A method for preparing active polyphenols and co-producing functional polysaccharides by solid-state fermentation and its application

Abstract

This invention discloses a method and application for preparing active polyphenols and co-producing functional polysaccharides through solid-state fermentation. Using a mixed grain matrix of black beans, Job's tears, rice, millet, corn, and wheat, solid-state fermentation is carried out by inoculating Ganoderma lucidum mycelium. Highly active crude polyphenol extract (FGP) is obtained by ethanol extraction, and the fermentation residue is subjected to water extraction and phosphoric acid modification to obtain phosphoric acid-modified polysaccharide (PPS). This invention significantly improves the bioavailability and bioactivity of grain polyphenols through a specific fermentation process. Experiments show that FGP not only has excellent antioxidant and anti-aging effects but also significant antibacterial activity against common pathogenic bacteria; PPS exhibits good in vitro antioxidant capacity. Furthermore, this invention uses sodium alginate (SA) as a film-forming matrix, adds PPS and FGP, and cross-links with calcium chloride to obtain a fully biodegradable active preservation film with excellent mechanical properties and barrier properties.

Description

Technical Field

[0001] This invention belongs to the technical fields of bio-fermentation engineering, functional food and active packaging materials, specifically a method and application for preparing active polyphenols and co-producing functional polysaccharides through solid-state fermentation. Background Technology

[0002] Grains, represented by black beans, Job's tears, rice, millet, corn, and wheat, are the core staple ingredients in the Chinese diet, rich in phenolic acids, flavonoids, and other natural polyphenolic compounds. These natural polyphenols possess significant antioxidant, antibacterial, and anti-inflammatory physiological activities, showing broad industrial application prospects in functional foods, biomedicine, and food preservation. However, the vast majority of polyphenols in natural grains exist in bound form, tightly bound to components such as cellulose, hemicellulose, and lignin in the grain cell wall through covalent bonds. This directly leads to poor water solubility and low bioavailability, making it difficult to fully exert their intended physiological activities in conventional applications. This has become a core bottleneck restricting the industrial development and high-value application of grain polyphenols.

[0003] To achieve the efficient release and extraction of polyphenols from grains, various extraction processes have been developed, with traditional physical extraction, chemical hydrolysis, and various physical-assisted enhanced extraction technologies being the mainstream. While traditional acid-base hydrolysis can break down cell wall structures and release some bound polyphenols, the reaction conditions are harsh, easily causing structural damage and degradation of the active polyphenol components, leading to a significant decrease in the physiological activity of the product. Furthermore, the large-scale use of acid and alkali reagents generates highly polluting wastewater, posing serious environmental pollution risks and increasing subsequent environmental treatment costs, which is inconsistent with the industry's trend towards green production.

[0004] To overcome the shortcomings of traditional extraction methods, existing technologies have successively developed various physical enhancement extraction techniques, such as ultra-high pressure pretreatment, electron beam irradiation, and ultrasonic-assisted extraction, and related patent solutions have been formed. For example, patent CN115154555A discloses a green and sustainable method and application for extracting millet polyphenols, which improves the extraction yield of millet polyphenols by pretreating millet under ultra-high pressure, combined with extraction using a hydrophilic eutectic solvent and β-cyclodextrin-assisted grinding; patent CN119925451A discloses a method and application for extracting polyphenols from persimmon peel, which uses electron beam irradiation to bombard the cellulose and lignin components of the raw material cell wall to break chemical bonds and improve polyphenol extraction efficiency; patent CN120550446A discloses a method and application for ultrasonic-assisted extraction of red grapefruit polyphenols, which achieves efficient dissolution of polyphenols through low-temperature pulsed ultrasonic-assisted extraction; and patent CN116898938B discloses a method for extracting polyphenols from cardamom, which improves the extraction rate and purity of cardamom polyphenols by combining ultra-fine grinding with ultrasonic treatment.

[0005] However, during the research process, the inventors discovered that the aforementioned existing technologies still have unavoidable objective technical defects: First, this type of physical enhancement extraction technology can only destroy the macroscopic structure of the cell wall through physical action, and cannot achieve efficient breaking of the covalent bonds between bound polyphenols and cell wall polysaccharides. Its ability to convert and release bound polyphenols is limited, making it difficult to fundamentally solve the core problem of low polyphenol bioavailability. Second, this type of technology lacks the ability to biomodify and directionally transform polyphenol structures, and cannot generate polyphenol metabolites with higher physiological activity, making it difficult to further enhance the application value of the products. Third, existing related technical solutions are all focused on polyphenol extraction from single raw materials, with very little research and industrial application of polyphenol extraction from multi-grain mixed matrices. This makes it unsuitable for the complex nutritional system of multi-grain raw materials, resulting in significant bottlenecks in extraction efficiency and product activity improvement.

[0006] Microbial solid-state fermentation technology is considered a highly efficient, green, and effective bioprocessing method for significantly enhancing the activity of natural products. During solid-state fermentation, microorganisms secrete various extracellular enzymes such as cellulase, ligninase, and esterase, which can efficiently dismantle the dense structure of plant cell walls under mild conditions, precisely breaking the chemical bonds between bound polyphenols and cell wall polysaccharides, thus significantly promoting the conversion of bound polyphenols to free polyphenols. Simultaneously, the biotransformation process of microorganisms can directionally modify the polyphenol molecule structure through demethylation and deglycosylation, generating novel metabolites with higher physiological activity. This technology combines the dual advantages of improved extraction efficiency and enhanced product activity, making it a highly promising polyphenol preparation technology.

[0007] However, through in-depth research, the inventors have discovered that existing research and technical solutions for solid-state fermentation extraction of polyphenols still have significant objective technical shortcomings: On the one hand, existing research and patented technologies still focus on the fermentation extraction of single raw materials, with very little research on solid-state fermentation enrichment of polyphenols in multi-grain mixed matrices. The nutrient matrix of a single raw material is insufficient to fully meet the growth, metabolism, and enzyme production needs of microorganisms, failing to maximize the biotransformation capacity of microorganisms, thus limiting the potential for improving polyphenol enrichment efficiency and product activity. On the other hand, existing solid-state fermentation technologies focus only on the single extraction and preparation of polyphenols from fermentation products, completely ignoring the high-value utilization of fungal polysaccharides produced by microbial metabolism during fermentation and fermentation residues. Fungal polysaccharides themselves possess excellent film-forming properties and antioxidant activity; their biological activity can be further enhanced after chemical modification (such as phosphorylation). The current technology's abandonment of this high-value component not only results in a serious waste of biomass resources but also increases the cost and environmental pressure of fermentation by-product treatment, failing to achieve the resource utilization and high-value utilization of all fermentation components.

[0008] Furthermore, in the field of food preservation, traditional plastic preservation films suffer from industry pain points such as difficulty in natural degradation and easy white pollution. They also lack active antibacterial and antioxidant functions, making it difficult to meet the long-term preservation requirements of high-quality fruits and vegetables. Sodium alginate, as a natural polysaccharide, possesses excellent film-forming properties and biocompatibility, making it an ideal matrix for preparing fully biodegradable packaging films. However, sodium alginate films alone have inherent defects such as poor mechanical properties, weak water resistance, and lack of antibacterial and antioxidant biological activity. Existing technologies have failed to synergistically combine natural polyphenols (natural antibacterial agents) derived from grain fermentation with modified fungal polysaccharides (antioxidant enhancers and mechanical property modifiers), making it impossible to develop a fully bio-based active packaging material with a three-in-one effect of "antibacterial-antioxidant-high barrier." This hinders the industrialization of polyphenol extraction technology and downstream high-value applications, and also fails to simultaneously solve the dual industry problems of post-harvest fruit and vegetable losses and plastic pollution.

[0009] In summary, existing polyphenol extraction technologies generally suffer from a series of objective drawbacks, including resource waste due to single-target extraction, low polyphenol conversion efficiency, limited improvement in product activity, narrow range of raw material compatibility, low comprehensive utilization rate of biomass resources, easy environmental pollution, and insufficient connection with downstream high-value applications. These drawbacks severely restrict the industrial development and large-scale application of cereal polyphenols. Therefore, developing a green and efficient preparation technology that enables high-value utilization of all components from multiple grains while simultaneously achieving efficient enrichment and activity enhancement of polyphenols has become an urgent technical problem to be solved in this field. Summary of the Invention

[0010] This application aims to solve the problems existing in the above-mentioned background technology, and innovatively breaks the limitation of single target extraction. It utilizes the fermentation residue after polyphenol extraction for polysaccharide water extraction and modification, realizing the tiered high-value utilization of grain matrix.

[0011] To achieve the above objectives, the present invention provides a method for preparing active polyphenols and co-producing functional polysaccharides by solid-state fermentation, comprising the following steps: (1) substrate preparation: weigh black beans, Job's tears, rice, millet, corn and wheat, and mix them in a mass ratio of 20-30:10-18:8-14:10-18:8-14:20-30; (2) Sterilization treatment: After washing and soaking the mixed grains in step (1), drain them until the moisture content is 50%-55%, and sterilize them under high pressure at 115℃-125℃ and 0.10MPa-0.15MPa for 120-240 min; (3) Inoculation and fermentation: Inoculate the mixed grains obtained in step (2) with Ganoderma lucidum mycelium and place them at 25℃ in the dark for 30-50 days for fermentation; (4) Extraction and preparation: The fermentation product obtained in step (3) is freeze-dried at -40℃ to -60℃ for 24-48h, pulverized and passed through a 40-100 mesh sieve, and then extracted with an aqueous ethanol solution with a volume concentration of 40-50%. The extract is extracted by rotating and shaking at 100-140rpm for 20-28h. The extraction is repeated at least twice, and the extracts are combined. The extract was first concentrated to 1 / 5 to 1 / 3 of its original volume, then subjected to alcohol precipitation, and then freeze-dried again at -40℃ to -60℃ for 24-48h to obtain the crude extract of active polyphenols, FGP; polysaccharides were obtained by water extraction from the fermentation residue. The alcohol precipitation procedure is as follows: add 3 times the volume of anhydrous ethanol to the concentrated extract and precipitate for 10-14 hours. The yield of the highly active crude polyphenol extract (FGP) was 17.0±1.2%, and the polyphenol content in the crude extract was 123.14±8.64 mg / g. The yield of crude polysaccharide from the fermentation residue was 8.37±0.23%, and the polysaccharide content in the crude polysaccharide extract was 170.98±6.82 mg / g.

[0012] Furthermore, the fermentation method provided by the present invention also includes the following steps: the polysaccharide product obtained from fermentation is reacted with a phosphate modifier under alkaline conditions of pH 8-10 at 30℃-60℃ for 2-6 hours, and modified polysaccharide PPS is obtained by dialysis, alcohol precipitation, and freeze drying. The yield of modified polysaccharide PPS with high antioxidant activity obtained by phosphorylation modification is 6.25±0.31% (based on the dry weight of fermentation material), and the polysaccharide content is 254.32±12.15 mg / g.

[0013] Furthermore, the polyphenol extracts and polysaccharide extracts obtained by the method of the present invention have changed from the prior art, therefore the polyphenol extracts and polysaccharide extracts obtained by the method of the present invention are also within the scope of protection of the present invention.

[0014] This invention also provides applications of the polyphenol extract, specifically the fermentation product polyphenols, for the preparation of antibacterial and anti-stress products, and for the preparation of functional preservative films; the antibacterial properties include those against *Escherichia coli* (…). E. coli Staphylococcus aureus S. aureus It has a significant inhibitory effect; the anti-stress includes anti-thermal stress and / or anti-oxidative stress.

[0015] The modified polysaccharide extract is used in the preparation of plastic wrap.

[0016] Specifically, when preparing the plastic wrap, the raw material film-forming solution formula includes: sodium alginate with a mass-volume concentration of 1.5%-2.5%, the amount of modified polysaccharide added from the fermentation product is 8%-12% of the mass of sodium alginate, the amount of polyphenol added from the fermentation product is 12%-20% of the mass of sodium alginate; and the crosslinking agent is a 1%-3% calcium chloride solution. Preparation method: (1) Prepare sodium alginate (SA) aqueous solution as film-forming matrix; (2) Add the fermentation product-modified polysaccharide to the SA solution as an antioxidant enhancer; (3) Add the fermentation product polyphenols to the solution as an antibacterial agent; (4) After thorough mixing, the film is cast and dried at 25℃-45℃ for 12-24 hours. Then, calcium chloride solution is sprayed to crosslink the film, and the film is dried again at room temperature to obtain the composite preservation film.

[0017] The present invention also provides an antibacterial and / or anti-stress product containing the above-mentioned polyphenol extract, which may be food, medicine, cosmetics, etc.

[0018] The present invention also provides a food preservation film containing the modified polysaccharide extract and / or the polyphenol extract.

[0019] The above-mentioned functions of the plastic wrap include its application in delaying the ripening of bananas, reducing weight loss, and maintaining titratable acid content.

[0020] In summary, by adopting the above technical solution, the beneficial effects of this invention are as follows: 1. By using Ganoderma lucidum solid-state fermentation of multigrains, not only is the total phenol content increased (second only to high-purity tea polyphenols), but more importantly, the composition or state of polyphenols is significantly changed, making them exhibit comprehensive biological activity superior to unfermented grain polyphenols (UGP) and even some commercial polyphenols (such as grape seed polyphenols and blueberry polyphenols).

[0021] 2. The preparation process is simple and environmentally friendly, and the raw materials are widely available and inexpensive, making it suitable for industrial production.

[0022] 3. Systematic in vitro and in vivo experiments have confirmed that the fermented polyphenols have definite anti-aging and anti-stress functions, providing a scientific basis for the development of novel cereal-based functional foods.

[0023] 4. Experiments have shown that FGP has a significant inhibitory effect on both Escherichia coli and Staphylococcus aureus, thus broadening the functionality of polyphenols.

[0024] 5. Phosphorylation modification significantly improved the in vitro antioxidant capacity of polysaccharides.

[0025] 6. After adding PPS and FGP, the tensile strength of the plastic wrap was significantly enhanced, the water vapor permeability was reduced, and the swelling rate decreased, indicating that the film structure was more compact and stable.

[0026] 7. Effective Fruit and Vegetable Preservation: When applied to banana preservation, this composite film effectively inhibits respiration, reduces moisture loss, delays browning of the peel and softening of the flesh, and significantly extends shelf life. Attached Figure Description

[0027] Figure 1 Comparison of total phenol content in different phenolic samples.

[0028] Figure 2 Comparison of in vitro antioxidant capacity of different phenolic samples (including DPPH, ABTS, hydroxyl radicals, and FRAP).

[0029] Figure 3 Survival curves showing the effects of different phenolic samples on the lifespan of Caenorhabditis elegans.

[0030] Figure 4 : Effect of different phenolic samples on the reproductive capacity of nematodes.

[0031] Figure 5 : The effect of different phenolic samples on the motility of nematodes during aging.

[0032] Figure 6 : Effect of different phenolic samples on swallowing rate during nematode aging.

[0033] Figure 7 Fluorescence micrographs and fluorescence intensity analysis of different phenolic samples on the accumulation of lipofuscin in nematodes.

[0034] Figure 8 Survival curves of nematodes in different groups under heat stress conditions.

[0035] Figure 9 Survival curves and lifespan statistics of nematodes in different groups under juglone-induced oxidative stress conditions.

[0036] Figure 10 Fluorescence patterns and intensity analysis of the effects of different phenolic samples on ROS levels in nematodes.

[0037] Figure 11 Effects of different phenolic samples on the activity of antioxidant enzymes (SOD, CAT, GSH-Px) and MDA content in nematodes.

[0038] Figure 12 Comparison of in vitro antioxidant capacity (DPPH, hydroxyl radical scavenging rate) between phosphate-modified polysaccharide (PPS) and unmodified polysaccharide.

[0039] Figure 13 Determination of inhibition zones and minimum inhibitory concentrations (MICs) of fermented polyphenols (FGP) against common pathogenic bacteria (Escherichia coli, Staphylococcus aureus).

[0040] Figure 14 Changes in cell growth curve (OD600) during the antibacterial process of fermented polyphenols (FGP).

[0041] Figure 15 In vitro antioxidant capacity test of composite membrane after PPS addition.

[0042] Figure 16 The effect of different PPS addition amounts on the mechanical properties (tensile strength, elongation at break) of composite films.

[0043] Figure 17 Effects of different PPS addition amounts on the water content and swelling rate of composite membranes.

[0044] Figure 18 Effect of different PPS addition amounts on water vapor transmission rate (WVP) of composite membrane.

[0045] Figure 19 The effects of composite preservation film wrapping on the weight loss, titratable acid and pH value of bananas during storage.

[0046] Figure 20 Comparison of banana appearance and quality changes after treatment with composite plastic wrap: physical comparison images. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0048] Example 1: Preparation of Crude Extract of Active Polyphenols (FGP) 1. Culture medium preparation: Mix commercially available black beans, Job's tears, rice, millet, corn, and wheat in a mass ratio of 25:14:11:14:11:25. Wash and soak for 12 hours, then drain to a moisture content of approximately 50%-55%. Fill each bottle with 130g of the mixture and compact it. Autoclave at 121℃ for 180 minutes and allow to cool naturally.

[0049] 2. Fermentation: Under aseptic conditions, inoculate an equal area of ​​Ganoderma lucidum mycelium into the cooled solid culture medium. Incubate at 25°C in the dark for 40 days for static fermentation.

[0050] 3. Pretreatment: After fermentation, the sample was freeze-dried at -40℃ for 48 hours, pulverized and passed through a 40-mesh sieve, and then stored at low temperature in the dark.

[0051] 4. Extraction: Weigh 1.0 g of lyophilized powder, add 10 mL of 45% (v / v) ethanol-water mixture, and extract for 24 h in a rotary shaker (120 rpm). Repeat the extraction twice, combine the extracts, centrifuge at 10,000 rpm for 10 min, and collect the supernatant.

[0052] 5. Purification: The supernatant was concentrated by rotary evaporation at 45℃, and precipitated by adding 3 times the volume of anhydrous ethanol for 12 h. The precipitate was removed by centrifugation, and the supernatant was freeze-dried at -80℃ to obtain the crude active polyphenol extract (FGP).

[0053] The following are tests on the content and various properties of the polyphenols obtained by the present invention and the polyphenols of the comparative examples. Comparative example 1 is: Unfermented cereal polyphenols (UGP): prepared by using the same cereal ratio as in Example 1, but without inoculating Ganoderma lucidum, and directly using the same extraction process. Comparative Examples 2-6: Other polyphenol samples They are tea polyphenols (TP), purple sweet potato polyphenols (SPP), blueberry extract (BP), amla extract (AEP), and grape seed polyphenols (GSP).

[0054] (a) Determination of total phenol content: The total phenol content of the FGP prepared in Example 1 and the comparative sample was determined using the Folin-Ciocalteu method.

[0055] The results are as follows Figure 1 As shown, the total phenolic content of FGP was significantly higher than that of the unfermented group (UGP) and other plant extracts, second only to high-purity tea polyphenols (TP). This indicates that Ganoderma lucidum fermentation effectively promotes the release of bound phenols from grains. After multiple tests, the yield of the highly active crude polyphenol extract (FGP) obtained in this invention was determined to be 17.0 ± 1.2%, and the polyphenol content in this crude extract was 123.14 ± 8.64 mg / g.

[0056] Experimental results show that although TP showed good results, FGP showed comparable or even better potential in terms of comprehensive antioxidant index and stress resistance, and was superior to SPP, BP, AEP and GSP.

[0057] (II) In vitro antioxidant activity assay The scavenging rates of DPPH, ABTS, and hydroxyl radicals, as well as the iron-reducing capacity of FRAP, obtained from Example 1 were determined.

[0058] The results are as follows Figure 2 As shown, FGP exhibited the strongest antioxidant capacity across all four indicators, significantly outperforming the unfermented group (UGP) and commercially available grape seed polyphenols (GSP), purple sweet potato polyphenols (SPP), etc. P<0.01). In particular, the activity was also improved compared with the TP group in the DPPH and FRAP tests, indicating that the polyphenols produced by fermentation have a unique electron donor capacity.

[0059] (III) Verification of anti-aging function: (1) Effects on the lifespan of Caenorhabditis elegans Synchronized N2 nematodes were cultured in NGM medium containing 0.5 mg / mL FGP, and their lifespan was recorded.

[0060] The results are as follows Figure 3 As shown in Table 1, the average lifespan of the nematodes in the FGP group obtained in Example 1 was 18.9 ± 0.88 days, which was 25.41% longer than that of the blank control group (CK, 15.07 days), comparable to and slightly better than that of the positive control group TP (18.0 days), and significantly higher than that of the UGP group (16.47 days). The maximum lifespan reached 26 days. This indicates that FGP has a significant effect in delaying aging.

[0061] Table 1. Effects of different phenolic samples on the lifespan of nematodes.

[0062] (2) Effects on anti-aging-related phenotypes (exercise, swallowing, lipofuscin) 1. Athletic ability ( Figure 5 On day 12 of aging, 65.3% of the nematodes in the FGP group remained in Grade A (autonomous movement) state, which was significantly better than the CK and UGP groups.

[0063] 2. Swallowing rate ( Figure 6 On days 8 and 12, the swallowing rate of nematodes in the FGP group was significantly higher than that in the control group, indicating that it delayed the decline of neuromuscular function.

[0064] 3. Lipofuscin ( Figure 7 Fluorescence microscopy showed that the amount of lipofuscin (blue fluorescence) accumulated in the nematodes of the FGP-treated group was significantly lower than that of the CK group.

[0065] In addition, the egg production experiment ( Figure 4 The results showed that FGP had no negative impact on the reproductive capacity of nematodes.

[0066] (iv) Testing of stress resistance: 1. Resistance to heat stress ( Figure 8 Table 2): At 35℃, the average lifespan of the FGP group was 9.93 days, which was 34.19% longer than that of the CK group, and significantly better than all other groups.

[0067] Table 2 Relative lifespan in heat stress tests

[0068] 2. Antioxidant stress ( Figure 9 (Table 3): Under juglone induction, the average survival time of the FGP group was prolonged by 42.71%, showing extremely strong oxidative tolerance.

[0069] Table 3. Lifetime of FGP-treated nematodes under juglone oxidative stress in experiments.

[0070] 3. In vivo mechanisms ( Figure 10 , Figure 11 The fluorescence intensity of ROS in nematodes in the FGP group was reduced by approximately 38% compared to the CK group. Simultaneously, SOD activity increased to 176%, CAT activity increased to 207%, GSH-Px activity increased by 57%, and MDA content decreased by 42%. This confirms that FGP delays aging by activating the in vivo antioxidant enzyme system and scavenging ROS.

[0071] As can be clearly seen from the above, the experimental results of FGP obtained by this invention and Comparative Example 1 show that UGP is significantly weaker than FGP in terms of antioxidant capacity, lifespan extension (only 9.29%) and stress resistance, proving the key role of fermentation process.

[0072] The experimental results of FGP obtained in this invention and Comparative Examples 2-6 show that although TP showed better performance, FGP showed comparable or even better potential in terms of comprehensive antioxidant index and stress resistance, and was superior to SPP, BP, AEP and GSP.

[0073] Example 2: Preparation of Phosphate-Modified Polysaccharide (PPS) Preparation: The steps from substrate preparation to fermentation are the same as in Example 1. The fermented crude polysaccharide was taken and esterified using the phosphate mixing method (sodium tripolyphosphate: sodium trimetaphosphate) at pH 8-10. The reaction was carried out at 30°C for 6 hours, and then PPS was obtained by dialysis, alcohol precipitation, and freeze drying.

[0074] Table 1. Comparison of the effects of "unfermented" and "Ganoderma lucidum mycelium fermentation" processes on extraction indicators.

[0075] The following is a verification of the content and function of polyphenol FGP and modified polysaccharide PPS obtained in this embodiment: (I) Content Determination: After multiple tests, it was determined that the crude polysaccharide yield from the fermentation residue extracted with water in this invention was 8.37±0.23%, and the polysaccharide content in the crude polysaccharide extract was 170.98±6.82 mg / g. The PPS yield was 6.25±0.31%, and the PPS content was 254.32±12.15 mg / g. Compared with the prior art, this invention utilizes enzymes generated by solid-state fermentation of Ganoderma lucidum to degrade the matrix cell wall, further improving the yield of the traditional direct water extraction method; the method of "first extracting polyphenols with alcohol and then extracting polysaccharides from the residue with water" reduces costs and component loss; and the use of compound phosphate for mild esterification modification effectively avoids excessive degradation of the polysaccharide backbone.

[0076] (II) Verification of antioxidant properties: Antioxidant experiment ( Figure 12 The results showed that, compared with unmodified polysaccharides, PPS significantly improved the scavenging rate of DPPH and hydroxyl radicals. P <0.05), and the effect is concentration-dependent. The introduction of phosphate groups enhances the hydrogen-donating capacity of the polysaccharide.

[0077] (III) Evaluation of the antibacterial activity of the polyphenols (FGP) obtained in the embodiments of the present invention: inhibition zone test ( Figure 13 ): Using the filter paper disc method, FGP for E. coli and S. aureus All showed clear and transparent inhibition zones, with a diameter significantly larger than that of unfermented grain extracts.

[0078] OD600 growth curve ( Figure 14 FGP was added to the culture medium to culture bacteria, and OD600 was measured every 2 hours. The results showed that FGP significantly prolonged the lag phase of bacteria and suppressed the peak value of the logarithmic growth phase, confirming that it can effectively disrupt bacterial cell membrane structure or inhibit metabolism.

[0079] Example 3: PPS application example obtained from Example 2: (I) Preparation and characterization of active composite food preservation film: Preparation process: Prepare a 2% (w / v) sodium alginate (SA) solution. Add different proportions of PPS (0%, 5%, 10%, 15%) to the solution, and determine the optimal addition amount through experiments. Then add 16% (w / w, relative to the mass of SA) of FGP. After stirring evenly, degas the solution, cast it into a film, and spray it with a 2% CaCl2 solution for crosslinking and curing.

[0080] Membrane antioxidant properties Figure 15 The composite membrane immersion solution exhibited extremely strong DPPH scavenging ability, confirming that the active ingredients in the membrane can be released and exert their effects. Mechanical properties ( Figure 16As the amount of PPS added increases, the tensile strength (TS) of the membrane first increases and then decreases, while the elongation at break (EAB) shows an increasing trend. When the amount of PPS added is 10%, TS reaches its maximum value, indicating that an appropriate amount of PPS, as a filler, forms a tight hydrogen bond network with the SA matrix. Hydration properties ( Figure 17 Adding 10% PPS resulted in a membrane with moderate water content and a significantly lower swelling ratio compared to pure SA membranes. This is attributed to the interaction between PPS, SA, and Ca. 2+ The resulting dense cross-linked structure restricts the entry of water molecules. Barrier performance ( Figure 18 Water vapor transmission rate (WVP) tests showed that the composite membrane with 10% PPS and 16% FGP had the lowest WVP value. Excellent water resistance is key to food preservation.

[0081] Application of the above-mentioned composite film in banana preservation: Bananas of uniform ripeness were selected and divided into a control group (no packaging), a PE film group (commercially available plastic wrap), and the composite film group of this invention (SA+10% PPS+16% FGP).

[0082] weight loss rate ( Figure 19 On the 7th day of storage, the weight loss rate of the control group was as high as 15%, and the peel was severely wrinkled; the weight loss rate of the composite film group was only about 6%, which significantly locked in moisture.

[0083] pH and titratable acid ( Figure 19 During banana ripening, acidity decreases and pH increases. The composite membrane effectively slows down the degradation rate of titratable acids and inhibits the rapid increase in pH, indicating that it slows down the respiration and metabolic processes of the fruit.

[0084] Sensory evaluation ( Figure 20 On day 7, the bananas in the control group turned brown all over and the flesh became soft and mushy; the bananas in the composite film group had only a few sesame-like spots on the peel, the flesh remained firm and golden in color, and the preservation effect was significantly better than that of the control group and ordinary PE film.

[0085] As can be seen from the embodiments of the present invention, the FGP and PPS obtained by the innovative method of the present invention have changed from the prior art in terms of antioxidant and anti-aging functions. Therefore, the FGP and PPS obtained by the present invention are also requested to be included in the scope of protection of the present invention. In addition, this invention also discovered that FGP has antibacterial and anti-stress effects; Products containing the aforementioned FGP and PPS that have functional properties, such as food, cosmetics, pharmaceuticals, and insurance products, should also be within the scope of protection of this invention; Meanwhile, the modified PPS of this invention has improved performance compared to existing technologies when used in the manufacture of food preservation films. Therefore, the application of the PPS of this invention in food preservation film products should also be within the scope of protection of this invention.

[0086] It should be noted that as long as the ratio of each component of the matrix used in this invention is within the range of 20-30:10-18:8-14:10-18:8-14:20-30, such as 20:10:8:10:8:20, the yield and content of polyphenols and polysaccharides obtained are within the range listed in Examples 1 and 2. In order to reduce the length and avoid unnecessary repetition, they will not be described one by one. The operating conditions are the same. For example, during sterilization, high-pressure sterilization is performed at 115℃-125℃ and 0.10MPa-0.15MPa for 120-240 min; fermentation time is 30-50 days; freeze-drying is performed at -40℃ to -60℃ for 24-48 h; the pulverized material is 40-100 mesh; extraction is performed with a 40-50% (v / v) ethanol aqueous solution, and the extract is extracted by rotary shaking at 100-140 rpm for 20-28 h; and during polysaccharide modification, the phosphate modifier is reacted at 30℃-60℃ for 2-6 hours under alkaline conditions at pH 8-10; and in the preparation of the cling film, the sodium alginate (v / v) concentration is 1.5%. The amount of modified polysaccharide added to the fermentation product is 8%-2.5% of the mass of sodium alginate, and the amount of polyphenol added to the fermentation product is 12%-20% of the mass of sodium alginate; the crosslinking agent is a 1%-3% calcium chloride solution; the drying at 25℃-45℃ for 12-24 hours used in the preparation method, etc., the yield and content of polyphenols and polysaccharides obtained under these conditions are all within the range listed in Examples 1 and 2. In order to reduce the length and avoid unnecessary repetition, they will not be described one by one.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for the production of active polyphenols coproduct with functional polysaccharides by solid state fermentation, characterized in that, The following steps are included: (1) Matrix preparation: weigh black beans, Job's tears, rice, millet, corn and wheat, and mix them in a mass ratio of 20-30:10-18:8-14:10-18:8-14:20-30; (2) Sterilization treatment: After washing and soaking the mixed grains in step (1), drain them until the moisture content is 50%-55%, and sterilize them under high pressure for 120-240 min at 115℃-125℃ and 0.10MPa-0.15MPa. (3) Inoculation and fermentation: Inoculate the mixed grains obtained in step (2) with Ganoderma lucidum mycelium and place them at 25℃ in the dark for 30-50 days for fermentation; (4) Extraction and preparation: The fermentation product obtained in step (3) is freeze-dried at -40℃ to -60℃ for 24-48h, pulverized and passed through a 40-100 mesh sieve, and then extracted with an aqueous ethanol solution with a volume concentration of 40-50%. The extract is extracted by rotating and shaking at 100-140rpm for 20-28h. The extraction is repeated at least twice, and the extracts are combined. The extract was first concentrated to 1 / 5 to 1 / 3 of its original volume, then subjected to alcohol precipitation, and then freeze-dried again at -40℃ to -60℃ for 24-48h to obtain the crude extract of active polyphenols, FGP; polysaccharides were obtained by water extraction from the fermentation residue. The alcohol precipitation procedure is as follows: add 3 times the volume of anhydrous ethanol to the concentrated extract and precipitate for 10-14 hours. The yield of the highly active crude polyphenol extract (FGP) was 17.0±1.2%, and the polyphenol content in the crude extract was 123.14±8.64 mg / g. The yield of crude polysaccharide from the fermentation residue was 8.37±0.23%, and the polysaccharide content in the crude polysaccharide extract was 170.98±6.82 mg / g.

2. The method of claim 1, wherein, The process also includes the following steps: the polysaccharide product obtained from fermentation is reacted with a phosphate modifier under alkaline conditions of pH 8-10 at 30℃-60℃ for 2-6 hours, and modified polysaccharide PPS is obtained by dialysis, alcohol precipitation, and freeze drying. The yield of modified polysaccharide PPS with high antioxidant activity obtained by phosphorylation modification is 6.25±0.31% (based on the dry weight of fermentation material), and the polysaccharide content is 254.32±12.15 mg / g.

3. A polyphenol characterized in that, It was prepared by the extraction method of claim 1.

4. A modified polysaccharide characterized in that, It is prepared according to claim 2.

5. Use of the polyphenol according to claim 3, characterized in that The fermentation product polyphenols are used in the preparation of antibacterial and anti-stress products, as well as in the preparation of functional food preservation films. The bacteriostasis includes significant inhibition on Escherichia coli ( E. coli ), Staphylococcus aureus ( S. aureus ); and the stress resistance includes heat stress resistance and / or oxidation stress resistance.

6. The application of the modified polysaccharide of claim 4, characterized in that, The modified polysaccharide is used in the preparation of plastic wrap.

7. The application according to claim 5 or 6, characterized in that, When preparing the plastic wrap, the raw material film-forming solution formula includes: sodium alginate with a mass-volume concentration of 1.5%-2.5%, the amount of modified polysaccharide added from the fermentation product is 8%-12% of the mass of sodium alginate, the amount of polyphenol added from the fermentation product is 12%-20% of the mass of sodium alginate; and the crosslinking agent is a 1%-3% calcium chloride solution. Preparation method: (1) Prepare sodium alginate (SA) aqueous solution as film-forming matrix; (2) Add the fermentation product-modified polysaccharide to the SA solution as an antioxidant enhancer; (3) Add the fermentation product polyphenols to the solution as an antibacterial agent; (4) After thorough mixing, the film is cast and dried at 25℃-45℃ for 12-24 hours. Then, calcium chloride solution is sprayed to crosslink the film, and the film is dried again at room temperature to obtain the composite preservation film.

8. A product for inhibiting bacteria and / or relieving stress, characterized in that, It contains the polyphenols described in claim 4.

9. A type of food preservation film, characterized in that, It contains the polysaccharide of claim 5 and / or the polyphenol of claim 4.

10. The application according to claim 7, characterized in that, The application of the plastic wrap in delaying the ripening of bananas, reducing weight loss, and maintaining titratable acid content.

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