JP6723427B1 - Lactic Acid Bacteria with Sour Taste Inhibition and Fermented Pickle Production Method Using the Same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lactic acid bacterium capable of suppressing sour expression and a method for producing a fermented pickle using the same. SOLUTION: A method for producing a fermented pickle, which comprises fermenting a pickle raw material using Leuconostoc fallax A-1 strain lactic acid bacteria having a deposit number NITE P-03040, and the fermentation thereof. pickles. [Selection diagram] Fig. 9

Description

TECHNICAL FIELD The present invention relates to a lactic acid bacterium that suppresses sour expression and a method for producing fermented pickles using the same.

Lactic acid fermentation with lactic acid bacteria has long been used for the production of various fermented pickles. In the production of fermented pickles using lactic acid fermentation, as fermentation progresses, the production of organic acids such as lactic acid lowers the pH and causes acidity. However, the fermentation rate varies depending on the type of lactic acid bacteria contributing to the fermentation and the fermentation conditions, and if the fermentation becomes excessive, the sourness becomes too strong, so it is difficult to produce a fermented pickle having a stable taste. In addition, fermented pickles that are sold without being sterilized are still in a situation where excessive sourness is likely to develop because the fermentation proceeds even after the purchase by the consumer. Therefore, development of a fermented pickle that does not easily develop an excessive sourness is desired.

In the production of fermented pickles such as fermented kimchi, the lactic acid bacterium Leuconostoc mesenteroides (Leuconostoc mesenteroides) starts rapid growth from the early stage of fermentation and produces various metabolites containing organic acids such as lactic acid and acetic acid. Carbon dioxide is discharged to maintain the inside of the fermented pickles under anaerobic conditions to strongly suppress the growth of aerobic bacteria. However, in the middle stage of fermentation, Leuconostoc mesenteroides will decrease, and other lactic acid bacteria including Lactobacillus plantarum will grow vigorously. It is known that Lactobacillus plantarum produces a large amount of lactic acid, so in the latter half of fermentation, low acid resistance Leuconostoc mesenteroides disappears, and Lactobacillus plantarum and other plants The acidity-expressing lactic acid bacterium becomes dominant, and the acidity of the fermented pickles is likely to rapidly increase (Non-Patent Document 1). In order to suppress excessive expression of sourness in fermented pickles, it is desired to suppress the growth of sourness-expressing lactic acid bacteria.

In kimchi fermentation, mannitol is produced by the conversion of fructose to mannitol by mannitol dihydrogenase or the like. Mannitol brings a mellow sweetness to kimchi, and since it is a sugar alcohol that is difficult for bacteria to assimilate, it can suppress fermentation and prevent an excessive sourness from occurring (Patent Document 1).

Patent Document 1 discloses an acid-resistant Leuconostoc mesenteroides DRC0512 strain excellent in mannitol production ability and a method for producing kimchi using the same.

Patent Document 2 discloses a lactic acid bacterium for fermenting kimchi belonging to Leuconostoc citrium, a method for producing kimchi using the same, and that the lactic acid bacterium can effectively control deterioration of fermentation quality in summer.

Patent Document 3 discloses a method for producing a pickle which has a good taste and is rich in L-lactic acid, by fermenting vegetables using Lactobacillus sake HS1 strain.

However, there is still a need for lactic acid bacteria that are less likely to produce excessive sourness and are useful in the production of fermented pickles.

Japanese Patent Publication No. 2009-518031 JP, 2011-19504, A JP, 2001-120173, A

Shigeo Miyao, “Useful microorganisms active in everyday life, food and useful microorganisms-Japanese food culture and microorganisms 5. Pickles and microorganisms”, Modern Media, Vol. 61, No. 11 (2015) 18-25

An object of the present invention is to provide a lactic acid bacterium that suppresses sourness and a method for producing a fermented pickle using the same.

As a result of repeated intensive studies to solve the above problems, the present inventors found that certain strains of lactic acid bacterium Leuconostoc fallax (Leuconostoc fallax) have properties effective in suppressing sour expression, The present invention has been completed.

That is, the present invention includes the following.
[1] A method for producing a fermented pickle, which comprises fermenting a pickled raw material using a Leuconostoc fallax A-1 strain lactic acid bacterium having a deposit number NITE P-03040 as a starter.
[2] The method according to [1] above, wherein the fermented pickles are fermented kimchi.
[3] The method according to the above [1] or [2], wherein the pickled raw material is fermented at 3°C to 15°C.
[4] Leuconostoc fallax A-1 strain lactic acid bacterium having a deposit number NITE P-03040.
[5] A starter composition comprising a culture of Leuconostoc fallax A-1 strain lactic acid bacteria having a deposit number NITE P-03040.
[6] The starter composition according to the above [5], which is used for producing fermented pickles.
[7] The starter composition according to the above [6], wherein the fermented pickles are fermented kimchi.
[8] Fermentation comprising the Leuconostoc fallax A-1 strain lactic acid bacterium having a deposit number NITE P-03040, which is produced by the method according to any one of the above [1] to [3] pickles.
[9] The fermented pickle according to the above [8], which is a fermented kimchi.

The lactic acid bacterium according to the present invention can effectively suppress the expression of excessive sourness during fermentation.

FIG. 1 is a diagram showing changes over time in the number of lactic acid bacteria in a Kimchi medium during cultivation of strains A-1 and X, respectively. Open squares indicate strain X and black circles indicate strain A-1. FIG. 2 is a diagram showing the organic acid contents (lactic acid and acetic acid) of the Kimchi medium during the cultivation of strains A-1 and X, respectively. FIG. 3 is a diagram showing the sugar contents (fructose and mannitol) of the Kimchi medium during the cultivation of strains A-1 and X, respectively. FIG. 4 is a diagram showing changes over time in the pH of the Kimchi medium during the culture of the A-1 strain, the NBRC 100496 strain, and the NBRC 113243 strain. Black circles indicate A-1 strain, open circles indicate NBRC 100496 strain, and black squares indicate NBRC 113243 strain. FIG. 5 is a diagram showing the content of mannitol in the medium after culturing for 5 days and 10 days for strain A-1 and strain NBRC 113244. FIG. 6 is a diagram showing a change with time of pH of the culture medium of the test plots 2, 3, 5, and 7. Black triangles are L. plantarum NBRC 15891 strains (Test plot 2), open circles are A-1 strains (Test plot 3), black circles are A-1 strains + L. plantarum NBRC 15891 strains (Test plot 5), and shaded. The circle (dotted line) indicates A-1 strain + small amount L. plantarum NBRC 15891 strain (test group 7). FIG. 7: is a figure which shows the organic acid content of the culture medium of the test plots 1-7. NBRC 113244 strain (test plot 1), L. plantarum NBRC 15891 strain (test plot 2), A-1 strain (test plot 3), NBRC 113244 strain + L. plan in order from left to right for each number of culture days Talam NBRC 15891 strain (Test plot 4), A-1 strain + L. plantarum NBRC 15891 strain (Test plot 5), NBRC 113244 strain + small amount L. plantarum NBRC 15891 strain (Test plot 6), and A-1 strain + Shows a graph of a small amount of L. plantarum NBRC 15891 strain (test plot 7). FIG. 8: is a figure which shows the mannitol content of the culture medium of the test plots 1-7. NBRC 113244 strain (test plot 1), L. plantarum NBRC 15891 strain (test plot 2), A-1 strain (test plot 3), NBRC 113244 strain + L. plan in order from left to right for each number of culture days Talam NBRC 15891 strain (Test plot 4), A-1 strain + L. plantarum NBRC 15891 strain (Test plot 5), NBRC 113244 strain + small amount L. plantarum NBRC 15891 strain (Test plot 6), and A-1 strain + Shows a graph of a small amount of L. plantarum NBRC 15891 strain (test plot 7). FIG. 9 is a diagram showing changes in pH of kimchi produced using strains A-1 and X as starters, respectively.

Hereinafter, the present invention will be described in detail.
TECHNICAL FIELD The present invention relates to a lactic acid bacterium that suppresses sour expression and the production of fermented pickles using the same. In the present invention, the sourness-inhibiting lactic acid bacterium means a lactic acid bacterium having a property effective in suppressing (reducing) the expression (generation) of sourness during fermentation. The lactic acid bacterium that suppresses sourness expression expresses a moderate sourness, but it is preferable that it is possible to create a fermentation environment in which excessive sourness is unlikely to be expressed.

The sourness-inhibiting lactic acid bacterium used in the present invention is specifically a Leuconostoc fallax lactic acid bacterium, and particularly a Leuconostoc fallax A-1 strain lactic acid bacterium. ..

Leuconostoc fallax A-1 strain lactic acid bacterium (hereinafter, also referred to as A-1 strain lactic acid bacterium or A-1 strain) is an independent administrative agency product evaluation technology institute patent on October 3, 2019. It has been deposited at the Microorganism Deposition Center (NPMD) (Kazusa Kamasa City, Chiba Prefecture, Japan, Room 2-5-8, Room 122, Postal Code 292-0818) with Accession No. NITE P-03040.

The lactic acid bacterium of Leuconostoc phallus A-1 strain is typically a coccus having the following mycological properties.
・Cell morphology: S. aureus (0.8-1.0 x 1.0-1.7 μm)
・Gram stainability: + (Positive)
・Spore formation: − (negative)
・Colony color: Milky white (MRS agar plate medium at 30℃ for 48 hours, aerobic culture)

The A-1 strain lactic acid bacterium can be anaerobically cultured at 30° C. for 24 hours in an MRS medium (for example, one having the composition shown in Table 6 below).

The A-1 strain lactic acid bacterium has a relatively low lactic acid producing ability. The ability of lactic acid bacteria to produce lactic acid can be evaluated using the amount of lactic acid produced in the fermentation medium as an index. Here, the "lactic acid production amount" refers to a value obtained by subtracting the initial lactic acid amount (the lactic acid compounding amount when lactic acid is mixed as a medium component) from the lactic acid content measured in the fermentation medium.

The A-1 strain lactic acid bacterium has a relatively high acetic acid-producing ability. The acetic acid-producing ability of lactic acid bacteria can be evaluated using the amount of acetic acid produced in the fermentation medium as an index. Here, the "acetic acid production amount" refers to a value obtained by subtracting the initial acetic acid amount (the acetic acid compounding amount when acetic acid is mixed as a medium component) from the acetic acid content measured in the fermentation medium. Acetic acid is less likely to increase the acidity than lactic acid, and is generally considered to show a growth inhibitory effect on lactic acid bacteria that express a strong sour taste such as Lactobacillus plantarum, which is predominant in the latter half of fermentation.

The A-1 strain lactic acid bacterium has a high mannitol producing ability. The mannitol-producing ability of lactic acid bacteria can be evaluated using the ratio of the amount of mannitol produced to the amount of fructose consumed in the fermentation medium (conversion rate of fructose to mannitol) as an index, as described in Example 5 below. Here, the “mannitol production amount” refers to a value obtained by subtracting the initial mannitol amount (the mannitol blending amount when mannitol is blended as a medium component) from the mannitol content measured in the fermentation medium.

It is known that mannitol is produced by conversion from fructose, and in a fermentation system using a lactic acid bacterium of A-1 strain, the fructose amount decreases (fructose consumption amount increases) as the mannitol production amount increases. Mannitol not only provides a mellow sweetness to soften the sour taste, but it is a sugar alcohol that is difficult for bacteria to assimilate, and therefore can have the effect of suppressing lactic acid fermentation that produces an organic acid from sugar such as fructose. In one embodiment, the A-1 strain lactic acid bacterium is cultured at 2 to 45° C., preferably 5 to 40° C., more preferably 5 to 30° C., further preferably 5 to 15° C., and fermented (lactic acid fermentation). According to this, mannitol can be produced at a high level in the fermentation system (fermented pickles).

A-1 strain lactic acid bacteria are high even if the pH of the fermentation system is lowered by lactic acid fermentation (but not limited to the following, preferably pH 4.2 to 5.0, for example pH 4.2 to 4.5), Shows viability. Therefore, in a preferred embodiment, the A-1 strain lactic acid bacterium can survive and continue to contribute to the fermentation even at the stage when the fermentation has progressed.

Strain A-1 lactic acid bacteria are difficult to be eliminated by expansion of growth of sourness-expressing lactic acid bacteria even if coexisting (coculture) with sourness-expressing lactic acid bacteria in the fermentation system, and rather strongly suppress growth of sourness-expressing lactic acid bacteria. You can Therefore, A-1 strain lactic acid bacteria, when used as a starter in the production of fermented pickles, suppress the growth of various sourness-expressing lactic acid bacteria that contribute to fermentation, and maintain a high ratio in lactic acid bacteria even when fermentation progresses. be able to. In a preferred embodiment, the lactic acid bacterium strain A-1 can maintain its predominance in the microflora in the production of fermented pickles using it as a starter, and particularly preferably exists as a dominant species even in the latter stage of fermentation. it can.

Here, the sourness-expressing lactic acid bacterium refers to a lactic acid bacterium known to contribute to the expression of excessive sourness by the production of a large amount of lactic acid, for example, Lactobacillus plantarum, Lactobacillus brevis ( Lactobacillus brevis) and the like, and is usually a bacillus.

Therefore, the A-1 strain lactic acid bacterium, while survival in a fermentation system for a longer period of time, suppresses lactic acid production, enhances mannitol production, and suppresses the growth of sourness-expressing lactic acid bacterium, thereby continuing to suppress sourness expression. it can. Thereby, the expression of excessive sourness in the fermentation system can be effectively suppressed. The present invention provides such a sour taste expression-inhibiting A-1 strain lactic acid bacterium.

The present invention also provides a method for producing a fermented pickle, which comprises fermenting a pickle raw material using a lactic acid bacterium strain A-1 as a starter. In the present invention, “starter” (sometimes referred to as “fermentation starter”) means a microbial culture used for stably inducing (initiating) fermentation.

In the present invention, the “fermented pickles” mean foods that have a unique flavor and are improved in preservability by fermenting food materials. The present invention particularly refers to fermented pickles obtained by fermentation involving lactic acid fermentation. The fermented pickles according to the present invention may be those obtained by using vegetables as a main raw material (raw material having the highest weight ratio), but are not limited thereto. The fermented pickles in the present invention include, but are not limited to, fermented kimchi, fermented shiba pickles, sukiki, sunki, nozawana pickles, sauerkraut, pickles and the like. In the present invention, the “fermented kimchi” refers to a fermented pickle produced by salting vegetables (such as Chinese cabbage and cucumber) that are main ingredients, adding pepper and other ingredients, and lactic-fermenting. As an example of fermented kimchi, fermented kimchi is added after lactic acid bacteria are fermented for a certain period of time, and then lactic acid bacteria are sterilized to be distributed after lactic acid fermentation is completed, or lactic acid bacteria are added (inoculated) and then distributed at 5 to 10°C. There is a fermented kimchi that slowly fermentes lactic acid during the process. Examples of the fermented kimchi include fermented Chinese cabbage kimchi (mainly made of Chinese cabbage) and oikimchi (mainly made of cucumber).

In the present invention, the "pickled raw material" means a food material other than the starter used for producing the fermented pickled vegetables, and includes at least the fermentation raw material. The pickled raw material may be any foodstuff or a mixture thereof that can be used in the production of fermented pickles, and is not limited to, but is optional such as vegetables, seafood, meat, salt, seasonings other than salt, condiments, preservatives, etc. Food additives and the like. The fermentation raw material means an organic substance containing a substrate such as sugar, protein and amino acid which is decomposed by fermentation and includes, but is not limited to, vegetables, seafood, meat, condiments and the like.

For example, pickled raw materials used in the production of fermented kimchi include salted vegetables (eg salted Chinese cabbage, salted cucumber), pepper (typically red pepper), salted salt (typically seafood salted salt, such as salted sardine of ami). , Liquid squid made from squid and krill) and fermented seasonings such as fish sauce, garlic, green onions, ginger, carrots, radish, other vegetables such as sesame seeds, fruits such as apples, vegetable extracts (such as Chinese cabbage extract), Examples include, but are not limited to, honey, sugar, reduced starch syrup, sugars such as fructose, sesame oil, salt, lactic acid, acetic acid, sodium L-glutamate, and food additives.

The production of fermented pickles using the lactic acid bacterium of A-1 strain as a starter can be carried out according to a known production procedure for fermented pickles, but is not limited thereto. The fermentation period of the pickled raw material for the production of fermented pickles may vary depending on the type of fermented pickles, for example, 3 days or more, preferably 4 days or more, for example 3 to 60 days, 3 to 30 days, 3 to 14 days. , 10 to 30 days, or 4 to 25 days. In one embodiment, when the fermented pickles are circulated after fermenting for a certain period of time and finishing the fermentation by sterilization or the like, the fermentation period is a fermentation process for the certain period (a fermentation process under a manufacturer. ) Means. In another embodiment, the fermented pickles are circulated after being fermented for a certain period of time, and when the fermentation is continued in the distribution process, the fermentation period is to be eaten by the consumer through the distribution process from the start of the fermentation process. It refers to the period until the time (specifically, the expiration date). In yet another embodiment, when lactic acid bacteria are added immediately before distribution and the fermented pickles are fermented during the distribution process, the fermentation period is from the time when the lactic acid bacteria are added to the time when the product is eaten by the consumer through the distribution process (specifically, The term is until the expiration date). The fermentation temperature for producing the fermented pickles (temperature of the fermentation environment) is not limited to the following, but is typically in the range of 0 to 40°C, preferably 2 to 30°C, more preferably 2 -20°C, for example 3-15°C, 4-13°C, or 5-15°C.

As an example, fermented kimchi is produced by draining salted vegetables (such as salted Chinese cabbage) and then chilling pepper and other ingredients (for example, fermented seafood such as salted fish and fish sauce, garlic, green onions, ginger, radish, etc.). Other vegetables, sugars such as reduced starch syrup and fructose), and A-1 strain lactic acid bacteria as a starter are added, mixed, and then immersed for a certain period of time. The soaking time (fermentation period) for producing the fermented kimchi is not limited to the following, but is typically 3 days or more, preferably 4 days or more, for example, 3 to 60 days, 3 to 30 days, 3 to 14 days. , 10 to 30 days, or 4 to 25 days. The pickling temperature of fermented kimchi (temperature of fermentation environment) is not limited to the following, but is typically in the range of 0 to 40°C, preferably 2 to 30°C, more preferably 2 to 20°C, For example, it may be 3-15°C, 4-13°C, or 5-15°C.

In the production of such fermented pickles, in addition to the lactic acid bacteria used as a starter, other microorganisms derived from the pickled raw material or the surrounding environment (for example, other lactic acid bacteria, yeast, etc.) may be involved in fermentation, for example, The sourness-expressing lactic acid bacteria as described above can also participate in fermentation. That is, the production of the fermented pickles according to the present invention includes acidity-expressing lactic acid bacteria such as Lactobacillus spp. such as Lactobacillus plantarum (typically, for example, derived from pickled raw materials such as Chinese cabbage or the surrounding environment. In the presence of such lactic acid bacteria), fermenting the pickled raw material with A-1 strain lactic acid bacteria can be included. Even in that case, the A-1 strain lactic acid bacterium can suppress the growth of the sourness-expressing lactic acid bacterium and maintain a high ratio in the lactic acid bacterium even when the fermentation proceeds. That is, A-1 strain lactic acid bacteria used as a starter, it is possible to maintain a more predominant in the microflora of the fermentation system, preferably, in the latter stage of fermentation or to maintain the predominance in the microflora of the fermented pickles produced. it can. In a preferred embodiment, the lactic acid bacterium strain A-1 used as a starter can be present as a predominant species in the fermentation system or fermented pickles. In the present invention, “existing as a dominant species” means that among living microorganisms (bacteria and fungi) existing in the fermentation system (fermented pickles), the A-1 strain lactic acid bacterium is present at the highest ratio. The A-1 strain lactic acid bacterium used as a starter in the present invention can continue to survive in a fermented pickle for a longer period of time. Therefore, in a preferred embodiment, the fermented pickles produced according to the present invention comprise strain A-1 lactic acid bacteria. The fermented pickles produced according to the present invention may contain sourness-expressing lactic acid bacteria (eg, Lactobacillus plantarum, Lactobacillus brevis, etc.) together with the A-1 strain lactic acid bacteria. The fermented pickles produced according to the present invention contain lactococcus (coccus) containing lactic acid bacterium of A-1 strain at a ratio higher than that of lactobacillus (bacillus) containing sourness-expressing lactic acid bacterium (eg, Lactobacillus plantarum). It is preferable. In the fermentation process of fermented pickles such as kimchi, it is common that even if a Leuconostoc genus is inoculated as a starter, the lactic acid bacteria that express acidity such as Lactobacillus plantarum gradually change their flora. The lactic acid bacterium of the A-1 strain inoculated as a starter can maintain the superiority to the sourness-expressing lactic acid bacterium such as Lactobacillus plantarum even when the fermentation proceeds. In a preferred embodiment of the present invention, for example, when the above fermentation period is completed, the lactic acid bacterium containing the A-1 strain lactic acid bacterium is a fermentation system or a fermented pickle, and the ratio of the number of cocci to the total number of cocci + rods ( %), preferably 50% or more, such as 70% or more, 80% or more, 90% or more, or 95% or more.

The fermented pickles (for example, fermented kimchi) produced as described above have a relatively low lactic acid content. The pH value of the produced fermented pickles (for example, fermented kimchi) is not limited to the following, for example, may be pH 4.2 or more and less than 5.0, for example, pH 4.5 to pH 4.9, and Even if fermentation progresses, the pH is unlikely to drop sharply. The fermented pickles produced preferably contain mannitol produced by fermentation. The fermented pickles according to the present invention are less likely to cause excessive fermentation and are less likely to exhibit excessive sourness, and thus have the advantage that the taste (particularly sourness) is easily maintained stably. The present invention also relates to such fermented pickles.

The produced fermented pickles may be refrigerated below the fermentation temperature, frozen, dried, and/or freeze-dried for the purpose of delaying or preventing the subsequent progress of fermentation. Alternatively, the produced fermented pickles may be maintained at the fermentation temperature as they are. The produced fermented pickles may be sterilized before distribution, and in that case, they may be refrigerated, frozen, dried, and/or freeze-dried for the purpose of preventing spoilage.

The present invention also relates to a starter composition comprising a culture of strain A-1 lactic acid bacteria. Such a starter composition can be advantageously used as a starter for producing fermented pickles, for example, in the production of fermented pickles as described above. The culture of the A-1 strain lactic acid bacterium contained in the starter composition may be in any form as long as the A-1 strain lactic acid bacterium is alive. The culture of the A-1 strain lactic acid bacterium contained in the starter composition may be, for example, one obtained by drying and pulverizing the culture of the A-1 strain lactic acid bacterium, or freezing the culture of the A-1 strain lactic acid bacterium. It may be dried. The starter composition may also include other ingredients such as food additives. In the present invention, the food additive may be any of preservatives, excipients, carriers, thickeners, flavors and the like. The invention also provides the use of such a starter composition or A-1 strain lactic acid bacteria in fermentation, in particular lactic acid fermentation, for example in the production of fermented products such as fermented pickles.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Screening for lactic acid bacteria 35 kinds of handmade fermented kimchi (hereinafter, also simply referred to as kimchi) manufactured in Fukushima prefecture, Tokyo prefecture, Kanagawa prefecture, Osaka prefecture, or Kyoto prefecture (Japan) were obtained. 10 g of each kimchi sample was cut into small pieces, and 90 ml of sterilized water was added. The sample was homogenized using a stomacher (streamer treatment) to obtain an extract.

The extract was plated on LUSM medium and anaerobically cultured at 30° C. for 1 to 3 days. According to the composition shown in Table 1, the LUSM medium was mixed with components other than L-cysteine hydrochloride and antibiotics, filled up to 98.8 ml with water, dissolved by heating, and the temperature was adjusted to 48°C. Prepared by adding L-cysteine hydrochloride solution filtered with a μm filter and each antibiotic solution.

After culturing, cocci having the same color tone and shape as Leuconostoc mesenteroides were selected from the appeared colonies by microscopic observation to obtain 31 colonies.

The obtained 31 colony strains were cultured in Kimchi medium having the composition shown in Table 2. This kimchi medium is prepared by adding Chinese cabbage extract AK (diluted 15.8 times with water) to pepper (powder), MSG, liquid salted syrup, reduced salt and salt while filling up to 89.5 ml, and then adding BCP. After autoclave sterilization, 10 ml of a 25% fructose solution was added, and 0.5 ml of a mixed solution of lactic acid and acetic acid was further added and mixed.

The color change of the Kimchi medium during the culture was observed, and the pH was also measured. When BCP is added at the concentration shown in Table 2, the Kimchi medium is colored deep green, but as the growth of lactic acid bacteria progresses, the organic acid produced causes the Kimchi medium to change from pale red to pale yellow. Therefore, the discoloration level of the Kimchi medium during fermentation of lactic acid bacteria reflects the degree of fermentation and the degree of pH decrease. Based on the color change of the Kimchi medium and the results of pH measurement, four strains with a small pH decrease were selected.

Next, the four strains were separately inoculated into kimchi, the pH of the medium was measured over time, and the A-1 strain showing the smallest pH decrease was selected.

[Example 2] Analysis of strain A-1
When the strain A-1 was observed under a microscope, the results shown in Table 3 were obtained.

In addition, the 16S rRNA gene (16S rDNA) of the A-1 strain was subjected to nucleotide sequence determination (about 1500 bp), and homology search and phylogenetic analysis by BLAST (Basic Local Alignment Search Tool). As a result, it showed the highest homology (99.9%) to the 16S rRNA gene sequence of Leuconostoc fallax (type culture) DSM 20189 (NCBI accession number AF360738). The strain was identified as belonging to the lactic acid bacterium Leuconostoc fallax.

[Example 3] Fermentability of A-1 strain in kimchi medium
A-1 strain and a kimchi-producing bacterium belonging to Leuconostoc citreum (hereinafter referred to as strain X) are inoculated into 10 ml of kimchi medium having the composition shown in Table 4 and allowed to stand. The cells were cultured under the conditions at 10°C for 4 weeks. This kimchi medium is mixed with pepper (powder), MSG, liquid salted rice, reduced starch syrup, and salt while adding water and filling up to 89.5 ml, sterilized by autoclave, and then added 10 ml of a 25% fructose solution, Further, 0.3 ml of a mixed solution of lactic acid and acetic acid was added and mixed to prepare.

In order to compare the fermentability in the Kimchi medium between the A-1 strain and the X strain, the Kimchi medium was sampled at the start of culture (0 week) and at 1, 2, 3 and 4 weeks after the start of culture. , Lactic acid bacteria count, organic acid content, and sugar content were measured. The number of lactic acid bacteria was measured by the pour-in culture method using BCP agar medium.

The time course of the number of lactic acid bacteria in the Kimchi medium during culture is shown in FIG. As shown in FIG. 1, the number of strains of the A-1 strain in the Kimchi medium did not decrease after 4 weeks, whereas the number of strains of the X strain showed a decrease after 3 weeks. From this result, it was shown that the survival rate of strain A-1 was considerably high for at least 4 weeks.

To measure the organic acid content and sugar content in kimchi medium, dilute the kimchi medium sample approximately 10 times with distilled water, filter the diluted solution with a 0.45 μm filter, and analyze the resulting filtrate. I served.

The organic acid content was measured by using ion-exclusion chromatography and post-column pH buffered conductivity detection method. The analysis conditions are as follows.
・Column: Shim-pack SCR-102H 2 (8 mm x 300 mm) with guard column ・Pump 1: Mobile phase: 5 mM p-toluenesulfonic acid aqueous solution
Flow rate: 0.8 ml/min
・Pump 2: Reaction liquid: 20 mM Bis-Tris aqueous solution containing 5 mM p-toluenesulfonic acid and 100 μM EDTA
Flow rate: 0.8 ml/min
・Column temperature: 40℃
・Detector: POLARITY: +, DISPLAY: BACK GROUND, RESPONSE: SLOW, GAIN: 1
・Sample injection volume: 10 μl
・1 cycle: 35 minutes

The sugar content is measured by measuring the refractive index of the compound separated by the size exclusion/ligand exchange separation method of liquid chromatography and quantitatively analyzing each sugar (sugar, sugar alcohol, and polyhydric alcohol). Went by. The analysis conditions are as follows.
・Column: Shim-pack SCR-101C with guard column ・Pump: Mobile phase: Water
Flow rate: 1.0 ml/min
・Column temperature: 70℃
・Sample injection volume: 10 μl
・1 cycle: 20 minutes

FIG. 2 shows the results of measuring the organic acid content, and FIG. 3 shows the results of measuring the fructose and mannitol contents. The A-1 strain showed a marked decrease in the lactic acid content in the Kimchi medium and a slight increase in the acetic acid content as compared with the X strain. Since lactic acid has a higher acidity than acetic acid, it was considered that strain A-1 was unlikely to increase the acidity in kimchi medium.

The A-1 strain also showed a decrease in fructose content in the Kimchi medium (ie, increased fructose consumption) and a significant increase in mannitol content (ie, mannitol production) compared to the X strain. It was Since mannitol is a sugar alcohol that is not readily assimilated by resident bacteria in kimchi, it was thought that strain A-1 could create a kimchi environment in which excessive fermentation does not easily occur.

From the above results, it was shown that the strain A-1 has a property of hardly causing excessive sourness and has a high survival rate (viability) in kimchi medium. If the A-1 strain is used as a starter bacterium for kimchi production, it is thought that a fermented kimchi having a stable quality by suppressing abnormal fermentation can be produced by long-term survival of a starter bacterium that does not cause excessive sourness. ..

[Example 4] Comparison of medium pH change with other Leuconostoc spp. Leuconostoc fallax A-1 strain, Leuconostoc mesenteroides NBRC 100496 strain, and With respect to Leuconostoc citreum NBRC 113243 strain, changes in medium pH, which is an index of sourness, were examined. The NBRC 100496 strain and the NBRC 113243 strain are available under the NBRC numbers 100496 and 113243, respectively, from the Japan Institute for Product Evaluation Technology, Biotechnology Center (NBRC).

Kimchi medium having the composition shown in Table 5 was prepared. This kimchi medium is mixed with red pepper (powder), MSG, fish sauce, reduced starch syrup, salt, garlic, and ginger, Chinese cabbage extract AK (diluted 15.8 times with water) while filling up to 89.75 ml, After autoclave sterilization, 10 ml of a 25% fructose solution was added, and 0.25 ml of a mixed solution of lactic acid and acetic acid was further added, followed by mixing.

Lactic acid bacteria were inoculated into the prepared Kimchi medium and cultured at 10°C under static conditions. The medium was sampled with time until 20 days after the culture, and the pH of the medium was measured.

The pH was measured using a pH meter (Toa DKK Corporation model number: HM-42X).

The results are shown in Fig. 4. The Leuconostoc farax A-1 strain showed a gradual decrease in pH as compared with the Leuconostoc mesenteroides NBRC 100496 strain and the Leuconostoc citrus NBRC 113243 strain. It was shown that the A-1 strain has a property of hardly expressing excessive sourness even when compared with other Leuconostoc sp.

[Example 5] Characterization of strain A-1 1) Evaluation of mannitol-producing ability Mannitol is produced by conversion from fructose. Therefore, the conversion rate of mannitol from fructose of A-1 strain (mannitol-forming ability) is calculated as follows by Leuconostoc fallax reference strain NBRC 113244 strain (Japan Independent Administrative Agency Product Evaluation Technology Base). It was available from the NITE Biological Resource Center (NBRC); Leuconostoc fallax DSM 20189 and equivalent strains) and evaluated.

Fructose was added to the MRS liquid medium (Table 6) to a final concentration of 2%, and mixed and dissolved. 10 ml of the medium was poured into a test tube and sterilized by an autoclave (121°C, 15 minutes). The MRS liquid medium (Table 6) is a synthetic medium raw material (peptone, meat extract, yeast extract, glucose, sorbitan monooleate, dipotassium hydrogen phosphate, sodium acetate trihydrate, triammonium citrate, magnesium sulfate 7). Hydrate, and manganese sulfate tetrahydrate) were added to 100 ml of water and mixed.

The cooled sterilized medium was inoculated with A-1 strain or NBRC 113244 strain, and cultured at 10° C. under static conditions. After the start of the culture, the medium was sampled with time, and the fructose content and the mannitol content in the medium were measured by liquid chromatography in the same manner as in Example 3.

FIG. 5 shows the mannitol contents in the medium 5 days and 10 days after the start of culture of the A-1 strain and the NBRC 113244 strain. The mannitol content in the medium obtained with the strain A-1 was significantly increased as compared with the strain NBRC 113244.

Based on the measurement results, the mannitol forming ability was calculated based on the following formula.

Mannitol production capacity = (Mannitol content in the medium 10 days after the start of culture (mg/100g))/Fructose consumption 10 days after the start of culture ^* (mg/100g)
^* Fructose consumption = Fructose medium addition (mg/100g)-Fructose content in the medium on the 10th day of culture (mg/100g)

As shown in Table 7, the mannitol-producing ability of the strain A-1 was significantly higher than that of the leuconostoc farax standard strain NBRC 113244 strain.

2) Evaluation of viability in co-culture with bacillus As described in Example 4, Kimchi medium having the composition shown in Table 5 was prepared.
In order to evaluate the viability in the prepared Kimchi medium, in addition to the Leuconostoc Fallax A-1 strain (coccus), and the Leuconostoc Fallax reference strain NBRC 113244 strain (coccus), sourness expressing lactic acid bacteria Lactobacillus plantarum NBRC 15891 strain (bacillus) (available from NITE Biological Resource Center (NBRC) of Japan Incorporated Administrative Agency Product Evaluation Technology Foundation) is used. Each lactic acid bacterium was prepared by inoculating MRS liquid medium (Table 6) and precultured at 30° C. for 24 hours under static conditions.

The Kimchi medium prepared as described above was dispensed into test tubes in an amount of 10 ml, and each test section was inoculated with the lactic acid bacteria shown in Table 8 and cultured at 10° C. under static conditions. After the start of the culture, the medium was sampled with time to measure the coccus ratio, pH, organic acid content, and mannitol content in the medium.

The coccus ratio is determined by observing a gram-stained medium sample of the bacteria under a phase-contrast microscope for the co-cultured cocci and bacilli, counting the number of each form of cocci and bacilli. + Calculated as the ratio (%) of the number of cocci to the total number of bacilli.

The pH, organic acid content and mannitol content of the medium were measured in the same manner as in Example 3.

Table 9 shows the coccus ratio of the test plots 4 to 7.

The Leuconostoc farax A-1 strain has a higher survival rate in a kimchi medium simulating kimchi, and the sourness expressing lactic acid lactobacillus lactobacillus It was shown that the growth inhibitory effect on plantarum was high. From these results, when manufacturing fermented pickles such as kimchi, lactic acid bacteria expressing acidity (lactobacillus) such as Lactobacillus plantarum derived from the pickling raw material and the surrounding environment were identified as Leuconostoc farax A-1 strain. Even when coexisting, since the A-1 strain according to the present invention can exist as a dominant species in the fermented pickles, it is unlikely to produce excessive sourness even when the fermentation period is long, and the fermentation has stable quality. It has been shown that it is possible to produce pickles.

FIG. 6 shows the changes in the pH of the culture medium of the test plots 2, 3, 5, and 7 (changes over time). By co-culturing the sourness-expressing lactic acid bacterium Lactobacillus plantarum and the A-1 strain (test groups 5 and 7), it was shown that the pH decrease due to the sourness-expressing lactic acid bacterium Lactobacillus plantarum was significantly suppressed (reduced). Was done.

FIG. 7 shows the organic acid content in the medium in test sections 1 to 5, and FIG. 8 shows the mannitol content in the medium. By co-culturing the sourness-expressing lactic acid bacterium Lactobacillus plantarum with the strain A-1 (test groups 5 and 7), lactic acid production was significantly suppressed as compared with the case of Lactobacillus plantarum alone. Further, when co-cultured with Lactobacillus plantarum and A-1 strain, as compared with the case of Lactobacillus plantarum alone and A-1 strain alone, a significant promotion of acetic acid production and mannitol production is shown, It was thought that this led to the predominance of A-1 strain in kimchi medium in which Lactobacillus plantarum and A-1 strain were co-cultured.

From the results shown in the above examples, it was shown that the Leuconostoc farax A-1 strain is a lactic acid bacterium having a high effect of suppressing sour expression in the production of fermented pickles such as fermented kimchi.

[Example 6] Kimchi production and analysis Chinese cabbage was cut into bite-sized pieces, immersed in salt water having a concentration of 9%, and then salted overnight. Subsequently, the pickled Chinese cabbage extracted from salt water (3% salt concentration in Chinese cabbage) was washed with water and then dehydrated for about 5 hours under its own weight.

A mixed seasoning for kimchi called Yannyom was prepared by mixing the raw materials shown in Table 10 and storing it in a refrigerator overnight.

The prepared medicine was added to dehydrated salted Chinese cabbage at a weight ratio of medicine:dehydrated salted Chinese cabbage=20:80 and mixed well.

Filling a container with 200g of Chinese cabbage mixed with medicinal herbs, inoculating Leuconostoc phallax A-1 strain or Leuconostoc cytorium X strain into it, and fermenting it by storing at 10°C to produce kimchi did.

The pH of kimchi during fermentation was measured with time. The kimchi was squeezed with a juicer, and the pH of the squeezed liquid was measured using a pH meter (Toa DKK Co., Ltd. model number: HM-42X).

The results are shown in Fig. 9. It was shown that the kimchi inoculated with the lactic acid bacteria of the A-1 strain showed a slower decrease in pH compared with the kimchi inoculated with the lactic acid bacteria of the X strain.

[Example 7] Sensory test of kimchi Regarding the kimchi at the start of fermentation (0 week) and the kimchi after 8 weeks fermentation (storage period: 8 weeks) produced according to Example 6, the fermentation odor, sourness, and comprehensive evaluation were performed. A sensory test was conducted.

The evaluation score of kimchi at the start of fermentation was set to 5, and the change in kimchi after fermentation for 8 weeks as compared with kimchi at the start of fermentation was sensory-evaluated as follows.

5: No change 4: Slightly changed 3: Slightly changed, but within reasonable range 2: Product quality limit: 1: Out of allowable range of product quality

The results are shown in Table 11.

Also, as a result of the sensory evaluation, Kimchi after fermentation for 8 weeks inoculated with A-1 strain is of good quality with moderate sourness and fermentation odor, while Kimchi after fermentation for 8 weeks inoculated with X strain. Showed a sharp sour taste and strong fermentation odor, indicating that it was outside the permissible range as a kimchi product.

Since the lactic acid bacterium according to the present invention has the property of effectively suppressing the expression of sourness, it is unlikely to produce an excessive sourness during fermentation and has a high survival rate. Therefore, by using the lactic acid bacterium according to the present invention as a starter in the production of fermented pickles, it is possible to suppress the expression of excessive sourness and enable the production of fermented pickles (such as kimchi) having a more stable taste.

Claims (9)

A method for producing a fermented pickle, which comprises fermenting a pickle raw material using Leuconostoc fallax A-1 strain lactic acid bacteria having a deposit number NITE P-03040 as a starter. The method according to claim 1, wherein the fermented pickles are fermented kimchi. The method according to claim 1 or 2, wherein the pickled raw material is fermented at 3°C to 15°C. Leuconostoc fallax A-1 strain lactic acid bacterium with deposit number NITE P-03040. A starter composition comprising a culture of Leuconostoc fallax A-1 strain lactic acid bacteria having accession number NITE P-03040. The starter composition according to claim 5, which is used for producing fermented pickles. The starter composition according to claim 6, wherein the fermented pickles are fermented kimchi. A fermented pickle containing the Leuconostoc fallax A-1 strain lactic acid bacterium having a deposit number NITE P-03040, which is produced by the method according to any one of claims 1 to 3. The fermented pickle according to claim 8, which is a fermented kimchi.

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