JP5703513B1 - Novel lactic acid strain and fermentation composition and fermentation extract thereof - Google Patents

Abstract

An object of the present invention is to provide a lactic acid bacterium that does not have an amino acid biosynthetic circuit deleted, particularly a lactic acid bacterium that biosynthesizes an amino acid constituting a protein, a culture of lactic acid bacterium, and a fermented extract. A lactose that biosynthesizes one or more amino acids among lysine, histidine, aspartic acid, alanine, and glycine, which are amino acids constituting proteins. Lactobacillus casei LC-Ikematsu strain. [Selection figure] None

Description

  The present invention relates to a novel lactic acid strain, a fermentation composition obtained using the lactic acid strain, and a fermentation extract thereof.

  Okinawa Prefecture belongs to the subtropical region, and because of the warm and rainy climate, there are a wide variety of plants and animals that have been adapted to that climate. Among these microorganisms, attention is also focused on lactic acid bacteria that have been used for many fermented foods, health foods, etc. from the past.

  In recent years, it has been reported that ingestion of lactic acid bacteria shows an intestinal regulating action, an antioxidant action, an immunoregulatory action, and a blood pressure lowering action, and the probiotic action of lactic acid fermented foods has attracted attention.

  In Japanese Patent Application Laid-Open No. 2008-54555, a lactic acid bacterium having high production ability of γ-aminobutyric acid (GABA), a method for producing GABA using the lactic acid bacterium, a method for producing a GABA-containing lactic acid fermented food, and glutamic acid desorption obtained from the lactic acid bacterium A novel lactic acid bacterium Lactobacillus sp. Having a high ability to produce GABA for the purpose of providing carbonic acid enzyme (GAD) and a nucleic acid encoding the same. L13 (NITE P-253), a method for producing GABA using the lactic acid bacterium and a method for producing a GABA-containing lactic acid fermented food, GAD obtained from the lactic acid bacterium, and a nucleic acid encoding the same are disclosed.

  JP 2006-262724 A aims to provide a lactic acid bacterium capable of highly expressing a protein, more specifically, a lactic acid bacterium having an appropriate promoter for highly expressing a protein in a wider range of lactic acid strains. The lactic acid bacterium with high expression of this protein was linked to a promoter for high expression in which a promoter sequence derived from lactic acid bacteria and a 5′-untranslated region sequence of the S-layer protein gene were combined, and under the control of the promoter for high expression It has DNA encoding the protein of interest Suitably the S-layer protein is derived from Lactobacillus acidophilus. Further, the DNA encoding the target protein may be linked to a secretory signal sequence or a surface layer display sequence.

JP 2004-357535 A, in order to provide a new lactic acid bacterium having a high immune enhancing effect, efficiently producing a high concentration of γ-aminobutyric acid (GABA) in a short period of time, derived from fish soy sauce, Lactobacillus sp. Having GABA production ability and immune enhancing effect Y-3 (FERM P-19317). In this strain, the production of GABA and the immune enhancing effect are promoted by the soybean extract (soybean broth). By adding this lactic acid bacteria culture, a food with pharmacological effects such as suppression of blood pressure rise can be obtained.

JP 2008-54555 A JP 2006-262724 A JP 2004-357535 A

  Thus, lactic acid bacteria exhibiting various probiotic actions such as GABA production and protein expression have been disclosed.

The genome size of lactic acid bacteria is usually 1.7 to 3.3 Mb, which is relatively small as a free-living bacterial genome. This is because many biosynthetic genes have been deleted. In particular, the lactic acid bacteria genome is highly auxotrophic because it lacks many of the genes that make up the TCA cycle.
Therefore, lactic acid bacteria secure energy by making full use of glycolysis, convert the accumulated pyruvic acid into lactic acid and release it outside the cells.

  In addition, the TCA cycle is a source of intermediates required for amino acid synthesis, and the lactic acid bacteria genome that has lost the TCA cycle lacks many genes in the amino acid synthesis system. Instead, there are many genes that encode peptidases, amino acid transporters, etc. in order to obtain amino acids from the outside.

  An amino acid is generally used as a general term for compounds having an amino group and a carboxyl group in one molecule. However, depending on the type of amino acid, the amino group may be an imino group. In some cases, it may be a sulfono group or a phosphono group.

  Amino acids may exist in free form in nature or may be combined with other amino acids to form peptides. However, most amino acids are present as constituents of proteins (polypeptides) constituting the body of an organism. Since foods are mostly organisms and their metabolites, the amino acids contained in foods are mostly amino acids that constitute proteins.

An object of the present invention is to provide a lactic acid bacterium that does not have an amino acid biosynthetic circuit deleted, particularly a lactic acid bacterium that biosynthesizes an amino acid constituting a protein. Moreover, let it be a subject to provide the fermentation composition by the lactic acid bacteria, its fermentation extract, and the food-drinks using them.

  The present invention can solve the above problems by the following means.

  Claim 1 of the present invention comprises one or more amino acids among lysine, histidine, aspartic acid, alanine and glycine, which are amino acids constituting the protein. Lactobacillus casei LC-Ikematsu strain (deposit number: NITE P-01946) to be biosynthesized.

  Claim 2 of the present invention is a method for producing a culture of lactic acid bacteria having an amino acid biosynthesis ability, which comprises inoculating the lactic acid bacterium of claim 1 into a medium and culturing.

  Claim 3 of the present invention is a fermentation composition obtained by fermenting pineapple fruit with the Lactobacillus casei LC-Ikematsu strain lactic acid bacterium according to claim 1 or claim 2 and a culture thereof, and the fermentation composition thereof It is a fermented extract.

  According to claim 4 of the present invention, pineapple juice is fermented with Lactobacillus casei LC-Ikematsu strain lactic acid bacteria according to claim 1 or claim 2 and a fermentation product thereof, and a fermentation composition thereof It is a fermented extract.

In Claim 5 of this invention, the fermented composition obtained by fermenting with the Lactobacillus casei LC-Ikematsu strain lactic acid bacteria of the said Claim 3 or Claim 4, and food-drinks using the fermented extract thereof It is what.

The present invention has the following effects.
(1) Lactic acid bacteria capable of biosynthesis of amino acids and cultures thereof can be provided.
(2) Biosynthesis of one or more amino acids among lysine, histidine, aspartic acid, alanine, and glycine, which are amino acids constituting proteins. Possible lactic acid bacteria and cultures thereof can be provided.
(3) A lactic acid bacterium derived from pineapple fruit capable of biosynthesizing amino acids can be provided.
(4) A new fermented composition obtained by fermenting with an amino acid biosynthetic lactic acid bacterium and a fermented extract thereof can be provided.
(5) A new fermented composition obtained by fermenting a pineapple fruit or fruit juice with an amino acid biosynthetic lactic acid bacterium and a fermented extract thereof can be provided.
(6) A food or drink using the fermented composition and the fermented extract thereof can be provided.

  The form of the lactic acid bacteria of this invention is demonstrated below.

  The lactic acid bacterium in the present invention is Lactobacillus casei LC-Ikematsu strain. The Lactobacillus casei LC-Ikematsu strain is as follows.

<Acquisition of Lactobacillus casei LC-Ikematsu stock>
Spin the pineapple and leave it for about a week to recover the fully fermented pineapple fruit. 1g of the recovered fruit pieces were added to a tube containing 9ml of sterilized water and stirred vigorously to obtain a pour solution.

  A sterilized cotton swab was immersed in the pour solution and smeared on an MRS agar medium (Becton Dickinson) containing 0.1% calcium carbonate.

  Pick up colonies that formed halos (calcium carbonate dissolved by acid and transparently removed) after anaerobic culture for 48 hours in a container containing anero pack (manufactured by AS ONE) in a 37 ° C incubator did.

  Furthermore, a catalase test using 5% hydrogen peroxide solution was performed on the picked-up colonies, and negative colonies were selected as lactic acid bacteria. Obtained).

  Since lactic acid bacteria do not have catalase, no bubbles are generated when hydrogen peroxide is decomposed into oxygen and water by catalase.

<Identification of Lactobacillus casei LC-Ikematsu strain>
(Morphological properties)
The morphological properties of this strain were confirmed by microscopic observation of colonies when cultured in the above medium.

  As a result, the cell form was Neisseria gonorrhoeae, and, like the Lactobacillus casei standard strain (hereinafter sometimes abbreviated as “reference strain”), it had no spores and no motility.

The scanning electron microscope image is shown below.
The scale bar (broken line) is 10 μm, acceleration voltage 5.0 kV, magnification 5000 times, working distance 4.6 mm.
FIG. Scanning electron microscope image of Lactobacillus casei LC-Ikematsu strain

  The following genetic tests were carried out as a new strain identification method.

(Base sequence)
Classification of Lactic Acid Bacteria by Sequencing of 16S rRNA Region The flow up to sequencing of 16S rRNA region of isolated lactic acid bacteria is as follows. First, the isolated lactic acid bacteria were inoculated on MRS agar medium.

  After anaerobic culture at 37 ° C. for 24 hours, DNA was extracted from the grown lactic acid bacteria colonies using PrepMan Ultra Sample Preparation Reagent (manufactured by ABI).

  Using the extracted DNA as a template, the 16S rRNA region (about 1500 bp) on the lactic acid bacteria genome was amplified by PCR.

As the DNA polymerase for PCR reaction, KOD FX (manufactured by TOYOBO) was used, and 9F and 1541R (manufactured by Greiner Japan) were used as universal primers.

Table 1. Base sequence of universal primers used for amplification of 16S rRNA region

  After the PCR product was subjected to agarose gel electrophoresis, the gel was stained with GelRed Nucleic Acid Gel Stain (manufactured by Wako) to confirm the amplification of a specific region.

  Thereafter, the PCR product was purified using a DNA cleaner (manufactured by Wako).

  A cycle sequence reaction using the dye terminator method was performed using the purified PCR product as a template.

The reaction was carried out according to the protocol of DTCS Quick Start Kit (Beckman Coulter), using 4 to 6 primers (Table 3) designed in the 16S rRNA region, and dividing the 16S rRNA region to perform a cycle sequence reaction.

Table 2. Base sequences of primers used in 16S rRNA region sequencing

  After the reaction, the product was purified by ethanol precipitation and subjected to a DNA sequencer gene analysis system CEQ 8800 (manufactured by Beckman Coulter).

  For the analysis of sequence data, free software Chromas Lite and SeqConv_v081 were used. The obtained data sequence is converted to FASTA format, and homology with the base sequence of 16S rRNA region on the base sequence database is confirmed using BLAST of DDBJ (DNA Data Bank of Japan). Classification was performed.

  Since LC-Ikematsu strain showed 100% homology with Lactobacillus casei by homology search of 16S rRNA region of sequence data using BLAST, LC-Ikematsu strain was classified as Lactobacillus casei.

The sequence results of the 16S rRNA region (9-1512) of the LC-Ikematsu strain isolated from pineapple fruit are shown below.

gagtttgatc ctggctcagg atgaacgctg gcggcgtgcc taatacatgc aagtcgaacg
agttctcgtt gatgatcggt gcttgcaccg agattcaaca tggaacgagt ggcggacggg
tgagtaacac gtgggtaacc tgcccttaag tgggggataa catttggaaa cagatgctaa
taccgcatag atccaagaac cgcatggttc ttggctgaaa gatggcgtaa gctatcgctt
ttggatggac ccgcggcgta ttagctagtt ggtgaggtaa tggctcacca aggcgatgat
acgtagccga actgagaggt tgatcggcca cattgggact gagacacggc ccaaactcct
acgggaggca gcagtaggga atcttccaca atggacgcaa gtctgatgga gcaacgccgc
gtgagtgaag aaggctttcg ggtcgtaaaa ctctgttgtt ggagaagaat ggtcggcaga
gtaactgttg tcggcgtgac ggtatccaac cagaaagcca cggctaacta cgtgccagca
gccgcggtaa tacgtaggtg gcaagcgtta tccggattta ttgggcgtaa agcgagcgca
ggcggttttt taagtctgat gtgaaagccc tcggcttaac cgaggaagcg catcggaaac
tgggaaactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg aaatgcgtag
atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct
cgaaagcatg ggtagcgaac aggattagat accctggtag tccatgccgt aaacgatgaa
tgctaggtgt tggagggttt ccgcccttca gtgccgcagc taacgcatta agcattccgc
ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc gcacaagcgg
tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatctttt
gatcacctga gagatcaggt ttccccttcg ggggcaaaat gacaggtggt gcatggttgt
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttatgact
agttgccagc atttagttgg gcactctagt aagactgccg gtgacaaacc ggaggaaggt
ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga
tggtacaacg agttgcgaga ccgcgaggtc aagctaatct cttaaagcca ttctcagttc
ggactgtagg ctgcaactcg cctacacgaa gtcggaatcg ctagtaatcg cggatcagca
cgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgagagtttg
taacacccga agccggtggc gtaacccttt tagggagcga gccgtctaag gtgggacaaa
tgat

(Deposit of lactic acid bacteria)
The Lactobacillus casei LC-Ikematsu strain, which is a lactic acid bacterium of the present invention, was deposited on October 2, 2014 at the Patent Microorganism Depositary Center of the National Institute for Product Evaluation Technology.
Deposit Number NITE P-01946

(Genome sequencing)
Genome sequencing was performed to understand the entire genome structure of the LC-Ikematsu strain.

  The procedure is described below.

  The genome of the LC-Ikematsu strain was sequenced using the next-generation sequencer GS junior (Roche).

  Sequencing required library preparation and emPCR pretreatment.

  The library was prepared according to the GS Junior Titanium Series Rapid Library (Shotgun) Adjustment Manual.

  emPCR was performed according to the GS Junior Titanium series emPCR (Lib-L) manual.

  As a result of assembling using the analysis software Newbler2.7, the sequencing data was 179 contigs and the genome size was 3.0MB.

(Comparison of metabolic function genes)
The following experiment was conducted using this 179 contig.

As comparative symmetry, five strains of Lactobacillus, L. casei W56 (lcw), L. rhamnosus (lra), L. sakei (lsa), L. brevis (lbr), and L. gasseri (lga) were used.

  The auto-annotation pipeline MiGAP (National Institute of Genetics) for microorganisms was used for comparison of metabolic function genes.

All contigs were annotated and the data was collected into one Excel file. MiGAP annotation includes the functional classification of COG (Clusters of Orthologous Groups of proteins), which is an ortholog database, and metabolic function genes were compared based on the functional classification code of COG. The function classification codes related to the metabolic system are as shown in Table 3 below.

Table 3. COG function classification table

The number of the above functional classification codes in the file was counted, and the genes involved in the metabolic system were compared between the five comparison targets and the LC-Ikematsu strain.
These were tabulated using analysis software R. The analysis results are shown below.

Figure 2. Comparison of genes involved in COG metabolic function

(Comparison of amino acid synthesis routes)
The amino acid synthesis pathway of IM-1 strain was analyzed using KEGG (Kyoto Encyclopedia of Genes and Genomes). Pathway reconstruction was performed using KAAS (KEGG automatic annotation server), and amino acid metabolic pathways were stored as images (see FIG. 3). The amino acid metabolic pathway was visually confirmed, and the classification was made by classifying into those in which the metabolic pathway was retained, those in which some genes were deleted, and those in which they were disrupted. The same treatment was performed for five types of comparative symmetry, and the results were summarized as a comparison of amino acid synthesis pathways.

Figure 3. Metabolic pathways of glycine, serine and threonine

The analysis results are shown below.

Figure 4. Comparison of amino acid synthesis pathways

(Amino acid analysis)
In order to confirm whether the function inferred using the bioinformatics technology is actually the same, the amino acids produced in the culture solution of the lactic acid bacteria LC-Ikematsu strain were analyzed.

Using an ultrahigh performance liquid chromatography system manufactured by Waters, a lactic acid bacteria culture solution sample was derivatized and measured. A calibration curve was drawn from the amino acid standard, and the amino acid content of the sample was determined individually.
The analysis results are shown below.

Figure 5. Results of amino acid analysis

(Amino acid requirement test)
Whether the amino acid synthesis pathway of the LC-Ikematsu strain predicted by KEGG pathway reconstruction actually functions in the fungus body was verified using a completely synthetic medium.

When an amino acid deficient medium in which each amino acid is deleted from a completely synthetic medium is prepared and the growth of an LC-Ikematsu strain is confirmed in a certain amino acid deficient medium, the LC-Ikematsu strain retains its amino acid synthesis pathway. Indicates that

[Test method]
A completely synthetic medium for Lactobacillus casei was prepared with the following composition.
Table 4. Fully synthetic medium composition

  Each amino acid was removed from the complete synthetic medium one by one to prepare each amino acid deficient medium. Lactic acid bacteria solution was added to the complete synthetic medium and each amino acid deficient medium and cultured at 37 ° C. for 48 hours. The lactic acid bacteria solution was prepared by centrifuging the LC-Ikematsu strain cultured in liquid and washing with sterilized water. The growth of each culture solution was measured at OD660, and the growth rate (%) was determined in comparison with the control.

[Test results]
It was Pro, Met, His, Lys, Asp, and Ser that the growth rate exceeded 50% (Table 5).

Table 5. Growth rate of LC-Ikematsu strain in each amino acid deficient medium

  Six amino acids, Gly, Ala, Lys, His, Asp, and Thr, were predicted to retain the synthetic pathway from the results of genetic analysis of the LC-Ikematsu strain. On the other hand, as a result of actually investigating using a deficient medium, Pro, Met, His, Lys, Asp, and Ser showed 50% or more growth compared to the control. It was confirmed that 8 types of amino acids, including Gly and Ala, which are not included in the composition of the synthetic medium, grow even if they are individually deleted.

  Comparison of synthetic pathway predictions made from genetic analysis results and synthetic ability tests using amino acid-deficient media revealed that the five types of Gly, Ala, His, Asp, and Lys had the same results and retained the amino acid synthesis pathway. It was shown that

  Although Thr was predicted to retain the synthetic pathway, the LC-Ikematsu strain did not grow well in Thr deficient medium. This is probably because some enzymes in the synthetic pathway are pseudogenes.

  Three types, Met, Pro, and Ser, which were predicted to be difficult to synthesize, showed growth of 50% or more in the deficient medium. When the amino synthesis pathway was reconstructed using the computer again for these three types, a pathway different from the synthesis pathway of interest was confirmed. Ser was synthesized from Gly, Met was synthesized from Asp, and Pro was synthesized from Glu. It was confirmed that

  It was confirmed that the LC-Ikematsu strain actually has five kinds of synthetic ability of Gly, Ala, Lys, His, and Asp among the six kinds of amino acids estimated from the gene analysis. Regarding Thr, it seems that synthesis was impossible as a result of the gene in the synthesis pathway not functioning for some reason.

  As described above, this strain has a 100% homology in the base sequence of 16S rRNA compared to the reference strain, and the morphological property is about 3.0 Mb in genome size, which is relatively large as a lactic acid bacterium. Have physiological properties.

  Compared to other Lactobacillus genera, it has many metabolic function genes, especially many genes related to carbohydrate and amino acid metabolism, and the lactic acid bacteria genome usually has many amino acid synthesis pathways disrupted. However, this strain has a remarkably excellent characteristic that it has few major disruptions and retains many genes that are involved in amino acid synthesis although there are five types of amino acids that can be synthesized. It was clearly different from the lactic acid strain belonging to Lactobacillus casei.

  From the above results, this strain was shown to be a novel lactic acid strain belonging to Lactobacillus casei.

  Next, a milk fermentation product was prepared using the lactic acid strain described above.

  For culturing lactic acid bacteria, any medium capable of culturing lactic acid bacteria may be used, but an MRS liquid medium is preferred. When cultivating lactic acid bacteria, the culture temperature is preferably 25 to 45 ° C and more preferably 35 to 40 ° C under various conditions. The culture time is preferably 12 to 72 hours, more preferably 20 to 30 hours. The culture conditions can be appropriately adjusted in either aerobic culture or anaerobic culture, but anaerobic culture is more preferable.

  In the following experiments, the isolated lactic acid bacteria were inoculated in an MRS liquid medium, and after anaerobic culture at 37 ° C. for 24 hours, they were used for fermented products and trial production.

  After anaerobic culture of lactic acid bacteria in MRS liquid medium at 37 ° C. for 24 hours, the culture solution was centrifuged to remove the supernatant, and the cells were washed with 0.85% sodium chloride solution. 30 ml of milk (component-free milk, Meiji Dairy Products) was added to 1 g (wet weight) of the cells and cultured at 37 ° C. for 24 hours. Furthermore, the same Lactobacillus genus as the LC-Ikematsu strain, and using the experienced Lactobacillus brevis, milk fermentation products were prepared in the same way, and the fermentation characteristics were compared for coagulation, color, smell and taste. did.

As a result of preparing a milk fermented product using the lactic acid bacterium Lactobacillus casei LC-Ikematsu strain of the present invention and Lactobacillus brevis of the same genus, the fermented product prepared with the LC-Ikematsu strain is white as a whole and has whey at the top. Was observed, and the protein coagulated in the milk to form small grains. The taste was sour like yogurt. Compared with the fermented milk prepared with Lactobacillus brevis, the coagulation, odor and taste of milk were similar.

FIG. Prepared fermented milk (when using Lactobacillus brevis (left) and when using LC-Ikematsu strain (right))

  Next, jelly was manufactured using said lactic acid strain.

(Raw material combination)
As a gelling agent, 2.0% by weight of xanthan gum, 10% by weight of glucose, 0.1% by weight of citric acid, 0.1% by weight of ascorbic acid, and 65% lactic acid fermented extract of Sikhwasa and papaya 1% by weight of Lactobacillus casei LC-Ikematsu strain and 21.8% by weight of water.

(Production method)
Among the above raw materials, after mixing the glucose and the gelling agent, water is added and heated at 80 ° C. for 10 minutes to dissolve the gelling agent. To the above raw material liquid, add sequwacer heated to 60 ° C and lactic acid fermented papaya extract, Lactobacillus casei LC-Ikematsu strain, and other auxiliary materials (citric acid, ascorbic acid) to correct the evaporation water. , Heat treatment up to 95 degrees. After the heat treatment, each retort pack was filled with 30 g, sealed and sealed, sterilized at 85 ° C. for 30 minutes, cooled in water, and made into a stick jelly containing lactic acid bacteria.

  Lactobacillus brevis (NBRC 12005) is a lactic acid fermented extract that produces γ-aminobutyric acid (GABA) using Lactobacillus brevis NBRC 12005. The active action includes improvement of cerebral blood flow, blood pressure drop, mental stability, renal / liver function activity, alcohol metabolism promoting action, deodorization and the like. It is also expected to have a suppressive action on colorectal cancer.

  In addition, Lactobacillus casei LC-Ikematsu strain is effective in improving the intestinal environment by lactic acid bacteria, preventing colds and influenza, activating and regulating immunity, preventing and improving hay fever, atopic dermatitis It has been reported to be effective in improving cancer, preventing cancer, lowering cholesterol, and improving high blood pressure.

  In addition, it has the effects of active ingredients (nobiletin, tangeretin, etc.) contained in sequwercer skin, which has been attracting attention for its cancer-suppressing effect and blood glucose-suppressing effect, etc. It is a lactic acid bacterium derived from pineapple and is a reliable and healthy jelly food that can be eaten safely and safely with natural and natural ingredients.

  In addition, it is not limited to said Example, For example, in manufacture of jelly, the compounding quantity of a lactic acid fermentation extract is 50-80 weight%, and the compounding quantity of a gelatinizer is 1.0-3. The amount of 0% by weight of Lactobacillus casei LC-Ikematsu strain is preferably about 0.1 to 10% by weight.

  Moreover, the Lactobacillus casei (Lactobacillus casei) LC-Ikematsu strain alone may be used, or a fermented extract obtained by lactic fermentation of Lactobacillus casei (Lactobacillus casei) LC-Ikematsu strain. As the gelling agent, commercially available gelling agents such as gellan gum, fercellan, low methoxyl pectin, guar gum, locust bean gum, xanthan gum, carrageenan, agar, gelatin and the like can be used.

Claims (5)

Lactobacillus casei (Lactobacillus) that biosynthesizes one or more amino acids among the protein-constituting amino acids Lysine, Histidine, Aspartic acid, Alanine, and Glycine casei) Lactic acid bacteria which are LC-Ikematsu strain (deposit number: NITE P-01946).
A method for producing a culture of lactic acid bacteria having an amino acid biosynthesis ability, wherein the lactic acid bacteria according to claim 1 are inoculated into a medium and cultured.
The fermentation composition obtained by fermenting a pineapple fruit with the Lactobacillus casei (Lactobacillus casei) LC-Ikematsu strain | stump | stock lactic acid bacteria of the said Claim 1 or Claim 2, and its culture, and its fermentation extract.
The fermentation composition obtained by fermenting a pineapple fruit juice with the Lactobacillus casei (Lactobacillus casei) LC-Ikematsu strain | stump | stock lactic acid bacteria of the said Claim 1 or Claim 2, and its culture, and its fermentation extract.
  The fermentation composition obtained by making it ferment with the Lactobacillus casei (Lactobacillus casei) LC-Ikematsu stock | strain lactic acid bacteria of the said Claim 3 or Claim 4, and food-drinks using the fermentation extract.