KR102242298B1 - Bacillus coagulans with excellent butter and cheese flavor productivity - Google Patents

Abstract

In the present specification, Bacillus coagulans having at least one flavor-producing ability of butter and cheese, and a lysate thereof; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. Microbial preparations or perfume compositions and the like comprising one or more of them are disclosed. In one aspect, the strain of the present disclosure has excellent ability to produce butter and cheese flavor, so it can be utilized to enhance the taste of consumers, and at the same time, it is possible to feel the flavor of butter without containing saturated fatty acids and trans fats. Has an advantage. Thus, as an example, the strain is utilized in various products such as foods and drugs to enable the production of high value-added products with enhanced flavor.

Description

Bacillus coagulans with excellent butter and cheese flavor productivity}

In the present specification, Bacillus coagulans having at least one flavor-producing ability of butter and cheese, and a lysate thereof; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. Microbial preparations or perfume compositions and the like comprising one or more of them are disclosed.

Butter is a variety of pastry, baked, and stir-fried. It is used in various dishes such as, and plays a role in enhancing the taste and flavor of the dish, but it is made by separating only fat from milk, and has the disadvantage of high price and increased blood saturated fatty acid due to animal fat. To solve this problem, margarine, which was made through processes such as decolorization, hardening, and deodorization, using vegetable oil that does not have to worry about saturated fatty acids, came out as a substitute. However, vegetable oils also have a problem with trans fats that are made through the hardening process.

Trans fats occur in hardening processes and high-temperature treatment processes in which hydrogen is added by double bonds in unsaturated fatty acids having a high content in oils, and these trans fats increase the level of saturated fatty acids in the body and at the same time reduce the level of unsaturated fatty acids. Known as fat. Currently, various advanced countries, including Korea, have established and strictly managed the content standards for trans fats of margarine. Accordingly, various studies for suppressing or removing trans fat production are being conducted, but due to an additional process and a deodorizing process for removing off-flavor generated, the overall buttery flavor and a decrease in savory flavor appear as a problem.

Flavors play a very important role in food, cosmetics, beverages, surfactants, feed, chemicals, biopharmaceuticals, and pharmaceutical industries. Such perfume production methods are largely divided into chemical synthesis and biological methods.The production of perfume ingredients through chemical synthesis allows mass production of scents with various types of chemical structures, but unwanted racemic mixtures. In addition to the possibility of being made of, there is a disadvantage that the substrate specificity is low. In addition, recently, consumers' consumption tendency is changing to prefer foods, medicines, chemicals, and pharmaceutical products that contain scents that are naturally friendly and harmless to the human body, and accordingly, rather than fragrances or scents produced by chemical synthesis. There is a lot of interest in perfume ingredients produced by biological methods.

Biological production methods include extraction method and microbial fermentation method.The extraction method that produces natural flavoring ingredients by extracting from plant cells or tissues has a very long cultivation time of plant cells and difficult cultivation conditions, so the yield is not high, and the concentration of the produced perfume ingredients is not high. Several limitations have been reported, such as low, complicated extraction process, and difficult separation process. On the other hand, the biotechnological method through microbial fermentation is in the spotlight as an alternative method that can overcome the shortcomings of the chemical synthesis method because it is nature-friendly and can produce high value-added products at a low cost.

Based on this, while conducting various studies to realize the unique flavor of butter including cheese, it was confirmed that the Bacillus coagulation strain newly discovered by the present inventors can produce flavors peculiar to cheese and butter, and the present invention Was completed.

KR Patent Publication No. 10-2008-0063035 US Patent Publication No. 5,783,247

As. J. Food Ag-Ind. 2012, 5(05), 355-363

In one aspect, the object of the present disclosure is Bacillus coagulans strain and a lysate thereof having at least one flavor-producing ability of butter and cheese; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. It is to provide a microbial preparation or perfume composition, etc. comprising at least one of.

In order to achieve the above object, the present disclosure in one aspect has a flavor producing ability of at least one of butter and cheese, the flavor producing ability is acetoin (acetoin), 2,3-butanediol (2,3-butanediol), 2-hep At least one selected from the group consisting of tanone (2-Heptanone), 2-nonanone (2-nonanone), 3-methylbutanoic acid (3-methylbutanoic acid), octanoic acid and hexanoic acid It provides a Bacillus coagulans strain, which is the ability to produce a compound containing.

In one aspect, the present disclosure is the strain; Its crushed material; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. It provides a microbial preparation comprising at least one of.

In one aspect, the present disclosure is the strain; Its crushed material; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. It provides a fermented coconut, which is a fermented product of one or more of the coconuts.

In one aspect, the present disclosure provides a perfume composition comprising the fermented coconut.

In one aspect, the present disclosure is coconut; And the strain; Its crushed material; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. It provides a fragrance composition containing one or more of;

In one aspect the present disclosure is the strain in the coconut; Its crushed material; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. It provides a method for producing a perfume composition comprising; treating one or more of the above.

In one aspect, the strain of the present disclosure is excellent in the ability to produce butter and cheese flavor, so it can be utilized to enhance the taste of consumers, and at the same time, it is possible to feel the flavor of butter without containing saturated fatty acids and trans fats. Has an advantage. Thus, as an example, the strain is utilized in various products such as foods and drugs to enable the production of high value-added products with enhanced flavor.

1 is a diagram showing a schematic diagram of a coagulans strain SMB 753 (Bacillus coagulans SMB 753) deposited with the accession number KCCM12474P at the Korea Microbiological Conservation Center on March 29, 2019 according to the present disclosure.
FIG. 2 is a diagram showing the results of confirming the production ability of compounds exhibiting butter and cheese flavors of coagulation strain SMB 753 through a GC-MS ion chromatogram according to the present disclosure.
3 is a diagram showing a manufacturing process for producing a flavoring flavor of butter and cheese using coconut and coagulation strain SMB 753.

Hereinafter, it will be described in detail.

In one aspect, the present disclosure provides a Bacillus coagulans strain having at least one flavor-producing ability of butter and cheese.

In one aspect, the flavor producing ability is acetoin (acetoin), 2,3-butanedione (2,3-butanedione), 2-heptanone (2-Heptanone), 2-nonanone (2-Nonanone), 3- It may be the ability to produce a compound containing at least one selected from the group consisting of methylbutanoic acid, octanoic acid, and hexanoic acid.

For example, acetoin is a compound that exhibits cheese flavor and may be derived from sugar. For example, 2,3-butanedione is a compound that exhibits a buttery flavor. For example, 2-heptanone is a compound that exhibits a cheese flavor. For example, 2-nonanone is a compound that exhibits cheese flavor. For example, 3-methylbutanoic acid is a compound that exhibits a cheese flavor. For example, octanoic acid is a compound that exhibits cheese flavor. For example, hexanoic acid is a compound that exhibits cheese flavor.

In one aspect, the concentration of acetoin produced when cultured at 37° C. for 48 hours after inoculation with 2% by weight of the strain of the present disclosure relative to the total weight of the medium containing 15% by weight of coconut is 1500 or more, 1600 or more, 1700 or more, 1800 or more, 1900 or more, 2000 or more, 2100 or less, 2200 or less, 2300 or less, 2400 or less, 2500 or less ppb ((parts per billion); the concentration of 2-butanedione produced is 20 or more, 21 or more, 22 More than, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or less, 31 or less, 32 or less, 33 or less, 34 or less, 35 or less, 36 or less, 37 or less, 38 or less, 39 or less, 40 or less ppb; The resulting 2-heptanone concentration is 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or less, 41 or less, 42 or less, 43 or less, 44 or less, 45 or less, 46 or less , 47 or less, 48 or less, 49 or less, 50 or less ppb; The resulting 2-nonanone concentration is 65 or more, 66 or more, 67 or more, 68 or more, 69 or more, 70 or more, 71 or less, 72 or less, 73 or less, 74 Or less, 75 or less ppb; The resulting 3-methylbutanoic acid concentration is 400 or more, 410 or more, 420 or more, 430 or more, 440 or more, 450 or less, 460 or less, 470 or less, 480 or less, 490 or less, 500 or less ppb; The concentration of octanoic acid produced is 1500 or more, 1600 or more, 1700 or more, 1800 or more, 1900 or more, 2000 or more, 2100 or less, 2200 or less, 2300 or less, 2400 or less, 2500 or less ppb; or the concentration of hexanoic acid produced is 750 It may be more than, 760 or more, 770 or more, 780 or more, 790 or more, 800 or more, 810 or less, 820 or less, 830 or less, 840 or less, 850 or less ppb, but is not limited thereto.

In one aspect, the strain is 1 or more, 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 5.5 or less, 6 or less, 6.5 or less, 7 or less, 7.5 or less, 8 Below, 8.5 or less, 9 or less, 9.5 or less, 10 or less% (v/v) salt concentration conditions may be possible to grow.

In one aspect, the strain may have at least one activity of esterase and lipase, and preferably, the activity has 4 or more, 5 or more, 6 or more, 7 or more, 8 or less, 9 or less carbon atoms. , 10 or less, 11 or less, 12 or less, 13 or less, may be for a fatty acid of 14 or less, but is not limited thereto.

In one aspect, the strain may be a strain having 16S rDNA comprising the nucleotide sequence of SEQ ID NO: 1.

In one aspect, the strain may be Bacillus coagulation SMB 753, preferably a strain having an accession number of KCCM12474P, but is not limited thereto.

In one aspect, the present disclosure is the strain; Its crushed material; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. It provides a microbial preparation comprising at least one of.

In one aspect, the fermented product may be a material obtained by fermenting the strain, a lysate thereof, or a culture thereof.

In one aspect, the lysate, culture, fermentation or extract may include sterilized. In addition, the culture may include a supernatant obtained by centrifuging the culture.

In one aspect, the present disclosure is the strain; Its crushed material; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. It provides a fermented coconut, which is a flame retardant of one or more of the coconuts. In one aspect, the fermented coconut may be obtained by separating only the oil layer, for example, fermented coconut oil.

In one aspect, coconut has a high content of saturated fatty acids among plants that can produce vegetable oils, has a relatively short fatty acid length of C12 (Lauric acid), has a flavor similar to milk, and is used in various ways in various foods. will be. Thus, as an example, a coconut fermented product having a butter or cheese flavor obtained by using the strain of the present disclosure newly discovered by the present inventors instead of butter having a high animal fat content was prepared to have excellent flavor and at the same time as described above. Materials with all of the same health benefits can be manufactured.

In one aspect, the present disclosure provides a perfume composition comprising the fermented coconut. As an example, the flavoring may represent one or more flavors of butter and cheese.

In one aspect, in the perfume composition of the present disclosure, cations such as fatty acids such as palmitic acid and stearic acid, fatty alcohols, linear and branched long chain alkyl quaternary ammonium salts, etc. Formulation can be facilitated by mixing with a cationized polymer such as a cationized surfactant, a cationized cellulose, and a cationized polyvinylpyrrolidone, a silicone, and the like. In addition, for example, for formulation purposes, components for formulation such as solvents, surfactants, thickeners, stabilizers, preservatives, colorants, pH adjusters, sequestering agents, colorants, pearlizing agents, appearance improving agents, pigments, powder particles, etc. It may be included as, but is not limited thereto.

In one aspect, the fragrance may be a food additive, a cosmetic additive, a fiber additive, or a pharmaceutical additive, and includes without limitation any type of product capable of imparting a fragrance.

As an example, the flavoring may be used as an additive in a food composition and may be used to enhance flavor in various foods by displaying butter or cheese flavor instead of butter.For example, the food is not limited to being consumed by humans, but also to animals other than humans. It includes all of the feed that is ingested.

For example, the fragrance is used as an additive that imparts fragrance in cosmetics to prepare and protect the skin (tooth, serum, essence, lotion, cream, etc.), color cosmetics (makeup base, foundation, powder, eye shadow, lip gloss). Rose, lipstick, lip balm, etc.), makeup for hands and toenails (manicure, nutrients, reinforcement, top coat, etc.), cosmetics for cleaning (foam cleansing, cleansing oil, cleansing lotion, cleansing cream, cleansing gel, pack, mask, soap, etc.) Cleansing tissues, etc.), sun care cosmetics, and body cosmetics (body lotion, shower gel, body cream, body oil, etc.). It may be added to all formulations such as hair essence, wax, gel, spray, etc. to exhibit butter or cheese flavor, but is not limited thereto.

In one aspect, the fragrance is a fabric softener, liquid detergent, powder detergent, sheet-type detergent, textile dyeing agent, textile treatment, fiber rinse, bleach, partial contamination treatment agent, textile styling agent, ironing aid, clothing depending on the intended use as a textile additive. It may be added to a storage agent, but is not limited thereto.

For example, the fragrance may be used as a pharmaceutical additive to enhance consumer preference by covering up unpleasant scents that may appear in pharmaceutical compositions or by giving special scents to pharmaceutical compositions.

In one aspect the present disclosure is the strain in the coconut; Its crushed material; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. It provides a method for producing a perfume composition comprising; treating one or more of the above.

In one aspect, the method for preparing the perfume composition is 90 or more, 95 or more, 100 or more, 105 or more, 110 or less, 115 or less, 120 or less, 125 or less, 130 or less ℃ after mixing purified water in the coconut before the processing step. 5 minutes or more, 6 minutes or more, 7 minutes or more, 8 minutes or more, 9 minutes or more, 10 minutes or more, 11 minutes or more, 12 minutes or more, 13 minutes or more, 14 minutes or more, 15 minutes or more, 16 minutes or more, 17 minutes More than, 18 minutes or more, 19 minutes or less, 20 minutes or less, 21 minutes or less, 22 minutes or less, 23 minutes or less, 24 minutes or less, 25 minutes or less, 26 minutes or less, 27 minutes or less, 28 minutes or less, 29 minutes or less, It may further include a heat treatment step of maintaining and heat treatment for 30 minutes or less, and for example, after the heat treatment step, it may further include a step of leaving at a temperature of 35 or more, 36 or more, 37 or less, 38 or less, 39 or less. .

For example, the purified water is 500 or more, 505 or more, 510 or more, 515 or more, 520 or more, 525 or more, 530 or more, 535 or more, 540 or more, 545 or more, 550 or more, 555 or more, 560 or more, based on 100 parts by weight of coconut , 565 or less, 570 or less, 570 or less, 580 or less, 580 or less, 590 or less, 595 or less, 600 or less by weight.

In one aspect, the strain; Its crushed material; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. At least one of them may be treated with 11 or more, 12 or more, 13 or more, 15 or less, 14 or less, or 16 parts by weight based on 100 parts by weight of coconut.

In one aspect, the treating may include the strain; Its crushed material; Its culture; Its fermented product; And extracts of the strain, lysate, culture or fermentation product. After at least one treatment of 35 or more, 36 or more, 37 or less, 38 or less, 39 or less ℃ 40 or more, 41 or more, 42 or more, 43 or more, 44 or less, 45 or less, 46 or less, 47 or less, 48 or less hours It may include the step of.

In one aspect, the method for producing the perfume composition is 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 or less, 61 or less after the treating step , 62 or less, 63 or less, 64 or less, 65 or more, 66 or less, 67 or less, 68 or less, 69 or less, 70 or less 30 minutes or more, 32 minutes or more, 34 minutes or more, 36 minutes or more, 38 minutes or more, 40 Minutes or more, 42 minutes or more, 44 minutes or more, 46 minutes or more, 48 minutes or more, 50 minutes or more, 52 minutes or more, 54 minutes or more, 56 minutes or more, 58 minutes or more, 60 minutes or less, 62 minutes or more, 64 minutes or less , 66 minutes or less, 68 minutes or less, 70 minutes or less, 72 minutes or less, 74 minutes or less, 76 minutes or less, 78 minutes or less, 80 minutes or less, 82 minutes or less, 84 minutes or less, 86 minutes or less, 88 minutes or less, 90 Sterilizing by holding for a period of minutes (3 hours) or less; A separation step of separating only the oil layer from the fermented product obtained in the sterilization step; And a solidification step of cooling and solidifying the coconut fermented oil obtained in the separation step at 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less ℃. It may further include;

Redundant content is omitted in consideration of the complexity of the present specification, and terms that are not otherwise defined herein have meanings commonly used in the technical field to which the present disclosure belongs. In addition,'or' used in this specification is a concept including'and' unless explicitly stated otherwise.

Hereinafter, examples and experimental examples will be described in detail to aid understanding of the present disclosure. However, the following Examples and Experimental Examples are merely illustrative of the contents of the present disclosure, and the scope of the present disclosure is not limited to the following Examples and Experimental Examples. Examples and experimental examples of the present disclosure are provided to more completely describe the present disclosure to those of ordinary skill in the art.

Example 1. Isolation and identification of strains

1-1. Isolation of strains

Among the traditional fermented foods of Korea, 4 kinds of traditional fast-stirring paste with a relatively short fermentation time (makjang, bijijang, jangjang, sigeumjang (barley bran paste)) are finely ground, and then 10 ³ ~10 in sterile physiological saline using a serial dilution method. ^{After 5} times dilution, 0.1 ml of the diluted solution was plated on MRS (Difco, USA) agar medium, an agar medium for separating lactic acid bacteria, and cultured at 30° C. for 48 hours under anaerobic conditions to first isolate microorganisms. At this time, the four types of fast-growing seeds were collected from Gangwon-do, Chungcheong-do, Jeolla-do, and Gyeongsang-do of Korea, respectively.

The first isolated microorganism is cultured at 30°C for 48 hours in MRS medium added with 5% NaCl to secondarily separate the salt-resistant microorganism, and finally, BCP agar (Eiken, Japan) medium that turns yellow when the pH is less than 5.2. After inoculation, cultured at 30° C. for 48 hours to isolate a yellow ring-forming microorganism (Table 1).

1-2. Genetic identification of strains

For (genetic) identification through DNA analysis of 39 microorganisms isolated in Example 1, 16S rDNA sequence analysis was requested to Solgent Co., Ltd. (Daejeon) to obtain DNA information. Using the obtained DNA information, identification was carried out by comparing the homology of base sequences in EzBioCloud (ChunLab, Inc.). Based on the identification results, 11 strains were finally isolated (Table 2).

Example 2. Selection of strains

2-1. Enzyme activity comparison

API zym (biomerieux, France) kit, which can confirm lipase activity, was used to select microorganisms with fat-degrading ability among 11 isolated microorganisms, and together with this, acetoin production can be confirmed. The activity of each enzyme was confirmed using the API 20E (biomerieux, France) kit (Table 3). At this time, in Table 3, positive is +, weakly positive is W (weak), and negative is -.

Based on the KIT test results, DKL1, YKL5, YKL15, and BBL10 strains that have lipolytic enzyme activity that can decompose medium-chain fatty acids (C12, Lauric acid) contained in coconut into short-chain fatty acids, and at the same time have the ability to produce acetoin were first tested. Were selected.

2-2. Comparison of aroma and sensory properties after fermentation of coconut

Coconut fermentation (refer to Experimental Example 1 described below for fermentation method) was performed to select a strain to be applied to coconut fermentation among the four selected strains, and the resultant coconut fermentation product was subjected to sensory evaluation. And the strain BBL10, which shows a strong butter flavor and a cumcum flavor, was finally selected, and the selected strain was named as Lactobacillus coagulans SMB 753 (Bacillus coagulans SMB 753) (Table 4).

2-3. Identification of selected strains

The final selected Bacillus coagulation SMB 753 strain was further identified. For the genetic identification of the firstly selected strain, cross-comparison was performed on 16s rDNA sequences using NCBI Blast (US National Library of Medicine) and EzBioCloud (ChunLab, Inc.), and as a result, Bacillus coagulation ( ATCC 7050=NCIB 9365=NCTC 10334=BCRC 10606=CCM 2013=CCUG 7417=CFBP 4225=CIP 66.25=DSM 1=HAMBI 1931=IAM 1115=JCM 2257=LMG 6326=KCCM 11715=KCTC 3625) and 99.79% similar Was confirmed.

After collecting 16s rRNA information of strains similar to Bacillus coagulation SMB753 strain, the nucleotide sequences were sorted using the MEGA program, and after visual observation and manual correction were made, a schematic was created using the Maximum Likelihood method. The created system diagram is shown in FIG. 1.

Additionally, for biochemical identification, differences between the SMB753 strain and the Bacillus coagulation KCCM 11715 strain were compared using API 50CH (biomerieux, France), a glycolytic enzyme activity assay kit (Table 5). At this time, in Table 5, positive is +, weakly positive is W (weak), and negative is -.

From the above results, Bacillus coagulation SMB753 strain was identified as a strain that is genetically close to Bacillus coagulation (ATCC 7050 = KCCM 11715), but among the glycolytic abilities, L-Arabinose, D- The difference was shown in D-Lactose, D-Melibiose, D-Saccharose, D-Raffinose, and D-Turanose. It was confirmed that they are different strains. Accordingly, Bacillus coagulation SMB 753 strain was deposited with the Korean Microbial Conservation Center, a microbial deposit organization, on March 29, 2019 under the accession number KCCM12474P.

Example 3. Analysis of Changes in Flavor Components of Coconut Fermented Products according to Strains

For the coconut fermented product prepared in the manner described in Experimental Example 1 below, fermentation was performed with the SMB753 strain and the Bacillus coagulans (ATCC 7050 = KCCM 11715) strain corresponding to the reference strain under the same conditions, and then described in Experimental Example 2. GC-MS was performed in one way. As a result, the difference in the production ability of the fragrance component compound is shown in FIG. 2.

As shown in Figure 2, acetoin and 2,3-butanediol (2,3) in the coconut fermented product using the Bacillus coagulus SMB753 strain compared to the coconut fermented product using the Bacillus coagulus (ATCC 7050 = KCCM 11715) strain. -butanediol) content was significantly different, and in addition, 2-Heptanone, 2-Nonanone, 3-methylbutanoic acid, and hexanoic acid ), and the like, also showed a difference in content.

Example 4. Analysis of changes in fragrance components before and after fermentation of fermented coconut

For a more accurate quantitative analysis based on the above results, an additional experiment was performed on the change of fragrance components before and after fermentation of the fermented coconut.

4-1. Quantitative analysis of volatile fragrance component profile alc (GC-MS)

Quantitative analysis and GC-O analysis were performed on the flavor of the fermented coconut prepared in the manner described in Experimental Example 1 below, and each of the experiments was performed in the manner described in Experimental Example 2 below.

Before fermentation, a total of 57 types of volatile fragrance components were identified, and if classified by group, 14 alcohols, 11 ketones, 11 acids, 8 aldehydes, 5 esters, 3 phenols, and 2 benzenes. There were two species, two kinds of pyrazines, and one kind of other compounds (Table 6). The highest volatile fragrance component was delta-octalactone, followed by delta-decalactone, ethyl octanoate, and ethyl decanoate. In order.

In the coconut fermented product, a total of 71 types of volatile fragrance components were identified, and classified by group, 17 alcohols, 12 ketones, 12 acids, 7 esters, 6 benzenes, 5 hydrocarbons, and aldehydes. There were 3 species, 2 phenols, 2 pyrazines, and 5 other compounds. The highest volatile fragrance component was acetoin, followed by octanoic acid, hexanoic acid, delta-octalactone, and 3-methylbutanoic acid. ) In order.

Coconut fermented products increased 14 kinds of volatile aroma components than before fermentation, of which 5 kinds of hydrocarbons and 3 kinds of alcohols were produced. Specifically, after fermentation, acetoin content increased significantly from 9.6 ppb to 1,989.7 ppb, and octanoic acid content increased from 23.1 ppb to 1,958.1 ppb.Hexanoic acid, 3-methylbutanoic acid, and acetic acid were not confirmed before fermentation, but after fermentation. The contents were 790.7, 446.2 and 264.7 ppb, respectively.

4-2. Fragrance Active Compound Analysis (GC-O)

A total of 15 fragrant active compounds were detected in the fermented coconut. By group, 6 ketones, 5 acids, 2 alcohols, 1 pyrazine, 1 aldehyde, 1 ester and 1 unknown. It was composed of (Table 7).

Specifically, the compound showing the strongest flavor is 3-methylbutanoic acid (methylbutanoic acid, cheesy, log2FD=8), acetoin (yogurt-like, log2FD=6), and delta-octalactone (coconut-like, log2FD). =6), delta-decalactone (coconut-like, log2FD=6), in that order, showed a strong scent. 3-Methylbutanoic acid has the strongest scent strength, showing cheesy scent characteristics, and delta-octalactone and delta-decalactone, which have coconut scent characteristics, also have strong scent strength, so the coconut fermented product has strong cheesy and coconut scent characteristics. appear.

As a result of the above results, the coconut fermented product using Bacillus coagulation SMB753 showed a high content of acetoin and octanoic acid, and the content increased significantly during the fermentation process, and hexanoic acid and 3-methylbutane showing Cheesy flavor. Acid was not detected in the raw material before fermentation, but it was confirmed that it was produced through the fermentation process.

Accordingly, the oil separated after fermentation of coconut using Bacillus coagulation SMB753 according to the present specification contains a large amount of ingredients that give the flavor of cheese, so it can be used as vegetable butter. It seems to be available in the source.

Experimental Example 1. Coconut fermentation and oil separation

After mixing 85 g of purified water with 15 g of coconut powder (supplied from Komida, Korea), sterilization and oil extraction were performed by heating at 121° C. for 20 minutes. Then, after cooling to 37°C, 2 g of a microorganism strain culture solution was inoculated, and fermentation was performed for 2 days (40 to 48 hours) at 37°C at 150 rpm. After the coconut fermentation was completed, it was heated at 65° C. for 3 hours to perform sterilization and separation of the oil layer. The separated oil was cooled to 4℃ to solidify and stored and used.

Experimental Example 2. Fragrance component analysis method-1

2-1. Volatile component SPME (Solid Phase Microextraction) extraction

The SPME extraction method is a method of extracting the analyte by maintaining the analyte at a constant temperature, moving volatile components to the headspace, and adsorbing it to a fiber coated with polymer. Divinylbenzene/Carboxen/polymethylsiloxane (DVB/CAR/PDMS, 50/30 μm) fiber ( Supelco, Bellefonte, PA, USA) was used. Put 4g of sample in a 20ml vial and maintain the equilibrium at 250 rpm for 10 minutes at 50˚C, and then expose the SPME fiber to adsorb volatile components for 10 minutes, and the desorption of volatile components is performed at the GC-MS inlet (250˚C). ) For 5 minutes.

2-2. Volatile component analysis using GC/MS

Volatile fragrance components of the sample extracted using SPME were analyzed by ISQ LT mass spectrometer (ThermoFisher scientific co.) connected to TRACE 1310 gas chromatography, and the column was DB-WAX (30m length x 0.25mm id x 0.25μm film thickness, J&W Scientific, Folsom, CA, USA) was used, and the GC oven temperature was maintained at 40°C for 5 minutes and raised to 110°C to 5°C per minute and maintained for 5 minutes.

After that, the temperature of the injector and ion source is 250°C and 250°C, respectively, and the mass scan range is 29°C, and the temperature of the injector and ion source is 250°C and 250°C. -180 amu And the mass spectra was obtained by electron ionization (EI) method at 70 eV. The carrier gas was helium at a flow rate of 0.8 mL/min and analyzed in splitless mode, and the total analysis run time was 48 minutes.

Experimental Example 3. Fragrance component analysis method-2

3-1. Conducted SAFE (Solvent assisted flavor evaporation)

In order to extract the volatile aroma components of the fermented coconut, the SAFE extraction method was used after solvent extraction was performed. 60 mL of dichloromethane re-distilled in 60 g of the sample was divided into three portions of 20 mL each, followed by solvent extraction for 1 hour, for a total of 3 hours. The extract was placed in a SAFE additional funnel and extracted by distillation at 40°C and 6×10-5 torr for 1 hour. In this case, 8.1 μg of 3-heptanol was used as an internal standard. The extract was frozen at -20°C for 12 hours to remove moisture, and the solvent layer was passed through 3 g of anhydrous sodium sulfate to remove excess moisture. The extract from which moisture was removed was concentrated to 200 μL using N2 gas and analyzed, and extraction was repeated three times.

3-2. Conducted gas chromatography-mass spectrometry (GC-MS)

The identification of volatile aroma components of the coconut fermentation was performed using an Agilent 7890B GC/Agilent 5977A mass selection analyzer (MSD) (Agilent Co., Palo Alto, CA, USA). DB-wax (60 m length × 0.25 mm id × 0.25 μm film thickness: J & W Scientific, Folsom, CA, USA) was used as the column, and the oven temperature was maintained at 40° C. for 5 minutes and then 5° C. to 200° C. The temperature was raised at a rate of /min and maintained for 30 minutes. The injector temperature was 200°C and the detector temperature was 250°C. Helium was used as the carrier gas and the flow rate was 1.0 mL/min. The ionization voltage was 70 eV, and the range (m/z) of the molecular weight to be analyzed was 33 to 350.

3-3. Conducted gas chromatography-olfactometry (GC-O)

GC-O was performed to analyze the aroma-active compound as a factor influencing the preference of coconut fermented products. In addition, aroma extract dilution analysis (AEDA) was performed to confirm the strength of the fragrance active compound. The SAFE extract was diluted by a factor of 2 with dichloromethane, and each dilution was sniffed to a dilution factor where no odor was detected anymore. The higher the flavor dilution (FD) factor, the higher the relative flavor intensity. GC-O used a Varian 3800 (Varian Instrument Group, Walnut Creek, CA, USA). As a detector, FID (Flame ionization detector) was used, branching from the column and sniffing was performed using a nose cone. DB-wax (30 m length × 0.25 mm i.d. × 0.25 μm film thickness: J & W Scientific, Folsom, CA, USA) was used as the column. The oven temperature was maintained at 40° C. for 5 minutes and then raised to 200° C. at a rate of 8° C./min and maintained for 10 minutes. The injector temperature was 200°C and the detector temperature was 250°C. Helium was used as a carrier gas, and the flow rate was 1.4 mL/min.

Korea Microorganism Conservation Center (overseas) KCCM12474P 20190329

<110> SEMPIO FOODS COMPANY <120> Bacillus coagulans with excellent butter and cheese flavor productivity <130> 19p118ind <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1424 <212> DNA <213> Bacillus coagulans <400> 1 tgcagtcgtg cggacctttt aaaagcttgc ttttaaaagg ttagcggcgg acgggtgagt 60 aacacgtggg caacctgcct gtaagactgg gataacgccg ggaaaccggg gctaatacca 120 gatagttttt tcctccgcat ggaggaaaaa ggaaaggcgg cttcggctgc cacttacaga 180 tgggcccgcg gcgcattagc tagttggcgg ggtaacggcc caccaaggca acgatgcgta 240 gccgacctga gagggtgatc ggccacattg ggactgagac acggcccaaa ctcctacggg 300 aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360 tgaagaaggc cttcgggtcg taaaactctg ttgccgggga agaacaagtg ccgttcgaac 420 agggcggcgc cttgacggta cccggccaga aagccacggc taactacgtg ccagcagccg 480 cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaagcg cgcgcaggcg 540 gcttcttaag tctgatgtga aatcttgcgg ctcaaccgca agcggtcatt ggaaactggg 600 aggcttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat 660 gtggaggaac accagtggcg aaggcggctc tctggtctgt aactgacgct gaggcgcgaa 720 agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct 780 aagtgttaga gggtttccgc cctttagtgc tgcagctaac gcattaagca ctccgcctgg 840 ggagtacggc cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 900 gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgacc 960 tccctggaga cagggccttc cccttcgggg gacagagtga caggtggtgc atggttgtcg 1020 tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgaccttag 1080 ttgccagcat tgagttgggc actctaaggt gactgccggt gacaaaccgg aggaaggtgg 1140 ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggatg 1200 gtacaaaggg ctgcgagacc gcgaggttaa gccaatccca gaaaaccatt cccagttcgg 1260 attgcaggct gcaacccgcc tgcatgaagc cggaatcgct agtaatcgcg gatcagcatg 1320 ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgta 1380 acacccgaag tcggtgaggt aaccttacgg agccagccgc cgaa 1424

Claims (18)

As a strain having at least one flavor-producing ability of butter and cheese,
The strain is Bacillus coagulans SMB 753 strain (accession number KCCM12474P),
The flavor-producing ability is acetoin, 2,3-butanedione, 2-heptanone, 2-nonanone, 3-methylbutanoic acid ( 3-methylbutanoic acid), octanoic acid and hexanoic acid.
The concentration of the compound produced when cultured at 37° C. for 48 hours after inoculating the strain at 2% by volume relative to the total volume of the medium containing 15% by weight of coconut is one or more of the following strains:
Acetoin from 1500 ppb to 2500 ppb;
2-butanedione is 20 ppb to 40 ppb;
2-heptanone is 35 ppb to 50 ppb;
2-nonanone is 65 ppb to 75 ppb;
3-methylbutanoic acid is 400 ppb to 500 ppb;
Octanoic acid is 1500 ppb to 2500 ppb; And
Hexanoic acid is 750 ppb to 850 ppb. delete The method of claim 1,
The strain is capable of growing in a salt concentration condition of 1 to 10% (v/v), a strain. The method of claim 1,
The strain has an activity of at least one of esterase and lipase, a strain. The method of claim 4,
The activity is for a fatty acid having 4 to 14 carbon atoms, strain. The method of claim 1,
The strain has a 16S rDNA comprising the nucleotide sequence of SEQ ID NO: 1, strain. delete delete The strain according to any one of claims 1 and 3 to 6; And a microbial preparation comprising at least one of the culture medium of the strain. As a fermented coconut,
The fermented product is a coconut fermented product obtained by fermenting coconut with the strain according to any one of claims 1 and 3 to 6. The method of claim 10,
The fermented product is that only the oil layer is separated, the coconut fermented product. A fragrance composition comprising the fermented coconut of claim 10. The method of claim 12,
The flavoring is to represent the flavor of at least one of butter and cheese, flavoring composition. The method of claim 12,
The perfume composition is one or more of food additives, cosmetic additives, fiber additives and pharmaceutical additives, perfume composition. The strain according to any one of claims 1 and 3 to 6 in the coconut; And treating one or more of the culture solutions of the strain; containing, a method for producing a perfume composition. The method of claim 15,
The method for preparing the perfume composition further comprises a heat treatment step of mixing purified water in the coconut before the treating step and then maintaining it at 90°C to 130°C for 5 to 30 minutes to heat treatment. The method of claim 15,
The treating step includes the strain; And After the treatment of at least one of the culture medium of the strain comprising the step of leaving for 40 to 48 hours at 35 to 40 ℃, a method for producing a perfume composition. The method of claim 15,
The method for preparing the perfume composition comprises the steps of sterilizing by maintaining at 50°C to 70°C for 30 to 90 minutes after the treating step;
A separation step of separating only the oil layer from the fermented product obtained in the sterilization step; And
A solidification step of cooling and solidifying the coconut fermented oil obtained in the separation step at 0 °C to 10 °C; further comprising, a method for producing a perfume composition.

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