JP4074006B2 - Method for producing IgE antibody production inhibitor and food using the same - Google Patents

Description

【００３４】
抗原刺激の前処理でＩｇＥ抗体産生を抑制したことは、菌体およびその分解物の摂取により生体の反応性乃至体質に影響していることを示した。また菌体自体より実施例４の分解物摂取群で強い抑制が認められたことは、酵素分解により活性が強まったこと、さらに、ＩｇＥ抗体産生抑制作用が実施例３の分解物摂取群で最も高かったことはその活性分解物は主として細胞壁の構成成分であることを示した。
【００３５】
実施例８
免疫調節活性分解物摂取のＩｇＧ抗体産生に及ぼす作用を卵白アルブミンで免疫したマウス血清中の抗卵白アルブミンＩｇＧ抗体産生により調べた。すなわち、平底９６穴マイクロプレート（Coster,Cambridge,MA,USA)に、０．０５ＭのｐＨ９．５の炭酸ナトリウム緩衝液に溶解した卵白アルブミン０．１ｍｇ／ｍｌの５０μｌを入れ、４℃に一晩置いた。洗浄液（０．９％ＮａＣｌ，０．０５％Ｔｗｅｅｎ２０）で洗浄後、牛血清アルブミン１ｍｇ／ｍｌを含有する燐酸緩衝生理食塩水（日水製薬、ＰＢＳ）２００を加え、３７℃、１時間おいてブロッキングし、洗浄後、牛血清アルブミン１０ｍｇ／ｍｌ、０．０５％Ｔｗｅｅｎ２０、食塩３％を含むＰＢＳ（溶液Ａ）にて希釈した実施例７のマウス血清を加え、３７℃に１時間置いた。洗浄後、ペルオキシダーゼ標識抗マウスＩｇＧヤギ抗体（生化学工業製）の溶液Ａによる１００００倍希釈液５０μｌを加え、３７℃に１時間置いた後、洗浄し、基質溶液（ｏ−フェニレンジアミン４０ｍｇ、３０％過酸化水素２０μｌ／クエン酸−リン酸ナトリウム緩衝液１００ｍｌ）１００μｌを加え、室温に置き、４９２ｎｍの吸光度で測定した。
【００３６】
抗体量は、卵白アルブミンをフロインドの完全アジュバントとともに免疫したマウス血清より、卵白アルブミン結合セファロース４Ｂアフィニテイーカラムを用いて精製したＩｇＧ抗体を用いて作成した検量線より求めた。実施例７で用いたプール血清中の抗卵白アルブミンＩｇＧ抗体（抗ＥＡＩｇＧ）の測定結果を表２に示した。
【００３７】
【表２】

【００３８】
この結果は納豆菌の菌体乃至酵素分解物の摂取により、ＩｇＧ抗体産生の増強（対照群の２倍以上）乃至増強傾向を示している。
【００３９】
実施例９
ＩｇＥ抗体産生抑制性分解物の安全性
４週齢のＳＤ系雄ラット一群５匹に実施例４で得られた酵素分解物の２ｇ／ｋｇ体重当てを強制経口投与し、単回投与における安全性を調べた。また、４週齢のＳＤ系雄ラット一群１０匹に５％含有する飼料で４週間に亘って飼育し、飼料のみを与えた群を対照群として投与群と比較した。その結果、単回投与では何らの急性症状も認められず、表３に示した如く、反復投与試験においても、体重増加、一般状態ともに対照群との間に差は認められなかった。
【００４０】
【表３】

【００４１】
実施例１０
実施例４で得られた納豆菌の卵白リゾチームおよびブロメラインを用いた酵素分解物９０ｍｇ、デンプン３０ｍｇ、アビセル（セルロース）１８０ｍｇの割合で用いて１錠３００ｍｇの食品を常法により製造した。
【００４２】
実施例１１
実施例４で得られた納豆菌の卵白リゾチーム及びブロメラインを用いた分解物２部、ココアバター１０部、グラニュー糖７部、牛乳７部、乳化剤０．０５部に精製水を加え、全量を１００部とし、常法によりココア飲料を製造した。
【００４３】
【発明の効果】
本発明によるとＩｇＧ抗体の産生を増強し、ＩｇＥ抗体の産生を選択的に抑制する免疫調節活性分解物を容易に製造することができ、そのような作用を有する食品を提供することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microbial cell degradation product having a novel immunoregulatory activity, a method for producing the same, and a food using the same.
[0002]
[Prior art]
Allergies are classified from type 1 to type 4 depending on the immunological mechanism of the onset. Of these, type 1 allergy is also known as immediate allergy or atopy, and is the main cause of the pathogenesis of hay fever, bronchial asthma, atopic dermatitis, food allergy, etc. Globulin is the cause. In contrast, type 2 and type 3 allergies are due to IgG and IgM type immunoglobulins.
[0003]
Usually, an allergic predisposition refers to a predisposition to easily develop type 1 allergy, but in patients with allergic predisposition, the concentration of IgE antibody in the blood is high, and IgE antibodies are easily administered against various antigens. It is known to produce. The produced IgE antibody binds to a receptor for IgE antibody on the surface of mast cells, which is abundant in mucosal tissues such as skin, eyes, nose, trachea and digestive tract. In the onset of type 1 allergy, when an antigen binds to this antibody, physiologically active substances (mediators) such as histamine, serotonin, and leukotriene are released from mast cells, and an allergic reaction is induced. In addition to mast cells, eosinophils and the like are activated through various cytokines produced from these cells in response to the reaction between antigen-specific lymphocytes and antigen, and an inflammatory reaction proceeds.
[0004]
For prevention and treatment of type 1 allergy, drugs that antagonize these mediators (antihistamines, antiserotonins, anti-leukotrienes), drugs that block the release of these mediators from mast cells, and activity of immunocompetent cells Adrenal hormone steroids or the like are used as pharmaceuticals to suppress the production of cytokines by inhibiting the production of cytokines or to suppress the activation by cytokines.
[0005]
Since the causative antibody of type 1 allergy is an IgE antibody as described above, a drug that suppresses the production of IgE antibody is considered to be effective as a causative therapy for type 1 allergy. Conventionally known drugs that suppress the production of antibodies include drugs that have anticancer effects, but these drugs are highly toxic and are therefore used for the treatment of allergic diseases. Absent. It is said that type 2 and type 3 allergies mentioned above are caused by IgG and IgM type immunoglobulins, but IgG and IgM antibodies together with IgA antibodies are important antibodies for protecting against viruses and pathogenic bacteria. In order to maintain the normal immune function of the living body, it is desirable to develop drugs or foods that rather enhance these antibodies.
[0006]
[Problems to be solved by the invention]
The object of the present invention is to provide food additives or health foods that can suppress or enhance the production of antibodies such as IgG antibodies while suppressing the production of IgE antibodies and improving atopy and allergic predisposition. It is.
[0007]
[Means for Solving the Problems]
As a result of diligent research to solve the above-mentioned problems, the present inventors do not affect the production of antibodies such as IgG antibodies by ingesting foods containing degradation products of Bacillus bacteria and / or lactic acid bacteria. The inventors have found that the production of IgE antibody can be suppressed while the present invention has been achieved.
[0008]
In order to efficiently produce immune antibodies in animals, the term “immune adjuvant” refers to what is administered parenterally mixed with an antigen. It has long been known that Mycobacterium tuberculosis dead cells have an immune adjuvant effect. As a result of pursuing the active ingredient of the immunoadjuvant action, the smallest structural unit was muramyl-L-alanyl-D-isoglutamine (MDP) which is a peptidoglycan constituent of the cell wall. MDP is known to be present not only in tuberculosis but also in all bacteria, including pathogenic, non-pathogenic, and Gram-stained positive-negative bacteria. In most bacteria examined in practice, the cell wall fraction containing MDP showed immunoadjuvant action. In addition, chemically synthesized MDP exhibited an immunopotentiating effect even when administered orally (Shozo Otani, Biochemistry, Vol. 48, No. 12, pp. 1081-1107, 1976).
[0009]
Based on the above background, various studies have been conducted for the purpose of enhancing immunity, and fractions containing cell walls are resistant to tumor (Bogdanov IG et al., Antitumor glycopeptides from Lactobacillus bulgaricus cell wall FEBS LETT. 1975 57 (3 ): 251-261) and enhancement of IgG antibodies that play an important role in activation of macrophages and cellular immunity related to resistance to bacterial infection and resistance to bacterial virus infection (Namba Y et al., Effect of oral administration of Isozyme or digested cell wall on immunostimulation in guinea pigs., Infect Immun 31: 580-583 1981). In addition, regarding the relationship between activity and structure, cell walls and enzyme digests were studied (Shozo Otani, Biochemistry, Vol. 48, No. 12, pp. 1081-1107, 1976).
However, previous studies have not yet known how the fraction containing MDP has an effect on the production of antibodies that cause type 1 allergies such as atopy.
[0010]
The present inventors prepared bacterial cells and enzyme digests thereof for natto and lactic acid bacteria, examined IgE antibody production when ingested orally, and administered both enzymes with enzyme digests containing MDP. Thus, it was found that the production of IgE antibody can be suppressed, and the activity itself is weak in the cell itself, but the activity of inhibiting IgE antibody production is enhanced by enzymatic degradation with cell wall lytic enzyme.
[0011]
The mucopeptide layer of the bacterial cell wall is composed of a polymer in which N-acetylglucosamine and muramic acid are bonded to a long chain by β-1,4 bonds and a carboxyl residue of muramic acid by peptide bonds to L-alanine or L-glycine, D- Amino acids are bound in the order of glutamic acid, L-lysine or memezodiaminopimelic acid, D-alanine and the like. The last D-alanine is peptide-bonded to D-glutamine or L-lysine or memezodiaminopimelic acid of the peptide chain similarly derived from the carboxyl residue of muramic acid of the adjacent sugar chain. The number of amino acids bound between sugar chains varies depending on the bacterium, but is an amino acid having 6 to 7 residues. If the sugar chains and sugar chains that are arranged in parallel are vertical threads, the peptide bonds that connect these sugar chains to each other are in the form of a horizontal thread, and both form a network structure and are also called peptidoglycans. Peptidoglycan is in a cytoplasmic form and forms a strong wall where the cells can physically cope with changes in osmotic pressure.
[0012]
Bacterial cell wall lytic enzyme is an enzyme that hydrolyzes peptidoglycan and is roughly divided from the mechanism of action of the enzyme to cleave sugar chains (eg egg white lysozyme), endopeptidase (eg Achromobacter protease I) that acts on peptide bonds, There are three types of amidases that break down the linkage between muramic acid and amino acids in sugar chains.
[0013]
The present invention relates to a cell lysate having immunomodulating activity obtained by dissolving cell walls of Bacillus bacteria and / or lactic acid bacteria (hereinafter referred to as immunomodulating activity lysate), a method for producing the same, a food containing the same or a food containing the same It is a beverage.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Bacillus genus bacteria and lactic acid bacteria are readily available bacteria. Among them, Bacillus natto has been recognized as a safe food and is an aerobic bacterium, so it can be cultured easily and the production efficiency of the cells. There is also an advantage that it is economical and inexpensive. Examples of Bacillus bacteria include Bacillus subtilis and Bacillus megaterium in addition to Bacillus natto. Examples of lactic acid bacteria include Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbruckii, Biphidobacterium longum, and Leuconostoc mesenteroides.
[0015]
What is necessary is just to culture | cultivate Bacillus genus bacteria and lactic acid bacteria by a well-known method using arbitrary culture media. As the microbial cells, any of the culture itself, live centrifuge cells, lyophilized cells, spray-dried cells, etc. can be used.
[0016]
The production of the immunomodulating activity degradation product of the present invention from the microbial cells is based on the lysis of the cell walls of the microbial cells, but the cell wall lysis methods include cell wall lytic enzyme methods, lysogen phage induction methods, and self-owned bacteria A method using a melting enzyme can be mentioned.
[0017]
Among these, an efficient method using a cell wall lytic enzyme is a method in which a cell wall lytic enzyme glucosidase type enzyme and / or an endopeptidase type protease is allowed to act. In the present invention, the combined use of both enzymes can produce a degradation product having an activity superior to that of both enzymes alone. Among them, degradation using a glucosidase type enzyme is efficient, and examples of the glucosidase type enzyme include egg white lysozyme, bacterial lysozyme, and N-acetylmuramidis SG. Egg white lysozyme used as is preferred.
[0018]
Endopeptidase-type proteolytic enzymes include bromelain, cerathiopeptidase, pronase (Streptomyces griseus f1-endopeptidase), nagase (Subtilisin BPN), seaprose S (Armillaria mellea protease), trypsin, Achromobacter protease I (lysyl endo) Peptidase), Grifola frondosa (maitake) metalloendopeptidase, and bromelain, which has a wide substrate specificity and is used for food processing, is most preferable.
[0019]
The treatment with these enzymes is performed near the optimum pH of each enzyme. Glycosidase type enzyme and endopeptidase type proteolytic enzyme treatment may be carried out at a normal neutral pH of 5 to 8, the enzyme may be added in an amount of 0.01 to 10 mg per 1 g of dried cells, and the temperature may be from room temperature to 30 to 70 ° C. The enzyme-treated product may contain 10 to 500 µg / ml of nucleolytic enzymes DNase and RNase as necessary to lower the viscosity.
[0020]
Bacterial lysis by induction of lysogen phage is performed by treating Bacillus bacteria for 5 minutes at 45 ° C and then culturing at 42 ° C. Moreover, the method by self-melting is performed by suspending bacterial cells in purified water and incubating at around 50 ° C. for 1 to 3 days.
[0021]
As a specific method for producing an immunomodulating activity degradation product, for example, Bacillus natto is cultured in an appropriate medium, and then collected by centrifugation, for example, cells in an isotonic medium such as physiological saline. Glycosidase-type enzyme such as egg white lysozyme or endopeptidase-type protease such as bromelain is added to the suspension to dissolve the cell wall, leaving the cell membrane of the cell body and the cytoplasm surrounded by it as a protoplast state. After separation and removal by means such as centrifugation or molecular sieve, the other enzyme, ie, endopeptidase-type protease or glycosidase-type enzyme, can be applied to the supernatant liquid. It is more efficient and preferable to perform endopeptidase-type proteolytic enzyme treatment after dissolving and removing protoplasts By separating and removing protoplasts, the active degradation product can be efficiently purified in the medium-solubilized fraction. Concentration of the resulting immunomodulating activity degradation product can also be achieved by fractionation of ethanol and acetone.
[0022]
As another production method, for example, after natto bacteria are cultured, separated and collected, suspended in purified water, glycosidase type enzyme is added and stirred to dissolve the cell wall, and then endopeptidase type protease is added. Further, a method of further stirring may be used.
[0023]
The standard intake of the immunomodulating activity degradation product of the present invention is equivalent to 0.2 to 2 g per day, preferably about 1 g in terms of dry dead cells.
[0024]
The immunomodulating activity degradation product of the present invention can be made into a food or beverage in the form of powder, powder, tablet, capsule, confectionery, bread, noodles, drink and the like. What is necessary is just to manufacture these foodstuffs by a well-known method.
[0025]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. In the examples, both% and parts are based on weight.
[0026]
Example 1
An L-shaped tube containing 10 ml of soy bean casein digest (SCD) medium was inoculated with Bacillus natto (BN-2 strain) and cultured with shaking at 37 ° C. for 7 hours. Each 1 ml of this culture solution was inoculated into 4 Sakaguchi flasks containing 100 ml of SCD medium and cultured with shaking at 37 ° C. for 24 hours. The cells were collected by centrifugation to obtain about 5 g of wet cells. After centrifugal washing once with physiological saline, 20 ml of purified water is added and suspended in the cells, 5 mg of egg white lysozyme is added while stirring, and after stirring for 1 hour at 37 ° C., 10 mg of seratiothiopeptidase is added. The mixture was stirred at 37 ° C. for 1 hour to obtain a decomposed product. The decomposed product was heated at 80 ° C. for 30 minutes and then freeze-dried to obtain 1 g of a dried product.
[0027]
Example 2
A Sakaguchi flask containing 100 ml of soy bean casein digest (SCD) medium was inoculated with Bacillus natto (BN-2 strain) and cultured with shaking at 37 ° C. for 18 hours. Each 1 ml of this culture solution was inoculated into 40 Sakaguchi flasks containing 100 ml of SCD medium, and cultured with shaking at 37 ° C. for 8 hours. The cells were collected by centrifugation to obtain about 50 g of wet cells. After centrifugal washing once with physiological saline, 200 ml of physiological saline is added to the cells, suspended, 50 mg of egg white lysozyme is added with stirring, the mixture is stirred at 37 ° C. for 1 hour, and then centrifuged at 13,000 rpm for 30 minutes to precipitate. The supernatant was collected except for the protoplast. Cerathiopeptidase 50 mg was added to the supernatant, and the mixture was further stirred at 37 ° C. for 1 hour to obtain a decomposed product. The decomposition product was heated at 80 ° C. for 30 minutes, dialyzed to remove sodium chloride, and then freeze-dried to obtain 1.2 g of a dried product.
[0028]
Example 3
A dried product 1.0 g was obtained in the same manner as in Example 2 except that 50 mg of bromelain was used instead of 50 mg of serratiopeptidase.
[0029]
Example 4
After suspending 1 kg of lyophilized natto (Miura strain) in 20 liters of physiological saline, stirring at 30 ° C, adding 100 ml of physiological saline containing 5 g of egg white lysozyme and stirring at 30 ° C for 1 hour Then, 200 ml of physiological saline containing 10 g of bromelain was added and further stirred at 30 ° C. for 1 hour to obtain a decomposed product. The decomposed product was spray-dried to obtain 1 kg of a dried product.
[0030]
Example 5
1 kg of freeze-dried microbial cells of Bacillus natto (Miura strain) was suspended in 20 liters of purified water and allowed to stand at 50 ° C. for 24 hours for self-thawing. The self-melted product was spray-dried to obtain 1 kg of a dried product.
[0031]
Example 6
Lysis by induction of Bacillus subtilis lysogen phage was performed as follows.
Bacillus subtilis marburg strain was inoculated into a Sakaguchi flask containing 100 ml of soy bean casein digest (SCD) medium and cultured with shaking at 37 ° C. for 18 hours. 1 ml of each culture was inoculated into 40 Sakaguchi flasks containing 100 ml of SCD medium and cultured with shaking at 37 ° C. for 8 hours. The cells were collected by centrifugation to obtain about 50 g of wet cells. The cells were suspended in 2 liters of fresh medium at 45 ° C. After culturing for 5 minutes with aeration, an ice-cooled medium was added to bring the culture temperature to 42 ° C., and the culture was continued for 60 minutes. The concentration of the bacterial solution was measured by absorbance at 610 nm. Although _OD610nm = 28 at the start of aeration culture, _OD610nm = 6.5 after 42 minutes at 42 ° C., and lysis was confirmed.
[0032]
Example 7
A 6-week-old BALB / c female mouse group of 6 mice was prepared by adding each of the Bacillus natto heated cadaver and the dried product obtained in Examples 3 and 4. After 2 weeks of feeding on a mixed diet, immunization was performed with 0.1 ml containing 10 μg of ovalbumin and 1 mg of aluminum hydroxide. 14 days after immunization, blood was collected from the fundus venous plexus and serum was separated. Six sera were pooled and the IgE antibodies of the pooled sera were measured by passive skin reaction in rats. That is, the back of 15-20 week old male SD rats was shaved, and under Nembutal anesthesia, 0.1 ml of serum diluted with physiological saline for injection was injected into two animals with each diluted serum. Administered. 24 hours after intradermal administration, 1 ml of physiological saline containing 10 mg of Evans blue dye and 1 mg of ovalbumin was intravenously administered under Nembutal anesthesia, and blue leakage observed at the site of intradermal administration 30 minutes after intravenous administration The major axis and minor axis of the dye were measured, and an average diameter of 5 mm or more was determined as positive. The antibody titer was expressed as the dilution factor of serum, and the high antibody titer value of two animals was defined as the antibody titer of the serum. The results are shown in Table 1. In addition, the determination of the effect in a table | surface determined the difference more than 2 pipe | tubes by the serum dilution 2-fold dilution multiple as the significance, and determined it as + per difference of 1 pipe | tube.
[0033]
[Table 1]

[0034]
Suppression of IgE antibody production by antigen stimulation pretreatment indicated that ingestion of bacterial cells and their degradation products affected the reactivity and constitution of the living body. Moreover, the strong suppression was recognized in the degradation product ingestion group of Example 4 from the microbial cells per se, that the activity was enhanced by enzymatic degradation, and that the IgE antibody production inhibitory effect was the most in the degradation product ingestion group of Example 3. It was high that the active degradation product was mainly a constituent of cell wall.
[0035]
Example 8
The effect of intake of immunomodulating activity degradation product on IgG antibody production was examined by production of anti-ovalbumin IgG antibody in the serum of mice immunized with ovalbumin. That is, 50 μl of ovalbumin 0.1 mg / ml dissolved in 0.05 M pH 9.5 sodium carbonate buffer was placed in a flat bottom 96-well microplate (Coster, Cambridge, MA, USA) overnight at 4 ° C. placed. After washing with a washing solution (0.9% NaCl, 0.05% Tween 20), phosphate buffered saline (Nissui Pharmaceutical, PBS) 200 containing 1 mg / ml of bovine serum albumin was added and left at 37 ° C. for 1 hour. After blocking and washing, the mouse serum of Example 7 diluted with PBS (solution A) containing 10 mg / ml bovine serum albumin, 0.05% Tween 20, and 3% sodium chloride was added and placed at 37 ° C. for 1 hour. After washing, 50 μl of a 10,000-fold diluted solution of peroxidase-labeled anti-mouse IgG goat antibody (manufactured by Seikagaku Corporation) in solution A was added and placed at 37 ° C. for 1 hour, followed by washing and substrate solution (o-phenylenediamine 40 mg, 30 % Hydrogen peroxide 20 μl / citrate-sodium phosphate buffer 100 ml) was added, and the mixture was placed at room temperature and measured by absorbance at 492 nm.
[0036]
The amount of antibody was determined from a calibration curve prepared using IgG antibody purified from ovalbumin-conjugated Sepharose 4B affinity column from mouse serum immunized with ovalbumin with Freund's complete adjuvant. Table 2 shows the measurement results of anti-ovalbumin IgG antibody (anti-EA IgG) in the pooled serum used in Example 7 .
[0037]
[Table 2]

[0038]
This result shows an increase in IgG antibody production (more than twice that of the control group) or an increase tendency due to ingestion of natto bacteria or enzyme degradation products.
[0039]
Example 9
Safety of IgE antibody production-inhibiting degradation product Five groups of 4 week-old SD male rats were forcibly orally administered with 2 g / kg body weight of the enzyme degradation product obtained in Example 4, and safety in a single administration I investigated. In addition, 10 groups of 4 week-old SD male rats were reared for 4 weeks with a feed containing 5% and compared with the administration group as a control group. As a result, no acute symptom was observed in a single administration, and as shown in Table 3, there was no difference between the control group and the weight gain and general state in the repeated administration test.
[0040]
[Table 3]

[0041]
Example 10
A 300 mg food product was prepared in a conventional manner using the enzyme-degraded product 90 mg, starch 30 mg, and avicel (cellulose) 180 mg using the egg white lysozyme and bromelain obtained in Example 4.
[0042]
Example 11
Purified water is added to 2 parts of a decomposition product using natto egg white lysozyme and bromelain obtained in Example 4, 10 parts of cocoa butter, 7 parts of granulated sugar, 7 parts of milk, and 0.05 part of emulsifier. A cocoa beverage was produced by a conventional method.
[0043]
【The invention's effect】
According to the present invention, it is possible to easily produce an immunomodulating activity degradation product that enhances the production of IgG antibody and selectively suppresses the production of IgE antibody, and can provide a food having such action.

Claims (4)

A method for producing an IgE antibody production inhibitor, comprising dissolving a cell wall of Bacillus natto with egg white lysozyme and then treating the cell wall with bromelain or cerathiopeptidase . Perform lysis of the cell walls by egg white lysozyme in a medium like Cho圧, resulting protoplasts were separated off, IgE antibody production according to claim 1, wherein processing the solubilized fraction of the medium in bromelain or serratiopeptidase suppressed A method for producing a substance . An IgE antibody production inhibitor obtained by the production method according to claim 1 or 2 . A food or beverage containing the IgE antibody production-suppressing substance according to claim 3 .