Compound chocolate with fat-reducing effect and preparation method thereof

Abstract

The application discloses a compound chocolate with a fat-reducing effect and a preparation method thereof, and raw materials of the compound chocolate include pure black chocolate, pure milk, erythritol, dihydromyricetin and berberine, and the proportion is 50-70 mL:5-40 mL:10-45 g:0.3-2.5 g:0.3-2.5 g. The compound chocolate can significantly reduce the weight of inguinal adipose tissue and the diameter of adipocytes of obese mice, and the reduction amplitude of the compound chocolate is greater than the linear addition of the reduction amplitudes of the DB group and the EMC group, which indicates that the black chocolate base and dihydromyricetin and berberine have a synergistic effect beyond the simple superposition of respective effects. The compound chocolate has better palatability than high-purity black chocolate.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, specifically to a compound chocolate with fat-reducing effects and its preparation method. Background Technology

[0002] Chocolate is a traditional food made primarily from cocoa beans. Cocoa solids are rich in polyphenolic compounds such as flavanols and flavonoids, as well as alkaloids like theobromine, giving chocolate certain antioxidant, anti-inflammatory, insulin-sensitivity-improving, and fatty acid metabolism-regulating effects. Based on this, high-purity chocolate itself possesses certain potential for lipid metabolism regulation.

[0003] However, existing chocolate products generally suffer from the drawback of not being able to balance palatability and health benefits: on the one hand, in order to mask the bitterness of pure cocoa solids, most commercially available chocolates add large amounts of sucrose, lipids and industrial emulsifiers, which significantly increases energy density. Long-term consumption can easily lead to excess energy and fat accumulation, and the effective proportion of cocoa solids is diluted, thus impairing its health value; on the other hand, although high-purity dark chocolate with a cocoa content of more than 85% retains a high level of cocoa solids, it has a strong bitter taste, resulting in low consumer acceptance and long-term consumption compliance.

[0004] Dihydromyricetin is a dihydroflavonol compound reported to possess various biological activities, including antioxidant, anti-inflammatory, and hypoglycemic effects, with a relatively high safety profile. Berberine is a natural quaternary ammonium isoquinoline alkaloid reported to reduce intestinal lipid absorption and improve insulin resistance. Both are active ingredients in traditional Chinese medicine and possess inherent sensory qualities such as bitterness, making them compatible with chocolate. Furthermore, to improve chocolate flavor, natural and healthy flavoring ingredients such as milk and erythritol can be added. Milk, as a natural dairy product, can impart a milky aroma and a smoother texture to chocolate, effectively neutralizing the bitterness of high-purity cocoa solids. Erythritol, as a natural sweetener, provides sweetness with extremely low calories and has negligible impact on blood sugar and insulin levels. To date, there are no reports of combining dihydromyricetin and berberine with a dark chocolate base in a specific ratio to maintain chocolate flavor while synergistically enhancing weight-loss effects. Summary of the Invention

[0005] The purpose of this invention is to provide a compound chocolate with fat-reducing effects and its preparation method, thereby solving the problem that existing chocolate products cannot simultaneously achieve both "good sensory flavor" and "fat-reducing efficacy." The compound chocolate of this invention, while maintaining good flavor and texture, exhibits significantly better effects on improving blood lipid levels and inhibiting subcutaneous fat deposition than the combined effects of using each active ingredient individually, demonstrating a synergistic effect.

[0006] To achieve the above objectives, the present invention provides the following technical solution: On the one hand, the present invention provides a compound chocolate with fat-reducing effect, the raw materials of which include: pure dark chocolate, pure milk, erythritol, dihydromyricetin and berberine, in the ratio of 50-70 mL: 5-40 mL: 10-45g: 0.3-2.5g: 0.3-2.5g.

[0007] Furthermore, the cocoa solids content in the pure dark chocolate is not less than 85% by mass.

[0008] Furthermore, the pure milk has a protein content ≥3.2 g / 100 mL and a fat content ≥3.6 g / 100 mL.

[0009] Furthermore, the mass ratio of dihydromyricetin to berberine is 1:1.

[0010] Furthermore, the ratio of the pure dark chocolate, pure milk, erythritol, dihydromyricetin, and berberine is 55-65 mL: 30-40 mL: 10-16 g: 1.0-1.5 g: 1.0-1.5 g.

[0011] Furthermore, the ratio of the pure dark chocolate, pure milk, erythritol, dihydromyricetin, and berberine is 60 mL: 40 mL: 12 g: 1.2 g: 1.2 g.

[0012] On the other hand, the present invention also provides a method for preparing a compound chocolate, comprising the following steps: (1) Take pure dark chocolate and heat it at 50-55℃ until it is completely melted; (2) Add pure milk, erythritol, dihydromyricetin and berberine to the melted dark chocolate, and stir at 60-100 rpm for 3-5 minutes at 50-55℃ to obtain a chocolate mixture; (3) After sieving the chocolate mixture, shape it and let it stand at room temperature until it solidifies to obtain the compound chocolate.

[0013] Furthermore, the time for standing at room temperature in step (3) is 12-24 h.

[0014] Furthermore, in step (3), the sieving is performed through a 60-mesh sieve.

[0015] Furthermore, it also includes: filling the sieved chocolate mixture from step (3) into a mold or 3D printing cylinder before solidification.

[0016] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: (1) Synergistic fat reduction effect: Animal experiments showed that compared with the intake of the same dose of dihydromyricetin + berberine (DB group) or the artificial sweetener chocolate without Chinese medicine active ingredients (EMC group), the weight of inguinal adipose tissue (iAT) and the diameter of adipocytes in obese mice treated with the compound chocolate of the present invention (EMC-DB group) were significantly reduced. Moreover, the reduction was greater than the linear sum of the reductions in the DB group and the EMC group, indicating that the combination of dark chocolate base and dihydromyricetin + berberine produced a synergistic effect that went beyond the simple superposition of their individual effects.

[0017] (2) Improve blood lipid status: The compound chocolate of the present invention can significantly reduce the serum triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels in obese mice (P<0.05).

[0018] (3) Good palatability: Milk and erythritol effectively neutralize the bitterness of high-purity cocoa solids. Sensory evaluation shows that the milky aroma and sweetness scores of the compound chocolate of the present invention are significantly higher than those of pure dark chocolate, while the bitterness, sourness and hardness scores are significantly reduced, the moisture content is improved, and the overall taste score is significantly better than that of pure dark chocolate (P<0.05).

[0019] (4) Good safety: Acute toxicity test showed that the compound chocolate of the present invention had no significant effect on mouse blood cell indicators, liver / heart / kidney function serological indicators and major organ tissue morphology. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0021] Figure 1 These are top and front views of chocolates containing different proportions of milk according to Embodiment 2 of the present invention; Figure 2 These are top and front views of chocolate containing different proportions of erythritol according to Embodiment 3 of the present invention; Figure 3 These are top and front views of chocolates containing different proportions of active ingredients from traditional Chinese medicine, as described in Example 4 of this invention. Figure 4 These are top views, front views, and cross-sectional photographs of chocolates prepared at different stirring times according to Example 5 of the present invention; Figure 5 These are top views, front views, and cross-sectional photographs of chocolates prepared at different stirring speeds according to Example 6 of the present invention; Figure 6 These are the serum TC(a), TG(b), HDL-C(c), and LDL-C(d) levels of mice in different intervention groups in Example 7 of this invention; Figure 7 These are representative photographs of isolated inguinal adipose tissue (iAT) and epididymal adipose tissue (eAT) from mice in different intervention groups in Example 7 of this invention; Figure 8 These are H&E staining images of iAT and eAT tissues from mice in different intervention groups in Example 7 of this invention; Figure 9 The sensory evaluation radar chart (a) and acceptability rating chart (b) of DC and EMC-DB chocolates in Embodiment 8 of the present invention are shown. Figure 10 The levels of hemoglobin (a), erythrocytes (b), and platelets (c) in mice in different intervention groups in Example 9 of this invention; Figure 11 The serum TBIL(a), ALT(b), LDH(c), CK(d), UREA(e), and UA(f) levels of mice in different intervention groups in Example 9 of this invention; Figure 12 These are H&E staining images of heart, lung, liver, spleen, kidney, and stomach tissues from mice in different intervention groups according to Example 9 of this invention. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Pure dark chocolate: Commercially available with a cocoa solids content of ≥85%, purchased from Valrhona; Pure milk: Commercially available whole milk sterilized with a protein content ≥3.2 g / 100 mL and a fat content ≥3.6 g / 100 mL, purchased from Mengniu; Erythritol: Standard grade, purity ≥99.0%, purchased from Sigma-Aldrich; Dihydromyricetin: purity ≥95.0% (detected by HPLC), purchased from Sigma-Aldrich; Berberine: Berberine Hydrochloride, purity ≥99.0%, purchased from Sigma-Aldrich.

[0028] Example 1: Basic Preparation of Compound Chocolate Weigh out pure dark chocolate (99.0% cocoa solids content) and place it in a beaker. Heat at 50°C until completely melted. The density of molten dark chocolate at 50°C is approximately 1.267 g / mL, and the density of pure milk is approximately 1.030 g / mL. Measure 60 mL of the molten dark chocolate, add 40 mL of pure milk, 12 g of erythritol, 1.2 g of dihydromyricetin, and 1.2 g of berberine. Stir at 50°C and 60 rpm for 4 minutes until uniformly combined. Pass the resulting chocolate mixture through a 60-mesh sieve and pour it into a mold. Let it stand at room temperature for 24 hours until solidified to obtain the finished composite chocolate product.

[0029] Example 2: The effect of milk dosage on product appearance Weigh out pure dark chocolate (99.0% cocoa solids content) and place it in a beaker. Heat at 50°C until completely melted. Set the mixing ratio of dark chocolate to milk as follows: 60:0, 60:5, 60:20, 60:40, 60:60, 60:80 (mL:mL). The remaining procedures are the same as in Example 1. The results are as follows... Figure 1As shown, with the increase of milk ratio, the color of chocolate gradually changes from pure black to brown. When the milk ratio is 0-40 mL (based on 60 mL of dark chocolate), the product has a uniform appearance without obvious color patches or spots; when the milk ratio exceeds 40 mL, the product color changes significantly from pure black to brown. Therefore, the preferred milk amount is 5-40 mL, more preferably 20-40 mL.

[0030] Example 3: Effect of erythritol dosage on product appearance Weigh out pure dark chocolate (99.0% cocoa solids content) and place it in a beaker. Heat at 50°C until completely melted. Set up a gradient with 60 mL of dark chocolate, 20 mL of milk, and 16 g, 24 g, 32 g, 40 g, and 48 g of erythritol. After passing the prepared chocolate sample through a 60-mesh sieve, fill it into a silicone mold and let it stand at room temperature for 24 hours until it solidifies. Demold the mold and observe its appearance.

[0031] The results are as follows Figure 2 As shown, when the ratio of erythritol to the liquid phase is ≤0.5 g / mL, the appearance is uniform; when it reaches 0.6 g / mL, brown spots appear on the product surface (possibly due to supersaturation precipitation). Therefore, the ratio of erythritol mass to the total volume of dark chocolate and milk is limited to 0.1-0.5 g / mL.

[0032] Example 4: Effect of dosage of active ingredients in traditional Chinese medicine on product appearance Weigh out pure dark chocolate (99.0% cocoa solids content) and place it in a beaker. Heat at 50°C until completely melted. Set up a gradient with 60 mL of dark chocolate, 20 mL of milk, 16 g of erythritol, and 0.4 g, 0.8 g, 1.2 g, 1.6 g, and 2.0 g of dihydromyricetin and berberine (each with a concentration of 5-25 mg / mL). After passing the prepared chocolate sample through a 60-mesh sieve, fill it into a silicone mold and let it stand at room temperature for 24 hours until it solidifies. Demold the mold and observe its appearance.

[0033] The results are as follows Figure 3 As shown, within the concentration range of 5-25 mg / mL, as the ratio of dihydromyricetin to berberine increased, the appearance of the chocolate did not change significantly in color, and no spots or patches appeared, indicating good uniformity.

[0034] Example 5: Effect of stirring time on dispersion uniformity Weigh out pure dark chocolate (99.0% cocoa solids content) and place it in a beaker. Heat at 50°C until completely melted. Add milk, erythritol, dihydromyricetin, and berberine according to the following ratio: pure dark chocolate (mL): milk (mL): erythritol (g): dihydromyricetin (g): berberine (g) = 60:40:12:1.2:1.2.

[0035] Chocolate samples were prepared by stirring at 50℃ and a constant speed of 60 rpm for 1, 2, 3, 4, and 5 minutes respectively. The chocolate samples were then passed through a 60-mesh sieve, filled into molds, and left at room temperature for 24 hours until solidified. After demolding, the samples were cut open to observe their uniformity.

[0036] The results are as follows Figure 4 As shown, when stirred for 1-2 minutes, the cross-section showed agglomeration and uneven distribution of Chinese herbal medicine particles; when stirred for ≥3 minutes, all components were fully mixed, with no obvious particles, spots, or patches. This result indicates that a stirring time of ≥3 minutes is a necessary condition to ensure the uniform dispersion of active ingredients in the lipid matrix.

[0037] Example 6: Effect of stirring speed on dispersion uniformity Weigh out pure dark chocolate (99.0% cocoa solids content) and place it in a beaker. Heat at 50°C until completely melted. In the beaker, add milk, erythritol, dihydromyricetin, and berberine in the following ratio: pure dark chocolate (mL): milk (mL): erythritol (g): dihydromyricetin (g): berberine (g) = 60:40:12:1.2:1.2.

[0038] Chocolate samples were prepared by adjusting the stirring speed to 20, 40, 60, 80, and 100 rpm at 50℃ for a fixed stirring time of 4 minutes. The chocolate samples were then passed through a 60-mesh sieve, filled into molds, and left at room temperature for 24 hours until solidified. After demolding, the samples were cut open to observe their uniformity.

[0039] The results are as follows Figure 5 As shown, at a rotation speed of 20-40 rpm, the cross-section showed agglomeration of Chinese herbal medicine particles, which failed to fully dissolve in the chocolate, potentially affecting the subsequent efficacy. At a rotation speed of 60-100 rpm, the dispersion was uniform, and no obvious particles, spots, or patches were observed. This result indicates that a rotation speed of 60-100 rpm is a necessary condition to ensure uniform dispersion.

[0040] Example 7: Efficacy Evaluation Samples: EMC-DB (ratio: 60 mL pure dark chocolate, 40 mL milk, 12 g erythritol, 1.2 g dihydromyricetin, 1.2 g berberine); EMC (ratio: 60 mL pure dark chocolate, 40 mL pure milk, 12 g erythritol).

[0041] Methods: Twenty 6-8 week old male C57BL / 6 mice were fed a 60% high-fat diet for 12 weeks to establish an obesity model. They were randomly divided into 4 groups (n=5), with 5 mice in each group, and the intervention lasted for 28 days. Control group: 60% high-fat diet; DB group: Daily intake of dihydromyricetin 18 mg + berberine 18 mg (equal to the EMC-DB group) + 60% high-fat diet; EMC group: Daily intake of 1.5 mL EMC + 60% high-fat diet; EMC-DB group: Daily intake of 1.5 mL EMC-DB + 60% high-fat diet.

[0042] (1) Effects on blood lipids in mice After the intervention, mice were fasted for 12 hours, anesthetized, and blood was collected from the retro-orbital venous plexus. After standing for 2-4 hours, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant serum was collected and stored at -20°C for subsequent determination of serum-related indicators. The levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in mouse serum were measured using a fully automated biochemical analyzer.

[0043] The results are as follows Figure 6 As shown, compared with the control group, the serum TG and LDL-C levels in the EMC-DB group and the serum TG level in the EMC group were significantly decreased (P<0.05); the TC and HDL-C levels in each group showed no significant changes (P<0.05). Figure 6 This indicates that compound chocolate can improve lipid metabolism to some extent.

[0044] (2) Effects on mouse adipose tissue weight and adipocyte morphology After blood collection from mice, they were euthanized by cervical dislocation. Inguinal adipose tissue (iAT) and epididymal adipose tissue (eAT) were harvested. The tissues were rinsed with pre-cooled physiological saline to remove blood, blotted dry with filter paper, photographed, and weighed. Subsequently, specific amounts of iAT and eAT were fixed in 4% paraformaldehyde, dehydrated stepwise with an ethanol-water solution of increasing concentration, cleared with xylene, and embedded in paraffin to obtain tissue blocks. The paraffin-embedded tissue blocks were cut into approximately 5 μm sections using a microtome. After dewaxing with xylene, they were rehydrated stepwise with an ethanol-water solution of decreasing concentration, and then stained with hematoxylin and eosin (H&E). The stained sections were again dehydrated and cleared with an ethanol-water solution of increasing concentration and xylene, and finally mounted with neutral resin. The obtained adipose tissue sections were observed and photographed under an optical microscope to examine their structure and cell morphology.

[0045] Table 1. Weights of isolated iAT and eAT in mice from different intervention groups (n = 5)

[0046] Note: a. indicates a statistically significant difference compared to the control group, P<0.05; b. indicates a statistically significant difference compared to the EMC-DB group, P<0.05.

[0047] like Figure 7 As shown in Table 1, there was no significant difference in eAT weight among the groups; the iAT weight of the DB, EMC, and EMC-DB groups was significantly lower than that of the control group (P<0.05). Key results: The iAT weight of the EMC-DB group (1.15±0.68 g) was significantly lower than that of the DB group (2.25±0.28 g) and the EMC group (2.31±0.67 g) (P<0.05); the decrease in iAT weight of the EMC-DB group (2.03 g) relative to the control group was greater than the linear sum of the decreases in the DB group (0.93 g) and the EMC group (0.87 g) (1.80 g), indicating that the dark chocolate base and the active ingredients of traditional Chinese medicine have a synergistic effect in inhibiting subcutaneous fat growth.

[0048] Table 2. Cell diameters of isolated iAT and eAT cells in mice from different intervention groups (n = 200)

[0049] Note: a. indicates a statistically significant difference compared to the control group, P<0.05; b. indicates a statistically significant difference compared to the EMC-DB group, P<0.05.

[0050] like Figure 8 As shown in Table 2, the diameter of iAT adipocytes in the EMC-DB group (77.21±31.66 μm) was significantly smaller than that in the control group, DB group, and EMC group (P<0.05). Moreover, the reduction in iAT adipocyte diameter in the EMC-DB group (87.19 μm) compared to the control group was greater than the sum of the reductions in the DB group (35.30 μm) and the EMC group (39.98 μm) (75.28 μm), further confirming the synergistic effect between the dark chocolate base and the active ingredients of traditional Chinese medicine.

[0051] The synergistic effect of this invention is speculated to be due to the following mechanisms: dihydromyricetin activates the AMPK pathway via the calcium signaling pathway; berberine activates the AMPK pathway via the cellular energy metabolism pathway; and the cocoa polyphenols (such as flavanols) abundant in dark chocolate can further amplify the downstream effects of AMPK via fatty acid oxidation. These three factors converge at multiple targets to achieve synergistic inhibition of subcutaneous fat growth. Furthermore, the lipid matrix environment of dark chocolate is conducive to improving the dispersion of the poorly water-soluble dihydromyricetin and berberine in the digestive tract, potentially increasing their bioavailability. It should be noted that the technical solution and scope of protection of this invention do not depend on the correctness of the above mechanism explanation.

[0052] To visually demonstrate the inventive contribution of the compound chocolate of this invention compared to the individual or simple combination of each active component, a quantitative analysis is conducted based on the experimental data of Example 7, focusing on the deviation between the expected and actual effects of the cumulative fat-reducing efficacy: 1. Analysis of the expected cumulative effect of fat loss Using inguinal adipose tissue (iAT) weight as the core indicator of fat reduction efficacy, the reduction in iAT weight of the DB group (treated with dihydromyricetin + berberine alone) and the EMC group (treated with artificial sweetener chocolate without Chinese herbal active ingredients alone) relative to the control group are considered as their independent contributions to fat reduction efficacy. The linear superposition of the two groups should be the arithmetic sum of the two reductions mentioned above.

[0053] Specifically: the weight reduction of iAT in the DB group relative to the control group was 3.18 - 2.25 = 0.93 g; the weight reduction of iAT in the EMC group relative to the control group was 3.18 - 2.31 = 0.87 g. If there is only a simple additive effect between the dark chocolate base and dihydromyricetin + berberine, then the weight reduction of iAT in the EMC-DB group relative to the control group should not exceed 0.93 + 0.87 = 1.80 g, that is, the weight of iAT in the EMC-DB group should not be less than 3.18 - 1.80 = 1.38 g.

[0054] 2. Comparison of actual fat loss efficacy and expected cumulative effect Table 1 of Example 7 shows that the actual iAT weight of the EMC-DB group was 1.15 ± 0.68 g, a decrease of 3.18 - 1.15 = 2.03 g compared to the control group. This actual decrease (2.03 g) was not only significantly greater than the decreases in the DB group (0.93 g) and the EMC group (0.87 g) (P < 0.05), but more importantly, 2.03 g > 0.93 g + 0.87 g = 1.80 g, meaning the actual fat-reducing efficacy exceeded the linear sum of the contributions from the DB group and the EMC group.

[0055] Similarly, cross-validation was performed using iAT adipocyte diameter as an indicator: the reduction in adipocyte diameter in the DB group compared to the control group was 164.40 - 129.10 = 35.30 μm; the reduction in the EMC group was 164.40 - 124.42 = 39.98 μm, and the sum of the two was 75.28 μm. The actual reduction in the EMC-DB group was 164.40 - 77.21 = 87.19 μm. 87.19 μm > 35.30 μm + 39.98 μm = 75.28 μm, further confirming the existence of a synergistic effect at the cellular level.

[0056] 3. Quantitative characterization of synergistic effects To quantify the degree of synergy, the Synergy Index (SI) is defined as the ratio of actual effectiveness to the expected combined effectiveness: SI = ΔEMC - DB / (ΔDB + ΔEMC). When SI > 1, it indicates the existence of synergy; when SI = 1, it is a simple summation; when SI < 1, it is antagonistic.

[0057] Using iAT weight as an indicator: SI1 = 2.03 / 1.80 ≈ 1.13; Using the diameter of iAT adipocytes as an indicator: SI2 = 87.19 / 75.28≈1.16.

[0058] Both SI1 and SI2 are greater than 1, exceeding the expected linear summation by approximately 13% and 16%, respectively. This indicates that the combination of dark chocolate base and dihydromyricetin + berberine produces a synergistic effect beyond the simple additive effect of their individual components. For the compounding of functional food components, this positive synergistic effect across multiple target metabolic pathways is something that those skilled in the art could not reasonably anticipate based on the known individual efficacy of each component.

[0059] 4. Synergistic characteristics of lipid improvement Example 7 Figure 6 The results further corroborated the aforementioned synergistic effect: compared with the control group, serum triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels in the EMC-DB group were significantly reduced (P<0.05), while intervention with DB or EMC alone failed to significantly improve both TG and LDL-C simultaneously. This result indicates that only when the dark chocolate base is combined with dihydromyricetin and berberine in the specific ratio of this invention can a comprehensive improvement on multiple lipid metabolism indicators be achieved, further ruling out a simple additive explanation of the independent effects of each component.

[0060] In summary, the compound chocolate of this invention combines dark chocolate base, dihydromyricetin and berberine in a specific ratio, which improves palatability and produces a synergistic fat-reducing effect that exceeds the linear sum of the effects of each component used alone, resulting in unexpected technical effects.

[0061] Example 8 Sensory Evaluation Sample: Dark chocolate (DC); EMC-DB.

[0062] Pure dark chocolate (DC): Take commercially available pure dark chocolate (same as in Example 1), melt it at 50°C until it is free of particles, fill it into the printing cylinder while it is still molten, shake it to remove the gas, let it stand at room temperature for 24 hours until it solidifies, and then 3D print it to obtain a DC cube.

[0063] EMC-DB: Take 60 mL of molten dark chocolate, 40 mL of pure milk, 12 g of erythritol, 1.2 g of dihydromyricetin, and 1.2 g of berberine in a beaker. Stir at 60 rpm for 3-5 minutes at 50℃ until uniformly blended. Pass the resulting chocolate mixture through a 60-mesh sieve, fill it into a printing cylinder, agitate to remove gas, and allow it to stand at room temperature for 24 hours until solidified. Then, 3D print the resulting EMC-DB cube.

[0064] Both samples were 3D printed into cubes to ensure shape consistency.

[0065] We are recruiting 24 volunteers (12 men and 12 women, aged 21-35). The inclusion / exclusion / removal criteria are as follows: Inclusion criteria: age 18-35, good health, and no history of serious or chronic illnesses; Exclusion criteria: history of allergy or severe adverse reactions to the main research ingredients such as cocoa, dairy products, or nuts; acute / chronic illnesses affecting smell, taste, or swallowing; consumption of strong spices or alcohol within the past 24 hours; or being pregnant or breastfeeding. Removal criteria: failure to complete the sensory evaluation questionnaire or experiencing adverse reactions.

[0066] Sensory evaluation analysis used a 5-point scoring system, with 1 point representing the lowest intensity and 5 points representing the highest intensity. Overall acceptability was also scored on a 5-point scale, with 1-5 points indicating acceptability from low to high.

[0067] The results are as follows Figure 9 As shown, compared with DC, EMC-DB showed significantly higher scores in shape, milky aroma, sweetness, and wettability, and significantly lower scores in gloss, bitterness, sourness, and hardness (P<0.05). Figure 9 a) The overall taste acceptability score was significantly better than DC (P<0.05). Figure 9 b). This demonstrates that the present invention significantly improves the palatability of high-purity dark chocolate.

[0068] Example 9 Safety Evaluation The compound chocolate (hereinafter referred to as EMC-DB) was prepared according to the optimal ratio (Example 1). Ten 6-8 week old male ICR mice were randomly divided into a control group (standard diet) and an EMC-DB group (1.5 mL EMC-DB + standard diet per day), with 5 mice in each group. The intervention lasted for 7 consecutive days, and the weight and condition of the mice were recorded daily.

[0069] (1) Assessment of hematologic cytotoxicity After the intervention, mice were fasted for 12 hours, anesthetized, and blood was collected from the retroorbital venous plexus into anticoagulant tubes. The whole blood in the anticoagulant tubes was thoroughly mixed with the anticoagulant and stored at 4°C for subsequent measurements. Hemoglobin (HGB), red blood cell (RBC), and platelet (PLT) levels in the mouse whole blood were measured using a blood cell analyzer.

[0070] During the observation period, the mice grew normally, no obvious poisoning reactions were observed, and no mice died. Blood cell indicators were as follows: Figure 10 As shown, there were no statistically significant differences in hemoglobin (HGB), red blood cell (RBC), and platelet (PLT) levels between the EMC-DB group mice and the control group.

[0071] (2) Organ toxicity assessment After the intervention, mice were fasted for 12 hours, anesthetized, and blood was collected from the retro-orbital venous plexus. After standing for 2-4 hours, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant serum was collected and stored at -20°C for subsequent determination of serum-related indicators. The levels of total bilirubin (TBIL), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine kinase (CK), urea (UREA), and uric acid (UA) in mouse serum were measured using a fully automated biochemical analyzer.

[0072] Serum biochemical indicators such as Figure 11 As shown, there were no statistically significant differences in serum TBIL, ALT (liver function), LDH, CK (cardiac function related), UREA, and UA (kidney function) levels compared to the control group.

[0073] After blood collection from mice, they were euthanized by cervical dislocation. Heart, lung, liver, spleen, kidney, and stomach tissues were collected. These tissues were rinsed with pre-cooled physiological saline to remove blood and other impurities, blotted dry with filter paper, and weighed. A portion of each tissue was fixed with 4% paraformaldehyde. The fixed tissues were dehydrated stepwise with an ethanol-water solution of increasing concentration, cleared with xylene, and then embedded in paraffin. The paraffin tissue blocks were cut into approximately 5 μm sections using a microtome. After dewaxing with xylene, the sections were rehydrated stepwise with an ethanol-water solution of decreasing concentration, and then stained with hematoxylin and eosin (H&E). The stained sections were again dehydrated and cleared with an ethanol-water solution of increasing concentration and xylene, and then mounted with neutral resin. The prepared tissue sections were observed and photographed under an optical microscope.

[0074] The results are as follows Figure 12 As shown, no pathological changes were observed in the heart, lung, liver, spleen, kidney, and stomach tissues after H&E staining, indicating that the EMC-DB group did not produce acute toxicity to the major organs of mice compared with the control group.

[0075] This indicates that no acute toxicity was observed in mice during the dosage and duration of this experiment using the compound chocolate of the present invention.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A compound chocolate with fat-reducing properties, characterized in that, Its ingredients include: pure dark chocolate, pure milk, erythritol, dihydromyricetin and berberine, in the following ratio: 50-70 mL: 5-40 mL: 10-45 g: 0.3-2.5 g: 0.3-2.5 g.

2. The compound chocolate with fat-reducing effect according to claim 1, characterized in that, The cocoa solids content in the pure dark chocolate shall not be less than 85% by mass.

3. The compound chocolate with fat-reducing effect according to claim 1, characterized in that, The pure milk has a protein content ≥3.2 g / 100 mL and a fat content ≥3.6 g / 100 mL.

4. The compound chocolate with fat-reducing effect according to claim 1, characterized in that, The mass ratio of dihydromyricetin to berberine is 1:

1.

5. The compound chocolate with fat-reducing effect according to claim 1, characterized in that, The ratio of pure dark chocolate, pure milk, erythritol, dihydromyricetin, and berberine is 55-65 mL: 30-40 mL: 10-16 g: 1.0-1.5 g: 1.0-1.5 g.

6. The compound chocolate with fat-reducing effect according to claim 5, characterized in that, The ratio of pure dark chocolate, pure milk, erythritol, dihydromyricetin, and berberine is 60 mL: 40 mL: 12 g: 1.2 g: 1.2 g.

7. A method for preparing the compound chocolate according to claim 1, characterized in that, Includes the following steps: (1) Take pure dark chocolate and heat it at 50-55℃ until it is completely melted; (2) Add pure milk, erythritol, dihydromyricetin and berberine to the melted dark chocolate, and stir at 60-100 rpm for 3-5 minutes at 50-55℃ to obtain a chocolate mixture; (3) After the chocolate mixture is sieved and shaped, it is left to stand at room temperature until it solidifies to obtain the compound chocolate.

8. The preparation method according to claim 7, characterized in that, In step (3), the time for standing at room temperature is 12-24 h.

9. The preparation method according to claim 7, characterized in that, In step (3), the sieving is done through a 60-mesh sieve.

10. The preparation method according to claim 7, characterized in that, Also includes: Before solidification, fill the sieved chocolate mixture from step (3) into a mold or 3D printing cylinder to form the mixture.

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