KR20180056053A - Use of 2-hydroxyisocaproic acid for indicator of fermentation - Google Patents

Abstract

The present invention relates to the use of 2-hydroxy isocaproic acid as a fermentation indicator, and since it is possible to confirm changes in the microflora of microorganisms involved in fermentation by comparing the amount of 2-hydroxy isocaproic acid produced in fermented food before and after fermentation, the fermentation degree (ripening degree) of the fermented food can be promptly grasped by using the changes in the microflora of microorganisms as an index.

Description

Use of 2-hydroxyisocaproic acid as an indicator of fermentation {Use of 2-hydroxyisocaproic acid for indicator of fermentation}

The present invention relates to a novel use as a fermentation index of 2-hydroxyisocaproic acid.

Kimchi, which is a typical fermented food, is an excellent food which produces a unique flavor by natural fermentation of microorganisms derived from materials and supplies dietary fiber, vitamins and minerals. The types and distribution patterns of lactic acid bacteria involved in kimchi fermentation are significantly affected by the materials, salinity, fermentation temperature and the like, while those of Leuconostoc sp.), in way Cellar (Weissella sp.), it is known that the lactic acid bacteria belonging to genus like Lactobacillus (Lactobacillus sp.) involved in zymogen state. In order to identify Kimchi lactic acid bacteria, morphological characteristics and biochemical characteristics have been mainly focused. Recently, however, attempts have been made to identify microorganisms involved in kimchi fermentation through molecular genetic methods during kimchi fermentation. For example, PCR, protein-SDS-PAGE, PCR-DGGE technology, genome probing microarray, and 16S rRNA sequencing are used for microbial species-specific primers. However, these methods require a lot of time and cost, and a method for confirming microbial changes that are involved in kimchi fermentation quickly and efficiently is needed.

2-Hydroxyisocaproic acid (2-HICA), on the other hand, is a mixture of leucic acid, 2-hydroxy-4-methylvaleric acid, 2- Also known as 2-hydroxy-4-methylpentanoic acid, it is known as the final metabolite of leucine. Hydroxyisocaproate dehydrogenase (2-hydroxyisocaproate dehydrogenase) is produced from 2-ketoisocaproic acid by the action of amino acid transferase (leucine) Hydroxyisocaproic acid by the action of a hydroxyl group (non-patent document 1). 2-hydroxyisocaproic acid is mainly produced by Lactobacillus plantarum , which is found in many fermented foods such as cheese, wine, soy sauce and the like (non-patent documents 2 and 3) or isolated from animal samples, Staphylococcus aureus , Pseudomonas aeruginosa , Fusobacterium < RTI ID = 0.0 > nucleatum), Candida (Candida), has been reported to exhibit antibacterial activity against bacteria in Aspergillus (Aspergillus sp.) (Non-Patent Document 4 and 5).

In addition, microbial-derived metabolites that can estimate the change of microorganism through the change of specific substance production from kimchi during the kimchi fermentation process have not been studied. Currently, changes in pH or acidity caused by lactic acid bacteria- And the degree of fermentation of kimchi is estimated.

Accordingly, the present inventor intends to predict the degree of fermentation of kimchi by using the metabolites produced from lactic acid bacteria of Kimchi as fermentation index through the present invention.

Mero et al. Journal of the International Society of Sports Nutrition, 2010, 7: 1 Van Wyk et al. Journal of Food Science, 1967, Vol. 32, Issue 6, pp 664-668 B. A Smit et al. Applied Microbiology and Biotechnology, 2004, Vol. 64, Issue 3, pp 396-402 M. Sakko et al. International Journal of Antimicrobial Agents, 2012, Vol. 39, Issue 6, pp 539-540 M. Sakko et al. Mycoses, 2013, Vol. 57, Issue 4, pp 214-221

The present invention is intended to provide the use of 2-hydroxyisocaproic acid as a fermentation index.

The present inventors have made efforts to find a new fermentation index by replacing the pH or acidity which was conventionally used as an fermentation index of kimchi. As a result, it has been found that the fermentation efficiency of 2-hydroxyisocaproic acid 2-Hydroxyisocaproic acid, 2-HICA) can be used as a fermentation index, thereby completing the present invention.

The present invention provides a method for monitoring the microbial flora change during fermentation period, comprising comparing the amount of 2-hydroxy isocaproic acid produced before and after fermentation from a fermented food sample.

According to the above method, it is confirmed that the amount of 2-hydroxyisocaproic acid produced after fermentation is remarkably increased compared with that before fermentation, so that the increase in the dominance rate of the lactic acid bacteria involved in fermentation in the fermented food can be predicted, Hydroxyisocaproic acid can be used as a fermentation index to monitor the degree of fermentation of the fermented food.

In the present invention, a fermented food means a food in which microorganisms beneficial for fermentation are increased according to the fermentation period. Although the present invention uses Kimchi as a fermented food, it is not limited thereto. Microbes beneficial to the fermentation in Lactobacillus (Lactobacillus sp.) And the flow in the Pocono stock (Leuconostoc sp.) Can include a strain, e.g., stock flow Pocono lactis (Leuconostoc but is not limited to, one or more lactic acid bacteria selected from the group consisting of lactis , Lactobacillus plantarum , and Leuconostoc mesenteroides . In the present invention, the fermented food may be fermented at a temperature of 1 to 10 ° C, for example, 2 to 8 ° C and 3 to 6 ° C for at least 7 days, and fermentation conditions such as temperature and fermentation period may be appropriately adjusted according to the fermented food have.

Further, in the present invention, 2-hydroxyisocaproic acid may be produced by a microorganism involved in fermentation, and may be produced by a strain of Lactobacillus sp. And Leuconostoc sp. Lt; / RTI > In one embodiment, the Lactobacillus and Leuconostoc sp. Strains are selected from the group consisting of Leuconostoc lactis , Lactobacillus plantarum , and Leuconostoc mesenteroides . But is not limited thereto.

The 2-hydroxyisocaproic acid is a compound represented by the following Formula 1, and the IUPAC name is 2-Hydroxy-4-methylpentanoic acid, which is lecic acid or 2-Hydroxy-4-methylvaleric acid. -4-methylvaleric acid).

[Chemical Formula 1]

In general, 2-hydroxyisocaproic acid is known as a component exhibiting antimicrobial activity and muscle-assisting effect. However, since the amount of 2-hydroxyisocaproic acid produced in the fermented food is measured as described in the present invention, The use as an fermentation index which can be confirmed is revealed by the present inventors. Therefore, the present invention can provide a use as a fermentation index of 2-hydroxyisocaproic acid.

In the present invention, the production amount of 2-hydroxyisocaproic acid can be measured through mass spectrometry of a fermented food sample or can be measured through a probe capable of detecting 2-hydroxyisocaproic acid.

In one embodiment, the amount of 2-hydroxyisocaproic acid produced through mass spectrometry can be determined by liquid chromatography-mass spectrometry (LC / MS / MS), MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) , SELDI-TOF (Sulfate Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, liquid chromatography-mass spectrometry (LC-MS) and ultra high performance liquid chromatography (UPLC) And can be applied to any method which is usually used for mass spectrometry of a compound.

According to another embodiment, the amount of 2-hydroxyisocaproic acid produced can be measured using a probe capable of detecting 2-hydroxyisocaproic acid. The probe capable of detecting 2-hydroxyisocaproic acid may be a detection aptamer that specifically binds to 2-hydroxyisocaproic acid. The aptamer means a detection agent having high specificity and affinity for a specific substance. In the present invention, the aptamer may be an agent specifically binding to 2-hydroxyisocaproic acid.

According to the present invention, the amount of 2-hydroxyisocaproic acid produced in the fermented food can be measured using the probe, and compared with that before fermentation, the change in the microflora of the fermenting microorganisms present in the fermented food can be monitored. The fermenting microorganism may be a lactobacillus genus or a leuconoblast genus.

In one embodiment of the invention, the fermented food may be a kimchi.

In the following examples, the amount of 2-hydroxyisocaproic acid produced according to the fermentation cycle of commercially available kimchi was measured and compared with the change in microbial culture according to the fermentation period, and the fermentation of kimchi through 2-hydroxyisocaproic acid The results of this study are as follows.

Therefore, by confirming the degree of production of 2-hydroxyisocaproic acid, the present invention can predict changes in the microflora of Lactobacillus genus and Leuconostoc strain, which are involved in fermentation in fermented foods, Can provide the use of isocaproic acid.

The present invention provides a composition for the detection of 2-hydroxyisocaproic acid for monitoring the degree of fermentation through microbial culture changes in fermented foods, including a probe capable of detecting 2-hydroxyisocaproic acid.

The present invention also provides a kit for the detection of 2-hydroxyisocaproic acid for monitoring the degree of fermentation through microbial changes in a fermented food, comprising the composition for detection.

In the detection composition and the detection kit according to the present invention, the content of the probe capable of detecting the 2-hydroxyisocaproic acid, 2-hydroxyisocaproic acid, the fermented food, and the fermentation microorganism is The content can be applied or applied as it is.

By detecting the 2-hydroxyisocaproic acid in the fermented food through the composition for detection and the kit for detection, it is possible to quickly confirm the change in the microflora of the fermented microorganism and to grasp the degree of fermentation of the fermented food.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

The present invention relates to the use of 2-hydroxyisocaproic acid as a fermentation index and it is possible to confirm the change in the microflora of microorganisms involved in fermentation by comparing the amount of 2-hydroxyisocaproic acid produced in the fermented food before and after fermentation, The fermentation degree of the fermented food (aging degree) can be grasped quickly by using this as an index.

1 is a graph showing the amount of 2-hydroxyisocaproic acid produced in a fermented food according to a fermentation cycle.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

[ Example ]

Example  1: Microorganisms according to fermentation period of commercial kimchi Lichen  Confirm change

Four commercially available kimchi products were collected and analyzed for microbial microflora in the early stage of kimchi fermentation (fermentation 1 week). For each kimchi fermentation cycle, DNA was extracted from FastDNA spin kit for soil (MP Biomedical, Santa Ana, CA). PCR was performed using the extracted genomic DNA as a template. The PCR conditions were 25 cycles of denaturation at 94 DEG C for 3 minutes, annealing at 55 DEG C for 30 seconds, extension at 72 DEG C for 30 seconds, Lt; RTI ID = 0.0 > 72 C < / RTI > for 5 minutes. Sequence analysis of the amplified DNA was carried out in a shear lap using the Illumina MiSeq Sequencing system (Illumina, USA). Microbial flora were analyzed by comparing the dominance rates of Lactobacillus sp. Strain and Leuconostor sp. Strain in kimchi samples. The results are shown in Table 1 below.

Kimchi One 2 3 4 Cycle 0 One 0 One 0 One 0 One Lactobacillus 11.7 28 4.5 44.6 6.3 34.2 5.7 54 Leuconostoc 17.3 4.2 17.2 25.6 5.5 26.4 6.1 6.3 Others 60.2 67.7 62.7 29.3 83.6 39 80 39.6

As shown in Table 1, it was confirmed that the ratio of Lactobacillus sp. Strain to Leuconostor sp. Strain in kimchi was remarkably increased at the early stage of kimchi fermentation. Especially, all strains of Lactobacillus sp. Were increased in fermentation period in all kimchi. As a result, it was confirmed that as the fermentation progressed, the dominance rate of the strain of the genus Lactobacillus and the strain of the genus Leuconostoc was increased.

Example  2: 2- Hydroxyisocaproic acid  Production amount measurement

The amount of 2-hydroxyisocaproic acid produced at the beginning of the fermentation of four types of kimchi currently marketed was confirmed. Each kimchi sample according to fermentation cycle was centrifuged at 12,000 rpm for 10 minutes, and the supernatant, excluding the precipitate, was extracted with a seq-pak cartridge (Waters Co.). LC-MS / MS analysis was performed in negative mode using electrospray ionization using an AB SCIEX TripleTOF® 5600+ system (AB SCIEX) equipped with AB SCIEX analyst software. Water and acetonitrile containing 10 mM ammonium acetate were used in an ACQUITY UPLC BEH C18 column (130 ANGSTROM, 1.7 mu m, 2.1 mm x 100 mm), 95 to 60% (v / v) v / v) for 8 minutes, 95% (v / v) of water for 5 minutes and separated at a flow rate of 0.5 ml / min. The yield of 2-hydroxyisocaproic acid was quantified using multiquant (AB SCIEX) software and shown in FIG. In addition, the pH and acidity of the same kimchi samples were measured, and the results are shown in Table 2.

pH Acidity Products Kimchi Cycle 0 One 0 One One 5.54 4.17 0.33 0.74 2 5.79 4.48 0.29 0.68 3 5.7 4.44 0.33 0.66 4 5.93 4.43 0.35 0.58

As shown in Fig. 1 and Table 2, it was found that the production of 2-hydroxyisocaproic acid also increased in the early stage of kimchi fermentation, as in the case where the pH was steadily lowered and the acidity became higher as the kimchi fermentation progressed there was. By confirming that the production of 2-hydroxyisocaproic acid increases in the first week of fermentation in the commercially available kimchi samples through this Example, it was confirmed that lactic acid bacteria belonging to the genus Lactobacillus, which is the fermented lactic acid bacteria of kimchi, We can predict the increase of the dominance rate of the strain in stock and the degree of fermentation of Kimchi.

Claims (11)

Comparing the amount of 2-hydroxyisocaproic acid produced before and after fermentation of the fermented food sample with the fermented food sample. The method according to claim 1,
The amount of 2-hydroxyisocaproic acid produced was determined by liquid chromatography-mass spectrometry (LC / MS / MS), MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI- Wherein the microorganism is measured by at least one method selected from the group consisting of ionization time of flight mass spectrometry analysis, liquid chromatography-mass spectrometry (LC-MS) and ultra high performance liquid chromatography (UPLC) How to monitor changes. The method according to claim 1,
Wherein the amount of 2-hydroxyisocaproic acid produced is measured by a probe capable of detecting 2-hydroxyisocaproic acid. The method of claim 3,
Wherein the probe capable of detecting 2-hydroxyisocaproic acid is an aptamer that specifically binds to 2-hydroxyisocaproic acid. The method according to claim 1,
The fermented food is a method of monitoring microbial changes of microorganisms during fermentation. The method according to claim 1,
Wherein the fermented food is fermented at 1 to 10 DEG C for at least 7 days. The method according to claim 1,
Wherein the 2-hydroxyisocaproic acid is produced by a strain of Leuconostoc sp. And / or a strain of Lactobacillus sp.. The method according to claim 1,
2-hydroxyisocaproic acid is one produced by one or more lactic acid bacteria selected from the group consisting of Leuconostoc lactis, Lactobacillus plantarum and Leuconostoc mesenteroides Phosphorus, and fermentation period. A composition for the detection of 2-hydroxyisocaproic acid for monitoring the degree of fermentation through microbial pathogen changes in a fermented food product, comprising a probe capable of detecting 2-hydroxyisocaproic acid. 10. The method of claim 9,
Wherein the probe capable of detecting 2-hydroxyisocaproic acid is a detection aptamer that specifically binds to 2-hydroxyisocaproic acid. A kit for the detection of 2-hydroxyisocaproic acid for monitoring the degree of fermentation through microbial culture changes in fermented foods, comprising the composition according to claim 9.

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