Preparation method of high-purity natural capsaicin and product thereof

Abstract

The invention provides a preparation method of high-purity natural capsaicin and a product of the high-purity natural capsaicin, which adopts a reverse extraction method, that is, most impurities are removed by microbial fermentation extraction, and then the capsaicin is extracted. Wherein in the fermentation process, the strains can remove impurities, the proportion of capsaicin in the chili powder is increased, and subsequent extraction, separation and purification are facilitated; according to the method, hydrogen peroxide is used as a reagent for fading materials, oxidation and chain scission can be carried out on part of macromolecular substances, the extraction rate of impurities is increased, the used concentration is low, the safety is high, and the structure of capsaicin cannot be damaged; the ultrafiltration membrane can intercept and remove most alkali-soluble impurities, and the nanofiltration membrane can filter and remove small molecular substances and intercept capsaicin, so that the content of capsaicin in the solution is increased; finally, through crystallization treatment, solvent residues are avoided, and then high-purity capsaicin can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to a method for preparing high-purity natural capsaicin and its product. Background Technology

[0002] Natural capsaicin (capsaicin) is a vanillamide alkaloid (total alkaloid) extracted and isolated from chili pepper fruits. It appears as a white or off-white crystalline powder and is the main source of the spiciness of chili peppers. Capsaicin mainly includes various alkaloids such as capsaicin (CAS No. 000404-86-4), dihydrocapsaicin (CAS No. 019408-84-5), nordihydrocapsaicin (CAS No. 028789-35-7), and homodihydrocapsaicin-1 (CAS No. 020279-06-5).

[0003] Natural capsaicin has a wide range of uses. In the pharmaceutical field, it can be used as an analgesic, anti-inflammatory, drug addiction treatment, and antibacterial and preservative ingredient. In the industrial field, it can be used as a stain remover, a corrosion protectant, and a security equipment. In the food and daily chemical fields, it can be used as a spicy food flavoring agent, a heat-sensitive agent in cosmetics / skincare products, and an environmentally friendly biological pesticide repellent.

[0004] CN118955319A discloses a method for extracting and purifying capsaicin. The method includes: mixing pulverized chili powder with a complex enzyme, dispersing the mixture in a plasma-active eutectic solvent, and simultaneously performing enzymatic hydrolysis and extraction purification; after extraction, centrifuging and collecting the supernatant, concentrating, and then freeze-drying. This invention utilizes a plasma-active eutectic solvent with high selectivity for capsaicin for targeted extraction and purification, resulting in high-purity extracts. The yield and purity of capsaicin extracted using this method are 222.04% and 36.36% higher, respectively, than those extracted with 70% (v / v) ethanol, while avoiding the large-scale use of organic reagents, meeting the requirements of green chemistry, and facilitating the industrial production and application of capsaicin.

[0005] CN108276301A discloses a method for extracting capsaicin. The method includes the following steps: weighing dried chili powder with a mesh size of 200; preparing a mixture of 2% NaOH solid and 95% ethanol solution; soaking the dried chili powder in the mixture; adsorption extraction using chitosan resin; elution with a water / ethanol solution; and concentration extraction under reduced pressure to obtain capsaicin. This invention uses dried chili powder as raw material, and through alkali treatment, chitosan resin adsorption, ethanol elution, and recrystallization with petroleum ether, yields capsaicin with a purity of 91.20% and an extraction rate of 2%, reaching the domestic production level for similar capsaicin products.

[0006] Existing technologies for preparing high-purity capsaicin include steps such as extraction, separation, purification, and crystallization. Literature and related patents indicate that extraction solvents can be organic solvents such as ethanol, ethyl acetate, and acetone, or alkaline aqueous solutions such as ammonia and sodium hydroxide. Separation and purification steps typically employ column chromatography, such as silica gel column chromatography or macroporous resin column chromatography. Crystallization methods include cooling crystallization and acid-base neutralization crystallization. Regardless of the method used, a large amount of organic solvent is required for extraction or elution, posing a risk of organic solvent residue in the product and generating substantial amounts of hazardous solvent waste. Chromatography column packing materials are expensive and require replacement after a certain period of use. Therefore, the preparation process for high-purity natural capsaicin has significant room for improvement. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for preparing high-purity natural capsaicin and the resulting product. The method involves no organic solvents, and does not require adsorption materials such as chromatography or decolorization. The prepared product is characterized by high purity and is environmentally friendly and safe.

[0008] To achieve this objective, the present invention employs the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing high-purity natural capsaicin, the method comprising:

[0010] (1) Crush the dried chili peppers and then add the inoculum for microbial fermentation;

[0011] (2) After fermentation, hydrogen peroxide is added to the fermentation material and stirred. Then, the treated fermentation material is separated into solid and liquid, and the solids are retained.

[0012] (3) Mix the solid with water, then adjust the pH to alkaline and perform ultrasonic extraction;

[0013] (4) After extraction, solid-liquid separation is performed, the extract is collected, and then the extract is filtered using an ultrafiltration membrane and the permeate is collected.

[0014] (5) The permeate is concentrated and filtered using a nanofiltration membrane, the retentate is collected, the pH value is adjusted, and after standing, the solid and liquid are separated. The filter residue is the crude capsaicin.

[0015] (6) Add crude capsaicin to a buffer solution, mix and then hot filter, collect the filtrate, then let it stand and cool to crystallize, and collect the crystals after filtration.

[0016] (7) After recrystallizing the crystals, wash them and then vacuum dry them to obtain the high-purity natural capsaicin.

[0017] The process of this invention employs a reverse extraction method, that is, firstly, microbial fermentation is used to remove most of the impurities, and then capsaicin is extracted. As is known in the art, capsaicin is readily soluble in methanol, ethanol, acetone and alkaline aqueous solutions, but poorly soluble in pure water or acidic liquids, and poorly soluble in liquids with a pH less than 10. In this invention, Bacillus subtilis is used to ferment chili powder. The fermentation broth is slightly alkaline, which can dissolve a large number of water-soluble components in the chili powder, while capsaicin is retained in the chili powder. During the fermentation process, the bacteria decompose and metabolize large molecules such as pectin, cellulose, and protein into small water-soluble substances, further removing impurities and increasing the proportion of capsaicin in the chili powder, which is beneficial for subsequent extraction and purification. Hydrogen peroxide is used as a decolorizing agent for the material, and it can also oxidize and break the chains of some high molecular weight substances, improving the extraction rate of impurities. The concentration used is low, which is safe and does not damage the structure of capsaicin. The ultrafiltration membrane can retain and remove most of the alkaline-soluble impurities, while the nanofiltration membrane filters out small molecules and retains capsaicin, increasing the capsaicin content in the solution. Finally, crystallization is performed to avoid solvent residue, thereby obtaining high-purity capsaicin.

[0018] Preferably, the high-purity capsaicin refers to capsaicin with a purity of ≥99%.

[0019] Preferably, the pulverization in step (1) to 80-100 mesh can be, for example, 82 mesh, 85 mesh, 88 mesh, 90 mesh, 92 mesh, 95 mesh or 98 mesh.

[0020] Preferably, the bacterial strain in step (1) is an alkaline probiotic.

[0021] Preferably, the bacterial strain is Bacillus subtilis.

[0022] Preferably, the Bacillus subtilis strain platform number is Bio-52499.

[0023] Capsaicin is readily soluble in methanol, ethanol, acetone, and alkaline aqueous solutions, but particularly difficult to dissolve in liquids with a pH less than 10. In this invention, Bacillus subtilis is used to ferment the chili powder, resulting in a slightly alkaline fermentation broth that can dissolve a large amount of water-soluble components from the chili powder.

[0024] Preferably, the fermentation step (1) includes: mixing chili powder with water and adding it to a fermentation tank, then adding culture medium, sterilizing, and then inoculating with bacteria for fermentation.

[0025] Preferably, the mass ratio of chili powder to water is 1:(10-15), for example, it can be 1:11, 1:12, 1:13 or 1:14, etc.

[0026] Preferably, the amount of culture medium added is 0.7-1.5% of the total mass of chili powder and water, for example, it can be 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3% or 1.4%, etc.

[0027] Preferably, the amount of inoculated microorganisms is 3-5% of the total mass of the material to be fermented, for example, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5% or 4.8%, etc.

[0028] Preferably, the fermentation conditions are: pH 7-9 (e.g., 7.2, 7.5, 7.8, 8, 8.2, 8.5, or 8.8), temperature 25-35℃ (e.g., 26℃, 28℃, 30℃, 32℃, or 34℃), and time 12-15h (e.g., 12.5h, 13h, 13.5h, 14h, or 14.5h).

[0029] Preferably, the amount of hydrogen peroxide added in step (2) is 1-3% of the total amount of fermentation material, for example, it can be 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5% or 2.8%, etc.

[0030] Preferably, the stirring time is 30-60 minutes, for example, 35 minutes, 40 minutes, 45 minutes, 50 minutes or 55 minutes.

[0031] Preferably, the mass ratio of the solid to water in step (3) is 1:(5-10), for example, it can be 1:6, 1:7, 1:8 or 1:9, etc.

[0032] Preferably, the pH is adjusted to 12-13.

[0033] Preferably, the ultrasonic extraction time is 1-2 hours, for example, 1.1 hours, 1.3 hours, 1.5 hours, 1.7 hours or 1.9 hours.

[0034] Preferably, the molecular weight cutoff of the ultrafiltration membrane in step (4) is greater than 1 kDa, for example, it can be 1.1 kDa, 1.2 kDa, 1.3 kDa or 1.4 kDa.

[0035] Preferably, the nanofiltration membrane in step (5) has a molecular weight cutoff greater than 150 Da, for example, it can be 160 Da, 180 Da, 200 Da, 220 Da, 240 Da, 260 Da or 280 Da.

[0036] Capsaicin has a molecular weight of 305.4. A 1kDa ultrafiltration membrane can remove most alkali-soluble impurities with a molecular weight greater than 1kDa, while a 150Da nanofiltration membrane can filter out small molecules smaller than 150Da, retain capsaicin, and increase the content of capsaicin in the solution.

[0037] Preferably, the concentration in step (5) is 10-20% of the total mass of the permeate, for example, it can be 11%, 13%, 15%, 17% or 19%, etc.

[0038] Preferably, after collecting the retentate in step (5), the pH of the retentate is adjusted to 10.2-10.5.

[0039] Preferably, the temperature for standing in step (5) is 15-20℃ (for example, it can be 16℃, 17℃, 18℃ or 19℃, etc.), and the time is 5-6h.

[0040] Preferably, the buffer solution in step (6) is an aqueous solution of disodium hydrogen phosphate and sodium acetate;

[0041] Preferably, the mass concentration of the disodium hydrogen phosphate-sodium acetate aqueous solution is 3-5% (e.g., 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, or 4.8%), and the mass ratio of disodium hydrogen phosphate to sodium acetate is 1:(0.3-0.5), for example, 1:0.32, 1:0.35, 1:0.38, 1:0.4, 1:0.42, 1:0.45, or 1:0.48.

[0042] In this invention, a buffer solution composed of disodium hydrogen phosphate, sodium acetate, and water is used as the crystallization solvent. The system is alkaline and has excellent thermal solubility for capsaicin, meaning that the solubility increases with increasing temperature and decreases with decreasing temperature. This property is related to the solubility of sodium acetate itself.

[0043] Preferably, the mass ratio of crude capsaicin to buffer solution is 1:(5-10), for example, it can be 1:6, 1:7, 1:8 or 1:9, etc.

[0044] Preferably, the temperature of the heat filtration in step (6) is 75-85°C, for example, it can be 76°C, 78°C, 80°C, 82°C or 84°C.

[0045] Preferably, the temperature after cooling in step (6) is 15-20℃ (e.g., 16℃, 17℃, 18℃ or 19℃, etc.), and the standing time is 5-6h (e.g., 5.1h, 5.3h, 5.5h, 5.7h or 5.9h, etc.).

[0046] Preferably, the recrystallization step in step (7) is as follows:

[0047] Add the crystals to a buffer solution, mix, let stand and cool to recrystallize, filter and collect the recrystallized crystals;

[0048] The mixing temperature is 75-85℃, for example, 76℃, 78℃, 80℃, 82℃ or 84℃; the temperature is cooled to 15-20℃ (for example, 16℃, 17℃, 18℃ or 19℃), and the standing time is 5-6h (for example, 5.1h, 5.3h, 5.5h, 5.7h or 5.9h).

[0049] Preferably, the vacuum drying temperature in step (7) is 40-45°C.

[0050] Preferably, the vacuum drying temperature in step (6) is 40-45°C (for example, it can be 41°C, 42°C, 43°C or 44°C, etc.).

[0051] All the specific point values ​​within the above range can be selected, and will not be elaborated on here.

[0052] Secondly, the present invention provides a high-purity natural capsaicin, which is prepared by the method described in the first aspect.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The process of this invention employs a reverse extraction method, that is, firstly, microbial fermentation is used to remove most of the impurities, and then capsaicin is extracted. As is known in the art, capsaicin is readily soluble in methanol, ethanol, acetone and alkaline aqueous solutions, but poorly soluble in pure water or acidic liquids, and poorly soluble in liquids with a pH less than 10. In this invention, Bacillus subtilis is used to ferment chili powder. The fermentation broth is slightly alkaline, which can dissolve a large number of water-soluble components in the chili powder, while capsaicin is retained in the chili powder. During the fermentation process, the bacteria decompose and metabolize large molecules such as pectin, cellulose, and protein into small water-soluble substances, further removing impurities and increasing the proportion of capsaicin in the chili powder, which is beneficial for subsequent extraction and purification. Hydrogen peroxide is used as a decolorizing agent for the material, and it can also oxidize and break the chains of some high molecular weight substances, improving the extraction rate of impurities. The concentration used is low, which is safe and does not damage the structure of capsaicin. The ultrafiltration membrane can retain and remove most of the alkaline-soluble impurities, while the nanofiltration membrane filters out small molecules and retains capsaicin, increasing the capsaicin content in the solution. Finally, crystallization is performed to avoid solvent residue, thereby obtaining high-purity capsaicin. Attached Figure Description

[0055] Figure 1 The appearance of the capsaicin product prepared in Example 1;

[0056] Figure 2 The chromatogram is of capsaicin prepared in Example 1. Detailed Implementation

[0057] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0058] In Examples 1 and 2 of this invention, the bacterial strain used is Bacillus subtilis, strain platform number Bio-52499;

[0059] The bacterial strain used in Example 3 was Lactobacillus acidophilus, platform number Bio-53058;

[0060] The bacterial strain used in Example 4 is Thermophilic Bacterium, platform number ATCC33923.

[0061] Example 1

[0062] This embodiment provides a high-purity natural capsaicin, and the preparation method of the high-purity natural capsaicin is as follows:

[0063] (1) Take about 5 kg of dried ghost peppers, crush them (capsaicin content 5.1%), and pass them through an 80-mesh sieve to obtain chili powder;

[0064] (2) Weigh 0.6 kg of chili powder and 7 kg of pure water and add them to the fermentation tank. Then add 15.2 g of peptone, 38 g of glucose, 3.8 g each of potassium dihydrogen phosphate, sodium chloride, and magnesium chloride. Sterilize the above total material at 121°C for 15 min. After the material is cooled to below 40°C, inoculate it with 360 g of Bacillus subtilis culture medium. The fermentation conditions are: pH 8 ± 0.3, temperature 30°C, and time 12 hours.

[0065] (3) Add hydrogen peroxide to the fermentation material until the content reaches 3% of the total material, and stir for 45 minutes;

[0066] (4) Filter the above material until no more liquid seeps out, and obtain 480g of solid filter residue;

[0067] (5) The obtained filter residue was put into an ultrasonic extraction tank, 2.4 kg of pure water was added, and the pH was adjusted to 12.5 with sodium hydroxide. Ultrasonic extraction was performed for 1 hour.

[0068] (6) Filter the above material until no more liquid seeps out, and obtain 412g of filter residue;

[0069] (7) The filtrate is separated using a 1kDa ultrafiltration membrane. The retentate is discarded and the permeate is retained. The retentate is washed and filtered again with a strongly alkaline aqueous solution with a pH greater than 12. The amount of retentate used is approximately equal to that of the retentate.

[0070] (8) The permeate was separated and concentrated using a 200Da nanofiltration membrane. The permeate was discarded and the retentate was retained to obtain 492g.

[0071] (9) The pH of the retentate was adjusted to 10.2 with citric acid, and the mixture was allowed to stand at 15°C for 6 hours. The residue was collected by filtration to obtain 31.6g of crude capsaicin.

[0072] (10) Add 221.2g of buffer solvent (4% disodium hydrogen phosphate-sodium acetate aqueous solution, with a mass ratio of disodium hydrogen phosphate to sodium acetate of 1:0.4) to crude capsaicin, stir to dissolve at 80°C, and perform hot filtration. Discard the insoluble matter, let the filtrate stand at 15°C for 6 hours to cool and crystallize, and filter to collect 28.5g of crystals.

[0073] (11) Add 199.5g of buffer solvent (4% disodium hydrogen phosphate-sodium acetate aqueous solution, with a mass ratio of disodium hydrogen phosphate to sodium acetate of 1:0.4) to the obtained crystals, stir at 80°C to dissolve until saturated, let stand at 15°C for 6 hours to cool and crystallize, filter out the crystals, wash the obtained crystals with pure water 3 times, and vacuum dry at 45°C to obtain 26.5g of capsaicin product.

[0074] Example 2

[0075] This embodiment provides a high-purity natural capsaicin, and the preparation method of the high-purity natural capsaicin is as follows:

[0076] (1) Take about 5 kg of dried ghost peppers, crush them (capsaicin content 5.1%), and pass them through a 90-mesh sieve to obtain chili powder;

[0077] (2) Weigh 0.5 kg of chili powder and 6 kg of pure water and add them to the fermentation tank. Then add 13 g of peptone, 32.5 g of glucose, and 2.6 g each of potassium dihydrogen phosphate, sodium chloride, and magnesium chloride. Sterilize the above-mentioned total material at 121℃ for 15 min. After the material is cooled to below 40℃, inoculate it with 325 g of Bacillus subtilis culture medium. The fermentation conditions are: pH 8.5 ± 0.3, temperature 35℃, and time 14 hours.

[0078] (3) Add hydrogen peroxide to the fermentation material until the content reaches 2% of the total material, and stir for 45 minutes;

[0079] (4) Filter the above material until no more liquid seeps out, and obtain 495g of solid filter residue;

[0080] (5) The obtained filter residue was put into an ultrasonic extraction tank, 2.4 kg of pure water was added, and the pH was adjusted to 12 with sodium hydroxide. Ultrasonic extraction was performed for 1 hour.

[0081] (6) Filter the above material until no more liquid seeps out, and obtain 430g of filter residue;

[0082] (7) The filtrate is separated using an 800kDa ultrafiltration membrane. The retentate is discarded and the permeate is retained. The retentate is washed and filtered again with a strongly alkaline aqueous solution with a pH greater than 12. The amount of retentate used is approximately equal to that of the retentate.

[0083] (8) The permeate was separated and concentrated using a 250Da nanofiltration membrane. The permeate was discarded and the retentate was retained to obtain 451g.

[0084] (9) Adjust the pH of the retentate to 7 with citric acid, let it stand at 20°C for 5 hours, filter and collect the residue to obtain 28.2g of crude capsaicin;

[0085] (10) Add 253.8g of buffer solvent (5% disodium hydrogen phosphate-sodium acetate aqueous solution, with a mass ratio of disodium hydrogen phosphate to sodium acetate of 1:0.5) to crude capsaicin, stir to dissolve at 85°C, and perform hot filtration. Discard the insoluble matter, let the filtrate stand at 20°C for 5 hours to cool and crystallize, and filter to collect 26.5g of crystals.

[0086] (11) Add 238.5g of buffer solvent (4% disodium hydrogen phosphate-sodium acetate aqueous solution, with a mass ratio of disodium hydrogen phosphate to sodium acetate of 1:0.4) to the obtained crystals, stir at 75°C until saturated, let stand at 20°C for 6 hours to cool and crystallize, filter out the crystals, wash the obtained crystals with pure water 3 times, and vacuum dry at 40°C to obtain 24.2g of capsaicin product.

[0087] Example 3

[0088] This embodiment provides a high-purity natural capsaicin. The preparation method of the high-purity natural capsaicin differs from that of Example 1 only in that the strain used in step (2) is Lactobacillus acidophilus, the fermentation pH is adjusted to 5-7, and the inoculum amount remains unchanged. The remaining steps and process parameters are the same as those in Example 1, and 29.6g of capsaicin product is obtained.

[0089] Example 4

[0090] This embodiment provides a high-purity natural capsaicin. The preparation method of the high-purity natural capsaicin differs from that of Example 1 only in that the strain used in step (2) is thermophilic bacteria, and the inoculum amount remains unchanged. The remaining steps and process parameters are the same as those in Example 1, and 27.1g of capsaicin product is obtained.

[0091] Example 5

[0092] This embodiment provides a high-purity natural capsaicin. The preparation method of the high-purity natural capsaicin differs from that of the embodiment only in that the buffer solvent used for crystallization and recrystallization is a 4% disodium hydrogen phosphate aqueous solution, and the amount of buffer solution is kept constant. Other steps are the same, and 27.8g of capsaicin product is obtained.

[0093] Example 6

[0094] This embodiment provides a high-purity natural capsaicin. The preparation method of the high-purity natural capsaicin differs from that of the embodiment only in that the buffer solvent used for crystallization and recrystallization is a 4% sodium acetate aqueous solution, and the amount of buffer solution remains unchanged. The other steps are the same, and 21.4g of capsaicin product is obtained.

[0095] Comparative Example 1

[0096] This comparative example provides a high-purity natural capsaicin. The preparation method of the high-purity natural capsaicin differs from that of the example only in that steps (2), (3), and (4) are missing. The amount of chili powder weighed is the same, and steps (5) to (11) are the same as in Example 1, yielding 30.9g of capsaicin product.

[0097] Comparative Example 2

[0098] This comparative example provides a high-purity natural capsaicin. The preparation method of the high-purity natural capsaicin differs from that of the example only in that steps (7)-(9) are not included. The filtrate obtained in step (6) is adsorbed by porous resin D101 and eluted by 0.5% NaOH aqueous solution. The pH of the resulting eluent is adjusted to 10.2. Crystals are filtered out and then steps (10)-(11) are performed according to Example 1 to obtain 20.6g of capsaicin product.

[0099] Comparative Example 3

[0100] This comparative example provides a high-purity natural capsaicin. The preparation method of the high-purity natural capsaicin differs from that of the example only in that step (3) is missing. The other steps are the same, and 31.8g of capsaicin product is obtained.

[0101] Comparative Example 4

[0102] This comparative example provides a high-purity natural capsaicin. The preparation method of the high-purity natural capsaicin is as follows: take the same chili powder as in Example 1, put it into an ultrasonic extraction tank, add 2.4 kg of 95% ethanol, and extract with ultrasonic assistance for 1 hour; then perform steps (6), (7), and (8) in Example 1, recover the ethanol by rotary evaporation under reduced pressure, dissolve the extract in 0.5% NaOH aqueous solution, and then perform steps (9), (10), and (11) in Example 1 to finally obtain 36.8 g of capsaicin product.

[0103] Test Example 1

[0104] The capsaicin obtained in the examples and comparative examples was subjected to a full component test using LC-MS (liquid chromatography-mass spectrometry). The results are shown in Tables 1-9.

[0105] The chromatographic conditions are as follows:

[0106] I. Liquid Chromatography (LC) Parameters

[0107] 1. Chromatographic column: ACQUITY UPLC BEH C18 column (1.7μm, 2.1×100mm) or equivalent C18 column.

[0108] 2. Mobile phase: Phase A: 0.1% formic acid aqueous solution or pure water; Phase B: acetonitrile or 0.1% formic acid acetonitrile solution.

[0109] 3. Elution program (gradient mode): Initial: 10%-30% phase B; increase to 90%-95% phase B within 3-5 minutes; maintain for 1-2 minutes and then equilibrate to the initial ratio.

[0110] 4. Flow rate: 0.2–0.3 mL / min;

[0111] 5. Column temperature: 30~40℃;

[0112] 6. Injection volume: 1–5 μL.

[0113] II. Mass Spectrometry (MS) Parameters

[0114] 1. Ion source: Electrospray ionization source (ESI), positive ion mode (ESI+);

[0115] 2. Monitoring mode: Multiple response monitoring (MRM).

[0116] 3. Key mass spectrometry parameters:

[0117]

[0118]

[0119] Table 1: Capsaicin obtained in Example 1

[0120] Serial Number Ingredient name CAS Peak area % 1 Oleamide 000301-02-0 0.0018 2 stearamide 000124-26-5 0.0042 3 dihydrocapsaicin 028789-35-7 3.0546 4 Capsaicin 000404-86-4 85.2615 5 Dihydrocapsaicin 019408-84-5 9.1284 6 High in capsaicin I (capsaicin impurity 4) 058493-48-4 1.8679 7 High dihydrocapsaicin 1 020279-06-5 0.6816

[0121] The purity of the capsaicin = serial number (3+4+5+6+7) = 99.994%.

[0122] Table 2: Capsaicin obtained in Example 2

[0123]

[0124]

[0125] The purity of the capsaicin = serial number (3+4+5+6+7+8) = 99.465%.

[0126] Table 3: Capsaicin obtained in Example 3

[0127] Serial Number Ingredient name CAS Peak area % 1 benzaldehyde 000100-52-7 0.1167 2 Benzaldehyde diethyl acetal 000774-48-1 1.5089 3 Benzaldehyde propylene glycol acetal 002568-25-4 3.0015 4 N-Hexadecylacetamide 014303-96-9 1.2347 5 dihydrocapsaicin 028789-35-7 3.0210 6 Capsaicin 000404-86-4 80.1754 7 Dihydrocapsaicin 019408-84-5 8.5847 8 Capsaicin impurities - 0.1167 9 High in capsaicin I (capsaicin impurity 4) 058493-48-4 1.9567 10 High dihydrocapsaicin 1 020279-06-5 0.2837

[0128] The purity of capsaicin = serial number (5+6+7+8+9+10) = 94.1382.

[0129] Table 4: Capsaicin obtained in Example 4

[0130]

[0131]

[0132] The purity of capsaicin = serial number (5+6+7+8+9+10) = 96.6585%.

[0133] Table 5: Capsaicin obtained in Example 5

[0134] Serial Number Ingredient name CAS Peak area % 1 Benzaldehyde diethyl acetal 000774-48-1 1.0067 2 84 147 175 - 1.0194 3 nordihydrocapsaicin 028789-35-7 1.4501 4 Capsaicin 000404-86-4 80.6517 5 Dihydrocapsaicin 019408-84-5 13.3058 6 Capsaicin impurities - 0.7405 7 High in capsaicin I (capsaicin impurity 4) 058493-48-4 0.7695 8 High dihydrocapsaicin 1 020279-06-5 0.8755 9 Long-chain alkanes - 0.1808

[0135] The purity of capsaicin = serial number (3+4+5+6+7+8) = 97.9739%.

[0136] Table 6: Capsaicin obtained in Example 6

[0137]

[0138]

[0139] The capsaicin purity = serial number (5+6+7+8+9+10) = 98.1392%.

[0140] Table 7: Capsaicin obtained from Comparative Example 1

[0141]

[0142]

[0143] The purity of capsaicin = serial number (10+11+12+13+14+15) = 92.7899%.

[0144] Table 8: Capsaicin obtained from Comparative Example 2

[0145] Serial Number Ingredient name CAS Peak area % 1 d-Limonene 005989-27-5 0.0068 2 Leaf ester propionate 033467-74-2 1.0057 3 Benzaldehyde propylene glycol acetal 002568-25-4 1.0022 4 Triacetin 000102-76-1 1.0047 5 8-Methyl-6-nonenamide 1000293-20-9 0.0232 6 lauramide 001120-16-7 1.0135 7 Oleamide 000301-02-0 1.0068 8 stearamide 000124-26-5 0.0026 9 Capsaicin impurities - 0.1976 10 nordihydrocapsaicin 028789-35-7 3.4543 11 Capsaicin 000404-86-4 75.0080 12 Dihydrocapsaicin 019408-84-5 14.6048 13 High in capsaicin I (capsaicin impurity 4) 058493-48-4 0.7753 14 High dihydrocapsaicin 1 020279-06-5 0.8945

[0146] The purity of capsaicin = serial number (9+10+11+12+13+14) = 94.9345%.

[0147] Table 9: Capsaicin obtained from Comparative Example 3

[0148] Serial Number Ingredient name CAS Peak area % 1 benzaldehyde 000100-52-7 1.0079 2 Benzaldehyde diethyl acetal 000774-48-1 2.0600 3 nordihydrocapsaicin 028789-35-7 3.6891 4 Capsaicin 000404-86-4 75.8308 5 Dihydrocapsaicin 019408-84-5 15.1627 6 Capsaicin impurities - 0.6262 7 High in capsaicin I (capsaicin impurity 4) 058493-48-4 0.6520 8 High dihydrocapsaicin 1 020279-06-5 0.9713

[0149] The purity of capsaicin = serial number (3+4+5+6+7+8) = 96.9321%.

[0150] Table 10: Capsaicin obtained in Comparative Example 4

[0151]

[0152]

[0153] The purity of capsaicin = serial number (8+9+10+11+12+13) = 89.1132%.

[0154] As can be seen from the comparison between Comparative Example 4 and Example 1, the preparation method of the present invention can prepare high-purity capsaicin without the need for organic solvents.

[0155] As can be seen from Examples 3-4, when the fermentation strain is not the strain specified in this invention, the decomposition effect of macromolecular pectin, cellulose and protein in chili powder is poor, resulting in a lower purity of capsaicin.

[0156] As can be seen from Examples 5-6, only when the buffer solution is the disodium hydrogen phosphate-sodium acetate aqueous solution as defined in this invention can it have better thermal solubility for capsaicin, and thus the purity of the product after crystallization can be higher.

[0157] As can be seen from the comparison between Comparative Examples 1-3 and Example 1, when any one or more steps are missing in the method of the present invention, the purity of the capsaicin is poor.

[0158] The applicant declares that this invention illustrates a method for preparing high-purity natural capsaicin and its product through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0159] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0160] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing high-purity natural capsaicin, characterized in that, The preparation method includes: (1) Crush the dried chili peppers and then add the inoculum for microbial fermentation; (2) After fermentation, hydrogen peroxide is added to the fermentation material and stirred. Then, the treated fermentation material is separated into solid and liquid, and the solids are retained. (3) Mix the solid with water, then adjust the pH to alkaline and perform ultrasonic extraction; (4) After extraction, solid-liquid separation is performed, the extract is collected, and then the extract is filtered using an ultrafiltration membrane and the permeate is collected. (5) The permeate is concentrated and filtered using a nanofiltration membrane, the retentate is collected, the pH value is adjusted, and after standing, the solid and liquid are separated. The filter residue is the crude capsaicin. (6) Add crude capsaicin to a buffer solution, mix and then hot filter, collect the filtrate, then let it stand and cool to crystallize, and collect the crystals after filtration. (7) After recrystallizing the crystals, wash them and then vacuum dry them to obtain the high-purity natural capsaicin.

2. The method for preparing high-purity natural capsaicin as described in claim 1, characterized in that, The step (1) involves pulverizing the material to 80-100 mesh. Preferably, the bacterial strain described in step (1) is an alkaline probiotic; Preferably, the bacterial strain is Bacillus subtilis.

3. The method for preparing high-purity natural capsaicin as described in claim 1 or 2, characterized in that, The fermentation steps described in step (1) include: mixing chili powder with water and adding it to a fermentation tank, then adding culture medium, sterilizing, and then inoculating with bacteria for fermentation; Preferably, the mass ratio of chili powder to water is 1:(10-15); Preferably, the amount of culture medium added is 0.7-1.5% of the total mass of chili powder and water; Preferably, the amount of inoculum introduced is 3-5% of the total mass of the material to be fermented; Preferably, the fermentation conditions are: pH 7-9, temperature 25-35℃, and time 12-15h.

4. The method for preparing high-purity natural capsaicin according to any one of claims 1-3, characterized in that, The amount of hydrogen peroxide added in step (2) is 1-3% of the total amount of fermentation material; Preferably, the stirring time is 30-60 minutes.

5. The method for preparing high-purity natural capsaicin according to any one of claims 1-4, characterized in that, The mass ratio of the solid to water in step (3) is 1:(5-10). Preferably, step (3) involves adjusting the pH to 12-13; Preferably, the ultrasonic extraction time is 1-2 hours.

6. The method for preparing high-purity natural capsaicin according to any one of claims 1-5, characterized in that, The ultrafiltration membrane described in step (4) has a molecular weight cutoff greater than 1 kDa; Preferably, the nanofiltration membrane in step (5) has a molecular weight cutoff greater than 150 Da; Preferably, step (5) involves concentrating the solution to 10-20% of the total mass of the permeate.

7. The method for preparing high-purity natural capsaicin according to any one of claims 1-6, characterized in that, Step (5) involves adjusting the pH to 10.2-10.5; Preferably, the temperature for standing in step (5) is 15-20°C and the time is 5-6 hours.

8. The method for preparing high-purity natural capsaicin according to any one of claims 1-7, characterized in that, The buffer solution mentioned in step (6) is an aqueous solution of disodium hydrogen phosphate and sodium acetate; Preferably, the mass concentration of the disodium hydrogen phosphate-sodium acetate aqueous solution is 3-5%, and the mass ratio of disodium hydrogen phosphate to sodium acetate is 1:(0.3-0.5). Preferably, the mass ratio of crude capsaicin to buffer solution is 1:(5-10).

9. The method for preparing high-purity natural capsaicin according to any one of claims 1-8, characterized in that, The temperature of the heat filtration in step (6) is 75-85℃; Preferably, the temperature after cooling in step (6) is 15-20℃, and the standing time is 5-6 hours; Preferably, the recrystallization step in step (7) is as follows: Add the crystals to a buffer solution, mix, let stand and cool to recrystallize, filter and collect the recrystallized crystals; Preferably, the vacuum drying temperature in step (7) is 40-45°C.

10. A high-purity natural capsaicin, characterized in that, The high-purity natural capsaicin is prepared using the method described in any one of claims 1-9.

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