KR102212909B1 - Use of 2-hydroxyisocaproic acid for indicator of fermentation - Google Patents

Abstract

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

Description

Use of 2-hydroxyisocaproic acid for indicator of fermentation

The present invention relates to a novel use of 2-hydroxyisocapronic acid as a fermentation indicator.

Kimchi, a representative fermented food, produces its own flavor through natural fermentation of microorganisms derived from ingredients, and is an excellent food that supplies dietary fiber, vitamins, and minerals. The types and distribution patterns of lactic acid bacteria involved in kimchi fermentation are significantly affected by ingredients, salinity, and fermentation temperature, but Leuconostoc genus ( Leuconostoc sp.) , Weissella sp., and Lactobacillus sp. are known to be involved as main fermenting bacteria. For the identification of kimchi lactic acid bacteria, morphological and biochemical characteristics have traditionally been the main focus, but recently, attempts have been made to identify microorganisms involved in kimchi fermentation through molecular genetic methods during kimchi fermentation. As an example, methods such as PCR using microbial species-specific primers, protein-SDS-PAGE, PCR-DGGE technology, genome probing microarray, and 16S rRNA sequencing are used. However, these methods require a lot of time and cost, so a method of quickly and efficiently confirming changes in the microbial flora involved in kimchi fermentation is needed.

On the other hand, 2-Hydroxyisocaproic acid (2-HICA) is leucic acid, 2-hydroxy-4-methylvaleric acid, or 2- Also called hydroxy-3-methylpentanoic acid (2-hydroxy-4-methypentanoic acid), it is known as the final metabolite of leucine. 2-Hydroxyisocaproic acid is produced from leucine, a precursor, by the action of aminotransferase, and then hydroxyisocaproate dehydrogenase (Hydroxyisocaproate dehydrogenase) It is produced as 2-hydroxyisocaproic acid by the action (Non-Patent Document 1). 2-hydroxyisocaproic acid is mainly found in various fermented foods such as cheese, wine, and soy sauce (Non-Patent Documents 2 and 3) or is produced by Lactobacillus plantarum isolated from animal samples, and Staphylo Staphylococcus aureus , Pseudomonas aeruginosa , Fusobacterium nucleatium nucleatum), Candida (Candida), has been reported to exhibit antibacterial activity against bacteria in Aspergillus (Aspergillus sp.) (Non-Patent Document 4 and 5).

In addition, metabolites derived from microorganisms that can estimate the change of microbial flora through changes in the amount of specific substances produced from kimchi during kimchi fermentation have not been studied. Currently, changes in pH or acidity caused by organic acids produced by lactic acid bacteria are not studied. The degree of kimchi fermentation is estimated through this method.

Accordingly, the present inventor intends to predict the degree of fermentation of kimchi by using metabolites generated from lactic acid bacteria of kimchi as a 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 to provide the use of 2-hydroxyisocapronic acid as a fermentation indicator.

Accordingly, the present inventors have tried to find new fermentation indicators by replacing pH or acidity, which were conventionally used as fermentation indicators of kimchi, and as a result, 2-hydroxyisocapronic acid (2-hydroxyisocapronic acid) produced by fermenting microorganisms that increases as kimchi is fermented ( The present invention was completed by confirming that 2-Hydroxyisocaproic acid, 2-HICA) can be used as a fermentation indicator.

The present invention provides a method for monitoring changes in microbial flora according to a fermentation period, comprising comparing the amount of 2-hydroxyisocapronic acid produced before and after fermentation from a fermented food sample.

According to the above method, by confirming that the production amount of 2-hydroxyisocapronic acid significantly increases after fermentation compared to before fermentation, it is possible to predict an increase in the dominant rate of lactic acid bacteria involved in fermentation in fermented foods. The degree of fermentation of fermented foods can be monitored using hydroxyisocapronic acid as a fermentation indicator.

In the present invention, the fermented food refers to a food in which microorganisms beneficial for fermentation increase according to the fermentation period. Although kimchi is used as a fermented food in the present invention, it is not limited thereto. Microorganisms beneficial for the fermentation may include Lactobacillus sp. and Leuconostoc sp. strains, such as Leuconostoc Lactis ( Leuconostoc ). lactis ), Lactobacillus plantarum , and Leuconostoc mesenteroides may be one or more lactic acid bacteria selected from the group consisting of, but is not limited thereto. In the present invention, the fermented food may be fermented at 1 to 10°C, such as 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.

In addition, in the present invention, 2-hydroxyisocapronic acid may be produced by microorganisms involved in fermentation, and produced by strains of Lactobacillus sp. and Leuconostoc sp. Can be. In one embodiment, the Lactobacillus and Leuconostoc strains are selected from the group consisting of Leuconostoc lactis , Lactobacillus plantarum , and Leuconostoc mesenteroides . It may include one or more, but is not limited thereto.

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

[Formula 1]

In general, 2-hydroxyisocaproic acid is known as a component exhibiting antibacterial activity and an auxiliary effect of muscle formation, but the degree of fermentation is determined by measuring the production amount of 2-hydroxyisocaproic acid in fermented foods as suggested in the present invention. The identifiable, use as a fermentation indicator has been discovered by the present inventors. Accordingly, the present invention can provide the use of 2-hydroxyisocapronic acid as a fermentation indicator.

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

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

According to another embodiment, the production amount of 2-hydroxyisocapronic acid may be measured using a probe capable of detecting 2-hydroxyisocapronic acid. The probe capable of detecting 2-hydroxyisocapronic acid may be an aptamer for detection that specifically binds to 2-hydroxyisocapronic acid. The aptamer refers to a detection agent having high specificity and affinity for a specific substance, and in the present invention may mean an agent that specifically binds to 2-hydroxyisocapronic acid.

According to the present invention, by measuring the production amount of 2-hydroxyisocapronic acid in the fermented food using the probe, and comparing it with before fermentation, it is possible to monitor the change in the colony of fermenting microorganisms present in the fermented food. The fermenting microorganisms may be strains of the genus Lactobacillus and genus Leukonostock.

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

In the following examples, by measuring the amount of 2-hydroxyisocapronic acid produced according to the fermentation cycle of commercially available kimchi, and comparing this with the change in the microbial flora according to the fermentation cycle, fermentation of kimchi through 2-hydroxyisocapronic acid It was confirmed that the degree can be predicted.

Accordingly, the present invention can predict changes in the colony of Lactobacillus and leukonostock strains involved in fermentation in fermented foods by confirming the degree of production of 2-hydroxyisocapronic acid, and thus 2-hydroxy as a fermentation indicator The use of isocapronic acid can be provided.

The present invention provides a composition for detecting 2-hydroxyisocapronic acid for monitoring the degree of fermentation through changes in microbial flora in fermented food, including a probe capable of detecting 2-hydroxyisocapronic acid.

In addition, the present invention provides a kit for detection of 2-hydroxyisocapronic acid for monitoring the degree of fermentation through changes in microbial flora in fermented food, including the detection composition.

In the detection composition and detection kit according to the present invention, the information on the probe capable of detecting the 2-hydroxyisocapronic acid, 2-hydroxyisocapronic acid, fermented food and fermented microorganisms is all described above. The content can be applied as it is or applied mutatis mutandis.

By detecting 2-hydroxyisocapronic acid in the fermented food through the detection composition and the detection kit, it is possible to quickly check the change in the flora of the fermented microorganism and the degree of fermentation of the fermented food.

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only these embodiments are intended to complete the disclosure of the present invention, and to provide ordinary knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to those who have it, and the invention is only defined by the scope of the claims.

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

1 is a graph showing the measurement of the amount of 2-hydroxyisocapronic acid produced in fermented foods according to the fermentation cycle.

Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[ Example ]

Example 1: Microorganisms according to fermentation period of commercially available kimchi Flora Check for change

Four types of kimchi that are currently on the market were collected, and the microbial population analysis was performed in the early stage of kimchi fermentation (week 1 of fermentation). Each kimchi according to the fermentation cycle was extracted with DNA through FastDNA spin Kit for soil (MP Biomedical, Santa Ana, CA). PCR was carried out using the extracted genomic DNA as a template, and the PCR conditions were 25 cycles with denaturation 94°C for 3 minutes, annealing 55°C for 30 seconds, and extension 72°C for 30 seconds, and the last cycle Performed the stretching step at 72° C. for 5 minutes. Sequencing of the amplified DNA was performed in CheonLab using the Illumina MiSeq Sequencing system (Illumina, USA). Microbial colonies were analyzed by comparing the dominance ratios of the strains of Lactobacillus genus and the strains of Leukonostock genus in the kimchi sample through the above method. 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 can be seen in Table 1, it was confirmed that the ratio of Lactobacillus sp. strain and Leukono Stock sp. strain in most kimchi at the beginning of kimchi fermentation significantly increased. Among them, strains of the genus Lactobacillus in all kimchi significantly increased with the fermentation cycle. Through this, it was confirmed that the dominance rate of the strains of the genus Lactobacillus and the strains of genus Leukonostock increased as the fermentation proceeded.

Example 2: Commercially available kimchi 2- Hydroxyisocapronic acid Measurement of production

The production amount of 2-hydroxyisocapronic acid at the beginning of fermentation of four kinds of kimchi on the market was confirmed. Each kimchi sample according to the fermentation cycle was centrifuged at 12,000 rpm for 10 minutes, and the supernatant excluding the sediment was extracted using a seq-pak cartridge (Waters Co.). LC-MS/MS analysis was performed in negative mode by electrospray ionization using the AB SCIEX TripleTOF® 5600+ system (AB SCIEX) equipped with AB SCIEX analyst software. Water containing 10 mM ammonium acetate and acetonitrile were used in ACQUITY UPLC BEH C18 Column (130 ÅA, 1.7 μm, 2.1 mm×100 mm), and 95 to 60% (v/v) of water for 17 minutes, 45 to 10% of water ( v/v) was performed for 8 minutes and water 95% (v/v) for 5 minutes, followed by separation at a flow rate of 0.5 ml/min. The amount of 2-hydroxyisocapronic acid produced was quantified using multiquant (AB SCIEX) software and shown in FIG. 1. In addition, the pH and acidity of the same kimchi sample were measured and compared and shown in Table 2.

pH Acidity Product 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 can be seen that the production of 2-hydroxyisocapronic acid also increases at the beginning of kimchi fermentation, in the same way as the result of steadily decreasing pH and increasing acidity as kimchi fermentation proceeds. there was. By confirming that the production of 2-hydroxyisocapronic acid is increased in the first week of fermentation in a commercial kimchi sample through this example, the strain of the genus Lactobacillus, which is the fermentation lactic acid bacteria of kimchi, and leukono in the fermentation period (week 1 of fermentation). It is possible to predict an increase in the dominance rate of strains in the stock and to determine the degree of fermentation of kimchi.

Claims (11)

Comprising the step of comparing the production amount of 2-hydroxyisocaproic acid before and after fermentation from the fermented food sample, a method for monitoring microbial flora change according to the fermentation period. The method of claim 1,
The amount of 2-hydroxyisocapronic acid produced was determined by liquid chromatography-mass spectrometry (LC/MS/MS), Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, and SELDI-TOF (Sulface Enhanced Laser Desorption/ Ionization Time of Flight Mass Spectrometry) analysis, liquid chromatography-mass spectrometry (LC-MS), and ultra-high performance liquid chromatography (UPLC), which is measured by one or more methods selected from the group consisting of microbial colonies according to fermentation period How to monitor change. The method of claim 1,
The production amount of 2-hydroxyisocapronic acid is measured by a probe capable of detecting 2-hydroxyisocapronic acid, a method of monitoring changes in microbial flora according to fermentation period. The method of claim 3,
A probe capable of detecting 2-hydroxyisocapronic acid is an aptamer that specifically binds to 2-hydroxyisocapronic acid, a method for monitoring changes in microbial flora according to fermentation period. The method of claim 1,
Fermented food is kimchi, a method of monitoring changes in microbial flora according to fermentation period. The method of claim 1,
Fermented food will be fermented for at least 7 days at 1 to 10 ℃, a method of monitoring changes in microbial flora according to the fermentation period. The method of claim 1,
2-Hydroxyisocapronic acid is produced by the Leuconostoc sp. and/or Lactobacillus sp. strain, a method for monitoring changes in microbial flora according to the fermentation period. The method of claim 1,
2-hydroxyisocapronic acid is produced by one or more lactic acid bacteria selected from the group consisting of Leuconostoc lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides. Phosphorus, a method of monitoring microbial flora changes according to fermentation period. A composition for detection of 2-hydroxyisocapronic acid for monitoring the degree of fermentation through a change in microbial flora in fermented food, comprising a probe capable of detecting 2-hydroxyisocapronic acid. The method of claim 9,
A probe capable of detecting 2-hydroxyisocapronic acid is a detection aptamer that specifically binds to 2-hydroxyisocapronic acid, a composition for detection. A kit for detecting 2-hydroxyisocapronic acid for monitoring the degree of fermentation through a change in microbial flora in fermented food, comprising the composition according to claim 9.

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