Formula and preparation method of medicinal and edible Chinese wolfberry fruit composite primary pulp

Abstract

The invention relates to the technical field of food processing, and discloses a medicinal and edible Chinese wolfberry fruit composite primary pulp formula and a preparation method thereof, the medicinal and edible Chinese wolfberry fruit composite primary pulp formula comprises the following components by weight: 65-75% of dried Chinese wolfberry fruit, 18-22% of cordyceps militaris mycelium, and 8-12% of fresh emblic leafflower fruit; the method further comprises the following steps: by taking the cleaning and crushing of medlar and emblic leafflower fruit as a starting point, degrading cell walls by stages through gradient enzymolysis, and dynamically regulating and controlling the carbon nitrogen ratio by combining with the liquid fermentation of cordyceps militaris to activate the synthesis of cordycepin; heat-sensitive components are protected through cold plasma non-thermal sterilization, polysaccharide-protein self-assembly is induced at low temperature to form nanoparticles to construct a stable system, and finally filling and storage are performed. Activity synergistic interaction is achieved through molecular compatibility and a gradient regulation and control process, thermosensitive activity is accurately protected through cold plasma-nitrogen combined sterilization, a nano stable system is constructed through polysaccharide-protein self-assembly, and a natural anti-corrosion barrier is formed through polyphenol multi-target bacteriostasis.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, in particular to a formula of a composite wolfberry puree which is both medicinal and edible and a preparation method thereof. Background Art

[0002] Liquid fermented products have important application value in the field of health food. Through microbial fermentation, they can enrich active ingredients such as cordycepin, polysaccharides, polyphenols, etc., and have both nutritional and functional properties. However, in the prior art, the preparation of such products generally faces bottleneck problems such as easy degradation of active ingredients, poor system stability, and high dependence on chemical additives, which seriously restricts product quality and market acceptance. How to achieve efficient sterilization without damaging heat-sensitive ingredients and at the same time build a natural and stable active ingredient delivery system has become a technical problem that needs to be broken through in this field.

[0003] In current technology, although high-temperature sterilization processes can effectively inactivate microorganisms, they can lead to the breakage of glycosidic bonds of heat-sensitive ingredients such as cordycepin and the intensification of Maillard reactions, resulting in loss of activity and color deterioration; and chemical preservatives (such as sodium benzoate) added to extend the shelf life not only pose potential health risks, but their one-dimensional antibacterial mechanism makes it difficult to inhibit secondary contamination during storage. In addition, liquid systems generally rely on synthetic emulsifiers to maintain stability, further weakening the natural properties of the product. More importantly, the traditional segmented process design causes the release sequence of Cordyceps militaris fermentation metabolites and plant active ingredients to be misaligned, resulting in a lack of synergistic effects between components and an inability to fully exert the functional advantages of the composite formula.

[0004] The above-mentioned technical defects are interrelated, forming a vicious cycle that restricts the upgrading of liquid fermentation products: the low efficiency of active ingredient extraction forces the process temperature to increase, while high temperature aggravates the degradation of ingredients, which in turn leads to dependence on more chemical additives, ultimately leading to a decline in both the naturalness and efficacy of the product. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a formula of a composite wolfberry puree that is both medicinal and edible and a preparation method thereof, which solves the problems of thermal degradation of active ingredients, dependence on chemical preservatives, insufficient system stability and lack of multi-component synergistic effect in the preparation process of liquid fermented products.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: The first aspect of the present invention provides a formula of a composite wolfberry puree that is both medicinal and edible, comprising the following components by weight percentage: Wolfberry dried fruit 65%-75%, Cordyceps militaris mycelium 18%-22%, Emblica emblica fresh fruit 8%-12%.

[0007] Dried wolfberry fruit, as the main ingredient, is rich in wolfberry polysaccharides and carotenoids; Cordyceps militaris mycelium metabolizes cordycepin, adenosine and other active substances through liquid fermentation; fresh emblica fruit contains high concentrations of gallic acid and polyphenols, and has both natural antibacterial and flavor regulation functions.

[0008] The three achieve functional synergy through metabolic complementarity: the extracellular enzymes secreted by Cordyceps militaris can break down wolfberry polysaccharides into low molecular weight fragments, thereby increasing the absorption rate; and the polyphenols in Phyllanthus emblica inhibit the growth of miscellaneous bacteria during the fermentation process, reducing dependence on chemical preservatives.

[0009] Preferably, the following auxiliary components are also included: Glucose, potassium dihydrogen phosphate and magnesium sulfate, wherein glucose accounts for 2% to 6% of the total weight of the raw pulp, potassium dihydrogen phosphate accounts for 0.08% to 0.12% of the total weight of the raw pulp, and magnesium sulfate accounts for 0.04% to 0.06%.

[0010] Glucose, as the initial carbon source for liquid fermentation, together with potassium dihydrogen phosphate and magnesium sulfate, constructs a dynamic C / N ratio control system, and induces directional metabolism of Cordyceps militaris mycelium through staged feeding. Phosphate and magnesium ions serve as coenzyme activators to ensure the proliferation of mycelium and the activity of cordycepin synthase system.

[0011] Preferably, the active ingredients in the puree include 0.5-1.0 mg / g cordycepin and 8%-12% wolfberry polysaccharides.

[0012] The second aspect of the present invention provides a method for preparing the edible and medicinal wolfberry composite puree formula, which is prepared using the edible and medicinal wolfberry composite puree formula, comprising the following steps: Raw material pretreatment: washing and crushing fresh wolfberry and emblica emblica fruits; Gradient enzymatic hydrolysis: wolfberry and emblica were treated with cellulase and pectinase in stages; Cordyceps militaris liquid fermentation: inoculation of bacteria and dynamic regulation of carbon-nitrogen ratio; Cold plasma pulse sterilization: non-thermal sterilization of fermentation broth; Self-emulsification stabilization treatment: forming nanoparticles and homogenizing; Filling and storage.

[0013] Preferably, in the raw material pretreatment step, the fresh fruit of Phyllanthus emblica is pitted and pulped, and the solid content of the pulp is 12% to 18%.

[0014] Preferably, in the gradient enzymatic hydrolysis step: The conditions for enzymatic hydrolysis of wolfberry cellulose are as follows: enzyme addition amount 0.2%-0.5%, temperature 45-55℃, pH 4.8-5.2, enzymatic hydrolysis time 1.5-2.5h, and 25-30kHz ultrasonic treatment for 8-12min after enzymatic hydrolysis; The enzymatic hydrolysis conditions of emblica pectin are as follows: enzyme addition amount 0.1%-0.3%, temperature 38-42℃, pH 3.2-3.8, and enzymatic hydrolysis time 1.0-2.0h.

[0015] Cellulose enzymatic hydrolysis stage: Prioritize the degradation of cellulose and hemicellulose in the wolfberry cell wall to release intracellular polysaccharides, while avoiding the inactivation of polyphenol oxidase caused by high temperature.

[0016] Pectin enzymatic hydrolysis stage: Selectively degrade pectin from the cell wall of Phyllanthus emblica under acidic conditions, release polyphenols, and inhibit browning reaction.

[0017] Ultrasonic-assisted enhancement mechanism: 25-30kHz ultrasound is applied after cellulose enzymatic hydrolysis to destroy the residual cell wall structure through the cavitation effect, thereby improving the substrate accessibility of the subsequent Cordyceps militaris liquid fermentation.

[0018] Preferably, the dynamic regulation of the carbon-nitrogen ratio in the Cordyceps militaris liquid fermentation step comprises: Initial C / N ratio 20-30:1, fermentation 0-48h; Glucose was added to adjust the C / N ratio to 15-20:1 and fermented for 49-72 hours; Add corn syrup powder to adjust the C / N ratio to 25-35:1 and ferment for 73-120 hours.

[0019] Phased C / N ratio regulation drives the switching of Cordyceps militaris metabolic pathways: Mycelial proliferation stage: High C / N ratio promotes the accumulation of mycelial biomass, providing a basis for the subsequent synthesis of metabolites.

[0020] Cordycepin synthesis period: reducing the C / N ratio activates key enzymes for cordycepin synthesis (such as nucleotide phosphorylase).

[0021] Polysaccharide-protein co-metabolism period: corn syrup is added to provide an organic nitrogen source to promote mycelium autolysis and release polysaccharide and protein complexes.

[0022] Preferably, the cold plasma pulse sterilization parameters in the cold plasma pulse sterilization step are: The voltage is 8-12 kV, the frequency is 4-6 kHz, the pulse mode is 4-6 kHz high-frequency discharge alternating with 0.5-2.0 ms intermittently, the sterilization time is 12-18 min, and nitrogen is introduced throughout the process to maintain the oxygen content <1.0%.

[0023] The pulsed discharge design reduces the continuous generation of reactive oxygen species (ROS) through intermittent energy input, thus avoiding the oxidative degradation of polysaccharides and cordycepin. Nitrogen protection (oxygen content <1.0%) further inhibits the oxidation reaction while maintaining the stability of plasma discharge.

[0024] Preferably, in the self-emulsification stability treatment step: The nanoparticles were placed at 4-10°C for 18-30h. Homogenizing pressure 40-60MPa, cycle 1-3 times.

[0025] The polysaccharide-protein complex in the metabolites of Cordyceps militaris spontaneously assembles into nanoparticles (50-100 nm) through hydrophobic interactions and hydrogen bonds during the static process. The particles maintain the stability of the system through steric hindrance and electrostatic repulsion, without the need for exogenous emulsifiers. Homogenization (40-60 MPa) refines the particle distribution and prevents stratification during storage.

[0026] The present invention provides a formula of a composite wolfberry puree that is both medicinal and edible and a preparation method thereof. It has the following beneficial effects: 1. The present invention adopts a molecular-level compatibility design of wolfberry polysaccharides, cordycepin and emblica polyphenols, and utilizes a gradient enzymatic hydrolysis process to release the active ingredients in stages, so that the wolfberry cellulose degradation products and the metabolites of the Cordyceps militaris mycelium form a hydrogen bond network. At the same time, the emblica polyphenols stabilize the composite system through hydrophobic interaction. The three synergistically activate the TLR4 receptor on the surface of macrophages and the Nrf2 antioxidant pathway, thereby achieving cross-dimensional synergistic enhancement of immunoregulation and free radical scavenging functions, breaking through the efficacy limitations of traditional single components.

[0027] 2. The present invention is based on a time-controlled enzymatic hydrolysis strategy and dynamic C / N regulation. In the early stage of Cordyceps militaris fermentation, a high-nitrogen environment is provided to promote the proliferation of mycelium. In the middle stage, it is switched to high-carbon conditions to activate the cordycepin synthase system. The ultrasonic cavitation effect is used to directionally break the cross-linked structure of wolfberry lignin-hemicellulose, forming an efficient extraction system of active ingredients integrating enzymatic hydrolysis, fermentation and physical wall breaking, which significantly improves the bioavailability of cordycepin and polysaccharides.

[0028] 3. The present invention adopts cold plasma pulse sterilization-nitrogen protection combined technology, which generates active oxygen clusters through high-energy electron excitation to instantly inactivate microorganisms. At the same time, the inert nitrogen environment blocks the oxidative cross-linking of polysaccharide side chains and the thermal breakage of cordycepin glycosidic bonds. Combined with the low-temperature sterilization process, the Maillard reaction is completely avoided, so that the molecular conformation and biological activity of heat-sensitive components remain in a natural state.

[0029] 4. The present invention is based on the self-assembly mechanism of polysaccharide-hydrophobic protein complexes. During the low-temperature standing process, hydrophobic interactions and hydrogen bonds are used for directional arrangement to form core-shell structured nanoparticles. The surface negative charge and steric hindrance effect synergistically inhibit Ostwald ripening, thereby achieving the kinetic stability of the liquid system during long-term storage and completely getting rid of the dependence on synthetic emulsifiers.

[0030] 5. The present invention utilizes the multi-target antibacterial network of emblica polyphenols, through the triple effects of phenolic hydroxyl groups inserting into microbial cell membranes to induce ion leakage, catechol structures chelating metal cofactors to block enzyme activity, and quinone metabolites inducing oxidative damage to microbial DNA, to construct a full-process biological preservation barrier from production to storage, completely replacing benzoic acid chemical preservatives, and achieving a technological breakthrough in self-preservation of natural components. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The present invention is a flow chart of the preparation method. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Please refer to the attached Figure 1 The present invention provides a formula of a composite wolfberry puree having both medicinal and edible properties and a preparation method thereof through multiple embodiments. The specific embodiments are as follows: Embodiment 1-3: Embodiment 1: Raw material ratio (weight percentage): Dried wolfberry fruit 70%; Cordyceps militaris mycelium (liquid fermentation product) 20%; Phyllanthus emblica fresh fruit 10%; Auxiliary components: glucose 4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%.

[0034] Preparation steps: Raw material pretreatment: The dried wolfberry fruit was soaked in 0.5% citric acid solution (30°C), cleaned with 45kHz ultrasonic wave for 5 min, and crushed to a particle size of 2 mm; Fresh emblica fruit is pitted and pulped through a 60-mesh sieve, with a solid content of 15%; Gradient enzymatic hydrolysis: Lycium barbarum cellulose enzymatic hydrolysis: enzyme addition amount 0.35% (w / w), 50℃, pH 5.0, enzymatic hydrolysis for 2h, followed by 28kHz ultrasonic treatment for 10min (power density 50W / L); Enzymatic hydrolysis of emblica pectin: enzyme addition amount 0.2% (w / w), 40℃, pH 3.5, enzymatic hydrolysis for 1.5h; Cordyceps militaris liquid fermentation: Mix the enzymatic hydrolysate, adjust the initial C / N to 25:1, and inoculate 8% Cordyceps militaris liquid culture (dissolved oxygen content 30%); C / N dynamic control: 0-48h: maintain C / N=25:1, stir at 200rpm; 49-72h: add glucose to C / N=17.5:1; 73-120h: Add corn syrup powder to C / N=30:1; Cold plasma pulse sterilization: The fermentation broth was filtered through a 50 kDa ceramic membrane (0.3 MPa, 25 °C).

[0035] Sterilization parameters: voltage 10kV, frequency 5kHz, pulse mode (5kHz discharge + 1.25ms interval), treatment 15min, nitrogen protection (oxygen content 0.5%); Self-emulsifying stability treatment: The sterilized stock was left to stand at 7°C for 24 h to form nanoparticles (particle size of about 75 nm); Homogenization pressure 50MPa, 2 cycles; Embodiment 2: Raw material ratio (weight percentage): Lycium barbarum dried fruit 65%; Cordyceps militaris mycelium 18%; Phyllanthus emblica fresh fruit 8%; Auxiliary components: glucose 2%, potassium dihydrogen phosphate 0.08%, magnesium sulfate 0.04%.

[0036] Preparation steps: Raw material pretreatment: The wolfberries were soaked in 0.3% citric acid solution (25°C), cleaned with 40kHz ultrasonic waves for 6 min, and crushed to a particle size of 3 mm; The emblica fruit pulp was passed through a 50-mesh sieve, and the solid content was 12%; Gradient enzymatic hydrolysis: Lycium barbarum cellulose enzymatic hydrolysis: enzyme addition amount 0.2% (w / w), 45℃, pH4.8, enzymatic hydrolysis for 2.5h, followed by 25kHz ultrasonic treatment for 12min (power density 40W / L); Enzymatic hydrolysis of emblica pectin: enzyme addition amount 0.1% (w / w), 38℃, pH 3.2, enzymatic hydrolysis for 2.0h; Cordyceps militaris liquid fermentation: Initial C / N=20:1, inoculated with 6% bacteria (dissolved oxygen 20%); C / N Control: 0-48h: C / N=20:1; 49-72h: Supplement sugar until C / N=15:1; 73-96h: add corn syrup until C / N=25:1; Cold plasma pulse sterilization: 30kDa ceramic membrane filtration (0.2MPa, 20℃); Sterilization parameters: voltage 8kV, frequency 4kHz, pulse mode (4kHz discharge + 2.0ms interval), treatment 18min, oxygen content 1.0%; Self-emulsifying stability treatment: Let stand at 4°C for 30 hours, homogenize at 40 MPa pressure, and cycle once.

[0037] Embodiment 3: Raw material ratio (weight percentage): Dried wolfberry 75%; Cordyceps militaris mycelium 22%; Phyllanthus emblica fresh fruit 12%; Auxiliary components: glucose 6%, potassium dihydrogen phosphate 0.12%, magnesium sulfate 0.06%.

[0038] Preparation steps: Raw material pretreatment: The wolfberries were soaked in 0.7% citric acid solution (35°C), cleaned with 50kHz ultrasonic waves for 4 min, and crushed to a particle size of 1 mm; The emblica fruit pulp was passed through a 70-mesh sieve, and the solid content was 18%; Gradient enzymatic hydrolysis: Lycium barbarum cellulose enzymatic hydrolysis: enzyme addition amount 0.5% (w / w), 55℃, pH 5.2, enzymatic hydrolysis for 1.5h, followed by 30kHz ultrasonic treatment for 8min (power density 60W / L); Enzymatic hydrolysis of emblica pectin: enzyme addition amount 0.3% (w / w), 42℃, pH 3.8, enzymatic hydrolysis for 1.0h; Cordyceps militaris liquid fermentation: Initial C / N=30:1, inoculated with 10% bacteria (dissolved oxygen 40%); C / N Control: 0-48h: C / N=30:1; 49-72h: Supplement sugar until C / N=20:1; 73-144h: Add corn syrup until C / N=35:1; Cold plasma pulse sterilization: 100kDa ceramic membrane filtration (0.5MPa, 30℃); Sterilization parameters: voltage 12kV, frequency 6kHz, pulse mode (6kHz discharge + 0.5ms interval), treatment 12min, oxygen content 0.3%; Self-emulsifying stability treatment: Stand at 10℃, 18h, homogenization pressure 60MPa, cycle 3 times; Comparative Examples 1-7: Comparative Example 1: Compared with Example 1, the difference is that the proportion of Cordyceps militaris mycelium is 25% (exceeding the upper limit of 18% to 22% of Claim 1), the proportion of wolfberry dried fruit is adjusted to 65%, and the proportion of fresh emblica fruit remains unchanged (10%). The remaining steps and parameters are the same.

[0039] Comparative Example 2: Compared with Example 1, the difference is that the pectin enzymatic hydrolysis step in the gradient enzymatic hydrolysis is omitted, only the wolfberry is subjected to cellulose enzymatic hydrolysis, and the fresh emblica fruit is directly pulped and then added to the fermentation system. The remaining steps and parameters are the same.

[0040] Comparative Example 3: Compared with Example 1, the difference is that: no dynamic C / N ratio control is performed during the liquid fermentation of Cordyceps militaris, the initial C / N=25:1 is maintained throughout the process, and no glucose or corn syrup powder is added. The remaining steps and parameters are the same.

[0041] Comparative Example 4: Compared with Example 1, the difference is that cold plasma pulse sterilization is replaced by traditional high temperature sterilization (121° C., 20 min). The remaining steps and parameters are the same.

[0042] Comparative Example 5: Compared with Example 1, the difference is that the standing step is omitted in the self-emulsification stability treatment, and the homogenization treatment is directly performed after sterilization. The remaining steps and parameters are the same.

[0043] Comparative Example 6: Compared with Example 1, the difference is that the fresh fruit of Phyllanthus emblica is replaced by an equal amount of citric acid (the pH is adjusted to the same value), and the fresh fruit component of Phyllanthus emblica is omitted. The remaining steps and parameters are the same.

[0044] Comparative Example 7: Compared with Example 1, the difference is that the ultrasonic-assisted treatment step is omitted and only conventional enzymatic hydrolysis is performed. The remaining steps and parameters are the same.

[0045] Test example 1-5: Test Example 1: Component Ratio and Active Ingredient Retention Rate Test Experimental purpose: To verify the necessity of the composition ratio specified in claim 1 (lycium barbarum 65%-75%, Cordyceps militaris 18%-22%, Emblica officinalis 8%-12%) for retaining the active ingredients.

[0046] Description of experimental steps: Sample preparation: Example 1: Prepare the stock paste according to the intermediate ratio of claim 1 (70% wolfberry, 20% cordyceps militaris, 10% emblica).

[0047] Comparative Example 1: Cordyceps militaris mycelium accounts for 25%, wolfberry is adjusted to 65%, and emblica emblica accounts for 10%.

[0048] Comparative Example 6: The fresh fruit of Emblica officinalis was replaced with an equal amount of citric acid (the pH was adjusted to 3.5), without the Emblica officinalis component.

[0049] Active ingredient extraction: Cordycepin extraction: Take 5 g of puree and use 50 mL of 70% ethanol for ultrasonic extraction (40°C, 30 min). After centrifugation, take the supernatant and filter it through a 0.22 μm filter membrane.

[0050] Extraction of Lycium barbarum polysaccharides: Take 5 g of the original pulp, add 50 mL of deionized water and extract in a boiling water bath for 1 hour, and take the supernatant after centrifugation.

[0051] Extraction of polyphenols from emblica: Take 5 g of puree and extract with 50 mL of 60% methanol under shaking (200 rpm, 25°C, 1 h), and take the supernatant after centrifugation.

[0052] Detection method: Cordycepin content: HPLC detection (C18 chromatographic column, mobile phase methanol-water gradient elution, detection wavelength 260nm).

[0053] Lycium barbarum polysaccharide content: phenol-sulfuric acid method (490nm wavelength colorimetry, calculated based on glucose standard curve).

[0054] Retention rate of polyphenols in Phyllanthus emblica: Folin phenol method (765nm wavelength colorimetry, calculated based on gallic acid standard curve).

[0055] Data processing: Each experiment was repeated 3 times, and the mean ± standard deviation was taken.

[0056] Experimental data: Table 1: Effect of group ratio on active ingredient retention rate Data description: Comparative Example 1 (Excessive dosage of Cordyceps militaris): The cordycepin content decreased by 37.8% (due to excessive mycelium leading to metabolic inhibition and reduced cordycepin synthase activity); Lycium barbarum polysaccharides decreased by 23.6% (a high proportion of mycelium competed for carbon sources, and the polysaccharide dissolution rate decreased).

[0057] Comparative Example 6 (Emblica officinalis replaced by citric acid): Emblic polyphenols were not detected (verifying that Emblic polyphenols were the only source of polyphenols); The cordycepin and polysaccharide contents were close to those in Example 1 (acidity adjustment did not affect fermentation, but the natural antibacterial function was lost).

[0058] Experimental conclusion: This experiment revealed the irreplaceable nature of the specific ratio of wolfberry, Cordyceps militaris mycelium and fresh fruit of Phyllanthus emblica for retaining active ingredients. When the proportion of Cordyceps militaris mycelium exceeded 22% (Comparative Example 1), the content of cordycepin in its metabolites decreased significantly, which is directly related to the carbon source competition inhibition caused by excessive proliferation of mycelium - high-density mycelium accelerates the consumption of intracellular glycogen, resulting in insufficient substrate supply for key enzymes in cordycepin synthesis (such as nucleotide phosphorylase). At the same time, the dissolution rate of wolfberry polysaccharides decreased, which was due to the unbalanced effect of cellulase secreted by mycelium and wolfberry cell wall: excessive mycelium prematurely depleted the surface polysaccharides of wolfberry, hindering the continuous release of deep polysaccharides.

[0059] The replacement experiment of fresh emblica fruit (Comparative Example 6) further verified the lack of its polyphenol-polysaccharide synergistic protection mechanism. Gallic acid and other polyphenolic substances in emblica inhibit the activity of polyphenol oxidase by chelating metal ions in the early stage of fermentation, while citric acid only provides an acidic environment and cannot block the oxidative degradation chain reaction of polysaccharides. In addition, the hydrogen bond interaction between emblica polyphenols and Cordyceps militaris metabolites is destroyed, resulting in a decrease in the antioxidant capacity of the system, which indirectly affects the half-life of cordycepin.

[0060] The ratio design of the core components essentially builds a metabolic balance network: Lycium barbarum polysaccharides as the main carbon source support the proliferation of Cordyceps militaris mycelium, Phyllanthus emblica polyphenols ensure the stability of the active ingredients by inhibiting bacteria and oxidation reactions, and mycelium metabolites reversely activate the bioavailability of Lycium barbarum polysaccharides. The deviation of the ratio of the three will break this balance, such as the "metabolic siphon effect" caused by excessive mycelium in Example 1, or the oxidation cascade reaction caused by the lack of polyphenols in Example 6, which confirms the strict necessity of the component ratio.

[0061] Test Example 2: Effect of preparation process steps on the release of active ingredients Experimental purpose: To verify the synergistic effect of gradient enzymatic hydrolysis, dynamic C / N regulation and ultrasound-assisted release on the active ingredient release.

[0062] Description of experimental steps: Sample preparation: Example 1: Prepared according to the original process (gradient enzymatic hydrolysis, dynamic C / N regulation, ultrasonic assistance).

[0063] Comparative Example 2: The step of enzymatic hydrolysis of emblica pectin was omitted, and the fresh emblica fruits were directly pulped and then added into the fermentation system.

[0064] Comparative Example 3: No dynamic C / N regulation was performed during the liquid fermentation of Cordyceps militaris, and the initial C / N=25:1 was maintained throughout the entire process.

[0065] Comparative Example 7: The ultrasonic-assisted treatment was eliminated and only conventional enzymatic hydrolysis was performed.

[0066] Active ingredient release efficiency test: Lycium barbarum polysaccharide dissolution rate: Take 10 mL of the enzymatic hydrolysate, centrifuge and take the supernatant, and determine the soluble polysaccharide content by the phenol-sulfuric acid method (calculate the dissolution rate based on the ratio with the total polysaccharide in the raw material).

[0067] Cordycepin synthesis efficiency: The fermentation broth was sampled every 24 h, and the cordycepin content (mg / g) was detected by HPLC, and the synthesis rate per unit time (mg / g·h) was calculated.

[0068] Fermentation broth residue amount: After the fermentation is terminated, 100 mL of the fermentation liquid is taken, filtered through a 100-mesh sieve, and the weight of the solid residue is weighed (g / L) after drying.

[0069] Detection method: HPLC conditions: C18 column, mobile phase methanol-water (70:30), flow rate 1.0 mL / min, detection wavelength 260 nm.

[0070] Centrifugation parameters: 4000 rpm, 15 min, 25 °C.

[0071] Data processing: Each experiment was repeated 3 times, and the mean ± standard deviation was calculated and analyzed for significant differences (p < 0.05).

[0072] Experimental data: Table 2: Effect of process steps on active ingredient release Data description: Comparative Example 2 (without pectin enzymolysis): The undegraded emblica pectin resulted in viscous slurry, which hindered the dissolution of Lycium barbarum polysaccharides (the dissolution rate decreased by 20.7%); The efficiency of cordycepin synthesis decreased by 25.6% (pectin residues inhibited the metabolic activity of mycelium).

[0073] Comparative Example 3 (without dynamic C / N regulation): The efficiency of cordycepin synthesis dropped sharply by 44.8% (the mycelium did not switch to the cordycepin synthesis metabolic pathway); The amount of residue increased by 46.7% (the lack of feed resulted in incomplete autolysis of mycelium).

[0074] Comparative Example 7 (without ultrasonic assistance): The amount of residue increased by 126.4% (cell wall residues prevented mycelium from contacting the substrate); The cordycepin efficiency was still partially retained (conventional enzymatic hydrolysis had a certain effect, but it was lower than ultrasound-assisted hydrolysis).

[0075] Experimental conclusion: This experiment confirmed the key role of the process synergy of gradient enzymatic hydrolysis, dynamic C / N regulation and ultrasonic-assisted treatment in the release of active ingredients. When the enzymatic hydrolysis step of emblica pectin was omitted (Comparative Example 2), the undegraded pectin formed a viscous colloidal barrier in the fermentation system, hindering the physical contact between the Cordyceps militaris mycelium and the Lycium barbarum polysaccharide substrate, resulting in a decrease in the efficiency of cordycepin synthesis. This phenomenon is directly related to the temporal synergistic mechanism of gradient enzymatic hydrolysis: pectinase degrades the cell wall of emblica under acidic conditions, and the released polyphenols can neutralize the metal ions in the fermentation broth to avoid its inhibitory effect on cellulase, thereby ensuring sufficient enzymatic hydrolysis of Lycium barbarum cellulose.

[0076] The lack of dynamic C / N regulation (Comparative Example 3) leads to the failure of the directional nature of the metabolic pathway of Cordyceps militaris. Although the initial high C / N ratio (25:1) can promote mycelial proliferation, the continuous high carbon environment inhibits the induced expression of cordycepin synthase. Dynamic feeding triggers the transition of mycelium from proliferation to secondary metabolism (cordycepin synthesis) through phased carbon and nitrogen adjustments, while the mycelium continues to consume carbon sources under a single C / N ratio, and eventually the metabolic stagnation occurs due to substrate depletion.

[0077] The omission of ultrasonic-assisted treatment (Comparative Example 7) exposed the defect of incomplete enzymatic hydrolysis. The cavitation effect of ultrasound can destroy the unenzymatically hydrolyzed lignin-cellulose composite structure in the cell wall of wolfberry, while relying solely on enzymatic hydrolysis can only degrade loose fibers, resulting in a large amount of residue accumulation. The lack of this step directly weakened the utilization rate of the substrate by Cordyceps militaris, confirming the physical-biochemical synergistic mechanism of ultrasound and enzymatic hydrolysis.

[0078] Test Example 3: Test on the protective effect of sterilization process on heat-sensitive components Experimental purpose: To verify the advantages of cold plasma pulse sterilization over traditional high temperature sterilization in protecting heat-sensitive components such as cordycepin and polysaccharides.

[0079] Description of experimental steps: Sample preparation: Example 1: Prepared according to the original process, using cold plasma pulse sterilization.

[0080] Comparative Example 4: The same preparation process, but the sterilization step was replaced by high-temperature sterilization at 121°C (20 min).

[0081] Sterilization and sampling: The fermentation broths of Example 1 and Comparative Example 4 were both subjected to the same pretreatment (50 kDa ceramic membrane filtration).

[0082] Samples were taken before sterilization (marked as T0), immediately after sterilization (marked as T1), and after refrigeration for 24 h (marked as T2).

[0083] Active ingredient testing: Cordycepin loss rate: The cordycepin content at time points T0, T1, and T2 was determined by HPLC, and the loss rate (%) was calculated as (T0-T1) / T0×100%.

[0084] Polysaccharide molecular weight distribution: T1 sample was taken and the weight average molecular weight (Mw) of polysaccharide was determined by gel permeation chromatography (GPC).

[0085] Browning index: Take T1 sample and measure the absorbance (OD value) at 420nm using a spectrophotometer to reflect the degree of Maillard reaction.

[0086] Detection method: HPLC conditions: C18 column, mobile phase methanol-water (65:35), flow rate 1.2 mL / min, detection wavelength 260 nm.

[0087] GPC parameters: TSKgelG3000PWxl column, mobile phase 0.1MNaNO3, flow rate 0.8mL / min, RID detector.

[0088] Spectrophotometer: quartz cuvette, optical path 1 cm, reference solution is deionized water.

[0089] Data processing: The cordycepin loss rate was calculated as the mean ± SD of three replicates; The molecular weight of the polysaccharide was taken as the main peak Mw value.

[0090] Experimental data: Table 3: Effect of sterilization process on heat-sensitive ingredients Data description: Cordycepin loss rate: Example 1 Due to cold plasma non-thermal sterilization, the cordycepin loss rate is only 8.3%; In Comparative Example 4, high temperature sterilization resulted in a cordycepin loss rate of more than 34%, and the molecular structure was destroyed (HPLC peak tailing).

[0091] Polysaccharide molecular weight distribution: The Mw of the polysaccharide in Example 1 remained at 182.4 kDa (close to the native state); The Mw of the polysaccharide in Comparative Example 4 dropped to 112.6 kDa (high temperature caused the sugar chain to break).

[0092] Browning index difference: Example 1 OD value 0.121 (no significant Maillard reaction); The OD value of Comparative Example 4 was 0.398 (high temperature promoted the reaction between reducing sugar and amino acid).

[0093] Experimental conclusion: This experiment clarified the core role of cold plasma pulse sterilization in the protection of heat-sensitive ingredients. The reactive oxygen species (ROS) generated by cold plasma through high-frequency pulse discharge have instantaneous sterilization characteristics, and their action time (milliseconds) is much shorter than the half-life temperature threshold of cordycepin (above 60°C for 5 minutes), thus avoiding the breakage of glycosidic bonds in cordycepin molecules caused by traditional high-temperature sterilization. At the same time, the nitrogen protection environment controls the oxygen content of the system below 0.5%, blocking the oxidative cross-linking of the polysaccharide side chain hydroxyl groups, so that the weight-average molecular weight of Lycium barbarum polysaccharide remains above 180kDa, close to the natural conformation.

[0094] The loss of cordycepin caused by high temperature sterilization (Comparative Example 4) is not only the result of thermal degradation, but also related to the synergistic destructive effect of the Maillard reaction. Under 121°C, reducing sugars in the fermentation broth react with free amino acids to produce brown substances such as melanoidins (browning index increased by 228%). These products wrap the cordycepin molecules to form steric hindrance, further inhibiting its bioavailability. Cold plasma sterilization is carried out at 25°C, which not only avoids heat sources triggering the Maillard reaction, but the ozone generated by its pulse discharge can also selectively degrade free amino groups in the fermentation broth, fundamentally cutting off the browning reaction chain.

[0095] The coordinated design of process parameters ensures sterilization-stabilization integration: the intermittent pulse mode of cold plasma (5kHz discharge and 1.25ms intermittent alternation) makes the ROS concentration fluctuate, which can inactivate microorganisms in stages and provide a repair time window for the cordycepin-polysaccharide complex. This dynamic balance is completely lost in high-temperature sterilization, resulting in a 38.2% decrease in the molecular weight of the polysaccharide in Comparative Example 4 and irreversible destruction of its tertiary structure with immunomodulatory function.

[0096] Test Example 4: Verification of the effect of self-emulsification stability treatment Experimental purpose: To verify the effect of low-temperature static self-assembly step on the stability and storage performance of nanoparticles.

[0097] Description of experimental steps: Sample preparation: Example 1: Prepared according to the original process, after sterilization, allowed to stand (4°C, 12h) for self-emulsification treatment.

[0098] Comparative Example 5: High-pressure homogenization (50 MPa, 3 cycles) was performed directly after sterilization without a standing step.

[0099] Nanoparticle Characterization: Particle size determination: Take 1 mL of sample and dilute it 10 times with deionized water, and measure the particle size distribution (25°C) by dynamic light scattering (DLS).

[0100] Delamination rate test: Take 50 mL of sample and put it into a transparent glass bottle, store it at 25℃ away from light for 30 days, and record the volume percentage (%) of the delamination.

[0101] Viscosity change rate: using a rotational rheometer (shear rate 100s -1 ) Determination of initial viscosity (η 0 ) and the viscosity after 30 days (η 1 ), calculate the change rate (%) = (η 1 -η 0 ) / η 0 ×100%.

[0102] Testing conditions: DLS parameters: detection angle 173°, wavelength 633nm, temperature 25°C, and the average value of three measurements.

[0103] Rheometer parameters: cone and plate diameter 40 mm, gap 0.5 mm, temperature 25 °C.

[0104] Data processing: The particle size was taken as Z-Average value, and the stratification rate and viscosity change rate were repeated three times and the mean ± standard deviation was taken.

[0105] Experimental data: Table 4: Effect of self-emulsification treatment on stability Data description: Particle size difference: Example 1 forms uniform nanoparticles (76.3 nm) due to static self-assembly, while Comparative Example 5 results in coarsened particles (214.5 nm) due to forced homogenization.

[0106] Comparison of stratification rate: The delamination rate of Example 1 is only 5.2%, while the delamination rate of Comparative Example 5 exceeds 40% (particle sedimentation leads to phase separation).

[0107] Viscosity Change: The viscosity of Example 1 decreased slightly (-8.1%), while the viscosity of Comparative Example 5 increased by 23.4% due to particle aggregation.

[0108] Experimental conclusion: This experiment reveals the core role of low-temperature static self-assembly in constructing a nanoparticle stability system. During the static process, the hydrophobic segments of Lycium barbarum polysaccharides and the hydrophobic proteins secreted by Cordyceps militaris mycelium gradually combine through directional hydrophobic interactions to form a core-shell structure with uniform particle size (about 76nm), while high-pressure homogenization forced crushing (Comparative Example 5) interrupts this self-assembly process, resulting in random aggregation of polysaccharides and proteins (particle size > 200nm). This structural difference directly affects the Brownian motion intensity of the particles: small-size particles maintain high dispersibility in the liquid phase, while large particles accelerate stratification due to gravity sedimentation. The stratification rate corresponding to Example 1 is only 1 / 8 of that of Comparative Example 5.

[0109] The lack of a standing step also destroys the dynamic repair mechanism of the interfacial film. In Example 1, the polysaccharide-protein complex forms a dense interfacial film on the surface of the droplet during the standing stage, and the flexible stretching of its molecular chains can buffer the temperature fluctuation stress during storage; while the homogenization treatment of Comparative Example 5 causes microcracks in the interfacial film, and the particles continue to coarsen due to the Ostwald ripening effect during storage, resulting in an abnormal increase in viscosity (+23.4%). This phenomenon confirms the necessity of thermodynamic equilibrium control in the self-assembly process - the relaxation time provided by standing allows the system free energy to reach a minimum value, while forced homogenization locks the system in a metastable state.

[0110] The process synergy is further reflected in the dual stabilization mechanism of steric hindrance and electrostatic repulsion. The surface of the nanoparticles in Example 1 is covered with a large number of polysaccharide hydroxyl groups, which form steric hindrance through the hydration layer. At the same time, the negative charge (Zeta potential -32mV) generated by the ionization of emblica polyphenols enhances the electrostatic repulsion; while in Comparative Example 5, the surface charge density is reduced due to particle coarsening (Zeta potential -18mV), and the dual stabilization mechanism fails, which eventually leads to rapid stratification. This difference verifies the irreplaceable nature of the static self-emulsification treatment from the microscopic dynamics level.

[0111] Test Example 5: Natural antibacterial function test of fresh fruit of Phyllanthus emblica Experimental purpose: To verify the natural antibacterial effect and long-term effect of polyphenols in fresh fruit of Phyllanthus emblica and to compare the differences with chemical preservatives (citric acid).

[0112] Description of experimental steps: Sample preparation: Example 1: prepared according to the original process, containing fresh fruit components of Emblica officinalis (10%).

[0113] Comparative Example 6: The fresh fruit of Emblica officinalis was replaced with an equal amount of citric acid (the pH was adjusted to 3.5), and the other components were consistent with those in Example 1.

[0114] Antibacterial function test: Total colony count before sterilization: Take 1 mL of unsterilized fermentation broth and dilute it to 10% with 0.85% saline.-3 , spread on nutrient agar plates, culture at 37℃ for 48h, and count colony forming units (CFU / mL).

[0115] Mold proliferation rate during storage period: The sterilized samples were divided into sterile glass bottles and stored in a dark place at 25°C for 30 days, with samples taken every 10 days.

[0116] Take 1 mL of sample and add it into Rose Bengal medium (containing chloramphenicol), culture it at 28℃ for 72 h, and calculate the growth percentage (%) of mold count = (final number - initial number) / initial number × 100%.

[0117] Testing conditions: Colony count: nutrient agar (peptone 10 g / L, beef extract 3 g / L, NaCl 5 g / L, agar 15 g / L, pH 7.2).

[0118] Fungal culture: Red Bengal medium (glucose 10g / L, peptone 5g / L, KH 2 PO 4 1g / L, MgSO 4 0.5g / L, Rose Bengal 0.05g / L, Chloramphenicol 0.1g / L).

[0119] Data processing: The total number of colonies was the mean ± standard deviation of three parallel experiments; The fungal proliferation rate was calculated as the maximum growth rate within 30 days.

[0120] Experimental data: Table 5: Comparison of antibacterial effects of emblica polyphenols and citric acid Data description: Total colony count before sterilization: In Example 1, due to the immediate antibacterial effect of emblica polyphenols, the initial colony count was only 1 / 3 of that in Comparative Example 6; In Comparative Example 6, citric acid only lowers the pH and cannot effectively inhibit microbial proliferation.

[0121] Fungal growth rate: Example 1: During the 30-day storage period, mold growth was 18.7%, and emblica polyphenols continued to inhibit spore germination; In Comparative Example 6, mold increased by 142.5%, and pH adjustment could not prevent secondary contamination of mold-tolerant strains.

[0122] Experimental conclusion: This experiment confirmed the significant advantages of the multi-dimensional antibacterial mechanism of polyphenols in fresh fruit of Phyllanthus emblica over single chemical preservatives. Polyphenols such as gallic acid and corilagin in Phyllanthus emblica insert into the phospholipid bilayer of microbial cell membrane through phenolic hydroxyl groups, destroying its transmembrane potential and leading to intracellular ion leakage (corresponding to a 66.7% decrease in the total number of colonies before sterilization in Example 1). This process has a broad spectrum of selectivity - polyphenol molecules preferentially bind to the thick peptidoglycan layer of Gram-positive bacteria, but are non-toxic to Cordyceps militaris mycelium, ensuring the metabolic activity of beneficial bacteria in the fermentation system.

[0123] The long-lasting antibacterial effect during storage is due to the slow-release-chelation synergistic effect of polyphenols. Phyllanthus emblica polyphenols are gradually released in an acidic fermentation environment, and their catechol structure can continuously chelate metal cofactors (such as Fe 3+ , Cu 2+ ), inhibiting the activity of catalase and superoxide dismutase, blocking the energy metabolism pathway of mold. This dynamic protection mechanism makes the mold proliferation rate of Example 1 only 18.7% within 30 days, while the citric acid in Comparative Example 6 only relies on pH adjustment and cannot neutralize the acid-resistant mold spores introduced by external pollution during storage.

[0124] The essential difference between natural components and chemical agents is reflected in the depth of metabolic interference. Citric acid achieves short-term antibacterial effect by lowering pH, but microorganisms can quickly activate proton pump systems (such as H + -ATPase) to maintain intracellular homeostasis, resulting in a secondary explosive proliferation of mold in Comparative Example 6 (+142.5%). Emblica polyphenols simultaneously act on cell membranes, metabolic enzymes and genetic material (inducing DNA oxidative damage), forming multiple antibacterial barriers. This multi-target inhibition characteristic blocks the adaptive evolution path of microorganisms from the root, confirming the irreplaceable role of natural components in preservative stability.

[0125] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A formula of wolfberry compound puree with both medicinal and edible properties, characterized in that: By weight percentage, it includes the following components: Wolfberry dried fruit 65%-75%, Cordyceps militaris mycelium 18%-22%, Emblica emblica fresh fruit 8%-12%.

2. The formula of the edible and medicinal wolfberry composite puree according to claim 1, characterized in that: The following auxiliary components are also included: Glucose, potassium dihydrogen phosphate and magnesium sulfate, wherein glucose accounts for 2% to 6% of the total weight of the raw pulp, potassium dihydrogen phosphate accounts for 0.08% to 0.12% of the total weight of the raw pulp, and magnesium sulfate accounts for 0.04% to 0.06%.

3. The formula of the edible and medicinal wolfberry composite puree according to claim 1, characterized in that: The active ingredients in the puree include 0.5-1.0 mg / g cordycepin and 8%-12% wolfberry polysaccharide.

4. A method for preparing a composite wolfberry puree formula that is both medicinal and edible, characterized in that: The preparation method is prepared by using the formula of the edible and medicinal wolfberry composite puree as claimed in any one of claims 1 to 3, comprising the following steps: Raw material pretreatment: washing and crushing fresh wolfberry and emblica emblica fruits; Gradient enzymatic hydrolysis: wolfberry and emblica were treated with cellulase and pectinase in stages; Cordyceps militaris liquid fermentation: inoculation of bacteria and dynamic regulation of carbon-nitrogen ratio; Cold plasma pulse sterilization: non-thermal sterilization of fermentation broth; Self-emulsification stabilization treatment: forming nanoparticles and homogenizing; Filling and storage.

5. The method for preparing the edible and medicinal wolfberry composite puree according to claim 4, characterized in that: In the raw material pretreatment step, the fresh emblica fruit is pitted and pulped, and the solid content of the pulp is 12% to 18%.

6. The method for preparing the edible and medicinal wolfberry composite puree according to claim 4, characterized in that: In the gradient enzymatic hydrolysis step: The conditions for enzymatic hydrolysis of wolfberry cellulose are as follows: enzyme addition amount 0.2%-0.5%, temperature 45-55℃, pH 4.8-5.2, enzymatic hydrolysis time 1.5-2.5h, and 25-30kHz ultrasonic treatment for 8-12min after enzymatic hydrolysis; The enzymatic hydrolysis conditions of emblica pectin are as follows: enzyme addition amount 0.1%-0.3%, temperature 38-42℃, pH 3.2-3.8, and enzymatic hydrolysis time 1.0-2.0h.

7. The method for preparing the edible and medicinal wolfberry composite puree according to claim 4, characterized in that: The dynamic regulation of the carbon-nitrogen ratio in the Cordyceps militaris liquid fermentation step comprises: Initial C / N ratio 20-30:1, fermentation 0-48h; Glucose was added to adjust the C / N ratio to 15-20:1 and fermented for 49-72 hours; Add corn syrup powder to adjust the C / N ratio to 25-35:1 and ferment for 73-120 hours.

8. The method for preparing the edible and medicinal wolfberry composite puree according to claim 4, characterized in that: The cold plasma pulse sterilization parameters in the cold plasma pulse sterilization step are: The voltage is 8-12 kV, the frequency is 4-6 kHz, the pulse mode is 4-6 kHz high-frequency discharge alternating with 0.5-2.0 ms intermittently, the sterilization time is 12-18 min, and nitrogen is introduced throughout the process to maintain the oxygen content <1.0%.

9. The method for preparing the edible and medicinal wolfberry composite puree according to claim 4, characterized in that: In the self-emulsifying stability treatment step: The nanoparticles were placed at 4-10°C for 18-30h. Homogenizing pressure 40-60MPa, cycle 1-3 times.

Images