EPSF / polygonatum sibiricum polysaccharide / casein submicron composite preservative as well as preparation method and application thereof

Abstract

The invention discloses an EPSF / polygonatum sibiricum polysaccharide / casein submicron composite preservative as well as a preparation method and application of the EPSF / polygonatum sibiricum polysaccharide / casein submicron composite preservative. The compound is prepared by compounding EPSF, polygonatum polysaccharide and sodium caseinate according to a mass ratio of 1: (0.2-0.8): (2-8), wherein the EPSF is synthesized by catalyzing an endogenous enzyme from aspergillus niger, and the compound is prepared by ultrasonic treatment and freeze-drying. The compound has excellent oxidation resistance, the superoxide anion clearance rate is as high as 88.36%, meanwhile, the compound preservative has broad-spectrum antibacterial activity, and the diameter of an inhibition zone is as high as 23.25-26.5 mm. When being applied to fresh-keeping of fruits and vegetables such as fresh-cut apples and potatoes, the fresh-keeping agent can remarkably inhibit browning, the browning index is reduced by about 60-80%, the weight loss ratio is reduced by 20-30%, and the fresh-keeping agent has the advantages of maintaining hardness, effectively prolonging the fresh-keeping period, being efficient, safe, nutritional and the like.

Description

TECHNICAL FIELD

[0001] The application discloses a preparation method of an EPSF / polygonatum sibiricum / casein sub-micron composite preservative and application of the EPSF / polygonatum sibiricum / casein sub-micron composite preservative in fruit and vegetable preservation, and belongs to the technical fields of embedding and food preservation. BACKGROUND

[0002] Fresh-cut fruits and vegetables refer to instant edible fruit and vegetable products prepared by taking fresh fruits and vegetables as raw materials, and then performing processing treatments such as cleaning, peeling, cutting or trimming, and then being transported at low temperature into a refrigerator for sale. Due to the convenience and nutrition, the demand for fresh-cut fruits and vegetables in the catering industry, retail industry and household consumption is increasing day by day. However, due to the fact that the fruit and vegetable tissues are damaged after being cut, and are directly exposed to the air, juice is prone to flow out, nutrient components are prone to be lost, and problems such as enzyme browning, microbial breeding and enhanced respiratory metabolism are accelerated, thereby causing quality deterioration and shelf life shortening. Therefore, it is crucial to develop efficient and safe preservation technologies for maintaining the sensory quality of fresh-cut fruits and vegetables and prolonging the storage period thereof.

[0003] At present, fresh-cut fruit and vegetable preservation technologies mainly include physical preservation (such as low-temperature storage, modified atmosphere packaging), chemical preservation (such as bacteriostatic agents, antioxidants) and biological preservation (such as natural extracts, edible film coating). Among them, chemical synthetic preservatives (such as sodium hypochlorite, chlorine dioxide) have a significant bacteriostatic effect, but there are risks of chemical residues and low consumer acceptance; and natural preservatives are more concerned due to safety and environmental friendliness, and components such as chitosan, whey protein and tea polyphenol have been widely studied. In recent years, through the synergistic effect of multiple active ingredients, composite preservation technology has become an important direction to improve the preservation performance. For example, patent CN 106578027 A discloses a melon and fruit preservative, which comprises beeswax, polygonum hydropiper extract, houttuynia cordata extract, xanthan gum, algal polysaccharide, hempseed oil, tea polyphenol and soybean protein. Although the preservative has certain bactericidal and antioxidant effects, the preparation method is relatively complex, and special treatment such as magnetic field treatment of mixed hempseed oil beeswax is required. Patent 108157744B discloses a preservative prepared by co-culturing polysaccharides of agaricus bitorquis and pholiota nameko, and then compounding the polysaccharides with tea polyphenol. Although the preparation method of the preservative is relatively simple, the bacteriostatic effect of the preservative is poor, and it is difficult to meet the actual demand. Patent CN 115226767 A discloses a composite preservative composed of malic acid, chitosan, aloe polysaccharide and protamine. The preservative also has a problem that the preservation effect needs to be improved.

[0004] Therefore, there is an urgent need to develop a safe, healthy, efficient and low-cost natural composite preservative for fresh-cut fruits and vegetables. SUMMARY

[0005] In view of the above problems, the application provides a preparation method of an EPSF / polysaccharide from Polygonatum multiflorum / casein sub-micron composite preservative. First, an EPSF with improved antioxidant performance is generated by using an enzyme-mediated mild reaction, and then a high-performance sub-micron composite is prepared by using a raw material self-assembly property by ultrasonic method. The sub-micron composite has strong antioxidant, high water retention and antibacterial properties, and can effectively prolong the storage quality of fresh-cut fruits and vegetables, thereby prolonging the edible and commodity nature of fresh-cut fruits and vegetables.

[0006] The application first provides a preparation method of an EPSF / polysaccharide from Polygonatum multiflorum / casein sub-micron composite, comprising the following steps:

[0007] The polysaccharide from Polygonatum multiflorum is dissolved in water, EPSF is added and stirred until completely dissolved, and then sodium caseinate is added and stirred to form a complex solution. The sub-micron solution is obtained by continuing ultrasonic treatment, and the EPSF / polysaccharide from Polygonatum multiflorum / casein sub-micron composite preservative is obtained by freeze-drying.

[0008] In an embodiment of the application, the sub-micron solution needs to be dialyzed by using a dialysis bag with a molecular weight cut-off MW = 500 before freeze-drying. The dialysis is performed at 4℃ for 8h with stirring.

[0009] In an embodiment of the application, the polysaccharide from Polygonatum multiflorum is a purified polysaccharide powder from Polygonatum sibiricum Red., and the mass / volume ratio of the polysaccharide from Polygonatum multiflorum to water is 0.5-1.0:0.1 (g / L), and the purity is more than 80%.

[0010] In an embodiment of the application, the mass / volume ratio of the added EPSF to water is 0.2-0.4:0.1 (g / L).

[0011] In an embodiment of the application, the mass / volume ratio of the added sodium caseinate to water is 2.0-4.0:0.1 (g / L), and the purity is 99%.

[0012] In an embodiment of the application, the mass ratio of the added EPSF and sodium caseinate to the polysaccharide from Polygonatum multiflorum is 0.2-0.8:1 and 2-8:1, respectively, and preferably 0.4-0.8:1 and 4-8:1.

[0013] In an embodiment of the application, the power during ultrasonic treatment is 200-300W, and the ultrasonic duration is 20-30min.

[0014] In an embodiment of the application, the freeze-drying is performed by pre-freezing the sub-micron solution at-20℃ for 12h and then freeze-drying by using a vacuum freeze-drying machine.

[0015] In an embodiment of the present application, the preparation method of the EPSF is as follows: EGCG and theanine are dissolved in distilled water, a crude enzyme solution of endogenous enzyme of Aspergillus niger is added for enzymatic reaction, the reaction solution obtained by the enzymatic reaction is dialyzed in a dialysis bag with a molecular weight cut-off of 500 at 4°C for 8-16 hours, and then freeze-dried to obtain the EPSF.

[0016] In an embodiment of the present application, the crude enzyme solution of endogenous enzyme is a mixed enzyme system derived from broken Aspergillus niger which is a dominant strain in the post-fermentation process of Pu'er tea.

[0017] In an embodiment of the present application, the preparation method of the crude enzyme solution of endogenous enzyme of Aspergillus niger is as follows: activated Aspergillus niger is inoculated in a sterilized agar-free PDA liquid medium at 5%, and seed bacteria are obtained by culturing at 30°C for 24 hours; 100 mL of seed liquid is inoculated in 100 g of bran medium dry base, and then uniformly stirred and placed in a constant-temperature and constant-humidity ventilated culture at 30°C; after culturing for 5 days, 3 times of 0.9% NaCl solution is added for homogenization, and then the supernatant is obtained by centrifugation after stirring overnight at 4°C, thereby obtaining the crude enzyme solution.

[0018] In an embodiment of the present application, the Aspergillus niger is a commercially available strain with a preservation number of CCTCC NF 20082077.

[0019] In an embodiment of the present application, the molar ratio of EGCG to theanine is 1:1.2-2.

[0020] In an embodiment of the present application, the temperature of the enzymatic reaction is 30-40°C, and the reaction time is 4-48 hours.

[0021] The present application also provides an EPSF / polygonatum sibiricum / casein sub-micron composite preservative prepared by the above method.

[0022] The present application also provides an application of the above EPSF / polygonatum sibiricum / casein sub-micron composite preservative in fresh-cut fruit and vegetable preservation.

[0023] In an embodiment, the application comprises dissolving the prepared EPSF / polygonatum sibiricum / casein sub-micron composite in pure water to prepare a solution with a mass concentration of 0.5-2%; and immersing or spraying the EPSF / polygonatum sibiricum / casein sub-micron composite solution in fresh-cut fruit and vegetable, wherein the fresh-cut fruit and vegetable is at least one selected from apple, pear, watermelon, polo, potato or broccoli.

[0024] Beneficial effects:

[0025] 1. Epigallocatechin gallate (EGCG), the most active catechin in tea, often suffers from poor stability (easily oxidized, sensitive to light and heat) and low oral bioavailability (intestinal degradation, weak permeability) due to its strong antioxidant and bioactivity. This invention addresses this by combining EGCG with theanine to form EPSF (8-CN-ethyl-2-pyrrolidone-substituted flavan-3-ol). EPSF exhibits stronger antioxidant and stability than EGCG, and its yield is significantly increased through a separate enzymatic system, overcoming the difficulties in extraction and application caused by the extremely low EPSF production in tea under natural conditions. Furthermore, an EPSF / Polygonatum polysaccharide / casein submicron composite preservative is prepared by reacting EPSF, Polygonatum polysaccharide, and sodium casein. This preservative possesses excellent antioxidant and antibacterial properties, extending the shelf life of fresh-cut fruits and vegetables.

[0026] 2. Polygonatum polysaccharides are natural high-molecular-weight compounds extracted from plants of the genus *Polygonatum* (such as *Polygonatum yunnanensis*, *Polygonatum multiflorum*, and *Polygonatum* var. *yunnanensis*) in the Liliaceae family. They possess complex structural characteristics and diverse biological activities. Primarily composed of fructans (accounting for over 80%), they exhibit significant free radical scavenging capabilities and can protect biomolecules from oxidative damage by providing hydrogen atoms to terminate oxidation chain reactions, demonstrating great potential as a green preservative. Casein, as a nutritional fortifier containing all essential amino acids, possesses excellent self-assembly properties. Leveraging its amphiphilic structure and multiple non-covalent interactions, it can efficiently form self-assembly carriers, forming stable encapsulation complexes with polysaccharides and polyphenols for food preservation and enhancing nutritional value. This invention selects natural *Polygonatum* polysaccharides and casein in combination with EPSF. The raw materials used are natural, safe, and non-toxic. The encapsulation technology is simple and easy to operate.

[0027] 3. Currently, to obtain EPSF, industrial processes typically involve extraction from tea leaves or chemical synthesis. Extraction from raw tea leaves is very difficult, as the tea content is only about 0.2%. Chemical synthesis usually involves many biochemical reagents, leading to potential toxicity in the product. This invention utilizes an enzymatic method to generate EPSF with high antioxidant and antibacterial activity, improves EGCG stability, and significantly increases EPSF yield. Furthermore, this invention does not involve any chemical additives in the EPSF synthesis process, facilitating its application in the food industry. This invention uses Polygonatum polysaccharide with good antioxidant and moisturizing properties to enhance binding sites through physical ultrasound; it also adds high-nutrient casein with self-assembly properties to form a stable complex via ultrasound. The submicron complex obtained by this invention exhibits high stability and strong activity, effectively delaying the increase in weight loss and browning index of fresh-cut fruits and vegetables, thus helping to maintain the firmness and moisture of fresh-cut fruits and vegetables and extending their edibility.

[0028] 4. The submicron complex prepared by this invention is rich in nutrients and can be consumed directly, showing great application potential. Attached Figure Description

[0029] Figure 1 The effect of different substrate concentrations on the yield of enzymatically catalyzed EPSF.

[0030] Figure 2 The effect of different reaction temperatures on the yield of enzymatically catalyzed EPSF.

[0031] Figure 3 The effect of different reaction times on the yield of enzymatically catalyzed EPSF.

[0032] Figure 4 A comparison of the antioxidant activities of different materials.

[0033] Figure 5 Images of fruits and vegetables after 10 days of storage with different treatments. Detailed Implementation

[0034] To illustrate the technical content, objectives, and effects of this invention in detail, the following description, in conjunction with embodiments and accompanying drawings, provides a clearer understanding of the invention. However, those skilled in the art will readily understand that the descriptions in the embodiments are merely illustrative and should not, and will not, limit the scope of the invention as detailed in the claims.

[0035] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are food-grade and commercially available. The EGCG (>98%) and theanine (>98%) used in the examples and comparative examples of this invention were derived from green tea and purchased from Zhejiang Yicun Biotechnology Co., Ltd.; the Polygonatum polysaccharide (>80%) was derived from Polygonatum multiflorum and purchased from Chizhou Jiuhuafu Jinlian Smart Agriculture Co., Ltd.; and the casein (>99%) was derived from milk and purchased from Zhejiang Yicun Biotechnology Co., Ltd.

[0036] The preparation of EPSF is as follows:

[0037] The Aspergillus niger involved in the embodiments and comparative examples of this invention was obtained from the China Center for Type Culture Collection, accession number CCTCC NF 20082077.

[0038] PDA medium: 5 g / L peptone, 2 g / L yeast extract, 20 g / L D-glucose, 1 g / L K2HPO4, 0.5 g / L MgSO4 and 15 g / L potato dextrose agar.

[0039] Bran culture medium: 80% wheat bran, 20% soybean meal powder, feed-to-water ratio 1:1.

[0040] Preparation of crude enzyme solution from endogenous enzymes in Aspergillus niger: Aspergillus niger was inoculated at 5% in sterile agar-free PDA liquid medium and cultured at 30℃ for 24 h to obtain seed culture. 100 mL of seed culture was inoculated per 100 g of dry bran medium, stirred thoroughly, and then cultured at 30℃ with constant temperature, humidity, and aeration. After 5 days of culture, 3 times the volume of 0.9% NaCl solution was added to homogenize the mixture. After stirring overnight at 4℃, the mixture was centrifuged, and the supernatant was obtained as the crude enzyme solution.

[0041] EPSF content determination: The reaction solution was concentrated, centrifuged, and passed through a 0.22 μm membrane before HPLC detection. Chromatographic column: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); mobile phase: Phase A: 0.1% formic acid aqueous solution, Phase B: acetonitrile; gradient elution program: 0 min, B 8%; 15 min, B 15%; 20 min, B 20%; 25 min, B 40%; 30 min, B 8%. Flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 280 nm; injection volume: 10 μL.

[0042] EPSF yield (%) = EPSF content / (EGCG content + Theanine content) × 100%

[0043] Physicochemical characterization: Particle size distribution was measured using a Malvern Nano-ZS laser particle size analyzer. Dynamic light scattering was performed at a scattering angle of 173°, a temperature of 25°C, and a wavelength of 633 nm. The polydispersity index (PDI) was used to assess the particle size distribution of the nanocomposite in solution, and the zeta potential was used to assess the stability of the nanocomposite solution.

[0044] Antioxidant activity: 0.1 mL of the analyte solution was mixed with 2.8 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer (100 mM, pH 8.0) and incubated at 25 °C for 10 min. Then, 0.1 mL of 3 mM pyrogallol solution was added, and the mixture was preheated in a 25 °C water bath and reacted for 3 min. The absorbance at 420 nm was measured using a spectrophotometer.

[0045] O 2- The clearance capacity is calculated as: O 2- Clearance rate % = (Blank absorbance value - Sample absorbance value) / Blank absorbance value × 100%

[0046] The blank absorbance value is calculated using purified water as a substitute for the analyte.

[0047] Antibacterial effect: The inhibitory effects of different samples on Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Listeria were determined using the Oxford cup method. Under aseptic conditions, 100 μL of bacterial suspension was added to LB agar plates, spread evenly, and then placed in Oxford cups. 20 μL of the diluted sample to be tested was added and incubated at 37℃ for 12 h. The diameter of the inhibition zone was then measured.

[0048] Example 1

[0049] Preparation of EPSF: EGCG and theanine were dissolved in 100 mL of distilled water at a molar ratio of 1:2 to form a supersaturated solution. Then, 1 mL of crude endogenous enzyme solution from Aspergillus niger was added for enzymatic hydrolysis. The reaction was carried out at 30 °C for 4 h to obtain the reaction solution. The reaction solution was dialyzed overnight at 4 °C in a dialysis bag with a molecular weight cutoff of 500 MW, and then lyophilized to obtain EPSF powder.

[0050] Preparation of EPSF / Polygonatum polysaccharide / casein submicron complex preservative:

[0051] First, Polygonatum polysaccharide was added to 100 mL of purified water and placed in an ultrasonic bath at 300 W for 20 min to prepare an aqueous solution. Then, EPSF was added until completely dissolved, followed by the addition of 2% sodium caseinate and stirring until dissolved. The solution was then ultrasonicated at 300 W for 20 min to form a submicron solution. The resulting solution was dialyzed at 4°C for 8 h using a dialysis bag with a molecular weight cutoff of 500 (MW) to obtain the target micro / nano solution. This solution was further pre-frozen at -80°C and then freeze-dried in a vacuum freeze dryer to obtain an EPSF / Polygonatum polysaccharide / sodium caseinate submicron composite preservative.

[0052] Before preparing the compound preservative, this example first explored the optimal reaction conditions for EPSF formation. The effects of EGCG to theanine molar ratios of 1:1, 1:1.2, 1:1.5, 1:2, and 2:1 on the amount of EPSF generated were investigated. The results are as follows: Figure 1 As shown, the yield of EPSF increases significantly with the increase of the theanine proportion in the reaction mixture. When the EGCG:theanine ratio changes from 1:1 to 1:2, the yield increases more than threefold. The concentration of theanine is the key factor limiting EPSF formation. The optimal substrate molar ratio for EPSF formation is EGCG:theanine = 1:2, where the EPSF yield reaches its highest level (14.85%). Excess EGCG has an inhibitory effect on the reaction because it leads to competitive side reactions.

[0053] Next, the effect of temperature on the enzymatic reaction was investigated while maintaining a fixed molar ratio of EGCG to theanine of 1:2. Specifically, the effects were studied at temperatures of 20℃, 25℃, 30℃, 35℃, 40℃, and 45℃. The results are as follows: Figure 2As shown, the production of EPSF is highly temperature-dependent, with a defined optimal temperature range between 30°C and 35°C for this enzymatic reaction. Reacting under these conditions maximizes EPSF yield. Temperatures that are too low (<25°C) result in insufficient reaction rate and extremely low production efficiency. Temperatures that are too high (≥40°C) lead to reversible or irreversible denaturation and inactivation of the enzyme, oxidative loss of EGCG, and severely reduce EPSF yield.

[0054] This embodiment also investigated the effect of enzymatic reaction time, specifically examining the influence of reaction times of 2, 4, 8, 24, 36, and 48 hours on the amount of EPSF generated. The results are as follows... Figure 3 As shown, the enzymatic reaction for EPSF production reaches its endpoint at approximately 24 hours, and extending the reaction time beyond 24 hours does not significantly increase the yield. Based on the combined data from the three sets, the optimized enzymatic reaction parameters for EPSF are: a temperature of 30℃-35℃, using a molar ratio of EGCG:theanine of 1:2, and a reaction time of 4-8 hours, which can efficiently obtain EPSF products close to the maximum yield.

[0055] This embodiment studied the effect of the mass ratio of Polygonatum polysaccharide, EPSF and sodium caseinate on the physicochemical characteristics of the final preservative. As shown in Table 1, when the mass ratio of Polygonatum polysaccharide, EPSF and sodium caseinate is 1:0.8:8, the obtained complex has excellent properties such as small particle size (~201nm), uniform distribution (PDI=0.20) and extremely high stability (Zeta≈-36mV).

[0056] Table 1 Physicochemical characterization of different material ratios

[0057] Group Particle size (nm) PDI Zeta 1:0.4:4 375.93±4.37 0.29±0.02 -26.23±1.85 1:0.6:6 395.17±29.22 0.25±0.04 -36.25±1.54 1:0.8:8 152.26±18.19 0.16±0.06 -33.15±0.83 1:0.2:2 634.82±20.53 0.41±0.08 -21.98±1.66 1:0.3:3 512.26±18.19 0.26±0.06 -23.15±0.83 0:1:10 142.24±10.75 0.14±0.02 -32.97±0.64 1:0:2 642.65±20.10 0.42±0.08 -20.15±0.83 1:0.4:0 742.82±24.45 0.11±0.03 -13.15±0.42

[0058] Example 2

[0059] Example 2, based on Example 1, fixed the mass ratio of Polygonatum polysaccharide, EPSF and sodium caseinate at 1:0.8:8, and investigated the effects of ultrasonic power and time.

[0060] The experimental results are shown in Table 2. Under ice bath conditions, ultrasonic treatment was performed at a power of 300W for 20 minutes. Under these conditions, the energy input of the ultrasound was balanced with the treatment time, which resulted in the production of submicron composites with the smallest particle size (~152nm), the most uniform distribution (PDI=0.16), and extremely high stability (Zeta≈-33mV).

[0061] Table 2 Physicochemical characterization under different ultrasound conditions

[0062] Group Particle size (nm) PDI Zeta 200 W, 10 min 275.93±4.37 0.29±0.02 -26.23±1.85 200 W, 20 min 172.24±10.75 0.18±0.02 -35.97±0.96 200 W, 30 min 195.17±29.22 0.25±0.04 -32.25±1.54 300 W, 10 min 234.82±20.53 0.31±0.08 -21.98±1.66 300 W, 20 min 152.26±18.19 0.16±0.06 -33.15±0.83 300 W, 30 min 212.64±12.75 0.26±0.08 -33.27±2.57

[0063] Example 3

[0064] The difference between Example 3 and Example 1 is that the mass ratio of Polygonatum polysaccharide, EPSF and sodium caseinate is fixed at 1:0.8:8.

[0065] Figure 4 The antioxidant capacity test results of the EPSF / Polygonatum polysaccharide / casein submicron complex in Example 3, along with EGCG, theanine, Polygonatum polysaccharide, EPSF, and casein, are presented. As shown in the figure, the submicron complex exhibits synergistically enhanced antioxidant activity (88.36%) in scavenging superoxide anion free radicals. Its activity is not only significantly higher than that of any single component, but also far exceeds the combined antioxidant activity of the classic antioxidant EGCG and the combined antioxidant activity of ECGC and theanine. This indicates that after the reaction to form ESPF, it not only combines the advantages of ECGC and theanine, but also generates new groups with antioxidant activity. The submicron composite system prepared in this invention leverages the bioactivity of the functional component EPSF, achieving dual optimization of physicochemical properties and functional activity.

[0066] The antibacterial activity of the submicron complex preservative prepared in Example 3 against EGCG, theanine, Polygonatum polysaccharide, EPSF, and casein is shown in Table 3. Table 3 shows that Polygonatum protein and casein alone had no antibacterial effect against *Escherichia coli* and *Bacillus subtilis*. EPSF showed antibacterial effects of only 16.5 and 20.5 against *E. coli* and *Bacillus subtilis*, respectively. However, the submicron complex preservative composed of Polygonatum protein, casein, and EPSF achieved antibacterial effects of 23.25 and 24.8 against *E. coli* and *Bacillus subtilis*, respectively, significantly higher than the sum of the individual effects of Polygonatum protein, casein, and EPSF. This indicates that the combination of these three substances creates new antibacterial sites. The submicron complex preservative prepared in Example 2 exhibited broad-spectrum antibacterial activity against the four bacteria. Its inhibition zone diameter (23.25–26.5 mm) was significantly larger than that of the single component EPSF and the positive control EGCG, demonstrating a strong synergistic antibacterial effect.

[0067] Table 3 Antibacterial effects of different materials

[0068]

[0069] Example 4: Application of EPSF / Polygonatum polysaccharide / casein submicron complex preservative

[0070] Complex dissolution: The freeze-dried EPSF / Polygonatum polysaccharide / casein submicron complex was dissolved in purified water to prepare a preservative; wherein, the preparation method of the freeze-dried EPSF / Polygonatum polysaccharide / casein submicron complex was the same as in Example 3.

[0071] Soaking: Peel and cut apples without mechanical damage, disease spots and pests into pieces. Place the apple pieces into 0.5% and 1% EPSF / Polygonatum polysaccharide / casein submicron complex preservative solutions and soak them evenly for 1 minute. Drain the solution to obtain the treated fresh-cut apple pieces, and store them in a sealed container at 4°C.

[0072] Spraying: Peel potatoes that are free from mechanical damage, disease spots and pests, slice them, and spray them evenly with 1% and 2% EPSF / Polygonatum polysaccharide / casein submicron complex preservative solution. Drain the solution to obtain the treated sliced ​​potatoes, and store them separately in storage boxes at 4℃.

[0073] To demonstrate the preservation effect of the EPSF / Polygonatum polysaccharide / casein submicron complex, the preservation effect of the treated fruits and vegetables was analyzed over the following 10-day period:

[0074] Color difference analysis: The surface color difference of fresh-cut fruits and vegetables was measured using a GR / SC-80C benchtop colorimeter (manufactured by Beixin Keyuan Instruments) to determine the L*, a*, and b* values ​​at different locations on the surface of the fruits and vegetables, and the average value was taken. The color difference value was evaluated using the ΔE value.

[0075]

[0076] Table 4 shows the results of the color difference analysis of treated fruits and vegetables during storage.

[0077]

[0078] Table 4 shows the color difference changes of apples and potatoes during storage after treatment with the submicron composite preservative prepared using the method of Example 3. As shown in Table 4, the color difference ΔE value of fresh-cut apples and potatoes treated with the composite preservative was significantly reduced during 10 days of storage at 4℃, and the higher the concentration, the more significant the preservation effect, indicating that the composite can effectively delay the discoloration of fruits and vegetables.

[0079] Browning index: Determined using a grading method. Fresh-cut fruits and vegetables were randomly selected, and the proportion of browned area to the total area of ​​the cut surface was divided into 0 to 3 levels: Level 0: No browning; Level 1: Browned area 0-30%; Level 2: Browned area 30-60%; Level 3: Browned area greater than 60%. Browning index = ∑(level number × number of samples) / (highest level number × total number of samples).

[0080] Table 5 Results of browning index of treated fruits and vegetables during storage

[0081]

[0082]

[0083] Table 5 shows the browning index results of apples and potatoes after treatment with the submicron composite preservative prepared using the method of Example 3. As shown in Table 5, the browning index of fresh-cut apples and potatoes treated with the composite preservative was significantly reduced, and the higher the concentration, the more significant the inhibitory effect. Apples treated with a 1% concentration and potatoes treated with a 2% concentration had the lowest browning index after 10 days of storage, indicating that the preservative can effectively delay the browning of fruits and vegetables.

[0084] Weight loss rate: Take a certain amount of sample, record its original weight, weigh it again after processing, and calculate the weight loss rate of samples with different treatments.

[0085] Table 6 shows the results of weight loss during storage of treated fruits and vegetables.

[0086]

[0087] Table 6 shows the changes in weight loss rate of apples and potatoes after treatment with the submicron composite preservative prepared using the method of Example 3. As shown in Table 6, the weight loss rate of fresh-cut apples and potatoes treated with the composite preservative was lower than that of the control group, and the higher the concentration, the more significant the effect. The 1% concentration of apples and the 2% concentration of potatoes showed the lowest weight loss rate after 10 days of storage, proving that the preservative can effectively reduce water loss from fruits and vegetables and delay wilting.

[0088] Hardness: The Universal TA texture analyzer (Shanghai Tengba Instruments) applies vertical pressure to the surface of fresh-cut fruits and vegetables through its measuring head, records the pressure values ​​that fruits and vegetables with different treatments can withstand, and obtains hardness data.

[0089] Table 7 shows the results of changes in storage hardness of treated fruits and vegetables.

[0090]

[0091] Table 7 shows the changes in firmness of apples and potatoes after treatment with the submicron composite preservative prepared in Example 3. As shown in Table 7, the firmness of fresh-cut apples and potatoes treated with the submicron composite preservative was significantly better than that of the control group, and the effect was positively correlated with the concentration. After 10 days of storage, the firmness of apples treated with a 1% concentration remained at 5.1 × 10⁻⁶. 5 The hardness of potatoes treated with a 2% concentration of Pa was 3.87 × 10⁻⁶ Pa. 5 Pa was significantly higher than its respective control groups. This indicates that the compound preservative can form a protective film on the surface of fruits and vegetables, reducing moisture loss, effectively slowing down the softening of fruit and vegetable tissues, and better maintaining texture.

[0092] Figure 5 The images show the storage appearance of fresh-cut fruits and vegetables treated with submicron complexes. Compared with the untreated control, the appearance quality of the treated fresh-cut fruits and vegetables was better protected, browning was significantly inhibited, and the quality of fresh-cut fruits and vegetables was effectively maintained.

[0093] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing an EPSF / polygonatum polysaccharide / casein submicron complex, characterized in that, The method comprises the following steps: Dissolve the polysaccharide in water, add EPSF and stir until completely dissolved, then add sodium caseinate and stir to form a complex solution, continue to ultrasonicate to form a submicron solution, and freeze-dry the submicron solution to obtain the EPSF / polysaccharide / sodium caseinate submicron complex preservative.

2. The production method according to claim 1, characterized by, The submicron solution needs to be dialyzed using a dialysis bag with a molecular weight cut-off MW=500 before freeze-drying.

3. The production method according to claim 1 or 2, characterized by, The polysaccharide is a purified polysaccharide powder from raw polygonatum, the mass / volume ratio of the polysaccharide to water is 0.5-1.0g:0.1L, the purity is more than 80%, and the mass ratio of the added EPSF and sodium caseinate to the polysaccharide is 0.2-0.8:1 and 2-8:1, respectively.

4. The production method according to claim 1 or 2, characterized by, The ultrasonic power is 200-300W, and the ultrasonic time is 20-30min.

5. The production method according to claim 1 or 2, characterized by, The freeze-drying is carried out by pre-freezing the submicron solution at-20℃ for 12h and then using a vacuum freeze-drying machine.

6. The production method according to claim 1 or 2, characterized by, The preparation method of the EPSF is as follows: dissolve EGCG and theanine in distilled water, add crude enzyme solution of endogenous enzyme of Aspergillus niger for enzymatic reaction, dialyze the reaction solution obtained by the enzymatic reaction using a dialysis bag with a molecular weight cut-off MW=500 at 4℃ for 8-16h, and then freeze-dry to obtain the EPSF.

7. The production method according to claim 6, wherein The preparation method of the crude enzyme solution of endogenous enzyme of Aspergillus niger is as follows: inoculate activated Aspergillus niger into sterilized agar-free PDA liquid medium at 5%, cultivate at 30℃ for 24h to obtain seed bacteria, inoculate 100mL seed solution into 100g bran medium dry basis, stir uniformly, and then place in a constant-temperature and constant-humidity ventilated culture at 30℃, add 3 times of 0.9% NaCl solution after 5 days of cultivation, stir overnight at 4℃, centrifuge, and then the supernatant is the crude enzyme solution.

8. The preparation method according to claim 6, characterized in that, The molar ratio of EGCG to theanine is 1:1.2-2, the temperature of the enzymatic reaction is 30-40℃, and the reaction time is 4-48h.

9. The EPSF / polysaccharide / sodium caseinate submicron complex preservative prepared by the preparation method according to any one of claims 1-8.

10. The application of the EPSF / polysaccharide / sodium caseinate submicron complex preservative according to claim 9 in fresh-cut fruit and vegetable preservation.

Images