CN106038610B - Use of bacillus coagulans for preparing medicament for preventing and/or treating anaphylaxis - Google Patents
Use of bacillus coagulans for preparing medicament for preventing and/or treating anaphylaxis Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Medicinal Chemistry (AREA)
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- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses an application of Bacillus coagulans (Bacillus coagulans) in preparing medicines or foods for preventing and/or treating organism anaphylactic reaction, wherein the preservation number of the Bacillus coagulans (Bacillus coagulans) is CCTCC NO: m2013193. Compared with other bacillus coagulans in the same genus, the preservation number is CCTCC NO: the bacillus coagulans M2013193 can effectively regulate the immunity of organisms, and the experimental result shows that the preservation number is CCTCC NO: the live bacterial agent prepared from the bacillus coagulans of M2013193 can effectively relieve aquatic product anaphylactic reaction caused by tropomyosin, can effectively promote polarization of Th0 cells to Th1 and Treg, and can reduce IgE antibody level and mast cell degranulation.
Description
Technical Field
The invention relates to an application of bacillus coagulans, in particular to bacillus coagulans CCTCC NO: the invention also relates to an application of M2013193 in regulating and controlling the immune function of an organism, and a functional food composition and a preparation method thereof.
Background
The IgE-mediated immediate food allergy mainly comprises two stages, namely an allergic activation phase and an effect phase of food allergens. The allergen of the first exposure stimulates the activation of the intestinal mucosa antigen presenting cells and effectively activates the juvenile CD4+The proliferation and differentiation of T cells into helper T cell (Th cells) subset, wherein the Th2 cell subset mainly mediates the humoral immune response related to anaphylaxis and parasitic infection, secretes cytokines such as IL-4, IL-5 and IL-13, induces the class switching of B cells and produces allergen-specific IgE, and the specific IgE can be combined with the high affinity receptor FcRI on the surfaces of mast cells and basophils, thereby enabling the body immunity to be in a sensitized state. When the allergen is re-exposed, FcRI cross-links with the IgE-allergen complex, rapidly initiating a signal transduction cascade, triggering degranulation of mast cells and basophils, releasing large amounts of allergic inflammatory mediators (histamine, leukotrienes, etc.), thereby causing IgE-mediated immediate hypersensitivity.
The penaeus vannamei boone is deeply favored by consumers due to the characteristics of being delicious and rich in nutritional protein, but the muscle protein of the penaeus vannamei boone contains a large amount of tropomyosin, so that anaphylactic reaction is easily caused. Tropomyosin is an allergic glycoprotein with a sugar content of about 4%, an isoelectric point of 4.5 and a molecular weight of about 35-40 kDa. Shrimp tropomyosin contains 8 IgE binding sites and each binding site is about 5-14 amino acid residues in length.
Bacillus coagulans, a spore-forming lactic acid bacterium, is a probiotic isolated from yellow croaker. The study shows that the bacterium is a kind of bacterium beneficial to the host, and the name list of safe microbial strains published by the FDA in the United states is also included. The bacillus coagulans has good probiotic characteristics, can effectively inhibit anaphylactic reaction, and plays an important role in regulating and controlling the health of a host.
Disclosure of Invention
The invention aims to provide a new application of bacillus coagulans.
The invention provides an application of Bacillus coagulans (Bacillus coagulans) in preparing medicines or foods for regulating the organism immune function, wherein the preservation number of the Bacillus coagulans (Bacillus coagulans) is CCTCC NO: m2013193.
The preservation number is CCTCC: the strain of C201351 is classified and named as Bacillus coagulans LL1103(Bacillus coagulans LL1103), the preservation date is 5 months and 9 days in 2013, the preservation unit is China center for type culture Collection, the preservation address is the university of Wuhan, China, and the strain can be obtained by purchasing from the preservation center.
The diameter of a colony of the bacillus coagulans strain on a nutrient agar plate is 2.0-2.5mm, the bacillus coagulans strain is platform-shaped, the edge is neat, the bacillus coagulans strain is opaque, glossy and pigment-free, and the bacillus coagulans strain is soft in texture; under the microscope, the cells of the strain are in straight rod shape, the cell size is 0.5 multiplied by 1.0 mu m to 1.5 multiplied by 4.0 mu m, and the cells have round ends and spore ellipse ends; the suspension was uniformly turbid in the liquid medium with a slight amount of precipitate, and the precipitate was dispersed by gentle shaking.
The research of the invention finds that the medicine or food prepared by Bacillus coagulans can prevent and/or treat the allergic reaction of organisms by adjusting the immune function of the organisms.
Therefore, the invention also provides an application of Bacillus coagulans (Bacillus coagulans) in preparing medicines or foods for preventing and/or treating organism anaphylactic reaction, wherein the preservation number of the Bacillus coagulans (Bacillus coagulans) is CCTCC NO: m2013193.
The anaphylaxis is the anaphylaxis caused by tropomyosin in the muscle protein of the penaeus vannamei boone, and the mechanism of the anaphylaxis caused by the tropomyosin is mainly as follows: primary exposure of tropomyosin stimulates activation of intestinal mucosal antigen presenting cells (mainly dendritic cells), effectively activating proliferation and differentiation of naive CD4+ T cells into helper T cell (Th cells) subsets, wherein the Th2 cell subset mainly mediates humoral immune response related to anaphylaxis and parasitic infection; when tropomyosin is re-exposed, FcRI cross-links with the IgE-allergen complex, rapidly initiating a signal transduction cascade, triggering degranulation of mast cells and basophils, releasing large amounts of allergic inflammatory mediators (histamine, leukotrienes, etc.), thereby causing IgE-mediated immediate hypersensitivity. The Bacillus coagulans (Bacillus coagulans) can promote polarization of Th0 cells to Th1 cells and Treg cells, further inhibit IgG1 antibody generation and mast cell degranulation, and promote inflammatory antibody IgE to be converted into non-inflammatory antibody IgG4, so that anaphylactic reaction caused by tropomyosin in the muscle protein of the penaeus vannamei is inhibited.
Therefore, the invention also provides an application of Bacillus coagulans (Bacillus coagulans) in preparing medicines or foods for promoting polarization of Th0 cells to Th1 cells and Treg cells, wherein the preservation number of the Bacillus coagulans (Bacillus coagulans) is CCTCC NO: m2013193.
The Th0 cells were derived from spleen.
The invention also provides an application of Bacillus coagulans (Bacillus coagulans) in preparing medicines or foods for inhibiting IgE antibody or/and IgG1 production, wherein the preservation number of the Bacillus coagulans (Bacillus coagulans) is CCTCC NO: m2013193.
The IgE and IgG1 antibodies are derived from serum.
The invention also provides an application of the Bacillus coagulans in preparing medicines or foods for inhibiting mast cell degranulation, wherein the preservation number of the Bacillus coagulans is CCTCC NO: m2013193.
The mast cells are derived from the intestinal tract.
The invention also provides a culture medium prepared from the components with the preservation number of CCTCC NO: m2013193 Bacillus coagulans (Bacillus coagulans) for preparing active agent for preventing and/or treating anaphylaxis.
When the viable bacteria agent is in a liquid state, the content of the bacillus coagulans in the viable bacteria agent is 108-109CFU。
The live bacterial agent is prepared by a conventional method, for example, the liquid live bacterial agent can be prepared by the following method:
the preservation number is CCTCC NO: m2013193 Bacillus coagulans is cultured in NB liquid medium at 37 deg.C for 12 hr, centrifugating the cultured Bacillus coagulans at 3000rpm/min for 10min, collecting thallus, suspending in sterile physiological saline, and adjusting thallus concentration to 2 × 108-109CFU/mL.
CFU (Colony-Forming Units) refers to the number of viable bacteria. During viable bacteria culture counting, a colony formed by growing and propagating a single thallus or a plurality of aggregated thallus on a solid culture medium is a colony forming unit.
The invention also provides a functional food composition for preventing and/or treating the anaphylactic reaction of the organism, wherein the functional food composition contains the components with the preservation number of CCTCC NO: m2013193. When the functional food is liquid, the bacteria content is 109The amount of CFU added is a preferred condition. The preferred conditions are most effective in regulating the body. The functional food has wide coverage and can comprise dairy products, fruit and vegetable juice products and the like. For example, the dairy product may contain cow milk, fruit granules, etc., and has a bacteria content of 109The preservation number of the CFU is CCTCC NO: m2013193.
Compared with other bacillus coagulans in the same genus, the preservation number is CCTCC NO: the bacillus coagulans M2013193 can effectively regulate the immunity of organisms, and the experimental result shows that the preservation number is CCTCC NO: the live bacterial agent prepared from the bacillus coagulans of M2013193 can effectively relieve aquatic product anaphylactic reaction caused by tropomyosin, can effectively promote polarization of Th0 cells to Th1 and Treg, and can reduce IgE antibody level and mast cell degranulation.
Drawings
FIGS. 1 and 2 are graphs showing the results of detection of specific IgE, IgG1 levels in serum in example 1.
FIGS. 3, 4 and 5 are graphs showing the results of flow cytometry for the detection of T cell subsets in example 1.
FIG. 6 is a graph showing the results of detecting mouse lymphocyte proliferation by the MTT method in example 1.
FIGS. 7, 8 and 9 are graphs showing the results of the expression levels of mRNA of various cytokines and transcription factors under the co-culture condition of Treg cells, Th2 cells and Bacillus coagulans in example 2.
FIG. 10 is a graph showing the results of in vitro co-culture of Bacillus coagulans with Treg cells and B cells under allergen stimulation in example 2.
FIGS. 11 and 12 are graphs showing the results of co-culture of B.coagulans with P815 mast cells.
Detailed Description
Example 1
The invention evaluates the regulation and control effect of bacillus coagulans on anaphylactic reaction by indexes such as specific IgE and IgG levels in serum, histamine content, cytokine content in cell supernatant, cell RNA expression level and the like.
1.1 construction of sensitized animal models
Experimental animals: female Balb/c mice, 6 weeks old, 18 + -2 g in weight, purchased from the Experimental animals center of Hangzhou university and continuously fed with special feed without shrimp protein for two generations. The temperature of the mouse room is 20 +/-2 ℃, the humidity is 50 +/-10%, the circulation is carried out for 12 hours day and night, the ventilation is ensured, and the mouse cage is cleaned and disinfected twice per week. A blank control group, an allergy group and a bacteria experimental group are respectively arranged by adopting a random grouping method, and each group comprises 8 mice.
Strain culture: the preservation number is CCTCC NO: bacillus coagulans strain of M2013193 used NBCulturing in liquid culture medium at 37 deg.C for 12 hr, centrifuging at 3000rpm/min for 10min, collecting thallus, suspending in sterile physiological saline, and adjusting thallus concentration to 2 × 109CFU/mL。
Injecting PBS buffer solution into abdominal cavity of control group mouse, injecting 1.2mg tropomyosin into each abdominal cavity of experimental group mouse, mixing with equal volume Freund complete adjuvant and fully emulsifying for the first injection, then using Freund incomplete adjuvant every time, immunizing 1 time every 7 days for 4 times, and performing intragastric lavage excitation by increasing dose of each group 1 time than the original dose in 35 days. And (3) starting from the 38 th day, performing intragastric administration on 0.5mL of bacillus coagulans every day in the experimental group, performing intragastric administration on the control group and the sensitized group with the same volume of sterile normal saline, and continuously performing intragastric administration for 20 days. Sensitization and gastric lavage were performed again on day 61, and after 1 week of the last challenge, the mice were fasted for 12 h and sacrificed. Orbital veins were bled on days 7, 28, 35, 48, 61, and 68. The blood collected each time is put into a 1.5mL EP tube, incubated for 1h at room temperature, coagulated overnight at 4 ℃, then centrifuged for 15min at 4 ℃ and 10000r/min, and the supernatant is taken as serum, and frozen and stored at-20 ℃.
1.2 serum levels of specific IgE, IgG1
Orbital veins were bled on days 7, 28, 35, 48, 61, and 68. The blood collected each time is put into a 1.5mL EP tube, incubated for 1h at room temperature, coagulated overnight at 4 ℃, and then centrifuged for 15min at 4 ℃ and 10000r/min, and the supernatant is the serum. The indirect ELISA method measures the specific IgE and IgG1 antibody level in the mouse serum at each time point: diluting the allergen-coated plate with substrate buffer solution, and standing overnight at 4 ℃; washing the plate, adding 200 mul of confining liquid into each hole, and incubating for 2h at 37 ℃; washing the plate, diluting the mouse serum by a diluent in a multiple ratio (1:20), adding the plate, and incubating for 1h at 37 ℃; washing the plate, adding enzyme-labeled secondary antibody (diluted 1: 8000), and incubating for 1h at 37 ℃; washing the plate, adding a developing solution, and incubating for 15min at 37 ℃ in a dark place; plus 2M H2SO4The reaction was stopped with 50. mu.L. The OD values of IgE and IgG1 were measured at 490nm and 450nm, respectively. The results are shown in FIGS. 1 and 2.
The aquatic product allergy belongs to type I anaphylactic reaction, and IgE and IgG1 antibodies are important action factors for the generation of the type I anaphylactic reaction and can be combined with receptors of mast cells to trigger a series of anaphylactic reactions such as degranulation and the like. As can be seen from the figure, the level of specific antibody in the serum of the mice in the sensitized group is obviously increased compared with that of the mice in the normal control group. The preservation number is CCTCC NO: the antibody level of M2013193 after bacillus coagulans intervention is reduced, and the result shows that bacillus coagulans has the effect of inhibiting the production of IgE and IgG1 antibodies and can play a role in preventing allergy.
1.3 levels of cytokine content in cell supernatants
Aseptically taking spleen tissues of mice, grinding the spleen tissues into cell suspension, and adjusting the cell concentration to 107And/ml. Taking 1ml of cell suspension into 24-well plate, performing secondary allergen in vitro stimulation (50 μ g/ml), 37 deg.C, and 5% CO2After 48h of incubation, the supernatant was centrifuged. The kit protocol was followed, and the absorbance of each well was measured at a wavelength of 450nm using a microplate reader, and the IL-4, IL-5, IL-13 and IFN-. gamma.concentrations of each sample were determined from the corresponding standard curve. The results are shown in Table 1.
TABLE 1
Note: in the table, a, b and c represent the significance of the difference, letters are different to represent the significance of the difference, and the same represents that the difference is not significant (P < 0.05).
IFN-gamma is one of main cytokines secreted by Th1 cells, and can promote the organism to generate antibodies such as IgG2a and the like and inhibit the generation of inflammation. IL-4, IL-5 and IL-13 are representative cytokines of Th2 cells, are mainly secreted by Th2 cells and mast cells, promote further differentiation of Th2 cells in a positive feedback manner, and play an important role in mediating immediate allergy. As can be seen from table 1, the preservation number provided by the drench is CCTCC NO: after the bacillus coagulans is M2013193, the secretion amount of Th1 type cytokines is obviously increased, the IL-4, IL-5 and IL-13 of Th2 type cytokines are respectively reduced by 69.73 percent, 17.92 and 71.90 percent, the reduction level is obvious, and the preservation number is CCTCC NO: the microbial inoculum prepared from the bacillus coagulans M2013193 can effectively relieve the anaphylactic reaction caused by the original globulin.
1.5 flow cytometry detection of T cell subpopulation numbers
Taking spleen of mouse aseptically, grinding glass needle core on 200 mesh sieve, adding RPMI1640 culture solution to make into single cell suspension. Centrifuging to obtain cell precipitate, adding 5ml erythrocyte lysate, mixing, standing for 5min, centrifuging, discarding supernatant, suspending the cell precipitate in RPMI1640 culture solution, centrifuging, collecting cell precipitate, and washing with RPMI1640 culture solution (1500rpm, centrifuging for 5min) for 2 times. Adjusting the concentration of splenocytes to 107One per ml. Taking 100 mu l of cell suspension, adding 1 mu l of FITC-anti-mouse CD69 and 1.25 mu l of PE-CD183(CXCR3) into the cell suspension, uniformly mixing, keeping the mixture at room temperature in the dark for 30min, centrifuging, removing supernatant, suspending in 300 mu l of PBS solution, and measuring the level of Th1 cells on a flow cytometer. Mu.l of the cell suspension was added with 1. mu.l of FITC-anti-mouse CD69 and 1.251. mu.l of PE-anti-mouse ST2(IL-33R), and after incubation for 30min, the Th2 cell level was examined. Taking 100 μ l of cell suspension, adding 0.5 μ l of FITC-anti-mouse CD4 and 0.625 μ l of PE-anti-mouse CD25, and after incubation for 30min, detecting the level of Treg cells. The results are shown in table 2, fig. 3, fig. 4 and fig. 5.
TABLE 2
From the above results, it was found that the immune response of the sensitized mice was unbalanced in Th1/Th2 type, and the response of Th2 type was too strong and the response of Th1 type was suppressed. After the bacillus coagulans is subjected to dry pretreatment, the ratios of splenic lymphocytes Th1/Th2 and Treg/Th2 of the mice are increased, and the results are remarkably different from those of mice in an sensitized group. By combining the results of the differentiation levels of the Th1, the Th2 and the Treg cells, the microbial inoculum prepared from the bacillus coagulans is found to be capable of effectively promoting the polarization of organisms to the directions of Th1 and Treg, inhibiting the Th2 immune response and adjusting the balance among the Th1, Treg and Th2 reactions.
1.6 MTT method for detecting mouse lymphocyte proliferation
Extracting spleen lymphocytes of mice, and adjusting the cell concentration to 107one/mL. Adding 100 μ L of cell suspension and tropomyosin diluent (protein final concentration of 1mg/mL) into 96-well plate, and finishing with RPMI1640The whole culture was adjusted to a final volume of 200. mu.L, and 3 replicates were set for each sample. A blank control (without cell suspension) was also set at 37 ℃ with 5% CO2Culturing for 44 h. mu.L of MTT solution was added to each well and incubation was continued for 4 h. Centrifuging at 1500rpm/min at 4 deg.C for 5min, and discarding the supernatant. 150 mu L DMSO solution is added into each hole, shaken evenly and then cultured for 15min in the dark under the condition of room temperature. The OD value at 490nm wavelength was measured by a microplate reader. The results are shown in FIG. 6.
Bacillus coagulans significantly increased proliferation of splenic lymphocytes compared to the sensitized group (P < 0.05).
Example 2
This example illustrates CCTCC NO: effect of bacillus coagulans M2013193 on Treg cells, mast cells and B cells.
2.1 sources of Treg, B and mast cells
Female Balb/c mice, 6 weeks old, weighing 18-22g were selected. After cervical vertebra death, spleen was aseptically taken and ground to obtain spleen lymphocytes. Flow cytometry was used to sort the two cell types.
P815 mast cells were purchased from shanghai ge van ltd under the following culture conditions: 5% CO at 37 deg.C2。
2.2 Co-culture of Bacillus coagulans with Treg cells and Th2 cells
The concentration of Treg cells and Th2 cells is adjusted to 10 respectively6one/mL. In a 6-well plate, 1mL each of Treg cells and Th2 cells was added, and 100. mu.L of Bacillus coagulans (concentration: 10)8CFU/mL), in vitro stimulation (stimulation concentration 50. mu.g/mL) after filtered sterilization of tropomyosin. 37 ℃ and 5% CO2Culturing under the condition for 48 h. 1500rpm/min, 4 ℃ centrifugation for 5 min. The cell pellet was dissolved in Trizol and total RNA was extracted according to the kit instructions. The primers were synthesized by Enwei fundi (Shanghai) trade Co., Ltd, see Table 2. The reaction system is 20 mul, and the system is prepared as follows: SYBR Mix 10. mu.l; upstream and downstream primers were 0.4. mu.l each; 2 μ l of cDNA; DEPC water 7.2. mu.l. Reaction conditions are as follows: at 95 ℃ for 10 min; at 95 ℃ for 20 s; 20s at 55 ℃; 72 ℃ for 20 s; a total of 40 cycles; extension at 72 ℃ for 7 min. Taking hprt as an internal reference gene, and adopting 2 for the relative expression quantity of each detection gene-ΔΔCtMethod calculation, knotThe results are shown in fig. 7, 8 and 9.
TABLE 3 real-time fluorescent quantitative PCR primer parameters
FIGS. 7 to 9 show the expression levels of mRNA of different cytokines and transcription factors under the co-culture condition of Treg cells, Th2 cells and Bacillus coagulans. As can be seen from the figure, the preservation number is CCTCC NO: after the bacillus coagulans of M2013193 is co-cultured, the mRNA expression levels of IL-10, Foxp3 and TGF-beta are obviously increased and have significant difference (P <0.001), which indicates that the bacillus coagulans has a regulating effect on Treg cells.
2.3 Co-culture of Bacillus coagulans with Treg cells and B cells
Adjusting the concentration of Treg cells and B cells to 10 respectively6one/mL. In 6-well plates, 1mL each of Treg cells and B cells was added, and 100. mu.L of Bacillus coagulans was added (concentration: 10)8CFU/mL), in vitro stimulation (stimulation concentration 50. mu.g/mL) after filtered sterilization of tropomyosin. 37 ℃ and 5% CO2Culturing under the condition for 48 h. 1500rpm/min, 4 ℃ centrifugation for 5 min. Taking supernatant for measuring IgE and IgG4Antibody content. The specific procedures are described in ELISA kit, and the results are shown in FIG. 10.
FIG. 10 shows the results of in vitro co-culture of B cells and Treg cells with Bacillus coagulans under allergen stimulation. As a result, it was found that Bacillus coagulans inhibited IgE antibody content and increased IgG content4Secretion of antibody, and statistical significance of difference from control group (P)<0.05). The results show that the bacillus coagulans can effectively promote Treg cells to generate direct effects on B cells and inhibit the generation of inflammatory antibodies.
2.4 Co-culture of Bacillus coagulans with P815 mast cells
Adjusting mast cell concentration to 106one/mL. To a 6-well plate, 1mL of a cell culture solution and 100. mu.L of Bacillus coagulans (10 concentration) were added8CFU/ml), tropomyosin filter-sterilized afterbodyExternal stimulation (stimulation concentration 50. mu.g/mL). 37 ℃ and 5% CO2Culturing under the condition for 48 h. 1500rpm/min, centrifuging for 5min, collecting supernatant, and determining histamine content. The cell pellet was washed twice with sterile PBS, 0.3% methyl red dye was added, mixed gently and cultured at 37 ℃ for 3 min. Randomly selecting 100 cells under an inverted microscope, and observing the degranulation condition of the cells. The vacuolated, unstained cells were degranulated and the results are shown in FIGS. 11 and 12.
Sensitized mast cells were co-cultured in vitro with Bacillus coagulans, and mast cell degranulation (FIGS. 4-6A) and histamine release (FIGS. 4-6B) were observed. From the results of the above figures, it was found that after the bacillus coagulans is dried, the degranulation number of mast cells is reduced, and histamine secretion is reduced, which is significantly different from that of the control group (P < 0.05).
The above description is only an embodiment of the present invention, but the technical features of the present invention are not limited thereto, and any person skilled in the relevant art can change or modify the present invention within the scope of the present invention.
Claims (1)
1. Bacillus coagulans (A)Bacillus coagulans) Use of Bacillus coagulans (A) for preparing a medicament for preventing and/or treating allergic reactions in the bodyBacillus coagulans) The preservation number of (A) is CCTCC NO: the number of the M2013193,
the anaphylaxis is the anaphylaxis caused by tropomyosin in the muscle protein of the penaeus vannamei,
the Bacillus coagulans（Bacillus coagulans) By promoting polarization of Th0 cells to Th1 cells and Treg cells, IgG1 antibody production and mast cell degranulation are inhibited, inflammatory antibody IgE is promoted to be converted into non-inflammatory antibody IgG4, and anaphylactic reaction caused by tropomyosin in muscle protein of Penaeus vannamei Boone is inhibited.
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