A monk fruit residue extract containing multiple active ingredients and its preparation method

Abstract

This invention relates to a monk fruit residue extract containing multiple active ingredients, comprising the following components: 10ppm-20wt% squalene, 1ppm-10wt% monk fruit glycosides, 10ppm-20wt% linoleic acid, 10ppm-20wt% sitosterol, and excipients. This invention fully utilizes monk fruit residue, resulting in an extract rich in various economically valuable active ingredients, with high yields of each. The process is continuous, the equipment is simple, the operation is highly operable, and the production cost is low, making it suitable for industrial production and achieving comprehensive utilization of monk fruit residue resources.

Description

Technical Field

[0001] This invention relates to a monk fruit extract and its preparation method, specifically to a monk fruit residue extract containing multiple active ingredients and its preparation method. Background Technology

[0002] Monk fruit, the fruit of a perennial vine belonging to the Cucurbitaceae family, is produced in the mountainous areas of Guilin in Guangxi, Shaoyang, Huaihua, Yongzhou in Hunan, and southeastern Guizhou. It is a precious local specialty of my country and one of the first batch of materials approved by the state for both medicinal and edible uses. Monk fruit is highly nutritious, containing abundant vitamin C, as well as mogrosides, fructose, glucose, protein, and lipids. According to the *Chinese Materia Medica*, monk fruit is sweet and cool in nature, entering the lung and large intestine meridians. It has the effects of moistening the lungs and relieving cough, promoting body fluid production and quenching thirst, and is suitable for coughs due to lung heat or dryness, whooping cough, and thirst due to summer heat. It also has the effect of moistening the intestines and promoting bowel movements. Modern medical research has confirmed that the mogrosides contained in monk fruit are a natural sweetener 400 times sweeter than sucrose, but it does not produce calories, making it an ideal alternative beverage for diabetics, obese individuals, and others who should not consume sugar. Mogrosides are safe and non-toxic as food. The national mandatory standard GB2760, "Standard for the Use of Food Additives", stipulates that mogrosides can be used in various foods without limit.

[0003] Currently, the cultivation and processing of monk fruit in my country is booming. However, in practice, the industrial utilization of monk fruit mainly focuses on the extraction and separation of glycosides from the fruit. The residue after glycoside extraction (mainly monk fruit seeds and shells) is directly treated as waste and not utilized. Due to the increasing scale of monk fruit cultivation and processing, a large amount of monk fruit residue is directly discarded every year, causing environmental pollution and wasting resources.

[0004] With in-depth research on monk fruit resources, scholars have discovered that monk fruit residue contains a variety of active ingredients that are beneficial to human health, such as squalene, linoleic acid, sitosterol, monk fruit glycosides, flavonoids, and so on.

[0005] Existing research generally uses a single solvent to extract or classify 1-3 active ingredients from monk fruit residue. Due to the limited polarity of a single solvent, it generally has the drawback of not being able to leach or utilize other active ingredients.

[0006] CN106860503A discloses a method for extracting flavonoids from monk fruit residue. The method uses monk fruit residue as raw material and involves steps such as drying, crushing, adding water, treating in a magnetic field, ultrasonic extraction, centrifugation, filtration, concentration, and drying to obtain monk fruit flavonoids. In this method, water can only leach water-soluble flavonoids from the monk fruit residue; it cannot leach the fat-soluble components.

[0007] CN111393400A discloses a method for preparing squalene, vitamin E, and sterols from monk fruit seeds. The method uses monk fruit seeds as raw material and involves steps such as crushing, drying, organic solvent extraction, chemical treatment, and repeated crystallization to obtain squalene and other components. However, this method only extracts and utilizes the seeds, neglecting the extraction and utilization of the seed coat and pericarp. Furthermore, the method suffers from inconvenient material sourcing, as monk fruit seeds are difficult to separate, thus significantly limiting the availability of raw materials.

[0008] CN1670005A discloses plant-derived squalene and its preparation method, which uses the kernels or seeds of *Siraitia grosvenorii* (monk fruit) as raw material, and obtains high-quality *Siraitia grosvenorii* squalene through steps such as crushing, extraction with a fat-soluble organic solvent, concentration, silica gel column chromatography, and elution. This method only extracts squalene as an active ingredient and does not utilize other active ingredients in the kernels or seeds of *Siraitia grosvenorii*, thus failing to achieve comprehensive resource utilization.

[0009] CN111848708A discloses a method for separating squalene and other active ingredients from monk fruit extract residue. The method involves extracting the monk fruit extract residue with an alkaline low-carbon alcohol / aqueous solution. The crude extract is then extracted using a supercritical carbon dioxide extractor. The initial extract is diluted with a solvent, back-extracted with alkaline water, washed with water, and then diatomaceous earth is added. The mixture is filtered, concentrated, and squalene is obtained. The supercritical extract is then added to a silica gel column for elution. Eluent I is concentrated to obtain sitosterol. Eluent II is added to neutral alumina, filtered, concentrated, and dried to obtain mogroflavin. This patent obtains various active ingredients from monk fruit separately, but it is not a method suitable for industrial-scale production to simultaneously obtain a monk fruit extract rich in multiple beneficial active ingredients.

[0010] CN111393400A discloses a method for preparing squalene, vitamin E, and sterols from monk fruit seeds. The method involves crushing the monk fruit seeds, extracting them with an organic solvent to obtain an extract, recovering the organic solvent to obtain monk fruit oil, saponifying it, adding an alcohol / petroleum ether mixed solvent to the unsaponifiable matter, stirring at a constant temperature, filtering, recovering the petroleum ether, obtaining precipitate A and mother liquor A; adding precipitate A to a ketone / water mixed solvent, stirring at a constant temperature, filtering, recovering the ketone, obtaining precipitate B and mother liquor B; washing precipitate B with cold water and drying to obtain squalene; cooling mother liquor A to obtain precipitate C and mother liquor C; washing precipitate C with cold alcohol and drying to obtain sterols; adding mother liquor C to water, stirring at a constant temperature, recovering the alcohol, cooling to obtain precipitate D; separating precipitate D, washing with cold water and drying to obtain vitamin E. This patented method involves multiple crystallization steps to obtain various active ingredients, but it is not suitable for large-scale industrial production of monk fruit extract rich in various beneficial active ingredients.

[0011] Currently, there is an urgent need for a method to fully extract and utilize the various natural active ingredients in monk fruit residue, thereby achieving comprehensive resource utilization and turning waste into treasure. This is of great practical significance for promoting environmental protection and increasing the added value of the monk fruit industry. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a monk fruit residue extract containing squalene and other active ingredients, which can fully extract a variety of active ingredients, comprehensively utilize monk fruit extract residue resources, has a continuous process, simple equipment, strong operability, low production cost, is suitable for industrial production, and has a high yield of various active ingredients.

[0013] The technical solution adopted by this invention to solve its technical problem is as follows:

[0014] A monk fruit residue extract containing multiple active ingredients is characterized by comprising the following components: 10ppm-20wt% squalene, 1ppm-10wt% monk fruit glycosides, 10ppm-20wt% linoleic acid, 10ppm-20wt% sitosterol, and excipients.

[0015] Furthermore, in the monk fruit residue extract containing multiple active ingredients, the mass ratio of squalene, monk fruit glycosides, linoleic acid, and sitosterol is 200-300:1:500-700:350-450, preferably 200-220:1:560-600:380-410.

[0016] Furthermore, the monk fruit residue extract containing multiple active ingredients includes the following components: 100ppm-10wt% squalene, 10ppm-1wt% monk fruit glycosides, 100ppm-10wt% linoleic acid, 100ppm-10wt% sitosterol, and excipients.

[0017] Furthermore, the monk fruit residue extract containing multiple active ingredients includes the following components: 0.5-5 wt% squalene, 10 ppm-1 wt% monk fruit glycosides, 100 ppm-10 wt% linoleic acid, 100 ppm-10 wt% sitosterol, and excipients.

[0018] The mogrosides mentioned are one or more selected from mogroside I, mogroside II, mogroside III, mogroside IV, mogroside V, 11-oxo-mogroside V, mogroside VI, and symmenidine. The excipients are one or more selected from maltodextrin, corn starch, rice protein, fructooligosaccharides, β-cyclodextrin, microcrystalline cellulose, sugar alcohols, stearic acid, magnesium stearate, calcium stearate, talc, and silicon dioxide. Those skilled in the art should understand that the mogroside residue extract may also contain unavoidable impurities originating from the mogroside residue raw material itself.

[0019] This invention also provides a method for preparing the above-mentioned monk fruit residue extract containing multiple active ingredients, comprising the following steps:

[0020] (1) After crushing the monk fruit residue, add ligninase and cellulase to carry out enzymatic hydrolysis to obtain enzymatic hydrolysate.

[0021] (2) The enzymatically hydrolyzed raw material is put into the extraction tank, and the mixture of low-polarity organic solvent and ethyl acetate is stirred and heated for extraction; the extract is filtered, and the extracts are combined to obtain the extract; the extraction residue is kept in the extraction tank for later use;

[0022] (3) Pass the extract through a silica gel column, collect the effluent from the silica gel column, concentrate under reduced pressure until no solvent is present, and obtain a decolorized concentrate for later use;

[0023] (4) After the residual organic solvent in the extraction residue is washed away by steam distillation, an alcohol solution is added to the extraction tank and the extraction is continued by heating; the extracts are filtered and combined to obtain an alcohol extract.

[0024] (5) Add activated carbon to the alcohol extract, heat and stir, filter, and concentrate the activated carbon filtrate under reduced pressure until no alcohol is present to obtain the alcohol extract decolorized concentrate for later use.

[0025] (6) Mix the decolorized concentrate from step (5) and the alcohol-extracted decolorized concentrate from step (4), add (or do not add) excipients, and dry to obtain a monk fruit residue extract containing active ingredients such as squalene.

[0026] Preferably, in step (1), the monk fruit residue refers to the waste residue after extracting monk fruit glycosides from fresh or dried monk fruit with water, including but not limited to monk fruit seeds and monk fruit shells.

[0027] Preferably, in step (1), the amount of ligninase used is 0.5-1 wt% of the monk fruit residue, and the amount of cellulase used is 0.1-0.5 wt% of the monk fruit residue. More preferably, the amount of ligninase used is 0.60-0.75 wt% of the dry weight of the monk fruit residue, and the amount of cellulase used is 0.15-0.3 wt% of the dry weight of the monk fruit residue. The purpose of using ligninase and cellulase is to soften the structure of the monk fruit residue, which is beneficial for solvent penetration and helps to improve the leaching rate in subsequent extraction steps. If the amount of enzyme used is too small, the enzymatic hydrolysis will be insufficient, resulting in a low yield in subsequent extraction steps; if the amount of enzyme used is too large, it will lead to material waste.

[0028] Preferably, in step (1), the enzymatic hydrolysis temperature is room temperature and the enzymatic hydrolysis time is 3-6 hours.

[0029] Preferably, in step (1), the enzymatic hydrolysis involves first adding ligninase and hydrolyzing for 1-3 hours, then adding cellulase and hydrolyzing for 2-4 hours. The inventors unexpectedly discovered that by performing ligninase hydrolysis first followed by cellulase hydrolysis on the monk fruit residue, the extraction efficiency is higher, which is more conducive to the extraction of active ingredients such as squalene, thereby increasing the yield of these less abundant but economically and research-valued active ingredients.

[0030] Preferably, in step (2), the low-polarity organic mixed solvent is a mixture of petroleum ether, No. 6 solvent oil, No. 120 solvent oil, n-hexane, cyclohexane, and ethyl acetate, with a mixing ratio of low-polarity organic to ethyl acetate = 1-9:1, preferably 3-9:1 (V / V). The purpose of using the low-polarity organic mixed solvent for extraction is to leach oil-soluble active ingredients such as squalene, linoleic acid, and sitosterol from the monk fruit residue. The reason for adding ethyl acetate to the low-polarity organic mixed solvent is that ethyl acetate is miscible with water in trace amounts. Since monk fruit residue contains a certain amount of water, the addition of ethyl acetate is beneficial for the mixed solvent to penetrate into the raw material, thus fully completing the extraction process. If the proportion of ethyl acetate in the mixed solvent is too small, the penetration capacity of the mixed solvent will be low, resulting in incomplete extraction of oil-soluble active ingredients from the monk fruit residue. If the proportion of ethyl acetate in the mixed solvent is too large, on the one hand, it will increase the production cost, and on the other hand, it will significantly change the polarity of the mixed solvent, which is not conducive to the leaching of oil-soluble active ingredients.

[0031] Preferably, in step (2), the total amount of the low-polarity organic mixed solvent is 10-30 times (L / kg) of the mass of the monk fruit residue, and the reflux extraction is performed 1-5 times for 1-2 hours per extraction. More preferably, the amount of organic mixed solvent used in each reflux extraction gradually decreases, reducing to 70-90% of the volume of the solvent used in the previous reflux extraction.

[0032] Preferably, in step (3), the amount of silica gel used is 0.1-0.5 times the mass of the monk fruit residue (L / kg), the height-to-diameter ratio of the silica gel column is 4:1-8:1, and the flow rate of the material through the silica gel column is 2-10 BV / hour. The purpose of using silica gel is to remove impurities such as oil-soluble pigments from the low-polarity organic mixed solvent extract. If the amount of silica gel used is too small, the height-to-diameter ratio of the silica gel column is too small, or the flow rate is too fast, the above purpose will not be fully achieved; if the amount of silica gel used is too large, the height-to-diameter ratio of the silica gel column is too large, or the flow rate is too slow, it will result in a waste of materials and energy.

[0033] Preferably, in step (4), the alcohol solution is an aqueous solution of methanol and / or ethanol, with a volume fraction of 30%-100% (when the volume fraction of alcohol is 100%, alcohol is added without water). The total volume of the alcohol solution is 10-30 times (L / kg) of the dry weight of the monk fruit residue. The reflux extraction is performed 1-5 times, with a reflux extraction time of 1-2 hours per extraction. During each reflux extraction, the amount of organic mixed solvent used gradually decreases, reducing to 70-99% of the volume of the solvent used in the previous reflux extraction. The purpose of using methanol or ethanol for extraction is to leach out alcohol-soluble active ingredients such as monk fruit glycosides and flavonoids from the monk fruit residue.

[0034] Preferably, in step (5), the amount of activated carbon used is 0.2-0.5 wt% of the monk fruit residue, the heating temperature is 50-80℃, and the stirring time is 1-5 hours. The purpose of using activated carbon is to remove impurities such as alcohol-soluble pigments from the methanol or ethanol extract. If the amount of activated carbon used is too small, the heating temperature is too low, or the stirring time is too short, the above purpose will not be fully achieved; if the amount of activated carbon used is too large, the heating temperature is too high, or the stirring time is too long, it will result in a waste of materials and energy.

[0035] Preferably, in step (6), the excipients are one or more of the following: maltodextrin, corn starch, rice protein, fructooligosaccharides, β-cyclodextrin, microcrystalline cellulose, sugar alcohol, stearic acid, magnesium stearate, calcium stearate, talc, and silicon dioxide. The amount of the excipients is not particularly limited, as long as the content of each active ingredient in the final product is within the aforementioned range. The function of the excipients is to facilitate drying. Since the monk fruit residue extract contains both water-soluble and fat-soluble components, direct drying easily leads to moisture absorption. Adding excipients facilitates use and storage. The drying process is not particularly limited, as long as the moisture content of the resulting product extract is below 8 wt%, preferably below 5 wt%.

[0036] This invention relates to a monk fruit residue extract containing various active ingredients beneficial to the human body. It can be taken internally or applied externally and used in food, pharmaceuticals, health products, and cosmetics. It exhibits unique therapeutic effects, particularly in skincare and the prevention and treatment of arthritis.

[0037] In particular, based on the needs of the application field, functional ingredients such as squalene, mogrosides, linoleic acid, and sitosterol may be added to the monk fruit residue extract provided in this application, in addition to the ingredients provided in this application. These are also within the scope of protection of this application.

[0038] In this invention, oil extraction is performed first, followed by alcohol extraction, rather than alcohol extraction first, then oil extraction. This is because squalene is readily soluble in low-polarity organic solvents, has poor thermal stability, and is easily oxidized; therefore, it should be extracted and preserved preferentially. Even if high concentrations of methanol or ethanol partially leach squalene, the extraction is insufficient. Furthermore, the temperature of the methanol or ethanol extract rises significantly during the final stage of concentration (due to the increased proportion of water and higher boiling point), which exacerbates squalene degradation, resulting in a lower overall squalene yield.

[0039] The beneficial effects of this invention are as follows:

[0040] (1) This invention provides a method for classifying and fully extracting various active ingredients from monk fruit residue, and the yield of various active ingredients is relatively high;

[0041] (2) The process is continuous, the equipment is simple, the operation is strong, the production cost is low, and it is suitable for industrial production.

[0042] (3) After "oil extraction" and "alcohol extraction", the extract residue has been leached out, removing compounds such as oils that affect the shelf life and quality of the dry extract residue. Therefore, the alcohol extract residue in step (4) is first steam distilled to recover the solvent and then dried to constant weight to obtain dried monk fruit residue by-product. This by-product is rich in cellulose and hemicellulose, can be stored for a long time, and can be widely used in feed additives, veterinary drugs, fertilizers, and papermaking industries. In this way, the comprehensive utilization of monk fruit residue resources can be truly realized. Detailed Implementation

[0043] The present invention will be further described below with reference to the embodiments, which should not be construed as limiting the scope of protection of the present invention.

[0044] The monk fruit residue used in this embodiment of the invention comes from the monk fruit extraction production workshop of Hunan Huacheng Biological Resources Co., Ltd., and is the remaining seeds and shells (moisture content 56.1%) after hot water extraction of glycosides from fresh monk fruit. The residue contains 0.21% squalene, 0.001% monk fruit terpene glycosides, 0.59% linoleic acid, and 0.41% sitosterol. Unless otherwise specified, the excipients or chemical reagents used in this embodiment of the invention are obtained through conventional commercial channels.

[0045] In this embodiment of the invention, the detection methods for squalene, mogrosides, linoleic acid, and sitosterol are all high performance liquid chromatography (HPLC) external standard method.

[0046] Example 1

[0047] This embodiment includes the following steps:

[0048] (1) Enzymatic hydrolysis: Take 200 kg of monk fruit residue, crush it, add 1.2 kg of ligninase, and enzymatically hydrolyze at room temperature for 2 h. Then add 0.6 kg of cellulase and enzymatically hydrolyze at room temperature for 4 h to obtain the enzymatically hydrolyzed raw material.

[0049] (2) Oil extraction: The enzymatically hydrolyzed raw material was added to an extraction tank and extracted by stirring and reflux with a low-polarity organic mixed solvent (petroleum ether: ethyl acetate = 9:1). The volume of the mixed solvent used in the first extraction was 1000L, and the extraction time was 2 hours; the volume of the mixed solvent used in the second extraction was 900L, and the extraction time was 2 hours; the volume of the mixed solvent used in the third extraction was 800L, and the extraction time was 1 hour. After filtration, the extracts were combined to obtain the oil extract; the oil extraction residue was retained in the extraction tank for later use.

[0050] (3) Decolorization and concentration of oil extract: The oil extract is passed through a silica gel column (the amount of silica gel is 50L, the height-to-diameter ratio of the silica gel column is 6:1, and the flow rate of the material through the silica gel column is 6BV / hour). The effluent from the silica gel column is collected and concentrated under reduced pressure until there is no solvent, so as to obtain the decolorized concentrate of the oil extract for later use.

[0051] (4) Alcohol extraction: After rinsing off the residual organic solvent in the oil extraction residue with steam, add 85% ethanol (by volume) to the extraction tank and continue heating under reflux for extraction. The first extraction uses 900 L of alcohol and takes 2 hours; the second extraction uses 800 L of alcohol and takes 2 hours; the third extraction uses 700 L of alcohol and takes 1 hour. Filter and combine the extracts to obtain the alcohol extract.

[0052] (5) Decolorization and concentration of alcohol extract: Add 1 kg of activated carbon to the alcohol extract, heat to 70°C, keep warm and stir for 3 hours, filter, and concentrate the activated carbon filtrate under reduced pressure until no alcohol is present to obtain alcohol extract decolorized concentrate for later use.

[0053] (6) Mixing, blending and drying: The oil extract decolorizing concentrate and the alcohol extract decolorizing concentrate were mixed, 15 kg of maltodextrin was added as an excipient, and spray-dried to obtain 28.36 kg of monk fruit residue extract containing active ingredients such as squalene.

[0054] According to the high performance liquid chromatography (HPLC) external standard method, the content of squalene in the monk fruit residue extract obtained in the examples of this invention was 1.44%, and the yield of squalene was 99.57%; the content of mogrosides was 0.0069%, and the yield of mogrosides was 99.58%; the content of linoleic acid was 3.99%, and the yield of linoleic acid was 99.76%; and the content of sitosterol was 2.69%, and the yield of sitosterol was 98.70%.

[0055] Example 2

[0056] This embodiment includes the following steps:

[0057] (1) Enzymatic hydrolysis: Take 200 kg of monk fruit residue, crush it, add 1.5 kg of ligninase, and enzymatically hydrolyze at room temperature for 2 h. Then add 0.3 kg of cellulase and continue enzymatic hydrolysis at room temperature for 4 h to obtain the enzymatically hydrolyzed raw material.

[0058] (2) Oil extraction: The enzymatically hydrolyzed raw material was added to an extraction tank and extracted by stirring and reflux with a low-polarity organic mixed solvent (6# solvent oil: ethyl acetate = 8.5: 1.5). The volume of the mixed solvent used for the first extraction was 1000L, and the extraction time was 2 hours; the volume of the mixed solvent used for the second extraction was 900L, and the extraction time was 1.5 hours; the volume of the mixed solvent used for the third extraction was 700L, and the extraction time was 1 hour. After filtration, the extracts were combined to obtain the oil extract; the oil extraction residue was retained in the extraction tank for later use.

[0059] (3) Decolorization and concentration of oil extract: The oil extract was passed through a silica gel column (the amount of silica gel used was 40L, the height-to-diameter ratio of the silica gel column was 7:1, and the flow rate of the material through the silica gel column was 5BV / hour). The effluent from the silica gel column was collected and concentrated under reduced pressure until there was no solvent, so as to obtain the decolorized concentrate of the oil extract for later use.

[0060] (4) Alcohol extraction: After rinsing off the residual organic solvent in the oil extraction residue with steam, add 75% methanol (by volume) to the extraction tank and continue heating under reflux for extraction. The first extraction uses 900 L of alcohol and takes 2 hours; the second extraction uses 900 L of alcohol and takes 1.5 hours; the third extraction uses 700 L of alcohol and takes 1 hour. Filter and combine the extracts to obtain the alcohol extract.

[0061] (5) Decolorization and concentration of alcohol extract: Add 0.8 kg of activated carbon to the alcohol extract, heat to 60°C, keep warm and stir for 4 hours, filter, and concentrate the activated carbon filtrate under reduced pressure until no alcohol is present to obtain alcohol extract decolorized concentrate for later use;

[0062] (6) Mixing, blending and drying: The oil extract decolorizing concentrate and the alcohol extract decolorizing concentrate were mixed, 20 kg of corn starch as an auxiliary material was added, and spray drying was performed to obtain 32.98 kg of monk fruit residue extract containing active ingredients such as squalene.

[0063] According to the high performance liquid chromatography (HPLC) external standard method, the content of squalene in the monk fruit residue extract obtained in the examples of this invention was 1.24%, and the yield of squalene was 97.37%; the content of mogrosides was 0.006%, and the yield of mogrosides was 98.94%; the content of linoleic acid was 3.47%, and the yield of linoleic acid was 96.98%; and the content of sitosterol was 2.33%, and the yield of sitosterol was 93.71%.

[0064] Example 3

[0065] This embodiment includes the following steps:

[0066] (1) Enzymatic hydrolysis: Take 200 kg of monk fruit residue, add compound enzyme (1.2 kg of ligninase and 0.6 kg of cellulase), mix evenly, and place at room temperature for 6 hours of enzymatic hydrolysis to obtain enzymatic hydrolysate.

[0067] (2) Oil extraction: The enzymatically hydrolyzed raw material was added to an extraction tank and extracted by stirring and reflux with a low-polarity organic mixed solvent (120# solvent oil: ethyl acetate = 8:2). The volume of the mixed solvent used in the first extraction was 900L, and the extraction time was 2 hours; the volume of the mixed solvent used in the second extraction was 900L, and the extraction time was 2 hours; the volume of the mixed solvent used in the third extraction was 800L, and the extraction time was 1 hour. After filtration, the extracts were combined to obtain the oil extract; the oil extraction residue was retained in the extraction tank for later use.

[0068] (3) Decolorization and concentration of oil extract: The oil extract was passed through a silica gel column (the amount of silica gel used was 35L, the height-to-diameter ratio of the silica gel column was 8:1, and the flow rate of the material through the silica gel column was 4BV / hour). The effluent from the silica gel column was collected and concentrated under reduced pressure until no solvent was found to obtain the decolorized concentrate of the oil extract, which was then set aside for use.

[0069] (4) Alcohol extraction: After rinsing off the residual organic solvent in the oil extraction residue with steam, add 80% ethanol (by volume) to the extraction tank and continue heating under reflux for extraction. The first extraction uses 900 L of alcohol for 2 hours; the second extraction uses 900 L of alcohol for 1 hour; and the third extraction uses 600 L of alcohol for 1 hour. Filter and combine the extracts to obtain the alcohol extract.

[0070] (5) Decolorization and concentration of alcohol extract: Add 0.5 kg of activated carbon to the alcohol extract, heat to 75°C, keep warm and stir for 5 hours, filter, and concentrate the activated carbon filtrate under reduced pressure until no alcohol is present to obtain alcohol extract decolorized concentrate for later use;

[0071] (6) Mixing, blending and drying: The oil extract decolorizing concentrate and the alcohol extract decolorizing concentrate were mixed, 30 kg of excipient β-cyclodextrin was added, and spray drying was performed to obtain 43.03 kg of monk fruit residue extract containing active ingredients such as squalene.

[0072] According to the high performance liquid chromatography (HPLC) external standard method, the content of squalene in the monk fruit residue extract obtained in the examples of this invention was 0.92%, and the yield of squalene was 94.26%; the content of mogrosides was 0.0045%, and the yield of mogrosides was 96.82%; the content of linoleic acid was 2.58%, and the yield of linoleic acid was 94.08%; and the content of sitosterol was 1.82%, and the yield of sitosterol was 95.51%.

[0073] Example 4

[0074] Other conditions and operations are the same as in Example 1, except that step (1) is replaced with: enzymatic hydrolysis: Take 200 kg of monk fruit residue, crush it, add 0.6 kg of cellulase, and hydrolyze at room temperature for 2 hours. Then add 1.2 kg of ligninase and hydrolyze at room temperature for 4 hours to obtain the hydrolyzed raw material. That is, the enzymatic hydrolysis order is changed. Finally, 28.17 kg of monk fruit residue extract is obtained.

[0075] According to HPLC testing and calculation, the content of squalene in the monk fruit residue extract obtained in this example was 1.38%, with a squalene yield of 92.56%; the content of mogrosides was 0.0069%, with a mogroside yield of 97.19%; the content of linoleic acid was 3.94%, with a linoleic acid yield of 94.06%; and the content of sitosterol was 2.67%, with a sitosterol yield of 91.72%.

[0076] Example 5

[0077] Other conditions and operations were the same as in Example 1, except that no excipients were added in step (6), and the concentrated mixture was directly dried by microwave. A total of 13.56 kg of monk fruit residue extract was finally obtained.

[0078] According to the high performance liquid chromatography (HPLC) external standard method, the content of squalene in the monk fruit residue extract obtained in the examples of this invention was 3.02%, and the yield of squalene was 97.50%; the content of mogrosides was 0.0145%, and the yield of mogrosides was 98.31%; the content of linoleic acid was 8.42%, and the yield of linoleic acid was 96.76%; and the content of sitosterol was 5.73%, and the yield of sitosterol was 94.75%.

[0079] Comparative Example 1

[0080] The other conditions and operations were the same as in Example 3, except that in step (S1), 1.8 kg of compound enzyme was replaced with 1.8 kg of cellulase. Finally, 42.88 kg of monk fruit residue extract was obtained.

[0081] According to the high performance liquid chromatography (HPLC) external standard method, the content of squalene in the monk fruit residue extract obtained in the examples of this invention was 0.87%, and the yield of squalene was 88.82%; the content of mogrosides was 0.0044%, and the yield of mogrosides was 94.34%; the content of linoleic acid was 2.45%, and the yield of linoleic acid was 89.03%; and the content of sitosterol was 1.70%, and the yield of sitosterol was 88.90%.

[0082] Comparative Example 2

[0083] The other conditions and operations were the same as in Example 3, except that in step (S1), 1.8 kg of compound enzyme was replaced with 1.8 kg of ligninase. Finally, 42.95 kg of monk fruit residue extract was obtained.

[0084] According to the high performance liquid chromatography (HPLC) external standard method, the content of squalene in the monk fruit residue extract obtained in the examples of this invention was 0.86%, and the yield of squalene was 87.94%; the content of mogrosides was 0.0045%, and the yield of mogrosides was 96.64%; the content of linoleic acid was 2.39%, and the yield of linoleic acid was 86.99%; and the content of sitosterol was 1.67%, and the yield of sitosterol was 87.47%.

[0085] Comparative Example 3

[0086] The other conditions and procedures were the same as in Example 1, except that in step (S2), the low-polarity organic mixed solvent was replaced with an equal volume of petroleum ether. That is, ethyl acetate was not added. The final yield was 28.15 kg of monk fruit residue extract.

[0087] According to the high performance liquid chromatography (HPLC) external standard method, the content of squalene in the monk fruit residue extract obtained in the examples of this invention was 1.22%, and the yield of squalene was 81.77%; the content of mogrosides was 0.0066%, and the yield of mogrosides was 92.90%; the content of linoleic acid was 3.74%, and the yield of linoleic acid was 89.22%; and the content of sitosterol was 2.58%, and the yield of sitosterol was 88.57%.

Claims

1. A method for preparing a monk fruit residue extract containing multiple active ingredients, characterized in that, Includes the following steps: (1) After crushing the monk fruit residue, add ligninase and cellulase for enzymatic hydrolysis to obtain enzymatic hydrolysate; the amount of ligninase is 0.60-0.75 wt% of the dry weight of monk fruit residue, and the amount of cellulase is 0.15-0.3 wt% of the dry weight of monk fruit residue; the enzymatic hydrolysis is carried out by first adding ligninase and hydrolyzing for 1-3 hours, and then adding cellulase and hydrolyzing for 2-4 hours. (2) The enzymatically hydrolyzed raw material is put into the extraction tank, and the mixture of low polarity organic solvent and ethyl acetate is stirred and heated for extraction; the extract is filtered, and the extracts are combined to obtain the extract; the extraction residue is kept in the extraction tank for later use; the volume ratio of low polarity organic solvent to ethyl acetate is 3-9:1; (3) Pass the extract through a silica gel column, collect the effluent from the silica gel column, concentrate under reduced pressure until no solvent is present, and obtain a decolorized concentrate for later use; (4) After the residual organic solvent in the extraction residue is washed away by steam distillation, an alcohol solution is added to the extraction tank and the extraction is continued by heating; after filtration, the extracts are combined to obtain the alcohol extract; (5) Add activated carbon to the alcohol extract, heat and stir, filter, and concentrate the activated carbon filtrate under reduced pressure until no alcohol is present to obtain the alcohol extract decolorized concentrate for later use; (6) Mix the decolorized concentrate from step (3) and the alcohol-extracted decolorized concentrate from step (5), add excipients, and dry to obtain a monk fruit residue extract containing active ingredients such as squalene. The monk fruit residue extract containing multiple active ingredients includes the following components: 100ppm-10wt% squalene, 10ppm-1wt% monk fruit glycosides, 100ppm-10wt% linoleic acid, 100ppm-10wt% sitosterol, and excipients; the mass ratio of squalene, monk fruit glycosides, linoleic acid, and sitosterol is 200-300:1:500-700:350-450.

2. The preparation method according to claim 1, characterized in that, The mass ratio of squalene, mogrosides, linoleic acid and sitosterol is 200-220:1:560-600:380-410.

3. The preparation method according to claim 1, characterized in that, It includes the following ingredients: 10.5-5wt% squalene, 10ppm-1wt% mogrosides, 100ppm-10wt% linoleic acid, 100ppm-10wt% sitosterol, and excipients.

4. The preparation method according to claim 1, characterized in that, The mogrosides mentioned are one or more of mogroside I, mogroside II, mogroside III, mogroside IV, mogroside V, 11-oxo-mogroside V, mogroside VI, and symmonoside; the excipients are selected from one or more of maltodextrin, corn starch, rice protein, fructooligosaccharides, β-cyclodextrin, microcrystalline cellulose, sugar alcohols, stearic acid, magnesium stearate, calcium stearate, talc, and silicon dioxide.

5. The preparation method according to claim 1, characterized in that, In step (1), the monk fruit residue refers to the waste residue after extracting monk fruit glycosides from fresh or dried monk fruit with water; the enzymatic hydrolysis temperature is room temperature, and the enzymatic hydrolysis time is 3-6 hours.

6. The preparation method according to claim 1, characterized in that, In step (2), the low-polarity organic mixed solvent is a mixed solvent of petroleum ether, No. 6 solvent oil, No. 120 solvent oil, n-hexane, cyclohexane and ethyl acetate.

7. The preparation method according to claim 6, characterized in that, In step (2), the total amount of the low-polarity organic mixed solvent is 10-30 times the mass of the monk fruit residue, L / kg, the number of reflux extractions is 1-5 times, and the reflux extraction time is 1-2 hours / time.

8. The preparation method according to claim 6, characterized in that, Each time a reflux extraction is performed, the amount of organic mixed solvent used is gradually reduced to 70-90% of the volume of the solvent used in the previous reflux extraction.

9. The preparation method according to claim 1, characterized in that, In step (3), the amount of silica gel used is 0.1-0.5 times the mass of the monk fruit residue, L / kg, the height-to-diameter ratio of the silica gel column is 4:1-8:1, and the flow rate of the material through the silica gel column is 2-10 BV / hour. In step (4), the alcohol solution is an aqueous solution of methanol and / or ethanol; the total amount of alcohol solution used is 10-30 times the dry weight of the monk fruit residue, L / kg, the number of reflux extractions is 1-5 times, and the reflux extraction time is 1-2 hours / time. In step (5), the amount of activated carbon used is 0.2-0.5 wt% of the monk fruit residue, the heating temperature is 50-80℃, and the stirring time is 1-5 hours; In step (6), the excipients are one or more of the following: maltodextrin, corn starch, rice protein, fructooligosaccharides, β-cyclodextrin, microcrystalline cellulose, sugar alcohol, stearic acid, magnesium stearate, calcium stearate, talc, and silicon dioxide.