Method for accelerating healing damage of Chinese yam tubers

Abstract

The invention relates to a method for accelerating healing damage of Chinese yam tubers, which utilizes ozone to assist glycosylation modification of soybean protein isolate so as to improve functional properties of the soybean protein isolate and prepare suppository-loaded nano-emulsion with a function of healing damage of Chinese yam tubers. The method comprises the following steps: introducing ozone into a soybean protein isolate suspension to effectively improve the secondary structure of the soybean protein isolate, mixing the soybean protein isolate with Chinese yam polysaccharide, carrying out a Maillard reaction at a certain temperature, and carrying out freeze drying to obtain glycosylated protein. By analyzing the emulsification characteristics and interface characteristics of proteins with different sugar grafting degrees, proper glycosylated proteins are screened to prepare the nanometer emulsion rich in suberite, and the callus effect of the nanometer emulsion on damaged Chinese yam tubers is evaluated. The reagent used in the method is safe, non-toxic and pollution-free, the operation is simple, the prepared nano-emulsion can remarkably accelerate the callus process of the damaged Chinese yam tubers, and the bottleneck problem that the bombardment is poor in water solubility and cannot be industrially applied is solved.

Description

Technical Field

[0001] The present invention discloses a method for applying suberin to accelerate the wound healing of yam tubers, belonging to the technical field of postharvest preservation of fruits and vegetables. Technical Background

[0002] Yam is the fourth largest tuber crop after potato, cassava, and sweet potato, with a global annual output of approximately 89.35 million tons (FAO, 2023), providing a food source for 300 million people worldwide. Yam is rich in active ingredients such as polysaccharides, amino acids, allantoin, and diosgenin, and has physiological functions such as improving immunity, anti-tumor, and hypoglycemic effects. China is one of the important producing areas of yam, and Henan Province is one of the main cultivation areas of yam in China. The local specialty Tieguanyin yam ranks first among the four famous traditional Chinese medicines in Henan, and is well-known throughout the country for its authentic quality and rich nutrition. However, yam tubers are prone to mechanical damage during harvesting, storage, transportation, and breeding processes, leading to losses of water and nutrients and microbial infection, resulting in deterioration of tuber quality.

[0003] Suberin is a plant secondary metabolite, which is mainly cross-linked by the suberin phenolic region and the suberin aliphatic compound (SPA) region. Suberin phenolic substances mainly include ferulic acid, caffeic acid, and p-coumaric acid, and SPA mainly includes fatty acids, ω-hydroxy acids, α,ω-diacids, and glycerol with different carbon chain lengths (generally C16-C24). Suberin is not only widely distributed in tissues such as seed coats, fruit peels, barks, and roots, but also plays an important role in the wound healing of fruits and vegetables. Injury can induce the accumulation of suberin between the cell membranes and cell walls of damaged tissues in fruits and vegetables (such as potatoes, tomatoes, kiwifruits, melons, and yams) to prevent the loss of water and nutrients and the infection of pathogenic bacteria. Therefore, accelerating the synthesis of endogenous suberin is crucial for the postharvest wound healing of fruits and vegetables. Currently, there are many methods for inducing the synthesis of endogenous suberin in damaged fruits and vegetables. For example, treatment with high-concentration oxygen (50%), abscisic acid, and methyl jasmonate can promote the wound healing of kiwifruit; nitric oxide (NO) fumigation treatment promotes the wound healing of sweet potato roots; abscisic acid and oligogalacturonic acid treatment promote the wound healing of tomato fruits; abscisic acid, methyl jasmonate, and ferulic acid treatment can all accelerate the wound healing of potato tubers, etc. However, these methods are currently in the experimental research stage and have not been applied in actual production, mainly because their operation methods are relatively cumbersome, and most of the reagents used are insoluble in water, making their application difficult.

[0004] During the harvesting, post-harvest handling, and breeding processes of yam tubers, mechanical damage can occur. Currently, in production, lime water and fungicides (such as potassium permanganate, 75% ethanol, and carbendazim, etc.) are commonly used to treat the wound surface to extend its storage period. However, they can cause certain harm to the human body and the environment. Currently, methods for yam wound healing include heat treatment (35°C), treatment with β-aminobutyric acid combined with sodium bicarbonate, treatment with abscisic acid and methyl jasmonate, etc. But these methods have poor safety and operability in actual production. Suberin not only plays an important role in plants' resistance to external environmental stresses, but also monomer components of suberin (such as ferulic acid) can induce the synthesis of endogenous suberin to accelerate the wound healing of fruits and vegetables. In addition, suberin is also developed as a value-added material for biochemical agents and biodegradable polymers, such as moisture-proof materials, plasticizers, surface treatment materials, and pharmaceutical excipients. Currently, the water-insolubility of suberin is the main factor limiting its application in yam wound healing.

[0005] An emulsion is a dispersion system in which one phase (dispersed phase) is dispersed in the form of droplets in another phase (continuous phase), and it is divided into two types: oil-in-water (O / W) and water-in-oil (W / O). Emulsions not only exist in various natural and processed foods, including soft drinks, milk, cream, sauces, and dips, etc., but can also be used as delivery systems for functional ingredients (such as pigments, flavors, and nutrients, etc.) to encapsulate, protect, and control the release of active ingredients. The O / W type emulsion is a thermodynamically unstable system, composed of a dispersed phase (oil phase), an oil-water interface layer (emulsifier), and a continuous phase (water phase). The type and concentration of the emulsifier have an important regulatory effect on the characteristics of the oil-water interface (interface thickness, packing density, and charge characteristics), thus affecting the formation, stability, and properties of the emulsion. An ideal emulsifier has the ability to adsorb at the oil-water interface and rapidly reduce the interfacial tension and form a protective layer on the surface of the oil droplets. Proteins and polysaccharides have high biocompatibility, good biodegradability, and a wide range of technical functions, so they are widely used in the preparation of food emulsions. However, emulsions stabilized by proteins are more sensitive to the external environment (such as pH, ionic strength, and temperature, etc.), while interfacial active complexes formed by covalent and non-covalent bonding between proteins and polysaccharides can improve the stability of the emulsion. The Maillard reaction is a common method for covalent bonding between proteins and polysaccharides. The formed protein-polysaccharide conjugates have high antioxidant activity, antibacterial activity, solubility, emulsifying property, and thermodynamic stability, and are often used as emulsifiers to prepare O / W emulsions for post-harvest preservation of fruits and vegetables.

[0006] Although different treatment methods can rapidly induce the synthesis of endogenous suberin to resist the invasion of pathogenic bacteria and water loss after mechanical damage to yam tubers, suberin usually forms a relatively complete protective barrier after one day. Therefore, to accelerate the wound healing of fruits and vegetables, the present invention intends to develop a nano-preservative loaded with suberin, attempting to achieve the purpose of wound healing and preservation of yam tubers through exogenous suberin protection and induction of the endogenous suberin layer. The adoption of this technology can effectively accelerate the wound healing process of yam tubers, prevent the massive loss of water and nutrients and the invasion of pathogenic bacteria in damaged yam tubers, and has important application value for the post-harvest preservation and breeding of yam tubers. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides a method for rapid wound healing of yam tubers, solving the industrial problem of quality deterioration during post-harvest storage and breeding caused by mechanical damage to yam tubers due to post-harvest or post-harvest treatment.

[0008] To solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A method for accelerating the wound healing of yam tubers (the technical process is as Figure 1 shown), the method includes the following steps:

[0010] 1) Prepare a protein suspension A from soy protein isolate, continuously introduce ozone into the protein suspension, and freeze-dry to obtain soy protein isolate powder;

[0011] 2) Prepare a suspension B from the soy protein isolate powder obtained in step 1), add yam polysaccharide to the suspension B, heat and react, and then freeze-dry to obtain glycated protein powder;

[0012] 3) Prepare a suspension C from the glycated protein powder obtained in step 2), add suberin, stir, and then process with a high-speed dispersion homogenizer to obtain a nano-emulsion loaded with suberin;

[0013] 4) Immerse the damaged yam tubers with the nano-emulsion loaded with suberin obtained in step 3), and evaluate the wound healing effect of the tubers.

[0014] Furthermore, the concentration of the protein suspension A prepared in step 1) is 5 mg / mL, the ozone flow rate is 2.8 mg / L, and the ozone treatment time is 0.5 - 1.5 h, preferably 1 h.

[0015] Furthermore, the concentration of the suspension B in step 2) is 10 mg / mL, and the mass ratio of soy protein isolate powder to yam polysaccharide is 2:1 - 1:4.5, preferably 1:3.5.

[0016] Further, in the step 2), the reaction temperature is 75 - 95°C, preferably 80°C, and the reaction time is 0.5 - 2.5 h, preferably 1.5 h.

[0017] Further, in the step 3), the concentration of the glycosylated protein in the suspension C is 0.2 - 2 mg / mL, preferably 1 mg / mL; the content of the added suberin is 0.1 - 1 mg / mL, preferably 1 mg / mL.

[0018] Further, in the step 3), the stirring is carried out at 35°C for 3 h.

[0019] Further, in the step 3), the shear rate of the high-speed dispersion homogenizer is 5000 - 30,000 r / min, and the shear time is 1 - 2 min, preferably 20,000 r / min, 2 min.

[0020] The callus effect of the nanoemulsion of the present invention on yam tubers includes: yam tuber injury, treatment with the nanoemulsion by soaking, determination of callus-related indexes of yam tubers, and detection of parameters related to suberin synthesis.

[0021] The beneficial effects of the present invention: On the one hand, the present invention provides a method for modifying plant proteins. Using ozone pretreatment of soy protein isolate can significantly improve the protein glycosylation degree, and the interfacial properties of the glycosylated protein are significantly improved; on the other hand, using the glycosylated protein to prepare an O / W emulsion loaded with suberin breaks through the bottleneck problem that suberin cannot be industrially applied due to its water-insolubility. On the other hand, the present invention designs and prepares an O / W nanoemulsion loaded with suberin based on the oil-water interface, applies it to treat damaged yam tubers, analyzes the weight loss rate, ion leakage rate, disease index, and the expression levels of genes related to suberin synthesis, suberin polymerization degree, and suberin accumulation amount of yam tubers, and evaluates the callus effect of the nanoemulsion loaded with suberin on yam tubers. The present invention can greatly accelerate the callus process of yam tubers, well maintain the moisture and nutrient components of the damaged tubers, reduce the tuber corruption rate, and make up for the lack of fruit and vegetable callus preservatives in the market. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments. The following drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 Technical flow chart for accelerating the callus of damaged yam tubers;

[0024] Figure 2 Effect of the mass ratio of soy protein isolate to yam polysaccharide on the protein glycosylation degree;

[0025] Figure 3 Effect of different reaction temperatures on the degree of protein-sugar grafting;

[0026] Figure 4 Effect of different reaction times on the degree of protein-sugar grafting;

[0027] Figure 5 Effect of different degrees of sugar grafting on the emulsifying property of soy protein isolate;

[0028] Figure 6 Effect of different degrees of sugar grafting on the interfacial tension of soy protein isolate;

[0029] Figure 7 Effect of different degrees of sugar grafting on the interfacial thickness of soy protein isolate;

[0030] Figure 8 Effect of different glycoprotein contents and shear rates on the suberin retention rate in emulsions;

[0031] Figure 9 Effect of nanoemulsions loaded with different contents of suberin on the weight loss rate of tubers;

[0032] Figure 10 Effect of nanoemulsions loaded with different contents of suberin on the ion leakage rate of damaged tissues of tubers;

[0033] Figure 11 Effect of nanoemulsions loaded with different contents of suberin on the disease index of tubers;

[0034] Figure 12 Effect of nanoemulsions loaded with different contents of suberin on the expression of DoCYP86A1 gene;

[0035] Figure 13 Effect of nanoemulsions loaded with different contents of suberin on the expression of DoCYP94B3 gene;

[0036] Figure 14 Effect of nanoemulsions loaded with different contents of suberin on the expression of DoGELP38 gene;

[0037] Figure 15 Effect of nanoemulsions loaded with different contents of suberin on the degree of suberin polymerization;

[0038] Figure 16 Effect of nanoemulsions loaded with different contents of suberin on the accumulation of suberin. Detailed implementation manners

[0039] Next, the technical solutions in the embodiments of the present invention will be described in conjunction with the accompanying drawings in the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0040] Example 1: Modification of soy protein isolate by ozone treatment, the steps are as follows:

[0041] (1) Add soy protein isolate to deionized water, stir at 40 °C for 30 min to fully dissolve the protein, prepare a protein suspension of 5 mg / mL, cool to room temperature, and introduce ozone into the protein suspension at a flow rate of 2.8 mg / L. The treatment times are 0, 0.5, 1, and 1.5 h respectively. Freeze-dry the treated protein solution into powder.

[0042] (2) Detection of protein secondary structure

[0043] Dissolve the sample in 20 mol / L Tris-HCL buffer solution at a concentration of 0.2 mg / mL. Under a constant nitrogen flow rate, scan with a circular dichroism spectrometer in the wavelength range of 190 - 250 nm at a scan speed of 100 nm / min, with a 1.0 nm interval, and perform three scans. Select 20 mol / L Tris-HCL buffer solution as the blank control. Use CDNN processing software to calculate the content of the secondary structure of each sample.

[0044] Experimental results:

[0045] During the folding process of natural proteins, due to the locking of disulfide bonds, hydrophobic groups are generally wrapped inside the molecule to prevent the contact between hydrophobic groups and the aqueous solution, thus limiting the glycosylation degree of soy protein isolate. Ozone treatment can oxidize disulfide bonds to break them, thereby exposing the hydrophobic groups of proteins and increasing the glycosylation degree of proteins. Therefore, the effect of different ozone flow rates on the protein secondary structure was investigated. As shown in Table 1, different ozone treatment times can change the protein secondary structure, such as α-helix, β-sheet, β-turn, and random coil. Ozone treatment can reduce α-helix, β-turn, and random coil, increase β-sheet, and ozone treatment for 1 h can significantly change the protein secondary structure. Therefore, to improve the protein glycosylation degree, the ozone treatment time is preferably 1 h.

[0046] Table 1 Effect of ozone treatment on protein secondary structure

[0047]

[0048] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0049] Example 2: Glycosylation modification of soy protein isolate, the steps are as follows:

[0050] (1) Add soy protein isolate to deionized water, stir at 40 °C for 30 min to fully dissolve the protein, prepare a protein suspension of 5 mg / mL, cool to room temperature, introduce ozone into the protein suspension at a flow rate of 2.8 mg / L for 1 h, and freeze-dry the treated protein solution into powder. Prepare a suspension of 10 mg / mL from the ozone-treated soy protein isolate, then add yam polysaccharide, and the mass ratios of protein to polysaccharide are 2:1, 1:1, 1:2, 1:3, 1:3.5, 1:4, and 1:4.5 respectively. Stir magnetically for 3 h, hydrate at 4 °C for 8 h, heat the mixture at 75, 80, 85, 90, and 95 °C for 0.5, 1, 1.5, 2, and 2.5 h respectively, and immediately immerse in an ice bath for 5 min to terminate the reaction after the reaction ends.

[0051] (2) Detection of protein grafting degree

[0052] The determination of protein grafting degree adopts the method of o-phthalaldehyde (OPA). Mix 40 mg of o-phthalaldehyde dissolved in 1 ml of methanol with 25 ml of 0.1 mol / L sodium tetraborate buffer solution (pH = 9.5), 25 ml of 0.2 g / g sodium dodecyl sulfate solution, and 0.1 m β-mercaptoethanol. Make up the volume to 50 ml in a volumetric flask. Mix 4 ml of OPA reagent with 0.2 ml of the grafted product at 2 mg / ml, use distilled water as a control, react at 35 °C for 2 min, and measure the absorbance value at 340 nm with a UV spectrophotometer. Grafting degree (%) = (A0 - A1) / A0 × 100

[0053] In the formula: A0 and A1 are the absorbances of untreated soy protein isolate and glycosylated protein at 340 nm respectively.

[0054] (3) Detection of protein emulsifying properties

[0055] Mix 9 mL of the sample (1 mg / mL) with 3 mL of soybean oil, homogenize at 20 Kr / min for 1 min, and draw 50 μL of the solution from the bottom of the container to 5 mL of SDS solution (0.1%) at 0 min and 10 min. After shaking and mixing evenly, measure the absorbance value at 500 nm, denoted as A0, A 10 。

[0056] Emulsifying property

[0057] In the formula: A0, the absorbance of the emulsion at 0 min; A 10 , the absorbance after standing for 10 min; D, dilution factor; C, protein concentration (g / mL); Volume fraction of the oil phase; L, optical path of the cuvette.

[0058] (4) Determination of protein interfacial tension

[0059] The pendant drop method using a fully automatic interfacial rheometer was employed to determine the change of interfacial tension with time. A certain volume (0.005%) of the sample was aspirated with a microsyringe, the needle was inserted into a glass dish containing soybean oil, and about 12 μL of the droplet was dropped out. The interfacial tension of the droplet within 3500 s was collected.

[0060] (5) Determination of protein interfacial thickness

[0061] Polystyrene microspheres were used to determine the interfacial layer thickness formed by glycoproteins with different grafting degrees. The polystyrene microspheres (1%, w / v) were diluted 10 times with deionized water. Subsequently, 100 μL of the microsphere suspension was mixed with 900 μL of glycoprotein samples with different grafting degrees and allowed to stand for 1.5 h. Then, the particle sizes of the latex beads before and after the adsorption of the protein layer were measured, and the interfacial thickness of the glycoprotein on the surface of the latex beads was calculated based on the difference in the diameters of the latex beads with and without the adsorption layer.

[0062] Experimental results:

[0063] To optimize the grafting reaction conditions of soy protein and yam polysaccharide, different mass ratios of soy protein isolate and yam polysaccharide were selected, the reaction temperature was 80 °C, and the time was 1.5 h ( Figure 2 ). The results showed that when the mass ratio of soy protein isolate to yam was 1:3.5, the highest sugar grafting degree was 29.0%. Under the conditions of different reaction temperatures, with a protein-to-polysaccharide mass ratio of 1:3.5 and a reaction time of 1.5 h, the protein sugar grafting degree was the highest at 80 °C ( Figure 3 ). Under the conditions of different reaction times, with a protein-to-polysaccharide mass ratio of 1:3.5 and a reaction temperature of 80 °C, the protein sugar grafting degree was the highest when the reaction time was 1.5 h ( Figure 4 ). Therefore, the preferred grafting reaction conditions for soy protein isolate and yam polysaccharide are: a protein-to-polysaccharide mass ratio of 1:3.5, a reaction temperature of 80 °C, and a reaction time of 1.5 h.

[0064] The emulsifying properties, interfacial tension, and interfacial thickness of glycoproteins with different grafting degrees were compared. Glycoproteins with sugar grafting degrees of 9%, 19%, and 29% were prepared under the conditions of a reaction temperature of 80 °C, a reaction time of 1.5 h, and mass ratios of soy protein isolate to yam polysaccharide of 1:2, 1:3, and 1:3.5, respectively, and their emulsifying properties, interfacial tension, and interfacial thickness were measured ( Figures 5 - 7 ). The results showed that the emulsifying property of the glycoprotein increased significantly with the increase in the grafting degree. The glycoprotein with a grafting degree of 29% had the highest emulsifying property, reaching 45.5 m 2 / g; The interfacial tension of the glycoprotein decreased with the increase of the grafting degree, and the interfacial tension of the glycoprotein with a grafting degree of 29% was the lowest; while the interfacial thickness of the glycoprotein increased with the increase of the grafting degree, and the interfacial thickness of the glycoprotein with a grafting degree of 29% was the highest, reaching 33.6 mm. From the above results, it can be seen that the glycoprotein prepared with a mass ratio of soy protein isolate to yam polysaccharide of 1:3.5 had the highest grafting degree (29%) and showed the best emulsifying and interfacial properties. Therefore, the glycoprotein with a grafting degree of 29% was preferably used as an emulsifier to prepare the suberin-loaded nanoemulsion.

[0065] Example 3: Preparation of suberin-loaded nanoemulsion, the steps are as follows:

[0066] (1) Suberin extraction: The oak bark was finely ground with a grinder, sieved through a 0.25 mm sieve, 10 g of the powder was taken, 3 g of NaOH and 150 mL of isopropanol were added, and the mixture was boiled for 3 h. It was filtered while it was hot. After standing at room temperature, a precipitate was precipitated. After the precipitate was air-dried, it was boiled with 100 mL of hot water for 3 h, and the white solid insoluble in water was filtered out. The solution was adjusted to pH < 3.8 with 2 mol / L H2SO4, centrifuged at 8000 r / min for 20 min, the precipitate was washed twice with deionized water, freeze-dried, and stored under refrigerated conditions for later use.

[0067] Nanoemulsion preparation: Soy protein isolate was added to deionized water and stirred at 40 °C for 30 min to fully dissolve the protein, and a protein suspension with a concentration of 5 mg / mL was prepared. After cooling to room temperature, ozone was introduced into the protein suspension at a flow rate of 2.8 mg / L for 1 h, and the treated protein solution was freeze-dried into powder. The soy protein isolate treated with ozone was prepared into a suspension with a concentration of 10 mg / mL, and then yam polysaccharide was added. The glycoproteins prepared with a mass ratio of soy protein isolate to yam polysaccharide of 1:3.5 were respectively prepared into suspensions with concentrations of 0.2, 0.5, 1, and 2 mg / mL. While stirring magnetically for 3 h, 1 mg / mL of suberin was added to the glycoprotein solution dropwise. Subsequently, the mixture was treated with a high-speed dispersion homogenizer at 10,000, 20,000, and 30,000 r / min for 2 min to obtain the nanoemulsion.

[0068] (2) Determination of encapsulation efficiency

[0069] Take 1 mL of the emulsion sample, add 4 mL of dichloromethane, mix well, centrifuge at 10,000 r / min for 10 min, collect the organic phase, blow dry with nitrogen, redissolve with dichloromethane, add methyl heptadecanoate as an internal standard, and determine the suberin content by gas chromatography-mass spectrometry. Chromatographic conditions:

[0070] 7890B-5977AGC-MS gas chromatography-mass spectrometer (Agilent)

[0071] Chromatographic column: HP-5MS chromatographic column (30m × 0.25mm × 0.25μm)

[0072] Injection mode: splitless

[0073] Carrier gas: high-purity helium

[0074] Flow rate: 1.5 mL / min

[0075] Inlet temperature: 280 °C

[0076] Column oven conditions: Maintain at 80 °C for 1 min, then increase to 180 °C at a rate of 10 °C / min, maintain for 0.5 min, then increase to 250 °C at a rate of 2 °C / min, maintain for 0.5 min, then increase to 300 °C at a rate of 20 °C / min, and maintain for 10 min.

[0077] Electron impact energy: 70 eV.

[0078] Suberin monomers were identified by matching with the NIST14 database. The suberin content was calculated based on the peak area of the monomer and the peak area of the internal standard (methyl heptadecanoate). The embedding rate was calculated by dividing the measured amount of suberin by the added amount of suberin.

[0079] Experimental results:

[0080] The effects of different glycoprotein contents and shear rates of a high-speed dispersion homogenizer on the embedding rate of suberin in nanoemulsions were compared. Nanoemulsions were prepared by embedding 1 mg / mL of suberin with 0.2, 0.5, 1, and 2 mg / mL of glycoprotein at shear rates of 10,000 r / min, 20,000 r / min, and 30,000 r / min. The results showed that the nanoemulsion prepared at a glycoprotein concentration of 1 mg / mL and a shear rate of 20,000 r / min had the highest embedding rate ( Figure 8 ), so it is preferred to prepare nanoemulsions loaded with suberin at a protein concentration of 1 mg / mL and a shear rate of 20,000 r / min.

[0081] Example 4: A method for accelerating the healing of damaged yam tubers (the technical process is as Figure 1 shown), that is, the evaluation of the wound healing effect of nanoemulsions on yam tubers, the steps are as follows:

[0082] (1) Preparation of nanoemulsions:

[0083] Soybean protein isolate was added to deionized water and stirred at 40 °C for 30 min to fully dissolve the protein, and a protein suspension with a concentration of 5 mg / mL was prepared. After cooling to room temperature, ozone was introduced into the protein suspension at a flow rate of 2.8 mg / L for 1 h, and the treated protein solution was freeze-dried into powder. The ozone-treated soybean protein isolate was prepared into a suspension with a concentration of 10 mg / mL, and then yam polysaccharide was added. The glycoprotein prepared from soybean protein isolate and yam polysaccharide with a mass ratio of 1:3.5 was prepared into a suspension with a concentration of 1 mg / mL. When magnetic stirring was carried out for 3 h, 1 mg / mL of suberin (prepared in the same way as in Example 3) was added to the glycoprotein solution while stirring, and then the mixture was treated with a high-speed dispersing homogenizer at 20,000 r / min for 2 min to obtain a nanoemulsion.

[0084] (2) Yam tuber treatment

[0085] Evaluation of the callus effect of yam tubers: After harvesting yam tubers, yam tubers with uniform thickness, no pests and diseases, and no surface damage were selected for subsequent experiments. After washing the surface of the yam tubers, they were soaked in 0.5% sodium hypochlorite solution for 1 min for sterilization, and then rinsed with sterile distilled water. After natural air drying at room temperature, the yam tubers were transversely cut into stem segments with a length of 5 cm with a sterile scalpel in a clean bench, and the cut surfaces at both ends were mechanical injury wounds. The damaged tubers were randomly divided into two treatment groups: (1) control group (CK), the damaged yam tubers were soaked in a protein suspension without suberin for 5 min; (2) nanoemulsion treatment group containing suberin (1-SUB), the damaged yam tubers were soaked in a nanoemulsion containing 1 mg / mL of suberin for 5 min. After the tubers were treated, they were immediately placed in a sterile incubator at 22 °C and a relative humidity of 85% for wound healing in the dark. At 0, 1, 5, and 9 d, 2-mm-thick tuber discs were cut from the cut surfaces at both ends of the damaged tubers with a sterile scalpel, which were suberized tissues. Among them, the 2-mm-thick tuber discs obtained immediately after tuber damage at 0 d were used.

[0086] (2) Determination of weight loss rate

[0087] The weight of the tubers was measured at 0, 1, 5, and 9 d respectively. The weight loss rate of the tubers per day was calculated by dividing the weight of the tubers per day by the weight of the tubers at 0 d and then dividing by the initial weight (the weight of the tubers at 0 d).

[0088] (3) Conductivity detection

[0089] At 0, 1, 5, and 9 d of callus, tissue discs with a diameter of 10 mm and a thickness of 2 mm were taken from the wound. A certain amount of the discs was placed in deionized water at 25 °C and soaked for 30 min, and the conductivity E of the solution was measured i , and then the solution was boiled for 20 min. After the solution was cooled, deionized water equal to the amount before boiling was added to make up the volume, and the conductivity E was measured at 25 °C f, Relative conductivity = E i / E f ×100.

[0090] (4) Determination of disease index

[0091] Inoculate Penicillium oxalicum into potato dextrose agar medium (PDA), culture at 22 °C for 5 d, rinse the spores of Penicillium oxalicum with sterile water, and prepare a spore suspension with a concentration of 1×10 6 spores / mL after filtering with gauze. Uniformly inoculate 100 μL of the spore suspension on the wound surface of tubers at 0, 1, 5, and 9 d after callus formation. After air-drying, put them into a perforated polyethylene fresh-keeping bag and culture at room temperature for 7 days, then count the disease index. Disease index = [Σ (number of wounds at each disease level × disease level) / (total number of wounds × highest disease level)] × 100

[0092] (5) Analysis of gene expression levels

[0093] Extract total RNA from yam damaged tissues using an RNA extraction kit. Reverse the RNA into cDNA using a cDNA kit. Analyze the expression levels of suberin synthesis genes DoCYP86A1, DoCYP94B3, and DoGELP38 using the TB Green Premix Ex Taq TMII kit. The real-time fluorescence quantitative PCR system is: 2 μL of cDNA, 2 μL of RNase-free water, 2 μL of forward primer, 2 μL of reverse primer, and 2 μL of SYBR Green PCR Masteer Mix; the PCR program is: one cycle at 95 °C / 30 s, 40 cycles at 95 °C / 5 s, 58 °C / 30 s, 72 °C / 30 s, and one cycle at 72 °C / 5 min. Use the 2 -ΔΔCT method to calculate the relative expression levels of genes.

[0094] (6) Detection of suberin polymerization degree

[0095] Detect the suberin polymerization degree using Fourier transform infrared spectroscopy. Mix 3 mg of the dried sample with 300 mg of KBr and perform FTIR scanning in the range of 500 - 3500 cm -1 , the number of scans is 64 times, and the resolution is 4 cm -1 . Use OMNIC software to analyze the peak areas of ester carbonyl (C=O) and suberin carbon chain methylene (CH2), and calculate the ratio of the peak areas of C=O to CH2 (C=O / CH2) to represent the suberin polymerization degree.

[0096] (7) Observation of suberin accumulation

[0097] Use a blade to cut tissue blocks (2 cm × 2 cm × 2 cm) from damaged tubers. Subsequently, cut thin slices with a thickness of approximately 0.2 - 0.3 mm (including the damaged surface) from the tissue blocks and place them in a toluidine blue solution (0.5%) for staining for 2 min. Then wash with 75% ethanol for 5 min and observe under a laser scanning confocal microscope with an excitation light wavelength of 380 - 405 nm and an emission light wavelength of 430 - 470 nm. Observe the suberin accumulation amount at a magnification of 10×.

[0098] Comparative Example 1: The same as Example 4, the only difference is that the suberin content in the suberin-loaded nanoemulsion treatment group is 0.1 mg / mL (0.1-SUB).

[0099] Comparative Example 2: The same as Example 4, the only difference is that the suberin content in the suberin-loaded nanoemulsion treatment group is 0.5 mg / mL (0.5-SUB).

[0100] Experimental results:

[0101] The effects of nanoemulsions loaded with different contents of suberin on the callus formation effect and suberin accumulation amount of damaged yam tubers were compared. As Figures 9 - 11 shown, the suberin-loaded nanoemulsion can significantly reduce the weight loss rate, ion leakage rate, and disease index of damaged tubers. These three indicators are the smallest in the treatment group of the nanoemulsion containing 1 mg / mL suberin (1-SUB), indicating that the addition of suberin can form a protective barrier on the damaged surface, hindering the loss of water and ions and the infection of Penicillium oxalicum. The suberin-loaded nanoemulsion can significantly induce the expression of suberin synthesis genes (DoCYP86A1 and DoCYP94B3) and the polymerization gene DoGELP38, and ultimately promote the polymerization and accumulation of suberin, and the 1-SUB treatment group shows the best effect ( Figures 12 - 16 ). This is mainly because the monomer components or precursors of suberin (such as ferulic acid and phenylalanine) have the effect of promoting endogenous suberin synthesis. In summary, the suberin loaded in the nanoemulsion can not only act as a protective barrier to isolate the surface of the damaged yam tuber from the outside world, prevent the loss of water and ions and the infection of pathogenic bacteria, but also the suberin monomer can induce the accumulation of endogenous suberin to seal the wound of the yam tuber, achieving a dual protection effect, and ultimately achieving the purpose of callus formation and preservation of the yam tuber.

Claims

1. A method for accelerating the callus of yam tubers, characterized in that, The method includes: 1) Prepare a protein suspension A from soy protein isolate. Continuously introduce ozone into the protein suspension and freeze-dry to obtain soy protein isolate powder. 2) Prepare a suspension B from the soy protein isolate powder obtained in step 1). Add yam polysaccharide to suspension B, heat and react, and then freeze-dry to obtain glycoprotein powder. 3) Prepare a suspension C from the glycoprotein powder obtained in step 2). Add suberin to suspension C, stir, and then process with a high-speed dispersion homogenizer to obtain a nanoemulsion loaded with suberin. 4) Soak the damaged yam tubers with the nanoemulsion loaded with suberin obtained in step 3) and evaluate the callus effect of the tubers.

2. The method for accelerating the callus of yam tubers according to claim 1, wherein In step 1), the concentration of the prepared protein suspension A is 5 mg / mL, the ozone flow rate is 2.8 mg / L, and the ozone treatment time is 0.5 - 1.5 h.

3. The method for accelerating callus of yam tubers according to claim 1, characterized in that, In step 2), the concentration of suspension B is 10 mg / mL.

4. The method for accelerating callus of yam tubers according to claim 1, characterized in that, In step 2), the mass ratio of the soy protein isolate powder to the yam polysaccharide is 2:1 - 1:4.

5.

5. The method for accelerating the callus of yam tubers according to claim 1, characterized in that, In step 2), the reaction temperature is 75 - 95 °C and the reaction time is 0.5 - 2.5 h.

6. The method for accelerating the healing of yam tubers according to claim 1, characterized in that: In step 3), the concentration of the glycoprotein in suspension C is 0.2 - 2 mg / mL. According to the method for accelerating yam tuber callus as claimed in claim 1, wherein the content of suberin added to suspension C in step 3) is 0.1 - 1 mg / mL.

7. The method for accelerating callus of yam tubers according to claim 1, wherein In step 3), the stirring is carried out at 35 °C for 3 h.

8. The method for accelerating callus of yam tubers according to claim 1, characterized in that, In step 3), the shear rate of the high-speed dispersion homogenizer is 5000 - 30,000 r / min and the shear time is 1 - 2 min.

9. The method for accelerating the callus of yam tubers according to claim 1, characterized in that, In step 4), soak the damaged yam tubers with the prepared nanoemulsion loaded with suberin for 5 min and evaluate the callus effect of the nanoemulsion on the yam tubers.

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