An immunomodulatory material comprising heat-killed enterococcus faecalis and fermented rice bran and a preparation method thereof

Abstract

The present invention relates to an immunomodulatory material comprising heat-killed Enterococcus faecalis and fermented rice bran, and its preparation method. The concentration of heat-killed Enterococcus faecalis in the immunomodulatory material of the present invention is not less than 5*109 / g. The immunomodulatory material of the present invention is prepared by cultivation of Enterococcus faecalis in a culture medium containing fermented rice bran and followed heat-sterilization.

Description

[0001] An immunomodulatory material comprising heat-killed Enterococcus faecalis and fermented rice bran and a preparation method thereof

[0002] Technical field

[0003] The present invention relates to an immunomodulatory material comprising heat-killed Enterococcus faecalis and fermented rice bran and its preparation method.

[0004] Background and purpose of the invention

[0005] Since the discovery of chronic inflammation being the main symptom of allergic diseases including asthma and allergic rhinitis, anti-inflammatory remedy based on steroid inhalant has been major strategy in the treatment of allergic diseases. The steroid anti-inflammatory agents have strong anti-inflammatory and immunosuppressive effect by acting on various cells attending to inflammatory reaction. However, systematic effect through chronic oral administration may cause side effects like inflammation, renal disorder, osteoporosis, diabetes, cataract, etc., which is typical in the use of steroid anti-inflammatory agents.

[0006] As microbial products have no issues of raw material limitation and side effects, they have been widely used in different areas. In particular, their use has been regarded as an effective and safe way for the prevention and treatment of chronic inflammatory diseases and allergic diseases with complex pathology and many attended factors for their ability to adjust human immune system.

[0007] The purpose of the present invention is to provide an immunomodulating material based on microbes which modulate human immune system by activating human innate immune modulating system without causing side effects and medicine tolerance, thereby overcoming the shortages of previous medicines used to improve immune disorders including chronic inflammatory and allergic diseases.

[0008] Description of the invention

[0009] In a first aspect, there is provided an immunomodulatory material comprising heat-killed Enterococcus faecalis and fermented rice bran. Heat-killed Enterococcus faecalis have widely been used as feed additives in fish farming and livestock breeding. Fermented rice bran has also been used as food additives or livestock feed. These two materials are used to prepare an immunomodulatory material with high immunomodulatory effect in the present invention.

[0010] The bacterial DNA is an immune-stimulating factor that can induce the production of human Th1 cytokine. Heat-killed Enterococcus faecalis can increase the level of Th1 cytokine and reduce the level of Th2 cytokine, so that immunoreaction is converted from Th2 dominating into Th1 dominating. In the present invention, it was envisaged to compose an effective immunomodulatory material by combining the said immunomodulatory effect of the heat-killed Enterococcus faecalis and the anti-inflammatory effect of the fermented rice bran. The present invention provides the compatible composition and the preparation method of the immunomodulatory material.

[0011] In the technical embodiment of the invention, the activation of innate immunity by the release of Th1 cytokine which is induced by CpG motif of Enterococcus faecalis and the anti-inflammatory action through the inhibition of IL-17 production by bioactive ingredients of the fermented rice bran are synergistically coupled to take the high immunomodulatory effect.

[0012] The concentration of the heat-killed Enterococcus faecalis in the immunomodulatory material of the present invention is not less than 5x109 / g.

[0013] The fermented rice bran contained in the immunomodulatory material of the present invention is prepared through two-step fermentation. It is the rice bran fermented secondarily by inoculating Enterococcus faecalis into the culture medium including the rice bran fermented primarily by bacteria, yeast or mold.

[0014] In a second aspect, there is provided a preparation method of the immunomodulatory material of the present invention.

[0015] In order for the immunomodulatory material of the present invention containing heat-killed Enterococcus faecalis and fermented rice bran to take sufficient immunomodulatory effect by maintaining the Th1 / Th2 balance, it is required to keep the DNA content of the heat-killed Enterococcus faecalis in the immunomodulatory material not less than 0.21% and to degrade the rice bran through sufficient fermentation.

[0016] In order to ensure the high concentration of heat-killed Enterococcus faecalis through the high-density culture of Enterococcus faecalis under the favorable culture condition for its growth and to sufficiently ferment rice bran, there is provided the effective and suitable method for obtaining the immunomodulatory material including the high concentration of heat-killed Enterococcus faecalis and the sufficiently fermented rice bran as a product of the culture by a single process, which is to culture Enterococcus faecalis and ferment rice bran at once by inoculating Enterococcus faecalis into the culture medium including the rice bran fermented primarily by bacteria, yeast, mold, etc.

[0017] In the present invention, a culture heat-sterilized after the high-density culture of Enterococcus faecalis in the culture medium containing the fermented rice bran is used as an immunomodulatory material.

[0018] The content of the fermented rice bran in the culture medium of Enterococcus faecalis is 45~50% w / w and the temperature for its culture is 36-40 °C.

[0019] The culture medium of Enterococcus faecalis may further comprise corn steep liquor and sulphate, and the concentration of the corn steep liquor in the culture medium is 3-10% and the concentration of the sulphate in the culture medium is 0.2-0.6%.

[0020] The culture medium of Enterococcus faecalis may further comprise saccharose or glucose, and the concentration of the saccharose or glucose in the culture medium is 0.2-2%.

[0021] Example

[0022] The following examples provide a detailed description of the preparation method of the immunomodulatory material of the invention.

[0023] (T) Seed culture

[0024] — Medium composition for the seed culture:

[0025] Glucose 1%, Peptone 0.5%, Yeast extract 0.25%, K2HPO4 0.2%, MnSC +MgSC solution (for the preparation of MRS medium) 1%, pH 7.0

[0026] The seed culture medium was prepared in an Erlenmeyer flask with the composition mentioned above and the medium was sterilized at 1 ATM for 50 min. The colony taken by a platinum loop was inoculated into 200ml medium after cooling to 38°C.

[0027] — Seed culture

[0028] The medium was then shake-cultured for 48h at 150rpm, 38°C.

[0029] (2) Mass culture (solid culture)

[0030] — Corn steep liquor (dry mass 30%) with the amount of 1.5~5% of fermented rice bran was dissolved in water (90% of fermented rice bran). This mixture was mixed with fermented rice bran and sulphate (0.3% of fermented rice bran) and followed by autoclavation for 1h at 121 °C, 1 atm.

[0031] — The autoclaved mixture was crushed and cooled to 40 °C. 2% of seed culture was inoculated into the mixture and cultured in a fermentation tank for 24h at 38 °C.

[0032] — The fermentation medium was further cultured with the thickness of 2cm for 48h at 38 °C on a culture board.

[0033] (3) Heat-sterilization and drying

[0034] The fermentation medium was sampled for the counting of Enterococcus faecalis cells. After the sampling, the culture medium was heat-sterilized for 1h at 121 °C, 1 atm. After the sterilization, the medium was air-dried at 50°C until its water content become lower than 8%. The concentration of the heat-killed Enterococcus faecalis in the immunomodulatory material through above-mentioned method is 1 x1010 / g.

[0035] Immunomodulating activities of the immunomodulatory material of the present invention was evaluated by measuring cytokine level in AR mouse model.

[0036] Experimental Methods

[0037] © AR mouse model

[0038] Sixty mice with the weight between 18~25g were housed with standard 12 / 12 light / dark schedule and free access to water and diet. Mice were randomly divided into 5 groups. AR group: On days 0, 7, 14 and 21 , mice were first sensitized by intraperitoneal injection of 75pg Ovalbumin (OVA, Sigma) and 2mg aluminum hydroxide (Sigma) that were dissolved in 200pl phosphate-buffered saline (PBS). On days 23, 25 and 27, the mice were administrated with 500pg OVA that was dissolved in 20pl PBS by nasal cavities injection. Control group: the mice were under the same schedule of AR mice, but were treated using PBS instead of OVA. Test groups: on day 28, the AR mice were confirmed by the AR symptom scale in Table 1. The confirmed AR mice were then given the immunomodulatory material of the present invention added diet with different doses (0.25, 0.5 and 1g / kg) from day 28 to day 42 daily. The mice were sacrificed on day 43 for further detection.

[0039] Table 1. Allergic rhinitis symptom scale.

[0040] Score Nose scratching Sneezing times Clear nasal discharge flow

[0041] 0 None None None

[0042] 1 Occasionally 1-3 To the prenaris

[0043] 2 Frequently 4-10 Out of prenaris

[0044] 3 Ceaselessly >10 All over the face (2) Sneezing and nasal rubbing

[0045] On day 28, within 20 min after the final nasal injection of OVA, the sneezing and nasal rubbing numbers were counted in 10 min.

[0046] (3) Enzyme-linked immunosorbent assay (ELISA)

[0047] The levels of histamine, IFN-y, IL-2 and IL-4 in serum were detected by using commercial ELISA kits following manufacturer’s instructions. All ELISA kits are purchased from Minneapolis.

[0048] @ Statistical analysis

[0049] All data was presented as mean ± SD. The statistical significance was detected using the R software. *p < 0.05, **p < 0.01 compared to control. *p < 0.05, **p < 0.01 compared to AR mice, p < 0.05 was considered as significant.

[0050] Results of Experiments

[0051] (D Allergic symptoms

[0052] To explore the potential of the immunomodulatory material of the present invention in attenuating AR, AR model was first established by intraperitoneal injection with OVA and AR mice were treated using the immunomodulatory material of the present invention that was added to the diet.

[0053] Table 2. Number of sneezing and nasal rubbing (Mean + SD, n=10)

[0054] Group Number of sneezing Number of nasal rubbing

[0055] Control 2.54+1.23 4.61±0.92

[0056] AR 22.45±2.06** 29.40±1.81**

[0057] **p<0.01 compared to control group, *p<0.05, **p<0.01 compared to AR group

[0058] As can be seen in table 2, in established AR mice, the sneezing and nasal rubbing in 10 min dramatically increased as compared with control mice. The treatment using the immunomodulatory material of the present invention with different doses significantly suppressed the sneezing and nasal rubbing in AR mice, indicating that the immunomodulatory material of the present invention attenuated allergic symptoms in a dose-dependent manner.

[0059] (2) Histamine level in serum

[0060] As the release of histamine is crucial for AR development, the histamine level in serum was detected. (Table 3)

[0061] Table 3. Histamine level in serum (Mean ± SD, n=5)

[0062] Group Histamine level in serum (ng / mL)

[0063] Control 0.44±0.03

[0064] AR 1.85±0.41*#

[0065] Test 1 (0.25g / kg) 1.47±0.57*

[0066] Test 2 (0.5g / kg) 1 ,23±0.46“

[0067] Test 3 (1g / kg) 0.86±0.39”

[0068] **p<0.01 compared to control group, *p<0.05, "p<0.01 compared to AR group

[0069] As shown in table 3, histamine level in serum was much higher in AR group than in control group while the treatment using the immunomodulatory material of the present invention significantly suppressed the histamine level in serum. This result suggests that the immunomodulatory material of the present invention attenuated histamine level in serum of AR mice.

[0070] (3) Serum cytokine levels

[0071] The effect of the immnunomodulatory material of the present invention on seum cytokine levels was determined. (Table 4,5,6)

[0072] Table 4. IFN-y level in serum (Mean ± SD, n=5)

[0073] Group IFN-y level in serum (ng / L) Control 46.14±3.24

[0074] Test 1

[0075] Test 2 (0.5g / kg) 36.12±2.14**

[0076] Test 3 (1 g / kg) 38.42+3.22**

[0077] #p<0.05 compared to control group, *p<0.05, **p<0.01 compared to AR group

[0078] As can be seen in table 4, the level of IFN-y in AR group was much lower than in control group. The treatment using the immunomodulatory material of the present invention significantly increased the IFN-y level in test groups compared to AR group.

[0079] Table 5. IL-2 level in serum (Mean ± SD, n=5)

[0080] Group IL-2 level in serum (ng / L)

[0081] Control 20.52+2.12

[0082] AR

[0083] Test 1 (0.25g / kg)

[0084] Test 2 (0.5g / kg) 16.26+0.84

[0085] Test 3 (1 g / kg) 16.82+1.24

[0086] -: undetectable (below 15.6pg / mL)

[0087] As can be seen in table 5, IL-2 in AR group was not detected while IL-2 level in control group was about 20ng / L. The treatment using the immunomodulatory material of the present invention dramatically increased the IL-2 level in test groups (except for test group 1) compared to AR group.

[0088] Table 6. IL-4 level in serum (Mean + SD, n=5)

[0089] Group IL-4 level in serum (ng / L)

[0090] Control 16.62±4.34

[0091] AR 34.78+3.46## Test 1 (0.25g / kg) 28.64+4.52

[0092] Test 2 (0.5g / kg) 20.64+3.24"

[0093] Test 3 (1g / kg) 18.5814.86"

[0094] **p<0.01 compared to control group, "p<0.01 compared to AR group

[0095] As can be seen in table 6, IL-4 level in AR group was much higher than in control group. The treatment using the immunomodulatory material of the present invention significantly lowered the IL-4 level in test groups compared with AR group.

[0096] The results suggest that the immunomodulatory material of the present invention attenuated the level of inflammatory cytokine IL-4, while elevated the level of anti-inflammatory cytokines IFN-y and IL-2.

Claims

Claims1. An immunomodulatory material, comprising heat-killed Enterococcus faecalis and fermented rice bran.

2. The immunomodulatory material of claim 1, wherein the concentration of the heat-killed Enterococcus faecalis is not less than 5xio9 / g.

3. The immunomodulatory material of claim 1, wherein the fermented rice bran is the rice bran fermented secondarily by inoculating Enterococcus faecalis into the culture medium including the rice bran fermented primarily by bacteria, yeast or mold.

4. A preparation method of the immunomodulatory material according to any one of claim 1 to 3 is characterized by the high-density culture of Enterococcus faecalis in the culture medium containing the fermented rice bran and followed heat-sterilization.

5. The preparation method of the immunomodulatory material according to claim 4, wherein the content of the fermented rice bran in the culture medium of Enterococcus faecalis is 45-50% w / w and the temperature for its culture is 36~40°C.

6. The preparation method of the immunomodulatory material according to claim 4 or claim 5, wherein the culture medium of Enterococcus faecalis further comprises corn steep liquor and sulphate, and the concentration of the corn steep liquor in the culture medium is 3-10% and the concentration of the sulphate in the culture medium is 0.2-06%.

7. The preparation method of the immunomodulatory material according to claim 4 or claim 5, wherein the culture medium of Enterococcus faecalis further comprises saccharose or glucose, and the concentration of the saccharose or glucose in the culture medium is 0.2-2%.