KR101912299B1 - Saccharomyces exiguus having capacity of inhibiting growth and development of Saccharomyces, composition comprising the same and method for manufacturing fermented food using the same - Google Patents

Abstract

Disclosed herein is a novel strain of Saccharomyces axigus, a composition comprising the same, and a method of producing a fermented food using the same. The strain, which is an aspect of the present invention, can produce toxins and inhibit the growth of other yeast bacteria. In addition, since the strain is less sensitive to the pH environment, toxins can be produced in a wide range of pH environments. Therefore, the use of the strain, the culture, the lysate thereof, or the extract thereof of the present invention is useful because it can minimize the spoilage and alteration of the food by removing the contaminated yeast in a wide pH environment.

Description

[0001] The present invention relates to a Saccharomyces exucus strain having the ability to inhibit the growth of Saccharomyces cerevisiae, a composition containing the same, and a method for producing fermented food using the same, for manufacturing fermented food using the same}

Disclosed herein is a novel Saccharomyces axigus strain, a composition comprising the same, and a method for producing a fermented food using the same.

Yeast is a major cause of food corruption and deterioration, and corruption and deterioration caused by yeast are viewed as important challenges for a variety of food companies, including beverages, dairy products, ready-to-eat foods, and fermented foods. In order to solve such problems, various attempts have been made to dehydrate and dry, such as moisture control methods such as salting, temperature control methods such as refrigeration or freezing, pH adjustment methods, heat sterilization and oxygen removal through vacuum packing, But did not achieve a satisfactory effect. In addition, the use of anti-yeast (ie, preservative) has been used regularly. However, as consumers are increasingly interested in health, studies on yeast control using natural products, rather than chemical additives, have been conducted. However, in order to control yeast using natural products, it is necessary to extract, purify and concentrate natural products. Therefore, there is a need for research on a method which is harmless to the body and can economically improve the corruption and alteration of the fermented food.

KR 2014-0032978 A

In one aspect, an object of the present invention is to minimize corruption and degeneration of fermented foods.

In another aspect, an object of the present invention is to remove contaminated yeast which is a factor of food deterioration.

In another aspect, an object of the present invention is to provide a strain toxic to Saccharomyces cerevisiae.

In another aspect, an object of the present invention is to provide a strain capable of inhibiting the growth of contaminated yeast at a wide pH.

In another aspect, an object of the present invention is to inhibit the growth of contaminated yeast at low cost.

In one aspect, the present invention provides a method of producing toxins that are toxic to Saccharomyces sp. Yeast within the entire pH range of pH 3.0 to pH 5.0, wherein the strain itself is resistant to the toxin, Saccharomyces exiguus strain.

In another aspect, the present invention provides a composition comprising the strain, a culture thereof, a lysate thereof, or an extract thereof.

In another aspect, the present invention provides a food manufacturing method comprising culturing the strain.

MRS to the one aspect of the invention Saccharomyces eksi Goose (Saccharomyces exiguus strains produce toxins that can inhibit the growth of other yeast strains. Since the strain of the present invention is less sensitive to the pH environment, toxins can be produced in a wide range of pH environments. In addition, the strain of the present invention is not toxic to microorganisms other than Saccharomyces cerevisiae. Therefore, the use of the strain, the culture, the lysate thereof, or the extract thereof of the present invention is useful because it can minimize the spoilage and alteration of the food by removing the contaminated yeast while preserving the beneficial bacteria in a wide pH environment.

FIG. 1 is a graph showing the results of confirming the ability of the present invention yeast seeds to be produced.
2 is a flow diagram of the yeast of the present invention.

In one aspect, the present invention provides a method of producing toxins that are toxic to Saccharomyces sp. Yeast within the entire pH range of pH 3.0 to pH 5.0, wherein the strain itself is resistant to the toxin, Saccharomyces exiguus .

For example, the strain may have a pH of 3.0 or higher, a pH of 3.1 or higher, a pH of 3.3 or higher, a pH of 3.5 or higher, a pH of 3.7 or higher, a pH of 3.9 or higher, a pH of 4.0 or higher, a pH of 4.1 or higher, a pH of 4.3 or higher, pH less than 4.9, less than pH 4.9, less than pH 4.7, less than pH 4.5, less than pH 4.3, less than pH 4.1, less than pH 4.0, less than pH 3.9, less than pH 3.7, less than pH 3.5, The toxin can be produced at pH 3.3 or below or pH 3.1 or below. In particular, the toxin can be produced in the range of pH 3.0 to pH 3.5, or pH 4.5 to pH 5.0.

As used herein, the term " toxin " may refer to a substance that is secreted by a strain and inhibits or prevents the metabolism, reproduction or growth of another organism, resulting in its death.

In one embodiment, the toxin may be a proteolytic toxin. Specifically, the protein toxin may be zymocide.

Killer yeast also produces proteinaceous toxins that kill other yeast. These toxins are called killer factors or killer toxins, but they are called gemocides because they are toxic only to yeast and do not show toxicity to other higher organisms or bacteria. The production capacity of killer yeast is known to be determined by the killer plasmid present in yeast cytoplasm.

Glycoside proteins can inhibit the growth of other yeasts by inhibiting protein synthesis and nucleic acid synthesis of other yeasts even in very small amounts. In addition, it may be toxic to yeast, but not toxic to other higher organisms or bacteria.

In one embodiment, the Saccharomyces axigus strain can produce the toxin at a temperature of 20 to 30 < 0 > C. For example, at least 20 占 폚, at least 21 占 폚, at least 23 占 폚, at least 25 占 폚, at least 27 占 폚, at least 29 占 폚, or at least 30 占 폚, at least 29 占 폚, at least 27 占 폚, at least 25 占 폚, The following toxins can be produced.

In one embodiment, the Saccharomyces sp. May be, but is not limited to, Saccharomyces cerevisiae .

The strain may be Saccharomyces exigus SMY76 strain or Saccharomyces exigus SMY77 strain. In one embodiment, the strain may be S. cerevisiae SMY76 strain.

In addition, the strain may be a strain having the accession number KCCM11818P.

Also, in one embodiment, the strain may be derived from a fermentation of a vegetable comprising at least one of pepper and radish. The fermented product may be a natural fermented product. In the present specification, 'natural fermentation product' may mean the product obtained through fermentation without artificially adding yeast or the like for fermentation of the sample. In one embodiment, the sample used in the natural fermentation may include one or more of pepper and radish. For example, the natural fermented product may be obtained by adding the sample, garlic, and salt, and fermenting at 20 to 30 DEG C for 2 to 4 days at room temperature.

The strain may include one or more of the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6. For example, the sequence of SEQ ID NO: 5 may include the ITS sequence of the SACRAMESES EXIGUSS SMY76 strain, and the sequence of SEQ ID NO: 6 may include the sequence of 26s rDNA of the SACRAMESES EXIGUS SMY76 strain.

In another aspect, the present invention may be a composition comprising the strain, its culture, its lysate or an extract thereof. The culture broth, crushed product, or extract may contain a sterilized product. Further, the culture liquid may include a supernatant obtained by centrifuging the culture liquid. The method for producing the above extract is not limited, and can be carried out by well-known methods known in the art.

In one embodiment, the composition may be a food composition or a pharmaceutical composition.

In one embodiment, the food may be a fermented food, and further, the fermentation may include lactic acid fermentation. Fermented foods herein may include, for example, soybean fermented foods such as soy sauce, soybean paste, hot pepper paste, fermented milk products such as cheese, yogurt, flavoring agents such as salt pickles or condiments such as kimchi, It is not.

The formulation of the food composition according to one aspect of the present invention is not particularly limited, but may be formulated into, for example, tablets, granules, powders, liquid preparations such as drinks, caramels, gels, bars and the like. The food composition may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. The food composition of each formulation can be blended with the ingredients commonly used in the field in addition to the active ingredient without difficulty by those skilled in the art depending on the purpose of formulation or use, and synergistic effect can be obtained when the composition is applied simultaneously with other ingredients.

The pharmaceutical composition may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc. These solid preparations may contain one or more excipients such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may be administered parenterally or orally, and may be administered in an amount of 0.1 to 500 mg, preferably 1 to 100 mg per kg of body weight per day, Lt; / RTI > The dosage for a particular patient may vary depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, severity of disease, and the like.

The pharmaceutical composition may be formulated into tablets, capsules, tablets, capsules, capsules, tablets, capsules, capsules, tablets, pills, capsules, Or may be formulated into a formulation.

The pharmaceutical composition may also be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes including parenteral, oral, and the like, all manner of administration being expected, for example oral, transdermal trandermally, intravenous, intramuscular, subcutaneous injection.

In one aspect, the present invention is a food manufacturing method comprising culturing the strain. The food manufacturing method may further include a step of adding the strain, the culture, the lysate thereof, or an extract thereof to the food.

The microorganism (Saccaharomyces exiguous SMY76) was deposited on March 15, 2016 with the deposit number KCCM11818P at the Korean Society for Microbiological Conservation.

Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the following examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example 1] Preparation of sample for isolation of wild yeast

Natural fermentation samples were prepared for the isolation of wild yeast producing gymoside. First, 0.001 to 0.01% of a liquid saccharifying enzyme is added to 15 to 30% of rice or brown rice, followed by liquification at 70 ± 5 ° C. for 14 to 18 hours. After sterilization at 85 ° C. for 1 hour and cooling, ) For 7 days. The samples were prepared by adding 2% ~ 5% of garlic and 2% of salt to pepper or 70% ~ 90% of non - fermented soybeans, followed by natural fermentation at room temperature (20 ~ 30) for 3 days.

[Example 2] Separation and selection of wild yeast

[Example 2-1] First separation

After injecting 10 g of the sample prepared in Example 1 into 90 ml of sterilized physiological saline (0.85% NaCl), the homogenization was performed for 30 minutes, and the homogenized sample was diluted to 10 ³ to 10 ⁶ by the continuous dilution method. 0.1m of the diluted solution was plated on a PDB (Potato Dextrose Broth) agar medium supplemented with 50mM of ampicillin, and cultured in a 25 ° C incubator for 2 days to isolate a yeast that formed milky colonies.

[Example 2-2] Secondary separation

Yeast isolated in Example 2-1 was inoculated into a liquid medium containing YEPD (2% yeast extract, 2% peptone, 2% glucose / dextrose) containing 5% NaCl and cultured for 30 to 24 hours to select yeast having salt resistance And yeast with acid resistance was selected by inoculation in a liquid medium of YEPD (2% yeast extract, 2% peptone, 2% glucose / dextrose) adjusted to pH 3.5 and culturing for 30 to 24 hours. The five yeasts selected (SMY73, SMY74, SMY75, SMY76, SMY77) are as shown in Table 1 below.

Natural fermentation sample PDA (50 mM Amp) Salt resistance (5% NaCl) Acid resistance (pH 3.5) Selected strains Rice / brown rice 27 species 11 species 3 species SMY 73
SMY 74
SMY 75 Pepper / radish 5 species 5 species 2 species SMY 76
SMY 77

[Example 2-3] Screening of yeast producing yeast seeds

Saccharomyces YEPD a cerevisiae) KCTC7296 strain (2% yeast extract, 2% peptone, 2% glucose / dextrose) YEPD for 24 hours culture at 25 were inoculated into the liquid medium, it takes a 100ml adjusted to pH 4.2 ± 0.1 (2% yeast extract , 2% peptone, 2% glucose / dextrose), 2% agar was added to the solid medium, and sterilized 8 mm diameter paper disks (Toyo Roshi CO. LTD., Japan) were placed. Thereafter, the yeast selected in Example 2-2 was inoculated into a liquid medium of YEPD (2% yeast extract, 2% peptone, 2% glucose / dextrose) adjusted to pH 4.2 ± 0.1 and cultured at 25 to 24 to 48 hours The supernatant was separated by centrifugation at 13000 rpm, the separated supernatant was sterilized by a 0.20 mu m filter, and 150 mu l was dispensed on the paper disk. Thereafter, the paper disk on which the supernatant liquid was dispensed was incubated in a 25 ° C incubator for 24 hours, and the diameter of the transparent pores formed around the paper disk was measured. As a result, transparent rings due to the generation of zymoside were observed in both SMY76 and SMY77 yeasts. The size of the transparent ring of SMY76 was about 12 mm, and the size of transparent ring of SMY77 was about 11 mm. SMY76 and SMY77, which are the best yeast growth inhibiting yeast, were finally selected.

[Example 3] Identification of yeast sorted

In order to identify two SMY76 and SMY77 yeasts with the ability to produce glycoside synthase, the ITS region and 26s rDNA were amplified to obtain nucleotide sequences. Specifically, the ITS region was amplified using the universal primer ITS1F (5'-TCCGTAGGTGAACCTGCGG-3 ') (SEQ ID NO: 1) and ITS4R (5'-TCCTCCGCTTATTGATATGC-3' 26s rDNA was amplified using NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3 ') (SEQ ID NO: 3) and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') (SEQ ID NO: 4). Amplified ITS and 26s rDNA were purified using a Wizard DNA purification kit (Promega, Madison, Wisconsin, USA), and analyzed by sequencing with Solgent. Comparing the homology in BLAST, and by using the program MEGA (MEGA program) was subjected to analysis system diagram (FIG. 2) As a result, the MRS in both yeast Saccharomyces eksi Goose (Saccharomyces exiguus .

Retaining ring size (mm) Culture temperature (캜) 20 25 30 35 SMY 76 10.5 12 11 - SMY 77 9 11 9.5

Retaining ring size (mm) Culture pH 2 2.5 3 3.5 4 4.5 5 5.5 6 SMY 76 - - 9 12 13 11.5 10.5 - - SMY 77 - - - 9.5 10 9.5 - - -

Formulation examples of compositions according to one aspect of the present invention are described below, but may be applied to various other formulations, which are not intended to be limiting but merely illustrative of the present invention.

[Production Example 1] Fermented milk

ingredient Content (% by weight) crude oil 94.0 Skim milk powder 3.0 Lactic acid bacteria powder 0.1 Culture medium of SMY 76 strain according to Example 2-3 0.1 glucose 1.0 Flavor handful total 100.0

[Manufacturing Example 2] Fermented beverage

ingredient Content (% by weight) Rice or brown rice 3.0 Purified water 92.0 Liquid saccharification enzyme 0.03 Lactic acid bacteria powder 0.1 Culture medium of SMY 76 strain according to Example 2-3 0.1 Flavor 0.05 pectin 0.1 total 100.0

[Preparation Example 3] Preparation of kimchi sprout sauce using vegetable fermented product

30% by weight of non-fermented product, 30% by weight of fermented red pepper, 0.5% by weight of vinegar, 5% by weight of onion, 20% by weight of brown rice fermentation, 3% by weight of chopped garlic, 0.05% by weight of xanthan gum, 1% by weight of the culture medium of the strain was added to prepare a sauce.

Korea Microorganism Conservation Center (overseas) KCCM11818P 20160315

<110> SAMPIO FOOD COMPANY <120> Saccharomyces exiguous capacity for inhibiting growth and          development of Saccharomyces, composition comprising the same and          method for manufacturing fermented food using the same <130> 16P175IND <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> primer_ITS1_foward <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_ITS4_reverse <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_NL1 <400> 3 gcatatcaat aagcggagga aaag 24 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer_NL4 <400> 4 ggtccgtgtt tcaagacgg 19 <210> 5 <211> 697 <212> DNA <213> ITS sequence of SMY76 <400> 5 ttcggaagga tcattaaaga aaatgatgaa taattggtag aggaggagtg atcgagcctg 60 cgcttaagtg cgcggcgagg ttgttctttt tcgttacgcc gataattctt tacacacact 120 ggagttttat ttctacagta tcggaggtag caataccgcc aaaacaaaac acaaacaatt 180 attttttatt attttaattt gtcatttcaa aatctgcttt attgcagtaa ccaaaatatt 240 caaaactttc aacaacggat ctcttggttc tcgcatcgat gaagaacgca gcgaaatgcg 300 atacgtaatg tgaattgcag aattccgtga atcatcgaat ctttgaacgc acattgcgcc 360 ccttggtatt ccagggggca tgcctgtttg agcgtcattt ccttctcaaa tacttgtatt 420 tggttgtgag tgacactcag tcttgcattg agttaacttg aaattgttgg ccgtagcggt 480 tgttgcagct tatagttttt gtgtaatggt atgatttctt tactattaaa caggatgctt 540 gagcatgtcg tattaggttt taccaactcc ggcagactcg gtatcttgga agagcgtact 600 ggcattagaa aattgagact gtcgactgtg gcaaacagta ctctttaagt ttgacctcaa 660 atcagctagg aatacccgct gaacttaagc atatcaa 697 <210> 6 <211> 972 <212> DNA <213> 26s rDNA sequence of SMY76 <400> 6 ttaagcatat caataagcgg aggaaaagaa accaaccggg attgccttag taacggcgag 60 tgaagcggca aaagctcaaa tttgaaatct ggtaccttcg gtgcccgagt tgtaatttgt 120 agagggcgac tttggggcgg ctccttgtct atgttccttg gaacaggacg tcatagaggg 180 tgagaatccc gtgtggcgag gagtgcggtt ccgtgtaaag cgctctcgaa gagtcgagtt 240 gtttgggaat gcagctctaa gtgggtggta aattccatct aaagctaaat attggcgaga 300 gaccgatagc gaacaagtac agtgatggaa agatgaaaag aactttgaaa agagagtgaa 360 aaagtacgtg aaattgttga aagggaaggg catttgatca gacatggtgt tttgtgcccc 420 tcgctccttg tgggtggggg aatctcgcag ctcactgggc cagcatcagt tttggcggtc 480 ggataaaacc aggggaacgt agcttgcttc gggaagtatt atagcctctg ggaatacggc 540 cagccgggac tgaggaacgc gattcgtcaa ggatgctggc ataatggtta tatgccgccc 600 gtcttgaaac acggaccaag gagtctaacg tctatgcgag tgtttgggtg tgaaacccat 660 acgcgtaatg aaagtgaacg taggttgggg cctgtcaaag ggtgcacaat cgaccgatcc 720 tgatgttttc agatggattt gagtaagagc atagctgttg ggacccgaaa gatggtgaac 780 tatgcctgaa tagggtgaag ccagaggaaa ctctggtgga ggctcgtagc ggttctgacg 840 tgcaaatcga tcgtcgaatt tgggtatagg ggcgaaagac taatcgaacc atctagtagc 900 tggttcctgc cgaagtttcc ctcaggatag cagaagctcg tatcagtttt atgaggtaaa 960 gcgaatgatt ag 972

Claims (17)

Produce toxins that are toxic to Saccharomyces sp. Yeast,
The strain itself is a strain of Saccharomyces exiguus deposited with the deposit number KCCM11818P which is resistant to the toxin. The method according to claim 1,
Wherein the strain produces a toxin at a temperature of 20 to 30 占 폚. The method according to claim 1,
The toxin is a proteolytic toxin, Saccharomyces axigus strain. The method of claim 3,
Wherein the proteolytic toxin is a zymocide, Saccharomyces axigus strain. The method according to claim 1,
The Saccharomyces cerevisiae strain is a Saccharomyces cerevisiae strain, Saccharomyces axigus strain. The method according to claim 1,
Wherein the strain is derived from a fermentation product of a vegetable comprising at least one of pepper and radish. The method according to claim 1,
Wherein the strain comprises at least one of the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6. The method according to claim 1,
Wherein said toxin is produced in a pH range of 4.2 +/- 0.1. The method according to claim 1,
The strain is Saccharomyces axigus strain, which is a strain of Saccharomyces axigus SMY76. A food composition comprising the strain of any one of claims 1 to 9, a culture thereof, a lysate thereof, or an extract thereof. delete 11. The method of claim 10,
Wherein the composition is a composition for inhibiting yeast activity. 11. The method of claim 10,
Wherein the food is a fermented food. 14. The method of claim 13,
Wherein the fermentation comprises lactic acid fermentation. A method for producing a food, comprising culturing the strain of any one of claims 1 to 9. 16. The method of claim 15,
And adding the strain, the culture, the lysate thereof, or an extract thereof to the food. delete

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