Method for extracting raspberry ketone through raspberry enzymolysis and ultrasonic wave

Abstract

The invention provides a method for extracting raspberry ketone through raspberry enzymolysis and ultrasonic wave, and belongs to the technical field of natural product extraction. The method comprises the following steps: selecting cyan raspberry fruits which are about to be ripe and free from diseases and insect pests, washing, freeze-drying and crushing to 30 meshes; then, carrying out composite enzymolysis, and carrying out enzymolysis on cellulase, pectinase and hemicellulase at the mass ratio of 0.8%-1.2% to 3: 1: 2 for 2-4 hours under the conditions that the temperature is 50 DEG C and the pH is 5.0; and extracting for 20-60 minutes by using a 80% ethanol solution under the assistance of the ultrasonic power of 220W and the frequency of 30kHz. And finally, carrying out vacuum concentration and silica gel column chromatography separation to obtain a high-purity product. The method obviously improves the extraction rate, has the advantages of environmental protection and economy, and meets the requirements of industrial production on raspberry ketone extraction.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and particularly relates to a method for extracting raspberry ketone from raspberries. Background Art

[0002] Raspberry (Rubus idaeus L.), as a traditional Chinese medicinal material and a highly valuable natural plant resource, has the effects of tonifying the kidney, consolidating essence, reducing urination, nourishing the liver and improving eyesight. It is commonly used to improve symptoms such as spermatorrhea, frequent urination, urgency of urination, and unclear vision caused by deficiency of the liver and kidney. In modern nutritional research, raspberries are rich in vitamin C, vitamin E, dietary fiber, and various minerals, and are nutritious health foods.

[0003] Raspberry ketone, as the key natural active ingredient in raspberry fruits, has the chemical name of 4-(p-hydroxyphenyl)-2-butanone, the English name of 4-(4-hydroxyphenyl)-2-butanone, and the structural formula of C 10 H 12 O 2 , and has a unique and strong sweet fruit aroma. In the food field, it is a widely used natural flavor, which can add raspberry fruit aroma to candies, beverages, baked foods, etc., enhance the flavor level of products, and meet consumers' pursuit of natural and delicious foods. In the cosmetics industry, raspberry ketone has antioxidant properties, can effectively fight free radicals, delay skin aging, and at the same time has a certain whitening effect, can inhibit melanin production, and helps the skin to maintain fair and bright, so it is commonly found in various high-end skin care products. In the medical field, preliminary studies show that it has potential effects on the regulation of fat metabolism and is expected to open up a new path for the treatment of obesity-related diseases.

[0004] At present, the extraction methods of raspberry ketone mainly include chemical synthesis method, biosynthesis method and natural extraction method. The common chemical synthesis method uses p-hydroxybenzaldehyde and acetone as raw materials, conducts Claisen-Schmidt condensation in an alkaline system, and then catalytic hydrogenation to synthesize raspberry ketone. This method has a high yield, can realize batch production, and meet the large-scale industrial demand. However, this method involves complex organic chemical reactions and requires the use of a large amount of toxic and harmful chemical reagents, which may cause environmental pollution. The biosynthesis method synthesizes raspberry ketone in Saccharomyces cerevisiae, but the current yield is still low and it is difficult to meet the demand of large-scale industrial production. Natural raspberry ketone products are pollution-free and free of chemical components, have high biological activity, purer aroma and quality, and can better meet the high-quality requirements of natural flavors for food, cosmetics, etc.

[0005] Therefore, it is of great practical significance to develop an efficient, low-cost and simple-operation natural raspberry ketone extraction method. Summary of the Invention

[0006] The object of the present invention is to overcome the defects of the prior art and provide an enzymatic hydrolysis and ultrasonic extraction method for raspberry ketone, so as to significantly improve the extraction rate of raspberry ketone, reduce production costs, reduce environmental pollution, and meet the requirements of industrial production.

[0007] Specifically, the method includes the following steps:

[0008] (1) Raw material pretreatment

[0009] Select fresh, nearly ripe, pest-free and disease-free green palmleaf raspberry fruits, wash them and freeze-dry them. After reducing the fruit moisture to less than 5%, further reducing the fruit moisture content can reduce the subsequent drying time and energy consumption. Subsequently, perform comminution treatment until the particle size reaches 30 mesh. Compared with the previous range of 20-60 mesh, this particle size range can ensure a certain surface area while reducing the subsequent filtration difficulties caused by too fine powder, making the material have better fluidity in subsequent processing.

[0010] Freeze-drying: Remove the moisture in the raspberry fruits through freeze-drying technology to obtain dried raspberry fruits. Freeze-drying can be carried out at low temperature, avoiding the destruction of raspberry ketone by high temperature and maintaining the active ingredients in the raspberry fruits at the same time.

[0011] (2) Composite enzymatic hydrolysis

[0012] Add a composite enzyme solution to the comminuted raspberry raw material. The composite enzyme solution contains cellulase, pectinase and hemicellulase, and its mass ratio is optimized to 3:1:2, further precisely adapting to the proportion of the raspberry fruit cell wall components. The enzymatic hydrolysis temperature is precisely controlled at 50 °C, which can make the enzyme activity reach a higher peak value. The pH value is maintained at 5.0, the enzymatic hydrolysis time is adjusted to 2-4 h, and the enzyme addition amount is 0.8%-1.2% of the raw material mass. This addition amount can not only ensure the enzymatic hydrolysis effect but also avoid the waste of enzymes.

[0013] (3) Ultrasonic-assisted extraction

[0014] After the enzymatic hydrolysis is completed, transfer the material to an ultrasonic extraction device and add an extraction solvent. The extraction solvent is an 80% ethanol solution. Appropriately increasing the ethanol concentration can enhance the solubility of raspberry ketone. The ultrasonic power is set at 220 W, the ultrasonic frequency is 30 kHz, the extraction time is 20-60 min, and the liquid-solid ratio is 10:1 (mL / g). The optimized parameters make the ultrasonic cavitation effect and the mass transfer process cooperate better.

[0015] (4) Concentration and separation

[0016] The extract is concentrated under reduced pressure to remove the solvent, obtaining a crude extract, which is then separated and purified by silica gel column chromatography using petroleum ether-ethyl acetate (volume ratio 8:2) as the eluent. This eluent ratio can precisely separate raspberry ketone. The eluent containing raspberry ketone is collected and further distilled under reduced pressure to obtain a high-purity raspberry ketone product.

[0017] The method for extracting raspberry ketone from raspberries provided by the present invention has the following advantages:

[0018] Through the optimization of complex enzymolysis, the synergistic effect of multiple enzymes is stronger, more precisely destroying the cell wall of raspberry fruits and greatly improving the dissolution efficiency of raspberry ketone.

[0019] After the optimization of ultrasonic-assisted extraction parameters, the ultrasonic cavitation effect is used to further enhance solvent penetration and mass transfer, shorten the extraction time, reduce costs, and the ultrasonic equipment is easy to operate and is conducive to industrial promotion.

[0020] After the entire technological process is optimized, compared with the conventional method, the extraction rate can be increased by 19.71%-37.13%. The usage amount of organic solvents is further reduced, the residual risk is lower, it is more environmentally friendly, and each step is closely connected, enabling the efficient and stable preparation of high-purity raspberry ketone products to meet the requirements of large-scale industrial production.

[0021] It drives the agricultural industrialization and large-scale development of raspberries in China, increases farmers' income; improves the added value of raspberry extracts, promotes the development of the plant extract industry, and creates good economic and social benefits for society. Specific Embodiments

[0022] (I) Raw Material Preparation

[0023] Pick fresh, nearly ripe, uniformly ripe and pest-free green palmleaf raspberry fruits, rinse them thoroughly with clean water to remove surface impurities, and then use freeze-drying technology to make the water content in the raspberry fruits less than 5%. The freeze-dried raspberry fruits are crushed using a pulverizer, screened, and the raspberry powder with a particle size of 30 meshes is collected for standby.

[0024] (II) Complex Enzymolysis

[0025] Prepare a complex enzyme solution: Weigh an appropriate amount of enzyme preparations according to the mass ratio of cellulase, pectinase and hemicellulase of 3:1:2, add them to an appropriate amount of buffer solution. The buffer solution uses 0.05mol / L acetic acid-sodium acetate buffer solution, and the pH value is adjusted to 5.0 with hydrochloric acid or sodium hydroxide, stir evenly to fully dissolve the enzymes, and prepare a complex enzyme solution.

[0026] Enzymatic hydrolysis operation: Add the pretreated raspberry powder into the enzymatic hydrolysis reaction vessel, add the complex enzyme solution according to 0.8%-1.2% of the raw material quality, stir well to make the raw material fully contact with the enzyme solution. Place the reaction vessel in a constant temperature water bath, control the temperature at 50°C, carry out the enzymatic hydrolysis reaction under the condition of a stirring speed of 120 r / min, and the enzymatic hydrolysis time is 2-4 h. During the enzymatic hydrolysis process, sample and detect the reducing sugar content or viscosity and other indicators of the enzymatic hydrolysis solution every 30 min to monitor the enzymatic hydrolysis process. When the indicators tend to be stable, it indicates that the enzymatic hydrolysis is basically completed.

[0027] (III) Ultrasonic-assisted extraction

[0028] Extraction preparation: After the enzymatic hydrolysis is completed, transfer the enzymatic hydrolysis material to the extraction kettle of the ultrasonic extraction device while it is hot, add the extraction solvent according to the liquid-solid ratio of 10:1 (mL / g), and the extraction solvent is a pre-prepared 80% ethanol solution.

[0029] Ultrasonic extraction: Close the extraction kettle, turn on the ultrasonic device, set the ultrasonic power to 220 W and the ultrasonic frequency to 30 kHz, and start ultrasonic extraction. During the extraction process, the stirring device can be appropriately turned on, and the stirring speed is controlled at 60 r / min to make the material and the solvent fully mixed. The extraction time is 20-60 min. After the extraction is completed, release the extraction solution, filter to remove the residue, and obtain a clear extraction solution.

[0030] (IV) Concentration and separation

[0031] Vacuum concentration: Transfer the extraction solution to a vacuum concentration device, such as a rotary evaporator, and carry out vacuum concentration under the conditions of a temperature of 45°C and a vacuum degree of 0.09-0.1 MPa to remove most of the solvent and obtain a thick crude extract.

[0032] Silica gel column chromatography separation: Select a silica gel column of appropriate specifications, dissolve the crude extract with a small amount of eluent and load the sample. The eluent is petroleum ether-ethyl acetate (volume ratio 8:2). Control the elution flow rate at 1.2-1.8 mL / min, collect the eluate with a test tube, detect the content of raspberry ketone in the eluate by high performance liquid chromatography (HPLC), and collect the eluate containing raspberry ketone. The collected eluate is subjected to vacuum distillation again under the conditions of a temperature of 55°C and a vacuum degree of 0.09-0.1 MPa to further remove the residual solvent, obtain a high-purity raspberry ketone product, and package and store it after drying, weighing, and detecting the purity.

[0033] To further illustrate the present invention, the following describes in detail a method for enzymatically hydrolyzing and ultrasonically extracting raspberry ketone provided by the present invention in combination with embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0034] Example 1

[0035] Raw materials: Select 100 g of nearly mature green fruits of Rubus chingii Hu, wash, freeze-dry, and pulverize them to obtain a powder with a particle size of 30 mesh.

[0036] Complex enzymatic hydrolysis: Weigh 0.6 g of cellulase, 0.2 g of pectinase, and 0.4 g of hemicellulase according to 1.2% of the mass of the raw materials, add them to 100 mL of 0.05 mol / L acetic acid-sodium acetate buffer solution (pH = 5.0), add 100 g of raspberry powder to the enzymatic hydrolysis reaction vessel, and carry out enzymatic hydrolysis at 50 °C and a stirring speed of 120 r / min for 2 h.

[0037] Ultrasound-assisted extraction: After enzymatic hydrolysis, transfer the material to an ultrasonic extraction device, add 1000 mL of 80% ethanol solution, with an ultrasonic power of 220 W, an ultrasonic frequency of 30 kHz, extract for 40 min, and a stirring speed of 60 r / min.

[0038] Concentration and separation: The extract is concentrated under reduced pressure, and then separated by silica gel column chromatography. Using petroleum ether-ethyl acetate (volume ratio 8:2) as the eluent, collect the eluent containing raspberry ketone, and obtain the product by reduced pressure distillation. After detection, the extraction rate of raspberry ketone is 78.2%.

[0039] Example 2

[0040] Raw materials: Take 180 g of nearly mature green fruits of Rubus chingii Hu, and obtain the corresponding powder according to the above method.

[0041] Complex enzymatic hydrolysis: Weigh 1.08 g of cellulase, 0.36 g of pectinase, and 0.72 g of hemicellulase according to 1.2% of the mass of the raw materials, add 180 mL of 0.05 mol / L acetic acid-sodium acetate buffer solution (pH = 5.0), add 180 g of the powder, and carry out enzymatic hydrolysis at 50 °C and a stirring speed of 120 r / min for 2.5 h.

[0042] Ultrasound-assisted extraction: Transfer the material, add 1800 mL of 80% ethanol solution, with an ultrasonic power of 220 W, an ultrasonic frequency of 30 kHz, extract for 40 min, and a stirring speed of 60 r / min.

[0043] Concentration and separation: The same operation as in Example 1. After detection, the extraction rate is 82.5%.

[0044] Example 3

[0045] Raw materials: Select 250 g of nearly mature green fruits of Rubus chingii Hu, and obtain the corresponding powder according to the above method.

[0046] Complex enzymatic hydrolysis: Weigh 1.5 g of cellulase, 0.50 g of pectinase, and 1.0 g of hemicellulase according to 1.2% of the raw material quality, add 250 mL of 0.05 mol / L acetic acid - sodium acetate buffer (pH = 5.0), add 250 g of the powder, and carry out enzymatic hydrolysis at 50 °C with a stirring speed of 120 r / min for 3 h.

[0047] Ultrasonic-assisted extraction: Transfer the material, add 2500 mL of 80% ethanol solution, with an ultrasonic power of 220 W, an ultrasonic frequency of 30 kHz, extract for 40 min, and a stirring speed of 60 r / min.

[0048] Concentration and separation: Operate in the same way as in Example 1. After detection, the extraction rate is 84.2%.

[0049] Example 4

[0050] Raw materials: Select 100 g of nearly ripe green Rubus chingii Hu fruits, and obtain the corresponding powder by treating according to the above method.

[0051] Complex enzymatic hydrolysis: Weigh 0.5 g of cellulase, 0.17 g of pectinase, and 0.23 g of hemicellulase according to 1.0% of the raw material quality, add them to 100 mL of 0.05 mol / L acetic acid - sodium acetate buffer (pH = 5.0), add 100 g of Rubus chingii Hu powder to the enzymatic hydrolysis reaction vessel, and carry out enzymatic hydrolysis at 50 °C with a stirring speed of 120 r / min for 3 h.

[0052] Ultrasonic-assisted extraction: After enzymatic hydrolysis, transfer the material to the ultrasonic extraction device, and add 1000 mL of 80% ethanol solution. Set the ultrasonic power to 220 W, the ultrasonic frequency to 30 kHz, extract for 40 min, and a stirring speed of 60 r / min.

[0053] Concentration and separation: Operate in the same way as in Example 1. After detection, the extraction rate of rubus ketone is 83.8%.

[0054] Example 5

[0055] Raw materials: Take 100 g of ripe Rubus chingii Hu fruits, and obtain the corresponding powder by treating according to the above method.

[0056] Complex enzymatic hydrolysis: Weigh 0.4 g of cellulase, 0.13 g of pectinase, and 0.27 g of hemicellulase according to 0.8% of the raw material quality, add 100 mL of 0.05 mol / L acetic acid - sodium acetate buffer (pH = 5.0). Add 100 g of the powder, and carry out enzymatic hydrolysis at 50 °C with a stirring speed of 120 r / min for 3 h.

[0057] Ultrasonic-assisted extraction: Transfer the material, add 1000 mL of 80% ethanol solution, with an ultrasonic power of 220 W, an ultrasonic frequency of 30 kHz, extract for 40 min, and a stirring speed of 60 r / min.

[0058] Concentration and separation: The same operation as in Example 1. After detection, the extraction rate is 79.7%.

[0059] Example 6

[0060] Raw materials: Take 100 g of mature raspberry fruits, and process them according to the above method to obtain the corresponding powder.

[0061] Compound enzymatic hydrolysis: Weigh 0.4 g of cellulase, 0.13 g of pectinase, and 0.27 g of hemicellulase according to 0.8% of the raw material mass, and add 100 mL of 0.05 mol / L acetic acid - sodium acetate buffer solution (pH = 5.0). Add 100 g of the powder, and carry out enzymatic hydrolysis at 50 °C and a stirring speed of 120 r / min for 3 h.

[0062] Ultrasound-assisted extraction: Transfer the material, add 1000 mL of 80% ethanol solution, with an ultrasonic power of 220 W, an ultrasonic frequency of 30 kHz, extract for 20 min, and a stirring speed of 60 r / min.

[0063] Concentration and separation: The same operation as in Example 1. After detection, the extraction rate is 69.5%.

[0064] Example 7

[0065] Raw materials: Take 100 g of mature raspberry fruits, and process them according to the above method to obtain the corresponding powder.

[0066] Compound enzymatic hydrolysis: Weigh 0.4 g of cellulase, 0.13 g of pectinase, and 0.27 g of hemicellulase according to 0.8% of the raw material mass, and add 100 mL of 0.05 mol / L acetic acid - sodium acetate buffer solution (pH = 5.0). Add 100 g of the powder, and carry out enzymatic hydrolysis at 50 °C and a stirring speed of 120 r / min for 3 h.

[0067] Ultrasound-assisted extraction: Transfer the material, add 1000 mL of 80% ethanol solution, with an ultrasonic power of 220 W, an ultrasonic frequency of 30 kHz, extract for 60 min, and a stirring speed of 60 r / min.

[0068] Concentration and separation: The same operation as in Example 1. After detection, the extraction rate is 80.3%.

[0069] Comparative example

[0070] Raw materials: Select 200 g of nearly mature green palmleaf raspberry fruits, and process them according to the above method to obtain the corresponding powder.

[0071] Conventional extraction (without using compound enzymatic hydrolysis and ultrasound assistance): Directly add 200 g of raspberry powder into 2000 mL of 80% ethanol solution, stir and extract at 50 °C for 3 h, and a stirring speed of 60 r / min.

[0072] Concentration and separation: The operation was the same as in Example 1. After detection, the extraction rate of raspberry ketone was only 61.4%.

[0073] Table 1 Extraction rate and purity of raspberry ketone in examples and comparative examples

[0074]

[0075] As can be seen from the above examples, the optimized improved method for extracting raspberry ketone of the present invention can stably achieve a higher extraction rate under different raw material dosages, further verifying the effectiveness and reliability of the method, and can better meet the requirements of industrial production for the extraction of raspberry ketone.

[0076] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting raspberry ketone by enzymatic hydrolysis and ultrasonic wave from raspberries, characterized in that: The following steps are involved: (1) Raw material pretreatment: fresh, ripe, disease-free and insect-free green palm-leaved raspberry fruits were selected, washed, freeze-dried, and crushed to a particle size of 30 mesh; (2) Composite enzymatic hydrolysis: adding a composite enzyme solution to the crushed raspberry raw material, wherein the composite enzyme solution comprises cellulase, pectinase and hemicellulase in a mass ratio of 3:1:2, the enzymatic hydrolysis temperature is controlled at 50° C., the pH value is maintained at 5.0, the enzymatic hydrolysis time is 2-4 h, and the amount of enzyme added is 0.8%-1.2% of the raw material mass; (3) Ultrasonic-assisted extraction: After the enzymatic hydrolysis is completed, for example, the material is transferred to an ultrasonic extraction device, an extraction solvent is added, the extraction solvent is an 80% ethanol solution, the ultrasonic power is set to 220 W, the ultrasonic frequency is 30 kHz, the extraction time is 20-60 min, and the liquid-to-solid ratio is 10:1 (mL / g); (4) Concentration and separation: The extract is concentrated under reduced pressure to remove the solvent to obtain a crude extract, which is then separated and purified by silica gel column chromatography using petroleum ether-ethyl acetate (volume ratio 8:2) as eluent, and the eluate containing raspberry ketone is collected. A high-purity raspberry ketone product is obtained by further distillation under reduced pressure.

2. The method for extracting raspberry ketone according to claim 1, characterized in that: In the composite enzymolysis step, the buffer solution adopts 0.05 mol / L acetic acid-sodium acetate buffer.

3. The method for extracting raspberry ketone according to claim 1, characterized in that: In the ultrasonic-assisted extraction step, the stirring speed is controlled at 60 r / min during the extraction process.

4. The method for extracting raspberry ketone according to claim 1, characterized in that: In the concentration and separation steps, the reduced pressure concentration temperature is 45°C, the vacuum degree is 0.09-0.1 MPa, the silica gel column chromatography elution flow rate is 1.2-1.8 mL / g, and the reduced pressure distillation temperature is 55°C, and the vacuum degree is 0.09-0.1 MPa.

5. The method for extracting raspberry ketone according to claim 1, characterized in that: In the raw material pretreatment step, the moisture content of the freeze-dried raspberry fruit is less than 5%.

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