JP2004101249A - Screening process of probiotics lactobacteriaceae - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To construct a system for screening intestinal tract system lactobacteriaceae having high intestinal adhesiveness compatible with the human blood type, especially L. acidophilus group lactobacteriaceae. <P>SOLUTION: This screening method, using a biosensor (BIACORE 1000), is established for L. acidophilus group lactobacteriaceae adhering to the human intestinal mucin of a nonhandicapped person of A blood type, which is most general in the Japanese. <P>COPYRIGHT: (C)2004,JPO

Description

【００２２】
この表面プラズモン共鳴スペクトルを利用したＢＩＡＣＯＲＥによる、乳酸菌の、両センサーチップに対する付着性を検証することにより、血液型糖鎖認識性Ｌ．　ａｃｉｄｏｐｈｉｌｕｓグループ乳酸菌のスクリーニングに極めて有効ではないかと考えられる。付着性を示すものはＲＵ値が上昇し、付着性を示さないものはＲＵ値の上昇が全く見られないといったＢＩＡＣＯＲＥによる結合性の評価法は、明確そのものであり、さらにセンサーグラムの形状より供試菌株がどの様な結合性を有するかについても有用な知見が得られる。
また、他の　Ｏ（Ｈ）型糖鎖　ＢＰ−ＰｒｏｂｅおよびＢ、Ｏ型ヒト大腸ムチンを用いたＢＩＡＣＯＲＥ解析を拡大することで、ヒトの全血液型に対応したヒト腸管付着性乳酸菌の新規スクリーニング法を確立することができる。当該方法によってＡＢＯ血液型別に腸管付着性の高い乳酸菌をスクリーニングすることができ、当該乳酸菌を用いて個人レベルに対応したオーダーメードの血液型別機能性ヨーグルトを作製することができる。
【００２３】
【発明の効果】
本発明により、表面プラズモン共鳴スペクトルを利用したＢＩＡＣＯＲＥによる、新機能をもったプロバイオティクス乳酸菌株の、新規なスクリーニング方法が提供された。本発明により得られた乳酸菌株は、ＡＢＯ血液型別に腸管付着性の高い乳酸菌であり、当該乳酸菌を用いて個人レベルに対応したオーダーメードの血液型別機能性ヨーグルトをはじめ、新しいプロバイオティクス食品、あるいは消化器プロバイオティクス、例えば感染症（歯周病、虫歯、慢性胃炎、胃十二指腸潰瘍、胃癌、腸管出血性大腸炎、溶血性尿毒症症候群、脳症、食中毒、偽膜性大腸炎）、アレルギー疾患（食物アレルギー、アトピー性皮膚炎、気管支喘息）、炎症性腸疾患（Ｃｒｏｈｎ　病、潰瘍性大腸炎）などの疾患の予防又は治療用食品の提供を可能とする。
【００２４】
【非特許文献１】
天野純子、生化学，　社団法人日本生化学会，　１９９９年，　第７１巻，　ｐ．２７４−２７７
【非特許文献２】
Ｆｕｊｉｓａｗａ，　Ｔ．　外，　Ｉｎｔ．　Ｊ．　Ｓｙｓｔ．　Ｂａｃｔｅｒｉｏｌ．，　１９９２年，　第４２巻，　ｐ．４８７−４９１
【非特許文献３】
Ｔａｋａｈａｓｈｉ，　Ｎ．　外，　Ｂｉｏｓｃｉ．　Ｂｉｏｔｅｃｈ．　Ｂｉｏｃｈｅｍ．，　１９９６年，　第６０巻，　第９号，　ｐ．１４３４−１４３８
【非特許文献４】
稲川淳一：実験医学別冊　クローズアップ実験法　総集編，　２００２年３月１日発行，　ｐ．１４３−１５０
【図面の簡単な説明】
【図１】ヒトＡＢＯ式血液型のＡ型とＢ型のエピトープ部位を有するＢＰプローブ糖鎖プローブの化学構造を示す。
【図２】図２−ＡはＡ型糖鎖ＢＰプローブの糖鎖部位、図２−ＢはＢ型糖鎖ＢＰプローブの糖鎖部位を示す。
【図３】表面プラズモン共鳴（ＳＰＲ）を利用したＢＩＡＣＯＲＥ　１０００測定法の原理とレゾナンスユニットの定義する。
【図４】ＢＩＡＣＯＲＥ　１０００解析で得られるセンサーグラムにおける結合・解離・再生のサイクルを示す。
【図５】図５−ＡはＡ型糖鎖ＢＰプローブへの結合を示したＬ．　ｃｒｉｓｐａｔｕｓＪＣＭ８７７８株のＢＩＡＣＯＲＥセンサーグラムを示し、図５−ＢはＡ型糖鎖ＢＰプローブへの結合を示した同株のＢＩＡＣＯＲＥセンサーグラム示し、そして図５−Ｃは対照を示す。
【図６】図６−ＡはセンサーチップＣＭへの、Ａ型血液型ヒト大腸粗精製ムチンの固定化センサーグラムを示し、図６−Ｂは同ムチンへの結合性を示したＬ．　ｃｒｉｓｐａｔｕｓ　ＪＣＭ８７７８株のＢＩＡＣＯＲＥセンサーグラムを示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel screening method for probiotic lactic acid bacteria having a new function.
[0002]
[Prior art]
In recent years, it has been reported that the chemical structure of the sugar chain constituting human colon mucin (HCM) differs depending on the ABO blood group (for example, see Non-Patent Document 1). This scientific fact strongly suggests that different blood types have different sugar chain structures in the digestive tract mucins of different individuals, and that the types of probiotic lactic acid bacteria that adhere and grow there are different.
[0003]
As a probiotic lactic acid bacterium, a representative strain of the genus Lactobacillus used in humans is L. lactis. acidophilus, L .; casei, L .; rhamnosus, and L. bulgaricus. L. isolated from human and animal feces Acidophilus group lactic acid bacteria are thought to live in the intestinal tract of the host, improve the intestinal flora, and perform functions such as intestinal regulation, similar to the Bifidobacterium group.
[0004]
L. acidophilus group lactic acid bacteria, as a result of DNA-DNA homology and cell wall composition analysis, L. acidophilus (A-1); crispatus (A-2); amylovorus (A-3); gallinarum (A-4); gasseri (B-1); Johnsonii (B-2) is classified into six bacterial species (for example, see Non-Patent Document 2).
[0005]
In order for these lactic acid bacteria to initially infect humans and colonize the intestinal tract, it is essential that they adhere to the surface of intestinal epithelial cells. Takahashi et al. Reported that L. s. To develop a colonic mucin (RCM) -coated polystyrene bead for screening lactic acid bacteria strains of the L. acidophilus group, L. lactis was developed. As a result of screening a lactic acid bacterium strain of L. acidophilus group, it has been reported that a surface protein (SLP) derived from a strain that strongly binds to RCM also binds to a mucous layer of human colon (see, for example, Non-Patent Document 3).
[0006]
On the other hand, a biosensor (BIACORE) using the surface plasmon resonance (SPR) phenomenon has been developed and has rapidly spread in the fields of biochemistry and molecular biology.
[0007]
[Problems to be solved by the invention]
The present invention relates to an intestinal lactic acid bacterium having extremely high intestinal adherence adapted to human blood groups, An object of the present invention is to construct a system for screening lactic acid bacteria of the acidophilus group.
[0008]
[Means for Solving the Problems]
Using this biosensor (BIACORE 1000), the present inventors have found that L. adenosine adhering to human intestinal mucin of the most common healthy group A blood group in Japan is used. A screening method for acidophilus group lactic acid bacteria was established.
[0009]
That is, the present invention
(1) A method for screening probiotic lactic acid bacteria by surface plasmon resonance spectrum, which comprises using an ABO blood group epitope portion as a ligand,
(2) a method for screening probiotic lactic acid bacteria by surface plasmon resonance spectrum, which comprises using mucin derived from the large intestine as a ligand;
(3) the screening method according to (2), wherein the mucin derived from the large intestine is a crudely purified mucin;
(5) Lactobacillus spp. acidophilus, L .; casei, L .; rhamnosus, and L. (4) a screening method selected from the group consisting of B. bulgaricus;
Consists of
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
BIOCORE is a surface plasmon resonance sensor developed and put into practical use by Pharmacia Biotech. The principle and outline are as follows.
Its basic structure consists of a light source and a prism, a detection system and a microchannel system (FIG. 3). A ligand is immobilized on the surface of a cassette-type sensor chip, and an analyte (measurement target) is injected there. If both have an affinity, the amount of binding is optically detected (FIG. 4). In summary, the detection principle is such that when polarized light is incident through the glass surface side prism of the sensor chip, a part of the reflected light where the reflected light intensity is reduced is observed. The angle of the dark line in the reflected light depends on the mass change (refractive index change) in the very vicinity of the gold thin film. That is, a change in mass caused when the analyte binds to the ligand immobilized on the chip is detected as a change in the angle of the dark line. The angle amount of the dark line is defined as RU (resonance unit), and is graphed (sensorgram) as an RU value with respect to time (second), and monitored in real time. The sensor chip is formed by depositing gold on a glass plate and further bonding carboxymethyl dextran (CM-dextran) (CM chip).
[0011]
<Materials and methods>
Test Lactic Acid Bacteria L. isolated from human infants, infants, adults and elderly stool. acidophilus (A-1) 6 strains, Crispatus (A-2) 3 strain, L. amylovorus (A-3) 3 strain, L. Gasseri (B-1) 7 strains and Johnsonii (B-2) 2 strains, a total of 21 strains, were used. Identification of each bacterial species was performed by a DNA-DNA hybridization method.
[0012]
2 Blood group sugar chain (trisaccharide) biotinylated polymer (BP-) probe experiments were performed using trisaccharide sugar chains (A type [GalNAcα1-3 (Fucα1-)), which are epitope partial structures of two types of human ABO blood group substrates. 2) Gal-] and B-type [Galα1-3 (Fucα1-2) Gal-]) BP-Probe (manufactured by Seikagaku Corporation) were used. As a control for checking non-specific adsorption, an α-L-Rhamnose BP-probe having a common spacer site for BP-linked sugar chain probes was used. The structure of this BP-probe is shown in FIG. 1, and the chemical structure of the sugar chain is shown in FIG.
[0013]
3. Preparation of Human Colon Mucin The human intestinal type A intestine (rectum) was distributed by Tohoku University Graduate School of Medicine as a sample. The mucus mucin layer was collected from a normal rectal site by a surface scraping method. The mucin layer was degreased with a Folch solvent and diethyl ether and then dissolved in 4 M guanidine hydrochloride. The solution was purified by gel filtration, and subjected to a test as crude human A-type intestinal mucin (HCM). The collection of these samples has been carried out by the ethics committee of the Graduate School of Medicine, with the patient's consent.
[0014]
4 L. Interaction Analysis of Lactobacillus acidophilus Group Lactic Acid Bacteria with Blood Group Sugar Chains and Human Intestinal Crude Mucin Interaction analysis between the acidophilus group lactic acid bacteria, blood group sugar chains and human intestinal crudely purified mucin was performed using a surface plasmon resonance analyzer (BIACORE 1000; Pharmacia Biotech). The interaction intensity in the sensorgram (binding / dissociation curve) was represented by a resonance unit (RU) value. The regeneration solution used was 0.75 to 3 M guanidine hydrochloride. The principle of the present device and the method of interpreting the sensorgram are shown in FIGS.
[0015]
4-1 Culture and Preparation of Analyte Cells Each strain was subcultured on MRS medium (Difco Laboratories) and then cultured at 37 ° C. for 12 hours. The cells collected from the culture solution (500 μl) were washed with running buffer HBS-EP buffer (containing 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20). The cells were suspended in the same buffer (500 μl) to obtain analyte cells.
[0016]
4-2 Analysis of Interaction with Blood Group Sugar Chain Three types of biotinylated ligands, that is, a type A blood group glycan BP probe are provided in each flow cell on a sensor chip SA of a type in which streptavidin (SA) is immobilized in advance. , The B-type blood group sugar chain BP probe and the α-L-Rhamnose BP-probe were respectively immobilized by binding to avidin via biotin. Next, each analyte cell prepared in 4-1 was injected into BIACORE 1000, and the interaction between the bound sugar chain and the cell was analyzed over time.
[0017]
4-3 Analysis of Interaction with Human Type A Intestinal Mucin A sensor chip CM5 into which carboxymethyl dextran was introduced was used. The partially purified human HCM (ligand) prepared in 3 was immobilized on each flow cell on the sensor chip by the amine coupling method. Ethanolamine was used for blocking the remaining active group not bound to the ligand. Subsequently, each analyte cell prepared in 4-1 was injected into BIACORE 1000, and the interaction between the human colon mucin and the cell was analyzed over time.
[0018]
<Results and Discussion>
1 Interaction analysis with BIACORE 1000 for blood group sugar chain probe The binding (affinity) between 21 strains and a ligand by BIACORE 1000 was examined by a sensorgram (reaction curve). As a result, the three strains were found to bind to the BP sugar chain probe. The remaining 18 strains showed no or very weak binding.
Binding to BP sugar chain probe was observed. 5A and 5B show sensorgrams of crispatus strain JCM8778. This JCM8778 strain was compared with the binding to B-trisaccharide BP-Probe (200 RU value), which is a B-type sugar chain (FIG. 5B), with respect to the A-trisaccharide BP-Probe (350 RU value), which is an A-type sugar chain. Showed stronger binding (FIG. 5A). On the other hand, it did not bind to α-L-Rhamnose BP-Probe as a control, which differs from BP-Probe only at the sugar chain epitope site (FIG. 5C). As a result, the binding of the JCM8778 strain to both sugar chain BP-Probes is not caused by non-specific binding to the spacer site of the sugar chain BP-Probe or the surface of the sensor chip SA, but by specific binding to the sugar chain epitope. It became clear that there was. That is, it was shown that the JCM8778 strain was one of the lactic acid bacteria that specifically recognizes and binds to blood group sugar chains.
[0019]
2 Analysis of interaction with human intestinal mucin type A This was a binding test of B. acidophilus lactic acid bacteria with an artificial sugar chain probe using BIACORE 1000. Therefore, the binding property of the JCM8778 strain to a type A human intestinal partially purified mucin prepared from the human intestine, which is considered to reflect the actual intestinal environment, was examined. As a result, although the binding was weaker than in the case of the A-type sugar chain BP-probe, binding to human A-type large intestine mucin was clearly observed. These experimental results suggested that the JCM8778 strain was highly likely to be strongly attached to the human intestinal tract. A-type sugar chain The binding was weaker than in the case of the BP-probe, because various types of proteins and sugar chain sites were contained in human A colon mucin in large amounts, and the type A sugar chain involved in the binding Was also considered as one of the causes.
[0020]
<Conclusion>
The biosensor BIACORE using surface plasmon resonance spectrum measures the interaction of biomolecules in real time, and can analyze each interaction from mere strength (affinity) analysis to speed (kinetics). Have. Moreover, difficulties in other analysis required for B / F separation, a very weak binding (low affinity, K _D value of 10 -3 ^M) can be analyzed (e.g., Non-Patent Document 4 reference.) .
A-type sugar chain Mass screening is performed using a sensor chip to which BP-Probe is bound, and further screening is performed using a sensor chip to which human A-type intestinal mucin is bound, thereby providing a useful protein with high human intestinal adhesion. Biotic lactic acid bacteria could be provided. Table 1 shows the lactic acid bacteria to be subjected to this screening.
[0021]
[Table 1]

[0022]
By verifying the adhesion of lactic acid bacteria to both sensor chips by BIACORE using this surface plasmon resonance spectrum, the blood group sugar recognition L. It is considered that this is extremely effective for screening lactic acid bacteria of the acidophilus group. The BIACORE evaluation method of binding, in which the RU value increases for those showing adhesiveness, and the RU value does not increase for those showing no adhesiveness, is very clear and furthermore, it is based on the shape of the sensorgram. Useful knowledge can also be obtained as to what kind of binding property the test strain has.
In addition, by expanding BIACORE analysis using other O (H) -type sugar chains BP-Probe and B, O-type human colon mucin, a novel screening method for human intestinal adherent lactic acid bacteria corresponding to human whole blood types Can be established. By this method, a lactic acid bacterium having a high intestinal adhesion can be screened for each ABO blood type, and a bespoke functional blood type yogurt corresponding to an individual level can be produced using the lactic acid bacterium.
[0023]
【The invention's effect】
According to the present invention, a novel screening method for a probiotic lactic acid bacterium strain having a new function by BIACORE using a surface plasmon resonance spectrum is provided. The lactic acid bacterium strain obtained by the present invention is a lactic acid bacterium having a high intestinal adherence for each ABO blood type, and using the lactic acid bacterium, a bespoke blood-type functional yogurt corresponding to an individual level and a new probiotic foodstuff Or gastrointestinal probiotics such as infections (periodontal disease, caries, chronic gastritis, gastroduodenal ulcer, gastric cancer, enterohemorrhagic colitis, hemolytic uremic syndrome, encephalopathy, food poisoning, pseudomembranous colitis), allergies It is possible to provide foods for preventing or treating diseases such as diseases (food allergy, atopic dermatitis, bronchial asthma) and inflammatory bowel diseases (Crohn's disease, ulcerative colitis).
[0024]
[Non-patent document 1]
Junko Amano, Biochemistry, The Biochemical Society of Japan, 1999, Vol. 71, p. 274-277
[Non-patent document 2]
Fujisawa, T .; Outside, Int. J. Syst. Bacteriol. , 1992, Vol. 42, p. 487-492
[Non-Patent Document 3]
Takahashi, N.M. Outside, Biosci. Biotech. Biochem. , 1996, Vol. 60, No. 9, p. 1434-1438
[Non-patent document 4]
Junichi Inagawa: Experimental Medicine Separate Volume, Close-up Experimental Method Summary, Published March 1, 2002, p. 143-150
[Brief description of the drawings]
FIG. 1 shows the chemical structure of a BP probe sugar chain probe having A and B epitope regions of human ABO blood group.
FIG. 2-A shows a sugar chain site of an A-type sugar chain BP probe, and FIG. 2-B shows a sugar chain site of a B-type sugar chain BP probe.
FIG. 3 defines the principle of a BIACORE 1000 measurement method using surface plasmon resonance (SPR) and the definition of a resonance unit.
FIG. 4 shows a binding / dissociation / regeneration cycle in a sensorgram obtained by BIACORE 1000 analysis.
FIG. 5-A shows binding to L-type A sugar chain BP probe. FIG. 5-B shows a BIACORE sensorgram of crispatus JCM8778 strain, FIG. 5-B shows a BIACORE sensorgram of the same strain showing binding to an A-type sugar chain BP probe, and FIG. 5-C shows a control.
Fig. 6-A shows a sensorgram of immobilized crude mucosa of human type A large intestine immobilized on a sensor chip CM, and Fig. 6-B shows the binding ability of L. mucin to the mucin. 2 shows a BIACORE sensorgram of crispatus JCM8778 strain.

Claims (5)

A method for screening probiotic lactic acid bacteria by surface plasmon resonance spectrum, wherein the method uses an ABO blood group epitope portion as a ligand. A method for screening a probiotic lactic acid bacterium by surface plasmon resonance spectrum, comprising using a large intestine-derived mucin as a ligand. 3. The screening method according to claim 2, wherein the colon-derived mucin is a crudely purified mucin. The screening method according to any one of claims 1 to 3, wherein the probiotic lactic acid bacterium is of the genus Lactobacillus or the genus Bifidobacterium. Lactobacillus sp. {Acidophilus, L .; {Casei, L .; Rhamnosus, and L. The screening method according to claim 4, wherein the screening method is selected from the group consisting of ｉｃbulgaricus.

Images