Method for producing dry-aged meat and novel filamentous fungal strain used therein

Abstract

To provide a method for producing aged meat having a flavor with high palatability, which is enriched in 1-hexanol among flavors expressed as the nut flavor.SOLUTION: A method for producing dry-aged meat includes the steps of: attaching a culture of filamentous fungi onto the surface of meat, the filamentous fungi being at least one selected from Helicostylum pulchrum KT1b strain (accession number: NITE P-03423) and Mucor flavus KT1a strain (accession number: NITE P-03422); and dry-aging the meat having the culture of filamentous fungi attached thereto.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing dry-aged meat and a novel filamentous fungus used in the method. [Background technology]

[0002] Aging is a processing method that can enhance the flavor, tenderness, and other palatability of meat, such as beef, by storing it at refrigerated temperatures for a certain period of time. Aging can be broadly divided into wet aging and dry aging. Wet aging is performed by storing vacuum-packed meat in a refrigerator, and allows for aging while avoiding microbial contamination and lipid oxidation. In contrast, dry aging is performed by storing meat in a refrigerator under air. In dry aging, filamentous fungi and yeasts present in the environment naturally attach and grow on the surface of the meat, and it is known that these microorganisms produce various substances such as proteolytic enzymes and lipolytic enzymes, which promote the aging process.

[0003] Dry aging produces a unique aroma (aged aroma) in meat, and as such, demand for this method is expanding as a method for producing highly palatable aged meat. Aged aromas are variously described, such as meaty, buttery, nutty, floral, mushroom-like, or earthy. Aroma components involved in aged aroma include nucleic acids, reducing sugars, amino acids, volatile alcohols, and volatile aromatic compounds, which are thought to be produced primarily by the action of microorganisms involved in aging (e.g., Non-Patent Documents 1 and 2).

[0004] In particular, a nutty aroma (nutty aroma) is a preferred aroma in dry-aged meat. Nutty aroma components are known to include 2-acetyl-1-pyrroline, 3-methylbutanal (isovaleraldehyde), 1-hexanol, and several other compounds. Mucor strictus, a type of filamentous fungus, has been reported to be involved in the nutty aroma of dry-aged meat (Non-Patent Document 2). However, the mechanism by which the nutty aroma is generated in dry-aged meat has not been elucidated, and the relationship between specific aroma components and the microorganisms that produce them is also unknown. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Lee et. al., Meat Science (2019) 153: 152-158. [Non-patent document 2] Nakagawa et al., FY2019 (Reiwa 1) Meat Research Grant Survey Results Report (Vol. 38), pp. 20-25, published November 2020, Ito Memorial Foundation, Public Interest Incorporated Foundation DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for producing aged meat having a highly palatable flavor, particularly aged meat enriched in 1-hexanol, which has a flavor described as a nutty aroma. [Means for solving the problem]

[0007] The present inventors have found that aged meat enriched in 1-hexanol can be produced by inoculating meat with a newly isolated and identified microorganism and carrying out dry aging, and have completed the following invention.

[0008] (1) A method for producing dry-aged meat, comprising attaching a culture of at least one filamentous fungus selected from the group consisting of Helicostylum pulchrum KT1b strain (Accession Number: NITE P-03423) and Mucor flavus KT1a strain (Accession Number: NITE P-03422) to the surface of meat, and dry-aging the meat to which the culture of the filamentous fungus has been attached. (2) The method according to (1), in which a culture of Helicostylum pulchrum KT1b strain (accession number: NITE P-03423) and Mucor flavus KT1a strain (accession number: NITE P-03422) is attached to the surface of meat. (3) The method according to (1) or (2), wherein the culture of the filamentous fungus is attached to the surface of the meat by rubbing it against the surface of the meat. (4) The method according to any one of (1) to (3), wherein the culture of the filamentous fungus is a spore. (5) The method according to any one of (1) to (4), wherein the dry-aged meat is dry-aged meat enriched in 1-hexanol. (6) A method for promoting 1-hexanol production in dry-aged meat, comprising attaching to the surface of meat a culture of at least one filamentous fungus selected from the group consisting of Helicostylum pulchrum KT1b strain (accession number: NITE P-03423) and Mucor flavus KT1a strain (accession number: NITE P-03422), and dry-aging the meat with the culture of the filamentous fungus attached. (7) The method according to (6), in which a culture of Helicostylum pulchrum KT1b strain (accession number: NITE P-03423) and Mucor flavus KT1a strain (accession number: NITE P-03422) is attached to the surface of meat. (8) The method according to (6) or (7), wherein the culture of the filamentous fungus is attached to the surface of the meat by rubbing it against the surface of the meat. (9) The method according to any one of (6) to (8), wherein the culture of the filamentous fungus is a spore. (10) Helicostylum pulchrum KT1b strain, a novel filamentous fungus strain (accession number: NITE P-03423). (11) Mucor flavus KT1a strain, a novel filamentous fungus strain (accession number: NITE P-03422). (12) A kit for producing dry-aged meat, comprising a culture of at least one filamentous fungus selected from the group consisting of Helicostylum pulchrum KT1b strain (accession number: NITE P-03423) and Mucor flavus KT1a strain (accession number: NITE P-03422). (13) The kit according to (12), comprising a culture of Helicostylum pulchrum KT1b strain (accession number: NITE P-03423) and Mucor flavus KT1a strain (accession number: NITE P-03422). (14) The kit according to (12) or (13), wherein the culture of the filamentous fungus is a spore. [Effects of the Invention]

[0009] According to the present invention, it is possible to stably produce dry-aged meat that is enriched in 1-hexanol and has excellent palatability. [Brief explanation of the drawings]

[0010] [Figure 1] The appearance of a colony of Helicostylum pulchrum KT1b strain cultured on potato dextrose agar medium (left) and low-magnification (center, scale bar 1 mm) and high-magnification (right, scale bar 500 μm) microscopic images are shown. [Figure 2] The figure shows the appearance of colonies when Mucor flavus KT1a strain was cultured on potato dextrose agar medium (left image), and low-magnification (center image, scale bar 1 mm) and high-magnification (right image, scale bar 500 μm) microscopic images. [Figure 3]The appearance of meat that has been dry-aged with the Helicostylum pulchrum KT1b strain and Mucor flavus KT1a strain rubbed on its surface (right image), and the appearance of meat that has been dry-aged without these strains rubbed on its surface (left image) are shown. [Figure 4] The figures show the amounts of 1-hexanol, 3-methylbutanal, and benzaldehyde produced in meat that has been dry-aged after being rubbed on the surface with Helicostylum pulchrum KT1b strain and Mucor flavus KT1a strain. DETAILED DESCRIPTION OF THE INVENTION

[0011] In a first aspect, the present invention provides a method for producing dry-aged meat, which comprises attaching a culture of at least one filamentous fungus selected from the group consisting of Helicostylum pulchrum KT1b strain and Mucor flavus KT1a strain to the surface of meat, and dry-aging the meat with the filamentous fungal culture attached.

[0012] The present inventors have newly isolated the Helicostylum pulchrum KT1b strain (hereinafter simply referred to as the KT1b strain) and the Mucor flavus KT1a strain (hereinafter simply referred to as the KT1a strain), which can be used for dry aging, and deposited the KT1b strain under accession number NITE P-03423 (identification: Hp) and the KT1a strain under accession number NITE P-03422 (identification: Mf) at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on February 24, 2021.

[0013] The KT1b and KT1a strains have the following characteristics.

[0014] KT1b strain Morphological properties On potato dextrose agar, it forms colonies with a fluffy white appearance. Under microscopic observation, sporangia arranged in a ring shape with hyphae and needle-like hyphae, and round sporangia at the tips of the aerial hyphae are confirmed. physiological properties It grows at temperatures between 4 and 15°C.

[0015] KT1a strain Morphological properties On potato dextrose agar, it forms colonies with a fluffy white appearance. Under microscopic observation, round sporangia are observed at the tips of the hyphae and aerial hyphae. physiological properties It grows at temperatures between 4 and 15°C.

[0016] In the present invention, the "culture" of a filamentous fungus includes not only the mycelium of the filamentous fungus, such as mycelia, but also spores produced by the filamentous fungus. The spores may be enclosed in sporangia. In a preferred embodiment of the present invention, the culture is a spore.

[0017] The filamentous fungal culture can be prepared by inoculating the KT1b strain and / or the KT1a strain into an appropriate microbial culture medium, for example, a fungal culture medium such as potato dextrose medium or brain heart infusion medium, and culturing at a temperature suitable for culturing the filamentous fungus, for example, 4 to 15°C. The medium may be a solid medium or a liquid medium. In addition to general microbial culture media, animal meat can also be used as the culture medium. The culture period may be adjusted appropriately depending on the growth of the filamentous fungus, and is, for example, 5 days or more, preferably 10 days or more, and more preferably 15 days or more. The filamentous fungal culture prepared by culturing on a solid medium may be trimmings of dry-aged meat having filamentous fungal cells or spores on the surface.

[0018] In a preferred embodiment of the present invention, the culture of filamentous fungi to be attached to meat is a culture of both the KT1b and KT1a strains of filamentous fungi. In this embodiment, the culture may be prepared by culturing the KT1b and KT1a strains separately, or may be prepared by co-cultivating both strains in a single medium.

[0019] In the present invention, the meat to be dry-aged may be any meat obtained from domesticated or wild animals, and examples thereof include, but are not limited to, beef, pork, chicken (chicken, duck, geese, turkey, guinea fowl, pigeon, etc.), horse meat, goat meat, mutton, wild boar, venison, rabbit meat, bear meat, and whale meat. Preferred meats are beef, pork, chicken, and horse meat, with beef from Holstein, Japanese Black, and Shorthorn cattle breeds being particularly preferred. There are no particular restrictions on the part or shape of the meat, but considering trimming loss after aging, so-called block meat is preferred.

[0020] The attachment of the culture to the surface of meat can be carried out, for example, by pressing or rubbing the surface of the meat to be aged with the fungal cells or spores cultured on a solid medium such as the aforementioned potato dextrose agar medium, either directly or transferred to a cloth or other medium. Attachment can also be carried out by pouring, spraying, or smearing a liquid medium in which the filamentous fungus has been cultured, or a suspension of the fungal cells or spores in water or other suitable liquid, onto the surface of the meat to be aged. In a preferred embodiment of the present invention, attachment is carried out by rubbing the surface of the meat to be aged with the filamentous fungal culture grown on a solid medium.

[0021] In order to avoid uneven aging, it is preferable that the attachment of the culture of filamentous fungi to the surface of the meat to be aged is carried out over as large a surface as possible of the meat to be aged. Furthermore, in an embodiment in which the culture of filamentous fungi is a culture of both the KT1b strain and the KT1a strain of filamentous fungi, a monoculture of either the KT1b strain or the KT1a strain of filamentous fungi may be attached first, and then a monoculture of the other filamentous fungus may be attached, or a mixture of monocultures of both strains may be attached, or a mixed culture of both strains may be attached.

[0022] The meat to which the filamentous fungal culture has been attached is subjected to dry aging. In the present invention, dry aging can be carried out under typical dry aging conditions, for example, by placing the meat to which the culture has been attached in an environment where the temperature is 1 to 15°C, preferably 1 to 10°C, and more preferably 1 to 4°C, and the relative humidity is 60 to 100%, preferably 65 to 100%, and more preferably 70 to 95%. The aging period can be adjusted appropriately depending on the desired degree of aging, and is, for example, 5 days or more, preferably 10 days or more, and more preferably 20 days or more. The air volume during aging may be approximately 0 to 2.5 m / sec.

[0023] The method for producing dry-aged meat of the present invention enables the production of dry-aged meat that is enriched in 1-hexanol, compared to dry-aging methods using conventional naturally attached microorganisms. 1-Hexanol is an aroma component that has been reported to have a nutty or popcorn-like aroma (Migita et al., Animal Science Journal (2017) 88: 2050-2056).

[0024] Furthermore, the method for producing dry-aged meat of the present invention can enrich the dry-aged meat with 3-methylbutanal (nutty aroma) and benzaldehyde (almond aroma) compared to conventional methods using naturally attached microorganisms. Furthermore, various aroma components such as 1-octen-3-ol (mushroom-like aroma), 3-hydroxy-2-butanone (buttery aroma), 1-heptanol (fruit aroma), and 2-butanone (sweet aroma, fruit aroma) may also be detected from the dry-aged meat produced by the method of the present invention. The various aroma components described in this paragraph, combined with the aroma of 1-hexanol, can have the effect of enhancing the palatability of the aged meat.

[0025] In another aspect, the present invention provides a method for promoting 1-hexanol production in dry-aged meat, which method comprises attaching a culture of at least one filamentous fungus selected from the group consisting of the KT1b strain and the KT1a strain to the surface of meat, and dry-aging the meat to which the filamentous fungal culture has been attached. Details of the filamentous fungus, its culture, the type and shape of the meat, the attachment method, the dry-aging conditions, etc., are as described in the first aspect. Furthermore, promoting 1-hexanol production means increasing the amount or rate of 1-hexanol production compared to conventional dry-aged meat using naturally attached microorganisms.

[0026] Furthermore, the present invention provides, as a further aspect, the aforementioned KT1b strain and KT1a strain, i.e., the microorganisms deposited on February 24, 2021, at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, under accession number NITE P-03423 (identification: Hp) and accession number NITE P-03422 (identification: Mf).

[0027] As described above, the KT1b and KT1a strains can be cultured in potato dextrose agar or other media, and spores can be formed by culturing. The cultures of the KT1b and / or KT1a strains, preferably spores, prepared in this manner can be used as kits for producing dry-aged meat, particularly dry-aged meat enriched in 1-hexanol. Such kits also constitute another aspect of the present invention. In addition to the cultures, such as spores contained in a suitable container, the kits may optionally contain components such as a manual for producing aged meat, tools for attaching the cultures to meat, and suitable media for further culturing the KT1b and KT1a strains.

[0028] To date, there are no clear standards or criteria for the production of dry-aged meat in Japan, and each producer carries out aging under different conditions. This has led to variations in the quality of aged meat distributed on the market, making it difficult to supply sufficient quantities of aged meat with stable quality. The kit containing the filamentous fungal strain or a culture thereof of the present invention can be used to stably produce dry-aged meat with an enriched production of 1-hexanol in a general aging chamber or the like where temperature, humidity, air volume, etc. can be controlled.

[0029] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Example]

[0030] Example 1 Isolation and identification of filamentous fungi (1) Collection of filamentous fungi Beef was dry-aged in a meat aging facility (Goto Reinetsu Co., Ltd., used for 16 months) at Kitaichi Meat Co., Ltd. (Hokkaido) under conditions of an average temperature of 2.9°C, relative humidity of 90%, and airflow of 1.8–2.5 m / s. Samples were scraped from the surface of the aged beef using a moistened cotton swab and immersed in 10 mL of sterile 0.9% NaCl solution. This solution was plated on potato dextrose agar medium (Becton Dickinson) and cultured aerobically at 4°C for 5–7 days. Appearance-distinct fungi were transferred to new media for single-colony isolation, and two types of filamentous fungi (filamentous fungi 1 and 2) were isolated.

[0031] (2) Internal transcribed spacer (ITS) sequence Filamentous fungi 1 and 2 were cultured at 4°C in potato dextrose broth (Becton Dickinson) supplemented with 0.1% yeast extract, and the resulting filamentous fungi were disrupted using Disruptor Genie (Scientific) to recover DNA.

[0032] Using the recovered DNA as a template, PCR was performed using the primer set ITS4 (sequence number 1) and ITS5 (sequence number 2) or the primer set NL1 (sequence number 3) and NL4 (sequence number 4). The resulting amplified fragments were sequenced to determine the base sequences of the internal transcribed spacer (ITS) region and the D1 / D2 region.

[0033] Using the determined ITS region sequence as a query, a search was performed using Nucleotide BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) against the public DNA database Nucleotide Collection (Update date: 2020 / 12 / 08, Number of sequences: 63493831). The ITS region of filamentous fungus 1 (SEQ ID NO: 5) was confirmed to be 100% identical to the ITS region of Helicostylum pulchrum (GenBank: AB614353.1). Furthermore, the ITS region of filamentous fungus 2 (SEQ ID NO: 6) was confirmed to be 99.12% identical to the ITS region of Mucor flavus CBS 992.68 clone c1 (GenBank: JN206067.1). The D1 / D2 region of filamentous fungus 1 had the nucleotide sequence shown in SEQ ID NO:7, and the D1 / D2 region of filamentous fungus 2 had the nucleotide sequence shown in SEQ ID NO:8.

[0034] Based on these results, filamentous fungus 1 was identified as Helicostylum pulchrum, and filamentous fungus 2 was identified as Mucor flavus, and they were designated strains KT1b and KT1a, respectively. Strains KT1b and KT1a were deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, under accession numbers NITE P-03423 (identification: Hp) and NITE P-03422 (identification: Mf).

[0035] (3) Morphological characteristics The KT1b and KT1a strains were cultured on potato dextrose agar medium at 4°C for 14 days, and then observed externally and under a microscope. For the KT1b strain, colonies with a fluffy white appearance were observed, and under a microscope, ring-shaped sporangia and needle-shaped hyphae were confirmed (Figure 1). For the KT1a strain, colonies with a fluffy white appearance were observed, and under a microscope, round sporangia were confirmed at the tips of the aerial hyphae (Figure 2).

[0036] Example 2: Production of dry-aged meat and component analysis (1) Preparation of filamentous fungal culture The fungal cultures were prepared from beef pieces on which both strains KT1b and KT1a were grown and sporulated.

[0037] (2) Production of aged meat A rump meat block (approximately 9 kg) prepared from an individual Holstein cow (18 months old) was prepared and divided into two equal parts to prepare a sample. The entire surface of one of the rump meat blocks was rubbed with the culture (1) and then stored for 20 days in the aging chamber described in Example 1 to prepare dry-aged meat. The other rump meat block was placed in the same aging chamber without being rubbed with the culture and stored for 20 days to prepare comparative dry-aged meat (comparison meat) to which microorganisms from the environment had naturally adhered.

[0038] The surface appearance of the control meat hardly changed during aging (Fig. 3, left). However, in the dry-aged meat that had been scraped with the culture, the growth of white filamentous fungi was visually confirmed on the surface of the meat within a short period of time, and eventually grew to cover the entire surface of the meat, becoming dominant (Fig. 3, right).

[0039] (3) Analysis of aroma components A 1 g specimen was collected from each of the dry-aged and control meat samples, minced, and then placed in a 15 mL glass vial, sealed with a PTFE / silicone septum, and stored at -80°C. Volatile components were extracted from the specimen by headspace solid-phase microextraction (Ai-Nong Yu et al., Food Chemistry (2008) 110: 233-238) using an SPME fiber (divinylbenzene / carboxene / polydimethylsiloxane, 50 / 30 μm thick, Supelco Co.).

[0040] The SPME fiber was heated at 260°C for 1 minute at the injection port of the GC-MS to thermally desorb the volatile components, and mass spectrometry was performed under the following conditions. Equipment: GCMS-QP2010 (Shimadzu Corporation) Column: monopolar InertCap I column (30 m × 0.25 mm inner diameter, 1.50 μm film thickness, GL Science Inc.) Column temperature: 40°C for 10 minutes, increase to 200°C at 5°C / min, increase to 250°C at 20°C / min, and hold at 250°C for 5 minutes. Inlet temperature: 260℃ Carrier: Helium gas, flow pressure 78.9 kPa Ion source temperature: 250℃ Ionization mode: electron impact ionization (70 eV) Measurement mode: Scan mode (30-550 m / z)

[0041] The mass spectra were deconvoluted using AMDIS GC / MS Analysis version 2.73 and matched with the Massbank of North America GC-MS Spectra (https: / / mona.fiehnlab.ucdavis.edu / ).Furthermore, the spectra were compared with commercial GC-MS libraries such as NIST05 and NIST05s using Shimadzu GCMS Solution Software.

[0042] Analysis of the volatile components by GC-MS confirmed that the amount of 1-hexanol produced in the dry-aged meat was more than 16 times higher than that in the control-aged meat, demonstrating a statistically significant increase. Furthermore, the amounts of 3-methylbutanal and benzaldehyde produced in the dry-aged meat were significantly higher than those in the control-aged meat (Figure 4; significance was tested using Student's t-test for both).

[0043] (4) Analysis of fatty acids A 0.5 g specimen was collected from each of the dry-aged and control meat samples, scraped with the culture medium, and minced. Total lipids were extracted according to the method of Bligh et al. (Canadian Journal of Biochemistry and Physiology (1959) 37(8): 911-917.) and the liquid was evaporated using nitrogen gas. 1 mL of hexane and 0.2 mL of 2N methanolic sodium hydroxide were added, followed by incubation at 50°C for 30 seconds. 0.2 mL of 2N methanolic hydrochloric acid was then added, stirred, and centrifuged at 3000 rpm for 5 minutes. The upper layer of fatty acid methyl esters was then collected.

[0044] Gas chromatographic analysis of fatty acid methyl esters was carried out under the following conditions. Equipment: GC-2014 (Shimadzu Corporation) Column: Zebron ZB-FAME capillary column (30 m × 0.25 mm inner diameter, 0.20 μm film thickness, Phenomenex Inc.) Column temperature: Increase from 140 to 240°C at 4°C / min, hold at 240°C for 15 min Inlet temperature: 250℃ Carrier: Nitrogen gas, flow rate 1.3 mL / min Detector: FID Detector temperature: 260℃

[0045] The obtained spectra were compared with a Supelco 37 Component FAME standard (Merck KGaA) to identify fatty acid methyl esters.

[0046] Gas chromatography analysis confirmed that the proportion of oleic acid, a fatty acid thought to be involved in the melt-in-the-mouth texture of beef, in the total amount of fatty acids was 47.2±0.7% in the dry-aged meat rubbed with the culture medium, a statistically significant increase compared to 45.2±0.2% in the control meat (significance test was performed using Student's t-test). [Sequence List Free Text]

[0047] SEQ ID NO: 1: Base sequence of primer ITS4 SEQ ID NO: 2: Base sequence of primer ITS5 SEQ ID NO: 3: Base sequence of primer NL1 SEQ ID NO: 4: Base sequence of primer NL4 SEQ ID NO: 5: Nucleotide sequence of the ITS region of Helicostylum pulchrum KT1b strain SEQ ID NO: 6: Nucleotide sequence of the ITS region of Mucor Flavus KT1a strain SEQ ID NO: 7: Nucleotide sequence of the D1 / D2 region of Helicostylum pulchrum KT1b strain SEQ ID NO: 8 Nucleotide sequence of the D1 / D2 region of Mucor Flavus KT1a strain

Claims

1. A method for producing dry-aged meat, comprising attaching cultures of Helicostylum pulchrum KT1b strain (accession number: NITE P-03423) and Mucor flavus KT1a strain (accession number: NITE P-03422) to the surface of meat, and dry-aging the meat with the cultures attached.

2. 10. The method of claim 1, wherein the culture is attached to the surface of the meat by scraping it against the surface.

3. 3. The method of claim 1 or 2, wherein the culture comprises spores.

4. The method according to any one of claims 1 to 3, wherein the dry-aged meat is dry-aged meat enriched in 1-hexanol.

5. A method for promoting 1-hexanol production in dry-aged meat, comprising attaching cultures of the Helicostylum pulchrum KT1b strain (accession number: NITE P-03423) and the Mucor flavus KT1a strain (accession number: NITE P-03422) to the surface of meat, and dry-aging the meat with the cultures attached.

6. 6. The method of claim 5, wherein the culture is attached to the surface of the meat by scraping it against the surface.

7. 7. The method of claim 5 or 6, wherein the culture comprises spores.

8. Helicostylum pulchrum KT1b strain, a novel filamentous fungus strain (accession number: NITE P-03423).

9. Mucor flavus KT1a strain, a novel filamentous fungus strain (accession number: NITE P-03422).

10. A kit for producing dry-aged meat, comprising a culture of Helicostylum pulchrum KT1b strain (accession number: NITE P-03423) and Mucor flavus KT1a strain (accession number: NITE P-03422).

11. The kit of claim 10 , wherein the culture comprises spores.

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