JP7204092B2 - Method for producing vegetable paste and strain therefor - Google Patents

Description

NPMD NITE P-02520

The present invention relates to a method for producing vegetable paste (particularly carrot paste) and strains therefor.

Vegetable pastes have a very wide range of uses, both as a food product per se and as an ingredient in freeze-dried curries. Vegetable pastes can vary in properties, characteristics and uses depending on the vegetables used. Among them, a vegetable paste made mainly from carrots is excellent in that it is rich in β-carotene and dietary fiber, and therefore has a high nutritional value and a bright color.

With the recent diversification of health consciousness and tastes, the demand for vegetable pastes exclusively based on carrots is increasing. On the other hand, since carrots have a peculiar odor and harshness, it is important to suppress these in the production and improvement of carrot paste. As means for producing a carrot paste with good aroma and taste, (1) a method of heating with fats and oils to reduce the amount of terpenes (Patent Document 1), (2) a method of enzymatic treatment, and (3) ) and the like using fermentation by microorganisms. (1) The method of heating with fats and oils requires heat energy for heating and addition of fats and oils, so it has drawbacks in terms of energy and cost. (2) Since the enzymes used in the enzymatic treatment are relatively expensive, there is a drawback in terms of cost. (3) Fermentation using microorganisms may be cheaper than other methods, but there is a problem that additional additives are required because no microorganisms that can ferment carrots using only carrots as nutrients are known. .

JP 2016-13092 A

The present invention provides a microbial method for producing and improving vegetable pastes, especially carrot pastes, simply and inexpensively. The invention also provides microorganisms for use in such microbiological techniques.

The inventors of the present invention have solved the above problems by providing a novel lactobacillus strain. The SB3 strain, which is a novel lactobacillus strain of the present invention, was identified as Lactobacillus paracasei from the results of a search against the NCBI nucleotide BLAST eubacterial/archaeal 16S rDNA database and the test results of the carbon source utilization ability. ). This SB3 strain was deposited on July 27, 2017 at the National Institute of Technology and Evaluation Biotechnology Center Patent Organism Depositary Center (NITE-IPOD) (acceptance number NITE P-02520).

For example, the present invention provides:
(Item 1)
SB3 strain (accession number NITE P-02520) belonging to Lactobacillus paracasei or a mutant strain thereof.
(Item 2)
2. The mutant strain of claim 1, which is capable of growing on carrots as a sole source of nutrition.
(Item 3)
A method for producing a fermented paste made from vegetables, using the SB3 strain (accession number NITE P-02520) according to item 1 or 2 or a mutant strain thereof.
(Item 4)
4. The method of item 3, wherein the vegetable is carrot.
(Item 5)
5. The method of item 4, wherein carrots are the main source of nutrition.
(Item 6)
5. The method of item 4, wherein carrots are substantially the only source of nutrition.
(Item 7)
A method for fermenting vegetables using the SB3 strain (accession number NITE P-02520) according to item 1 or 2 or a mutant strain thereof.
(Item 8)
8. The method of item 7, wherein the vegetable is carrot.
(Item 9)
9. The method of item 8, wherein carrots are the main source of nutrition.
(Item 10)
9. The method of item 8, wherein carrots are substantially the only source of nutrition.
(Item 11)
A composition containing SB3 strain belonging to Lactobacillus paracasei (accession number NITE P-02520) or a mutant strain thereof, for producing a fermented paste made from vegetables.
(Item 12)
A composition comprising strain SB3 belonging to Lactobacillus paracasei (accession number NITE P-02520) or a variant thereof for fermenting vegetables.

The novel bacterial strain SB3 of the present invention is considered to belong to Lactobacillus paracasei from the results of a carbohydrate fermentation test using an API50CHL kit (manufactured by bioMerieux Japan).

The present invention provides a novel Lactobacillus paracasei strain SB3. The present invention also provides a method for producing and/or improving vegetable-based fermented paste (especially carrot-based fermented paste) using the novel strain provided by the present invention.

FIG. 1 is a graph showing pH changes with the passage of culture time for a sample inoculated with the SB3 strain (filled circles) and a sample not inoculated (white circles). Figure 2 shows the results of boiling sterilization of carrot paste fermented with SB3 strain (Fig. 2 left) and the results of boiling sterilization of unfermented carrot paste (Fig. 2 right).

The present invention will be described below. It should be understood that throughout this specification, expressions in the singular also include the concept of the plural unless specifically stated otherwise. Also, it should be understood that the terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification (including definitions) will control. Also, "wt%" is used interchangeably with "mass percent concentration" herein. Also, unless otherwise defined in this specification, "%" means "wt%".

(Definition of terms)
Listed below are definitions of terms specifically used in this specification.

As used herein, the term "vegetable paste" refers to a paste prepared using vegetables as the main raw material. The vegetable paste is preferably a fermented paste fermented by microorganisms.

As used herein, "improving" or "modifying" the vegetable paste is used interchangeably to reduce and/or change the "smell" and/or "harshness" of the vegetable paste. Say things.

The term "carrot paste" as used herein means that all or substantially all of the vegetables used as raw materials in the manufacture of the vegetable paste, alternatively 100%, 99% or more, 98% or more, 97% % or more, 96% or more, 95% or more, 90% or more, 85% or more, 80% or more, 75% or more, 70% or more, 65% or more, 60% or more, 55% or more, or 50% or more carrots It refers to a vegetable paste that is

As used herein, the term "nutrient source" refers to substances (excluding water and gases) required for the growth of microorganisms.

As used herein, the term "primary nutrient source" refers to 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more of all nutrients, A nutrient source that accounts for 85% or more, or 90% or more.

As used herein, the term "substantially the only nutrient source" refers to 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of all nutrients A nutrient source that occupies

As used herein, the term "carrot" includes common edible carrots, such as Daucus carota L. var. sativa DC., which belongs to the genus Daucus of the family Umbelliferae. Any of these carrots can be used for fermentation of the SB3 strain.

As used herein, the term "fermentation" refers to a phenomenon in which components present in the system are degraded by enzymes present in the cells of microorganisms such as the SB3 strain.

(1. Culture of SB3 strain or its mutant strain)
The SB3 strain or its mutants can be cultured under well-known culture conditions used for Lactobacillus, preferably under well-known culture conditions used for Lactobacillus paracasei.

(2. Preparation of mutant strain)
Said mutants are produced, for example, by subjecting them to general mutagenesis, or by adaptation or natural mutation through subculturing.

The mutagenesis can be performed using common mutagens. Mutagens include, for example, agents having mutagenic action, ultraviolet rays, and the like. Examples of drugs having mutagenic activity include streptomycin, ofloxacin, ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil, and acridines. .

The mutant strains of the present invention are typically capable of growing on carrots as a major source of nutrition, carrots as substantially the only source of nutrition, and/or carrots as the sole source of nutrition.

(3. Production or modification of vegetable paste)
The SB3 strain of the present invention or its mutants can be used to produce or modify vegetable pastes (especially carrot pastes). Such production and/or modification can be achieved, for example, by bringing the bacterium of the present invention into contact with raw material, chopped vegetables (e.g., carrot) or vegetable paste (e.g., carrot paste), and applying conditions that allow the bacterium of the present invention to grow. This can be done by culturing under Culture conditions include all conditions in which the bacterium of the present invention can grow. Typically, culture conditions commonly used for Lactobacillus paracasei can be used. Such culture conditions are preferably, but not limited to, temperatures between 30°C and 37°C. For example, when a mutant strain having heat resistance is used as the mutant strain of the present invention, culturing at a higher temperature is also possible.

Any vegetable can be used as a raw material for making or modifying the vegetable paste.

The invention will now be described on the basis of examples, which are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the foregoing detailed description of the invention nor to the following examples, but only by the claims.

(Example 1: Isolation of SB3 strain)
The SB3 strain of the present invention was isolated from Aji no Narezushi (produced in Noto Peninsula, Ishikawa Prefecture). Specifically, a food sample (Aji no Narezushi) was suspended in sterilized physiological saline and added to de Man Rogosa and Sharpe (MRS) agar medium (with 4 (w/v)% sodium chloride added). The suspension was applied and cultured at 30°C. The cells that formed colonies were collected, purely cultured in MRS medium + 4% NaCl liquid medium, and cryopreserved in a cryogenic freezer at -80°C in the presence of 15% glycerin. The isolated SB3 strain was deposited on July 27, 2017 at the National Institute of Technology and Evaluation Biotechnology Center Patent Organism Depositary Center (NITE-IPOD) (accession number NITE P-02520).

(Example 2: Characterization of SB3 strain)
Regarding the mycological properties of the SB3 strain isolated in Example 1, a carbohydrate fermentation ability test was performed using an API50CHL kit (manufactured by bioMerieux Japan). When the results were applied to apiweb (https://apiweb.biomerieux.com) and searched, there was a 99% match with Lactobacillus paracasei ssp paracasei.
A partial sequence of 529 bases corresponding to the V1 to V3 variable regions in the 16S rDNA of the SB3 strain was sequenced. The sequence was subjected to Microbial Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastASTM Search&BLAST), which is a nucleotide sequence homology search service of NCBI (National Center for Biotechnology Information). Similarly, when a search against the NCBI nucleotide BLAST eubacterial/archaeal 16S rDNA database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch#) was performed, the lactobacillus lacto It was found to be most closely related to Lactobacillus paracasei. The homology within the searched sequence was 100%.
Based on the above mycological characteristics (biochemical test) and rDNA base sequence results, the SB3 strain was determined to be Lactobacillus paracasei.
(Example 3: Fermentation of carrot paste by SB3 strain)
Fermentation of carrot paste by strain SB3 was tested. A 100% carrot product (manufactured by Sugiyo Farm (1 kg/pack)) was used as the carrot paste. This product has been sterilized by boiling at 90°C for 30 minutes at the time of manufacture. The fermentation test procedure is as follows.
(1) 150 g of carrot paste was placed in a 200 ml flask, sealed with a silicon stopper, covered with aluminum foil, and sterilized in an autoclave at 121° C. for 15 minutes.
(2) A colony of SB3 strain cultured at 37° C. in BCP-added plate count medium (manufactured by Nissui) was collected with a platinum loop in a clean bench and inoculated into the carrot paste of (1). As a control, one without inoculation was prepared.
(3) Culture at 37°C for 5 days. Appropriate amounts were sampled on the 0th day and 5th day of culture in a clean bench to confirm the pH and growth of the SB3 strain.
(4) Growth of SB3 strain was carried out by suspending the sample from (3) in sterilized physiological saline on a clean bench and then adding it to BCP-added plate count medium. It was evaluated by culturing at 37° C. for 72 hours and calculating the Colony Forming Unit (CFU/g) from the colonies that appeared.
(5) Lactic acid and vitamin A were quantified by high-performance liquid chromatography at the Japan Food Research Laboratories (Japan Food Research Laboratories) for the juice of carrot paste (5 days after culture) inoculated with SB3 strain and without inoculated.
(6) The test was conducted with n=2.

The change in pH of the carrot paste was as follows.

The growth of the SB3 strain was confirmed by comparing the colony forming units (CFU/g) on day 0 and day 5 of culture. The results are as follows.

The growth of the SB3 strain was confirmed from the amount of lactic acid in the SB3 strain inoculated and non-inoculated carrot pastes (5th day of culture). The results are as follows.

The above results indicate that the SB3 strain can grow and assimilate carrot paste as the sole source of nutrition.

When fermenting vegetables using the SB3 strain of the present invention (for example, when producing a fermented paste), it is not necessary to add nutrients of lactic acid bacteria from the outside during fermentation (if necessary, sugar etc. can be added). good).

From the above results, the present invention provides the advantage of being able to control the production cost of fermentation products.

(Example 4: Sensory test)
It was demonstrated as follows that fermentation of Lactobacillus strain SB3 resulted in improved organoleptic test results for carrot paste.
1. Lactobacillus paracasei SB3 strain deposited under accession number NITE P-02520 was used as the lactic acid bacterium.
2. A 100% carrot product (manufactured by Sugiyo Farm (1 kg/pack)) was used as the carrot paste. This product was sterilized by boiling at 90°C for 30 minutes at the time of production.
3. A fermentation test was performed according to the following procedure.
(1) The test was performed with n=3;
(2) Place 150 g of carrot paste in a 200 ml flask, seal with a silicon stopper, cover with aluminum foil, and sterilize in an autoclave at 121 ° C. for 15 minutes;
(3) Inoculating and culturing according to the following procedure;
(i) SB3 strain cultured at 37° C. for 48 hours in GYP liquid medium and washed three times with sterile saline;
(ii) Suspend in sterilized physiological saline to adjust the number of cells, inoculate the paste of (2) to a final concentration of 1.0×10 ⁶ cells/g, and culture at 37°C. As a control, one without inoculation was prepared. In the SB3 strain suspension, all viable bacteria, heat-tolerant bacteria, yeast, and coliform bacteria were below the detection limit. In addition, the number of viable bacteria in the SB3 strain was below the detection limit by autoclaving.
(4) Until the 5th day of culture, the pH was measured every other day, and appropriate amounts of each of the SB3 strain inoculated and non-inoculated carrot pastes were sampled in a clean bench and stored frozen. Fig. 1 shows pH changes with the passage of culture time for carrot paste inoculated with SB3 strain (filled circles) and uninoculated carrot paste (white circles). In those inoculated with the SB3 strain, the pH decreased and a fermentation odor was also observed;
(5) Bactericidal test of SB3 strain was carried out as follows;
(i) After sampling on the fifth day, 100 g of the carrot paste inoculated with the SB3 strain was sealed in a vacuum bag in a clean bench, steamed and heated at 90 ° C. for 20 minutes,
(ii) After steaming and heating, an appropriate amount was suspended in sterilized physiological saline on a clean bench, added to each of the following media, and then cultured at a predetermined temperature and time. Next, by confirming the appearance of colonies, etc., the effectiveness of the sterilization method is evaluated.
(Sterilization test items)
General viable bacteria: Cultured at 37°C for 48 hours on a standard agar medium (manufactured by Eiken Chemical) Heat-resistant bacteria: After heating the sample at 75°C for 15 minutes, cultured at 37°C on a standard agar medium for 48 hours Lactic acid bacteria: BCP added Cultured on plate count agar medium at 37°C for 72 hours Yeast: PDA agar medium supplemented with chloramphenicol (100 mg/L) at 25°C for 120 hours
Cultivation Coliform bacteria: Cultured at 37 ° C. for 24 hours in blue light medium (manufactured by Nissui) (6) Sterilization of sample for tasting;
Since the sterilization test showed the effectiveness of steaming and heating at 90°C for 20 minutes, the samples of each culture time frozen and stored in (4) were steamed and heat sterilized at 90°C for 20 minutes.
(7) sensory test;
The samples sterilized in (6) at each culture time were tasted, and a sensory test was performed. The results are shown in Table 6 below (change in taste of carrot paste over time).

In the above experiment, it was felt that the SB3 strain was added to enhance the acidity associated with the decrease in pH. It was shown that the fishy smell disappeared with the addition of SB3 strain as the culture time increased, and the original taste of carrots combined with sourness gave a sour taste similar to citrus fruits.

The results show that carrot paste with SB3 strain is a superior product in taste and smell.

(Example 5: Comparison of fermented carrot paste)
Carrot paste fermented with strain SB3 according to the method described in Example 3 was compared to unfermented carrot paste. Specifically, both carrot pastes were sterilized by boiling (90° C. for 20 minutes), and the color change (browning) was compared. As a result, as shown in the photograph of Fig. 2, the carrot paste fermented with the SB3 strain (Fig. 2 left) showed less browning after boiling than the unfermented carrot paste (Fig. 2 right). It was less.

To provide an excellent method for producing or improving vegetable paste (especially carrot paste).

The SB3 strain was deposited on July 27, 2017 at the National Institute of Technology and Evaluation, Biotechnology Center, Patent Organism Depositary Center (NITE-IPOD) (accession number NITE P-02520).

Claims (11)

SB3 strain (accession number NITE P-02520 ) belonging to Lactobacillus paracasei. A method for producing a fermented paste made from vegetables, using the SB3 strain (accession number NITE P-02520 ) according to claim 1 . 3. The method of claim 2 , wherein the vegetable is carrot. 4. The method of claim 3 , wherein carrots are the primary source of nutrition. 4. The method of claim 3 , wherein carrots are substantially the only source of nutrition. A method for fermenting vegetables using the SB3 strain (accession number NITE P-02520 ) according to claim 1 . 7. The method of claim 6 , wherein said vegetable is carrot. 8. The method of claim 7 , wherein carrots are the primary source of nutrition. 8. The method of claim 7 , wherein carrots are substantially the only source of nutrition. A composition containing SB3 strain belonging to Lactobacillus paracasei (accession number NITE P-02520 ) for producing a fermented paste made from vegetables. A composition comprising strain SB3 belonging to Lactobacillus paracasei (accession number NITE P-02520 ) for fermenting vegetables.

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