Method for extracting bromelain by ultrasonic-assisted deep-eutectic solvent

Abstract

The invention belongs to the field of food science and engineering, and discloses a method for extracting bromelain by using an ultrasonic-assisted eutectic solvent. The method for extracting bromelain by using the ultrasonic-assisted deep-eutectic solvent comprises the following steps: heating and mixing a hydrogen bond acceptor and a hydrogen bond donor to obtain a deep-eutectic solvent, adding water, and uniformly mixing to obtain a deep-eutectic extracting agent; and then adding the pineapple peel powder into the eutectic extractant, and carrying out ultrasonic extraction to obtain bromelain. The highest enzyme activity of the bromelain can reach 1000U / mg or above, under the condition that the highest enzyme activity is obtained, the protein content of the bromelain reaches 10g / kg or above, and high purity is kept.

Description

Technical Field

[0001] The present invention belongs to the field of food science and engineering, and particularly relates to a method for extracting bromelain by ultrasonic-assisted deep eutectic solvents. Background Art

[0002] Pineapple, belonging to the genus Ananas in the family Bromeliaceae, is a perennial monocotyledonous plant widely cultivated in tropical and subtropical regions. A large number of by-products are generated during the processing of pineapples, such as pineapple stems, peels, and crowns, which account for about 70% of the total amount of pineapples and are good sources of proteins, peptides, and other bioactive compounds, including proteolytic enzymes such as bromelain. Bromelain is a sulfhydryl protease containing multiple different proteolytic enzyme components, which has hydrolytic effects on various polypeptides, esters, amides, and proteins and is widely used in beer clarification, cheese production, meat tenderization, and protein hydrolysis. Bromelain has pharmacological activities such as antioxidant, anti-inflammatory, anti-cancer, and anti-tumor effects and has good application prospects in the pharmaceutical industry.

[0003] Currently, traditional industrial extraction methods of bromelain, such as kaolin adsorption method, tannin extraction method, and ammonium sulfate precipitation method, have high enzyme impurity content, low enzyme activity, and are prone to cause serious environmental pollution. Chinese Patent CN117821430 A discloses a method for extracting bromelain. In this method, pineapple juice is placed in an enzyme separation tank and tannic acid is added to prepare bromelain, and the purity of the bromelain prepared by this method needs to be improved. Chinese Patent CN 100535110 C discloses a method for extracting bromelain, which uses ultrafiltration to purify bromelain. Since the pineapple juice becomes more and more viscous during the ultrafiltration process, it is easy to cause the ultrafiltration membrane to be blocked, and the obtained bromelain is relatively low and cannot meet the industrial requirements. Chinese Patent CN114107265B discloses a method for extracting bromelain from pineapple peels. In this method, the pineapple peels are juiced and then bromelain paste is obtained by adjusting the pH, and the bromelain finished product is obtained after freeze-drying. The content of bromelain extracted by this method is low. Chinese Patent CN 111876401 A provides a new method for extracting bromelain, which extracts bromelain in a reaction kettle by preparing extraction microspheres, and organic reagents such as 3-aminopropyltriethoxysilane and dimethyl sulfoxide need to be added. The whole process is relatively complex, and the large amount of use of organic reagents is not conducive to environmental friendliness.

[0004] In summary, the problems existing in the above-mentioned related bromelain extraction methods include: (1) the enzyme activity and enzyme purity need to be improved; (2) the operation process is complex; (3) the use of organic reagents is not conducive to environmental friendliness; (4) the extraction cost is relatively high. Therefore, it is of great significance to seek a low-cost, simple-process, and green and environmentally friendly extraction method to obtain bromelain with high enzyme activity. Summary of the Invention

[0005] To overcome the disadvantages and deficiencies of the above-mentioned prior art, the primary object of the present invention is to provide a method for extracting bromelain using an ultrasonic-assisted deep eutectic solvent.

[0006] The bromelain extracted by the method of the present invention has a relatively high enzyme activity, which can reach up to 1003.39 U / mg, far higher than the national standard of 800 U / mg; and high purity (protein content is 12.99 g / kg) is maintained under the conditions of obtaining the highest enzyme activity.

[0007] The object of the present invention is achieved by the following solutions:

[0008] A method for extracting bromelain using an ultrasonic-assisted deep eutectic solvent, comprising the following steps:

[0009] Heat and mix a hydrogen bond acceptor and a hydrogen bond donor to obtain a deep eutectic solvent, add water, and mix evenly to obtain a deep eutectic extraction agent; then add pineapple peel powder to the deep eutectic extraction agent and perform ultrasonic extraction to obtain bromelain.

[0010] The pineapple peel powder is obtained after pretreatment of pineapple peel, and the pretreatment includes washing, freeze-drying for 48 - 72 h, pulverizing, and sieving through a 40 - 60 mesh sieve.

[0011] After the ultrasonic extraction, the obtained mixture is centrifuged, the centrifugation speed is 8000 - 10000 rpm, and the time is 10 - 30 min.

[0012] The hydrogen bond acceptor includes at least one of choline chloride, betaine, and L-proline.

[0013] The viscosity of the sugar-based deep eutectic solvent is relatively high, and water needs to be added to promote its synthesis. When the hydrogen bond acceptor is betaine, water is added during the mixing process of the hydrogen bond acceptor and the hydrogen bond donor, and the amount of water used satisfies the molar ratio of the hydrogen bond acceptor, the hydrogen bond donor, and water of 1:1:1 - 2.

[0014] The hydrogen bond donor includes at least one of glycerol, ethylene glycol, propylene glycol, glucose, and sorbitol.

[0015] The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1 - 2:5.

[0016] The heating temperature is 80 - 85 °C.

[0017] The volume ratio of water to the deep eutectic solvent is 10 - 60:40 - 90.

[0018] The dosage ratio of the pineapple peel powder to the deep eutectic extraction agent is 1 - 3 g:20 mL.

[0019] The equipment for ultrasonic extraction is a cell ultrasonic disruptor.

[0020] The power of the ultrasonic extraction is 60 - 500 W, preferably 60 - 400 W; the time is 5 - 30 min.

[0021] The temperature of the ultrasonic extraction is 0 - 4 °C.

[0022] The present invention has the following advantages and beneficial effects compared with the prior art:

[0023] 1. The bromelain extracted from pineapple peel by the ultrasonic-assisted deep eutectic solvent in the present invention can reach a maximum enzyme activity of over 1000 U / mg, obtaining bromelain with high enzyme activity, which is beneficial to its application in industry.

[0024] 2. The present invention uses a deep eutectic solvent to extract bromelain from pineapple peel. The prepared deep eutectic solvent is green and safe, avoiding the environmental pollution caused by the large use of traditional organic reagents, and realizing the green extraction of bromelain by using this new solvent that is simple to prepare, green and pollution-free.

[0025] 3. The present invention uses ultrasonic-assisted deep eutectic solvent to extract bromelain. Through the treatment of this non-thermal physical field of ultrasound, the cavitation and implosion generated during the ultrasonic process cause the cell wall to rupture, increasing the contact area between the pineapple peel powder and the deep eutectic solvent, improving the dissolution rate of the protease by the deep eutectic solvent, shortening the extraction time. At the same time, the present invention is carried out under ice bath conditions to prevent the protease from denaturing under high temperature conditions. Description of the Drawings

[0026] Figure 1 Shows the effects of the water content of the deep eutectic extractant on the enzyme activity and protein content of bromelain.

[0027] Figure 2 Shows the effects of the ultrasonic time on the enzyme activity and protein content of bromelain.

[0028] Figure 3 Shows the effects of the solid-liquid ratio of pineapple peel powder to the deep eutectic extractant on the enzyme activity and protein content of bromelain.

[0029] Figure 4 Shows the effects of the ultrasonic power on the enzyme activity and protein content of bromelain. Detailed Embodiments

[0030] The present invention will be further described in detail below with reference to the embodiments and the drawings, but the embodiments of the present invention are not limited thereto. Those not specified in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0031] The reagents used in the examples can be routinely purchased from the market without special instructions.

[0032] Example 1

[0033] Wash the pineapple peel, freeze-dry it for 48 h, crush it and then pass it through a 40-mesh sieve to obtain pineapple peel powder.

[0034] Weigh choline chloride and ethylene glycol in a molar ratio of 1:2, mix them and heat to 80 °C until a homogeneous and stable transparent liquid is formed. After cooling to room temperature, a deep eutectic solvent is obtained. Add water according to the volume ratio of water to the deep eutectic solvent of 30:70, mix evenly to obtain a deep eutectic extractant, named DES-1.

[0035] Weigh 1 g of pineapple peel powder, add 20 mL of DES-1, and extract in a cell ultrasonic crusher for 15 min under the condition of ice bath at 4 °C and an ultrasonic power of 200 W. Then centrifuge at 10000 rpm for 10 min to obtain bromelain solution.

[0036] Example 2

[0037] The difference from Example 1 is that ethylene glycol is replaced by propylene glycol, and the deep eutectic extractant is named DES-2.

[0038] Example 3

[0039] The difference from Example 1 is that ethylene glycol is replaced by glycerol, and the deep eutectic extractant is named DES-3.

[0040] Example 4

[0041] The difference from Example 1 is that the preparation of the deep eutectic extractant is as follows:

[0042] Weigh betaine, glucose, and water in a molar ratio of 1:1:2, mix them and heat to 80 °C until a homogeneous and stable transparent liquid is formed. After cooling to room temperature, a deep eutectic solvent is obtained. Add water according to the volume ratio of water to the deep eutectic solvent of 30:70, mix evenly to obtain a deep eutectic extractant, named DES-4.

[0043] Example 5

[0044] The difference from Example 4 is that glucose is replaced by sorbitol, and the deep eutectic extractant is named DES-5.

[0045] Example 6

[0046] The difference from Example 1 is that the preparation of the deep eutectic extractant is as follows:

[0047] Weigh L-proline and glycerol in a molar ratio of 2:5, mix them, heat to 80 °C until a homogeneous and stable transparent liquid is formed, and after cooling to room temperature, a deep eutectic solvent is obtained. Add water in a volume ratio of water to the deep eutectic solvent of 30:70, mix evenly to obtain a deep eutectic extraction agent, named DES-6.

[0048] Example 7

[0049] The difference from Example 6 is that the volume ratios of water to the deep eutectic solvent are 10:90, 20:80, 40:60, 50:50, and 60:40 respectively, and the water contents in the corresponding deep eutectic extraction agents are 10%, 20%, 40%, 50%, and 60% respectively.

[0050] It can be seen from Figure 1 that when the water content of the deep eutectic extraction agent is 50%, the enzyme activity is the highest, which is 1003.39 U / mL, and the protein content is 12.99 mg / g.

[0051] Example 8

[0052] The difference from Example 6 is that the volume ratio of water to the deep eutectic solvent is 50:50; the ultrasonic extraction times are 5, 10, 15, 20, 25, and 30 min respectively.

[0053] It can be seen from Figure 2 that when the extraction time is 20 min, the enzyme activity is the highest, which is 905.56 U / mL, and the protein content is 16.36 mg / g.

[0054] Example 9

[0055] The difference from Example 6 is that the volume ratio of water to the deep eutectic solvent is 50:50; the ultrasonic extraction time is 20 min; the dosages of pineapple peel powder are 0.25, 0.5, 1, 2, and 3 g respectively.

[0056] It can be seen from Figure 3 that when the solid-liquid ratio is 2:20 (g / mL), the enzyme activity is the highest, which is 950.58 U / mL, and the protein content is 11.1 mg / g.

[0057] Example 10

[0058] The difference from Example 6 is that the volume ratio of water to the deep eutectic solvent is 50:50; the ultrasonic extraction time is 20 min; the dosage of pineapple peel powder is 2 g; the ultrasonic powers are 60, 100, 200, 300, 400, and 500 W.

[0059] It can be seen from Figure 4 that when the ultrasonic power is 200 W, the enzyme activity is the highest, which is 949.94 U / mL, and the protein content is 8.91 mg / g.

[0060] Comparative Example 1

[0061] The difference from Example 1 is that the eutectic extractant is replaced with 0.02 M sodium phosphate buffer solution at pH 6.0.

[0062] Comparative Example 2

[0063] The difference from Example 1 is that ethylene glycol is replaced with lactic acid, and the molar ratio of choline chloride to lactic acid is 1:1. The eutectic extractant is named DES-7.

[0064] Comparative Example 3

[0065] The difference from Example 4 is that glucose is replaced with lactic acid, and the molar ratio of betaine: lactic acid: water is 1:1:1. The eutectic extractant is named DES-8.

[0066] Comparative Example 4

[0067] The difference from Example 4 is that glucose is replaced with malic acid, and the molar ratio of betaine: malic acid: water is 1:1:1. The eutectic extractant is named DES-9.

[0068] Comparative Example 5

[0069] The difference from Example 6 is that glycerol is replaced with lactic acid, and the molar ratio of L-proline to lactic acid is 1:1. The eutectic extractant is named DES-10.

[0070] Result determination

[0071] Determination of bromelain enzyme activity: Take the bromelain enzyme solution obtained from each example and comparative example, use casein as the substrate, and determine the bromelain enzyme activity. The calculation formula for enzyme activity is shown in the following formula (1):

[0072]

[0073] Where: E is the bromelain enzyme activity / U / mL; A b -A s is the absorbance difference, A is the absorbance value of 50 mg tyrosine, V0 is the volume of the enzyme solution / L, and V k is the total volume of the reaction solution / L, and DF is the dilution factor.

[0074] Determination of the protein content in the bromelain enzyme solution obtained from each example and comparative example: The protein content is determined according to the BSA method.

[0075] Table 1 shows the bromelain enzyme activity and protein content extracted in Examples 1-6 and Comparative Examples 1-5

[0076]

[0077]

[0078] Note: 0 indicates that the bromelain enzyme activity was not detected.

[0079] As can be seen from Table 1, the bromelain enzyme activity and protein content extracted by DES-6 are the highest. However, due to the low pH values of DES-7, DES-8, DES-9, and DES-10, the structure of bromelain was damaged, resulting in the loss of its activity. Therefore, the bromelain activity could not be detected.

[0080] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for extracting bromelain by ultrasonic-assisted deep eutectic solvent, characterized in that, It includes the following steps: Heat and mix a hydrogen bond acceptor and a hydrogen bond donor to obtain a deep eutectic solvent, add water, and mix evenly to obtain a deep eutectic extractant; then add pineapple peel powder to the deep eutectic extractant and perform ultrasonic extraction to obtain bromelain.

2. The method for ultrasonic-assisted extraction of bromelain with a deep eutectic solvent according to claim 1, wherein: The pineapple peel powder is obtained after pretreatment of pineapple peel, and the pretreatment includes washing, freeze-drying for 48 - 72 h, pulverizing, and sieving through a 40 - 60 mesh sieve; After the ultrasonic extraction, the obtained mixture is centrifuged at a centrifugation speed of 8000 - 10000 rpm for 10 - 30 min.

3. The method for ultrasonic-assisted extraction of bromelain with a deep eutectic solvent according to claim 1, wherein: The hydrogen bond acceptor includes at least one of choline chloride, betaine, and L-proline; The hydrogen bond donor includes at least one of glycerol, ethylene glycol, propylene glycol, glucose, and sorbitol.

4. The method for ultrasonic-assisted extraction of bromelain with deep eutectic solvents according to claim 3, wherein: When the hydrogen bond acceptor is betaine, water needs to be added during the mixing process of the hydrogen bond acceptor and the hydrogen bond donor, and the dosage of water satisfies that the molar ratio of the hydrogen bond acceptor, the hydrogen bond donor, and water is 1:1:1 - 2.

5. The method for ultrasonic-assisted extraction of bromelain with deep eutectic solvents according to claim 1, wherein: The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1 - 2:

5.

6. The method for ultrasonic-assisted extraction of bromelain with deep eutectic solvents according to claim 1, wherein: The heating temperature is 80 - 85 °C.

7. The method for extracting bromelain by ultrasonic-assisted deep eutectic solvent according to claim 1, wherein: The volume ratio of water to the deep eutectic solvent is 10 - 60:40 - 90.

8. The method for extracting bromelain by ultrasonic-assisted deep eutectic solvent according to claim 1, wherein: The dosage ratio of the pineapple peel powder to the deep eutectic extractant is 1 - 3 g:20 mL.

9. The method for ultrasonic-assisted extraction of bromelain with deep eutectic solvents according to claim 1, wherein: The equipment for ultrasonic extraction is a cell ultrasonic disruptor; the temperature for ultrasonic extraction is 0 - 4 °C.

10. The method for ultrasonic-assisted extraction of bromelain with deep eutectic solvent according to claim 1, wherein: The power of ultrasonic extraction is 60 - 500 W; the time is 5 - 30 min.

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