Extraction method of momordica grosvenori and application thereof

Abstract

This invention discloses an extraction method for monk fruit and its application, belonging to the field of food processing technology. Addressing the technical problem of the easy enzymatic degradation of mogroside V during the hot air drying process of monk fruit, this invention provides an extraction method: Fresh monk fruit is harvested and ripened at 20-30℃; the ripened monk fruit is pretreated in a processing solution; then hot air drying is performed; finally, monk fruit extract is obtained through post-processing. This invention effectively inhibits the activity of β-glucosidase through combined pretreatment with citric acid and ascorbic acid, achieving a mogroside V retention rate of over 90%, while controlling the total content of bitter glycosides below 0.06 wt%, and significantly improving the product color and ascorbic acid retention rate. This method is simple to operate, low in cost, and the obtained extract can be used as a low-calorie sweetener raw material, widely applied in the food and health food fields.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a method for extracting monk fruit and its application. Background Technology

[0002] Monk fruit (Siraitia grosvenorii), commonly known as monk fruit, is a plant of the Cucurbitaceae family, native to southern China. Dried monk fruit can be steeped in water for drinking, used in soups, or used in medicinal cuisine. Its extracts are widely used in sugar-free beverages, desserts, and health supplements. Monk fruit not only contains nutrients such as amino acids, minerals, vitamins, polysaccharides, and volatile oils, but is also rich in mogrosides. Mogroside V, in particular, is about 300 times sweeter than sucrose and is a zero-calorie sweetener. It also possesses antioxidant properties and offers health benefits such as relieving coughs, expectorating phlegm, reducing inflammation, and regulating blood sugar and lipid levels. It is a recognized healthy sugar substitute for obese and diabetic patients.

[0003] Hot air drying is one of the most common drying methods for monk fruit processing due to its simple equipment, convenient operation, and low cost. After hot air drying, the moisture content of monk fruit is significantly reduced, allowing for long-term storage without mold growth, facilitating transportation and sales, and making it the main form of monk fruit products on the market. However, while dehydrating, hot air drying also causes changes in some components of the monk fruit. Studies have shown that the content of mogroside V decreases significantly after hot air drying. For monk fruit, where mogroside V is the core sweetener, the level of mogroside V directly determines its value as a natural sweetener—the higher the mogroside V content, the higher the sweetness and the better the sugar substitute effect. Therefore, minimizing the loss of mogroside V without sacrificing the efficiency of hot air drying is of significant practical importance for developing monk fruit into a high-quality, low-calorie sweetener.

[0004] Existing technologies have reported on the post-ripening and drying processes of monk fruit. For example, CN105852172A relates to a method for promoting the post-ripening of monk fruit by controlling temperature and light conditions to increase the content of glycosides in the fruit before processing. CN112056593B further addresses the endogenous mechanism of monk fruit post-ripening by using sterilization, shell breaking, ripening agent treatment, and microenvironmental factor regulation to increase the content of glycosides. In addition, there are process routes that combine post-ripening with blanching and drying; for example, the method disclosed in CN103622022A includes steps such as post-ripening, washing, blanching, and dynamic vacuum microwave drying. However, the above methods either focus on the accumulation of glycoside V before drying or use special drying methods to reduce losses. No effective targeted solutions have yet been found for the protection of glycoside V during conventional hot air drying.

[0005] Studies have shown that mogroside V possesses excellent thermal stability, remaining largely intact even after continuous heating in a neutral aqueous solution at 100°C for 25 hours. This characteristic indicates that the degradation of mogroside V during hot air drying is not solely driven by thermophysical factors, but most likely originates from enzymatic hydrolysis at the biochemical level. While existing blanching techniques can inactivate enzymes, they often involve high-temperature blanching, making it difficult to preserve heat-sensitive components (such as vitamin C). Therefore, how to specifically inhibit the enzyme activity mediating mogroside V degradation under conventional hot air drying conditions, while simultaneously maintaining drying efficiency and product color, is a pressing technical problem to be solved in this field. Summary of the Invention

[0006] In view of the above, it is necessary to provide an extraction method for monk fruit and its application. This method, through the synergistic combination of citric acid-ascorbic acid pretreatment and hot air drying, achieves a synergistic improvement in high glycoside V retention rate, low bitter glycoside content, and high vitamin C retention rate while taking into account drying efficiency. This provides a feasible technical path for the preparation of high-quality monk fruit low-calorie sweeteners.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for extracting monk fruit includes the following steps:

[0009] (1) Harvesting and ripening: Select fresh monk fruit 75-85 days after pollination and ripen it for 6-10 days at 20-30℃;

[0010] (2) Pretreatment: The ripened monk fruit is soaked in a treatment solution containing citric acid and ascorbic acid.

[0011] (3) Drying: The pretreated monk fruit is dried with hot air until the moisture content is ≤15%;

[0012] (4) Post-processing: After the dried monk fruit is crushed, a solvent is added for ultrasonic-assisted extraction to obtain an extract; the extract is purified by macroporous adsorption resin column chromatography to obtain monk fruit extract.

[0013] In this invention, the ripening time in step (1) is 8 days, and the fresh Luo Han Guo fruit is harvested 80 days after pollination; the temperature of hot air drying in step (3) is 60-80℃.

[0014] In this invention, further, the temperature of hot air drying in step (3) is 70°C; before hot air drying, the monk fruit is perforated with a hole diameter of 1.5-2.5 mm and a depth of 2-3 cm.

[0015] In this invention, the concentration of citric acid in the treatment solution is 1.0%–5.0% (w / v), and the concentration of ascorbic acid is 0.3%–1.0% (w / v); the immersion treatment temperature is 20–40°C, and the time is 10–60 min; the immersion treatment is performed under ultrasonic assistance.

[0016] In this invention, the treatment solution further contains gluconolactone and glutathione, wherein the concentration of glutathione is 0.05% to 0.2% (w / v) and the concentration of gluconolactone in the treatment solution is 0.1 to 5.0 mmol / L.

[0017] Furthermore, in this invention, the treatment solution also contains glutathione, and the concentration of glutathione is 0.05% to 0.2% (w / v).

[0018] In this invention, further, the post-processing in step (4) includes: adding a solvent for ultrasonic-assisted extraction to obtain an extract; purifying the extract by macroporous adsorption resin column chromatography to obtain a monk fruit extract.

[0019] The present invention also proposes a monk fruit extract prepared by the above-described method, wherein the retention rate of mogroside V in the extract is ≥90%; the retention rate is calculated according to the following formula: retention rate = total amount of mogroside V in dried fruit / total amount of mogroside V in fresh fruit × 100%.

[0020] The present invention also proposes the application of the above-mentioned monk fruit extract in the preparation of low-calorie sweeteners.

[0021] This invention also proposes the application of the aforementioned monk fruit extract in the preparation of low-sugar foods.

[0022] The present invention also proposes the application of the above-mentioned monk fruit extract in the preparation of anti-glycation drugs or anti-glycation health foods.

[0023] The present invention has at least the following beneficial effects:

[0024] 1. This invention discovers that during hot air drying, β-glucosidase hydrolyzes high-sweetness glycosides V and 11-oxo-glycoside V, regenerating bitter low-glucosides (glycosides III and IIe), leading to a decrease in the product's sweetness and a rebound in bitterness. This invention, by adding a combined pretreatment of citric acid and ascorbic acid before drying, synergistically inhibits β-glucosidase activity at three levels: citric acid lowers the microenvironment pH, causing the enzyme to deviate from its optimal reaction conditions; ascorbic acid reduces oxidative loss through reducing protection; and gluconolactone (Example 3) acts as a competitive inhibitor, directly occupying the enzyme's active site. At these three levels, the unfavorable pH environment created by citric acid macroscopically reduces the enzyme's catalytic efficiency, while ascorbic acid microscopically eliminates the damage to glycoside V caused by oxidative stress, both providing a fundamental guarantee for the stable retention of glycoside V; and the direct enzymatic inhibition by gluconolactone fundamentally cuts off the enzymatic hydrolysis pathway of glycoside V. These three mechanisms act on different stages of the glycoside V degradation chain—environmental conditions, oxidative damage, and enzyme catalysis—forming a multi-target synergistic effect. It is worth noting that this enzyme-inhibiting effect cannot be replaced by any enzyme inhibitor. If gluconolactone is replaced with nojirimycin (Comparative Example 4), which also has enzyme-inhibiting activity, the glycoside V retention rate significantly decreases from 96.8% to 66.4%. This indicates that while nojirimycin provides some antioxidant protection (Vc retention rate reaches 86.7%), it cannot effectively block the hydrolysis of the β-glycosidic bond of glycoside V. Based on the above triple synergistic mechanism, this invention achieves a glycoside V retention rate ≥90% under 70℃ hot air drying conditions, increasing the glycoside V content by approximately 4 times compared to traditional baking and drying. Simultaneously, the total content of bitter glycosides (glycosides III+IIe) is controlled below 0.06wt%, a reduction of 82.9% compared to the untreated control. The high glycoside V content endows the extract with approximately 300 times the sweetness potential of sucrose, while the extremely low bitter glycoside content ensures a pure taste quality—these two together constitute the core quality indicators of a high-quality sweetener.

[0025] 2. During hot air drying, the Maillard reaction is the main cause of browning in monk fruit. In the treatment solution of this invention, citric acid effectively inhibits the Maillard reaction by lowering the system pH, while ascorbic acid inhibits enzymatic browning through its strong reducing properties. The two work synergistically to reduce the ΔE value to below 17.8, a 45.2% reduction compared to the untreated control. Simultaneously, the reducing microenvironment of ascorbic acid and the acidic protection of citric acid result in a retention rate of over 88.9% of endogenous vitamin C in monk fruit, a 113.8% increase compared to the untreated control. The improved color enhances the product's appearance and acceptability, while the high retention of vitamin C allows the extract to be used as a sweetener while also possessing nutritional value. Furthermore, the addition of glutathione forms a redox buffer pair with ascorbic acid, further reducing the ΔE value to 15.5 and increasing the endogenous vitamin C retention rate to 92.2%, achieving a synergistic improvement in color protection and nutrient retention without affecting the retention rate of vitamin C glycosides.

[0026] 3. In existing technologies, to obtain a high glycoside V retention rate, low-temperature drying (e.g., 40℃) or microwave vacuum drying is usually required. The former has a drying time of up to 420 hours and is extremely inefficient, while the latter has high equipment costs. This invention completes drying in just 96 hours under 70℃ hot air drying conditions, improving efficiency by 77.1% compared to low-temperature drying at 40℃. It also achieves a superior glycoside V retention rate compared to low-temperature drying. This is due to the effective inhibition of β-glucosidase activity by pretreatment: after enzyme activity is inhibited, glycoside V is no longer degraded in large quantities due to enzymatic hydrolysis. Therefore, the accelerated enzyme activity effect caused by temperature increase is offset, allowing a high glycoside V retention rate to be maintained even at higher temperatures. Furthermore, the citric acid and ascorbic acid used in this invention are both food-grade safe ingredients, widely available and inexpensive. Only conventional soaking and hot air drying equipment are required, eliminating the need for large-scale modifications to existing monk fruit processing lines and making it easy to promote and implement.

[0027] 4. The monk fruit extract prepared by this invention has a high content of glycoside V, a low content of bitter glycosides, excellent color, and complete retention of vitamin C, making it suitable for direct use as a low-calorie sweetener raw material. The high content of glycoside V endows the extract with extremely high sweetness potential, far exceeding that of the same weight of sucrose. The extremely low content of bitter glycosides ensures a pure taste without unpleasant aftertaste. Its zero-calorie characteristic aligns with modern healthy eating trends, satisfying both the demand for natural sweetness and the need for controlled sugar intake. Based on these quality advantages, the extract of this invention can be widely used in the food industry, including sugar-free beverages, low-sugar pastries, and health foods. It can also be actively used as a natural sugar substitute in emerging health industries such as medical foods, sports nutrition products, and oral beauty products, meeting the diverse consumer demand for functional sweeteners and demonstrating excellent industrial promotion prospects and socio-economic benefits. Detailed Implementation

[0028] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0029] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0030] In this invention, the content of mogroside V was determined by the following method: High-performance liquid chromatography (HPLC) was used to determine the content of mogroside V. The chromatographic conditions were as follows: the column was an Agilent Poroshell 120 SB C18 column (150 mm × 2.1 mm, 2.7 μm); the mobile phase was water (A) and methanol (B); the gradient elution program was: 0-8 min, 50% B; 8-10 min, 50%-55% B; 10-34 min, 55% B; 34-37 min, 55%-62% B; 37-51 min, 62% B; 51-55 min, 62%-100% B; the flow rate was 0.5 mL / min, the injection volume was 1.0 μL, and the column temperature was 25℃. The detection wavelength was 203 nm. External standard method was used for quantification. The contents of mogroside III and mogroside IIe were also determined under the above HPLC conditions.

[0031] In this invention, the retention rate of mogroside V is calculated according to the following formula: Retention rate (%) = (Total mogroside V in dried fruit / Total mogroside V in fresh fruit) × 100%; where, the total mogroside V in dried fruit refers to the total mass of mogroside V measured after the dried whole fruit (including peel, pulp, and seeds) is crushed; the total mogroside V in fresh fruit refers to the total mass of mogroside V measured after the whole fresh fruit (including peel, pulp, and seeds) harvested in the same batch is homogenized. All content determinations are calculated based on fresh fruit harvested in the same batch.

[0032] Example 1

[0033] This embodiment provides a method for preparing monk fruit extract, including the following steps:

[0034] (1) Harvesting and Post-ripening: Monk fruit grown in Yongfu County, Guilin City, Guangxi Province was selected and harvested 80 days after pollination. Harvesting criteria: fruit diameter 55–58 mm, dark green peel, no mechanical damage, and no pests or diseases. After harvesting, the fruit was quickly transported to the laboratory, where bright-looking, uniformly sized fruits were selected for later use. The harvested fresh monk fruit was placed indoors and post-ripened for 8 days at 25±2℃ and 60%–70% relative humidity. During this period, the fruit gradually changed from dark green to yellowish-green, and the flesh gradually softened. The appearance of the fruit was regularly observed during the post-ripening process, and any moldy or rotten fruit was removed.

[0035] (2) Pretreatment: Prepare the treatment solution by dissolving citric acid and ascorbic acid in deionized water to obtain a treatment solution containing citric acid (concentration of 2.0% w / v) and ascorbic acid (concentration of 0.5% w / v). The pH of the treatment solution is approximately 2.8–3.2. Place the monk fruit, which has been ripening for 8 days, into the above treatment solution and treat it with ultrasonic-assisted soaking at 30°C for 30 minutes (ultrasonic power of 600W and frequency of 40kHz). The cavitation effect of ultrasound is used to promote the penetration of the treatment solution into the fruit. During the soaking process, the treatment solution should completely submerge the monk fruit. After soaking, remove the monk fruit and drain the surface liquid.

[0036] (3) Drying: The pretreated monk fruit was placed in an electric hot air drying oven and dried at 70℃. Before hot air drying, holes were punched at the top stem and bottom navel of the monk fruit, with a hole diameter of 2.0 mm and a depth of 2.5 cm. During the hot air drying process, the air velocity inside the drying oven was kept constant at 5 m / s. The moisture content was sampled and tested every 12 hours. Drying was stopped when the moisture content dropped below 15%, and the drying time was recorded.

[0037] (4) Post-processing: The dried whole monk fruit was crushed and passed through a 40-mesh sieve. 10g of monk fruit powder was weighed and added to 100mL of 70% (v / v) methanol aqueous solution. The mixture was ultrasonically extracted for 1h at an ultrasonic power of 600W and a frequency of 40kHz. After extraction, the mixture was filtered and the filtrate was collected. The residue was extracted once more, and the two filtrates were combined. The combined filtrate was purified by column chromatography using a macroporous adsorption resin (D101 type). First, deionized water was used to elute until colorless, and then 70% (v / v) ethanol aqueous solution was used to elute. The ethanol eluent was collected. The eluent was concentrated to dryness under reduced pressure at 50℃ to obtain monk fruit extract.

[0038] HPLC analysis, based on the same batch of fresh fruit, showed that the retention rate of mogroside V in the monk fruit extract obtained in this example was 94.2%, with a content of 2.95 g / 100 g on a dry basis. The total content of mogroside III and mogroside IIe was 0.06 wt%. The color difference value ΔE of the extract was 17.8, and the ascorbic acid (Vc) content was 182.6 mg / 100 g. The drying time was 96 h.

[0039] Example 2

[0040] This embodiment provides a method for preparing monk fruit extract, which differs from Example 1 only in the concentrations of citric acid and ascorbic acid in step (2) pretreatment. The treatment solution in this embodiment contains citric acid (concentration of 5.0% w / v) and ascorbic acid (concentration of 1.0% w / v), and the remaining steps and conditions are exactly the same as in Example 1.

[0041] HPLC analysis, based on the same batch of fresh fruit, showed that the retention rate of mogroside V in the monk fruit extract obtained in this example was 97.1%, with a content of 3.04 g / 100 g on a dry basis. The total content of mogroside III and mogroside IIe was 0.05 wt%. The color difference value ΔE of the extract was 17.1, and the ascorbic acid (Vc) content was 190.2 mg / 100 g. The drying time was 96 h.

[0042] Example 3

[0043] This embodiment provides a method for preparing monk fruit extract, which differs from Example 1 only in the composition of the pretreatment solution in step (2). The pretreatment solution in this embodiment contains citric acid (2.0% w / v), ascorbic acid (0.5% w / v), and gluconolactone (1.0 mmol / L), while the remaining steps and conditions are exactly the same as in Example 1.

[0044] HPLC analysis, based on the same batch of fresh fruit, showed that the retention rate of mogroside V in the monk fruit extract obtained in this example was 96.8%, with a content of 3.21 g / 100 g on a dry basis. The total content of mogroside III and mogroside IIe was 0.04 wt%. The color difference value ΔE of the extract was 16.2, and the ascorbic acid (Vc) content was 185.3 mg / 100 g. The drying time was 96 h.

[0045] Example 4

[0046] This embodiment provides a method for preparing monk fruit extract, which differs from Example 1 only in the concentrations of citric acid and ascorbic acid in step (2) pretreatment. The treatment solution in this embodiment contains citric acid (concentration of 1.0% w / v) and ascorbic acid (concentration of 0.3% w / v), and the remaining steps and conditions are exactly the same as in Example 1.

[0047] HPLC analysis, based on the same batch of fresh fruit, showed that the retention rate of mogroside V in the monk fruit extract obtained in this example was 92.5%, with a content of 2.87 g / 100 g on a dry basis. The total content of mogroside III and mogroside IIe was 0.08 wt%. The color difference value ΔE of the extract was 19.2, and the ascorbic acid (Vc) content was 174.5 mg / 100 g. The drying time was 96 h.

[0048] Example 5

[0049] This embodiment provides a method for preparing monk fruit extract, which differs from Example 1 only in the composition of the pretreatment solution in step (2). The pretreatment solution in this embodiment contains citric acid (2.0% w / v), ascorbic acid (0.5% w / v), and glutathione (0.1% w / v), while the remaining steps and conditions are exactly the same as in Example 1.

[0050] HPLC analysis showed that, based on the same batch of fresh fruit, the retention rate of mogroside V in the monk fruit extract obtained in this example was 94.5%, and the content of mogroside V on a dry basis was 2.98 g / 100 g. The total content of mogroside III and mogroside IIe was 0.06 wt%. The color difference value ΔE of the extract was 15.5 (significantly lower than 17.8 in Example 1), and the ascorbic acid (Vc) content was 189.4 mg / 100 g. The drying time was 96 h.

[0051] Comparative Example 1

[0052] This comparative example provides a method for preparing monk fruit extract. The only difference from Example 1 is that no pretreatment is performed in step (2), and the ripened monk fruit is directly dried in step (3). The remaining steps and conditions are exactly the same as in Example 1.

[0053] HPLC analysis showed that, based on the same batch of fresh fruit, the retention rate of mogroside V in the monk fruit extract obtained in this comparative example was 38.9%, with a content of 1.22 g / 100 g on a dry basis. The total content of mogroside III and mogroside IIe was 0.35 wt%. The color difference value ΔE of the extract was 32.5. The ascorbic acid (Vc) content was 85.4 mg / 100 g. The drying time was 96 h.

[0054] Comparative Example 2

[0055] This comparative example provides a method for preparing monk fruit extract. The only difference from Example 1 is that in step (2) pretreatment, the treatment solution contains only citric acid (concentration of 2.0% w / v) and no ascorbic acid is added. The remaining steps and conditions are exactly the same as in Example 1.

[0056] HPLC analysis showed that, based on the same batch of fresh fruit, the retention rate of mogroside V in the monk fruit extract obtained in this comparative example was 58.1%, with a content of 1.82 g / 100 g on a dry basis. The total content of mogroside III and mogroside IIe was 0.18 wt%. The color difference value ΔE of the extract was 22.3, and the ascorbic acid (Vc) content was 132.5 mg / 100 g. The drying time was 96 h.

[0057] Comparative Example 3

[0058] This comparative example provides a method for preparing monk fruit extract. The only difference from Example 1 is that in step (2) pretreatment, the treatment solution contains only ascorbic acid (concentration of 0.5% w / v) and no citric acid is added. The remaining steps and conditions are exactly the same as in Example 1.

[0059] HPLC analysis showed that, based on the same batch of fresh fruit, the retention rate of mogroside V in the monk fruit extract obtained in this comparative example was 55.2%, and the content of mogroside V on a dry basis was 1.73 g / 100 g. The total content of mogroside III and mogroside IIe was 0.20 wt%. The color difference value ΔE of the extract was 20.5, and the ascorbic acid (Vc) content was 163.8 mg / 100 g. The drying time was 96 h.

[0060] Comparative Example 4

[0061] This comparative example provides a method for preparing monk fruit extract, which differs from Example 1 only in that: in step (2) pretreatment, nojirimycin (concentration of 1.0 mmol / L) is used to replace gluconolactone, and the treatment solution contains citric acid (concentration of 2.0% w / v), ascorbic acid (concentration of 0.5% w / v) and nojirimycin (concentration of 1.0 mmol / L). The remaining steps and conditions are exactly the same as in Example 1.

[0062] HPLC analysis showed that, based on the same batch of fresh fruit, the retention rate of mogroside V in the monk fruit extract obtained in this comparative example was 66.4%, with a content of 2.08 g / 100 g on a dry basis. The total content of mogroside III and mogroside IIe was 0.14 wt%. The color difference value ΔE of the extract was 18.5, and the ascorbic acid (Vc) content was 178.2 mg / 100 g. The drying time was 96 h.

[0063] Comparative Example 5

[0064] This comparative example provides a method for preparing monk fruit extract, which differs from Example 1 only in the concentrations of citric acid and ascorbic acid in the pretreatment step (2). The treatment solution in this example contains citric acid (0.5% w / v) and ascorbic acid (0.1% w / v), and the remaining steps and conditions are exactly the same as in Example 1.

[0065] HPLC analysis, based on the same batch of fresh fruit, showed that the retention rate of mogroside V in the monk fruit extract obtained in this comparative example was 80.5%, with a content of 2.52 g / 100 g on a dry basis. The total content of mogroside III and mogroside IIe was 0.10 wt%. The color difference value ΔE of the extract was 20.3, and the ascorbic acid (Vc) content was 165.4 mg / 100 g. The drying time was 96 h.

[0066] Comparative experiments and results

[0067] To demonstrate the practical value of this invention, the inventors conducted the following comparative experiment:

[0068] I. Effects of different pretreatment methods on the content, retention rate, and bitter glycoside content of mogroside V

[0069] The results of the determination of mogroside V content in Examples 1-5 and Comparative Examples 1-5 are summarized in Table 1.

[0070] Table 1. Effects of different pretreatment methods on the content and retention rate of mogroside V.

[0071]

[0072] The results in Table 1 show that:

[0073] (1) In Comparative Example 1 (without pretreatment), the retention rate of glycoside V after hot air drying at 70℃ was only 38.9%, and the total amount of glycosides III+IIe was as high as 0.35wt%, indicating that a large number of high-glucosinolates underwent enzymatic hydrolysis during hot air drying, generating bitter glycosides. The retention rate of glycoside V in Comparative Example 2 (citric acid only) increased to 58.1%, and in Comparative Example 3 (ascorbic acid only) it increased to 55.2%. Both citric acid and ascorbic acid alone can inhibit the degradation of glycoside V to some extent, but the effect is limited. The inhibitory effect of citric acid is mainly due to its reduction of the pH value inside the monk fruit tissue, causing β-glucosidase to deviate from the optimal pH range (usually 4.5-6.0); the inhibitory effect of ascorbic acid is due to the inhibition of oxidative degradation by its reducing microenvironment. However, inhibition by a single pathway cannot completely block the degradation pathway of glycoside V.

[0074] (2) The retention rate of glycoside V in Example 1 (combined pretreatment with citric acid and ascorbic acid) reached 94.2%, which was 55.3 percentage points higher than that in Comparative Example 1, 36.1 percentage points higher than that in Comparative Example 2, and 39.0 percentage points higher than that in Comparative Example 3. The combined effect far exceeded the simple sum of the effects of the single components, indicating that there is a significant synergistic effect between citric acid and ascorbic acid. The mechanism is that citric acid inhibits enzyme catalytic activity through pH regulation, while ascorbic acid reduces oxidative loss through reducing protection. The two act on the degradation process of glycoside V through different pathways, achieving multiple blocking.

[0075] (3) In Example 3 (synergistic pretreatment with citric acid, ascorbic acid, and gluconolactone), the retention rate of glycoside V was further increased to 96.8%, and the total amount of glycosides III and IIe was reduced to 0.04 wt%. Gluconolactone, as a competitive inhibitor of β-glucosidase, can directly bind to the active site of the enzyme, further blocking the enzymatic hydrolysis of high-glycosides to low-glycosides. Based on citric acid and ascorbic acid, the addition of gluconolactone achieved a triple blockade on the degradation of glycoside V.

[0076] (4) The retention rate of Example 2 (high concentration) reached 97.1%, slightly higher than that of Example 3 (96.8%). This may be due to the fact that the high concentration of citric acid further lowered the pH of the system, enhancing the non-specific pH inhibition. However, Example 3 achieved a retention rate comparable to that of Example 2 (96.8% vs 97.1%) at a lower acid concentration (2.0% citric acid vs 5.0% of Example 2), and the content of bitter glycosides was lower (0.04 wt% vs 0.05 wt%), indicating that the direct enzymatic inhibition of gluconolactone has a higher targeting efficiency and can more effectively block specific enzymatic hydrolysis pathways under mild pH conditions, thereby optimizing the sensory quality of the product while ensuring a high glycoside V retention rate. (5) The glycoside V retention rate of Comparative Example 4 (nojirimycin replacing gluconolactone) was only 66.4%, significantly lower than that of Example 3 (96.8%). Nojirimycin is a potent inhibitor of α-glucosidase, which mainly acts on the hydrolysis of α-glycosidic bonds, while its inhibitory activity on β-glucosidase is weak. The glycosidic bond linking the sugar moiety in mogroside V is in the β-configuration, and its hydrolysis requires the catalysis of β-glucosidase. Therefore, nojirimycin cannot effectively inhibit the enzymatic hydrolysis of mogroside V. The results of Comparative Example 4 indicate that the specific inhibition of β-glucosidase by gluconolactone is the key to achieving high mogroside V retention and is irreplaceable.

[0077] (6) In Example 5, after further adding glutathione to Example 1, the retention rate of glycoside V was 94.5%, which was basically the same as that in Example 1 (94.2%). The total amount of bitter glycosides also remained at the same level of 0.06 wt%, indicating that the addition of glutathione did not interfere with the synergistic inhibitory effect of citric acid and ascorbic acid on β-glucosidase and did not affect the protection of glycoside V. At the same time, as shown in Table 2, the addition of glutathione further reduced the ΔE value from 17.8 in Example 1 to 15.5, and the color protection effect was significantly improved. This shows that glutathione and ascorbic acid formed an effective redox buffer pair in the antioxidant and color protection layer, which can further improve the color of the product without damaging the retention of glycoside V.

[0078] (7) The retention rate of glycoside V in Comparative Example 5 (pretreatment with low concentration of citric acid + ascorbic acid) was 80.5%, which was better than the control without pretreatment (38.9%) and the single-component treatment (55.2% to 58.1%), but still did not reach the target retention rate of 90%. Compared with Example 4 (1.0% citric acid + 0.3% ascorbic acid, retention rate 92.5%), the concentrations of citric acid (0.5%) and ascorbic acid (0.1%) in Comparative Example 5 were both about 50% lower, but the retention rate of glycoside V decreased by 12 percentage points (80.5% vs 92.5%), indicating that there is a certain threshold effect of the concentrations of citric acid and ascorbic acid in the pretreatment solution - below this threshold, even if used in combination, effective protection of glycoside V cannot be achieved.

[0079] II. The Influence of Different Pretreatment Methods on Color

[0080] Table 2. Effects of different pretreatment methods on the color of monk fruit extract.

[0081]

[0082] As shown in Table 2, the ΔE value of Comparative Example 1 (no pretreatment) was 32.5, indicating that hot air drying caused severe browning of the monk fruit. The ΔE values ​​of Comparative Example 2 (citric acid only) and Comparative Example 3 (ascorbic acid only) decreased to 22.3 and 20.5, respectively, indicating that both had some color-protecting effect, but their use alone was still insufficient to effectively inhibit browning. The ΔE value of Comparative Example 5 (low-concentration citric acid + ascorbic acid combined pretreatment, 0.5% + 0.1%) was 20.3, which was a slight improvement compared to Comparative Examples 2 and 3, but the improvement was limited, indicating that the color-protecting effect of the low-concentration combination was not sufficient. The ΔE value of Example 4 (citric acid 1.0% + ascorbic acid 0.3%) was 19.2, which was lower than that of Comparative Example 5, indicating that the color-protecting effect began to appear after increasing the concentration. The ΔE value of Example 1 (2.0% citric acid + 0.5% ascorbic acid) was further reduced to 17.8, a 45.2% decrease compared to Comparative Example 1, demonstrating significantly better performance than the low-concentration combination. The mechanism is as follows: citric acid inhibits the Maillard reaction by lowering the pH (this reaction is more active under alkaline to neutral conditions), while its chelating effect inhibits the metal ion cofactors required by polyphenol oxidase; ascorbic acid, as a strong reducing agent, consumes dissolved oxygen in the system and reduces early browning products. The combined use of both synergistically protects color from both enzymatic and non-enzymatic browning perspectives. Example 3 (citric acid + ascorbic acid + glucono-delta-lactone) had a ΔE value of 16.2, exhibiting the best color, possibly due to the addition of glucono-delta-lactone further optimizing the acidic environment of the system and enhancing the inhibitory effect on the browning reaction. The ΔE value of Example 2 (high concentration) was 17.1, which is close to that of Example 1 (17.8) and Example 3 (16.2), indicating that the color-protecting effect is good under high concentration conditions, but the marginal improvement effect of increasing concentration on color has tended to plateau. Furthermore, in Example 5, after adding glutathione to the pretreatment solution, the ΔE value further decreased from 17.8 in Example 1 to 15.5, and the color-protecting effect was significantly better than the group without addition. This is likely because glutathione and ascorbic acid can achieve redox regeneration cycles in cells through the dehydroascorbic acid reductase pathway, continuously consuming reactive oxygen species in the system and inhibiting the catalytic oxidation of polyphenol oxidase, thereby providing a more durable antioxidant microenvironment for mogroside V and vitamin C.

[0083] It should be noted that the ΔE value of Comparative Example 4 (nojirimycin replacing gluconolactone) was 18.5, which is close to that of Example 1 (17.8) and Example 3 (16.2), but its glycoside V retention rate (66.4%) was much lower than that of the examples of this invention (≥92.5%). The ΔE value of Comparative Example 5 was 20.3, which was not much different from that of Comparative Example 4, but its glycoside V retention rate was only 80.5%. This indicates that a good color-protecting effect (lower ΔE value) does not necessarily lead to a high glycoside V retention rate—color protection mainly acts on polyphenol oxidase and browning reaction, while the retention of glycoside V requires specific inhibition of β-glucosidase activity. This invention, through combined pretreatment with citric acid and ascorbic acid, achieves color protection, and more importantly, effectively inhibits the degradation of glycoside V, demonstrating a dual synergistic effect of color protection and glycoside retention.

[0084] III. Effects of different pretreatment methods on ascorbic acid (Vc) content

[0085] Table 3. Effects of different pretreatment methods on vitamin C content in monk fruit.

[0086]

[0087] Table 3 shows that the vitamin C content in the fresh fruit was 205.5 mg / 100g. In Comparative Example 1 (no pretreatment), the vitamin C retention rate after drying was only 41.6%, mainly because vitamin C is extremely sensitive to heat and oxygen and is easily oxidized and degraded during prolonged hot air drying. Comparative Example 2 (citric acid only) had a vitamin C retention rate of 64.5%, and Comparative Example 3 (ascorbic acid only) had a retention rate of 79.7%. The addition of ascorbic acid directly supplemented exogenous vitamin C and protected endogenous vitamin C from oxidation through its strong reducing properties; citric acid, by lowering the pH value, created an acidic microenvironment, inhibiting the oxidative degradation of vitamin C. However, protection through a single pathway is still insufficient to achieve the ideal vitamin C retention effect. The vitamin C retention rate of Comparative Example 5 (pretreatment with low concentration citric acid and ascorbic acid) was 80.5%, which was basically the same as that of Comparative Example 3 (79.7%). This indicates that ascorbic acid was the main contributor to vitamin C protection during low-concentration combined treatment, but the synergistic effect of citric acid was not fully realized. The vitamin C retention rate of Example 4 (1.0% citric acid + 0.3% ascorbic acid) was 84.9%, which was improved compared to Comparative Example 5, but did not reach the level of Example 1. The vitamin C retention rate of Example 1 (2.0% citric acid + 0.5% ascorbic acid) was further improved to 88.9%, Example 3 (citric acid + ascorbic acid + gluconolactone) reached 90.2%, and Example 2 (high concentration) reached 92.6%, indicating that the vitamin C retention rate gradually increased with the increase of the treatment solution concentration, showing a certain concentration dependence. The vitamin C retention rate of Comparative Example 4 (nojirimycin) was 86.7%, which is close to that of Example 1 (88.9%), Example 3 (90.2%), and Example 2 (92.6%). However, its glycoside V retention rate (66.4%) was significantly lower than that of the examples of this invention (≥92.5%). This result fully demonstrates that the retention of vitamin C mainly depends on the antioxidant protection of ascorbic acid, while the retention of glycoside V requires specific inhibition of β-glucosidase. Although nojirimycin can provide some antioxidant protection (making the vitamin C retention rate close to the level of the examples), it cannot effectively inhibit β-glucosidase, so the glycoside V retention rate is much lower than that of the examples of this invention. This comparison further proves that the selection of gluconolactone in this invention is based on a deep understanding of the degradation mechanism of glycoside V, rather than a byproduct of antioxidant activity. Furthermore, in Example 5, after the addition of glutathione, the vitamin C retention rate reached 92.2%, which was further improved from 88.9% in Example 1 and basically the same as that in Example 2 (92.6%). This indicates that the redox synergistic protective effect between glutathione and ascorbic acid significantly enhanced the stability of endogenous vitamin C.

[0088] IV. The Influence of Different Pretreatment Methods on Drying Time

[0089] Table 4. Effects of different pretreatment methods on drying time

[0090]

[0091] As shown in Table 4, the pretreatment in all embodiments and comparative examples of this invention did not prolong the hot air drying time, and all samples reached the target moisture content in 96 hours at 70℃. This indicates that neither the soaking treatment with citric acid / ascorbic acid, the addition of gluconolactone, nor the replacement of nojirimycin altered the physical mass transfer characteristics of monk fruit, and therefore did not affect the drying efficiency. For reference, if a low-temperature drying at 40℃ (a common method in existing technologies) is used to obtain a high glycoside V retention rate, the drying time would be 420 hours, which is extremely inefficient and not feasible for industrial production. This invention can complete the drying in only 96 hours under hot air drying conditions at 70℃, which is 77.1% more efficient than the low-temperature drying at 40℃, while achieving a glycoside V retention rate of over 92.5% (92.5% in Example 4, 94.2% in Example 1, 96.8% in Example 3, and 97.1% in Example 2), thus balancing drying efficiency and quality retention.

[0092] Based on the above experimental results, the combined pretreatment with citric acid and ascorbic acid in this invention can synergistically inhibit the activity of β-glucosidase at three levels, thereby reducing the enzymatic hydrolysis of mogroside V during hot air drying. First, citric acid lowers the pH of the microenvironment within the mogroside tissue to 2.8–3.2, causing β-glucosidase to deviate from its optimal pH range (typically 4.5–6.0). As a typical glycoside hydrolase, β-glucosidase contains catalytic nucleophiles and acid / base catalytic residues in its active site; the protonation state of these residues is directly regulated by pH. When the environmental pH deviates from the optimal range, the enzyme's conformation changes, and its catalytic efficiency decreases significantly, thereby reducing the hydrolysis of the β-1,6 glycosidic bond in mogroside V and 11-oxo-mogroside V. Second, ascorbic acid, as a strong reducing agent, can effectively scavenge reactive oxygen species generated during drying, creating a reducing microenvironment, thereby protecting potentially oxidized structural units in the mogroside V molecule and preventing the oxidative degradation of endogenous vitamin C in the mogroside. Furthermore, ascorbic acid can inhibit the activity of polyphenol oxidase, reducing enzymatic browning. Further, when the treatment solution also contains gluconolactone, this compound can act as a competitive inhibitor of β-glucosidase. Its molecular structure is similar to β-D-gluconolactone, enabling it to form hydrogen bonds and hydrophobic interactions with the active site of β-glucosidase, occupying the enzyme's catalytic site and directly blocking the binding of the enzyme to the substrate (glycoside V). Unlike the indirect pH regulation of citric acid, gluconolactone provides direct and specific inhibition of β-glucosidase.

[0093] The three layers mentioned above complement each other and work synergistically: citric acid creates a pH environment unfavorable to the enzymatic reaction, ascorbic acid eliminates oxidative stress, and gluconolactone directly occupies the enzyme's active site. This triple protection ensures that, under 70℃ hot air drying conditions, the enzymatic hydrolysis of mogroside V by β-glucosidase is maximally inhibited, resulting in an excellent mogroside V retention rate of ≥92%. It is worth noting that this direct enzyme inhibition exhibits strict structure-activity relationship specificity. If gluconolactone is replaced with nojirimycin, which also has enzyme-inhibiting activity, the mogroside V retention rate significantly decreases from 96.8% to 66.4% (Comparative Example 4). This is mainly because nojirimycin is a potent inhibitor of α-glucosidase, while its inhibitory activity against β-glucosidase is extremely weak. Given that the hydrolysis of mogroside V depends on the breaking of β-glycosidic bonds, nojirimycin cannot effectively block this degradation pathway. This result confirms that only gluconolactone with affinity for β-glucosidase can play a key "direct inhibition" role in the system of this invention, thereby achieving synergistic effects with the other two mechanisms.

[0094] V. Industrial Applicability

[0095] The method for extracting monk fruit provided by this invention is simple, controllable, and low-cost. The monk fruit extract prepared contains glycoside V content of over 2.87 g / 100 g with a retention rate of ≥90%, and bitter glycoside (glycoside III+IIe) content ≤0.08 wt%, far lower than the control group without pretreatment. The high content of glycoside V endows the extract with extremely high sweetness potential, far exceeding that of the same weight of sucrose, while the extremely low bitter glycoside content ensures a pure taste quality. This makes it suitable as a high-quality, low-calorie sweetener raw material, directly applicable to sugar-free beverages, low-sugar pastries, health foods, and other fields, balancing the demand for natural sweetness with the health requirement of zero calorie intake. It has good industrial practical value and broad market prospects.

[0096] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for extracting monk fruit, characterized in that, Includes the following steps: (1) Harvesting and ripening: Select fresh monk fruit 75-85 days after pollination and ripen it for 6-10 days at 20-30℃; (2) Pretreatment: The ripened monk fruit is soaked in a treatment solution containing citric acid and ascorbic acid. (3) Drying: The pretreated monk fruit is dried with hot air until the moisture content is ≤15%; (4) Post-processing: After the dried monk fruit is crushed, a solvent is added for ultrasonic-assisted extraction to obtain an extract; the extract is purified by macroporous adsorption resin column chromatography to obtain monk fruit extract.

2. The method according to claim 1, characterized in that, The ripening time in step (1) is 8 days, and the fresh Luo Han Guo fruit is harvested 80 days after pollination; the temperature of hot air drying in step (3) is 60-80℃.

3. The method according to claim 2, characterized in that, The temperature of hot air drying in step (3) is 70°C; before hot air drying, the monk fruit is perforated with a hole diameter of 1.5-2.5 mm and a depth of 2-3 cm.

4. The method according to claim 1, characterized in that, The concentration of citric acid in the treatment solution is 1.0%–5.0% (w / v), and the concentration of ascorbic acid is 0.3%–1.0% (w / v); the immersion treatment temperature is 20–40°C, and the time is 10–60 min; the immersion treatment is carried out under ultrasonic assistance.

5. The method according to claim 1, characterized in that, The treatment solution also contains gluconolactone and glutathione, wherein the concentration of glutathione is 0.05% to 0.2% w / v and the concentration of gluconolactone in the treatment solution is 0.1 to 5.0 mmol / L.

6. The method according to claim 1, characterized in that, The treatment solution also contains glutathione, and the concentration of glutathione is 0.05% to 0.2% w / v.

7. The method according to claim 1, characterized in that, The post-processing described in step (4) includes: adding solvent for ultrasonic-assisted extraction to obtain an extract; purifying the extract by macroporous adsorption resin column chromatography to obtain monk fruit extract.

8. The monk fruit extract prepared according to any one of claims 1-7, characterized in that, The retention rate of mogroside V in the extract is ≥90%.

9. The application of the monk fruit extract according to claim 8 in the preparation of low-calorie sweeteners or low-sugar foods.

10. The use of the monk fruit extract according to claim 8 in the preparation of anti-glycation drugs or anti-glycation health foods.

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