A method for removing channel proteins from starch granules using pulsed electric fields

Abstract

The present invention discloses a method for removing channel proteins from starch granules by using pulsed electric fields. The starch suspension is continuously treated with a direct current pulsed electric field at a constant temperature by a constant-speed peristaltic pump. The starch suspension after pulsed electric field treatment is transferred to a Buchner funnel, filtered by suction, washed, and then dried to obtain starch from which the channel proteins of starch granules have been removed. Compared with the protein denaturant method, this method does not require the introduction of reagents with potential toxicity such as mercaptoethanol. Compared with the alkali treatment method, this method does not require long-term treatment. Compared with the protease method, this method does not require the use of expensive proteases. In short, this method has the advantages of good removal effect, high efficiency, no residual toxic substances, and continuous treatment, etc.

Description

Technical Field:

[0001] The present invention relates to a method for removing starch granule channel proteins by using pulsed electric fields. Background Art:

[0002] At present, starch is a high-molecular carbohydrate existing in major food crops such as grains, tubers, and legumes in nature, and can be absorbed and utilized by the human digestive system to provide energy for human metabolic activities. In addition to a large number of amylose and amylopectin molecules, starch granules also contain trace components such as starch granule-bound proteins. According to their different distribution positions on starch granules (granule surface, granule pores, granule matrix), they can be divided into starch granule surface proteins, starch granule channel proteins, and starch granule intrinsic proteins. Existing research shows that although the mass fraction of starch granule-bound proteins in total starch is less than 1%, they have a significant impact on the processes of starch adsorption, modification, degradation, etc. Among them, the natural pores existing in starch granules provide reliable conditions for foreign substances to enter the interior of starch granules. However, there are a large number of starch granule channel proteins in the pores, which will hinder the sufficient combination of starch molecular chains with water, oil, active substances, modification reagents, degradation reagents, etc., and limit the application of starch in the fields of food, environment, materials, chemical engineering, etc.

[0003] Existing methods for starch granule channel proteins mainly include protein denaturant method, alkali treatment method, and protease method. Using the protein denaturant method can effectively remove starch granule channel proteins by changing the protein conformation, but the protein denaturants used in this method usually have certain potential toxicity and residue risks. For example, in Chinese invention patent ZL201810077828.5, protein denaturants such as tris(hydroxymethyl)aminomethane, glycerol, sodium dodecyl sulfate, and mercaptoethanol are used to strip starch granule-bound proteins from starch. This method can efficiently remove starch granule channel proteins, but the sodium dodecyl sulfate and mercaptoethanol used in this method have certain biological toxicity and may remain in the starch channels to cause potential risks. Although the alkali treatment method avoids introducing toxicity risks, the efficiency of the alkali treatment method is low and the time consumption is long. The protease treatment method has good specificity and high efficiency in removing starch granule channel proteins, but the protease used in the enzymatic hydrolysis method has a high preparation cost and is expensive, making it difficult to be used for large-scale industrial starch treatment. Therefore, in view of these problems, there is an urgent need in this field for an efficient and green method to remove starch granule channel proteins. Summary of the Invention:

[0004] The object of the present invention is to provide a method for removing starch granule channel proteins by using pulsed electric fields.

[0005] The present invention is realized by the following technical solutions:

[0006] A method for removing channel proteins from starch granules by using pulsed electric fields, the method comprising the following steps:

[0007] (1) Add starch to water and stir until a uniform starch suspension is formed;

[0008] (2) Continuously treat the starch suspension with pulsed electric fields at a constant temperature of 4 - 30 °C by a constant-speed peristaltic pump; the specific conditions for the pulsed electric field treatment are: using a DC power supply, an electric field strength of 6 - 8 kV / cm, preferably 8 kV / cm, a frequency of 50 - 200 Hz, a pulse width of 5 - 30 μs, and a treatment time of 10 - 60 min;

[0009] (3) Transfer the starch suspension after pulsed electric field treatment to a Buchner funnel, filter by suction, wash and then dry to obtain starch with channel proteins removed from starch granules.

[0010] Preferably, the starch includes at least one of corn starch, rice starch, wheat starch, potato starch, and cassava starch.

[0011] Preferably, in step (1), the water is distilled water; the mass fraction of starch in the suspension is 5 - 30%.

[0012] Preferably, in step (2), the constant speed of the flow rate is 100 - 400 mL / min.

[0013] Preferably, in step (3), the specific steps of the washing are: wash with distilled water 1 - 3 times and filter by suction under reduced pressure; then wash with 80 wt% ethanol 1 - 3 times and filter by suction under reduced pressure; finally wash with absolute ethanol 1 - 3 times until no liquid filters out.

[0014] Preferably, in step (3), the specific conditions for the drying are: drying in an oven at 40 - 50 °C for 24 - 48 h.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) Compared with the alkali treatment method, the present invention does not require long-term treatment, efficiently removes channel proteins from starch granules, the electric field treatment time is less than 30 min, and the highest continuous treatment flow rate of starch is 80 g / min, and the treatment efficiency is very high.

[0017] (2) Compared with the protein denaturant method, the present invention does not need to introduce protein denaturing reagents such as mercaptoethanol with potential toxicity, avoiding potential toxicity and reagent residue risks.

[0018] (3) Compared with the protease method, this method does not need to use expensive proteases, greatly reducing the treatment cost.

[0019] In summary, the present invention does not require the addition of reaction reagents such as protein denaturants, alkalis, and proteases, avoiding potential toxicity and reagent residue risks. At the same time, the processing efficiency is very high, significantly reducing the processing cost. Description of the Drawings:

[0020] Figure 1 It is a scanning electron micrograph of Example 1 of the present invention.

[0021] Figure 2 It is a scanning electron micrograph of Example 2 of the present invention.

[0022] Figure 3 It is a scanning electron micrograph of Comparative Example 1 of the present invention.

[0023] Figure 4 It is a scanning electron micrograph of Comparative Example 2 of the present invention.

[0024] Figure 5 It is a scanning electron micrograph of Comparative Example 3 of the present invention.

[0025] Figure 6 It is a scanning electron micrograph of Comparative Example 4 of the present invention.

[0026] Figure 7 It is a scanning electron micrograph of Comparative Example 5 of the present invention.

[0027] Figure 8 It is a scanning electron micrograph of Comparative Example 6 of the present invention. Detailed Embodiments:

[0028] The following is a further description of the present invention, rather than a limitation thereof.

[0029] Example 1: A method for removing starch granule channel proteins using pulsed electric fields

[0030] It includes the following steps:

[0031] (1) Add corn starch with a mass fraction of 20% to distilled water and stir until a uniform starch suspension is formed;

[0032] (2) Continuously treat the starch suspension with pulsed electric fields under constant temperature conditions using a constant-speed peristaltic pump. The flow rate is controlled at 200 mL / min, the temperature is controlled at 20 °C, and the pulsed electric field treatment conditions are: using a DC power supply, an electric field strength of 6 kV / cm, a frequency of 100 Hz, a pulse width of 10 μs, and a treatment time of 30 min;

[0033] (3) Transfer the starch suspension after pulsed electric field treatment to a Buchner funnel for vacuum filtration, then wash it 3 times with distilled water and perform vacuum filtration; then wash it 3 times with 80 wt% ethanol and perform vacuum filtration; finally, wash it 3 times with absolute ethanol until no liquid filters out from the filter cake;

[0034] (4) The filter cake was dried in an oven at 40 °C for 48 h, ground and sieved to obtain starch with starch granule channel proteins removed.

[0035] The protein content of the treated starch was determined by the Kjeldahl method, the pore volume of the treated starch was measured using a fully automatic specific surface area and pore size analyzer, and the microstructure of the treated starch was observed by field emission scanning electron microscopy. Figure 1 It is the scanning electron micrograph of Example 1 of the present invention. It can be clearly seen that there are a large number of nanoscale channels on the surface of the starch granules, and there are also a large number of protein particles with sizes smaller than the channel diameter on the particle surface. This indicates that after pulsed electric field treatment, most of the starch granule channel proteins have transferred from inside the channels to outside the channels.

[0036] Example 2

[0037] Referring to Example 1, the difference is that: the electric field strength in step (2) is 8 kV / cm.

[0038] (1) 20% by mass of corn starch was added to distilled water and stirred until a homogeneous starch suspension was formed;

[0039] (2) The starch suspension was continuously treated with pulsed electric field under constant temperature conditions by a constant speed peristaltic pump. The flow rate was controlled at 200 mL / min, the temperature was controlled at 20 °C, and the pulsed electric field treatment conditions were: using a DC power supply, the electric field strength was 8 kV / cm, the frequency was 100 Hz, the pulse width was 10 μs, and the treatment time was 30 min;

[0040] (3) The starch suspension after pulsed electric field treatment was transferred to a Buchner funnel for vacuum filtration, then washed 3 times with distilled water and vacuum filtered; then washed 3 times with 80 wt% ethanol and vacuum filtered; finally washed 3 times with absolute ethanol until no liquid filtered out from the filter cake;

[0041] (4) The filter cake was dried in an oven at 40 °C for 48 h, ground and sieved to obtain starch with starch granule channel proteins removed.

[0042] The protein content of the treated starch was determined by the Kjeldahl method, the pore volume of the treated starch was measured using a fully automatic specific surface area and pore size analyzer, and the microstructure of the treated starch was observed by field emission scanning electron microscopy. Figure 2 It is the scanning electron micrograph of Example 2 of the present invention. It can be clearly seen that there are a large number of nanoscale pores on the surface of the starch granules, and the pore size is slightly larger than that of Example 1. At the same time, there are also protein particles with sizes smaller than the channel diameter on the particle surface. This indicates that after pulsed electric field treatment, most of the starch granule channel proteins have transferred from inside the channels to outside the channels.

[0043] Comparative Example 1

[0044] Referring to Example 1, the difference is that in step (2), no pulsed electric field treatment is performed.

[0045] (1) Add corn starch with a mass fraction of 20% to distilled water and stir until a uniform starch suspension is formed;

[0046] (2) Flow-treat the starch suspension through a constant-speed peristaltic pump under constant temperature conditions, control the flow rate at 200 mL / min, and control the temperature at 20 °C;

[0047] (3) Transfer the treated starch suspension to a Buchner funnel for vacuum filtration, then wash it 3 times with distilled water and perform vacuum filtration; then wash it 3 times with 80 wt% ethanol and perform vacuum filtration; finally, wash it 3 times with absolute ethanol until no liquid filters out from the filter cake;

[0048] (4) Dry the filter cake in an oven at 40 °C for 48 h, grind it and sieve it to obtain starch from which the starch granule channel protein has been removed.

[0049] The protein content of the treated starch was determined by the Kjeldahl method, the pore volume of the treated starch was determined by a fully automatic specific surface area and pore size analyzer, and the microstructure of the treated starch was observed by a field emission scanning electron microscope. Figure 3 This is the scanning electron micrograph of Comparative Example 1 of the present invention. It can be clearly seen that there are a large number of nano-scale channels on the surface of the starch granules, but there are almost no protein particles on the particle surface. Comparing with Example 1, it can be known that under the condition of not being treated with pulsed electric field, the starch granule channel protein cannot be transferred from the inside of the channel to the outside of the channel.

[0050] Comparative Example 2:

[0051] Referring to Example 1, the difference is that in step (2), the electric field strength is relatively low, being 2 kV / cm.

[0052] (1) Add corn starch with a mass fraction of 20% to distilled water and stir until a uniform starch suspension is formed;

[0053] (2) Continuously treat the starch suspension with a pulsed electric field through a constant-speed peristaltic pump under constant temperature conditions, control the flow rate at 200 mL / min, control the temperature at 20 °C, and the pulsed electric field treatment conditions are: using a DC power supply, the electric field strength is 2 kV / cm, the frequency is 100 Hz, the pulse width is 10 μs, and the treatment time is 30 min;

[0054] (3) Transfer the starch suspension after pulsed electric field treatment to a Buchner funnel for vacuum filtration, then wash it 3 times with distilled water and perform vacuum filtration; then wash it 3 times with 80 wt% ethanol and perform vacuum filtration; finally, wash it 3 times with absolute ethanol until no liquid filters out from the filter cake;

[0055] (4) The filter cake was dried in an oven at 40 °C for 48 h, ground and sieved to obtain starch with starch granule channel proteins removed.

[0056] The protein content of the treated starch was determined by the Kjeldahl method, the pore volume of the treated starch was determined by a fully automatic specific surface area and pore size analyzer, and the microstructure of the treated starch was observed by a field emission scanning electron microscope. Figure 4 It is the scanning electron micrograph of Comparative Example 2 of the present invention. It can be clearly seen that there are a large number of nanoscale channels on the surface of the starch granules, and there are also some protein particles with sizes smaller than the channel diameter on the surface. This indicates that after treatment with a pulsed electric field with a relatively low electric field strength, a small part of the starch granule channel proteins are transferred from inside the channels to outside the channels.

[0057] Comparative Example 3:

[0058] Referring to Example 1, the difference is that: in step (2), the electric field strength is relatively high, being 10 kV / cm.

[0059] (1) 20% by mass of corn starch was added to distilled water and stirred until a uniform starch suspension was formed;

[0060] (2) The starch suspension was subjected to continuous pulsed electric field treatment through a constant-speed peristaltic pump under constant temperature conditions. The flow rate was controlled at 200 mL / min, the temperature was controlled at 20 °C, and the pulsed electric field treatment conditions were: using a DC power supply, an electric field strength of 10 kV / cm, a frequency of 100 Hz, a pulse width of 10 μs, and a treatment time of 30 min;

[0061] (3) The starch suspension after pulsed electric field treatment was transferred to a Buchner funnel for vacuum filtration, then washed 3 times with distilled water and vacuum filtered; then washed 3 times with 80 wt% ethanol and vacuum filtered; finally washed 3 times with absolute ethanol until no liquid was filtered out from the filter cake;

[0062] (4) The filter cake was dried in an oven at 40 °C for 48 h, ground and sieved to obtain starch with starch granule channel proteins removed.

[0063] The protein content of the treated starch was determined by the Kjeldahl method, the pore volume of the treated starch was determined by a fully automatic specific surface area and pore size analyzer, and the microstructure of the treated starch was observed by a field emission scanning electron microscope. Figure 5 It is the scanning electron micrograph of Comparative Example 3 of the present invention. It can be clearly seen that there are a large number of nanoscale channels on the surface of the starch granules, and there are some protein particles with relatively large sizes at some channel openings. This indicates that after treatment with a pulsed electric field with an excessively high electric field strength, the starch granule channel proteins aggregate and are blocked inside the pores at the same time.

[0064] Comparative Example 4:

[0065] Referring to Example 1, the difference is that in step (2), an alternating current power supply is used for the pulsed electric field treatment.

[0066] (1) Add corn starch with a mass fraction of 20% to distilled water and stir until a uniform starch suspension is formed.

[0067] (2) Continuously treat the starch suspension with pulsed electric field under constant temperature conditions through a constant-speed peristaltic pump. The flow rate is controlled at 200 mL / min, the temperature is controlled at 20 °C, and the pulsed electric field treatment conditions are: using an alternating current power supply, an electric field strength of 6 kV / cm, a frequency of 100 Hz, a pulse width of 10 μs, and a treatment time of 30 min.

[0068] (3) Transfer the starch suspension after pulsed electric field treatment to a Buchner funnel for vacuum filtration, then wash it 3 times with distilled water and perform vacuum filtration; then wash it 3 times with 80 wt% ethanol and perform vacuum filtration; finally, wash it 3 times with absolute ethanol until no liquid filters out from the filter cake.

[0069] (4) Dry the filter cake in an oven at 40 °C for 48 h, grind it and pass it through a sieve to obtain starch from which the starch granule channel protein has been removed.

[0070] The protein content of the treated starch is determined by the Kjeldahl method, the pore volume of the treated starch is determined by a fully automatic specific surface area and pore size analyzer, and the microstructure of the treated starch is observed by a field emission scanning electron microscope. Figure 6 It is the scanning electron microscope image of Comparative Example 4 of the present invention. It can be clearly seen that there are a large number of nano-scale channels on the surface of the starch granules, but there are almost no protein particles on the granule surface. This shows that under the condition of pulsed electric field treatment with an alternating current power supply, the starch granule channel protein cannot be transferred from the inside of the channel to the outside of the channel.

[0071] Comparative Example 5:

[0072] Referring to Example 2, the difference is that in step (2), an alternating current power supply is used for the pulsed electric field treatment.

[0073] (1) Add corn starch with a mass fraction of 20% to distilled water and stir until a uniform starch suspension is formed.

[0074] (2) Continuously treat the starch suspension with pulsed electric field under constant temperature conditions through a constant-speed peristaltic pump. The flow rate is controlled at 200 mL / min, the temperature is controlled at 20 °C, and the pulsed electric field treatment conditions are: using an alternating current power supply, an electric field strength of 8 kV / cm, a frequency of 100 Hz, a pulse width of 10 μs, and a treatment time of 30 min.

[0075] (3) Transfer the starch suspension after pulsed electric field treatment to a Buchner funnel for vacuum filtration, then wash it 3 times with distilled water and perform vacuum filtration again; then wash it 3 times with 80 wt% ethanol and perform vacuum filtration; finally, wash it 3 times with absolute ethanol until no liquid filters out from the filter cake;

[0076] (4) Dry the filter cake in an oven at 40 °C for 48 h, grind it and sieve it to obtain the starch from which the starch granule channel proteins have been removed.

[0077] The protein content of the treated starch was determined by the Kjeldahl method, the pore volume of the treated starch was determined by a fully automatic specific surface area and pore size analyzer, and the microstructure of the treated starch was observed by a field emission scanning electron microscope. Figure 7 It is the scanning electron micrograph of Comparative Example 5 of the present invention. It can be clearly seen that there are a large number of nano-scale channels on the surface of the starch granules, but there are almost no protein particles on the particle surface. This shows that under the condition of treatment with an alternating current power pulse electric field, the starch granule channel proteins cannot transfer from inside the channels to outside the channels.

[0078] Comparative Example 6:

[0079] Referring to Example 1, the difference is that: in step (2), the pulsed electric field treatment refers to the literature (Achayuthakan, P.; Wongsagonsup, R.; Sriprablom, J.; Suphantharika, M.; Intra, P. Effect of Pulsed Electric Field Treatment on the Protein, Digestibility, and Physicochemical Properties of Starch Granules in Wheat Flour. Polymers 2023, 15, 4087.) and uses static pulsed electric field treatment with an electric field strength of 3 kV / cm, a pulse width of 10 μs, a frequency of 1 Hz, 1400 pulses, and the temperature is controlled within 35 °C.

[0080] (1) Add corn starch with a mass fraction of 20% to distilled water and stir until a uniform starch suspension is formed;

[0081] (2) Place the starch suspension in a static treatment chamber for pulsed electric field treatment with the temperature controlled within 35 °C. The pulsed electric field treatment conditions are: using a DC power supply, an electric field strength of 3 kV / cm, a frequency of 1 Hz, a pulse width of 10 μs, and a treatment time of 1400 s (a total of 1400 pulses);

[0082] (3) Transfer the starch suspension after pulsed electric field treatment to a Buchner funnel for suction filtration under reduced pressure, then wash it 3 times with distilled water and perform suction filtration under reduced pressure; wash it 3 times with 80 wt% ethanol and perform suction filtration under reduced pressure; finally, wash it 3 times with absolute ethanol until no liquid filters out from the filter cake;

[0083] (4) Dry the filter cake in an oven at 40 °C for 48 h, grind it and sieve it to obtain the starch with starch granule channel proteins removed.

[0084] The protein content of the treated starch was determined by the Kjeldahl method, the pore volume of the treated starch was determined by a fully automatic specific surface area and pore size analyzer, and the microstructure of the treated starch was observed by a field emission scanning electron microscope. Figure 8 It is the scanning electron micrograph of Comparative Example 6 of the present invention. It can be clearly seen that there are a large number of nano-scale channels on the surface of the starch granules, but there are almost no protein particles on the particle surface. This shows that under the pulsed electric field treatment conditions of static pulsed electric field treatment with an electric field strength of 3 kV / cm, a frequency of 1 Hz, a pulse width of 10 μs, and a treatment time of 23.33 min (equivalent to 1400 pulses), the starch granule channel proteins cannot be transferred from the channels to the outside of the channels.

[0085] Table 1. Protein content and pore volume of different samples

[0086]

[0087] The protein content and pore volume of the original corn starch, Examples 1-2 and Comparative Examples 1-6 are shown in Table 1.

[0088] Comparing the original corn starch and Comparative Example 1, it can be seen that the protein content of the starch without pulsed electric field treatment in Comparative Example 1 is significantly lower than that of the original corn starch, but the pore volume has not changed significantly, indicating that mainly the surface proteins of the starch are removed.

[0089] Compared with Comparative Example 1, the protein content of Examples 1 and 2 decreased significantly, but the pore volume increased, indicating that treatment with a DC pulsed electric field of 6-8 kV / cm can remove the starch granule channel proteins.

[0090] Compared with Comparative Examples 2-3, the protein content in Examples 1-2 decreased significantly, and the pore volume of starch granules also increased significantly. The reason for this phenomenon is that within the extremely short time when the starch solution flows through the treatment chamber, the orientation of starch granules can be considered fixed. Starch granule channel proteins may escape from the pores through electrophoresis. However, when the electric field strength is low (e.g., 2 kV / cm), the electrophoretic migration speed of starch granule channel proteins is slow and it is difficult to quickly transfer to the outside of starch granules; while when the electric field strength is too high (e.g., 10 kV / cm), the strong polarization force generated by molecular orientation polarization causes the conformation of starch granule channel proteins to unfold, the size of protein molecules increases and the internal positive charges are exposed, resulting in electrostatic attraction between channel proteins and aggregation occurring in the starch granule channels, and it is also difficult to transfer from the channels to the outside of starch granules; only when the electric field strength is appropriate (6-8 kV / cm), within one electric pulse time, the electrophoretic migration distance of starch granule channel proteins just exceeds the depth of the pores where they are located, so a large amount of them can be removed.

[0091] Compared with Examples 1-2, in Comparative Examples 4-5, an alternating current power supply was used for pulsed electric field treatment, but their protein contents were both high and the pore volumes of starch granules were both low. The reason for this phenomenon is that due to the use of an alternating current power supply, the direction of the electric field will change, resulting in the change of the electrophoretic direction of starch granule channel proteins, so that the displacement of starch granule channel proteins to the outside of the granules is offset, making it difficult to transfer to the outside of starch granules.

[0092] Compared with Examples 1-2, in Comparative Example 6, a lower electric field strength and a stationary treatment chamber were used. Its protein content was high and the pore volume was low, indicating that the pulsed electric field under the conditions of Comparative Example 6 could not effectively remove the starch granule-bound proteins. The reasons for this phenomenon are as follows: (1) Within one electric pulse time (in the order of microseconds), the orientation of starch granules can be considered fixed. Starch granule channel proteins may escape from the pores through electrophoresis. However, when the electric field strength is low, the electrophoretic migration speed of starch granule channel proteins is slow and it is difficult to quickly transfer to the outside of starch granules; (2) When a stationary treatment chamber is used, starch is extremely likely to deposit and aggregate, resulting in the pores of starch granules being blocked by other starch granules.

Claims

1. A method for removing starch granule channel protein using a pulsed electric field, characterized in that: The method comprises the following steps: (1) Add starch to water and stir until a uniform starch suspension is formed; (2) subjecting the starch suspension to continuous pulse electric field treatment at a constant temperature of 4 to 30° C. through a constant speed peristaltic pump; the specific conditions of the pulse electric field treatment are: using a DC power supply, an electric field strength of 6 to 8 kV / cm, a frequency of 50 to 200 Hz, a pulse width of 5 to 30 μs, and a treatment time of 10 to 60 min; (3) The starch suspension treated with the pulse electric field is transferred to a Buchner funnel, filtered, washed and dried to obtain starch from which the starch granule channel protein is removed.

2. The method according to claim 1, characterized in that: The starch includes at least one of corn starch, rice starch, wheat starch, potato starch and tapioca starch.

3. The method according to claim 1, characterized in that In step (1), the water is distilled water.

4. The method according to claim 1, characterized in that: The mass fraction of starch in the suspension in step (1) is 5 to 30%.

5. The method according to claim 1, characterized in that In step (2), the constant flow rate is 100-400 mL / min.

6. The method according to claim 1, characterized in that In step (2), the electric field strength is 8 kV / cm.

7. The method according to claim 1, characterized in that In step (3), the specific steps of washing are: washing with distilled water for 1 to 3 times, filtering under reduced pressure; then washing with 80wt% ethanol for 1 to 3 times, filtering under reduced pressure; and finally washing with anhydrous ethanol for 1 to 3 times until no liquid is filtered out.

8. The method according to claim 1, characterized in that: In step (3), the specific conditions for drying are: drying in an oven at 40-50° C. for 24-48 hours.

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