing the antifungal substance of the present invention is preferably clavispora lucitanie, and particularly preferably clavispora species p-5 or clavispora lucitanie NBRC10059. Moreover, it is preferable that the Torula spora genus microbe which produces the antifungal substance of this invention is a Torulas pora delbrecky, and it is especially preferable that it is a Torulas pora delbrucky NBRC0955.

  The yeast of the present invention, that is, the Candida spp., Saccharomyces spp., Pichia spp., Rhodotorula spp., Clavispora spp. And Torlaspora spp. The culture conditions such as culture medium and temperature are not particularly limited as long as they are conditions for culturing yeasts such as Candida. Examples of the medium include YM medium and 10% (w / w) skim milk medium. Since there is little possibility that the activity of the antifungal substance of the present invention is inhibited and the purification of the antifungal substance becomes simple, a 10% (w / w) skim milk medium is preferable. The culture temperature is preferably 27 to 30 ° C.

  The antifungal substance of the present invention can be obtained by culturing the yeast of the present invention. For example, the antifungal substance of the present invention is present in a supernatant obtained by centrifuging a culture solution of the yeast of the present invention. The conditions for the centrifugation treatment are not particularly limited as long as the solid components such as the cells in the culture solution can be removed. However, in order to avoid contamination with other substances derived from the cells, 2 Centrifuging is preferably performed at a low speed of 500 to 5,000 × g. Moreover, low molecular impurities contained in the supernatant can be removed by subjecting the supernatant to ultrafiltration using an ultrafiltration membrane having a MWCO of 10 kD. The supernatant thus obtained can be used as an antifungal substance-containing solution of the present invention as an antifungal agent or a food preservative.

  The method for inhibiting the growth of the mold and / or yeast of the present invention is a method using the antifungal substance of the present invention or the yeast of the present invention, wherein the antifungal activity of the antifungal substance of the present invention is obtained. If it is, it will not specifically limit. As a method of using the antifungal substance of the present invention, for example, a method of adding the culture supernatant of the yeast containing the antifungal substance as a raw material to a food or the like, or spraying or There is a method of applying. Since the antifungal substance of the present invention is inactivated by heat treatment, a method of spraying or applying to the manufactured food is preferable. Moreover, as a method using the yeast of this invention, there exists the method of fermenting, for example, after adding the yeast of this invention to a raw material in manufacture of fermentable foodstuff. The fermentable food is preferably a fermentable food that does not include heat treatment.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Reference Example 1]
Preparation of Aspergillus niger spore suspension Aspergillus niger NBRC 9455 strain was applied to a plate medium of PDA (potato dextrose agar) and then cultured at 28 ° C. for 3 days, and 0.05% tween 80 was added. It was suspended in physiological saline, and the suspension was subjected to cotton filtration and then stored frozen at -80 ° C. The Aspergillus niger NBRC 9455 strain after the cryopreservation was diluted with physiological saline, and then a 1.0 × 10 ⁴ spores / mL Aspergillus niger spore suspension was prepared using Sabouraud medium.

[Example 1]
Candida maltosa strain O9-NP9 was inoculated into 10% (w / w) skim milk medium and cultured at 28 ° C. for 48 hours to prepare a culture solution. The culture solution was sonicated to disperse the cells uniformly, and then centrifuged at 3,000 × g for 10 minutes to obtain a supernatant.
The supernatant was adjusted to 7.0 with a 5% (w / v) aqueous sodium carbonate solution and then subjected to ultrafiltration using an ultrafiltration membrane having a MWCO of 10 kD. The solution held on the surface of the ultrafiltration membrane was used as an antifungal substance solution.

  Ten 1.5 mL tubes into which 1 mL of the antifungal substance solution was dispensed were prepared. The tube was heat-treated at 10, 30, 40, 45, 50, 55, 60, 80, and 90 ° C. for 15 minutes, and then cooled with ice. To each of the ice-cooled tubes, 100 μL of an antibacterial agent (Penicillium GK: 0.7 mg / mL, kanamycin: 60 μg / mL, streptomycin sulfate: 0.1 mg / mL) was added and mixed. Thereafter, 252 μL of the antifungal substance solution in each tube and 28 μL of Sabouraud medium were mixed to prepare an antifungal substance solution for activity measurement.

To each well of a 96-well microplate, a two-fold serial dilutions of the adjusted antifungal substance solution (2 ^{0-2 9} times) were each added in 130 [mu] L. A Sabouraud medium was used for dilution of the antifungal substance solution. Specifically, the antifungal substance solution is added to the first row of a 96-well microplate, the 2-fold dilution of the antifungal substance solution is added to the second row, and the third row A 4 (2 ² ) -fold diluted solution of the antifungal substance solution was added. In each row, a 2-fold serial dilution was added to the wells, and finally in the 10th row, a 512 (2 ⁹ ) dilution of the antifungal substance solution was added. As a control, only 130 μL of Sabouraud medium was added. Furthermore, 50 μL each of 1.0 × 10 ⁴ sports / mL Aspergillus niger spore suspension prepared by the method described in Reference Example 1 was added to each well. The 96-well microplate was cultured at 28 ° C. for 48 hours, and then each well was observed with a microscope.

Table 4 shows the results of microscopic observation of the wells with the lowest antifungal substance concentration among the wells in which bacterial growth suppression was observed in the 2-fold serial dilution series of each temperature-treated antifungal substance solution. The dilution factor, that is, the maximum dilution factor is shown. In the table, the description of "2 ^4", a solution of 2 ^4-fold dilution following dilution, such as anti-mycotic material solution, i.e., in 2 ⁰ 21 ^{to 24} times dilution wells were added, the growth inhibition of bacteria Is observed. Moreover, the descriptions in Table "2 ^9", a solution of 2 ^9-fold dilution following dilution, such as anti-mycotic material solution, i.e., in addition to wells of all dilutions of this embodiment, the growth of bacteria It shows that suppression was observed. In addition the table, "-" described indicates that no activity was observed in the wells with the addition of 2 ^0-fold dilutions (undiluted).
As is apparent from Table 4, in each temperature-treated antifungal substance solution at 10 ° C. and 40 ° C., inhibition of bacterial growth was observed in the wells to which all the diluted solutions of this example were added. Thus, in each temperature treated antifungal substance solution of 10 ° C. and 40 ° C., even 2 ⁹ magnification or diluent, may proliferation of bacteria was inhibited, have a high antifungal activity It is clear. On the other hand, it was found that when the heat treatment temperature exceeds 40 ° C., the antifungal activity suddenly decreases and is deactivated by heat treatment at 55 ° C. for 15 minutes.

  That is, from the results of Example 1, the antifungal substance of the present invention is produced by yeasts such as Candida spp. And has antifungal activity at pH 7.0 and 10% (w / w w) When the yeast is cultured in a skim milk medium, the supernatant of the culture liquid secreted into the culture medium and cultured in 10% (w / w) skim milk medium is ultrafiltered with a MWCO of 10 kD. When ultrafiltration is performed using a membrane, it is apparent that the antifungal activity is inactivated by heat treatment at 55 ° C. for 15 minutes, which is retained on the surface of the ultrafiltration membrane. is there.

[Example 2]
The antifungal activity of the antifungal substance of the present invention against various fungi was measured. Cycloheximide, a protein synthesis inhibitor for eukaryotic cells, was used as a positive control for antifungal activity.
First, an antifungal substance solution was prepared from the culture solution of Candida maltosa strain O9-NP9 in the same manner as in Example 1 except that no heat treatment was performed. Further, 1.0 × 10 ⁴ spores / mL spore suspensions of 18 strains described in Table 5 were prepared in the same manner as in Reference Example 1.

In the same manner as in Example 1, in 96-well microplates were prepared antifungal substance solution or 500ppm cycloheximide solution to prepare 2-fold serial dilutions (2 ^{0-2 9} times). Furthermore, 50 μL of each spore suspension per well was added to each 2-fold serial dilution series. After the 96-well microplate was cultured at 28 ° C. for 48 hours, it was observed with a microscope whether or not the growth of the bacteria was suppressed in each well.

  Table 5 shows the dilution ratio of the well having the lowest antifungal substance concentration among the wells in which bacterial growth suppression was observed in the 2-fold serial dilution series of each antifungal substance solution, as in Example 1. That is, the maximum dilution rate is shown.

The susceptibility of 18 strains listed in Table 5 to cycloheximide was different, but growth suppression was observed in all strains by adding a 500 ppm cycloheximide solution. On the other hand, the antifungal substance solution of the present invention was observed in all strains at a dilution ratio larger than that of the 500 ppm cycloheximide solution, and bacterial growth suppression was observed.
From the results of Table 5, the antifungal substance of the present invention has antifungal activity against various fungi, and the antifungal substance solution of the present invention prepared by the method described in Example 2 Apparently has higher antifungal activity than 500 ppm cycloheximide solution.

[Example 3]
Whether Candida sp. Other than Candida maltosa O9-NP9 strain and other yeasts produced the antifungal substance of the present invention produced by Candida maltosa O9-NP9 strain was examined. Cycloheximide was used as in Example 2 as a positive control for antifungal activity.
First, in the same manner as in Example 2, antifungal substance solutions were prepared from the yeast culture fluid described in Table 6, respectively. Further, in the same manner as in Reference Example 1, a 1.0 × 10 ⁴ sports / mL Aspergillus niger spore suspension was prepared.

In the same manner as in Example 2, in 96 well microplate, the antifungal substance solution or 500ppm of cycloheximide solution of the yeast prepared, to prepare 2-fold serial dilutions (2 ^{0-2 9} times). Further, 50 μL of Aspergillus niger spore suspension per well was added to each 2-fold serial dilution series. After the 96-well microplate was cultured at 28 ° C. for 48 hours, it was observed with a microscope whether or not the growth of the bacteria was suppressed in each well.

Table 6 shows the dilution of the well with the lowest antifungal substance concentration among wells in which fungal growth suppression was observed in the 2-fold serial dilution series of the antifungal substance solution of each yeast, as in Example 2. The magnification, that is, the maximum dilution magnification is shown. In the table, "-" described are like the Table 4, indicate that the activity was not observed in the wells with the addition of 2 ^0-fold dilutions (undiluted).

  From the results of Table 6, some yeasts other than the Candida maltosa O9-NP9 strain were observed to suppress the growth of Aspergillus niger as in the case of the Candida maltosa O9-NP9 strain. It became clear that the substance was produced. In particular, Candida maltosa IAM12247 and Candida maltosa IAM12248 also had significantly higher antifungal activity than other strains, as in Candida maltosa O9-NP9 strain. That is, it has been clarified that Candida maltosa has a very superior ability to produce the antifungal substance of the present invention compared to other yeasts. In addition, the antifungal substance solution of Candida tropicalis IAM4965 also had higher antifungal activity than the 500 ppm cycloheximide solution. In addition, among Candida spp., Candida pseudointermedia IAM12510 also had antifungal activity. In addition to Candida spp., Pichia fermentas JCM1824 and Rhodotorula acta JCM9494 were found to have antifungal activity.

[Example 4]
In addition to the yeast examined in Example 3, it was examined whether or not more yeasts produced the antifungal substance of the present invention produced by the Candida maltosa strain O9-NP9. Specifically, all were carried out in the same manner as in Example 3 except that the yeasts described in Tables 7 and 8 were used.

As in Example 3, Tables 7 and 8 show the wells with the lowest antifungal substance concentration among the wells in which the growth inhibition of the fungus was observed in the 2-fold serial dilution series of the antifungal substance solution of each yeast. The dilution ratio, that is, the maximum dilution ratio is shown. In the table, the description of “-” is the same as in Table 6.
From the results of Tables 7 and 8, it was found that among the Candida spp., Some fungi such as Candida saitana JCM1438 also have antifungal activity.

[Example 5]
The antifungal activities of Candida maltosa O9-NP9 strain, Saccharomyces cerevisiae H-1 strain, and Clavispora species P-5 strain were compared.
First, in the same manner as in Example 2, antifungal substance solutions were prepared from the culture solutions of the three strains. Further, in the same manner as in Reference Example 1, a 1.0 × 10 ⁴ sports / mL Aspergillus niger spore suspension was prepared.

In the same manner as in Example 2, in 96 well microplate, the antifungal substance solution of the 3 strains were prepared 2-fold serial dilutions (2 ^{0-2 9} times). Further, 50 μL of Aspergillus niger spore suspension per well was added to each 2-fold serial dilution series. After the 96-well microplate was cultured at 28 ° C. for 48 hours, it was observed with a microscope whether or not the growth of the bacteria was suppressed in each well.

Results of microscopic observation, the Candida maltosa O9-NP9 strain, be a well added with 2 ^9-fold dilutions of antifungal substance solution, Aspergillus niger growth inhibition was observed. On the other hand, in the Saccharomyces cerevisiae H-1 strain and Kurabisupora sp P-5 strain, the wells with the addition of 2 ⁰ 21 to ^24-fold dilutions of the antifungal substance solution only, Aspergillus niger growth inhibition was observed .

  From the results of Example 5, Saccharomyces cerevisiae H-1 strain and Clavispora sp. P-5 strain produce the antifungal active substance of the present invention in the same manner as the Candida maltosa O9-NP9 strain, and Among the three strains, it is clear that the Candida maltosa strain O9-NP9 has the highest ability to produce the antifungal substance of the present invention.

[Example 6]
It was investigated whether Saccharomyces spp. And related bacteria produced the antifungal substance of the present invention produced by Candida maltosa strain O9-NP9. Cycloheximide was used as in Example 2 as a positive control for antifungal activity.
First, in the same manner as in Example 2, antifungal substance solutions were prepared from the yeast culture fluid described in Table 9. Further, in the same manner as in Reference Example 1, a 1.0 × 10 ⁴ sports / mL Aspergillus niger spore suspension was prepared.

In the same manner as in Example 2, in 96 well microplate, the antifungal substance solution or 500ppm of cycloheximide solution of the yeast prepared, to prepare 2-fold serial dilutions (2 ^{0-2 9} times). Further, 50 μL of Aspergillus niger spore suspension per well was added to each 2-fold serial dilution series. After culturing the 96-well microplate at 28 ° C. for 3 days or 7 days, it was observed with a microscope whether or not the growth of bacteria in each well was suppressed.
Table 9 shows the dilution of the well with the lowest antifungal substance concentration among the wells in which the inhibition of bacterial growth was observed in the 2-fold serial dilution series of the antifungal substance solution of each yeast, as in Example 2. The magnification, that is, the maximum dilution magnification is shown. In the table, "-" described, as in Table 4, in the wells with the addition of 2 ^0-fold dilutions (undiluted) indicates that the activity was not observed, "ND" was not determined It means that. In the table, “after 3 days” and “after 7 days” indicate the results after 3 days of culture or after 7 days of culture, respectively.

From the results of Table 9, it is clear that some yeasts other than the Saccharomyces cerevisiae H-1 strain also inhibit the growth of Aspergillus niger, and these yeasts produce the antifungal substance of the present invention. became. In particular, after 3 days in culture, Saccharomyces cerevisiae NBRC0216 and Saccharomyces cerevisiae NBRC10181 had significantly higher antifungal activity than other strains, as did Saccharomyces cerevisiae H-1. In addition, Torlaspola del Brukki NBRC0955 ( Deposited with the Patent Microorganism Depositary Center for Product Evaluation Technology, Incorporated Administrative Agency. Deposit No. NITE BP-476 , date of deposit January 16, 2008), Saccharomyces pastrianus NBRC11024 Kluyveromyces lactis var. Drosophilarum NBRC1012, Kluyveromyces dobhanskii NBRC10603 also had high antifungal activity. In particular, it was revealed that Torlospola delbruecki NBRC0955 has a higher antifungal activity than cycloheximide even after 7 days of culture and is very superior.

[Example 7]
Whether Clavispora spp. And related bacteria produced the antifungal substance of the present invention produced by Candida maltosa strain O9-NP9 was examined.
Specifically, in place of the yeast described in Table 9, except that the yeast described in Table 10 was used, in the same manner as in Example 6, by adding the antifungal substance solution of each yeast, Aspergillus It was observed with a microscope whether the growth of niger was suppressed.
Table 10 shows the dilution ratio of the well having the lowest antifungal substance concentration among the wells in which bacterial growth suppression was observed in the 2-fold serial dilution series of the antifungal substance solution of each yeast, that is, the maximum dilution ratio. Is shown. In the table, “-”, “ND”, “after 3 days”, and “after 7 days” are the same as in Table 9.

From the results shown in Table 10, the suppression of growth of Aspergillus niger was observed in some yeasts other than the Clavis spora spices P-5 strain, similar to the Candida maltosa O9-NP9 strain, and the antifungal substance of the present invention It became clear to produce. After 3 days of culture, Clavispora species P-5 and Clavispora lucitanie NBRC10059 had significantly high antifungal activity, similar to Candida maltosa O9-NP9. In addition, Clavispora lucitanie NBRC10058 also had higher antifungal activity than cycloheximide. In particular, Clavispora Lucitanie NBRC10059 ( deposited with the Patent Microorganism Depositary of the National Institute of Technology and Evaluation, Accession No. NITE BP-475 , date of deposit January 16, 2008) is a candy even after 7 days of culture. It has been revealed that it has a high antifungal activity as well as Da Martosa O9-NP9 and is very excellent.

[Example 8]
The antifungal activity of various antifungal substances of the antifungal substance of the present invention produced by Clavispora lucitanie NBRC10059 strain and Torrospora delbruecki NBRC0955 strain was measured.
Specifically, in place of the Candida maltosa O9-NP9 strain, the antifungal properties of each yeast were all the same as in Example 2, except that the Clavispora lucitanie NBRC10059 strain or the Trurospola delbruecki NBRC0955 strain was used. It was observed with a microscope whether or not the growth of the bacteria described in Table 11 was suppressed by adding the substance solution.
Table 11 shows the dilution ratio of the well having the lowest antifungal substance concentration among the wells in which bacterial growth suppression was observed in the 2-fold serial dilution series of the antifungal substance solution of each yeast, that is, the maximum dilution ratio. Is shown. In the table, “-”, “ND”, “after 3 days”, and “after 7 days” are the same as in Table 9.

  In all of the 13 strains described in Table 11, the Clavispora lucitanie NBRC10059 strain showed higher antifungal activity than cycloheximide. On the other hand, although some of the Torlospola delbruecki NBRC0955 strains did not exhibit the growth-inhibiting effect, depending on the type of fungi, they exhibited higher antifungal activity than cycloheximide.

[Example 9]
The heat resistance of the antifungal active substance possessed by each yeast was measured.
First, 1 L of PD (potato dextrose) medium inoculated with each yeast listed in Table 12 was cultured by shaking at 30 ° C. for 48 hours, centrifuged at 3,000 × g for 5 minutes, and the supernatant was obtained. It was collected. The supernatant was treated at each temperature of 50 ° C., 60 ° C., 85 ° C., and 90 ° C. for 10 minutes and then cooled to obtain an antifungal substance solution. Note that the supernatant that was not heat-treated (non-heat-treated) was used as a control.
In the same manner as in Example 2, it was observed with a microscope whether or not the growth of Aspergillus niger was suppressed by adding these antifungal substance solutions.
Table 12 shows the dilution ratio of the well having the lowest antifungal substance concentration among the wells in which bacterial growth suppression was observed in the 2-fold serial dilution series of each antifungal substance solution, that is, the maximum dilution ratio. It is a thing. In the table, "-" it is described, indicating that the activity was not observed in the wells with the addition of 2 ^0-fold dilutions (undiluted), the description of "2 ^9", the 2 ^9-fold dilution This shows that suppression of bacterial growth was also observed in the added well.
As a result, it was found that all the antifungal active substances possessed by the yeasts listed in Table 12 were inactivated by heat treatment at 60 ° C. for 10 minutes or more.

[Example 10]
The antifungal effect was observed when the antifungal substance of the present invention produced by Candida maltosa strain O9-NP9 was added to bread as a raw material.
First, 1 L of PD (potato dextrose) medium inoculated with Candida maltosa O9-NP9 strain was cultured at 30 ° C. for 3 days. The entire culture as antifungal substance solution, in the same manner as in Example 2, whether of Aspergillus niger growth is inhibited was observed with a microscope, added 2 ^9-fold dilutions of the culture broth In the wells, suppression of bacterial growth was also observed.
The culture broth was used as a raw material for bread instead of the total amount of water raw material. The breads to which the commercially available antibacterial agent Neocabin P (sold by JT Foods Co., Ltd.) was added and the breads to which no antibacterial agent was added were compared. Specifically, bread was produced by a conventional method with the blending amounts shown in Table 12. In the table, “%” means parts by weight (%) with respect to 100% strong powder.

  The produced bread was sliced into the usual thickness of 6 slices. Twenty pieces of each bread were left in the room for 10 minutes to allow bacteria to fall on the surface of the bread. Thereafter, each was put in a bag and kept at 28 ° C. for 5 days, and then the presence or absence of mold and how to grow were observed. Mold growth was divided into 4 categories where the number of colonies was 0, 1 to several tens, several tens or more, and counting was impossible. “Uncountable” means that the mold covered most of the surface and the number of colonies could not be measured.

Table 14 shows the result of categorizing 20 breads by observation in terms of the number of pieces for each category. As a result, all 20 types of bread had mold on all 20 sheets. In the bread without additive, there were many colonies per sheet and there were 6 that could not be counted. However, the bread with neocabine had few colonies per sheet and there was no one that could not be counted. . On the other hand, the bread with the culture solution of Candida maltosa O9-NP9 strain tended to be larger than the one with neocabine added, but the number of colonies per plate was much higher than that without additive There was only one sheet that could not be counted. In particular, in the culture solution-added bread, mold was hardly grown at the center of the bread, and many molds were grown only on the ears of the bread.
As described above, in the bread to which the culture broth was added, the mold was more on the outside of the bread and less on the inside, because the antifungal substance of the present invention was vulnerable to heating and was inactivated in the step of baking the bread. It is inferred that this has happened. That is, from these results, it is clear that the antifungal substance of the present invention can be expected to have an antibacterial effect in the same manner as conventional antibacterial agents when used as an additive to foods and the like that are not subjected to heat treatment.

[Example 11]
When the antifungal substance of the present invention produced by Candida maltosa strain O9-NP9 was applied to bread, the antifungal effect against Aspergillus niger was observed.
First, 1 L of PD medium inoculated with Candida maltosa O9-NP9 strain was cultured at 30 ° C. for 3 days, and the whole culture was used as an antifungal substance solution in the same manner as in Example 2 to prepare Aspergillus niger. It was observed with a microscope whether or not the growth of the cells was suppressed. As a result, the growth inhibition of fungus was observed in wells with the addition of 2 ^9-fold dilutions of the culture solution. This culture broth was applied to the whole of commercially available bread slices (San Aroma, manufactured by Yamazaki Baking Co., Ltd.) by spraying, and spread over the whole using a large stick.
Inoculate 0.5 ml of Aspergillus niger spore suspension (2.0 × 10 ⁴ spores / mL) to each of the bread with and without the application of the culture solution (control). And kept at 28 ° C. The Aspergillus niger spore suspension was prepared in the same manner as in Reference Example 1.
As a result, three days after the inoculation, the control bread was moldy and green, but no mold was grown on the bread coated with the culture solution. Furthermore, 5 days after the inoculation, black mold colonies were generally observed in the control bread, but only white hairy hyphae were observed in one place in the bread to which the culture solution was applied.
From these results, it is clear that the antifungal substance of the present invention exhibits a very good antifungal effect under non-heated conditions.

[Example 12]
When the antifungal substance of the present invention produced by Candida maltosa strain O9-NP9 was applied to bread, the antifungal effect against falling bacteria was observed.
Bread bread prepared by applying the culture solution of Candida maltosa O9-NP9 strain prepared in the same manner as in Example 11 and uncoated bread (control) were left in the room for 10 minutes each for 10 minutes. Falling bacteria were allowed to adhere on the surface. Thereafter, each was put into a bag and kept at 28 ° C. for 5 days, and then the presence or absence of mold was observed.
As a result, all of the 6 control breads had mold, but none of the breads to which the culture solution had been applied. Also from these results, it is clear that the antifungal substance of the present invention exhibits a very good antifungal effect under non-heated conditions.

[Example 13]
The influence of the culture conditions on the antifungal activity of the culture medium in which the yeast of the present invention was cultured was observed. Specifically, the influence of shaking culture and stationary culture on the antifungal activity of the culture solution was examined.
1 L PD medium inoculated with Candida maltosa strain O9-NP9 and culture medium obtained by shaking culture at 25 ° C. for 24 hours or 48 hours, and 1 L inoculated with Candida maltosa strain O9-NP9 The PD medium was prepared by stationary culture at 25 ° C. for 24 hours or 48 hours. These culture solutions were centrifuged at 3,000 × g for 5 minutes, separated into supernatant and precipitate, and collected. The supernatant thus obtained was directly used as an antifungal substance solution. On the other hand, for the precipitate containing the bacterial cells, a solution in which the bacterial cells were uniformly dispersed by adding ultrasonic water equivalent to the supernatant and sonicating was used as an antifungal substance solution.
The antifungal activity of each antifungal substance solution was measured by observing with a microscope whether or not the growth of Aspergillus niger was suppressed in the same manner as in Example 2.

Table 15 shows the dilution ratio of the well having the lowest antifungal substance concentration among the wells in which bacterial growth suppression was observed in the 2-fold serial dilution series of each antifungal substance solution, that is, the maximum dilution ratio. It is a thing. As a result, regardless of the culture time and culture conditions, the antifungal substance solution from precipitation, growth inhibition of bacteria observed in the wells with the addition of 2 ^9-fold diluted solution is the maximum dilution ratio of this experiment, high Has antifungal activity. In contrast, in the antifungal substance solution from the supernatant, the differences due to culture time without a lot bacteria even in both, antifungal substance solution from shaking culture wells with the addition of 2 ^8-fold dilution growth inhibition but was observed, the antifungal substance solution from static culture growth inhibition of the fungus was observed only up well with the addition of 2 ^two-fold dilutions. Moreover, in any culture condition, the antifungal substance solution derived from the precipitate had higher antifungal activity than the antifungal substance solution derived from the supernatant.
That is, it was found that the antifungal substance of the present invention is more easily transferred to a culture solution by shaking culture than stationary culture, and that the precipitate (bacteria) has higher antifungal activity than the supernatant. . Therefore, it was suggested that the antifungal substance of the present invention may be more easily secreted out of the yeast during the shaking culture than in the stationary culture. Moreover, since the antifungal activity in which precipitation was higher than that in the supernatant was detected, it is presumed that the antifungal substance of the present invention has the property of being easily adsorbed on the yeast surface.

[Example 14]
In order to investigate the property of the antifungal substance of the present invention, after the crude purification, the neutral sugar of the substance in the crude purified product was quantitatively analyzed.
First, 1 L of PD medium inoculated with Candida maltosa strain O9-NP9 was cultured with shaking at 30 ° C. for 24 hours, centrifuged at 3,000 × g for 5 minutes, and 7769.95 g of supernatant was obtained. It was collected. Some of the supernatant as an anti-mycotic material solution, in the same manner as in Example 2, whether of Aspergillus niger growth is inhibited was observed with a microscope, a 2 ^10-fold dilutions of the culture broth Bacterial growth inhibition was also observed in the added wells.
The remaining 417.86 g of the supernatant was subjected to ultrafiltration using an ultrafiltration module SEP0031 (MWCO: 3 kD, manufactured by Asahi Kasei Corporation). At this time, the solution (S1) which permeated | transmitted the ultrafiltration membrane was 314.87g, and the residue (R1) hold | maintained on this ultrafiltration membrane surface was 128.34g. After deionized water was added to R1 and stirred to 500 g, ultrafiltration was again performed using the ultrafiltration module SEP0031. At this time, the solution (S2) which permeated | transmitted the ultrafiltration membrane was 305.52g, and the residue (R2) hold | maintained on this ultrafiltration membrane surface was 138.87g. After adding deionized water to R2 and stirring to 500 g, ultrafiltration was performed again using the ultrafiltration module SEP0031. At this time, the solution (S3) which permeated | transmitted the ultrafiltration membrane was 402.36g, and the residue (R3) hold | maintained on this ultrafiltration membrane surface was 59.28g.
The obtained residue R3 was stirred as it was, and as an antifungal substance solution, as in Example 2, it was observed with a microscope whether or not the growth of Aspergillus niger was suppressed. Bacterial growth inhibition was observed in the wells to which ⁰ to ^25- fold diluted solution was added.

Furthermore, the quantitative analysis of the neutral sugar of the substance in the residue R3 was performed.
Specifically, first, 100 μL of a suspension obtained by uniformly stirring the residue R3 as an antifungal substance solution was collected in a glass test tube and dried under reduced pressure. To the obtained residue, 200 μL of trifluoroacetic acid (2 mol / L) was added, and after a vacuum sealed tube, hydrolyzed at 100 ° C. for 6 hours. The residue obtained by drying the hydrolyzate under reduced pressure was dissolved in 100 μL of purified water, and then filtered through a filter having a pore size of 0.22 μm, to obtain a measurement sample stock solution. The measurement sample stock solution diluted 100-fold or 1000-fold with purified water is used as a measurement sample, and is measured by a high performance liquid chromatograph (HPLC) method under the following conditions. Neutral sugars (rhamnose, ribose, mannose, arabinose, galactose) , Xylose, and glucose). The peaks were identified by comparison with each neutral sugar standard.
HPLC system: LC-9A system (Shimadzu Corporation)
Detector: Spectrofluorometer RF-10A _XL (Shimadzu Corporation)
Column: TSK-gel Sugar AXG 150 × 4.6 mmI. D. (Tosoh)
Column temperature: 70 ° C
Mobile phase: 0.5 M potassium borate buffer pH 8.7
Mobile phase flow rate: 0.4 mL / min
Post-column labeling: Reaction reagent: 1% arginine / 3% boric acid
Reaction reagent flow rate: 0.5 mL / min
Reaction temperature: 150 ° C
Detection wavelength: Ex. 320 nm, Em. 430nm

As a result, the residue R3 was found to contain about 1% glucose and a small amount of mannose. That is, since the residue R3 contains many saccharides, it is suggested that the antifungal substance of the present invention may be a compound containing a large amount of sugars such as glycoproteins and polysaccharides.
Further, from the analysis result of this neutral sugar, the point that it can be easily transferred to the culture medium by shaking culture rather than the stationary culture, and the result of Example 16 described later, as in Example 13, It is suggested that the fungal substance may have the property of being easily adsorbed on the yeast surface by interacting with the glycoprotein on the yeast surface via the neutral sugar that it has.

[Example 15]
500 μL of Candida maltosa O9-NP9 strain culture solution and 500 μL of 2.4 × 10 ⁶ sports / mL Aspergillus niger spore suspension were mixed and incubated at 28 ° C. for 24 hours. The Candida maltosa O9-NP9 strain culture solution was prepared by inoculating the Candida maltosa O9-NP9 strain into a 10% (w / w) skim milk medium and culturing at 28 ° C. for 48 hours. Further, Aspergillus niger spore suspension was prepared in the same manner as in Reference Example 1.
To 1 mL of this culture solution, 200 μL of a fluorescent dye 0.5 μM SYTOX-Green (manufactured by Takara Bio Inc.) was added and further incubated for 0.5 hours. Then, this culture solution was centrifuged at 3,000 × g for 5 minutes, and the supernatant was removed to remove the fluorescent dye. Distilled water was added to the resulting precipitate to suspend it, and then centrifuged again to remove the supernatant and wash the precipitate.
An additional 1 mL of distilled water was added to the precipitate to prepare a yeast / spore suspension. When this suspension was observed with fluorescence and transmitted light using a normal optical microscope, spores stained with SYTOX-Green were observed. Further, it was observed that the fluorescently stained spores were expanded to a size equal to or larger than that at the time of water absorption.
Here, SYTOX-Green is a dye that can specifically bind to DNA, but cannot pass through the cell membrane. Nevertheless, the spores of Aspergillus niger incubated with the Candida maltosa O9-NP9 strain were stained because of the action of the antifungal substance possessed by the Candida maltosa O9-NP9 strain. Presumed to have been damaged. In addition, the fact that the fluorescently stained spores were expanded suggests that the cell walls of the spores were damaged.
A mixture of 500 μL of 10% (w / w) skim milk medium mixed with 500 μL of 2.4 × 10 ⁶ spores / mL of Aspergillus niger spore suspension was incubated in the same manner, and SYTOX-Green was added. Stained spores were not observed. In addition, in place of Candida maltosa O9-NP9 strain, the one using Candida utilis in which the antifungal activity of the present invention was not observed was similarly incubated, and SYTOX-Green was added, No fluorescently stained spores were observed. These results also suggest that the spore staining was due to the antifungal substance of the present invention possessed by the Candida maltosa strain O9-NP9.

[Example 16]
The activity of the antifungal active substance of the present invention when cultured in an agar medium was examined.
First, PDA medium mixed with Candida maltosa strain O9-NP9 was cultured at 28 ° C. for 48 hours and then hollowed out using a sterilized mold, and a column in which Candida maltosa strain O9-NP9 was grown. The shape of PDA medium (hereinafter referred to as “yeast pour agar medium”) was prepared.
On the other hand, using the same type, a PDA medium mixed with an Aspergillus niger spore suspension prepared in the same manner as in Reference Example 1 was hollowed out, and a cylindrical PDA medium containing Aspergillus niger spores (hereinafter referred to as spore mixing) Agar medium).
These two types of agar medium are (a) a state in which a spore mixed agar medium is directly superimposed on a yeast pour agar medium, and (b) a spore sandwiched between wraps on a yeast pouch agar medium. In a state where the pour agar medium was overlaid, and (c) a spore mixed agar medium overlaid on the yeast pour agar medium with a dialysis membrane having a pore diameter of 14 kD, respectively. And cultured for 48 to 72 hours, and the state of the spores was observed.
As a result, spore growth was observed in the spore pour agar medium (b). On the other hand, no spore growth was observed in the spore mixed agar mediums (a) and (c).
A part of the cultured spore mixed agar medium was added to a liquid PD medium and cultured at 28 ° C. for 24 hours, and then the culture was smeared on a PDA plate medium and further cultured. As a result, yeast was growing on the PDA plate medium derived from the spore blended agar medium in (a), and mold did not grow. On the other hand, fungi grew on the PDA plate medium derived from the spore mixed agar medium in (b), but yeast could not be confirmed. Furthermore, mold was grown on the PDA plate medium derived from the spore mixed agar medium in (c), but yeast could not be confirmed.
From these results, in (b), since two agar media were sandwiched between wraps, the Aspergillus niger spores in the spore-blended agar were not affected by the yeast-blended agar, and the spores grew. However, in (a), it is considered that the growth of Aspergillus niger spores was inhibited as a result of the growth of Candida maltosa strain O9-NP9 transferred to the spore pour agar medium. In contrast, in (c), the dialysis membrane prevented the yeast from moving to the spore-mixed agar medium as in (b), but the antifungal substance of the present invention produced from the yeast was dialyzed. It was considered that the growth of Aspergillus niger spores was inhibited because of the passage through the membrane to the spore mixed agar medium, and as a result, no spore growth was observed on the spore mixed agar medium. On the other hand, since the spores originally poured into the spore mixed agar medium did not die, it is considered that mold grew on the PDA plate medium derived from the spore mixed agar medium of (c).
Also from these results, it is clear that the Candida maltosa O9-NP9 strain produces the antifungal substance of the present invention.

  The antifungal substance of the present invention is safe because it is produced by cheese-derived yeast, and exhibits antifungal activity in neutrality. Therefore, it can be used particularly in the food field where mold growth is a problem.

A 2-fold serial dilution series of a sample solution derived from the O-9 fungus group culture solution and an Aspergillus niger spore suspension were added to each well of a 96-well microplate and cultured at 28 ° C. for 48 hours. The result of having observed each well with the microscope is shown. Lane A in the figure shows the dilution series of the supernatant sample solution derived from the O-9 fungus group, Lane B shows the dilution series of the precipitate sample solution derived from the O-9 fungal group, and Lane C shows the control. Yes. ○ in the figure represents each well, x in ○ indicates a well in which Aspergillus niger growth was observed, ○ in ○ indicates inhibition of Aspergillus niger growth Each well is represented. A 2-fold serial dilution series of a supernatant sample solution derived from a culture solution obtained by culturing P1 to P10 strains and Y1 to Y3 strains, respectively, and an Aspergillus niger spore suspension were added to each well of a 96-well microplate. The result of having observed each well with the microscope after culture | cultivation is shown. (A) and (b) are the results of observing each well with a microscope after culturing the 96-well microplate at 28 ° C. for 48 hours, and (c) and (d) are the observations after culturing for 36 hours. Each result is shown. ○ in the figure represents each well, x in ○ indicates a well in which Aspergillus niger growth was observed, ○ in ○ indicates inhibition of Aspergillus niger growth Each well is represented. It is an electron micrograph of O9-NP9 strain cultured at 25 ° C. for 2 days on a YM agar plate.

Claims (8)

Food-derived Candida maltosa O9-NP9 (Accession number NITE BP-297), Saccharomyces cerevisiae H-1 (Accession number NITE BP-473), Clavispora sp. P-5, Accession number NITE BP-474), Candida pseudointermedia IAM12510, Pichia membranifacens JCM1455, Pichia Nakasei Azia Actor (Rhodotorula acuta) JCM949 4. Selected from Filibasidium capsuligenum JCM 2273, Candida terebra JCM9566, N from Saccharomyces cerevisiae NBRC0216 An antifungal substance that inhibits the germination of mold spores produced by the yeast of
(1) having antifungal activity at pH 7.0,
(2) the culture solution obtained by culturing the yeast in 10% (w / w) skim milk medium has antifungal activity;
(3) The supernatant of the culture solution obtained by culturing the yeast in 10% (w / w) skim milk medium was subjected to ultrafiltration using an ultrafiltration membrane having a MWCO (Molecular Weight Cut Off) of 10 kD. In that case, it is retained on the surface of the ultrafiltration membrane,
(4) The antifungal activity is deactivated by heat treatment at 55 ° C. for 15 minutes. Fungicide containing an antifungal substance of claim 1 Symbol placement. Food preservative containing antifungal agent according to claim 1 Symbol placement. Method of inhibiting the growth of fungi, which comprises using an anti-mycotic material of claim 1 Symbol placement. Characterized by using the foodborne yeast to produce anti-mycotic material of claim 1 Symbol placement, a method of inhibiting the growth of mold.   Candida maltosa O9-NP9 (accession number NITE BP-297), which produces the antifungal substance according to claim 1. Claim 1 Saccharomyces cerevisiae H-1, characterized in that to produce the antifungal substance described (consignment number NITE BP -473). Claim 1 Kurabisupora sp P-5, characterized in that to produce the antifungal substance described (consignment number NITE BP -474).

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