Preparation method and application of mogroside composition

Abstract

The invention relates to the technical field of biotechnology and food chemical industry, in particular to a preparation method and application of a mogroside composition.The preparation method comprises the steps that pulp and peel of fresh momordica grosvenori are taken, enzymolysis is conducted through biological enzyme, pulse extraction is conducted, composite anion and cation resin column chromatography is conducted, elution, low-temperature concentration, biological enzyme method enzymolysis is conducted again, macroporous resin chromatography is conducted, and the mogroside composition is obtained. And carrying out gradient elution with ethanol, stirring at low temperature to remove impurities, drying and the like. According to the preparation method, pulse extraction and composite ion exchange resin are adopted, bitter taste in pulp is effectively removed, pH is neutralized, meanwhile, the decolorization effect is considered, and the purity of mogroside V is improved; the mogroside V is converted into mogroside IV by adopting a biological enzyme method, the mogroside IV is subjected to macroporous resin column chromatography and low-temperature concentration, so that effective components and impurities are separated, the purity of the mogroside IV is improved, and the preparation process is simple, safe, environmentally friendly, good in adaptability and suitable for the whole process of factory mass production. And the prepared mogroside composition has pure taste and color at the same time.

Description

Technical Field

[0001] The invention relates to the technical fields of biotechnology and food chemical industry, and in particular to a preparation method and application of a mogroside composition. Background Art

[0002] Momordica grosvenori is a perennial herbaceous vine of the Cucurbitaceae family. It is a precious Chinese medicinal material for both medicine and food, and is known as the "magic fruit". The cucurbitane triterpenoid glycosides in Momordica grosvenori include simoneoside I, mogroside II, mogroside III, mogroside IIE, mogroside IV, mogroside V, etc. Among them, the content of mogroside IV is extremely low, and the extraction is difficult and costly.

[0003] At present, foreign medical workers have found that mogroside IV has a good effect in treating hepatitis C, and can be used alone or in combination with other drugs to achieve good therapeutic effects. Mogroside IV has little toxicity and side effects, high bioavailability, and is an emerging antiviral natural plant ingredient with broad market value.

[0004] The current bottleneck restricting the application of mogroside IV is the preparation of mogroside IV. The content of mogroside IV in the raw powder of monk fruit is less than 0.05% (the content of mogroside V is about 2%), so the preparation of mogroside IV is difficult and costly, which seriously restricts the development and application of mogroside IV.

[0005] At present, there are few studies on the enzymatic conversion of mogroside V into IV. This method provides a preparation method for converting mogroside IV using a biological enzyme method, which is of great significance for the application of monk fruit products. Summary of the invention

[0006] The object of the present invention is to provide a preparation method of mogroside IV by using a biological enzyme method, and the preparation method comprises the following steps:

[0007] (1) taking the pulp and skin of fresh Momordica grosvenori, adding water, heating and inactivating, cooling, then adding biological enzymes for enzymolysis, then inactivating, and cooling;

[0008] (2) performing pulse extraction twice, combining the filtrates from the two extractions to obtain a total filtrate;

[0009] (3) passing the total filtrate through a composite anion and cation resin column for chromatography, eluting with pure water, and combining and collecting the effluent and the water washing liquid;

[0010] (4) concentrating the combined effluent and the water washing liquid at low temperature, performing enzymatic hydrolysis by biological enzyme method, and after the enzymatic hydrolysis is complete, heating to inactivate, cooling, and filtering;

[0011] (5) passing through a macroporous resin for chromatography, first eluting with pure water, then eluting with an ethanol gradient, and collecting the ethanol eluate;

[0012] (6) stirring at low temperature to remove impurities, and drying to obtain a mogroside composition.

[0013] The step (1) specifically comprises: crushing fresh monk fruit, removing the seeds, taking the pulp and skin, crushing them at high speed with 0.5-1 times the amount of pure water, pouring them into a container, heating them to >80°C, maintaining them for 10-30 minutes, cooling them to 45-50°C, adding 1-5% of the raw material weight of biological enzymes, performing enzymolysis for 4-6 hours, and using 10% citric acid to maintain the pH of the solution at 5.0-5.5. After the enzymolysis is completed, heating the temperature to >99°C, maintaining them for 1-5 minutes, inactivating them, and rapidly cooling them to 20-25°C.

[0014] Preferably, the biological enzyme described in step (1) is one or more of cellulase and plant extract enzyme; the biological enzyme described in step (4) is P-fructofuranosidase and tannin hydrolase in a mass ratio of 4-15:1-10, or UDP-glucosyltransferase and sucrose synthase in a mass ratio of 2-10:2-5.

[0015] The step (2) is specifically as follows: pulse extraction is performed twice, in which pure water 3 times the weight of the raw material is added each time for the first time, the extraction time is 1 minute, a rest period of 2 minutes is taken between each extraction, the filtrate is separated, and the filtrates from the two extractions are combined to obtain the total filtrate.

[0016] Preferably, the electromagnetic field strength of the pulse extraction is 0-12Kv, the pulse width is 1-10μs, and the frequency is 1-300Hz.

[0017] The composite anion and cation resin column described in step (3) consists of an upper layer and a lower layer, the upper layer is anion resin, the lower layer is cationic resin, the mass ratio of anion resin: cationic resin is 2:1, and the total amount of composite anion and cation resin and the weight ratio of raw materials are 1:3-5; the amount of pure water used for pure water elution in step (3) is 0.5-1 times the weight of the resin.

[0018] The step (4) specifically comprises: concentrating the effluent and washing liquid collected in step (3) by vacuum concentration equipment to a solid content of 10-15%, adding 0.4-1.5‰ of P-fructofuranosidase and 0.1-1‰ of tannin hydrolase by weight of the raw material, or adding 0.2-1‰ of UDP-glucosyltransferase and 0.2-0.5‰ of sucrose synthase by weight of the raw material, performing enzymolysis at 35-38°C for 4-6 hours, maintaining stirring throughout the process, and after the enzymolysis is complete, heating to a temperature greater than 98°C and maintaining for 5-10 minutes for inactivation, and cooling the liquid to a temperature of 40-50°C and filtering through a 200-mesh filter cloth.

[0019] The step (5) is specifically as follows: taking the filtrate obtained in step (4) and passing it through macroporous resin chromatography, after completion, eluting with pure water until the eluted liquid is clear and transparent; then gradient eluting, first eluting with 1.5-2% HCl 20% ethanol, the elution volume is 1:2-4 for the resin dosage volume ratio, after elution, washing the resin with pure water until it is neutral, and then eluting with 53-57% ethanol, the elution volume is 1:2-4 for the resin dosage volume ratio, collecting the ethanol eluate, and stopping the collection until the mogroside V in the eluate is less than 0.2%.

[0020] Preferably, the weight ratio of the macroporous resin used in step (5) to the raw material is 1:4-5; the model is one of LX-83S, LX-200, and LX-20B, and the manufacturer is Xi'an Lanxiao Co., Ltd.

[0021] The step (6) is specifically as follows: the ethanol eluate collected in step (5) is concentrated to be alcohol-free and to 40-45 BRIX at a temperature below 60° C. using a low-temperature concentrator, sealed, stirred continuously at 4-6° C., placed for 12 hours, centrifuged to remove the precipitate, and the supernatant is concentrated to 50-52 BRIX, spray-dried, and a mogroside composition is obtained.

[0022] The mogroside composition contains mogroside IV>20%, mogroside III 0.1-6% and mogroside II E 0.2-5%.

[0023] The mogroside composition prepared by the preparation method is used in the preparation of food, beverage, dietary supplement, medicine and pet food.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The preparation method adopts pulse extraction to shorten the extraction time, and the extracted mogroside content is higher, while retaining all nutrients; through the composite ion exchange resin, the bitter taste in the pulp is effectively removed and the pH is neutralized, while taking into account the decolorization effect, the purity of mogroside V is improved, and the subsequent conversion is easier; then the bioenzymatic method is used to convert mogroside V into mogroside IV, and large-pore resin column chromatography and low-temperature concentration are performed to separate the effective ingredients and impurities, thereby improving the purity of mogroside IV.

[0026] (2) The pulp and peel of the whole fruit are selected for production, and the total sugar and other nutrients in the kernel account for a very small proportion. If the whole fruit is extracted, some lipid components will be brought in, which will affect the subsequent purification and product color, and will also affect the taste of the subsequent product when it is used.

[0027] (3) The preparation process is simple, safe, environmentally friendly, and has good adaptability, and is suitable for the entire large-scale production process in a factory. The prepared mogroside composition has a pure taste and color and can be widely used in various aspects, such as food, beverages, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : HPLC detection spectrum of the mogroside composition obtained in Example 1;

[0029] Figure 2 : HPLC detection spectrum of the mogroside composition obtained in Example 2;

[0030] Figure 3 : HPLC detection spectrum of the mogroside composition obtained in Example 3. DETAILED DESCRIPTION

[0031] The specific implementation of the present invention is further described below in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art.

[0032] Example 1

[0033] Take 2kg of fresh monk fruit, crush it, remove the seeds, take the pulp and skin, crush it with 1 times the amount of pure water at high speed, pour it into a container, heat it to 85℃, keep it for 10 minutes, cool it to 45℃, add 20g of plant extract enzyme, and enzymolyze it for 4 hours. Use 10% citric acid to adjust the solution to keep the pH at 5.5. After the enzymolysis is completed, inactivate it, heat it to 99.5℃ for 2 minutes, and quickly cool it to 25℃.

[0034] The time was controlled within 20 minutes, and pulse extraction was performed twice at an electromagnetic field strength of 10Kv, a pulse width of 8μs, and a frequency of 150Hz. Pure water 3 times the amount of the raw material was added each time. The extraction time was 1 minute each time. There was a 2-minute break in between each extraction. The filtrate was separated and the filtrates from the two extractions were combined to obtain the total filtrate.

[0035] The total filtrate was chromatographed on a 500 g composite column. After completion, it was eluted with 500 mL of pure water. The effluent and the water washing were collected and combined. The composite column consisted of an upper layer and a lower layer. The upper layer was an anion resin and the lower layer was a cationic resin. The ratio of the two was 2:1.

[0036] The combined effluent and washing liquid were concentrated to 10% solids by vacuum concentration equipment, 2g P-fructofuranosidase and 0.2g tannin hydrolase were added, and the enzyme was hydrolyzed at 36℃ for 6 hours by biological enzymatic method, and stirring was maintained throughout the process. After the enzymatic hydrolysis was complete, it was heated to 98.5℃ and kept for 8 minutes for inactivation. When the liquid was cooled to 40℃, it was filtered through a 200-mesh filter cloth.

[0037] Then, the product was chromatographed with LX-83S macroporous resin, with a dosage of 500 g. After the elution, it was eluted with pure water until the eluted liquid was clear and transparent. Then, it was eluted with gradient fractionation, first with 1.5% HCl 20% ethanol, with an elution volume of 1000 mL. After the elution, the resin effluent was washed with pure water until it was neutral, and then eluted with 53% ethanol, with an elution volume of 1500 mL. The ethanol eluate was collected until the mogroside V in the eluate was <0.2%, and the collection was stopped.

[0038] The ethanol eluate was concentrated to 45 BRIX by using a low-temperature concentrator (temperature < 60°C) to be free of alcohol, and then sealed and stirred continuously at 5°C for 12 hours. The precipitate was removed by centrifugation, and the supernatant was concentrated to 52 BRIX and spray-dried to obtain a mogroside composition.

[0039] HPLC detection showed that the mogroside composition contained 24.5% of mogroside IV, 4.5% of mogroside III and 4.8% of mogroside IIE.

[0040] Example 2

[0041] Take 2kg of fresh monk fruit, crush it, remove the seeds, take the pulp and skin, crush it with 0.5 times the amount of pure water at high speed, pour it into a container, heat it to 90℃, keep it for 20 minutes, cool it to 48℃, add 100g of cellulase, and hydrolyze it for 5 hours. Use 10% citric acid to adjust the solution to keep the pH at 5.0. After the hydrolysis is completed, inactivate it, heat it to 100℃ for 5 minutes, and quickly cool it to 22℃.

[0042] The time was controlled within 20 minutes, and pulse extraction was performed twice at an electromagnetic field strength of 5Kv, a pulse width of 5μs, and a frequency of 200Hz. Pure water 4 times the amount of the raw material was added each time. The extraction time was 1 minute each time. There was a 2-minute break in between each extraction. The filtrate was separated and the filtrates from the two extractions were combined to obtain the total filtrate.

[0043] The total filtrate was chromatographed on a 700 g composite column. After completion, it was eluted with 700 mL of pure water. The effluent and the water washing were collected and combined. The composite column consisted of an upper layer and a lower layer. The upper layer was an anion resin and the lower layer was a cationic resin. The ratio of the two was 2:1.

[0044] The combined effluent and washing liquid were concentrated to 15% solids by vacuum concentration equipment, 2g UDP-glucose transferase and 0.4g sucrose synthase were added, and the enzyme was hydrolyzed at 35°C for 5 hours, with stirring throughout the process. After the hydrolysis was complete, it was heated to 99°C and kept for 10 minutes for inactivation. When the liquid was cooled to 45°C, it was filtered through a 200-mesh filter cloth.

[0045] Then, the product was chromatographed with LX-83S macroporous resin, with a dosage of 400 g. After the elution, it was eluted with pure water until the eluted liquid was clear and transparent. Then, it was eluted with gradient fractionation, first with 2% HCl 20% ethanol, with an elution volume of 1600 mL. After the elution, the resin effluent was washed with pure water until it was neutral, and then eluted with 55% ethanol, with an elution volume of 1600 mL. The ethanol eluate was collected until the mogroside V in the eluate was <0.2%.

[0046] The ethanol eluate was concentrated to 42 BRIX by using a low-temperature concentrator (temperature < 60°C) to be free of alcohol, and then sealed and stirred continuously at 4°C for 12 hours. The precipitate was removed by centrifugation, and the supernatant was concentrated to 52 BRIX and spray-dried to obtain a mogroside composition.

[0047] HPLC detection showed that the mogroside composition contained 20.6% of mogroside IV, 3.2% of mogroside III and 3.8% of mogroside IIE.

[0048] Example 3

[0049] Take 2kg of fresh monk fruit, crush it, remove the seeds, take the pulp and skin, crush it with 1 times the amount of pure water at high speed, pour it into a container, heat it to 95℃, keep it for 10 minutes, cool it to 50℃, add 50g of plant extract enzyme, and enzymolyze it for 4 hours. Use 10% citric acid to adjust the solution to keep the pH at 5.2. After the enzymolysis is completed, inactivate it, heat it to 99.5℃ for 2 minutes, and quickly cool it to 22℃.

[0050] The time was controlled within 20 minutes, and pulse extraction was performed twice at an electromagnetic field strength of 10Kv, a pulse width of 8μs, and a frequency of 150Hz. Pure water 3 times the amount of the raw material was added each time. The extraction time was 1 minute each time. There was a 2-minute break in between each extraction. The filtrate was separated and the filtrates from the two extractions were combined to obtain the total filtrate.

[0051] The total filtrate was chromatographed on a 500 g composite column. After completion, it was eluted with 500 mL of pure water. The effluent and the water washing were collected and combined. The composite column consisted of an upper layer and a lower layer. The upper layer was an anion resin and the lower layer was a cationic resin. The ratio of the two was 2:1.

[0052] The combined effluent and washing liquid were concentrated to 12% solids by vacuum concentration equipment, 3g P-fructofuranosidase and 0.2g tannin hydrolase were added, and the enzyme was hydrolyzed at 36℃ for 6 hours by biological enzymatic method, and stirring was maintained throughout the process. After the enzymatic hydrolysis was complete, it was heated to 100℃ and kept for 5 minutes to inactivate. When the liquid was cooled to 50℃, it was filtered through a 200-mesh filter cloth.

[0053] Then, the product was chromatographed with LX-83S macroporous resin, with a dosage of 500 g. After the product was eluted, it was eluted with pure water until the effluent was clear and transparent. Then, it was eluted with gradient fractionation, first with 1.5% HCL 20% ethanol, with an elution volume of 1000 mL. After the elution, the resin effluent was washed with pure water until it was neutral, and then eluted with 57% ethanol, with an elution volume of 1500 mL. The ethanol eluate was collected until the content of mogroside V in the eluate was <0.2%.

[0054] The ethanol eluate is concentrated to 40 BRIX by using a low-temperature concentrator (temperature < 60°C) to be free of alcohol, and is sealed, kept stirring at 6°C, left for 12 hours, centrifuged to remove the precipitate, and the supernatant is concentrated to 50 BRIX, spray-dried, and a mogroside composition is obtained.

[0055] HPLC detection showed that the mogroside composition contained 20.9% of mogroside IV, 3.1% of mogroside III and 3.6% of mogroside IIE.

[0056] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for preparing a mogroside composition, characterized in that: The preparation method comprises the following steps: (1) taking the pulp and skin of fresh Momordica grosvenori, adding water, heating and inactivating, cooling, then adding biological enzymes for enzymolysis, then inactivating, and cooling; (2) performing pulse extraction twice, combining the filtrates from the two extractions to obtain a total filtrate; (3) passing the total filtrate through a composite anion and cation resin column for chromatography, eluting with pure water, and combining and collecting the effluent and the water washing liquid; (4) concentrating the combined effluent and the water washing liquid at low temperature, performing enzymatic hydrolysis by biological enzyme method, and after the enzymatic hydrolysis is complete, heating to inactivate, cooling, and filtering; (5) passing through a macroporous resin for chromatography, first eluting with pure water, then eluting with an ethanol gradient, and collecting the ethanol eluate; (6) stirring at low temperature to remove impurities, and drying to obtain a mogroside composition.

2. The preparation method according to claim 1, characterized in that: The step (1) specifically comprises: crushing fresh monk fruit, removing the seeds, taking the pulp and skin, crushing them at high speed with 0.5-1 times the amount of pure water, pouring them into a container, heating them to >80°C, maintaining them for 10-30 minutes, cooling them to 45-50°C, adding 1-5% of the raw material weight of biological enzymes, performing enzymolysis for 4-6 hours, and using 10% citric acid to maintain the pH of the solution at 5.0-5.

5. After the enzymolysis is completed, heating the temperature to >99°C, maintaining them for 1-5 minutes, inactivating them, and rapidly cooling them to 20-25°C.

3. The preparation method according to claim 2, characterized in that: The biological enzyme described in step (1) is one or more of cellulase and plant extract enzyme; the biological enzyme described in step (4) is P-fructofuranosidase and tannin hydrolase in a mass ratio of 4-15:1-10, or UDP-glucose transferase and sucrose synthase in a mass ratio of 2-10:2-5.

4. The preparation method according to claim 1, characterized in that: The step (2) is specifically as follows: pulse extraction is performed twice, in which pure water 3 times the weight of the raw material is added each time for the first time, the extraction time is 1 minute, a rest period of 2 minutes is taken between each extraction, the filtrate is separated, and the filtrates from the two extractions are combined to obtain a total filtrate; the electromagnetic field strength of the pulse extraction is 0-12Kv, the pulse width is 1-10μs, and the frequency is 1-300Hz.

5. The preparation method according to claim 1, characterized in that: The composite anion and cation resin column described in step (3) consists of an upper layer and a lower layer, the upper layer is anion resin, the lower layer is cationic resin, the mass ratio of anion resin: cationic resin is 2:1, and the total amount of composite anion and cation resin and the weight ratio of raw materials are 1:3-5; the amount of pure water used for pure water elution in step (3) is 0.5-1 times the weight of the resin.

6. The preparation method according to claim 1, characterized in that: The step (4) specifically comprises: concentrating the effluent and washing liquid collected in step (3) by vacuum concentration equipment to a solid content of 10-15%, adding 0.4-1.5‰ of P-fructofuranosidase and 0.1-1‰ of tannin hydrolase by weight of the raw material, or 0.2-1‰ of UDP-glucosyltransferase and 0.2-0.5‰ of sucrose synthase by weight of the raw material, performing enzymolysis at 35-38°C for 4-6 hours, maintaining stirring throughout the process, and after the enzymolysis is complete, heating to a temperature greater than 98°C and maintaining for 5-10 minutes to inactivate, and after the liquid is cooled to a temperature of 40-50°C, filtering through a 200-mesh filter cloth; the total amount of the P-fructofuranosidase and tannin hydrolase added is 1% of the weight of the raw material.

7. The preparation method according to claim 1, characterized in that: The step (5) is specifically as follows: taking the filtrate obtained in step (4) and passing it through macroporous resin chromatography, after completion, eluting with pure water until the eluted liquid is clear and transparent; then gradient elution, first eluting with 1.5-2% HCl20% ethanol, the elution amount is a resin dosage volume ratio of 1:2-4, after elution, washing the resin with pure water until it is neutral, and then eluting with 53-57% ethanol, the elution amount is a resin dosage volume ratio of 1:2-4, collecting the ethanol eluate until the eluate contains mogroside V<0.2% and the collection is terminated; the resin dosage and raw material weight ratio of the macroporous resin is 1:4-5; the model is one of LX-83S, LX-200, and LX-20B, and the manufacturer is Xi'an Lanxiao Co., Ltd.

8. The preparation method according to claim 1, characterized in that: The step (6) is specifically as follows: the ethanol eluate collected in step (5) is concentrated to be alcohol-free and to 40-45 BRIX at a temperature below 60° C. using a low-temperature concentrator, sealed, stirred continuously at 4-6° C., placed for 12 hours, centrifuged to remove the precipitate, and the supernatant is concentrated to 50-52 BRIX, spray-dried, and a mogroside composition is obtained.

9. The preparation method according to claim 1, characterized in that: The mogroside composition contains mogroside IV>20%, mogroside III 0.1-6% and mogroside IIE 0.2-5%.

10. Use of the mogroside composition prepared according to the preparation method according to any one of claims 1 to 9 in the preparation of food, beverage, dietary supplement, medicine and pet food.

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