Ganoderma lucidum polysaccharide-fish scale peptide compound with high reducing power as well as preparation method and application thereof

Abstract

The invention discloses a ganoderma lucidum polysaccharide-fish scale peptide compound with high reducing power as well as a preparation method and application thereof, and belongs to the field of biological medicines. According to the method, firstly, by designing different test combinations, correlation between factors and test results (response values) is fitted through polynomials, the relation between the factors and the test results is functionalized, the interaction relation between the factors and the response values and the interaction relation between the factors are researched, the contribution degrees of the important factors to target values are further optimized, and the target values are optimized; and determining an optimal extraction process. The extracted ganoderan-fish scale peptide compound has a certain effect of high oxidation resistance or high reducing power, and has a better application effect in the aspects of oral digestion, stomach digestion, small intestine digestion and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a highly reducing Ganoderma lucidum polysaccharide-fish scale peptide complex, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Reishi mushroom (Ganoderma lucidum) belongs to the Polyporaceae family and the Ganoderma genus. Its cap is typically semi-circular or kidney-shaped, reddish-brown with a lacquer-like sheen, exhibiting annular ridges and radial wrinkles, with thinner edges that often curl inwards. Reishi is a fungus used in both medicine and food, containing abundant triterpenoids, polysaccharides, amino acids, and alkaloids, among other active ingredients, exhibiting significant effects in anti-oxidation, enhancing immunity, and anti-inflammation.

[0004] Polysaccharides, an important active ingredient in Ganoderma lucidum, possess various biological activities and broad health benefits. Due to their complex structure, Ganoderma lucidum endows it with significant immunomodulatory capabilities, activating immune cells, enhancing the phagocytic activity of macrophages and the activity of natural killer cells, thereby improving the body's immune function. Furthermore, Ganoderma lucidum polysaccharides are safe and non-toxic, and have been widely used in the fields of biomedicine, food, and cosmetics.

[0005] Furthermore, with the rapid development of the global seafood processing industry, the output of fish processing by-products such as fish scales, fish skin, and fish bones has been increasing year by year. As the world's largest aquaculture country, my country has ranked first globally in total aquatic product output for many consecutive years, generating over one million tons of processing by-products annually, but its utilization rate remains low. Large quantities of fish scales are directly discarded or simply processed, not only wasting resources but also easily causing environmental pollution. When extracted fish scale peptides are used alone, their antioxidant activity is limited, failing to meet the demand for highly active antioxidant products. Moreover, existing composite systems often target single effects, are limited to the functional development of materials such as film-forming agents, and focus primarily on fungal components without exploring their binding with exogenous substances. Furthermore, the polysaccharide types used lack significant synergistic potential in bioactivity, failing to fully utilize the synergistic antioxidant potential of polysaccharides and peptides. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a highly reducing Ganoderma lucidum polysaccharide-fish scale peptide complex, its preparation method, and its application. The present invention uses the Maillard method to prepare the Ganoderma lucidum polysaccharide-fish scale peptide complex, which has simple operation steps and good biological activity.

[0007] This invention aims to combine Ganoderma lucidum polysaccharide, a medicinal and edible active ingredient, with fish scale peptide, a bioactive component from aquatic processing byproducts. Response surface methodology is used to optimize the preparation process of the polysaccharide-peptide complex. By simulating the pH and enzyme environment of the oral cavity, stomach, and small intestine through in vitro digestion, the invention focuses on analyzing the reducing power stability of the complex under dynamic physiological conditions, exploring the synergistic effect of the two natural active substances, and overcoming the limitations of traditional single-component research. This invention prepares a novel natural complex with high reducing power, providing theoretical support for the development of natural high-reducing-power complexes with good long-lasting effects and stability. It also offers new ideas for the high-value utilization of aquatic processing waste, and has dual practical significance for promoting the development of the health industry and achieving the goal of a circular economy.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a highly reducing Ganoderma lucidum polysaccharide-fish scale peptide complex, which is composed of Ganoderma lucidum polysaccharide and fish scale peptide, wherein the mass ratio of fish scale peptide to Ganoderma lucidum polysaccharide is (1.8~3.4):1.

[0009] Secondly, the present invention provides a method for preparing a highly reducing Ganoderma lucidum polysaccharide-fish scale peptide complex, comprising the following steps: Ganoderma lucidum polysaccharide and fish scale protein peptide are mixed according to the mass ratio, water is added, and the mixture is heated at the set temperature and time. After the reaction is completed, it is freeze-dried to obtain the final product.

[0010] In one or more embodiments, the mass ratio of fish scale peptide to Ganoderma lucidum polysaccharide is (1.8~3.4):1, such as 1.8:1, 2:1, 2.2:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.86:1, 2.9:1, 3:1, 3.2:1, 3.4:1, etc., and specific values ​​between the above values, preferably (2.5~3):1, more preferably (2.8~2.9):1.

[0011] The heating temperature is 40~80℃, such as 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 80℃, etc., or specific values ​​between the above points. The heating temperature is preferably 60~80℃, and more preferably 70~75℃.

[0012] The heating time is 45-85 minutes, such as 45 minutes, 50 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 65 minutes, 70 minutes, 80 minutes, 85 minutes, etc., or specific values ​​between the above points. The heating time is preferably 50-70 minutes, and more preferably 55-60 minutes.

[0013] Response surface methodology revealed that the high reducing power of the Ganoderma lucidum polysaccharide-fish scale peptide complex is closely related to its mass ratio and the heating temperature and time of the binding reaction. The order of influence on the reducing power of the complex is: heating temperature > mass ratio > heating time. Only when the mass ratio, heating temperature, and heating time are within appropriate ranges can the prepared complex exhibit superior reducing power.

[0014] In one or more embodiments, the amount of water added is 2-10% of the total mass of Ganoderma lucidum polysaccharides and fish scale peptides, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. Preferably, it is 4-6%, and most preferably 5%. The intensity of the Maillard reaction is largely affected by the hydration of the medium. After freeze-drying, the polysaccharides and fish scale peptides have low internal water content, making it difficult to exert their reactivity. Furthermore, as the amount of water added increases, the concentration of the polysaccharide-peptide decreases accordingly, which also leads to a weakening of the reactivity. Therefore, to achieve maximum reactivity, in this invention, a 5% water content is used to prepare the polysaccharide-peptide solution.

[0015] In one or more embodiments, the preparation method was optimized using response surface methodology, and the quadratic polynomial regression equations relating the reducing power of the resulting complex to the mass ratio (A), heating temperature (B), and heating time (C) are as follows: Y=-3.20234+0.738781×A+0.056474×B+0.025201×C+0.002813×A×B–0.001406×A×C–0.000106×B×C–0.150937×A 2 –0.000404×B 2 –0.000119×C 2 The model established by this invention through response surface methodology has a good fit and produces small errors during the experiment.

[0016] In one or more embodiments, the preparation method of Ganoderma lucidum polysaccharide includes: pulverizing Ganoderma lucidum, sieving, and preparing polysaccharide by water extraction and alcohol precipitation. The specific preparation method of water extraction and alcohol precipitation includes: adding water according to the material-to-liquid ratio, heating and extracting, filtering and collecting the solution, centrifuging to obtain the supernatant and concentrating; mixing the solution with 95% ethanol evenly, allowing it to stand at low temperature, centrifuging to obtain the precipitate, and then freeze-drying to obtain Ganoderma lucidum polysaccharide. Further, the material-to-liquid ratio is 1:(30~50) (g / mL), heating and extracting at 80~100℃ for 2~6 h, centrifuging at 2000~6000 rpm for 5~20 min, the volume ratio of solution to 95% ethanol is 1:(2~4), and allowing it to stand at low temperature (2~5℃) for 10~15 h.

[0017] The water extraction and alcohol precipitation method selected in this invention has the advantages of being simple to operate, environmentally friendly, and causing less damage to the polysaccharide structure. The polysaccharide content obtained by this method is 12.4%.

[0018] 95% ethanol refers to an aqueous solution of ethanol with an ethanol content of 95 wt%.

[0019] In one or more embodiments, water is added at a material-to-liquid ratio of 1:(30~50) (g / mL), and the mixture is heated and extracted at 80~100℃ for 2~6h. After filtration and collection of the solution, the supernatant is obtained by centrifugation at 2000~6000 rpm for 5~20min and concentrated to an appropriate volume. The solution is mixed with 95% ethanol at a volume ratio of 1:(2~4), and allowed to stand at 2~5℃ for 10~15h. After centrifugation at 2000~6000 rpm for 5~20min, the precipitate is obtained and then freeze-dried to obtain Ganoderma lucidum polysaccharide.

[0020] Ganoderma lucidum polysaccharides have powerful antioxidant effects, which can scavenge free radicals, inhibit oxidative stress, thereby delaying aging and preventing a variety of oxidative stress-related diseases.

[0021] In one or more embodiments, the preparation process of fish scale peptides includes: decalcifying fish scale fragments, then removing impurities and proteins, filtering and washing, soaking them in acetic acid solution, filtering to obtain an extract, adding sodium chloride solution to the extract and salting out under low temperature conditions, centrifuging to collect the precipitate, resolving it with acetic acid solution, dialysis in a dialysis bag, and freeze-drying to obtain fish scale peptides.

[0022] Furthermore, the decalcification process involves immersing the dried fish scale fragments in an acid solution. The acid is hydrochloric acid, with a concentration of 0.5–2 mol / L, and the immersion time is 80–110 min. Using hydrochloric acid for decalcification effectively removes calcium from fish scales. Moreover, within the specified concentration range, as the hydrochloric acid concentration increases, the degree of hydrolysis of fish scale peptides gradually decreases, thus facilitating the extraction and separation of fish scale peptides.

[0023] Furthermore, the deproteinization treatment involves immersing the decalcified fish scales in a sodium carbonate solution at a material-to-liquid ratio of 1:30-50 (g / mL) to remove impurities. The concentration of the sodium carbonate solution is 0.2-0.5 mol / L, and the immersion time is 4-7 hours. Within this range, effective removal of impurities is more likely.

[0024] The extraneous proteins mainly include mucins and globulins, whose presence adversely affects the extraction process of fish scale peptides and increases the difficulty of subsequent purification. Therefore, pretreatment of these extraneous proteins is an essential step before extracting fish scale peptides.

[0025] Furthermore, the sample was soaked in 0.5-2 mol / L acetic acid solution at a material-to-liquid ratio of 1:5-15 (g / mL) for 15-25 h, filtered to obtain the extract, and then salted out at 2-5℃ for 20-25 h with 0.5-1 mol / L sodium chloride solution. The precipitate was collected by centrifugation, redissolved with 0.2-0.6 mol / L acetic acid solution, dialyzed in a dialysis bag for 24-48 h, and then freeze-dried.

[0026] Analyzing the above factors, when the material-to-liquid ratio is too high, the acetic acid solution cannot adequately wet the fish scale residue, leading to incomplete protein dissolution and thus reducing the extraction rate. Simultaneously, the solution viscosity may increase, making subsequent separation processes (such as centrifugation and filtration) more difficult. Conversely, if the material-to-liquid ratio is too low, although it helps with complete protein dissolution, it results in an excessively low solution concentration, increasing energy consumption in subsequent concentration processes. Furthermore, the proportion of impurities in the solution (such as inorganic salts and small molecules) may increase, affecting the purity of the final product.

[0027] Acetic acid, used as a solvent, affects the solubility of fish scale peptides by adjusting the pH and ionic strength of the solution. If the concentration is too low, the acidity is insufficient to break the hydrogen bonds and hydrophobic interactions between fish scale protein molecules, leading to decreased protein solubility and extraction rate. If the concentration is too high, it can cause protein molecular chain breakage, destroying its natural structure and activity; it may also dissolve more impurities, thus reducing product purity.

[0028] Soaking time affects the extraction efficiency by influencing the degree of reaction between proteins and acetic acid. If the soaking time is too short, the proteins will not dissolve sufficiently, leading to a decrease in extraction rate; while if the soaking time is too long, it may cause excessive hydrolysis of the proteins, reducing their molecular weight and thus affecting functional properties (such as gelling and emulsifying properties). Furthermore, long soaking times also increase the risk of microbial contamination.

[0029] In one or more embodiments, the preparation process of fish scale peptides includes: soaking dried fish scale fragments in a 0.5-2 mol / L acid solution for 80-110 min to remove calcium; soaking the decalcified fish scales in a 0.2-0.5 mol / L sodium carbonate solution for 4-7 h to remove impurities and proteins at a material-to-liquid ratio of 1:30-50 (g / mL); filtering and washing; soaking the treated fish scales in a 0.5-2 mol / L acetic acid solution at a material-to-liquid ratio of 1:5-15 (g / mL) for 15-25 h; filtering to obtain an extract; adding a 0.5-1 mol / L sodium chloride solution to the extract and salting out at 2-5℃ for 20-25 h; centrifuging to collect the precipitate; redissolving the precipitate in a 0.2-0.6 mol / L acetic acid solution; dialyzing in a dialysis bag for 24-48 h; and freeze-drying to obtain fish scale peptides.

[0030] Fish scale peptides are a class of bioactive small-molecule protein fragments extracted from fish scales. Compared to collagen peptides extracted from terrestrial animal bones, they have the advantages of smaller molecular weight, higher safety, and better absorption. Fish scale peptides possess various biological activities, including antioxidant, anti-inflammatory, whitening, and wound-healing effects. Among these, antioxidant activity is one of the most noteworthy benefits of fish scale peptides. Fish scale peptides exert their antioxidant effects through mechanisms such as scavenging free radicals and chelating metal ions. Furthermore, compared to other existing peptides, such as carnosine, the polysaccharide-fish scale peptide complex prepared using fish scale peptides in this invention exhibits superior reducing power.

[0031] Thirdly, this invention provides the application of the above-mentioned highly reducing Ganoderma lucidum polysaccharide-fish scale peptide complex in the preparation of highly reducing products, wherein the reduction includes reduction of digestive sites, including oral digestion, gastric digestion, and small intestinal digestion. Preferably, the products include pharmaceuticals, health products, and food.

[0032] Fourthly, the present invention provides a product comprising the above-mentioned highly reducing Ganoderma lucidum polysaccharide-fish scale peptide complex.

[0033] Fifthly, the present invention provides a composition comprising the above-described high-reducing-potency Ganoderma lucidum polysaccharide-fish scale peptide complex or the high-reducing-potency Ganoderma lucidum polysaccharide-fish scale peptide complex obtained by the above-described preparation method or the above-described product, and pharmaceutically acceptable excipients or carriers.

[0034] In a sixth aspect, the present invention provides an antioxidant method, wherein the method comprises applying the above-described high-reducing-potency Ganoderma lucidum polysaccharide-fish scale peptide complex or the above-described product or the above-described composition to an application subject.

[0035] One or more of the above technical solutions have the following advantages or beneficial effects: (1) Compared with individual fish scale peptides or individual polysaccharides, the Ganoderma lucidum polysaccharide-fish scale peptide complex prepared by the process provided in this invention has a synergistic effect in reducing power, and its reducing power is more effective. The reducing power of individual fish scale peptides is 0.05±0.01, the reducing power of individual polysaccharides is 0.36±0.01, while the reducing power of the prepared polysaccharide-peptide complex is 0.63±0.02.

[0036] (2) This invention first designs different experimental combinations, fits the relationship between factors and experimental results (response values) using a polynomial, functions the relationship between factors and experimental results, studies the interaction between factors and response values, and between factors themselves, further optimizes the contribution of these important factors to the target value, and determines the optimal extraction process. The extracted Ganoderma lucidum polysaccharide-fish scale peptide complex has a certain high antioxidant effect and good application results. The reducing power of the Ganoderma lucidum polysaccharide-fish scale peptide complex prepared by this invention is higher than 0.3, preferably higher than 0.4, and more preferably higher than 0.5.

[0037] (3) The model established by this invention through response surface methodology has a good fit and produces small errors during the experiment. Further significance analysis of the regression coefficients of the quadratic model revealed that the order of influence of the three linear factors on the reducing power of the complex is B>A>C, i.e., heating temperature>mass ratio>heating time. With reducing power as the indicator, the optimal preparation process of the Ganoderma lucidum polysaccharide-fish scale peptide complex is: mass ratio of 1:2.86, heating temperature of 73℃, heating time of 57 min, and three repeated experiments were conducted. The measured reducing power of the complex was 0.596±0.009.

[0038] (4) The polysaccharide-peptide complex prepared in this invention exhibits good adaptability to complex digestive environments. Its structure can achieve long-lasting antioxidant effects by balancing anti-degradation capacity and activity release efficiency. In the oral simulated digestion stage, the complex did not show a significant structural degradation trend during simulated oral digestion. In particular, after 90 min of in vitro simulated digestion, the reducing power of the complex was still close to the initial value, indicating that it can effectively maintain antioxidant activity in the oral digestion stage and has good structural stability and antioxidant capacity. In the gastric digestion stage, the complex showed a significant trend of increased reducing power. In the small intestinal digestion stage, the reducing power of the complex showed a fluctuating trend of first increasing, then decreasing, and then increasing again. The final reducing power at 6 h was 0.737±0.017, slightly higher than the initial value, suggesting that the complex can still retain some functional stability in the small intestinal environment. Attached Figure Description

[0039] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0040] Figure 1 For the accuracy analysis of the model in Embodiment 1 of the present invention; wherein, (A) predicted value and actual value; (B) external studentized residual normal probability; (C) residual and predicted value; Figure 2Outlier analysis for the model of this invention; wherein, (A) run number and studentized outlier residual; (B) run number and leverage level; Figure 3 These are response surface plots and contour plots showing the interaction between mass ratio and heating temperature in an embodiment of the present invention; where a is the response surface plot and b is the contour plot. Figure 4 The diagrams shown are the response surface plot and contour plot of the interaction between mass ratio and heating time in an embodiment of the present invention; where a is the response surface plot and b is the contour plot. Figure 5 These are response surface plots and contour plots showing the interaction between heating temperature and heating time in an embodiment of the present invention; where a is the response surface plot and b is the contour plot. Figure 6 The above is an optimized slope diagram for the preparation of the composite in this embodiment of the invention; where A is the mass ratio of 2.86, B is the heating temperature of 72.37℃, C is the heating time of 56.71 min, and D is the reducing power of the composite of 0.611493. Figure 7 The Fourier transform infrared spectra of protein (Pro), polysaccharide (PS), and protein-polysaccharide complex (Pro-PS) in the embodiments of the present invention are shown. Figure 8 The reducing power changes of the complex in simulated in vitro digestion in the embodiments of the present invention are shown; wherein, (A) oral digestion stage; (B) gastric digestion stage; (C) small intestinal digestion stage; Figure 9 The reducing power of different samples at a 5% concentration. Detailed Implementation

[0041] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0042] Fish scale protein peptides are extracted and prepared from the fish scales remaining after the industrial processing of grass carp.

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] Example 1 1. Sample processing After drying the fruiting bodies of Ganoderma lucidum, they were pulverized into granules, sieved, and polysaccharides were prepared using a water extraction and alcohol precipitation method. Deionized water was added at a material-to-liquid ratio of 1:40 (g / mL), and the mixture was heated and extracted in a water bath at 90℃ for 4 h. After filtration, the solution was collected and centrifuged at 3000 rpm for 10 min to obtain the supernatant, which was then concentrated to an appropriate volume. The solution was mixed with 95% ethanol at a volume ratio of 1:3, allowed to stand at 4℃ for 12 h, and centrifuged at 3000 rpm for 10 min to obtain the precipitate. The precipitate was then freeze-dried to obtain Ganoderma lucidum polysaccharides.

[0045] Dried fish scale fragments were soaked in 1 mol / L hydrochloric acid solution for 95 min to remove calcium. The decalcified fish scales were then soaked in 0.3 mol / L sodium carbonate solution for 6 h to remove impurities and proteins. After filtration and washing, the fish scales were soaked in 1 mol / L acetic acid solution for 24 h at a material-to-liquid ratio of 1:10 (g / mL). The extract was obtained by filtration. 0.9 mol / L sodium chloride solution was added to the extract and salted out at 4℃ for 24 h. The precipitate was collected by centrifugation, redissolved in 0.5 mol / L acetic acid solution, dialyzed in a dialysis bag for 48 h, and then freeze-dried to obtain fish scale peptides.

[0046] 2. Following the Box-Behnken design scheme in Design-Expert software, this study investigated the interaction between three factors: the mass ratio of fish scale peptides to Ganoderma lucidum polysaccharides (A), heating temperature (B, °C), and heating time (C, min). Using reducing power as an indicator of in vitro antioxidant activity, the effects of different preparation conditions on the in vitro antioxidant activity of the Ganoderma lucidum polysaccharide-fish scale peptide complex were explored, and the preparation process of the complex was optimized. The corresponding levels of the three factors are shown in Table 1.

[0047] Table 1. Factors and levels in Box-Behnken experimental design

[0048] A series of parallel experiments were conducted according to the Box-Behnken experimental design. Ganoderma lucidum polysaccharide and fish scale protein peptide were placed in conical flasks according to different addition mass ratios, and deionized water was added at a mass fraction of 5%. The mixtures were heated in a water bath at different temperatures and times to bind. After the reaction was completed, the mixtures were freeze-dried to obtain complexes under different binding conditions.

[0049] 3. In vitro antioxidant activity assessment The antioxidant capacity of Ganoderma lucidum polysaccharide-fish scale peptide complexes under different binding conditions was evaluated using the Prussian blue method. One mL of the complex sample was weighed into a test tube, and 2.5 mL of phosphate buffer (0.2 mol / L, pH 6.6) and 1 mL of 1% potassium ferricyanide solution were added sequentially. After mixing thoroughly, the mixture was reacted at 50℃ for 20 min. The mixture was then cooled under running water, and 2 mL of 10% trichloroacetic acid solution and 1.2 mL of 0.1% ferric chloride solution were added sequentially. After mixing, the mixture was centrifuged, and the supernatant was collected. All complex samples were prepared in triplicate, with deionized water used as a control for zeroing. The absorbance was measured at 700 nm. Higher absorbance indicates higher reducing power and stronger antioxidant capacity.

[0050] 4. Fourier transform infrared spectroscopy analysis Fourier transform infrared spectroscopy (FT-IR) was used to characterize the structure of the samples. After vacuum freeze-drying of Ganoderma lucidum polysaccharides, fish scale peptides, and polysaccharide-peptide complexes, 1.0 mg of each dried sample was accurately weighed and mixed with 150 mg of potassium bromide (KBr) in a mortar. The mixture was then ground uniformly to prepare thin slices, which were then analyzed using a Fourier transform infrared spectrometer in the wavenumber range of 500 cm⁻¹. -1 ~4000 cm -1 The system performs scanning. Based on the wavenumber and peak shape characteristics of the characteristic absorption peaks, it systematically analyzes the vibrational modes of characteristic functional groups in the three samples.

[0051] 5. BBD Data Analysis The results obtained from the response surface methodology are shown in Table 2. Using Design-Expert 13.0 for data processing, the quadratic polynomial regression equations between the reducing power of the composite and the mass ratio (A), heating temperature (B), and heating time (C) are as follows: Y=-3.20234+0.738781×A+0.056474×B+0.025201×C+0.002813×A×B–0.001406×A×C–0.000106×B×C–0.150937×A 2 –0.000404×B 2 –0.000119×C 2 .

[0052] Table 3 shows that the model's F-value is 690.11, and the P-value is less than 0.0001, indicating that the model shows a highly significant difference and a good fit. The P-value for the model's lack-fit term is 0.6766, which is greater than 0.05, indicating that the difference from the pure error is small and not significant. As shown in Table 4, the standard deviation is 0.0082, indicating low bias in the experimental association. The coefficient of variation is 2.01, indicating high reliability and low dispersion. The PRESS is 0.0027, and the noise ratio is 71.7952, which is greater than 4, indicating a significant linear relationship between the independent variable and the response value. The model's R-value... 2 The value is 0.9989, which is greater than 0.9, indicating a good fit. Corrected R-squared value. 2 The value is 0.9974, indicating that independent variables account for 99.74% of the total variation, but uncertainties such as random errors may have led to data misfit. In summary, the model has a good fit and produces small errors during the experiment, making it suitable for analyzing the optimal preparation process of the complex.

[0053] Based on the data analysis results in Table 5, it can be concluded that in the linear term, A and B have a highly significant effect on the reducing power index of the complex at the p-value < 0.01 level. In the interaction term, AB, AC, and BC all have highly significant effects at the p-value < 0.01 level. Furthermore, in the quadratic term, A... 2 B 2 and C 2 All of these results showed extremely significant levels. Therefore, based on the significance analysis of the regression coefficients in the quadratic model, the order of influence of the three linear factors on the reducing power of the complex is B>A>C, i.e., heating temperature>mass ratio>heating time.

[0054] Table 2 Results of Response Surface Design Experiments

[0055] Table 3. Centralized Portfolio Design Model and Analysis of Variance

[0056] Note: * indicates a significant difference (P<0.05); ** indicates a highly significant difference (P<0.01).

[0057] Table 4. Analysis of variance of the reducing power fit of the complex

[0058] Table 5. Significance test of regression coefficients in the quadratic model

[0059] Note: * indicates a significant difference (P<0.05); ** indicates a highly significant difference (P<0.01).

[0060] Model accuracy and outlier analysis: like Figure 1 As shown, the predicted and actual values ​​of the reducing power of the complex are evenly distributed, located on both sides of the straight line and close to the straight line, showing a good correspondence. Figure 1 (A). In a normal probability distribution, the points are randomly distributed on both sides of a straight line and are almost on the same straight line. This indicates that the response distribution plot follows a normal distribution, and the variance of the model is not biased. See [link to relevant documentation]. Figure 1 (B). In the residual and model prediction distribution plot, the response values ​​are randomly distributed and all lie within the marked red limit line, see... Figure 1 (C). In summary, the model is well-constructed and can be used for response analysis of the reducing power of complexes.

[0061] like Figure 2 As shown, in the external studentized residual normal probability distribution of the reducing power of the complex, all data points are within the red limit line. Figure 2 (A). Furthermore, the leverage values ​​for all data are less than 1, indicating the absence of outliers that could potentially affect model fit; see [link to relevant documentation]. Figure 2 (B). Therefore, this model can be used to analyze the relationships between variables that affect the reducing power of the complex.

[0062] Interaction analysis of factors: Response surface methodology (RSM) visually illustrates the interactions between factors, thereby optimizing conditions and predicting response values. Contour plots, on the other hand, quickly locate the optimal region through contour line distribution, simplifying complex relationships and aiding experimental design. Based on the regression equation analysis results, RSM and contour plots were drawn to illustrate the interaction between the mass ratio of fish scale protein active peptides and Ganoderma lucidum polysaccharides, heating temperature, and heating time on the reducing power of the complex. Figures 3-5 As shown. By Figure 3 As shown in Table 5, the interaction between the mass ratio of polysaccharides and peptides and the heating temperature of the binding reaction has a significant effect on the polysaccharide-peptide complex (P < 0.0001, F = 119.42). The response surface exhibits a non-linear trend of first increasing and then decreasing, with a relatively steep surface, indicating that the reducing power of the complex reaches its peak under the synergistic effect of the mass ratio and heating temperature; however, the reducing power of the complex then decreases, possibly due to polysaccharide degradation or inactivation of fish scale protein peptides. The contour lines are more densely packed along the mass ratio axis, indicating that small changes in the mass ratio are more sensitive to the results, but the analysis of variance shows that the single main effect of heating temperature (F = 3328.86) is much higher than that of mass ratio (F = 73.72), indicating that temperature has a stronger independent influence on the reducing power.

[0063] from Figure 4 The contour plots, response surface plots, and Table 5 show that the interaction between mass ratio and heating time significantly affects the reducing power of the polysaccharide-peptide complex (P=0.0009, F=29.85). The response surface plots show an initial increase followed by a gradual flattening, with a relatively gentle curve, indicating that the reducing power of the complex gradually increases under the synergistic effect of mass ratio and heating time, but tends to stabilize after reaching a certain threshold without a significant decrease. The contour lines are sparser along the heating time axis, indicating that the adjustment of heating time has a low sensitivity to the results. In the analysis of variance, the main effect of mass ratio alone (F=73.72) is much higher than that of heating time (F=4.25), indicating that the independent effect of mass ratio on reducing power is more critical.

[0064] from Figure 5 The results of the interaction analysis and Table 5 show that the interaction between heating temperature and heating time has a highly significant effect on the reducing power of the polysaccharide-peptide complex (P<0.0001, F=106.52). The response surface is steep, showing a trend of first increasing and then significantly decreasing, indicating that under the synergistic effect of temperature and time, the reducing power of the complex initially increases with increasing parameters, but weakens after exceeding the critical range, possibly due to polysaccharide thermal degradation or molecular structure destruction. The axial contour lines are more dense along the temperature axis, indicating that small adjustments in temperature are more sensitive to the results, which is consistent with the conclusion in the analysis of variance that the main effect of temperature alone (F=3328.86) is much stronger than that of time (F=4.25).

[0065] The above analysis shows that the interaction between the mass ratio of fish scale protein active peptides and Ganoderma lucidum polysaccharides, heating temperature, and heating time on the reducing power of the complex is consistent with the regression model. Figure 6 As shown, regression analysis revealed that the optimal preparation process for the Ganoderma lucidum polysaccharide-fish scale peptide complex, using reducing power as an indicator, was: a mass ratio of 1:2.86, a heating temperature of 72.37℃, and a heating time of 56.71 min. Under these conditions, the theoretically optimal reducing power was 0.611493. To ensure the reliability of the preparation process parameters, the optimized key parameters were adjusted to a mass ratio of 1:2.86, a heating temperature of 73℃, and a heating time of 57 min, and three repeated experiments were conducted. The measured reducing power of the complex was 0.596 ± 0.009, which is consistent with the theoretical value predicted by the model.

[0066] 6. Fourier transform infrared spectroscopy results: The figure shows the Fourier transform infrared (FT-IR) spectra of protein (Pro), polysaccharide (PS), and the protein-polysaccharide complex (Pro-PS). From the Pro spectrum, it can be seen that the protein is located at 3326 cm⁻¹. -1The absorption band in the vicinity is the stretching vibration peak of the OH group in the protein and water molecules in the amide A region. Due to the significant hydrogen bonding in the protein, this peak has a relatively broad width, located at 3079 cm⁻¹. -1 The absorption band nearby is the NH stretching vibration peak of the protein in the amide A region, located at 2929 cm⁻¹. -1 and 2878cm -1 The absorption band is mainly due to the vibrational absorption of CH, located at 1661 cm⁻¹. -1 The band in the vicinity is produced by the stretching vibration of the C=O group in amide I. The precise position of this peak reflects the protein's secondary structure—the α-helix at 1650-1658 cm⁻¹. -1 β-fold at 1620-1640cm -1 Random curls at 1640-1650cm -1 Located at 1550cm -1 The vibrations in the vicinity are caused by the coupled vibrations of the bending vibration of the NH group and the stretching vibration of the CN group in amide II. For amide III, the vibrations are located at 1400 cm⁻¹. -1 The absorption peak represents the bending vibration of CH, located at 1241 cm⁻¹. -1 The absorption peak represents the stretching vibration of CN, located at 1081 cm⁻¹. -1 The absorption peak represents the absorption vibration peak of CO, located at 1034 cm⁻¹. -1 The absorption peak represents the absorption vibration peak of CC, which mainly comes from the glycans in glycoproteins.

[0067] The graph from PS shows that it is located at 3426cm. -1 The absorption peak represents the stretching vibration peak of OH, located at 2942 cm⁻¹. -1 The absorption peak represents the stretching vibration peak of the sugar ring and the -CH2- group, located at 1617 cm⁻¹. -1 The vibrations in the vicinity are caused by the stretching vibrations of the C=O group, located at 1136 cm⁻¹. -1 and 1081cm -1 The main peaks are COC and CO stretching (glycosidic bond + cyclic ether bond) peaks, which are characteristic peaks of glycosidic bonds, located at 879 cm⁻¹. -1 The main characteristic peaks are those of β-glycosidic bonds.

[0068] It is clear from the graph that the NH in the original Pro peak has disappeared, and has formed a wider peak with a weaker intensity with the OH in the original Pro peak. This indicates that the NH in the protein and the OH in the polysaccharide form a hydrogen bond network. At the same time, the peak originally located at 1661 cm⁻¹ in Pro has disappeared. -1 The C=O at that point is shifted to a lower band (1629cm) after synthesis. -1The peak intensity decreased, indicating that the C=O (amide I) of Pro and the OH group of PS formed a hydrogen bond. This hydrogen bond formation signifies the successful synthesis of Pro-PS. Meanwhile, the synthesized spectrum shows peaks located in the 1150-1000 cm⁻¹ range. -1 The presence of characteristic peaks related to glycosidic bonds in the polysaccharide within the range indicates that the polysaccharide has successfully bound to the protein.

[0069] 7. In vitro digestion activity 7.1. In vitro simulation of complex digestion in saliva Prepare a simulated saliva fluid (SSF) with slight modifications. Accurately weigh 0.476 g of disodium hydrogen phosphate, 0.038 g of potassium dihydrogen phosphate, and 1.6 g of sodium chloride into an Erlenmeyer flask, add 200 mL of deionized water, and mix thoroughly. Accurately weigh 170 mg of α-amylase and add it to the above saliva electrolyte solution, mix thoroughly, and adjust the pH of the mixture to 6.7 using 0.5 mol / L hydrochloric acid.

[0070] Mix 30 mL of the 5 mg / mL complex solution with 30 mL of SSF until homogeneous. Take 2 mL of the reaction solution and heat in a boiling water bath for 10 min to inactivate the enzyme, storing the residue for later use. Incubate the remaining mixture at a constant temperature of 37℃. Monitor the pH of the reaction system dynamically throughout the experiment, maintaining a pH of 6.7 by adding 0.5 mol / L hydrochloric acid. The complex experimental group was run in triplicate. The blank control group was prepared by replacing the complex solution with an equal volume of deionized water; all other procedures were the same as the experimental group. At 10 min, 25 min, 45 min, 75 min, and 90 min after the start of the reaction, take 2 mL of the reaction solution and heat in a boiling water bath for 10 min to terminate enzyme activity. Centrifuge the resulting reaction samples and collect the supernatant for analysis.

[0071] 7.2. In vitro simulation of the digestion of the complex in gastric digestive juices Prepare and slightly adjust the simulated gastric fluid (SGF). Accurately weigh 0.62 g sodium chloride, 0.22 g potassium chloride, 0.03 g calcium chloride, and 0.12 g sodium bicarbonate into an Erlenmeyer flask. Add 200 mL of deionized water to the flask and mix thoroughly. Accurately weigh 150 mg of pepsin and 132 mg of acid lipase into the above gastric electrolyte solution. Mix thoroughly and adjust the pH of the mixture to 3.0 using 0.5 mol / L hydrochloric acid.

[0072] An equal volume of SGF was added to the mixture after saliva digestion for 90 min to adjust the pH of the reaction system to 3.0. Two mL of the reaction solution was then heated in a boiling water bath for 10 min to inactivate the SGF and stored for later use. The remaining mixture was reacted at a constant temperature of 37℃. The pH of the reaction system was dynamically monitored throughout the experiment and stabilized at 3.0 using 0.5 mol / L hydrochloric acid. At 0.5 h, 1 h, 2 h, 4 h, and 6 h after the start of the reaction, 2 mL of the reaction solution was taken and heated in a boiling water bath for 10 min. The resulting reaction samples were centrifuged, and the supernatant was collected for analysis.

[0073] 7.3. In vitro simulation of the digestion of the complex in small intestinal fluid An equal volume of simulated intestinal fluid (SIF) was added to the reaction system after 6 h of reaction with gastric digestive fluid. The pH of the reaction system was adjusted to 7.0 using 1 mol / L sodium bicarbonate solution. A 2 mL sample was taken and heated in a boiling water bath for 10 min to inactivate the enzyme. The remaining mixture was reacted at a constant temperature of 37℃. At 0.5 h, 1 h, 2 h, 4 h, and 6 h after the reaction, 2 mL samples were taken and heated in a boiling water bath for 10 min to terminate the enzymatic reaction. The supernatant was collected after centrifugation for analysis.

[0074] 7.4 Analysis of in vitro simulated digestion results Changes in the reducing power of polysaccharide-peptide complexes during oral simulated digestion, as follows: Figure 8As shown in (A), the reducing power of the complex was initially 1.431 ± 0.010, decreased to 1.412 ± 0.017 after 10 min, and then increased to 1.516 ± 0.012 at 25 min, reaching its highest value. Subsequently, the reducing power was 1.453 ± 0.008 at 45 min, 1.531 ± 0.014 at 75 min, and finally stabilized at 1.513 ± 0.017 at 90 min. Overall, it showed a trend of first decreasing, then increasing, and then stabilizing. In the initial stage of simulated oral digestion, the reducing power of the complex decreased slightly, possibly due to the influence of enzymes or physical processes in saliva on the surface structure of the complex, resulting in the temporary masking or alteration of some active sites. Over time, the reducing power of the complex gradually increased, possibly because the action of saliva further disrupted the surface structure of the complex, exposing more antioxidant active sites, thereby enhancing its reducing power. Ultimately, the reducing power stabilized at 90 min, indicating that the complex reached a dynamic equilibrium in the simulated oral digestion environment. Although a brief decrease in reducing power occurred at 10 min, overall, the complex did not exhibit a significant structural degradation trend during simulated oral digestion. In particular, after 90 min of in vitro simulated digestion, the reducing power of the complex remained close to its initial value, indicating that it effectively maintained its antioxidant activity during oral digestion and possessed good structural stability and antioxidant capacity.

[0075] During the digestive stage in the stomach, the complex exhibits a significant trend towards increased reducing power. From Figure 8 (B) It can be seen that the average reducing power continuously increased from 0.333 ± 0.014 at 0 h to 0.638 ± 0.017 at 6 h. This significant upward trend indicates that the low pH environment of gastric acid and the synergistic effect of pepsin gradually disrupted the internal structure of the complex. Under acidic conditions, hydrogen bonds or hydrophobic interactions between polysaccharides and peptide chains may be broken, releasing antioxidant components such as reduced peptide segments or active groups at the ends of sugar chains. In addition, the limited hydrolysis of peptide chains by pepsin may further generate exposed thiol groups, and the resulting short peptides have antioxidant properties. The brief decrease in reducing power to 0.264 ± 0.016 at 0.5 h may be due to the aggregation of the complex induced in the initial acidic environment, which obscured some active sites. However, as the digestion time gradually increased, the complex structure gradually dissociated, and the active components were fully released. The consistency of reducing power in the three repeated experiments at 6 h further verified the highly efficient promoting effect of gastric digestion on the activity of the complex.

[0076] During the small intestine digestion stage, the reducing power of the complex exhibits a fluctuating trend of first increasing, then decreasing, and then increasing again. For example... Figure 8As shown in (C), the initial value was 0.696 ± 0.014, which decreased to 0.642 ± 0.012 after 2 h, and then rebounded to 0.737 ± 0.017 after 6 h. The initial decrease may be related to the alkaline environment of the small intestine (pH approximately 7.4) and the effect of bile salts. Alkaline conditions may disrupt the molecular cross-linking structure of the complex, causing some active ingredients to bind with bile salts or be oxidized after exposure, temporarily inhibiting reducing power. The subsequent rebound indicates that as digestion time increases, more active components such as enzyme-resistant peptides or branched polysaccharides in the complex are gradually released, or degradation products such as oligosaccharides and short peptides continue to exert antioxidant activity through synergistic effects. The final reducing power of 0.737 ± 0.017 at 6 h is slightly higher than the initial value, suggesting that the complex can still retain some functional stability in the small intestinal environment.

[0077] In summary, this dynamic change reflects the adaptability of the polysaccharide-peptide complex in a complex digestive environment, and its structure can achieve long-lasting antioxidant effects by balancing anti-degradation capacity and activity release efficiency.

[0078] Comparative Example 1 Unlike Example 1, only fish scale peptides were prepared.

[0079] Comparative Example 2 Unlike Example 1, only polysaccharides were prepared.

[0080] The reducing power was determined at a sample concentration of 5% (w / v), and the following results were obtained. Figure 9 The results showed that at a concentration of 5%, the reducing power of the polysaccharide-peptide complex was 0.63 ± 0.02, significantly higher than that of the peptide (0.05 ± 0.01) or the polysaccharide (0.36 ± 0.01). This demonstrates that the polysaccharide-peptide complex obtained in this invention exhibits a synergistic effect compared to individual fish scale peptides or individual polysaccharides, resulting in a higher technical effect in terms of reducing power.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A highly reducing Ganoderma lucidum polysaccharide-fish scale peptide complex, characterized in that, The Ganoderma lucidum polysaccharide-fish scale peptide complex is composed of Ganoderma lucidum polysaccharide and fish scale peptide, with a mass ratio of fish scale peptide to Ganoderma lucidum polysaccharide of (1.8~3.4):

1.

2. The method for preparing a highly reducing Ganoderma lucidum polysaccharide-fish scale peptide complex according to claim 1, characterized in that, Includes the following steps: Ganoderma lucidum polysaccharide and fish scale peptide were mixed according to the mass ratio, water was added, and the mixture was heated at the set temperature and time. After the reaction was completed, the mixture was freeze-dried to obtain the final product.

3. The preparation method according to claim 2, characterized in that, The mass ratio of fish scale peptides to Ganoderma lucidum polysaccharides is (1.8~3.4):1, preferably (2.5~3):1, and more preferably (2.8~2.9):1; Preferably, the heating temperature is 40~80℃, more preferably 60~80℃, and even more preferably 70~75℃; Preferably, the heating time is 45-85 min, more preferably 50-70 min, and even more preferably 55-60 min.

4. The preparation method according to claim 2, characterized in that, The preparation method was optimized using response surface methodology. The quadratic polynomial regression equations between the reducing power of the resulting complex and the mass ratio (A), heating temperature (B), and heating time (C) are as follows: Y=-3.20234+0.738781×A+0.056474×B+0.025201×C+0.002813×A×B–0.001406×A×C–0.000106×B×C–0.150937×A 2 -0.000404×B 2 -0.000119×C 2 。 5. The preparation method according to claim 2, characterized in that, The preparation method of Ganoderma lucidum polysaccharides includes: crushing Ganoderma lucidum, sieving, and preparing polysaccharides by water extraction and alcohol precipitation; Preferably, the specific preparation method of water extraction and alcohol precipitation includes: adding water according to the material-liquid ratio, heating and extracting, filtering and collecting the solution, centrifuging to obtain the supernatant and concentrating; mixing the solution with 95% ethanol evenly, letting it stand at low temperature, centrifuging to obtain the precipitate and then freeze-drying it to obtain Ganoderma lucidum polysaccharide; Preferably, the material-to-liquid ratio is 1:(30~50) (g / mL), the extraction is carried out by heating at 80~100℃ for 2~6h, centrifugation at 2000~6000 rpm for 5~20min, the volume ratio of solution to 95% ethanol is 1:(2~4), and the solution is allowed to stand at a low temperature of 2~5℃ for 10~15h.

6. The preparation method according to claim 2, characterized in that, The preparation process of fish scale peptides includes: decalcifying fish scale fragments, removing impurities and proteins, filtering and washing, soaking them in acetic acid solution, filtering to obtain an extract, adding sodium chloride solution to the extract and salting out under low temperature conditions, centrifuging to collect the precipitate, resolving it with acetic acid solution, dialysis in a dialysis bag, and freeze-drying to obtain fish scale peptides. Preferably, the decalcification process involves soaking the dried fish scale fragments in an acid solution; preferably, the acid is hydrochloric acid with a concentration of 0.5~2 mol / L and a soaking time of 80~110 min. Preferably, the protein removal treatment is as follows: the decalcified fish scales are soaked in sodium carbonate solution at a material-to-liquid ratio of 1:30~50 (g / mL) to remove impurities and proteins, wherein the concentration of sodium carbonate solution is 0.2~0.5mol / L and the soaking time is 4~7h; Preferably, the sample is soaked in 0.5-2 mol / L acetic acid solution at a material-to-liquid ratio of 1:5-15 (g / mL) for 15-25 h, filtered to obtain an extract, and then salted out at 2-5℃ for 20-25 h by adding 0.5-1 mol / L sodium chloride solution. The precipitate is collected by centrifugation, redissolved in 0.2-0.6 mol / L acetic acid solution, dialyzed in a dialysis bag for 24-48 h, and then freeze-dried.

7. The application of the high reducing power Ganoderma lucidum polysaccharide-fish scale peptide complex according to claim 1 or the high reducing power Ganoderma lucidum polysaccharide-fish scale peptide complex obtained by any one of claims 2-6 in the preparation of high reducing power products.

8. The application according to claim 7, characterized in that, The reduction includes the reduction of digestive sites, including oral digestion, gastric digestion, and small intestinal digestion; Preferably, the products include pharmaceuticals, health products, and food.

9. A product characterized in that, The high reducing power Ganoderma lucidum polysaccharide-fish scale peptide complex according to claim 1 or the high reducing power Ganoderma lucidum polysaccharide-fish scale peptide complex obtained by the preparation method according to any one of claims 2-6.

10. A composition, characterized in that, The product includes the high reducing power Ganoderma lucidum polysaccharide-fish scale peptide complex of claim 1, or the high reducing power Ganoderma lucidum polysaccharide-fish scale peptide complex obtained by any one of claims 2-6, or the product of claim 9, and pharmaceutically acceptable excipients or carriers.

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