Method for inactivation or elimination of ricin contained in Ricinus communis seed, and method for producing Ricinus communis oil and Ricinus communis cake produced using Ricinus communis seed produced thereby

Abstract

The present invention relates to a method for inactivating and removing ricin contained in castor seeds, and a method for producing castor oil and castor seed meal using castor seeds produced according to this method, comprising: (A) a step of immersing castor (Ricinus communis) seeds in alkaline washing water; (B) a step of heating the castor seeds immersed in alkaline washing water under purified water at 70 to 90 ℃; and (C) a step of drying and aging the heated castor seeds; thereby, the content of ricin, a toxic plant protein contained in castor seeds, can be significantly reduced or removed.

Description

Technology Field

[0001] The present invention relates to a method for inactivating and removing ricin, a toxic plant protein contained in castor seeds, and a method for producing castor oil and castor seed meal using castor seeds produced thereby (a method for detoxification). Background Technology

[0002] Native to India, Asia, and North Africa, the castor plant is an annual herb belonging to the sumac family, but it grows as a perennial in tropical regions. India, China, and Brazil are the main production areas. Castor oil, derived from the seeds, is used for various industrial purposes such as lubricants, coatings, cosmetics, and detergents, as well as for traditional folk remedies and various internal and external treatments for conditions like constipation. Castor meal is used as fertilizer or fuel.

[0003] However, castor seeds contain highly toxic components such as ricin, so it is known that consuming the seeds themselves is dangerous. The aforementioned ricin is one of the most dangerous toxins in nature and is a toxic substance that can cause death in an adult with just a small amount of about 0.001 g.

[0004] The above castor seeds contain 30-50% lipids, protein (about 26%) globulin, nucleoalbumin, glycophortein, toxic albumin lysine, lipase, and 0.2% toxic alkaloid ricinine, etc. 85% of the lipids consist of glycerides of ricinoleic acid, mixed with small amounts of glycerides of 9,10-dihydroxystearic acid, oleic acid, and linoleic acid.

[0005] Castor oil has a laxative effect, which occurs when the seed oil is hydrolyzed into ricinoleic acid and glycerin by alkaline intestinal fluid. It is known that eating raw castor oil causes poisoning, and the toxic albumin lysine has a very strong toxic effect.

[0006] Since these toxic substances are mostly found in the shells of castor seeds, it is common to consume castor oil obtained by pressing the seeds for medicinal purposes.

[0007] Generally, castor oil is manufactured by pressing castor beans to efficiently separate it; however, it is predicted that even if toxic substances are present in the castor seeds before pressing, if the seeds are pressed after treatments such as heat treatment, the toxic substances will mainly remain in the castor meal, and only a minute amount of ricin will be present in the castor oil.

[0008] Due to past attempts to use ricin as a biological weapon, a persistent risk always exists, and the possibility of causing mass casualties cannot be ruled out. Furthermore, it is listed on the Biological Weapons Convention's regulatory list and the Australia Group's export control core list, and the U.S. Centers for Disease Control and Prevention classifies it as a Category B agent.

[0009] A method is required to inactivate such dangerous ricin in castor seeds and to obtain castor oil and castor meal from the inactivated castor seeds. Prior art literature

[0010] Republic of Korea Registered Patent No. 1625952 Republic of Korea Registered Patent No. 0615111 The problem to be solved

[0011] The objective of the present invention is to provide a method for inactivating ricin, a toxic plant protein contained in castor seeds.

[0012] In addition, another objective of the present invention is to provide a method for producing castor oil and castor seed meal using castor seeds in which ricin has been inactivated. means of solving the problem

[0013] The method for inactivating ricin contained in castor seeds according to the present invention for achieving the above-mentioned purpose may include: (A) a step of roasting castor (Ricinus communis) seeds and then immersing them in alkaline washing water; (B) a step of heating the castor seeds immersed in the alkaline washing water under purified water at 70 to 90 ℃; and (C) a step of drying and aging the heated castor seeds.

[0014] In step (A) above, the alkaline washing water may be purified water in which one or more alkaline substances selected from the group consisting of sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), potassium hydroxide (KOH), and ammonium hydroxide (NH4OH) are dissolved at a concentration of 0.01 to 3 weight%.

[0015] In step (A) above, immersion can be performed for 2 to 24 hours in alkaline washing water at 20 to 26 ℃.

[0016] In step (B) above, heating can be performed under a pressure of 0.3 to 0.8 atmospheres.

[0017] In step (C) above, drying and aging can be performed at 10 to 20 ℃ for 2 to 3 days while stirring.

[0018] In step (C) above, the drying and aging process can be carried out by mixing heated castor seeds and rice bran in a weight ratio of 1:0.3-0.9.

[0019] In addition, the method for producing castor oil according to the present invention for achieving the other objectives mentioned above may include a step of separating castor oil and castor meal using ricin-inactivated castor seeds obtained according to the above method.

[0020] The above-mentioned separated castor oil may further include a step of processing it in a counter-current flow manner.

[0021] The above-mentioned separated castor meal may further include a step of crushing it and mixing it with mica.

[0022] The above castor meal powder and mica can be mixed in a weight ratio of 1:0.1-5.0. Effects of the invention

[0023] The present invention inactivates ricin, a toxic plant protein contained in castor seeds, thereby almost completely removing it, and accordingly, the ricin content in castor oil and castor meal obtained from castor seeds in which the ricin has been inactivated can also be significantly reduced.

[0024] In addition, an additional process can be performed to further lower or remove the ricin content in the above castor oil and castor meal. Brief explanation of the drawing

[0025] FIG. 1 is a diagram showing the process of refining castor oil using an anti-current method according to one embodiment of the present invention. Specific details for implementing the invention

[0026] The present invention relates to a method for inactivating and removing ricin, a toxic plant protein contained in castor seeds, and a method for producing castor oil and castor seed meal using castor seeds produced thereby.

[0027] The aforementioned ricin is a toxic plant protein found in castor seeds and the like; due to its strong toxicity, it must be inactivated and removed for practical use.

[0029] The present invention will be described in detail below.

[0030] The method for inactivating ricin contained in castor seeds according to the present invention comprises: (A) a step of roasting castor (Ricinus communis) seeds and then immersing them in alkaline washing water; (B) a step of placing the castor seeds immersed in the alkaline washing water into purified water at 70 to 90 ℃ and heating them; and (C) a step of drying and aging the heated castor seeds.

[0031] First, in step (A) above, the castor seeds are immersed in alkaline washing water at 20 to 26°C, preferably 23 to 25°C, for 2 to 24 hours, preferably 8 to 15 hours, to reduce the ricin content contained in the castor seeds.

[0032] The above castor seeds may be roasted at 50 to 80°C, preferably 60 to 70°C, for 1 to 5 hours, preferably 1.5 to 2 hours. When using the above roasted castor seeds, 5 to 50 weight percent more ricin can be removed compared to when using untreated castor seeds.

[0033] By immersing the above castor seeds in alkaline washing water, about 50 to 70 weight percent of the total ricin contained in the castor seeds can be removed. The alkaline washing water is purified water in which an alkaline substance is dissolved at 0.01 to 3 weight percent, preferably 0.1 to 1.0 weight percent. If the content of the alkaline substance is below the lower limit, the ricin may not be removed, and if it exceeds the upper limit, the castor seeds may deteriorate, and the quality and yield of the castor oil may decrease.

[0034] The above alkaline substances may include one or more selected from the group consisting of sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), potassium hydroxide (KOH), and ammonium hydroxide (NH4OH); however, if sodium hydroxide (NaOH) is used, the quality of the castor meal may be degraded due to strong alkalinity.

[0035] When soaking castor seeds, if the temperature and time of the alkaline washing water are below the lower limit, the removal rate of ricin is significantly reduced, and if they exceed the upper limit, the quality of the castor oil obtained may be degraded.

[0036] Next, in step (B), the castor seeds immersed in the alkaline washing water are heated in purified water at 70 to 90°C, preferably 70 to 80°C, for 1 to 5 hours, preferably 2 to 3 hours.

[0037] By heating the castor seeds immersed in the above alkaline washing water in purified water, the deterioration of the physical properties of the castor seeds that have undergone step (A) due to alkalinity can be prevented.

[0038] Furthermore, if the heating process is performed under a pressure of 0.3 to 0.8 atmospheres, preferably 0.5 to 0.7 atmospheres, the ricin content in the castor seeds that have undergone step (A) can be further reduced by 15 to 20 weight percent, thereby removing 65 to 90 weight percent of the total ricin content in the untreated castor seeds, so the ricin content can be further reduced compared to when the heating process is performed under 1 atmosphere.

[0039] If the step of heating with the aforementioned purified water is not performed, the physical properties of the castor oil may be degraded by residual alkali, and off-flavors may already occur after drying and aging; therefore, it is desirable to perform the heating process.

[0040] When heating with purified water, if the temperature and time are below the lower limit, the effect of reducing lysine cannot be obtained, and if they exceed the upper limit, the physical properties may deteriorate.

[0041] Next, in step (C) above, the heated castor seeds are placed directly into a stirrer and dried and aged at 10 to 40°C for 2 to 3 days.

[0042] By introducing the castor seeds heated through the above heating process directly into a stirrer and stirring them, the temperature is naturally lowered, and by finally performing stirring at 10 to 40°C, the ricin content in the castor seeds that have undergone step (B) can be further reduced by 5 to 10 weight%.

[0043] When castor seeds heated through a heating process are cooled and then fed into a stirrer for stirring, the ricin content is not reduced, and an unpopular scent may develop in the resulting castor oil.

[0044] Furthermore, when the above dry aging process is performed by mixing castor seeds and rice bran, which have been heated through a heating process, in a weight ratio of 1:0.3-0.9, preferably 1:0.4-0.6, the ricin contained in the castor seeds can be almost completely removed. For example, if stirring aging is performed without using rice bran, about 90% by weight of the total ricin content contained in the untreated castor seeds is removed, but if stirring aging is performed using rice bran, more than 95% by weight of the total ricin content contained in the untreated castor seeds is removed, leaving only trace amounts of ricin.

[0045] If the content of rice bran based on castor seeds is below the lower limit mentioned above, ricin cannot be removed to a significant extent, and if it exceeds the upper limit mentioned above, an unpopular scent may occur in the resulting castor oil and castor meal.

[0046] If the temperature and time during drying and aging are below the lower limit mentioned above, the content of ricin removed may decrease, and if they exceed the upper limit mentioned above, the castor seeds may deteriorate, and the quality of the castor oil and castor meal obtained may decrease.

[0048] In addition, the present invention can provide a method for obtaining castor oil and castor meal using the castor seeds in which the obtained ricin has been inactivated.

[0049] The method for producing castor oil according to the present invention includes the step of separating castor oil and castor meal using the castor seeds in which ricin has been inactivated obtained.

[0050] The above method for obtaining castor oil and castor meal may use a conventional oil pressing method or a solvent extraction method.

[0051] The above-mentioned separated castor oil can be processed using a counter-current flow method to almost completely remove trace amounts of residual ricin.

[0052] As shown in FIG. 1, the above anti-current method uses a method in which alkaline water and castor oil are introduced into the upper and lower parts of the extraction tower, respectively, and contact is carried out in an anti-current manner. This eliminates the need for additional energy or additives during the extraction process, thereby reducing operating costs compared to conventional processes involving the addition of other compounds or distillation, and also allows for the removal of all trace amounts of ricin.

[0053] The alkaline water introduced into the extraction tower is purified water in which an alkaline substance is dissolved at a concentration of 0.01 to 0.1 weight%. If the content of the alkaline substance is below the lower limit, ricin may not be removed, and if it exceeds the upper limit, the castor oil may deteriorate and its marketability may be reduced.

[0054] The above alkaline substances may include one or more selected from the group consisting of sodium hydroxide (NaOH), sodium bicarbonate (NaHCO3), and sodium carbonate (Na2CO3).

[0055] Inside the extraction tower, the alkaline water and castor oil introduced from the top and bottom, respectively, are mixed and fused by a counter-current method in which the flows of the alkaline water and castor oil are opposite to each other, thereby allowing contact to proceed and removing trace amounts of ricin contained in the castor oil. The alkaline water and castor oil that have undergone the fusion process are separated into refined castor oil and washing weak alkaline water, respectively, inside the extraction tower.

[0056] In addition, since the separated castor meal also contains a trace amount of ricin, all of the trace amount of ricin can be removed by crushing the castor meal and mixing the castor meal powder with mica.

[0057] The above mica is a plate-shaped porous material in which a large amount of microorganisms are contained within the pores, which can remove trace amounts of ricin contained in the castor film. Accordingly, the mica can be used after being treated with beneficial microorganisms, etc.

[0058] The castor meal powder and mica are mixed in a weight ratio of 1:0.1-5.0, preferably 1:1.0-3.0, and stirred for 1 to 3 hours. If the mica content based on the castor meal powder is below the lower limit, trace amounts of ricin may not be completely removed, and if it exceeds the upper limit, only costs increase and there is no further increase in effectiveness.

[0059] The castor meal powder mixed with the mica mentioned above can be used in a state mixed with mica or as castor meal powder with the mica removed, and can be used as an animal feed additive, fertilizer, fuel, etc.

[0061] Hereinafter, preferred embodiments are presented to aid in understanding the present invention; however, the following embodiments are merely illustrative of the invention, and it is obvious to those skilled in the art that various changes and modifications are possible within the scope and spirit of the invention, and that such variations and modifications fall within the scope of the appended claims.

[0062] Example 1.

[0063] Castor seeds (Ricinus communis) were roasted at 60°C for 2 hours, then the roasted castor seeds were immersed in sodium bicarbonate (NaHCO3) washing water (0.3 wt% NaHCO3 dissolved in purified water) at 25°C for 12 hours, then removed and placed in purified water at 80°C for 2 hours, then heated, then placed the heated castor seeds directly into a stirrer without cooling and dried and aged at 18°C ​​for 3 days while stirring, and then washed to obtain castor seeds with ricin inactivated.

[0064] In addition, the above castor seeds were pressed to obtain castor oil and castor meal, respectively.

[0066] Example 2. Pressure during heating

[0067] The above Example 1 was carried out in the same manner, but castor seeds were added to purified water at 80°C and heated under a pressure of 0.7 atm to obtain inactivated castor seeds.

[0068] In addition, the above castor seeds were pressed to obtain castor oil and castor meal, respectively.

[0070] Example 3. Addition of rice bran

[0071] Inactivated castor seeds were obtained by carrying out the same procedure as in Example 1 above, but by mixing castor seeds heated through a heating process during stirring and aging with rice bran in a weight ratio of 1:0.5.

[0072] In addition, the above castor seeds were pressed to obtain castor oil and castor meal, respectively.

[0074] Example 4. Pressure + Rice Bran upon Heating

[0075] Castor seeds (Ricinus communis) were roasted at 60°C for 2 hours, then the roasted castor seeds were immersed in sodium bicarbonate (NaHCO3) washing water (0.3 wt% NaHCO3 dissolved in purified water) at 25°C for 12 hours, then removed and placed in purified water at 80°C and heated for 2 hours under a pressure of 0.7 atmospheres, then the heated castor seeds and rice bran were mixed in a weight ratio of 1:0.5, and without cooling, were placed directly into a stirrer and dried and aged while stirring at 18°C ​​for 3 days, and then washed to obtain castor seeds with ricin inactivated.

[0076] In addition, the above castor seeds were pressed to obtain castor oil and castor meal, respectively.

[0078] Comparative Example 1. Omission of heating process

[0079] The above Example 1 was carried out in the same manner, but castor seeds immersed in sodium bicarbonate washing water were placed in purified water at 25°C and stirred for 2 hours, after which drying and aging were performed to obtain inactivated castor seeds.

[0081] Comparative Example 2. Omission of dry aging process

[0082] The above example was carried out in the same manner as Example 1, but the heated castor seeds were washed to obtain castor seeds in which ricin was inactivated.

[0084] Comparative Example 3. Use of sodium hydroxide (NaOH)

[0085] The above Example 1 is carried out in the same manner, except that sodium bicarbonate (NaHCO3) washing water is replaced with sodium hydroxide (NaOH). Inactivated castor seeds were obtained using washing water.

[0087] <Test Example>

[0088] All experiments were repeated at least three times, and the results were expressed as mean and standard deviation. Significant differences in the results of each experiment were confirmed using one-way analysis of variance in SPSS Statistics 20.0 (IBM, Armonk, NY, USA), and in Duncan's multiple comparison test, a difference was considered significant when the significance level was 5% or less.

[0089] Test Example 1. of castor seeds Lysine content measurement

[0090] The lysine content in the castor seeds prepared in the above examples and comparative examples was measured.

[0091] Securing Lysine Crude Protein

[0092] ① Separate the inside of the castor seed.

[0093] ② (Lip removal: ②-⑥) Grind the inside of the castor beans with a blender.

[0094] ③ Put equal amounts of crushed castor seeds and ether into a beaker and mix well for 5 minutes.

[0095] ④ Place in a 50 ml conical tube and centrifuge at 4,800 rpm for 10 minutes.

[0096] ⑤ Collect the supernatant in a glass bottle and the pellet back into a beaker, then repeat steps ②-③ 3 more times (a total of 4 times).

[0097] ⑥ Finally, the remaining pellets are dried in the air at room temperature.

[0098] ⑦ Add 2 g of the sample (lipid-removed castor oil) and 10 ml of pH 4.0 distilled water (pH adjusted with acetic acid) to a 50 ml tube.

[0099] ⑧ Insert the tube into ice and, while it is cold, run the homogenizer at maximum speed and repeat “1 minute of grinding followed by 30 minutes of storage at 4℃” 10 times.

[0100] ⑨ Overnight at 4 ℃.

[0101] 10. Centrifuge at 4,800 rpm for 10 minutes and take the supernatant.

[0102] 11. Filter the supernatant through a 0.45 µm filter.

[0103] ⑫ Store the prepared lysine crude protein at 4 ℃.

[0104] ELISA analysis

[0105] ① (Capture antibody coating: ①-②) Add 100 µl of Anti-Ricin toxin A chain antibody ab27169 (rabbit, polyclonal) to an ELISA plate and leave at 4°C for one day. Use PBS to dilute Anti-Ricin toxin A chain antibody ab27169 (rabbit, polyclonal).

[0106] ② Shake the liquid off the plate vigorously and wash three times with 300 µl of PBS per well.

[0107] ③ (Blocking and Sample Addition: ③-⑥) Add 300 µl of blocking buffer per well and leave at room temperature for 1 hour.

[0108] ④ Shake the liquid off the plate vigorously and add 100 µl of sample per well. Use blocking buffer when diluting the sample.

[0109] ⑤ Leave at room temperature for 2 hours.

[0110] ⑥ Shake the liquid off the plate vigorously and wash three times with 300 µl of washing buffer per well.

[0111] ⑦ (Addition of detection antibody and secondary antibody: ⑦-⑩) Add 100 µl of Ricin alpha Antibody RA999 (mouse, monoclonal) and leave at room temperature for 1 hour and 30 minutes. Use blocking buffer when diluting Ricin alpha Antibody RA999 (mouse, monoclonal).

[0112] ⑧ Shake the liquid off the plate vigorously and wash three times with 300 µl of washing buffer per well.

[0113] ⑨ Add 100 µl of Anti-mouse IgG, HRP-linked Antibody and leave at room temperature for 1 hour and 30 minutes. Use blocking buffer when diluting Anti-mouse IgG, HRP-linked Antibody.

[0114] 10. Shake the liquid off the plate vigorously and wash three times with 300 µl of washing buffer per well.

[0115] 11 (Detection: ②-③) Add 100 µl of substrate solution (TMB) and leave at room temperature for 20 minutes.

[0116] ⑫ After adding 100 µL of 0.1 N H2SO4, measure the absorbance at 450 nm.

[0117] Untreated castor seeds were used as the control group.

[0118] division control group Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Lysine (ug / ml) 22.6±2.9 a 4.50±0.29 c 2.16±0.19 d 2.37±0.51 d 0.20±0.07 e 11.3±0.9 b 10.5±1.0 b 5.99±0.62 c

[0119] As shown in Table 1 above, it was confirmed that lysine is removed because the castor seeds prepared according to Examples 1 to 4 of the present invention have a lower lysine content compared to the control group and Comparative Examples 1 to 3.

[0120] In particular, it was confirmed that ricin is further removed because the castor seeds of Example 4 have a significantly lower ricin content compared to Examples 1 to 3.

[0122] Test Example 2. Measurement of ricin content in castor oil and castor meal

[0123] 2-1. Measurement of ricin content of castor oil: The ricin content of castor oil obtained in the above examples and comparative examples was measured by treating the castor oil with a countercurrent method.

[0124] For the countercurrent treatment, the alkaline water used was purified water in which 0.05 wt% sodium hydroxide (NaOH) was dissolved.

[0125] Qualitative analysis of ricin in castor oil through aqueous separation

[0126] ① Add pH 4.0 distilled water (pH adjusted with acetic acid) and castor oil to a fractionation flask in a weight ratio of 1:5, close the lid, and shake to mix well.

[0127] ② Let it stand for about a day to allow the oil layer and buffer layer to separate.

[0128] ③ Once the layers separate, open the stopcock to separate only the buffer layer. At this stage, separate the layers in three steps: beginning, middle, and end, and use the middle separation liquid for the test.

[0129] ④ The lysine content was measured according to the ELISA analysis method of Test Example 1 above.

[0130] 2-2. Measurement of ricin content of castor meal: The castor meal obtained in the above examples and comparative examples and mica were mixed in a weight ratio of 1:0.3 and stirred for 3 hours, after which the ricin content of the castor meal was measured.

[0131] The lysine content of the above castor meal was measured according to the ELISA analysis method of Test Example 1.

[0132] division Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Lysine (ug / ml) Castor oil 0.32±0.84 b ND ND ND 0.62±0.17 a 0.54±0.09 a 0.21±0.02 b Castor oil plant 2.18±0.29 b 0.88±0.11 c 0.90±0.43 c ND 7.95±0.57 a 6.67±0.28 a 3.06±0.43 b

[0133] As shown in Table 2 above, it was confirmed that the castor oil and castor film prepared according to Examples 1 to 4 of the present invention had a lower ricin content compared to Comparative Examples 1 to 3.

[0134] In particular, among the castor oils, ricin was not detected in the castor oils prepared according to Examples 2 to 4, and among the castor meal, ricin was not detected in the castor meal prepared according to Example 4.

Claims

Claim 1 A method for inactivating ricin contained in castor seeds, characterized by comprising: (A) a step of roasting castor (Ricinus communis) seeds and then immersing them in alkaline washing water; (B) a step of heating the castor seeds immersed in the alkaline washing water under purified water at 70 to 90 ℃; and (C) a step of drying and aging the heated castor seeds. Claim 2 A method for inactivating ricin contained in castor seeds according to claim 1, wherein in step (A), the alkaline washing water is characterized by having 0.01 to 3 weight% of one or more alkaline substances selected from the group consisting of sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), potassium hydroxide (KOH), and ammonium hydroxide (NH4OH) dissolved in purified water. Claim 3 A method for inactivating ricin contained in castor seeds according to claim 1, characterized in that the immersion in step (A) is performed for 2 to 24 hours in alkaline washing water at 20 to 26 ℃. Claim 4 A method for inactivating ricin contained in castor seeds according to claim 1, characterized in that the heating in step (B) is performed under a pressure of 0.3 to 0.8 atmospheres. Claim 5 A method for inactivating ricin contained in castor seeds according to claim 1, characterized in that the drying and aging in step (C) is performed at 10 to 20 ℃ for 2 to 3 days while stirring. Claim 6 A method for inactivating ricin contained in castor seeds according to claim 1, characterized in that, in step (C), the drying and aging is performed by mixing heated castor seeds and rice bran in a weight ratio of 1:0.3-0.

9. Claim 7 A method for producing castor oil characterized by including the step of separating castor oil and castor meal using castor seeds in which ricin obtained according to any one of the methods of claims 1 to 6 has been inactivated. Claim 8 A method for manufacturing castor oil according to claim 7, further comprising the step of treating the separated castor oil in a counter-current flow manner. Claim 9 A method for producing castor oil according to claim 7, further comprising the step of crushing the separated castor meal and mixing it with mica. Claim 10 A method for producing castor oil according to claim 9, characterized in that the castor meal powder and mica are mixed in a weight ratio of 1:0.1-5.0.

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