Compound natural sweetener, preparation method and application thereof

Abstract

The application discloses a compound natural sweetener, a preparation method thereof and application of the compound natural sweetener in low-sugar fruit juice, and belongs to the technical field of food additives. The compound sweetener is prepared from erythritol, mogroside, stevioside, thaumatin and monopotassium glycyrrhizinate, and has high synergistic coefficient, pure taste and no bad aftertaste. The preparation method is simple and stable, and adopts step-by-step mixing, low-temperature and low-humidity and ultraviolet sterilization processes. When applied to fruit juice, the compound natural sweetener can replace sucrose, realize low-sugar / sugar-free, stabilize blood sugar, improve storage stability, protect the intestinal tract, enhance polyphenol absorption, and optimize aroma and flavor. The compound natural sweetener is safe, compliant, metabolically friendly and suitable for industrialization, and provides a core sweetening solution for low-sugar healthy fruit juice.

Description

Technical Field

[0001] This invention belongs to the field of food additives and low-sugar healthy beverages, specifically relating to a compound natural sweetener, its preparation method, and its application. Background Technology

[0002] Sweeteners, as key functional ingredients that replace sucrose in providing sweetness, are experiencing explosive market demand. Currently, sweeteners are divided into artificial sweeteners and natural sweeteners. While artificial sweeteners such as aspartame, acesulfame potassium, and sucralose are high in sweetness and low in cost, they pose safety controversies, including carcinogenic risks and disruption of gut microbiota, leading to a decline in consumer trust. Natural sweeteners, derived from plants or microorganisms, are highly safe, low in calories or zero in calories, and some even possess antioxidant and other bioactivities, making them a core ingredient in the research and development of sugar-reduced foods. Currently widely used natural sweeteners include steviol glycosides, mogrosides, monopotassium glycyrrhizate, thomatase, and erythritol, which have been applied in various sub-sectors such as beverages, baked goods, and dairy products.

[0003] However, when used as a single component, the aforementioned natural sweeteners fail to achieve a taste experience similar to sucrose: steviol glycosides are 200-300 times sweeter than sucrose, but have a bitter aftertaste, a licorice-like off-flavor, and a short duration of sweetness; mogrosides have a milder bitter aftertaste, but at high concentrations they have an astringent taste and a cooling flavor, making them unsuitable for hot drinks; monopotassium glycyrrhizate has a long-lasting sweetness but a medicinal taste, which can be unpleasant; sematriol is extremely sweet but slow to take effect, and its sweetness diminishes under acidic conditions; erythritol has the sweetest taste closest to sucrose, but its sweetness is only 60%-70% of sucrose, and at high concentrations it can have a cooling or unpleasant sensation. Furthermore, some natural sweeteners exhibit poor stability under high-temperature processing, long-term storage, or acidic environments, easily leading to sweetness decay and flavor deterioration, thus limiting their widespread application.

[0004] To address the aforementioned shortcomings, the mainstream solution in the industry is synergistic blending of sweeteners. This involves mixing two or more sweeteners with different structures in a certain proportion, resulting in a synergistic effect where the perceived sweetness is greater than the sum of its parts (1+1>2). The principle is that different sweetener structures bind to different sites on sweet taste receptors, synergistically activating the receptors, enhancing sweetness, and masking unpleasant flavors. However, existing blending technologies still have shortcomings: First, the synergistic effect is sensitive to the ratio; deviations in the ratio can easily lead to the disappearance of the synergistic effect or antagonistic effects, requiring high precision in production formulations and quality control. Second, blending is mostly a physical mixing process, without considering the binding mechanism between components and sweet taste receptors at the molecular level, resulting in a difference in taste compared to sucrose. Third, blended systems exhibit significant differences in performance across different food matrices, lacking broad applicability, and the increased number of components leads to higher costs, hindering large-scale commercialization. Therefore, how to achieve a taste close to sucrose through scientific blending while ensuring safety, and balancing stability and cost control, is a pressing technical challenge in the current application of natural sweeteners. Summary of the Invention

[0005] This invention provides a compound natural sweetener, its preparation method, and its application, aiming to address the technical shortcomings of existing single-agent natural sweeteners, such as poor taste and weak stability, low tolerance for ratio errors in conventional compounding schemes, poor sweetness simulation, narrow matrix applicability, and high commercialization costs. This invention uses five natural sweeteners permitted by the national standard GB 2760-2024 as raw materials. Through optimized multi-component compounding and synergistic ratios, it effectively masks undesirable flavors and constructs a sweetness profile close to sucrose. Simultaneously, it verifies the food safety and metabolic regulation functions of the compound system, applying it to fruit juice products to improve the flavor and quality stability of beverages, balancing safety and economy, and providing technical support for the industrial development of low-sugar healthy fruit juices.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a compound natural sweetener, wherein the component ratio, based on the total mass of the compound sweetener, includes: erythritol: mogroside: sematriol sweetness = 6.11: 0.013: 0.00067.

[0007] Furthermore, the compound natural sweetener is compatible with acidic fruit juice matrix, and its sweetness does not decrease under long-term storage at 25°C, and it does not induce acidification, precipitation or deterioration of the fruit juice system.

[0008] This invention also provides a method for preparing a compound natural sweetener, the specific steps of which are as follows: Take the appropriate amounts of erythritol, mogroside, and thomaline, sieve them, and obtain a homogeneous powder. The sieved mogrosides, sematriol and 1 / 10 erythritol were premixed to prepare a premix; Add the remaining sieving erythritol to the premix, mix well, sterilize and seal to obtain a compound natural sweetener.

[0009] This invention also provides the application of a compound natural sweetener in the preparation of metabolic regulation functional foods. The compound natural sweetener serves as a sweetener substitute, giving the food the physiological effects of low postprandial sugar load and regulation of body lipid metabolism.

[0010] Furthermore, the compound natural sweetener inhibits insulin resistance induced by exogenous sugars, maintains insulin sensitivity, and reduces blood sugar fluctuations after consumption.

[0011] Furthermore, the compound natural sweetener reduces the accumulation of uric acid in the body's urine, reduces lipid droplet deposition in the liver, and alleviates liver lipid degeneration damage.

[0012] This invention also provides the application of a compound natural sweetener in the preparation of intestinal homeostasis maintenance functional foods, wherein the compound natural sweetener has both intestinal barrier repair and intestinal flora structure bidirectional regulatory effects.

[0013] Furthermore, the compound natural sweetener upregulates colonic tight junction protein. ZO-1 It expresses the level of repair of intestinal epithelial barrier rupture and damage caused by the sweet matrix of food.

[0014] Furthermore, the compounded natural sweetener enriches beneficial butyrate-producing bacteria and mucus-degrading probiotics in the gut, downregulates the abundance of harmful bacteria that promote metabolic disorders in the gut, and optimizes the gut microbiota community structure.

[0015] This invention also provides the application of compound natural sweeteners in improving the retention of nutrients and stabilizing the flavor of fruit juice. The compound sweeteners synergistically enhance the bioavailability of total phenols and hesperidin active substances in fruit juice, while inhibiting the formation of aldehyde off-flavor substances during fruit juice storage and locking in the natural fruit aroma of the fruit juice.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: This invention selects natural sweeteners permitted by the national standard GB 2760-2024, and screens the optimal ternary compound system through binary and ternary compounding experiments. The components produce a synergistic effect of sweetness, achieving a compounding effect of 1+1>2. It effectively masks the bitterness, irritating coolness and other off-flavors of single sweeteners, and the overall sweetness profile and sweetness release curve are highly close to sucrose. The whole preparation process is simple to operate, the powder is mixed evenly without layering, the raw materials are compliant and safe, and it is suitable for large-scale industrial production and processing of food.

[0017] The compound sweetener of this invention has excellent bio-metabolic safety. Long-term intake will not affect body weight, food intake, or liver and kidney function, nor will it induce insulin resistance. It can effectively reduce blood lipid and uric acid levels, reduce lipid droplet accumulation in the liver, alleviate liver lipid degeneration, and simultaneously upregulate the expression of intestinal tight junction proteins, repair intestinal barrier damage, enrich beneficial intestinal bacteria and inhibit the proliferation of harmful bacteria. Its metabolic regulation and intestinal care effects are significantly better than those of sucrose and single natural sweeteners.

[0018] The compound sweetener of this invention has excellent compatibility with sweet orange juice matrix, which can slow down acidity and pH fluctuations during juice storage, reduce system sedimentation rate, and improve the physicochemical stability of juice during shelf life. At the same time, it significantly improves the bioavailability of polyphenols and hesperidin active substances in the juice, enhances the absorption and utilization rate of nutrients, inhibits the surge of aldehydes induced by sucrose and the generation of caramel off-flavor, preserves the natural original fruit aroma of juice during long-term storage, and simultaneously optimizes the nutritional and flavor quality of juice. Attached Figure Description

[0019] Figure 1 Scatter plot showing the synergistic effect and sensory evaluation of binary compound sweeteners.

[0020] Figure 2 Scatter plot of the synergistic effect of ternary compound sweeteners and the overall sensory score.

[0021] Figure 3 The effects of compounded natural sweeteners on mouse body weight, food intake, water intake, and blood glucose changes were investigated.

[0022] Figure 4 To investigate the effect of compound natural sweeteners on oral glucose tolerance in mice, (A) oral glucose tolerance test; (B) area under the glucose tolerance curve.

[0023] Figure 5 The effect of exposure to compound natural sweeteners on insulin tolerance in mice: (A) Insulin tolerance test; (B) Area under the insulin tolerance curve.

[0024] Figure 6 To investigate the effect of compound natural sweeteners on uric acid in mouse urine.

[0025] Figure 7 The effect of compound natural sweeteners on lipid droplet accumulation in mouse liver tissue was calculated (×200).

[0026] Figure 8 To formulate natural sweeteners to target tight junction proteins in mouse colon ZO-1 The impact.

[0027] Figure 9 To investigate the effects of compound natural sweeteners on the composition and distribution of intestinal flora at the phylum level in mice.

[0028] Figure 10 To investigate the effect of compound natural sweeteners on the species abundance at the genus level in the intestinal flora of mice.

[0029] Figure 11 The effects of compound natural sweeteners on acidity, pH, and sedimentation during fruit juice storage were investigated.

[0030] Figure 12 To investigate the effect of compound natural sweeteners on the bioavailability of phenolic substances in fruit juice.

[0031] Figure 13 The effect of compound natural sweeteners on the main volatile substances in fruit juice.

[0032] Figure 14 The effect of fruit juice containing compound natural sweeteners on blood glucose in mice. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0034] This invention provides a compound natural sweetener. The formula specifies the mass ratio of each sweetener in the compound system, and the overall addition amount can equivalently replace the sweetness of 10% sucrose. That is, after compounding according to this ratio, adding it to fruit juice can achieve a sweetness level comparable to a 10% sucrose solution. The mass fractions of each sweetener in the sweetener system are: erythritol 6.11%, mogroside 0.013%, and thomaside 0.00067%.

[0035] The preparation method of the above-mentioned compound natural sweetener is as follows: Steps: 1) Pass each raw material through a 100-mesh sieve; 2) First, premix mogrosides, sematriol and 1 / 10 erythritol for 12 minutes; 3) Add the remaining erythritol and mix for 25 minutes; 4) Sterilize with ultraviolet light for 30 minutes, then seal to obtain the finished product.

[0036] The above-mentioned compound natural sweetener can be used in low-sugar fruit juices. The specific operating steps are as follows: Add the above-mentioned compound natural sweetener to the juice to achieve an equivalent 10% sucrose sweetness, then blend, homogenize, sterilize, and fill. Product characteristics: Blood glucose IAUC is not significantly different from pure juice; acidity, pH, and precipitation rate remain stable after 8 weeks of storage at 25℃; bioavailability of hesperidin and total phenols is improved; aroma is close to that of the original juice, resulting in high preference ratings.

[0037] The following is a verification of the effects of the compound natural sweetener of the present invention on lowering uric acid and regulating intestinal flora: Experiment 1. Construction and sensory evaluation of a binary compound system of natural sweeteners Erythritol (Er), steviol glycosides (St), mogrosides (Mo), monopotassium glycyrrhizate (Mg), and thomatose (Th) were used as research subjects. These five natural sweeteners were paired and mixed in mass ratios of 2:8, 4:6, 6:4, and 8:2, so that the total sweetness of each mixture was equivalent to a 10% sucrose solution. The synergy coefficient of each binary compound system was calculated. The synergy coefficient (SC) was determined using the isosweetness method. A sample solution with an equivalent sweetness of 10% sucrose was prepared using Wahaha purified water. The sweetness of the 10% sucrose solution was used as the total sweetness, and the theoretical sweetness of each sweetener was allocated to it. Based on this, the concentration of each sweetener in the compound system was calculated. The formula for calculating the synergy coefficient (SC) is as follows:

[0038] The sweetness of compound sweeteners was determined using quantitative descriptive analysis. Sucrose solutions of 5%, 9%, 10%, 13%, 15%, and 17% (w / v) were used as reference solutions, corresponding to sweetness values ​​of 5, 9, 10, 13, 15, and 17, respectively. The sweetness evaluation scale ranged from 1 to 20 points. All sensory evaluation experiments were performed in triplicate. Results for erythritol (Er), steviol glycosides (St), mogrosides (Mo), monopotassium glycyrrhizate (Mg), and thomatide (Th) are shown in [reference needed]. Figure 1 .

[0039] Depend on Figure 1 It can be seen that, except for Th, the other four sweeteners all exhibited certain synergistic effects in pairs. Among them, the Er (8.07%) + Mo (0.017%), Er (8.07%) + St (0.017%), and Mo (0.039%) + St (0.007%) compound systems showed good sensory compatibility and their sweetness characteristics were closer to sucrose.

[0040] Experiment 2: Construction and sensory evaluation of a ternary compound system of natural sweeteners Based on the synergy coefficient, three binary compound combinations with the most significant synergistic effects were screened for subsequent formulation development. Subsequently, the three selected binary compound combinations were combined with a third sweetener to form ternary compound systems, mixed at mass ratios of 2:8, 4:6, 6:4, and 8:2, to develop compound systems with richer flavor characteristics and superior quality. The optimal compound formulation was selected by combining the synergy coefficient and sensory evaluation scores. All evaluations were performed in triplicate. Results are shown below. Figure 2 .

[0041] Depend on Figure 2 It is evident that, based on this, these three groups can be further combined with other sweeteners to develop a variety of flavored compound sweeteners. Through screening the above sweetener mixtures, four formulations with excellent synergistic effects and economic benefits were selected: 1) Er(6.11%)+Mo(0.013%)+Th(0.00067%); 2) Er(6.11%)+Mo(0.012%)+St(0.013%); 3) Mo(0.032%)+St(0.0057%)+Mg(0.011%); 4) Mo (0.039%) + St (0.007%).

[0042] Mouse rearing methods: Healthy male C57BL / 6 mice (specific pathogen-free, 5 weeks old) were purchased from the Experimental Animal Center of Shaanxi Normal University. After acclimatizing for one week under standard housing conditions (12 / 12h light / dark cycle, 25±2℃, 60±5% relative humidity), the mice were randomly divided into 12 groups (n=8). A uniform blank control was used for the animal experiment. The group names and Chinese and English abbreviations were uniformly defined (abbreviations are shown in Table 1). The control group (Control) drank sterile water, while the other groups drank a sweetener solution of a certain concentration. Using sucrose as a reference, the equivalent sweetness was uniformly set at 3% sucrose sweetness. The estimated water intake was 4 mL / day, and the amount of sweetener added was adjusted according to the water intake to ensure uniform sweetness. The exposure time was 8 weeks.

[0043] All subsequent detection indicators and experimental analyses in this study were conducted based on the mice that had undergone the aforementioned feeding and treatment.

[0044] Table 1 Experimental Groups and Treatments

[0045] Experiment 3: Effects of compound natural sweeteners on blood glucose levels in mice Based on the above-described feeding method, fasting blood glucose levels (accurate to 0.1 mmol / L) were measured weekly during the feeding period. Mice were fasted for 12 hours but allowed free access to water. Blood was collected from the tail of a lancet to measure fasting blood glucose levels. Results are shown below. Figure 3 .

[0046] Depend on Figure 3 It can be seen that neither the single treatment group nor the compound treatment group of each sweetener caused abnormal fluctuations in fasting blood glucose in mice.

[0047] Experiment 4: Effect of compound natural sweeteners on oral glucose tolerance in mice Based on the mice raised as described above, an oral glucose tolerance test (OGTT) was performed at the end of week 7 of the mouse experiment. Mice were fasted but allowed free access to water for 12 hours. During the fasting period, all drinking water was replaced with sterile water. Mice were administered a 3g / kg glucose solution by gavage. Blood was collected from the tail of a lancet, and blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes. Blood glucose curves were plotted, and the area under the OGTT curve was calculated. Results are shown below. Figure 4 .

[0048] Depend on Figure 4 It was found that blood glucose levels in all groups reached their highest level at 15 minutes, then gradually decreased, returning to normal levels after 2 hours. No significant changes were observed in the other sweetener groups compared to the Control group.

[0049] Experiment 5: Effect of compound natural sweeteners on insulin tolerance in mice At the end of the mouse experiment, an insulin tolerance test (ITT) was performed. Mice were fasted for 4 hours but allowed free access to water. During the fasting period, all drinking water was replaced with sterile water. Mice were injected intraperitoneally with 0.75 U / kg insulin. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes. Blood glucose curves were plotted, and the area under the ITT curve was calculated. Results are shown below. Figure 5 .

[0050] Depend on Figure 5 It was found that after intraperitoneal injection of insulin, the blood glucose levels of mice in all groups showed a continuous decreasing trend until reaching the lowest value at 60 minutes, after which they began to rise. The area under the insulin tolerance curve in the sucrose group (Su) was significantly higher than that in the control group ( P <0.05; in contrast, the area under the insulin tolerance curve for other single-component treatments (Er, St, Mo, Mg, and Th) was not statistically different from that of the Control group. P >0.05 indicates that long-term sucrose exposure significantly reduces insulin sensitivity in mice and induces insulin resistance. Except for the sucrose group (Su), there was no statistically significant difference in the area under the insulin tolerance curve between the other compound natural sweetener groups and the Control group. P >0.05), indicating that exposure to the compound natural sweetener did not have a negative impact on the insulin sensitivity of mice, and its metabolic safety was comparable to that of the Control group; however, compared with other sweetener groups, the area under the insulin tolerance curve (AUC) of the erythritol + mogroside + sematriol (Er+Mo+Th) compound group (value 476.3) was closest to that of the Control group (484.1).

[0051] Experiment 6: Effects of compound natural sweeteners on urinary uric acid in mice Mouse urine was collected using sterile, enzyme-free tubes, and uric acid in the urine was detected using a fully automated biochemical analyzer. Results are shown below. Figure 6 .

[0052] Depend on Figure 6 It can be seen that, compared with the control group, there was no significant difference in urinary uric acid levels in mice treated with a single sweetener. P>0.05); while the uric acid levels in the four compound sweetener groups Er+Mo+Th (206.54±51.04), Er+Mo+St (216.20±65.04), Mo+St+Mg (202.70±61.98), and Mo+St (193.60±41.82) were significantly lower than those in the control group (298.38±56.64). Among them, the urinary uric acid level in the erythritol + mogroside + sematrandrol (Er+Mo+Th) compound group was significantly lower than that in the control group by 30.9%. P The value <0.05 indicates that this compound combination can effectively promote uric acid excretion, reduce uric acid accumulation in the body, and does not interfere with the homeostasis of kidney function, demonstrating good metabolic protection potential.

[0053] Experiment 7: Effects of compound natural sweeteners on fat accumulation in mouse liver tissue After the mouse experiment concluded, following an 8-week sweetener drinking water intervention in 12 treatment groups of male C57BL / 6 mice, the mice were uniformly sacrificed, and liver tissue was collected from each group. Liver tissue from the 12 treatment groups was prepared into approximately 1cm × 1cm pieces, fixed with 4% paraformaldehyde, and then frozen sections 6-10μm thick were prepared. After section preparation, the sections underwent fixation, Oil Red staining, background differentiation, hematoxylin staining, and section blocking. Images were then captured and analyzed using an inverted microscope at 200x magnification. Results are shown below. Figure 7 .

[0054] Depend on Figure 7 It was found that only a small number of lipid droplets were observed in the liver tissue of mice in the Control group, while the number and size of lipid droplets in the sucrose group (Su) were significantly increased, and they were distributed in obvious clusters, which is a typical morphological manifestation of fatty liver, suggesting that sucrose intervention may induce hepatic steatosis. Compared with the sucrose group (Su), only the mogroside + steviol glycoside + potassium glycyrrhizate (Mo+St+Mg) group and the erythritol + mogroside + sematate (Er+Mo+Th) group showed significantly reduced lipid droplet accumulation, which was close to the level of the control group. In particular, the erythritol + mogroside + sematate (Er+Mo+Th) group had almost no obvious lipid droplets. Compared with the Control group and the sucrose group (Su), the positive area of ​​lipid droplet accumulation in the liver of mice in this group was significantly reduced by 74.0% and 88.20%, respectively, indicating that this combination of sweeteners can effectively alleviate hepatic lipid deposition and exhibits the best protective effect against hepatic lipid metabolism.

[0055] Experiment 8: Effects of compound natural sweeteners on tight junction protein in mouse colon tissue ZO-1 Impact After the mouse experiment was completed, colon tissue samples from the 12 treatment groups were collected and fixed in 4% paraformaldehyde. Once the fixation was confirmed to be satisfactory, the samples were processed strictly according to the following steps: cutting, gradient dehydration, paraffin embedding, sectioning, staining, and sealing. After sample processing, panoramic section scanning was used for image acquisition. ImageJ software was used for quantitative analysis of the acquired images, measuring indicators such as the area of ​​inflammatory infiltration, the proportion of lipid droplets, the area of ​​positive expression, and the average optical density. The obtained data were used for subsequent statistical analysis and results discussion. See details below. Figure 8 .

[0056] Depend on Figure 8 It can be seen that the tight junction protein in the colon of the Control group mice... ZO-1 The sucrose group (Su) exhibits a continuous, clearly defined linear distribution along the boundaries of intestinal epithelial cells, indicating an intact intestinal barrier structure. ZO-1 Significantly weakened and fragmented signaling was observed, suggesting that sucrose intervention can disrupt the tight junction structure of the colon, leading to impaired intestinal barrier function. Compared with the sucrose group (Su), each compound sweetener group showed significantly reduced signaling. ZO-1 Expression intensity and continuity were close to those of the control group, indicating that the four groups of compound sweeteners maintained [the desired effect]. ZO-1 Expression and distribution showed superior intestinal safety compared to sucrose; among them, the erythritol + mogroside + thromycin (Er+Mo+Th) group was superior to the sucrose group (Su). ZO-1 The expression level increased the most, by 60.34%, and the colonic barrier was the most intact.

[0057] Experiment 9: Effects of compound natural sweeteners on the phylum-level community of intestinal flora in mice. After the mouse experiments were completed and the mice were euthanized, the contents of the colon and cecum were quickly dissected and scraped (fecal samples), approximately 200 mg per sample. These samples were placed in sterile cryovials, labeled with sample numbers, immediately flash-frozen in liquid nitrogen, and then transferred to a -80°C ultra-low temperature freezer for storage. After all samples were collected, 16S rRNA gene sequencing analysis was performed to elucidate the community structure and diversity characteristics of the gut microbiota. Results are shown below. Figure 9 .

[0058] Depend on Figure 9 It can be seen that the intestinal flora of mice is dominated by Bacteroidetes (Bacteroidetes phylum). Bacteroidota ) and Firmicutes ( Bacillota The th group showed an absolute advantage. Compared with the control group, the th group had a significantly higher prevalence of verrucous microbes (Th). Verrucomicrobiota The abundance of sematrandrine was significantly enriched (275.74%), suggesting that sematrandrine can be enriched through [the process]. Akkermansia muciniphila Strengthens intestinal barrier function. Erythritol + mogroside + sematriol group (Er+Mo+Th) Campylobacter phylum ( CampylobacterotaThe abundance of [a substance] increased significantly by 31.01%, which may exert its advantages in anti-inflammatory and in maintaining intestinal immune homeostasis by enhancing the production of short-chain fatty acids.

[0059] Experiment 10: Effects of compound natural sweeteners on the genus-level community of intestinal flora in mice. Depend on Figure 10 It can be seen that the core dominant bacterial genera in each group are uncultured bacterial genera. norank-f-Muribaculaceae), Ischemic bacteria (Allobaculum) Uncultured Prevotella (norank-f-Prevotellaceae), Duborella (Dubosiella) Clostridium (norank-f-Clostridia_UCG-014)、 genus *Trichophyton* (Lachnospiraceae- K4A136-group), genus Rocher (Roseburia) As a core butyric acid-producing bacterium, its abundance was significantly upregulated in the erythritol + mogroside + sematriol group (Er+Mo+Th) compared to the Control group; and *Proteus vulgaris* ( Faecalalimonas) and uncultured bacteria ( Lachnospiraceae_UCG-006) The abundance in the erythritol + mogroside + sematriol group (Er+Mo+Th) was generally similar to that in the Control group. (Rhodotorulaceae family) Coriobacteriaceae_UCG-002) It participates in bile acid and cholesterol metabolism. Its abundance was significantly downregulated in the erythritol + mogroside + sematriol group (Er+Mo+Th), suggesting that this compound sweetener combination may exert its cholesterol-regulating potential by modulating *Pseudomonas* species, and the perturbation amplitude is smaller than that of a single sweetener, further demonstrating its metabolic safety advantage. *Prevotella* spp. Prevotellaceae NK3B31 group) and candidate genera ( Candidatus Saccharimonas The abundance of SCFAs was significantly upregulated in the erythritol + mogroside + sematriol group (Er+Mo+Th), which may be achieved by promoting the generation of SCFAs and optimizing energy utilization efficiency.

[0060] Experiment 11: Effects of compound natural sweeteners on acidity, pH, and precipitation rate during storage. Using commercially available sweet orange concentrate as the raw material, a base juice with a sugar content of 6°Brix was prepared by diluting and fixing the volume with pure water. Sucrose was added to the base juice to obtain a sucrose control group juice with a sugar content of 12°Brix. For the other experimental groups, the equivalent sweetness was calculated based on the amount of sucrose added, and corresponding sweeteners (erythritol 3.67%, mogroside 0.0078%, thomaside 0.00041%) were added accordingly; this formulation can equivalently supplement 6% sucrose sweetness. After thorough mixing, the overall final sweetness of the juice reached 12°Brix. All prepared samples were stored at 25°C to simulate the product's shelf life environment, with a total storage time of 44 days.

[0061] (1) Determination of acidity: Accurately weigh 5.00 g of sweet orange juice sample into a 150 mL Erlenmeyer flask, add 100 mL of distilled water and 3 drops of 0.5% phenolphthalein indicator, and mix thoroughly. Titrate with 0.1001 mol / L sodium hydroxide standard solution until the solution turns pink and remains so for at least 30 s, and record the volume of sodium hydroxide standard solution consumed. Substitute into the calculation formula:

[0062] X-Total acid content in juice, g / 100g (or g / 100mL). V1 - The volume of sodium hydroxide standard solution consumed in the titration of the sample, in mL; V0 – The volume of sodium hydroxide standard solution consumed in the blank test, in mL; Concentration of C-sodium hydroxide standard solution, mol / L; m - Mass of the sample, in g (or mL); K-Conversion factor for the main acids in fruit juice; 0.07 (calculated as citric acid).

[0063] (2) pH value was measured using a pH meter.

[0064] (3) Determination of centrifugal sedimentation rate Accurately weigh 10 ml of sweet orange juice sample and centrifuge at 4000 r / min for 10 min. Discard the supernatant and accurately weigh the precipitate. Perform three parallel determinations for each sample and take the average value.

[88] The centrifugal sedimentation rate is calculated according to the following formula:

[0065] See results Figure 11 .

[0066] Depend on Figure 11 A, Figure 11 As shown in section B, all samples stored at 25℃ exhibited significant variations in acidity and pH, indicating that high temperatures drastically accelerate microbial metabolic activity. Under microbial action, sucrose is rapidly converted into organic acids such as lactic acid and acetic acid, directly leading to system acidification. Compared to the sucrose group (Su), the erythritol + mogroside + sematrandrol (Er+Mo+Th) compound sweetener group showed smaller changes in acidity and pH, indicating superior stability.

[0067] The physical stability of fruit juice is mainly reflected in the degree of dispersion of suspended particles, which can be evaluated by changes in centrifugal sedimentation rate. For example... Figure 11As shown in Figure C, the precipitation rate of the erythritol + mogroside + semathymidine (Er+Mo+Th) compound sweetener group was significantly lower than that of the control group (Control), sucrose group (Su), and erythritol + mogroside group (Er+Mo). In particular, on the 44th day of storage, the centrifugal precipitation rate of the erythritol + mogroside + semathymidine group (Er+Mo+Th) was reduced by 76.37%, 76.39%, and 72.98% compared with the control group (Control), sucrose group (Su), and sucrose + erythritol group (Su+Er), respectively. This indicates that the erythritol + mogroside + semathymidine sweetener combination (Er+Mo+Th) can effectively maintain the long-term stability of the fruit juice colloidal system.

[0068] Experiment 12: The effect of compound natural sweeteners on the bioavailability of phenolic substances in fruit juices. An in vitro digestion model simulating the oral cavity, stomach, and intestines was used to study the digestive stability of phenolic compounds. A blank sample without sweetener was also prepared and incubated under the same conditions for calibration in subsequent analyses.

[0069] Simulated oral digestion: Take 5 mL of sweet orange juice, add 3.5 mL of simulated saliva, 0.5 mL of α-amylase solution (1500 U / mL), and 25 μL of calcium chloride (0.3 M), and bring the volume to 10 mL with distilled water. Incubate in a constant temperature water bath at 37 °C for 2 min with continuous shaking. After simulated saliva digestion, take 2 mL of the sample from each sample.

[0070] Gastric stage: Add 6 mL of simulated gastric juice, 1.28 mL of pepsin solution (25000 U / mL), and 4 µL of calcium chloride (0.3 M) to the remaining digestive fluid after oral digestion. Adjust the pH to 3.0 with 6 M hydrochloric acid, and bring the volume to 8 mL with distilled water. Incubate at 37°C with continuous stirring for 2 h. After completion, take 2 mL aliquots from each sample.

[0071] Intestinal stage: 7.7 mL of simulated intestinal fluid, 3.5 mL of pancreatic enzyme solution (800 U / mL), 1.75 mL of bile solution (160 mM), and 28 µL of calcium chloride (0.3 M) were added to the remaining digestive fluid after gastric digestion. The pH was adjusted to 7.0 with 1 M sodium hydroxide, and the volume was brought up to 14 mL with distilled water. The mixture was incubated at 37 °C with continuous stirring for 2 h. After simulated intestinal digestion, the final 2 mL aliquots were collected.

[0072] All aliquots collected during the oral, gastric, and intestinal digestion stages were centrifuged at 23000×g for 10 min using a high-speed refrigerated centrifuge. After filtration of the supernatant, individual polyphenols were determined by high-performance liquid chromatography.

[0073] Chromatographic separation conditions: A Welchrom C18 column (150 mm × 4.6 mm, 5 µm) was used at a column temperature of 30 °C, a detection wavelength of 280 nm, and an injection volume of 20 µL. The mobile phase consisted of water-formic acid (99:1, v / v; A) and acetonitrile (B), and quantification was performed using the external standard method. Hesperidin standards were accurately weighed and dissolved separately in 1% formic acid aqueous solution, transferred to 10 mL amber volumetric flasks, and diluted to volume to prepare single stock solutions, which were stored at 4 °C protected from light. These stock solutions were mixed in appropriate proportions to prepare a series of mixed standard working solutions. Subsequently, under specified chromatographic conditions, the standard solutions were analyzed to determine the retention times, elution order, and relative retention times of phenolic compounds. Results are shown below. Figure 12 .

[0074] Depend on Figure 12 It can be seen that the combination of erythritol + mogroside + sematriol (Er+Mo+Th) achieved the highest bioavailability of hesperidin (66.98%) and total phenolic bioavailability (63.70%), significantly higher than the pure juice group (O), sucrose group (Su), and sucrose + erythritol group (Su+Er). P <0.05); Among them, the bioavailability of hesperidin and total phenols in the erythritol + mogroside + semathymidine group (Er+Mo+Th) was significantly higher by 22.76% and 29.52% compared with the sucrose + erythritol group (Su+Er), respectively.

[0075] Experiment 13: The effect of fruit juice containing compound natural sweeteners on the main volatile components The effects of compound sweeteners on specific aroma compounds in fruit juice were evaluated using gas chromatography-mass spectrometry. 4 mL of sweet orange juice sample and 1 g of sodium chloride were added to a 20 mL headspace vial, followed by 1 µL of 2-octanol (internal standard, 5 μg / mL, prepared in ethanol), and the vial was immediately sealed with a septum.

[0076] Extraction conditions: Equilibration at 40℃ for 20 min, solid-phase microextraction (SPME) fiber extraction for 30 min, desorption at 250℃ for 5 min before injection. Gas chromatography conditions: DB-WAX column; splitless injection; helium (99.99%) as carrier gas; flow rate 1.0 mL / min. Column temperature program: Hold at 40℃ for 5 min, increase to 120℃ at 5℃ / min and hold for 10 min, then increase to 220℃ at the same rate and hold for 5 min. Mass spectrometry conditions: Electron ionization (EI) mode; electron energy 70 eV; ion source temperature 230℃; transfer line temperature 230℃; mass scan range 35-500 m / z; solvent delay 3 min.

[0077] Aroma compound identification: The mass spectra of unknown volatile compounds were compared with those in the NIST14 and NIST14s databases. The relative abundance of each aroma compound was calculated using peak area normalization. See results below. Figure 13 .

[0078] Depend on Figure 13 It was found that after adding sucrose (Su) to the fruit juice, the aldehyde content increased dramatically, soaring from 16.85% to 67.04%. Correspondingly, the relative contents of other categories such as alcohols, esters, and carboxylic acids decreased significantly, resulting in a severe disruption of the natural flavor balance. Compared with other sweetener groups, including the sucrose group (Su), the erythritol + sucrose group (Er + Su), the mogroside + steviol glycoside + monopotassium glycyrrhizate group (Mo + St + Mg), and the mogroside + steviol glycoside group (Mo + St), the erythritol + mogroside + thomatose group (Er + Mo + Th) showed a significant reduction in volatile aldehydes, and was close to that of the pure fruit juice control group. The proportions of all volatile compound categories were relatively balanced, indicating that this compound sweetener can retain the original fruit flavor of the fruit juice to the greatest extent.

[0079] Experiment 14: Effect of fruit juice containing compound natural sweeteners on postprandial blood glucose in mice. Sweeteners for each group (glucose group, pure fruit juice group, sucrose group, erythritol + sucrose group, erythritol + mogroside + sematriol group, erythritol + steviol glycoside + mogroside group, mogroside + steviol glycoside + monopotassium glycyrrhizate group, mogroside + steviol glycoside group) were added to the fruit juice respectively. The volumetric flasks were brought to 10 mL. Mice were fasted for 10 h, with free access to water. Weight was recorded, and fasting blood glucose levels were measured using a blood glucose meter via tail sampling. Subsequently, the prepared solution was administered orally at a dose of 10 mL / kg body weight. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after administration. Blood glucose response curves were plotted, and the area under the curve (IAUC) was calculated. Results are shown below. Figure 14 .Depend on Figure 14 It can be seen that the area under the curve (IAUC) of the sweetener in the erythritol + mogroside + sematriol group (O+Er+Mo+Th) was the lowest, and significantly lower than that in the sucrose group (O+Su) with a decrease of 38.40%, indicating that adding compound sweeteners can achieve the goal of enjoying sweetness without raising blood sugar.

[0080] In summary, this invention selects five natural sweeteners permitted for use under GB2760-2024 "National Food Safety Standard for the Use of Food Additives" as experimental raw materials, constructs a sweetener dosage-sweetness correspondence evaluation system, and explores the synergistic effect between components based on binary and ternary compound experiments, optimizing compound formulation schemes and screening the optimal compound combination. This invention utilizes the synergistic effect of sweet components to eliminate the bitterness and off-flavor defects inherent in single sweeteners, optimizes the sweetness release curve, and makes the sweetness profile of the compound system highly similar to sucrose. The raw materials are compliant and meet standards, the preparation process is simple, the powder is uniformly mixed without layering, and it is suitable for large-scale industrial food production. Animal experiments have verified that this compound natural sweetener has excellent metabolic safety; long-term intake will not interfere with the body's basic physiological state and liver and kidney function, can avoid insulin resistance problems, effectively lowers blood lipid and uric acid levels, and reduces lipid droplet accumulation in the liver; simultaneously, it upregulates intestinal tight junction proteins. ZO-1 , Occludin This compound sweetener exhibits superior performance in regulating metabolism and maintaining gut health compared to sucrose and single natural sweeteners. When applied to sweet orange juice, it effectively inhibits acidification and precipitation during storage, improves the juice's physicochemical stability, and does not increase postprandial glycemic load. Simultaneously, it enhances the bioavailability of polyphenols, hesperidin, and other active substances within the juice, optimizes the volatile aroma components, inhibits the abnormal formation of caramel off-flavors and aldehydes, and preserves the juice's original fruity aroma during long-term storage. This invention completes the development of a healthy sucrose alternative compound sweetener, achieving a synergistic effect in sweetness while considering product flavor, storage stability, food safety, and nutritional benefits. It provides a solid theoretical basis and technical support for the industrialization research and application of low-sugar healthy fruit juices.

Claims

1. A compound natural sweetener, characterized in that, Based on the total mass of the compound sweetener, the component ratio includes: erythritol: mogroside: sematriol sweetness = 6.11: 0.013: 0.00067.

2. The compound natural sweetener according to claim 1, characterized in that, The compound natural sweetener is suitable for acidic fruit juice bases. Its sweetness does not decrease under long-term storage at 25°C and it does not induce acidification, precipitation or deterioration of the fruit juice system.

3. The method for preparing the compound natural sweetener according to claim 1, characterized in that, The specific steps are as follows: Take the appropriate amounts of erythritol, mogroside, and thomaline, sieve them, and obtain a homogeneous powder. The sieved mogrosides, sematriol and 1 / 10 erythritol were premixed to prepare a premix; Add the remaining sieving erythritol to the premix, mix well, sterilize and seal to obtain a compound natural sweetener.

4. The application of the compound natural sweetener according to any one of claims 1 to 3 in the preparation of metabolic regulation functional foods, characterized in that, The compounded natural sweetener serves as a sweetener substitute, imparting physiological effects such as low post-meal sugar load and regulation of lipid metabolism in food.

5. The application according to claim 4, characterized in that, The compound natural sweetener inhibits insulin resistance induced by exogenous sugars, maintains insulin sensitivity, and reduces blood sugar fluctuations after consumption.

6. The application according to claim 4, characterized in that, The compound natural sweeteners reduce the accumulation of uric acid in the body's urine, reduce lipid droplet deposition in the liver, and alleviate liver lipid degeneration damage.

7. The application of the compound natural sweetener according to any one of claims 1 to 3 in the preparation of intestinal homeostasis maintenance functional foods, characterized in that, The compounded natural sweetener has a dual effect of intestinal barrier repair and intestinal flora structure regulation.

8. The application according to claim 7, characterized in that, The compound natural sweetener upregulates colonic tight junction protein. ZO-1 It expresses the level of repairing the damage to the intestinal epithelial barrier caused by the sweet matrix of food.

9. The application according to claim 7, characterized in that, The compounded natural sweetener enriches beneficial butyrate-producing bacteria and mucus-degrading probiotics in the gut, downregulates the abundance of harmful bacteria that promote metabolic disorders in the gut, and optimizes the gut microbiota community structure.

10. The application of a compound natural sweetener according to any one of claims 1 to 3 in enhancing the retention of nutrients and stabilizing the flavor of fruit juice, characterized in that, The compound sweeteners synergistically enhance the bioavailability of total phenols and hesperidin in fruit juice, while inhibiting the formation of aldehydes and other off-flavors during fruit juice storage, thus locking in the natural fruit aroma of the juice.

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