Lactic acid bacteria growth promoter

Abstract

A lactic acid bacteria growth promoter which has a growth promotion effect on lactic acid bacteria is provided. This lactic acid bacteria growth promoter contains at least one type of peptide selected from a first peptide, in which the total number of residues is less than or equal to 9 and there is a hydrophobic amino acid residue on the C-terminal, and a second peptide, in which the total number of residues is less than or equal to 9 and the ratio of the total number of hydrophobic amino acid residues and proline residues to the total number of residues is greater than or equal to 2 / 3.

Description

[Technical Field]

[0001] The present invention relates to a lactic acid bacteria growth promoter. [Background technology]

[0002] Lactic acid bacteria are known to have complex nutritional requirements. In culturing lactic acid bacteria, variations in the quality of the culture medium raw materials can sometimes result in variations in the yield of the resulting lactic acid bacteria. In relation to this, factors promoting the growth of lactic acid bacteria have been proposed. For example, International Publication No. 2017 / 034011 proposes a growth promoter for beneficial intestinal bacteria containing a specific cyclodipeptide. Furthermore, International Publication No. 2011 / 027719 proposes a growth promoter for Lactobacillus lactic acid bacteria containing κ-caseinoglycomacropeptide as an active ingredient. Summary of the Invention [Problem to be solved by the invention]

[0003] An object of the present invention is to provide a lactic acid bacteria growth promoter having an effect of promoting the growth of lactic acid bacteria. [Means for solving the problem]

[0004] Specific means for solving the above problems are as follows, and the present invention encompasses the following aspects: A first aspect is a lactic acid bacteria growth promoter comprising at least one peptide selected from the group consisting of a first peptide having a total residue number of 9 or less and having a hydrophobic amino acid residue at the C-terminus, and a second peptide having a total residue number of 9 or less and in which the ratio of the total number of hydrophobic amino acid residues and proline residues to the total number of residues is 2 / 3 or more.

[0005] In one embodiment, the hydrophobic amino acid residue may be an amino acid residue selected from the group consisting of alanine (A), glycine (G), valine (V), isoleucine (I), leucine (L), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M) and cysteine ​​(C), or may be an amino acid residue selected from the group consisting of alanine (A), glycine (G), phenylalanine (F) and tyrosine (Y).

[0006] In one embodiment, the first peptide may have a total residue number of 6 or less, and the hydrophobic amino acid residue may be a residue of an amino acid selected from the group consisting of phenylalanine (F) and tyrosine (Y). In one embodiment, the first peptide may have a ratio of the total number of hydrophobic amino acid residues and proline residues to the total number of residues of 2 / 3 or more.

[0007] In one embodiment, the ratio of the total number of hydrophobic amino acid residues and proline residues to the total number of residues in the second peptide may be 3 / 4 or more. In one embodiment, the total number of residues in the second peptide may be 4 or less. In one embodiment, the second peptide may consist of hydrophobic amino acid residues and proline residues. In one embodiment, the second peptide may have a hydrophobic amino acid residue at the C-terminus. [Effects of the Invention]

[0008] According to the present invention, a lactic acid bacteria growth promoter having an effect of promoting the growth of lactic acid bacteria can be provided. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the growth-promoting effect of various peptides on Lactobacillus gasseri. [Figure 2] FIG. 1 shows the growth-promoting effect of a dipeptide and amino acid mixture on Lactobacillus gasseri. [Figure 3] FIG. 1 shows the growth-promoting effects of various peptides on Lactobacillus acidophilus. [Figure 4] FIG. 1 shows the growth-promoting effects of various peptides on Lactobacillus amylovorus. [Figure 5] FIG. 1 shows the growth-promoting effects of various peptides on Lactobacillus culbatus. [Figure 6] FIG. 1 shows the growth-promoting effects of various peptides on Lactobacillus plantarum. [Figure 7] FIG. 1 shows the growth-promoting effects of various peptides on Lactobacillus brevis. [Figure 8] FIG. 1 shows the growth-promoting effect of dipeptides on Lactobacillus gasseri. DETAILED DESCRIPTION OF THE INVENTION

[0010] In this specification, the content of each component in a composition means the total amount of the components in the composition unless otherwise specified, when the composition contains multiple substances corresponding to each component. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments shown below are examples of lactic acid bacteria growth promoters for realizing the technical concept of the present invention, and the present invention is not limited to the lactic acid bacteria growth promoters shown below.

[0011] Lactic acid bacteria growth promoter The lactic acid bacteria growth promoter may contain at least one first peptide having a total residue count of 9 or less and a hydrophobic amino acid residue at the C-terminus. The lactic acid bacteria growth promoter may also contain at least one second peptide having a total residue count of 9 or less and a ratio of the total number of hydrophobic amino acid residues and proline residues to the total number of peptide residues of 2 / 3 or more. Peptides with specific structures have a lactic acid bacteria growth promoting effect. By containing at least one peptide selected from the group consisting of the first peptide and the second peptide having a lactic acid bacteria growth promoting effect, the lactic acid bacteria growth promoter can exhibit an excellent lactic acid bacteria growth promoting effect.

[0012] Here, the amino acids in this specification include amino acids used in protein synthesis, and these are preferably L-amino acids. The amino acids used in protein synthesis are the 20 amino acids encoded by codons in genes. The amino acids used in protein synthesis include hydrophobic amino acids, hydrophilic amino acids, and proline (P). Hydrophobic amino acids include aliphatic hydrophobic amino acids consisting of alanine (A), glycine (G), valine (V), isoleucine (I), and leucine (L), aromatic hydrophobic amino acids consisting of phenylalanine (F), tyrosine (Y), and tryptophan (W), and sulfur-containing hydrophobic amino acids consisting of methionine (M) and cysteine ​​(C). Hydrophilic amino acids include basic hydrophilic amino acids consisting of lysine (K), arginine (R), and histidine (H), acidic hydrophilic amino acids consisting of aspartic acid (D) and glutamic acid (E), and neutral hydrophilic amino acids consisting of serine (S), threonine (T), asparagine (N), and glutamine (Q). Proline (P) is an imino acid and is sometimes referred to as a non-standard amino acid in this specification.

[0013] The amino acids that make up the peptides contained in the lactic acid bacteria growth promoter may include the above 20 amino acids used in protein synthesis; amino acids that can make up proteins, including amino acids generated by post-translational modification such as selenocysteine, pyrrolysine, N-formylmethionine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, and O-phosphoserine, in addition to the amino acids used in protein synthesis; and amino acids that are not normally found in proteins, such as β-alanine, sarcosine, ornithine, citrulline, creatine, γ-aminobutyric acid (GABA), opine, and trimethylglycine.

[0014] The total number of residues of the first peptide and the second peptide contained in the lactic acid bacteria growth promoter may each be 9 or less, and preferably 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less. The total number of residues of the first peptide and the second peptide may each be 2 or more, and preferably 3 or more, or 4 or more. The total number of residues of the first peptide and the second peptide may be the same or different.

[0015] The first peptide has a hydrophobic amino acid residue at its C-terminus. The hydrophobic amino acid residue at the C-terminus of the first peptide may be at least one selected from the group consisting of alanine (A), glycine (G), valine (V), isoleucine (I), leucine (L), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), and cysteine ​​(C). The hydrophobic amino acid residue at the C-terminus of the first peptide is preferably at least one selected from the group consisting of alanine (A), glycine (G), phenylalanine (F), and tyrosine (Y), and more preferably at least one selected from the group consisting of phenylalanine (F) and tyrosine (Y).

[0016] The first peptide may have a total residue number of 6 or less and may have at least one hydrophobic amino acid residue selected from the group consisting of phenylalanine (F) and tyrosine (Y) at the C-terminus. Furthermore, the ratio of the total number of hydrophobic amino acid residues and proline residues to the total number of residues ((number of hydrophobic amino acid residues + number of proline residues) / total number of residues; hereinafter also referred to as "specific residue ratio") may be 2 / 3 or more, and preferably the specific residue ratio is 3 / 4 or more.

[0017] Specific examples of the first peptide are shown below, but the present invention is not limited to these. For example, the first peptide may include a peptide in which any one or two residues have been inserted, deleted, substituted, or added in the amino acid sequence of the specific examples below, and which has the effect of promoting the growth of lactic acid bacteria. Furthermore, the lactic acid bacteria growth promoter may contain one type of first peptide alone, or two or more types in combination.

[0018] [Table 1]

[0019] The second peptide has a total residue number of 9 or less, and the ratio of the total number of hydrophobic amino acid residues and proline residues to the total number of residues in the peptide (specific residue ratio) is 2 / 3 or more, preferably 3 / 4 or more, or 1. Here, a specific residue ratio of 1 means that the second peptide is composed only of hydrophobic amino acid residues and proline residues. The total residue number of the second peptide may be 6 or less, preferably 4 or less. The total residue number of the second peptide may be 2 or more, preferably 3 or more. Furthermore, the second peptide may have a hydrophobic amino acid residue at the C-terminus.

[0020] The hydrophobic amino acid residue in the second peptide may be at least one selected from the group consisting of alanine (A), glycine (G), valine (V), isoleucine (I), leucine (L), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), and cysteine ​​(C). The hydrophobic amino acid residue in the second peptide is preferably at least one selected from the group consisting of alanine (A), glycine (G), phenylalanine (F), and tyrosine (Y), and more preferably at least one selected from the group consisting of phenylalanine (F) and tyrosine (Y).

[0021] Specific examples of the second peptide are shown below, but the present invention is not limited to these. For example, the second peptide may include a peptide in which any one or two residues have been inserted, deleted, substituted, or added in the amino acid sequence of the specific examples below, and which has the effect of promoting the growth of lactic acid bacteria. Furthermore, the lactic acid bacteria growth promoter may contain one type of second peptide alone or a combination of two or more types.

[0022] [Table 2]

[0023] The lactic acid bacteria growth promoter may further contain at least one other peptide having a lactic acid bacteria growth promoting effect in addition to at least one selected from the group consisting of the first peptide and the second peptide. Specific examples of other peptides are listed below, but the present invention is not limited to these. The lactic acid bacteria growth promoter may contain one other peptide alone or two or more other peptides in combination.

[0024] [Table 3]

[0025] The peptides contained in the lactic acid bacteria growth promoter may be selected, for example, by differential analysis of the components of lactic acid bacteria culture media from different production lots, which can be performed, for example, by liquid chromatography-mass spectrometry (LC-MS).

[0026] Lactic acid bacteria in this specification include species belonging to the genus Lactobacillus and Lactococcus, such as Lactobacillus gasseri (e.g., FERM BP-11331), Lactobacillus acidophilus (FERM BP-4981), Lactobacillus amylovorus (FERM BP-11255), Lactobacillus curvatus (NITE P-02033), Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus. rhamnosus, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus jensenii, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus zeae Examples of the bacterium include Lactobacillus zeae, Lactobacillus gallinarum, and Lactococcus lactis, and preferably at least one selected from the group consisting of these.More preferably, the lactic acid bacteria may be at least one species selected from the group consisting of Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus curvatus, Lactobacillus plantum, and Lactobacillus brevis.

[0027] The growth-promoting effect of the lactic acid bacteria growth promoter on lactic acid bacteria may be, for example, greater than 100%, preferably 105% or greater, 110% or greater, or 120% or greater, when the growth rate of the control is taken as 100%. The growth-promoting effect on lactic acid bacteria can be evaluated by adding 0.25 mg / mL of the lactic acid bacteria growth promoter to a medium and measuring the rate of increase in bacterial cell count after anaerobic culture at 37°C for 18 hours.

[0028] The peptides contained in the lactic acid bacteria growth promoter can be obtained, for example, by treating edible proteins with an appropriate protease. They can also be obtained by isolating them from meat extracts, yeast extracts, etc. Furthermore, they can also be obtained by chemical synthesis. Conventional methods can be appropriately used for chemically synthesizing peptides.

[0029] The lactic acid bacteria growth promoter can be used for the production of lactic acid bacteria, the production of fermented products using lactic acid bacteria, etc. In the production of lactic acid bacteria, for example, lactic acid bacteria are cultured in an appropriate medium to which a predetermined amount of the lactic acid bacteria growth promoter has been added. The amount of the lactic acid bacteria growth promoter added to the medium may be, for example, 0.001 mg / mL to 25 mg / mL, preferably 0.01 mg / mL to 10 mg / mL, and more preferably 0.1 mg / mL to 2.5 mg / mL. The medium may contain, as needed, meat extract, yeast extract, soy milk, skim milk powder, sucrose, inorganic salts, etc., appropriately selected and contained in a predetermined amount. The culture conditions may be, for example, a temperature of about 25°C to about 40°C, a pH of about 5 to about 6.5, and anaerobic conditions such as a nitrogen gas atmosphere. The initial concentration of lactic acid bacteria may be, for example, 1 x 10 4 The concentration may be 1×10 or more / mL of medium, preferably 1×10 5The culture medium is a medium containing lactic acid bacteria or more per mL. The culture time may be, for example, 4 hours or more, and preferably 6 hours to 48 hours. The culture method can be appropriately selected from commonly used culture methods such as static culture, agitation culture, and shaking culture. Lactic acid bacteria can be recovered from the culture medium after culture using a separation device such as a centrifuge.

[0030] Examples of fermented products using lactic acid bacteria include fermented milk and silage. Fermented milk can be obtained by adding lactic acid bacteria and a lactic acid bacteria growth promoter to milk and then subjecting the milk to lactic acid fermentation. Silage is animal feed made by packing green-harvested feed crops (such as grass and corn) into silos, fermenting them with lactic acid, and storing them. When producing silage, adding a lactic acid bacteria growth promoter to the feed crops can efficiently grow lactic acid bacteria and promote lactic acid fermentation. This effectively inhibits the growth of putrefactive bacteria, allowing high-quality silage to be produced.

[0031] Therefore, the present invention also encompasses the use of a lactic acid bacteria growth promoter in the production of lactic acid bacteria or the production of a fermented product using lactic acid bacteria. Further, in other aspects, the present invention also encompasses the use of at least one peptide selected from the group consisting of the first peptide and the second peptide in the production of a lactic acid bacteria growth promoter, the use of at least one peptide selected from the group consisting of the first peptide and the second peptide in lactic acid bacteria cultivation, and at least one peptide selected from the group consisting of the first peptide and the second peptide used in lactic acid bacteria cultivation. [Example]

[0032] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0033] (1) Preparation of evaluation medium Six strains of Lactobacillus gasseri (FERM BP-11331), Lactobacillus acidophilus (FERM BP-4981), Lactobacillus amylovorus (FERM BP-11255), Lactobacillus curvatus (NITE P-02033), Lactobacillus plantarum, and Lactobacillus brevis were cultured until the total number of bacteria reached 1.0 × 10 8 pcs / mL or more 1.0×10 9 A medium composition was developed by reducing the nitrogen source from MRS medium so that the number of cells / mL or less was maintained. This composition was used for evaluation as "modified MRS medium." The composition of MRS medium was based on the composition listed in the Becton, Dickinson and Company (BD) medium catalog. The compositions of MRS medium and modified MRS medium are shown below.

[0034] [Table 4]

[0035] Example 1 Growth promoting effect on Lactobacillus gasseri (FERM BP-11331) The 28 candidate peptides shown in the sequences below were prepared by chemical synthesis. The candidate peptides were added to modified MRS medium at a concentration of 0.25 mg / mL to prepare a medium for evaluation. 1.0 × 10 Lactobacillus gasseri was added to the prepared medium for evaluation. 7 The cells were seeded at 100p / mL and cultured at 37°C for 18 hours. After culture, the culture medium was collected and the particle concentration (particles / mL) was measured using a particle counter analyzer (CDA-1000, Sysmex) as a characteristic value that serves as an indicator of bacterial growth. The growth rate (%) was calculated, with the control value using medium without peptide added being set at 100%. The results are shown in the table below.

[0036] [Table 5]

[0037] Comparative Example 1 Using the dipeptide LF (Leu-Phe) and an equimolar mixture of leucine (L) and phenylalanine (F), cultivation was carried out in the same manner as in Example 1, and the growth-promoting effect of the dipeptide and amino acid mixture on lactic acid bacteria was evaluated. The results are shown in the table below.

[0038] [Table 6]

[0039] Example 2 Growth promoting effect on Lactobacillus acidophilus (FERM BP-4981) The eight candidate peptides, the sequences of which are shown below, were evaluated for their growth-promoting effect on lactic acid bacteria in the same manner as in Example 1, except that Lactobacillus acidophilus was used instead of Lactobacillus gasseri. The results are shown in the table below.

[0040] [Table 7]

[0041] Example 3 Growth promoting effect on Lactobacillus amylovorus (FERM BP-11255) The eight candidate peptides, the sequences of which are shown below, were evaluated for their growth-promoting effect on lactic acid bacteria in the same manner as in Example 1, except that Lactobacillus amylovorus was used instead of Lactobacillus gasseri. The results are shown in the table below.

[0042] [Table 8]

[0043] Example 4 Growth promoting effect on Lactobacillus curvatus (NITE P-02033) The eight candidate peptides, the sequences of which are shown below, were evaluated for their growth-promoting effect on lactic acid bacteria in the same manner as in Example 1, except that Lactobacillus culbatus was used instead of Lactobacillus gasseri. The results are shown in the table below.

[0044] [Table 9]

[0045] Example 5 Growth promoting effect on Lactobacillus plantarum The eight candidate peptides whose sequences are shown below were evaluated for their growth-promoting effect on lactic acid bacteria in the same manner as in Example 1, except that Lactobacillus plantarum was used instead of Lactobacillus gasseri. The results are shown in the table below.

[0046] [Table 10]

[0047] Example 6 Growth-promoting effect on Lactobacillus brevis The eight candidate peptides whose sequences are shown below were evaluated for their growth-promoting effect on lactic acid bacteria in the same manner as in Example 1, except that Lactobacillus brevis was used instead of Lactobacillus gasseri. The results are shown in the table below.

[0048] [Table 11]

[0049] Example 7 The growth-promoting effect on lactic acid bacteria was evaluated in the same manner as above, except that dipeptide (LF) was used as the peptide and the addition concentration was changed to 0.1 mg / mL, 0.25 mg / mL, or 1.0 mg / mL. The results are shown in the table below.

[0050] [Table 12]

[0051] The disclosure of Japanese Patent Application No. 2020-096288 (filing date: June 2, 2020) is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

[Claim 1] A lactic acid bacteria growth promoter comprising at least one peptide having the amino acid sequence VPAF, LPWG, APKPDYPIA, PVF, PSVF, AGPQTF, PVY, LPLG, SEYPPLG, IPGI, LF, IF, LPVL, VPI, FL, VPL, VPVG, IPIT, IPIQ, LVE, or LPT.

Images