Method for preparing softened grains and method for preparing chicken rice using softened grains

Abstract

This invention discloses a method for preparing softened grains and a method for preparing chicken-flavored rice from softened grains, belonging to the field of bio-cultured meat technology. The method for preparing softened grains includes a method for preparing chicken-flavored grains and collagen incubation; the method for preparing chicken-flavored rice from softened grains includes chicken myoblast inoculation, chicken myoblast culture, and harvesting the chicken-flavored rice. The production process of this invention does not rely on the use of commercial microcarriers, significantly reducing the complexity and cost of the production process; the production process of this invention is mature and controllable, enabling stable product quality in large-scale production; it is green and environmentally friendly, with no resource waste and no animal cruelty; by not using commercial microcarriers, not only is the cost of raw materials reduced, but the production process is also simplified, making the entire production process more efficient and economical; by using biomaterials such as fish collagen, the nutritional value and biocompatibility of the product are improved, making the final product more competitive in the market.

Description

Technical Field

[0001] This invention relates to the field of cultured meat technology, specifically to a method for preparing softened grains and a method for preparing chicken-flavored rice using softened grains. Background Technology

[0002] Chicken consumption has long ranked second only to pork in meat consumption. In recent years, with the changing consumption habits of Chinese residents, per capita chicken consumption has been continuously increasing. The upgrading of domestic and international consumption demands and the improvement of living standards have led to a growing demand for high-quality broiler chickens. Simultaneously, the booming catering industry and the expanding food delivery market have resulted in a steady increase in broiler chicken consumption. The chicken farming industry is large-scale, especially in Asia and North America, meeting the huge demand for chicken products. However, traditional chicken farming faces many environmental and ethical challenges, urgently requiring the search for sustainable alternatives.

[0003] Cell-cultured meat refers to meat obtained through cell culture without slaughtering livestock. This meat production model greatly alleviates problems such as resource waste, environmental pollution, and animal cruelty caused by the ever-increasing consumption of meat products due to population growth.

[0004] Traditional protein foods typically provide only a single source or type of protein, failing to fully meet consumers' demand for diverse and nutrient-rich proteins. While grains, as a primary energy source, provide carbohydrates, their protein content is low, at only about 7.5%. The protein level in grains is insufficient to meet the daily protein requirement of 60 to 90 grams for the average adult. Therefore, relying solely on grains cannot fully satisfy people's protein intake needs.

[0005] This invention organically combines grains and chicken cells, thus solving the above-mentioned problems. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing softened grains and a method for preparing chicken rice using softened grains, which solves problems such as environmental pollution, animal cruelty, and limited protein content.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing softened grains,

[0009] S1: Method for preparing cultivated grains

[0010] a. Grain softening treatment: Soak the grain in PBS solution;

[0011] b. Sterilization treatment: After the PBS solution is mixed evenly with the softened grains, sterilization is performed to obtain sterile grains;

[0012] S2: Collagen incubation

[0013] Fish collagen, hyaluronic acid, and bovine bone collagen are mixed evenly and prepared into a saturated concentration solution. The sterile grains are then incubated in the saturated concentration solution to obtain the incubated grains.

[0014] Preferably, the grain is any one of rice, millet, or purple rice.

[0015] Preferably, the grains in S1a are soaked for 2 hours.

[0016] Preferably, in step b of S1, the sterilization is performed under high temperature and high pressure for 20 minutes at a pressure of 0.1 MPa and a temperature of 121°C.

[0017] Preferably, in S1, the PBS solution in b is mixed with the softened grains at a mass ratio of 1:3.

[0018] Preferably, in S2, the fish collagen, the hyaluronic acid, and the bovine bone collagen are mixed in a mass ratio of 8:1:1; the saturated concentration solution is prepared by mixing the fish collagen, the hyaluronic acid, and the bovine bone collagen in an 8:1:1 ratio and then preparing a saturated solution using PBS.

[0019] Preferably, in S2, the sterile grains are incubated in the saturated concentration solution for 120 minutes.

[0020] A method for preparing chicken rice using softened grains,

[0021] F1: Chicken myoblast inoculation

[0022] The incubated grains were evenly spread on a culture plate, and chicken myoblast suspension was injected into each well of the culture plate.

[0023] F2: Chicken myoblast culture

[0024] The culture plate containing the chicken myoblast suspension in F1 was placed in an incubator for cell culture, and the culture medium was changed from bottom to top every other day.

[0025] F3: Harvest Chicken Rice

[0026] After the culture is completed, the culture plate is rinsed with PBS solution to remove residual culture medium, and chicken grain products are obtained.

[0027] Preferably, the concentration of the chicken myoblast suspension in F1 is 1 × 10⁻⁶.7 per mL.

[0028] Preferably, in F2, the incubator conditions are: temperature 41°C, CO2 content 5%, culture time 8 days; the culture medium is 10% FBS F12 DMEM medium; and the chicken myoblast suspension is replaced every two days.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] (1) The production process of this invention is mature and controllable, and can maintain stable product quality in large-scale production to meet human demand for chicken; it does not produce feces or harmful gases, making it green and environmentally friendly; it saves breeding space and eliminates resource waste. In addition, there is no slaughtering process and no animal abuse, making this invention more humane.

[0031] (2) Because commercial microcarriers are not used, not only is the cost of raw materials reduced, but the production process is also simplified, making the entire production process more efficient and economical.

[0032] (3) By using biomaterials such as fish collagen, hyaluronic acid and bovine bone collagen, the nutritional value and biocompatibility of the product are improved, making the final product more competitive in the market. Attached Figure Description

[0033] Figure 1 This is a schematic flowchart of the method for preparing softened grains and the method for preparing chicken rice using softened grains according to the present invention.

[0034] Figure 2 This is a magnified 10x image of the chicken-cultured rice product finally obtained in Example 1 of the present invention;

[0035] Figure 3 This is a magnified 10x image of the chicken-grown millet obtained in Example 1 of the present invention.

[0036] Figure 4 This is a magnified 10x image of the chicken-grown purple rice product finally obtained in Example 1 of the present invention;

[0037] Figure 5 This is a magnified 10x image of the chicken-cultured rice product finally obtained in Comparative Example 1 of Embodiment 1 of the present invention;

[0038] Figure 6 This is a magnified 10x image of the chicken-cultured millet obtained in Comparative Example 2 of Embodiment 2 of the present invention.

[0039] Figure 7 This is a magnified 10x image of the chicken-cultured purple rice product finally obtained in Comparative Example 3 of Embodiment 3 of the present invention;

[0040] Figure 8 The images show the fluorescence of cell attachment and proliferation on chicken-cultured meat rice obtained in Examples 1, 2, and 3 of this invention.

[0041] Figure 9 The figures show the RT-PCR detection results of chicken-cultured meat rice cells obtained in Examples 1, 2, and 3 of this invention.

[0042] Figure 10 These are WB experimental images of chicken-cultured meat rice cells obtained in Examples 1, 2, and 3 of this invention. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials can be obtained commercially.

[0044] Example 1

[0045] This embodiment provides a method for preparing chicken-cultured rice, the raw materials of which include a first component: rice, and a second component: fish collagen, hyaluronic acid, and bovine bone collagen. The mass ratio of fish collagen to hyaluronic acid to bovine bone collagen is 8:1:1.

[0046] This embodiment provides a method for preparing cultivated rice, including the following steps:

[0047] (1) Rice softening treatment: Soak rice in PBS solution for 2 hours to achieve softening effect.

[0048] (2) Sterilization: After softening, the rice is mixed with PBS solution at a mass ratio of 1:3 and then transferred to an autoclave. The mixture is then subjected to high-temperature autoclaving at 0.1 MPa and 121°C for 20 minutes to ensure sterility. Alternatively, this process can be performed using 1 hour of ultraviolet sterilization or irradiation sterilization. Irradiation sterilization involves 20 seconds of 60Co irradiation.

[0049] (3) Collagen incubation: Fish collagen, hyaluronic acid and bovine bone collagen were mixed in a mass ratio of 8:1:1 and the three species were dissolved in PBS to prepare a saturated solution. The sterilized rice was incubated in the solution for 120 minutes to fully combine these nutrients.

[0050] Here are the steps to prepare chicken rice using softened grain rice:

[0051] (1) Cell seeding: The incubated rice cereal products were evenly spread on a 24-well culture plate, and the 10% FBS F12 DMEM medium was diluted to a concentration of 1×10⁻⁶. 7 Chicken myoblast suspension of cells / mL was injected into each well to ensure uniform cell distribution.

[0052] (2) Cell Culture: Cells were cultured in an incubator at 41℃ and 5% CO2, with the culture medium changed from bottom to top every other day for 8 days. The culture medium was 10% FBS F12 DMEM. 10% FBS F12 DMEM can also be used instead of McCoy's 5A. The chicken myoblast suspension was changed every two days.

[0053] (3) Chicken Rice Harvesting: After cultivation, the culture plates were rinsed with PBS solution to remove residual culture medium, thereby obtaining chicken rice products. Chicken rice products have good texture and taste.

[0054] Example 2

[0055] This embodiment provides a method for preparing chicken-cultured millet, the raw materials of which include a first component: millet, and a second component: fish collagen, hyaluronic acid, and bovine bone collagen. The mass ratio of fish collagen:hyaluronic acid:bovine bone collagen is 8:1:1.

[0056] This embodiment provides a method for preparing cultivated millet, including the following steps:

[0057] (1) Millet softening treatment: Soak millet in PBS solution for 2 hours to achieve softening effect.

[0058] (2) Sterilization: After softening, the millet was mixed with PBS solution at a mass ratio of 1:3 and then transferred to an autoclave. The mixture was autoclaved at 0.1 MPa and 121°C for 20 minutes to ensure sterility. Alternatively, sterilization could be performed using 1 hour of ultraviolet light or irradiation. Irradiation sterilization involved 20 seconds of 60Co irradiation.

[0059] (3) Collagen incubation: Fish collagen, hyaluronic acid and bovine bone collagen were mixed in a mass ratio of 8:1:1 and the three species were dissolved in PBS to prepare a saturated solution. The sterilized millet was incubated in the solution for 120 minutes to fully combine these nutrients.

[0060] The following are the steps to prepare chicken rice using softened millet:

[0061] (1) Cell seeding: The incubated millet cereal product was evenly spread on a 24-well culture plate, and the 10% FBS F12 DMEM medium was diluted to a concentration of 1×10⁻⁶. 7 Chicken myoblast suspension of cells / mL was injected into each well to ensure uniform cell distribution.

[0062] (2) Cell Culture: Cells were cultured in an incubator at 41℃ and CO2 concentration of 5. The culture medium was changed from bottom to top every other day for 8 days. The culture medium was 10% FBS F12 DMEM. 10% FBS F12 DMEM can also be used instead of McCoy's 5A. The chicken myoblast suspension was changed every two days.

[0063] (3) Harvesting of Chicken Millet: After cultivation, the culture plates were rinsed with PBS solution to remove residual culture medium, thereby obtaining chicken millet products. Chicken millet products have good texture and taste.

[0064] Example 3

[0065] This embodiment provides a method for preparing chicken-cultured purple rice, the raw materials of which include a first component: purple rice, and a second component: fish collagen, hyaluronic acid, and bovine bone collagen. The mass ratio of fish collagen to hyaluronic acid to bovine bone collagen is 8:1:1.

[0066] This embodiment provides a method for preparing cultivated purple rice, including the following steps:

[0067] (1) Purple rice softening treatment: Soak purple rice in PBS solution for 2 hours to achieve softening effect.

[0068] (2) Sterilization: After softening, the purple rice was mixed with PBS solution at a mass ratio of 1:3, and then transferred to an autoclave. The mixture was autoclaved at 0.1 MPa and 121°C for 20 minutes to ensure sterility. Alternatively, sterilization could be performed using 1 hour of ultraviolet light or irradiation. Irradiation sterilization involved 20 seconds of 60Co irradiation.

[0069] (3) Collagen incubation: Fish collagen, hyaluronic acid and bovine bone collagen were mixed in a mass ratio of 8:1:1 and the three species were dissolved in PBS to prepare a saturated solution. The sterilized purple rice was incubated in the solution for 120 minutes to fully combine these nutrients.

[0070] The following are the steps to prepare chicken rice using softened purple rice:

[0071] (1) Cell seeding: The incubated cereal products were evenly spread on 24-well culture plates, and 10% FBSF12 DMEM medium was diluted to a concentration of 1×10⁻⁶. 7 Chicken myoblast suspension of cells / mL was injected into each well to ensure uniform cell distribution.

[0072] (2) Cell Culture: Cells were cultured in an incubator at 41℃ and 5% CO2, with the culture medium changed from bottom to top every other day for 8 days. The culture medium was 10% FBS F12 DMEM. 10% FBS F12 DMEM can also be used instead of McCoy's 5A. The chicken myoblast suspension was changed every two days.

[0073] (3) Chicken and millet harvesting: After cultivation, the culture plates were rinsed with PBS solution to remove residual culture medium, thereby obtaining chicken and millet products. Chicken and millet products have good texture and taste.

[0074] Comparative Example 1

[0075] This comparative example provides a chicken-based biocultured rice, which differs from Example 1 only in that the second component uses only fish collagen. Fish collagen was added to PBS until it became insoluble in the liquid, thus preparing a saturated fish collagen solution.

[0076] The only difference between the above-mentioned method for preparing chicken-based biocultured rice and Example 1 is that the sterilized rice is incubated in a saturated fish collagen solution for 120 minutes to fully combine these nutrients.

[0077] Comparative Example 2

[0078] This comparative example provides a chicken-based biocultured millet, which differs from Example 2 only in that the second component uses only fish collagen. Fish collagen was added to PBS until it could no longer dissolve in the liquid, thus preparing a saturated fish collagen solution.

[0079] The only difference between the above-mentioned method for preparing chicken-based biocultured millet and Example 2 is that the sterilized millet is incubated in a saturated fish collagen solution for 120 minutes to fully combine these nutrients.

[0080] Comparative Example 3

[0081] This comparative example provides a chicken-based cultured purple rice, which differs from Example 2 only in that the second component uses only fish collagen. Fish collagen was added to PBS until it could not be dissolved in the liquid, thus preparing a saturated fish collagen solution.

[0082] The only difference between the above-mentioned method for preparing chicken-based biocultured purple rice and Example 2 is that the sterilized purple rice is incubated in a saturated fish collagen solution for 120 minutes to fully combine these nutrients.

[0083] Example Detection and Results Discussion

[0084] (1) Verification of the adhesion efficiency of chicken-cultured rice cells

[0085] Immunofluorescence labeling was performed on adherent cells proliferating on chicken cultured meat to verify cell proliferation efficiency. PBST: PBS solution containing 0.5% Triton X-100 (volume percentage).

[0086] Specific operations:

[0087] 1) Cultivate chicken meat rice, discard the culture medium, and wash twice with PBS.

[0088] 2) Add 4% paraformaldehyde and fix at room temperature for 15 minutes.

[0089] 3) Wash 3 times with PBS, 5 minutes each time.

[0090] 4) Permeabilize with PBST solution at room temperature for 10 minutes.

[0091] 5) Wash 3 times with PBS, 5 minutes each time.

[0092] 6) Add the final concentration of DAPI / Hoechst and let stand at room temperature for 5 minutes.

[0093] 7) Wash 3 times with PBS, 5 minutes each time.

[0094] 8) Add 500 μl of PBS and take a picture under a fluorescence microscope.

[0095] 9) Observe the growth and proliferation morphology of cells on the scaffold using fluorescence microscopy to verify the scaffold cell culture efficiency.

[0096] The final fluorescence results showed that, after inoculation, a large number of rice, millet, and purple rice cells treated in Examples 1, 2, and 3 adhered and proliferated, with a layer of cells adhering to the surface of the rice. This indicates that the surface treatment of the grains in this invention is effective and has a beneficial effect on cell adhesion and growth. Fish collagen is a mild type I collagen and a major component of the extracellular matrix. By adding a certain proportion of hyaluronic acid and bovine bone collagen to the fish collagen solution, the surface of the rice grains more effectively mimics the extracellular matrix to promote cell adhesion and growth. By comparing the cell growth of the examples and the comparative examples, it was found that after treatment with the second component solution in the examples, cell adhesion was more easily achieved on the surface of the rice grains.

[0097] (2) Real-time quantitative RT-PCR detection of chicken and rice adherent cells

[0098] Real-time quantitative RT-PCR was performed on adherent and proliferating chicken meat cultured cells to detect their proliferation and differentiation status. The experimental steps are as follows:

[0099] 1) RNA extraction: After washing the chicken rice three times, add Trizol reagent to rinse and lyse the cells on the chicken rice, and extract RNA using an RNA rapid extraction kit.

[0100] 2) Reverse transcribed RNA: RNA concentration was measured using a Nanodrop differentiation spectrophotometer, and RNA was reverse transcribed into cDNA using reverse transcriptase.

[0101] 3) RT-PCR

[0102] ① Primer design for RT-PCR

[0103] Table 1 Gene Primer Sequences

[0104]

[0105] ②Preparation of RT-PCR components for PCR reaction solution

[0106] Table 2 RT-PCR Component Preparation Table

[0107]

[0108] ③RT-PCR procedure:

[0109] Stage 1: Prevariation

[0110] 95℃ 30 seconds 20℃ / second

[0111] 1 CycleStage 2: PCR reaction

[0112] 95℃ for 5 seconds 20℃ / second

[0113] 60℃ 20 seconds 20℃ / second

[0114] 40 Cycles Stage 3: Melting Curve Analysis

[0115] 95℃ 0 seconds 20℃ / second

[0116] 65℃ 15 seconds 20℃ / second

[0117] 95℃ 0 seconds 0.1℃ / second

[0118] according to Figure 8RT-PCR results showed significant differences in the expression of MYOD and MYOG genes among chicken-based rice, chicken-based millet, chicken-based purple rice, and adherent cells. Firstly, chicken-based rice exhibited the highest activity in both gene expression, with the highest contrast between MYOD and MYOG gene expression, indicating the most vigorous activity of myogenic genes on this substrate. In contrast, chicken-based millet and chicken-based purple rice showed slightly lower MYOD and MYOG gene expression, but still demonstrated good gene activity, indicating their potential to support muscle formation. Overall, chicken-based rice was the best cell culture substrate, while chicken-based millet and chicken-based purple rice also showed certain culturing advantages.

[0119] (3) Quantitative detection of WB protein in chicken and rice adherent cells

[0120] Western blot (WB) experiments were performed on adherent and proliferating chicken meat cultured on a culture vessel to detect the cell proliferation and differentiation status. The experimental steps are as follows:

[0121] 1) Protein extraction: After washing the chicken rice with PBS, soak the chicken rice in lysis buffer, rinse 3 times, and collect the protein lysis buffer.

[0122] 2) Protein concentration determination: Take a small amount of lysis buffer and use the BCA method to quantitatively analyze the protein.

[0123] 3) Preparation of SDS-PAGE gel: Determine the concentration, gel thickness, and quantity of separating and stacking gels based on the molecular weight of the target protein, its expression location, and the number of samples.

[0124] 4) Sample loading and electrophoresis: The total amount of protein loaded is 30 μg. The stacking gel is electrophoresed at 100V for 30 min, then the voltage can be increased to 150V for 1 h. Excess SDS-PAGE is cut off at the interface between the stacking and separating gels.

[0125] 5) Transfer: WB wet transfer method

[0126] 6) Blocking: After the transfer is completed, the PVDF membrane is taken out, washed in TBST for 5 minutes, and then transferred to blocking solution (5% skim milk powder) and blocked at room temperature for 1 hour.

[0127] 7) Primary antibody incubation: Dilute the primary antibody with antibody diluent according to the WB dilution ratio in the antibody instructions (β-actin: brand: abcam, catalog number: ab8226, dilution ratio: 1:1000; MyoD: brand: abcam, catalog number: ab16148, dilution ratio: 1:1000). Remove the membrane from the blocking solution, blot off excess liquid with filter paper, place it in the antibody incubation box, pour in the diluted primary antibody, and incubate overnight.

[0128] 8) Secondary antibody incubation: Select a suitable secondary antibody based on the source of the primary antibody, and dilute the secondary antibody with antibody diluent according to the WB dilution ratio in the antibody instructions. (Goat Anti-Mouse IgG H&L (HRP): Brand: abcam, Catalog No.: ab6789, Dilution ratio: 1:2000) Wash the PVDF membrane incubated with primary antibody three times with TBST, 5 min each time. Remove the membrane from the washing solution, absorb excess liquid with filter paper, place it in the antibody incubation box, pour in the diluted secondary antibody, and incubate at room temperature for 1 h.

[0129] 9) Washing: Wash the membrane with TBST 3 times, 5 minutes each time.

[0130] 10) Development and Exposure: Take equal volumes of ECL luminescent solution A and solution B, mix well, and store in the dark for later use. Place the PVDF film in a dark box containing the developing solution and incubate for 5 minutes, gently shaking the box continuously during incubation. Then place it in the instrument for exposure and photography.

[0131] Figure 9 The image shows a Western blotting (WB) analysis of myoblast-related proteins after cell lysis in fully cultured chicken-rice, chicken-millet, and chicken-purple rice grains. The bands correspond to the expression of the MYOD target protein. The darker bands in chicken-rice and chicken-millet indicate higher MYOD expression levels, while the lighter bands in chicken-purple rice indicate that the protein is expressed, but at a lower level. This demonstrates that cell growth and proliferation occurred in the rice grains, indicating successful construction of the chicken-rice system.

[0132] Chicken myoblasts can be replaced with cells from other species or other types of cells to prepare different types of cell-cultured meat products.

[0133] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing softened grains, characterized in that: S1: Method for preparing cultivated grains a. Grain softening treatment: Soak the grain in PBS solution; b. Sterilization treatment: After the PBS solution is mixed evenly with the softened grains, sterilization is performed to obtain sterile grains; S2: Collagen Incubation Fish collagen, hyaluronic acid and bovine bone collagen are mixed evenly and prepared into a saturated concentration solution. The sterile grains are incubated in the saturated concentration solution to obtain the incubated grains. The grain described in S1a is soaked for 2 hours; In S1b, the sterilization is performed under high temperature and high pressure at a pressure of 0.1 MPa and a temperature of 121°C for 20 minutes. In S2, the sterile grains are incubated in the saturated concentration solution for 120 minutes; In S1, the grain is any one of rice, millet, or purple rice.

2. The method for preparing softened grains according to claim 1, characterized in that: In S1b, the PBS solution is mixed with the softened grains at a mass ratio of 1:

3.

3. The method for preparing softened grains according to claim 1, characterized in that: In S2, the fish collagen, the hyaluronic acid, and the bovine bone collagen are mixed in a mass ratio of 8:1:1; the saturated concentration solution is prepared by mixing the fish collagen, hyaluronic acid, and bovine bone collagen in an 8:1:1 ratio and then preparing a saturated solution using PBS.

4. A method for preparing chicken-flavored rice using softened grains prepared as claimed in claim 1, characterized in that: F1: Chicken myoblast inoculation The incubated grains were evenly spread on a culture plate, and chicken myoblast suspension was injected into each well of the culture plate. F2: Chicken myoblast culture The culture plate containing the chicken myoblast suspension in F1 was placed in an incubator for cell culture, and the culture medium was changed from bottom to top every other day. F3: Harvest Chicken Rice After the culture is completed, the culture plate is rinsed with PBS solution to remove residual culture medium, and chicken grain products are obtained.

5. A method for preparing chicken-flavored rice using softened grains according to claim 4, characterized in that: The concentration of the chicken myoblast suspension in F1 is 1×10⁻⁶. 7 per mL.

6. A method for preparing chicken-flavored rice using softened grains according to claim 4, characterized in that: In F2, the incubator conditions are: temperature 41°C, CO2 content 5%, culture time 8 days; the culture medium is 10% FBS F12DMEM medium; the chicken myoblast suspension is changed every two days.

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