AU2020101185A4

AU2020101185A4 - Use of Fibroin and Method for Storing Fruits and Vegetables

Info

Publication number: AU2020101185A4
Application number: AU2020101185A
Authority: AU
Inventors: Junjia Chen; Liang GONG; Yueming Jiang; Lei Liang; Juan Liu
Original assignee: Guangdong Institute of Bioengineering Guangzhou Cane Sugar Industry Research Institute
Current assignee: Guangdong Institute of Bioengineering Guangzhou Cane Sugar Industry Research Institute
Priority date: 2019-07-17
Filing date: 2020-06-29
Publication date: 2020-07-30
Anticipated expiration: 2028-06-29
Also published as: CN110353038A

Abstract

The present invention discloses use of fibroin and a method for storing fruits and vegetables, and particularly, discloses use of fibroin in the preparation of an anti-chilling injury agent for fruits and vegetables. The method for storing fruits and vegetables includes the following step: coating an anti-chilling injury agent including fibroin for fruits and vegetables on the surface of fruits and vegetables. The anti-chilling injury agent for fruits and vegetables of the present invention has excellent application effects in the storage of fruits and vegetables such as bananas. The fibroin used in the present invention can form an edible protein film on the surface of fruits and vegetables, and abundant hydrogen bonds, disulfide bonds and hydrophobic interaction forces in fibroin can maintain the stable structure of the film; therefore, the fibroin can serve as an excellent mechanical barrier. The fibroin can significantly inhibit the reduction of cell membrane lipids of the peel tissue, thereby alleviating chilling injuries. Since the fibroin is naturally toxic-free and easily soluble in water and requires no treatment by other chemical additives, no harmful chemicals will remain on the surface of fruits and vegetables after the anti-chilling injury agent for fruits and vegetables of the present invention is used, making fruits and vegetables safer and healthier. Moreover, the fibroin forms an edible film that is completely soluble in water and does not pollute water. 2/5 ail) 0~ 00 U( 4cl CN _____O___ W00 co C XOUU 91[I)t~

Description

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USE OF FIBROIN AND METHOD FOR STORING FRUITS AND VEGETABLES

TECHNICAL FIELD The present invention relates to the technical field of fruit storage, and in particular, to use of fibroin and a method for storing fruits and vegetables. BACKGROUND Fruits and vegetables are natural nutritious foods that include various essential nutrients for human, such as vitamin, mineral, carbohydrate, crude fiber, protein and fat. Fruits and vegetables not only are delicious, but also can promote physical health, thereby preventing and treating diseases and maintaining beauty and youth. Fruits and vegetables are the most popular natural health foods for people nowadays. Harvested fruits and vegetables, while having left the plant body, are still living organisms. Therefore, there will be continuous physiological, physical and chemical changes in harvested fruits and vegetables. Therefore, how to achieve a longer shelf life for fruits and vegetables by appropriately storing fruits and vegetables has always been a major problem in the agricultural field. The cold preservation technique, one of the most widely used preservation techniques at present, inhibits the respiratory metabolism of fruits and vegetables, postpones the release of endogenous ethylene and delays the aging of fruits and vegetables by reducing the temperature, thereby extending the shelf life and maintaining more stable nutrients for fruits and vegetables. Moreover, the growth of microorganisms can also be effectively inhibited to avoid deterioration of fruits and vegetables caused by microorganism infection. However, for some fruits and vegetables originating in tropics or subtropics, at a temperature below the critical temperature for chilling injury, normal metabolic activities cannot be performed in tissues, thereby resulting in a reduced resistance and various physiological and biochemical disorders, and eventually leading to various chilling injuries. The phenomenon where fruits and vegetables exhibit physiological and metabolic incompatibility at a low temperature above 0°C, which is called chilling injury. After subjected to chilling injuries, the fruits and vegetables will have black, brown and dried tissues and pitted plaques on the surface, and become stinky, and some fruits with thinner and softer skins are prone to have water spots. Therefore, controlling chilling injuries is of great significance for the storage of fruits and vegetables. Guangdong of China is rich in famous Lingnan fruits that grow well in a warm and wet climate, and harvested Lingnan fruits tend to undergo quality deterioration, such as aging and disease. Banana is a major economic fruit worldwide, ranking first in the fresh fruit trade. Moreover, banana is a main fruit in South China that is transported to North China and exported. However, Lingnan fruits, such as banana, are sensitive to low temperatures and tend to undergo chilling injuries during low-temperature storage. Chilling injuries to banana are as follows: the upper epidermis turns brown or brownish black; the lower epidermis turns brown; and brown or black stripes can be seen on a longitudinal section. In severe cases, the fruit flesh also turns brown or black, and even loses its original flavor (Jiang Y M., Joyce D. C., Jiang W., Lu W. Effects of chilling temperatures on ethylene binding by banana fruit. Plant Growth Regulation, 2004, 43(2): 109-115). Edible coating is an effective method widely used in the field of fruit and vegetable preservation. The edible film wrapped around the surface of fruits and vegetables can not only prevent gas exchange, control the respiration rate of fruits and vegetables and reduce nutrient loss and water evaporation, but also prevent the invasion of microorganisms. In the prior art, common coating films have components, such as polysaccharide, peptide, resin, lipid and complexes thereof. Although the coating components in the prior art can play a certain role in keeping fresh, these coating components exhibit a poor effect in the anti-chilling injury technologies for fruits and vegetables sensitive to cold. SUMMARY A first technical problem to be solved in the present invention is to provide an anti-chilling injury agent for fruits and vegetables that can alleviate chilling injuries of harvested fruits and vegetables during low-temperature storage. A second technical problem to be solved in the present invention is to provide a storage method that can alleviate chilling injuries of harvested fruits and vegetables during low-temperature storage. To solve the first technical problem, the technical solution adopted in the present invention is as follows: use of fibroin in the preparation of an anti-chilling injury agent for fruits and vegetables. Preferably, the fibroin has a molecular weight of 200 Da to 4,000 Da. The present invention has the following beneficial effects: Fibroin in the anti-chilling injury agent of the present invention can efficiently reduce chilling injuries of harvested fruits and vegetables such as bananas. Fibroin that can prevent gas exchange and has excellent film-forming properties is adopted to alleviate chilling injuries of fruits and vegetables during low-temperature storage. To solve the second technical problem, the technical solution adopted in the present invention is as follows: a method for storing fruits and vegetables, including the following step: coating an anti-chilling injury agent including fibroin for fruits and vegetables on the surface of fruits and vegetables. Further, the coating operation is to coat the anti-chilling injury agent including fibroin for fruits and vegetables on the surface of fruits and vegetables by soaking, cold compress or spraying. Further, the method for storing fruits and vegetables includes the following steps: soaking harvested fruits and vegetables in a solution including fibroin, and then drying, and storing at a low temperature. Preferably, the drying operation is air-drying. Preferably, the solution including fibroin has a fibroin concentration of 0.5 g/L to 5 g/L. Further, the soaking operation is conducted for1 min to 10 min, and preferably for 5 min.

Further, the low temperature refers to 0°C to 10°C. Further, the fruits and vegetables include cold-sensitive fruits. Further, the cold-sensitive fruits include at least one of bananas and strawberries. The present invention has the following beneficial effects: The fibroin used in the present invention can form an edible protein film on the surface of fruits and vegetables, and abundant hydrogen bonds, disulfide bonds and hydrophobic interaction forces in fibroin can maintain the stable structure of the film, therefore, the fibroin can serve as an excellent mechanical barrier. Fibroin, also known as silk fibroin, is a natural high-molecular protein extracted from silk, accounting for about 70% of silk. Fibroin is a polymer composed of 18 amino acids with hydrophilic groups and hydrophobic groups. The hydrophobic chain fragment mainly includes 70% of the highly-recurrent sequence of Glly-X, composed of Gly-Ala-Gly-Ala-Gly-Ser. Fibroin is non-toxic, odorless, non-irritating and soluble in water, and has excellent mechanical, physical and chemical properties, such as prominent toughness and tensile strength, air and moisture permeability, and slow-release property. Therefore, using fibroin as an anti-chilling injury agent can achieve excellent anti-chilling injury effects. After the anti-chilling injury agent for fruits and vegetables in the present invention is used without other chemical additives, no harmful chemicals will remain on the surface of fruits and vegetables, making fruits and vegetables safer and healthier. Moreover, the fibroin forms an edible film that is completely soluble in water and does not pollute water. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a comparison diagram for bananas that are untreated and treated with fibroin in an example of the present invention; FIG. 2 is a peel browning index diagram for bananas that are untreated and treated with fibroin in an example of the present invention; FIG. 3 is an active oxygen change diagram for peels of bananas that are untreated and treated with fibroin in an example of the present invention during storage at 6°C; FIG. 4 is a saturated fatty acid change diagram for peels of bananas that are untreated and treated with fibroin in an example of the present invention during storage at 6°C; and FIG. 5 shows the relative contents of lipids in peels of bananas that are untreated and treated with fibroin in an example of the present invention during storage at 6°C, exhibiting significant difference. DETAILED DESCRIPTION In order to describe the technical contents and the objectives and effects achieved in the present invention in detail, the present invention is described below with reference to implementations and accompanying drawings. The example of the present invention is as follows: an anti-chilling injury agent for fruits and vegetables, where the anti-chilling injury agent for fruits and vegetables is fibroin. The anti-chilling injury agent for fruits and vegetables was used in the preservative storage of bananas for mitigating chilling injuries. The specific operations were as follows: 1. Materials Bananas: harvested green mature bananas Fibroin: provided by Jiangsu Fushengde Bioengineering Co., Ltd. Kits for detecting superoxide anion generation rate and hydroxyl radical content: provided by Nanjing Jiancheng Co., Ltd. 2. Instruments Ultra-low temperature freezer: 6°C, relative humidity: 85% to 90%, Thermo Scientific; Gas chromatograph-mass spectrometer (Thermo, DSQII); UHPLC Nexera LC-30A ultra-performance liquid chromatography system; Q Exactive plus mass spectrometer (Thermo ScientificTM). 3. Treating methods Bananas: Bananas having maturity of about 80% were harvested for researches on post-harvest chilling injuries. Fruits, having the same size, no obvious injuries, and no damages from pests and diseases, were selected and divided into two groups for treatment: (1) Fibroin treatment: 1.0 g L-1 fibroin was applied to the fruit. (2) Control treatment: water was applied to the fruit. Fruits of above two groups were soaked for 5 min separately, dried at room temperature, packed in 0.03 mm polyethylene bags, and then stored at 6°C. Samples were collected at Storage I (day 0), Storage II (day 2) and Storage III (day 4), and the obtained peel samples were treated with liquid nitrogen and then stored at -80°C in a refrigerator for use. 4. Effect of fibroin on chilling injuries of bananas (1) Determination of browning index: 50 bananas in each group were used for peel browning index determination, and the browning area on each fruit peel surface was estimated according to the following browning grade standard: 0: no browning; 1: 0% to 25% browning area; 2: 25% to % browning area; 3: 50% to 75% browning area; and 4: 75% to 100% browning area. The banana peel browning index was calculated according to the following formula: browning index= Y(browning grade x the proportion of fruits at each browning grade in total fruits). (2) Determination of active oxygen 1) Determination of H 2 0 2 content: 5 g of banana sample peel was taken and first ground with liquid nitrogen. Then 25 mL of cold acetone was added for grinding and extraction. The extract was centrifuged at 6,000 g for 15 min, and 1 mL of the supernatant was taken and added with 0.1 mL of % TiCl4 and 0.2 mL of 2 mol/L concentrated ammonia water for reaction. The reaction solution was centrifuged at 6,000 g for 15 min, and the precipitate was collected. The precipitate was dissolved in 3 mL of 1 mol/L H2 SO4 , and the resulting solution was centrifuged at 5,000 g for 20 min. The absorbance value was determined at 410 nm for the supernatant. The H 2 0 2 content in the peel tissue was calculated by H 2 0 2 standard curve. The result for hydrogen peroxide content was expressed in nmol-kg-1. 2) Superoxide anion generation rate and hydroxyl radical content were determined using kits (Nanjing Jiancheng Co., Ltd, Nanjing, China), and the measuring wavelengths are 530 nm and 550 nm respectively. The result for superoxide anion generation rate was expressed in nmol.kg--s-, and the hydroxyl radical content was expressed in U-mL-1. The activity unit (U), defined as 1 mmol-L-1 increase of H 2 0 2 concentration in the reaction solution per mg of tissue, was a unit indicating the hydroxyl radical producing ability. (3) Determination of saturated fatty acid 1) Extraction of samples: 200 mg of banana peel sample was suspended in 1,800 PL of methanol pre-cooled at -20°C, and the resulting suspension was thoroughly shaken and added with 200 pL of ribitol internal standard (0.2 mg mL-1 in water). The resulting mixture was thoroughly shaken and subjected to supersonic treatment at 4°C for 15 min, placed in 70°C water bath for 15 min, and then placed at -20°C to condense the solvent. The reaction solution was centrifuged at 4°C and 5,000 g for 15 min, and 100 pL of the supernatant was pipetted to a 1.5 mL centrifuge tube, then concentrated to dryness under vacuum at 30°C, and stored in an ultra-low temperature freezer at -80°C for use. 50 pL of methoxyamine hydrochloride (2 mg mL-1 in pyridine) was added to the sample concentrated to dryness under vacuum, and the resulting mixture was incubated in a vacuum oven at 50°C for 30 min. 50 pL of BSTFA+1%TMCS was added, and the resulting mixture was incubated in a vacuum oven at 60°C for 40 min. The sample was filtered using a 0.22 Pm microporous membrane and then placed at room temperature for loading. 2) Analysis by gas chromatograph-mass spectrometer: The gas chromatograph-mass spectrometer (Thermo, DSQ II) adopted the following sample loading method and specific parameters. i) Capillary column: DB-5MS (5% phenyl/methyl polysiloxane, 30 m x 0.25 mm x 0.25 m); ii) Ion source: 70 eV; iii) Scanning range: 45 m/z to 600 m/z; iv) Inlet temperature: 230°C; v) Transmission line temperature: 250°C; vi) Carrier gas: high-pure helium (99.999%); vii) Carrier gas flow rate: 1.2 mL min-'; viii) Temperature programming: starting at 100°C, keeping at this temperature for 1 min; increasing to 184°C at 3°C.min-1; increasing to 190°C at 0.5°C.min-1, and keeping at this temperature for 1 min; and then increasing to 280°C at 15°C-min- , and keeping at this temperature for 5 min; ix) Split ratio: 10:1; and x) Manual loading: with an injection volume of 1 pL. The mass spectrum obtained in the experiment was compared with the standard spectra in the NIST2008 and Wiley databases to obtain a preliminary qualitative analysis report for polar metabolites in banana peel. Column bleed and components having a relative peak area < 2% were eliminated in the report. Components with RSI and SI both less than 700 were eliminated. Components with a repeat number < 3 were eliminated. Components with a matching degree less than 70 in the report were manually analyzed once again. (4) Lipid extraction and analysis About 80 mg of the frozen banana peel sample was accurately weighed (5 replicates for each group) and added with 240 p L of pre-cooled methanol, and the resulting solution was vortexed. 800 pL of methyl tert-butyl ether (MTBE) was added, and the resulting solution was vortexed. 200 pL of water was added, and the resulting solution was vortexed. The resulting mixture was subjected to supersonic treatment in a low-temperature water bath for 20 min, placed at room temperature for 30 min, and then centrifuged at 10°C and 8,000 g for 15 min. The upper organic phase was dried under nitrogen, and added with 200 L of isopropanol solution for mass spectrometry analysis. The resulting solution was vortexed and centrifuged at 8,000 g and 10°C for 15 min, and the supernatant was loaded for analysis. LC-MS analysis: The sample was separated by UHPLC Nexera LC-30A ultra-performance liquid chromatography system. Column temperature: 45°C; flow rate: 300 L.min-'; and injection volume: 4 L. Mobile phase composition: A: a solution of 10 mM ammonium formate in acetonitrile and water (acetonitrile: water =6:4, v/v); B: a solution of 10 mM ammonium formate in acetonitrile and isopropanol (acetonitrile :isopropanol = 1:9, v/v). The gradient elution procedure was as follows: 0 min to 7 min: B was maintained at 30%; 7.1 min to 25 min: B changed linearly from 30% to 100%; 25.1 min to 30 min, B was maintained at 30%. The sample was placed in the °C autosampler throughout the analysis. In order to avoid influence caused by the fluctuation of instrument detection signals, the samples were continuously analyzed in a random sequence. One quality control (QC) sample was set up every 8 experimental samples in a sample queue to monitor and evaluate the stability of the system and the reliability of the experimental data. Electrospray ionization (ESI) positive and negative ion modes were used for detection. The samples were separated by UHPLC and then analyzed by Q Exactive plus mass spectrometer (Thermo ScientificTM). ESI source parameters were as follows: Positive ion mode: heater temperature: 300°C, sheath gas flow rate: 45 arb, auxiliary gas flow rate: 15 arb, sweep gas flow rate: 1 arb, spray voltage: 3.0 KV, capillary temperature: 350°C, S-lens RF level: 50%, MS1 scan ranges: 200-1,800. Negative ion mode: heater temperature: 300°C, sheath gas flow rate: 45 arb, auxiliary gas flow rate: 15 arb, sweep gas flow rate: 1 arb, spray voltage: 2.5 KV, capillary temperature: 350°C, S-lens RF level: 60%, MS1 scan ranges: 250-1,800. The mass-to-charge ratios for lipid molecules and lipid fragments were collected according to the following method: 10 fragment spectra (MS2 scan, HCD) were collected after each full scan. MS1 had a resolution of 70,000 at M/Z 200, and MS2 had a resolution of 17,500 at M/Z 200. 5. Experimental results and analysis 1) FIG. 1 shows physical pictures of bananas at different storage time, and bananas in the picture are always green. As shown in FIG. 1, compared with the control group, the peels of bananas treated with fibroin exhibit significantly alleviated browning, indicating that fibroin can remarkably reduce chilling injuries of bananas during low-temperature storage. 2) FIG. 2 shows determination results for browning index. As shown in FIG. 2, compared with the control group, the peels of bananas treated with fibroin exhibit significantly reduced browning indexes, indicating that fibroin can remarkably alleviate browning of bananas during low-temperaturestorage. 3) FIG. 3 shows determination results for active oxygen. In FIG. 3, A shows the relationship between the superoxide anion generation rate and the storage time, B shows the relationship between the hydrogen peroxide content and the storage time, and C shows the relationship between the hydroxyl radical content and the storage time. As shown in FIG. 3, compared with the control group, the peels of bananas treated with fibroin have a reduced active oxygen level, indicating that fibroin can alleviate oxidative damages of bananas during low-temperature storage, thereby reducing chilling injuries. 4) FIG. 4 shows determination results for saturated fatty acid. In FIG. 4, A shows the relationship between palmitic acid and storage time, and B shows the relationship between stearic acid and storage time. As shown in FIG. 4, on day 4 of storage, compared with the control group, the peels of bananas treated with fibroin exhibit a significantly-reduced saturated fatty acid content, indicating that fibroin can remarkably alleviate oxidation of membrane lipid in peels of bananas during low-temperature storage, thereby reducing chilling injuries. 5) FIG. 5 shows results for lipid extraction and analysis. In FIG. 5, A shows the relationship between the content of phosphatidic acid (PA), phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) and the storage time; B shows the relationship between the content of phospholipids glycerin (PG), phosphatidylserine (PS) and triglyceride (TG) and the storage time; C shows the relationship between the content of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) and the storage time; and D shows the relationship between the content of monogalactosyl diacylglycerol (MGDG), monogalactosyl monoacylglycerol (MGMG) and sulfoquinovosyl diacylglycerol (SQDG) and the storage time. As shown in FIG. 5, compared with the control group, the peels of bananas treated with fibroin exhibit significantly-increased content of PC, PE, PS, MGDG, MGMG and SQDG, substantially-unchanged content of PG, and significantly-reduced content of PA, LPC, LPE and TG, indicating that fibroin can significantly inhibit the reduction of cell membrane lipid in peel tissues, thereby reducing chilling injuries. It can be seen from above results that the fibroin provided in the present invention, if used for reducing chilling injuries of bananas during storage according to the molecular weight, concentration and soaking time for fibroin proposed in the present invention, can alleviate the browning of peels of bananas during low-temperature storage, thereby reducing chilling injuries of bananas. Therefore, using fibroin to alleviate chilling injuries of bananas is of great significance for studying on the occurrence mechanism of chilling injuries of harvested bananas during low-temperature storage and the development of preservation technologies for bananas. Similarly, fibroin, when used in the storage of other fruits and vegetables (such as strawberries), can also achieve excellent anti-chilling injury effects. In the preparation process of the anti-chilling injury agent for fruits and vegetables in the present invention, some other agents that enhance other properties or preservation properties of fruits and vegetables can also be added as needed. The foregoing is merely examples of the present invention and does not constitute a limitation on the scope of the present invention. Any equivalent change made by using the description and the accompanying drawings of the present invention, or direct or indirect application thereof in related technical fields, shall still fall in the protection scope of the patent of the present invention.

Claims

What is claimed is: 1. A method for storing fruits and vegetables, comprising the following step: coating an anti-chilling injury agent comprising fibroin for fruits and vegetables on the surface of fruits and vegetables.
2. The method for storing fruits and vegetables according to claim 1, wherein the coating operation comprises coating the anti-chilling injury agent comprising fibroin for fruits and vegetables on the surface of fruits and vegetables by soaking, cold compress or spraying.
3. The method for storing fruits and vegetables according to claim 2, wherein the method for storing fruits and vegetables comprises the following steps: soaking harvested fruits and vegetables in a solution comprising fibroin, and then drying, and storing at a low temperature.
4. The method for storing fruits and vegetables according to claim 3, wherein the solution comprising fibroin has a fibroin concentration of 0.5 g/L to 5 g/L; wherein the soaking operation is conducted for1 min to 10 min; wherein the low temperature refers to 0°C to 10°C.
5. The method for storing fruits and vegetables according to claim 1, wherein the fruits and vegetables comprise cold-sensitive fruits.