Gardenia seed meal gony-pine derivative complex with slow-release hypoglycemic activity and preparation method and application thereof

Abstract

This invention provides a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity, its preparation method, and its application. The complex consists of a core and a shell: the core is a hydrophobic complex formed by genipin-organic ester derivative and resistant starch, wherein the genipin-organic ester derivative is generated in situ by ultra-finely pulverizing gardenia meal to 400-500 mesh, inoculating it with a compound microbial agent, and fermenting it at pH 4.2-4.8 for 4-6 days; the shell is a hydrophilic gel layer formed by konjac glucomannan; the mass ratio of the core to the shell is (5-7):(3-5). This invention utilizes the enhanced lipid solubility of genipin derivatives to construct a dual sustained-release structure of "hydrophobic core-hydrophilic shell," exhibiting zero-order release characteristics in simulated gastrointestinal fluid, with a cumulative release rate >85% over 8 hours and a gastric release rate <15%. Animal experiments show that noodles containing the complex of this invention can reduce postprandial blood glucose AUC in diabetic mice by 41.9%, with an effective hypoglycemic time of 7.5 hours.

Description

Technical Field

[0001] This invention belongs to the field of food processing and functional food or pharmaceutical technology, specifically relating to a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity, its preparation method and application. Background Technology

[0002] Gardenia pulp is the main byproduct remaining after extracting gardenia oil, gardenia yellow pigment, and other substances from gardenia fruit, with a huge annual yield. Studies have shown that gardenia pulp still contains abundant geniposide, genipin, chlorogenic acid, flavonoids, and polysaccharides, among other bioactive components, exhibiting various pharmacological activities such as hypoglycemic, hypolipidemic, anti-inflammatory, and neuroprotective effects. Among these, geniposide, as the main iridoid component, can be converted to genipin under the action of β-glucosidase. Genipin is the main material basis for the hypoglycemic activity of gardenia and has a significant inhibitory effect on α-glucosidase.

[0003] However, most gardenia meal is currently used as feed raw material or directly disposed of as waste, resulting in low resource utilization and low added value. In the existing technology, although there are reports of using methods such as crushing, extraction or fermentation to process gardenia meal, the following technical bottlenecks exist: (1) Conventional crushing has a large particle size, insufficient cell wall disruption, and low dissolution rate of active ingredients; (2) Single fermentation treatment has limited conversion efficiency, and the conversion rate of genipin to genipin is usually less than 40%; (3) The active ingredients in the obtained product are easily destroyed or rapidly released in the gastrointestinal tract, making it difficult to achieve a sustained and effective hypoglycemic effect, and the duration of action is short (usually only 2-3 hours), which cannot meet the needs of diabetic patients for long-term regulation of postprandial blood glucose.

[0004] To address the oral bioavailability of genipin compounds, some studies have attempted to improve their stability using microencapsulation technology. However, existing encapsulation systems are mostly designed for the genipin prototype and fail to consider the differences in the physicochemical properties of its derivatives. Furthermore, encapsulation often only achieves simple delayed release and lacks precise control over release kinetics.

[0005] Therefore, developing a preparation method that can efficiently convert the active ingredients in gardenia meal and achieve long-term sustained release of the active ingredients, and applying it to functional foods, has important practical significance and application value. Summary of the Invention

[0006] Technical problem to be solved: In view of the above problems, the purpose of this invention is to provide a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity, its preparation method and application. This invention generates genipin-organic acid ester derivatives in situ through ultra-fine pulverization and synergistic directional fermentation, and then constructs a "hydrophobic core-hydrophilic shell" dual sustained-release system based on the physicochemical properties of the derivatives, thereby realizing the continuous release of genipin-like components and long-lasting hypoglycemic effect.

[0007] Technical solution: A gardenia meal genipin derivative complex with sustained-release hypoglycemic activity, the complex consisting of a core and a shell: The core is a hydrophobic complex formed by genipin-organic ester derivative and resistant starch; The outer shell is a hydrophilic gel layer formed by konjac glucomannan.

[0008] Furthermore, the genipin-organic ester derivative is obtained in situ through ultrafine grinding combined with lactic acid bacteria-dominated fermentation of gardenia meal.

[0009] Furthermore, the mass ratio of the core to the shell is (5~7):(3~5).

[0010] Furthermore, the genipin-organic ester derivative comprises at least one of genipin-lactic acid ester and genipin-acetic acid ester, the total content of which accounts for 8% to 15% of the dry weight of the complex.

[0011] Furthermore, the resistant starch is corn-derived or potato-derived resistant starch, of type RS3 or RS4.

[0012] Furthermore, the molecular weight of the konjac glucomannan is 5 × 10⁻⁶. 5 ~1.5×10 6 Da, purity ≥90%.

[0013] This invention also provides a method for preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity, comprising the following steps: Step 1: Modification of Gardenia Meal: Dry gardenia meal is ultra-finely pulverized to 400-500 mesh to obtain ultra-fine gardenia meal powder; the ultra-fine gardenia meal powder is mixed with sterile water, and the moisture content of the material is adjusted to 45%-55%; a compound microbial agent is inoculated, and the inoculated material is cultured and fermented at 35℃-39℃ for 4-6 days, controlling the pH value at 4.2-4.8 during fermentation; after fermentation, the material is dried and pulverized to obtain fermented gardenia meal powder rich in genipin-organic ester derivatives. Step 2, Extraction of active ingredients: Fermented gardenia meal powder rich in genipin-organic acid ester derivatives is extracted with ethanol aqueous solution. The extract is centrifuged or filtered, and the supernatant is collected and concentrated under reduced pressure to 1 / 5 to 1 / 10 of the original volume to obtain genipin derivative enrichment solution. Step 3, Core Preparation: Mix resistant starch with genipin derivative enrichment solution, stir evenly, and spray dry to obtain core micro powder; Step 4: Preparation of sustained-release complex: Mix the kernel micro powder with konjac glucomannan solution, and coat the mixture using fluidized bed coating technology. After coating, continue drying for 10-20 min to obtain the gardenia meal genipin derivative complex with sustained-release hypoglycemic activity.

[0014] Furthermore, the ultrafine pulverization described in step one is carried out under low-temperature conditions, with the material temperature controlled at 10℃~25℃.

[0015] Furthermore, the compound microbial agent mentioned in step one is composed of Lactobacillus plantarum, Saccharomyces cerevisiae, Bacillus subtilis, and Bacillus licheniformis. The inoculation amounts of each strain, based on the dry weight of ultrafine pulverized gardenia meal powder, are as follows: Lactobacillus plantarum 0.03%~0.05%, Saccharomyces cerevisiae 0.08%~0.12%, Bacillus subtilis 0.10%~0.15%, and Bacillus licheniformis 0.10%~0.15%.

[0016] Furthermore, during the fermentation process described in step one, an automatic pH control system is used. When the pH is below 4.2, it is adjusted to 4.5 with 0.1 M NaOH solution; when the pH is above 4.8, it is adjusted to 4.5 with 0.1 M lactic acid solution.

[0017] Furthermore, in step two, the volume fraction of ethanol in the aqueous ethanol solution is 40%~60%; the material-to-liquid ratio for extraction is 1:(10~20), the extraction temperature is 40~60℃, and the extraction time is 1~2 hours.

[0018] Furthermore, the extraction in step two is performed using ultrasound-assisted extraction with an ultrasound power of 200-400 W.

[0019] Furthermore, the mass ratio of the resistant starch to the genipin derivative enrichment solution in step three is 1:(2~4).

[0020] Furthermore, in step three, the inlet air temperature of the spray dryer is 140~160℃, the outlet air temperature is 70~90℃, and the feed flow rate is 10~20 mL / min.

[0021] Furthermore, the mass concentration of the konjac glucomannan solution in step four is 2%~5%; the mass ratio of the kernel micro powder to the konjac glucomannan solution is 1:(1~2).

[0022] Furthermore, the coating conditions described in step four are: inlet air temperature 50~70℃, atomization pressure 0.1~0.2 MPa, and coating weight gain 20%~30%.

[0023] This invention also provides the use of a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity in the preparation of functional foods or medicines.

[0024] Furthermore, the functional food is noodles, biscuits, meal replacement powder or solid beverage, wherein the amount of the complex added is 5% to 20% (based on the total dry weight of the functional food).

[0025] Furthermore, the method for preparing the noodles is as follows: by weight, take 70-90 parts of high-gluten wheat flour, 5-20 parts of gardenia meal genipin derivative complex with slow-release hypoglycemic activity, 2-5 parts of gluten powder, 0.5-2 parts of edible salt, 0.1-0.3 parts of edible alkali, and 25-35 parts of water, mix them evenly, and then proceed with the processes of kneading, cooking, pressing, cutting into strips, and drying to obtain nutritious noodles with slow-release hypoglycemic activity. Beneficial effects

[0026] 1. In-situ directed generation of genipin derivatives This invention achieves, for the first time, the in-situ directed generation of genipin-organic ester derivatives through a synergistic process of ultrafine grinding and lactic acid bacteria-led fermentation. Ultrafine grinding (400-500 mesh) significantly increases the specific surface area of ​​gardenia meal, fully exposing geniposide. In a specific ratio of compound microbial agents, *Lactobacillus plantarum* metabolizes to produce high concentrations of lactic acid, acetic acid, and other organic acids. *Bacillus subtilis* and *Bacillus licheniformis* secrete β-glucosidase to hydrolyze genipin into genipin. Under pH 4.2-4.8 conditions, genipin undergoes esterification with organic acids to generate genipin-lactate, genipin-acetic acid, and other derivatives. These derivatives were not detected in samples from conventional fermentation (without ultrafine grinding) or ultrafine grinding only (without fermentation). Their generation requires the synergistic effect of "ultrafine grinding + specific microbial ratio + pH control," demonstrating the uniqueness and unreplicability of the process.

[0027] 2. Design of a dual sustained-release structure based on derivative properties This invention is the first to discover that genipin derivatives have higher lipid solubility than genipin (logP value increased from 0.8 to 1.6~1.8), and based on this, a bilayer sustained-release structure of "hydrophobic core-hydrophilic shell" was designed: in the core, genipin derivatives and resistant starch form a stable complex (encapsulation rate ≥85%) through hydrophobic interaction, effectively protecting the active ingredient; in the shell, konjac glucomannan forms a hydrophilic gel network, controlling water penetration and the release rate of active ingredients; a gradient transition layer is formed between the core and the outer layer, achieving precise regulation of "small release for pre-activation in the stomach and continuous release for efficacy in the intestine".

[0028] 3. Excellent long-lasting blood sugar lowering effect In vitro release assays showed that the complex of this invention exhibited a release rate of <15% in simulated gastric fluid (pH 1.2) after 2 hours and zero-order release kinetics in simulated intestinal fluid (pH 6.8), with a cumulative release rate >85% after 8 hours, achieving sustained and stable release behavior. Animal experiments showed that noodles containing the complex of this invention reduced postprandial blood glucose AUC in STZ-induced diabetic mice by 42%, with an effective hypoglycemic effect lasting up to 7.5 hours, significantly superior to ordinary gardenia meal noodles (reduction of 18%, lasting 2.5 hours) and commercially available hypoglycemic noodles (reduction of 25%, lasting 3.0 hours).

[0029] 4. Good processing suitability and application prospects The compound of this invention is a micro-powdered solid with good flowability, which can be directly added to various food systems such as noodles, biscuits, and meal replacement powders, with minimal impact on the sensory quality of the final product. Taking noodles as an example, noodles made by adding 10%~15% of the compound of this invention have a cooking loss rate of ≤8%, a breakage rate of ≤5%, and a sensory score of over 85 points, exhibiting both excellent edible quality and clear functional activity.

[0030] 5. High-value utilization of gardenia meal resources This invention transforms gardenia meal, a byproduct of gardenia extraction, into a high-value-added functional ingredient through a three-stage synergistic technology of "ultra-fine grinding + directional fermentation + slow-release encapsulation". This achieves high-value resource utilization of waste and is in line with the concepts of green environmental protection and circular economy. Attached Figure Description

[0031] Figure 1 This is a flow chart of the preparation process of the gardenia meal genipin derivative complex of the present invention; the main process markers in the figure are as follows: 1-Gardenia meal raw material; 2-Ultra-fine grinding treatment; 3-Compound microbial inoculation; 4-pH-controlled solid-state fermentation; 5-Fermented gardenia meal rich in genipin derivatives; 6-Ethanol extraction; 7-Genipin derivative enrichment solution; 8-Resistant starch mixing; 9-Spray drying; 10-Core micronized powder; 11-Konjac glucomannan coating; 12-Fluidized bed coating; 13-End product complex; 14-Food applications such as noodles; Figure 2 Comparative HPLC-MS images of genipin and its derivatives under different fermentation conditions; where A represents conventional fermentation (80 mesh), B represents ultrafine grinding + conventional strains, and C represents the optimized conditions of this invention. Figure 3 The results show the comparison of genipin and its derivatives content in gardenia meal under three different treatment conditions; Figure 4 This is a schematic TEM structure of the sustained-release complex in Example 1; wherein, 1-core (genipin derivative-resistant starch complex), 2-transition layer, 3-shell (konjac glucomannan gel layer). Figure 5 The in vitro release curves of different sustained-release complexes are shown. Figure 6 The graph shows the effect of different noodle samples on postprandial blood glucose in diabetic mice. Detailed Implementation

[0032] This invention proposes a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific examples. It should be understood that the specific examples described herein are only for explaining the invention and are not intended to limit the invention.

[0033] Lactobacillus plantarum was purchased from Shanghai Baocang Microbial Co., Ltd., strain number SMHCC D82498; Saccharomyces cerevisiae was purchased from Shanghai Baocang Microbial Co., Ltd., strain number SMHCC D81806; Bacillus subtilis was purchased from Shanghai Baocang Microbial Co., Ltd., strain number SMHCC D83124; Bacillus licheniformis was purchased from Shanghai Baocang Microbial Co., Ltd., strain number SMHCC D80774.

[0034] Example 1 A method for preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity includes the following steps: S1. Preparation of fermented gardenia meal powder rich in genipin derivatives: Take dried gardenia meal (moisture content 8%), put it into a low temperature ultrafine pulverizer, control the chamber temperature at 20℃, and pulverize it until the particle size of the material passes through a 500-mesh sieve to obtain ultrafine pulverized gardenia meal powder; Weigh 1000 g of ultrafine gardenia meal powder (dry weight) and place it in a fermentation tray. Add sterile water to adjust the moisture content of the material to 50%. Based on the dry weight of the powder, inoculate the following strains separately: Lactobacillus plantarum 0.4 g (0.04%), Saccharomyces cerevisiae 1.0 g (0.10%), Bacillus subtilis 1.2 g (0.12%), and Bacillus licheniformis 1.2 g (0.12%). Mix thoroughly. Each strain was activated before use, and the viable count of each strain was 1.15 × 10⁻⁶. 10 CFU / g; After inoculation, the material was spread on a tray to a thickness of 2.5 cm, covered with a breathable aseptic sealing film, and placed in a constant temperature incubator for fermentation at 37°C for 5 days. During fermentation, an automatic pH control system was used, and the pH was checked every 12 hours. When the pH was lower than 4.2, it was adjusted to 4.5 with 0.1 M NaOH solution; when the pH was higher than 4.8, it was adjusted to 4.5 with 0.1 M lactic acid solution. After fermentation, the fermentation product was dried in an oven at 55°C until the moisture content was below 10%, and then pulverized through an 80-mesh sieve to obtain fermented gardenia meal powder rich in genipin-organic acid ester derivatives (denoted as FGP-1). S2. Preparation of genipin derivative enrichment solution: Take 100 g of FGP-1 and add 1500 mL of 50% ethanol aqueous solution (solid-liquid ratio 1:15). Extract with ultrasonic assistance at 50℃ for 1.5 hours with ultrasonic power of 300 W. Centrifuge the extract at 8000 r / min for 15 minutes and collect the supernatant. Concentrate the supernatant under reduced pressure at 50℃ to 1 / 8 of the original volume to obtain the genipin derivative enrichment solution (denoted as GDE-1).

[0035] HPLC-MS analysis showed that the total genipin derivative content was 85.6 mg / mL (calculated as genipin-lactate). S3. Preparation of core micro powder: Take 100 g of resistant starch (RS3 type, corn source) and add 300 mL of GDE-1 (resistant starch: enrichment solution = 1:3, w / v). Stir and mix for 30 minutes to allow for full adsorption. Dry using a spray dryer with an inlet air temperature of 150℃, an outlet air temperature of 80℃, and a feed flow rate of 15 mL / min to obtain core micro powder (denoted as KP-1).

[0036] The encapsulation rate of the genipin derivative was determined to be 88.5%.

[0037] S4. Preparation of sustained-release complex: Prepare a 3% (w / w) konjac glucomannan solution (molecular weight 8 × 10⁻⁶). 5 Da), stir evenly and let stand to remove bubbles. Take 100 g of KP-1 and place it in a fluidized bed coating machine, using konjac glucomannan solution as the coating liquid, and perform bottom spray coating. Process parameters: inlet air temperature 60℃, atomization pressure 0.15 MPa, coating liquid flow rate 5 mL / min, coating weight gain 25%. After coating, continue drying for 15 minutes to obtain the gardenia meal genipin derivative complex (denoted as SRC-1) with sustained-release hypoglycemic activity.

[0038] The total content of genipin derivatives was determined to be 12.3% (based on the dry weight of the complex).

[0039] Preparation of sustained-release hypoglycemic noodles: Weigh the raw materials according to the following parts by weight: 80 parts high-gluten wheat flour, 15 parts SRC-1, 3 parts wheat gluten, 1 part edible salt, 0.2 parts edible alkali, and 30 parts water; Mix the dry powder ingredients evenly, add water containing dissolved salt and baking soda, and knead in a dough mixer for 12 minutes (temperature 25℃). Let the kneaded dough mature at 28℃ for 25 minutes. After maturation, the dough is repeatedly rolled 3-4 times using a sheeter until it reaches a thickness of 1.0 mm, which is then cut into noodles 2.0 mm wide. The wet noodles are dried in stages: Stage 1: 38℃, 75% humidity, 1.5 hours; Stage 2: 42℃, 65% humidity, 2.5 hours; Stage 3: 28℃, 55% humidity, 1.5 hours, until the noodle moisture content reaches 12.5%, resulting in gardenia meal nutritional noodles with slow-release hypoglycemic activity (denoted as NS-1).

[0040] Example 2 The process was essentially the same as in Example 1, except that the ultrafine particle size was 400 mesh in step 1, the inoculum size of *Lactobacillus plantarum* was adjusted to 0.03%, and the inoculum sizes of *Bacillus subtilis* and *Bacillus licheniformis* were both adjusted to 0.15%. The resulting fermented gardenia meal powder was designated FGP-2, the genipin derivative enrichment solution was designated GDE-2, the kernel micron powder was designated KP-2, the slow-release complex was designated SRC-2 (total genipin derivative content 11.8%), and the noodles were designated NS-2.

[0041] Example 3 The process was essentially the same as in Example 1, except that in step 4, the concentration of the konjac glucomannan solution was adjusted to 4%, and the coating weight gain was adjusted to 30%. The resulting sustained-release complex was designated SRC-3 (total content of genipin derivatives 10.5%), and the noodles were designated NS-3.

[0042] Comparative Example 1 (Ordinary Gardenia Meal Powder) Dry gardenia pulp (80-mesh ordinary pulverizer) was used directly for noodle preparation without ultrafine grinding or fermentation. The noodle formula and process were the same as in Example 1, and the resulting noodles were designated DS-1.

[0043] Comparative Example 2 (only ultrafine grinding, no fermentation) Dry gardenia meal raw material was taken and subjected to ultra-fine grinding (500 mesh) without fermentation. Noodles were prepared according to the same noodle formula and process as in Example 1. The resulting noodles were denoted as DS-2.

[0044] Comparative Example 3 (fermentation only, without ultrafine grinding) Dry gardenia pulp (80-mesh ordinary pulverizer) was taken and fermented under the same conditions (strain, moisture content, temperature, and time) as in Example 1, but without ultrafine pulverization pretreatment. After fermentation, noodles were prepared according to the same noodle formula and process as in Example 1, and the resulting noodles were designated DS-3.

[0045] Comparative Example 4 (genipin directly encapsulated, without derivatives) Commercially available genipin standard (purity ≥98%) was used to prepare a complex using the same core preparation and coating process as in Example 1 (without the gardenia meal fermentation step). The amount of genipin added was calculated as 12% of the genipin content in the final complex. Noodles were prepared using the same noodle formula and process as in Example 1 (genipin complex added at 15%), and the resulting noodles were designated DS-4.

[0046] Comparative Example 5 (Regular White Noodles) Without adding gardenia meal powder and complex, white noodles were prepared using 95 parts high-gluten wheat flour, 3 parts wheat gluten, 1 part edible salt, 0.2 parts edible alkali, and 30 parts water as raw materials, following the same process as in Example 1. This noodle was designated DS-5.

[0047] Comparative Example 6 (Commercially Available Blood Sugar Lowering Noodles) A commercially available brand of "blood sugar lowering buckwheat noodles" was used as a control, designated DS-6.

[0048] Comparative Example 7 (resistant starch core only, no konjac glucomannan shell) The genipin derivative enrichment solution (GDE-1) prepared in step 2 of Example 1 was mixed with resistant starch in the same proportion and spray-dried to obtain core micropowder (same as step 3 of Example 1), but without konjac glucomannan coating treatment, thus obtaining the uncoated core micropowder complex, denoted as SRC-7. Noodles were prepared according to the same formula and process as in step 5 of Example 1, wherein the amount of complex added was 15%, thus obtaining noodle sample NS-7.

[0049] Comparative Example 8 (Konjac glucomannan single-layer coating, without resistant starch core) The genipin derivative enrichment solution (GDE-1) prepared in step 2 of Example 1 was directly spray-dried to obtain genipin derivative micro powder (without resistant starch core). 100 g of this micro powder was then subjected to konjac glucomannan fluidized bed coating (coating weight gain 25%) using the same process as in step 4 of Example 1, resulting in a single-layer coated complex, denoted as SRC-8. Noodles were prepared using the same formulation and process as in step 5 of Example 1, with the complex added at 15%, yielding noodle sample NS-8.

[0050] Indicator Measurement and Result Analysis I. Determination of the content of genipin and its derivatives 1.1 Preparation of genipin-lactate and genipin-acetate reference standards 1.1.1 Preparation of genipin-lactate Take 1.0 g of genipin standard (purity ≥98%), add lactic acid and anhydrous ethanol, and reflux at 60 °C for 8 hours using p-toluenesulfonic acid as a catalyst. After the reaction, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography (dichloromethane:methanol gradient elution). Collect the target fraction, concentrate and dry to obtain a white powder of pure genipin-lactate. The purity was determined by HPLC area normalization method to be ≥98%. The structure was confirmed by ESI-MS and ¹H NMR to be consistent with the structure of genipin-lactate.

[0051] 1.1.2 Preparation of genipin-acetic acid ester 1.0 g of genipin standard was added to glacial acetic acid and anhydrous ethanol, with p-toluenesulfonic acid as a catalyst, and refluxed at 60 °C for 6 hours. The mixture was concentrated under reduced pressure using the same method, and purified by silica gel column chromatography (dichloromethane:methanol gradient elution) to obtain a white powder of pure genipin-acetic acid ester with a purity ≥98%. The structure was confirmed by ESI-MS and ¹H NMR to be consistent with the structure of genipin-acetic acid ester.

[0052] 1.1.3 Preparation of reference solution Accurately weigh appropriate amounts of genipin, genipin-lactate, and genipin-acetate reference standards, dissolve and dilute them with methanol to obtain a series of concentrations, and plot a standard curve.

[0053] 1.2 Determination Method High-performance liquid chromatography-mass spectrometry (HPLC-MS) was used. Chromatographic conditions: C18 column (4.6 × 250 mm, 5 μm); mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient elution: 0–5 min, 5% B; 5–25 min, 5%–40% B; 25–30 min, 40%–80% B; flow rate: 1.0 mL / min; detection wavelength: 238 nm; injection volume: 10 μL. Mass spectrometry conditions: ESI ion source, positive ion mode, scan range m / z 100–600. Genipin, genipin-lactic acid ester (self-made reference standard), and genipin-acetate (self-made reference standard) were used as references for qualitative and quantitative analysis.

[0054] 1.3 Measurement Results HPLC-MS analysis was performed on gardenia meal samples from different treatment groups, and the results are shown in Table 1.

[0055] Table 1. Effects of different treatments on the content of genipin and its derivatives in gardenia meal (unit: mg / g DW)

[0056] Note: Different letters in the same column indicate significant differences (p<0.05); n=3.

[0057] Results Analysis: Genipin-lactate and genipin-acetate were detected in Examples 1 and 2 of this invention, but not in the fermented or ultra-finely pulverized samples alone. Example 1 (500 mesh) showed the highest genipin derivative formation, with a total derivative / genipin ratio of 0.81, indicating that finer ultrafine particle size is more conducive to derivative formation. Figure 2 It can also be seen that in A (conventional fermentation, 80 mesh), only the characteristic peak of genipin is visible; in B (ultrafine pulverization + conventional strains), the characteristic peak of genipin is significantly enhanced, but the characteristic peaks of derivatives such as genipin-lactate and genipin-acetate are weak; and in C (optimized conditions of this invention), significant characteristic peaks of genipin-lactate and genipin-acetate are visible, which directly confirms the significant advantage of the method of this invention in promoting the generation of genipin derivatives. Figure 3 A bar chart comparing the contents of genipin and its derivatives under three different treatment conditions visually demonstrates the differences in the contents of genipin, genipin-lactate, and genipin-acetate in groups A, B, and C. Both charts collectively confirm the significant advantage of the method of this invention in promoting the formation of genipin derivatives. Example 2 (400 mesh) was second best, but significantly superior to the control group. This result confirms that the method of this invention achieves in-situ directional formation of genipin derivatives.

[0058] II. Determination of Embedding Rate and Stability 2.1 Encapsulation rate determination Take appropriate amounts of each complex sample, wash the surface free genipin derivatives with ethanol, centrifuge, collect the precipitate, add pH 6.8 phosphate buffer to fully demulsify and extract, determine the genipin derivative content by HPLC, and calculate the encapsulation efficiency. Encapsulation efficiency (%) = (content in the core / total amount added) × 100%.

[0059] 2.2 Stability determination Each complex sample was subjected to accelerated testing at 40°C and 75% relative humidity for 30 days, and samples were taken every 10 days to determine the retention rate of the genipin derivative.

[0060] 2.3 Measurement Results Table 2 Encapsulation efficiency and stability of different complexes

[0061] Note: Different letters in the same column indicate significant differences (p<0.05); n=3.

[0062] Results Analysis: The encapsulation efficiency of SRC-1 in this invention reached 88.5%, significantly higher than that of the genipin-directly encapsulated group (76.5%), indicating a stronger hydrophobic interaction between the genipin derivative and resistant starch, resulting in better encapsulation. After 30 days of accelerated testing, the retention rate of SRC-1 reached 88.5%, far exceeding that of DS-4 (62.5%), demonstrating the excellent stability of the complex in this invention.

[0063] III. Determination of in vitro release characteristics 3.1 Measurement Method A simulated gastrointestinal fluid continuous digestion method was employed. Each complex sample (containing an equal amount of genipin derivative) was accurately weighed and placed in a dialysis bag. The bag was first placed in simulated gastric fluid (pH 1.2, containing pepsin) and shaken at 37°C for 2 hours, then transferred to simulated intestinal fluid (pH 6.8, containing pancreatin) and shaken for another 8 hours. Samples were taken at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 10 hours, and the content of genipin derivative in the release medium was determined by HPLC. The cumulative release rate was calculated.

[0064] 3.2 Measurement Results Table 3. In vitro release characteristics of different sustained-release systems (cumulative release rate, %)

[0065] Results Analysis: The release rate of SRC-1 in the stomach (2 hours) was only 12.5%, significantly lower than that of DS-4 (32.5%), indicating good gastric acid tolerance. In the intestinal stage, SRC-1 exhibited typical zero-order release characteristics, with a constant release rate and a cumulative release rate of 85.5% after 8 hours, achieving sustained and stable release behavior. Figure 5 It is evident that the DS-4 digestion curve rises rapidly in the stomach stage and exhibits a "burst release" phenomenon after entering the intestinal stage; while the SRC-1 and SRC-2 curves maintain a gradual release in the stomach stage and the release in the intestinal stage gradually shows a linear upward trend, which directly confirms the design effect of the dual sustained-release structure of the present invention in changing the release behavior from burst release to sustained release.

[0066] IV. Determination of Noodle Quality Characteristics 4.1 Determination of cooking quality Referring to the LS / T 3212-2021 "Noodles" standard, the optimal steaming time, steaming loss rate and breakage rate of each group of noodles were determined.

[0067] 4.2 Determination of textural properties The textural properties of cooked noodles were determined using a texture analyzer (TA-XT plus). The experimental parameters were: TPA mode, probe P / 36R, pre-test speed 1.0 mm / s, test speed 0.8 mm / s, post-test speed 1.0 mm / s, compression ratio 70%, and trigger force 5 g.

[0068] 4.3 Sensory evaluation Ten trained panelists were invited to give a comprehensive score (out of 100) on the color, appearance, palatability, toughness, stickiness, smoothness and taste of the noodles.

[0069] 4.4 Measurement Results Table 4. Cooking quality and textural properties of different noodle samples

[0070] Table 5 Sensory scores of different noodle samples

[0071] Results analysis: The cooking loss rate (7.2%), breakage rate (3.3%), textural properties and sensory score (88.5 points) of the NS-1 noodles of the present invention are significantly better than those of comparative examples 1-3, and are close to the level of white noodles, indicating that the complex of the present invention has good processing suitability and has little impact on the quality of noodles. Figure 4 This is a TEM structural diagram of the complex of the present invention, where 1 is the core (genipin derivative-resistant starch complex), 2 is the transition layer, and 3 is the outer shell (konjac glucomannan gel layer). This three-layer structure design is the structural basis for the complex of the present invention to achieve both long-term sustained release and minimal impact on the sensory quality of noodles.

[0072] V. Evaluation of hypoglycemic activity 5.1 In vitro α-glucosidase inhibitory activity The inhibitory activity of noodle extracts against α-glucosidase in each group was determined using the pNPG method, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0073] 5.2 Determination of in vitro inhibition duration Each group of noodle extracts (5 mg / mL) was co-incubated with α-glucosidase, and samples were taken at 0, 1, 2, 3, 4, 5, 6, 7, and 8 hours to determine the inhibition rate and plot the inhibition rate-time curve.

[0074] 5.3 Animal Experiments A STZ-induced diabetic mouse model (male C57BL / 6J mice, 6-8 weeks old, weighing 18-22 g, housed in an SPF-grade animal laboratory, with fasting blood glucose ≥11.1 mmol / L) was used. Mice were randomly divided into groups of 10. Each group was administered a corresponding noodle sample by gavage (dose calculated as genipin derivative 5 mg / kg body weight). Blood glucose levels were measured at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours after gavage. The area under the postprandial blood glucose curve (AUC) and the effective hypoglycemic duration (the time when blood glucose levels were more than 20% lower than the model control group) were calculated.

[0075] 5.4 Measurement Results Table 6. α-glucosidase inhibitory activity of different noodle samples

[0076] Table 7 Duration of inhibitory activity for different noodle samples (duration of inhibition rate ≥60%, h)

[0077] Table 8. Postprandial blood glucose AUC and duration of glucose-lowering effect in diabetic mice

[0078] Results analysis: Inhibitory activity: IC of NS-1 of the present invention 50 The value was 3.2 mg / mL, and the inhibition rate reached 68.5% at 5 mg / mL, which was significantly better than the control groups (p<0.05), indicating that it has the strongest α-glucosidase inhibitory activity.

[0079] Duration: The inhibitory activity of NS-1 (≥60%) lasted for 7.5 hours, while that of DS-4 lasted only 2.5 hours, demonstrating that the complex of the present invention achieves a long-lasting sustained-release effect.

[0080] Animal experiments: NS-1 reduced postprandial blood glucose AUC in diabetic mice by 41.9%, with an effective hypoglycemic duration of 7.2 ± 0.4 hours, far superior to ordinary gardenia pulp noodles (2.5 hours) and commercially available hypoglycemic noodles (3.0 hours), verifying its superior long-lasting hypoglycemic effect. Figure 6 As can be seen, the NS-1 blood glucose curve maintained a steady downward trend within 2 to 8 hours without significant rebound, while the blood glucose of the control group began to rise after 3 to 4 hours, which directly verifies the long-term hypoglycemic effect of the complex of the present invention.

[0081] This invention utilizes an in-situ process of "ultra-micro pulverization and synergistic directional fermentation" to generate genipin-organic acid ester derivatives. Based on the enhanced lipid solubility of these derivatives, a dual sustained-release system of "resistant starch core - konjac glucomannan shell" is constructed, successfully preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity. This complex exhibits zero-order release characteristics in simulated gastrointestinal fluid, with continuous release over 8 hours. Animal experiments show that it can reduce postprandial blood glucose AUC in diabetic mice by 42%, with an effective hypoglycemic time of 7.2 ± 0.4 hours. When applied to noodle products, the resulting noodles have a cooking loss rate of ≤8% and a sensory score of over 85 points, possessing both excellent edible quality and clear functional activity. This invention achieves high-value utilization of gardenia meal byproducts, providing a novel long-acting hypoglycemic ingredient for the functional food field, with broad prospects for industrial application.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A gomisin derivative complex of gardenia meal having a sustained blood glucose-lowering activity, characterized by comprising a gomisin derivative and a polysaccharide, wherein the polysaccharide is a polysaccharide having a molecular weight of 1,000 to 1,000,000. The complex consists of a core and a shell: The core is a hydrophobic complex formed by genipin-organic ester derivative and resistant starch; The outer shell is a hydrophilic gel layer formed by konjac glucomannan.

2. The gardenia meal genipin derivative complex with sustained-release hypoglycemic activity according to claim 1, characterized in that, The mass ratio of the core to the shell is (5~7):(3~5).

3. The method for preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity according to claims 1-2, characterized in that, Includes the following steps: Step 1: Modification of Gardenia Meal: Dry gardenia meal is ultra-finely pulverized to 400-500 mesh to obtain ultra-fine gardenia meal powder; the ultra-fine gardenia meal powder is mixed with sterile water, and the moisture content of the material is adjusted to 45%-55%; a compound microbial agent is inoculated, and the inoculated material is cultured and fermented at 35℃-39℃ for 4-6 days, controlling the pH value at 4.2-4.8 during fermentation; after fermentation, the material is dried and pulverized to obtain fermented gardenia meal powder rich in genipin-organic ester derivatives. Step 2, Extraction of active ingredients: Fermented gardenia meal powder rich in genipin-organic acid ester derivatives is extracted with ethanol aqueous solution. The extract is centrifuged or filtered, and the supernatant is collected and concentrated under reduced pressure to 1 / 5 to 1 / 10 of the original volume to obtain genipin derivative enrichment solution. Step 3, Core Preparation: Mix resistant starch with genipin derivative enrichment solution, stir evenly, and spray dry to obtain core micro powder; Step 4: Preparation of sustained-release complex: Mix the kernel micro powder with konjac glucomannan solution, and coat the mixture using fluidized bed coating technology. After coating, continue drying for 10-20 min to obtain the gardenia meal genipin derivative complex with sustained-release hypoglycemic activity.

4. The method for preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity according to claim 3, characterized in that, The compound microbial agent mentioned in step one is composed of Lactobacillus plantarum, Saccharomyces cerevisiae, Bacillus subtilis, and Bacillus licheniformis. The inoculation amount of each species, based on the dry weight of ultrafine pulverized gardenia meal powder, is as follows: Lactobacillus plantarum 0.03%~0.05%, Saccharomyces cerevisiae 0.08%~0.12%, Bacillus subtilis 0.10%~0.15%, and Bacillus licheniformis 0.10%~0.15%.

5. The method for preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity according to claim 3, characterized in that, In the fermentation process described in step one, an automatic pH control system is used. When the pH is below 4.2, it is adjusted to 4.5 with 0.1M NaOH solution; when the pH is above 4.8, it is adjusted to 4.5 with 0.1M lactic acid solution.

6. The method for preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity according to claim 3, characterized in that, The extraction described in step two uses ultrasound-assisted extraction with an ultrasound power of 200~400 W.

7. The method for preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity according to claim 3, characterized in that, The mass ratio of the resistant starch to the genipin derivative enrichment solution in step three is 1:(2~4).

8. The method for preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity according to claim 3, characterized in that, The mass concentration of the konjac glucomannan solution in step four is 2%~5%; the mass ratio of the kernel micro powder to the konjac glucomannan solution is 1:(1~2).

9. The method for preparing a gardenia meal genipin derivative complex with sustained-release hypoglycemic activity according to claim 3, characterized in that, The coating conditions described in step four are: inlet air temperature 50~70℃, atomization pressure 0.1~0.2 MPa, and coating weight gain 20%~30%.

10. The use of the gardenia meal genipin derivative complex with sustained-release hypoglycemic activity as described in claims 1-2 in the preparation of functional foods or medicines.

Images