Composition for fruit and vegetable preservation, chlamydomonas reinhardtii-based thin film and preparation method and application thereof

Abstract

The invention discloses a composition for fruit and vegetable preservation, a chlamydomonas reinhardtii-based thin film and a preparation method and application thereof. The composition for fruit and vegetable fresh keeping is prepared from chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate. Wherein the mass ratio of the chlamydomonas reinhardtii to the lipoic acid to the carboxymethyl chitosan to the sodium alginate is (0.1-0.3): (0.25-0.5): 15: (10-30). The edible microalgae chlamydomonas reinhardtii is used in the field of fruit preservation, and the prepared chlamydomonas reinhardtii-based film can effectively adjust the gas environment around fruits and vegetables and reduce the respiration rate of the fruits and vegetables, so that the aging and deterioration processes of the fruits and vegetables are delayed, and a good preservation effect is finally achieved.

Description

Technical Field

[0001] The present invention relates to a composition for preserving fruits and vegetables, a Chlamydomonas reinhardtii-based film, and a preparation method and application thereof, and in particular to a composition for preserving fruits and vegetables, a Chlamydomonas reinhardtii-based film, and Chlamydomonas reinhardtii, the composition for preserving fruits and vegetables, applications of the Chlamydomonas reinhardtii-based film in the field of fruit and vegetable preservation, and a fruit and vegetable preservation method, belonging to the technical field of fruit and vegetable storage and preservation. Background Art

[0002] Blueberries have garnered significant attention for their antioxidant content (particularly anthocyanins and phenolics), which are beneficial for human health and cognitive function, and for being a high-quality source of vitamins, minerals, and dietary fiber. However, blueberries have a short shelf life after harvest, lasting only a few weeks, largely due to their inherent characteristics. Their vigorous respiration and fragile structure make them susceptible to mechanical damage during harvesting, transportation, and storage, creating favorable conditions for microbial growth. Mechanical damage not only damages the appearance but also triggers physiological reactions such as increased respiration and ethylene release, leading to fruit softening, a significant loss of nutrients (such as sugars and vitamins), and accelerated aging and deterioration.

[0003] While traditional fungicides can inhibit microbial contamination, they pose a threat to the ecological environment and consumer health. Therefore, the development of new and efficient blueberry preservation methods is urgent. This requires overcoming the preservation challenges caused by mechanical damage and reducing postharvest losses while also reducing reliance on harmful chemicals. This ensures the long-term stability of blueberry quality and nutritional value, meets market demand, and protects consumer health. This has significant practical implications for blueberry market supply and industry optimization, and also provides new directions and insights for innovation in berry preservation technologies.

[0004] With the increasing requirements for food packaging safety and environmental protection, the demand for biomaterials has increased significantly due to their biocompatibility, degradability and non-toxicity. Polysaccharides such as cellulose and chitosan are often used in the production of active edible films. Carboxymethyl chitosan (CMCS), as a chitosan derivative, has good biocompatibility, excellent moisture retention and film-forming properties, can delay water loss and inhibit microbial growth, but its poor water vapor barrier and strong brittleness limit its direct use in food packaging. Currently, lipoic acid (α-lipoic acid, ALA) is mainly used in the field of clinical medicine. Although there have been studies on improving plant tolerance to abiotic stress, there are few reports on the inhibition of post-harvest browning and the improvement of nutritional quality of fruits and vegetables. It is urgent to further explore its potential in the preservation and nutritional enhancement of fruits and vegetables to provide new ideas and methods for the development of the fruit and vegetable industry. Summary of the Invention

[0005] The main purpose of the present invention is to provide a composition for preserving fruits and vegetables, a Chlamydomonas reinhardtii-based film, and a preparation method and application thereof, so as to overcome the deficiencies in the prior art.

[0006] To achieve the aforementioned object of the invention, the technical solutions adopted by the present invention include: An embodiment of the present invention provides a composition for preserving fruits and vegetables, comprising: Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate; wherein the mass ratio of Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate is 0.1-0.3:0.25-0.5:15:10-30.

[0007] An embodiment of the present invention further provides a method for preparing a Chlamydomonas reinhardtii-based film, which comprises: Dissolve carboxymethyl chitosan and sodium alginate in water, then add lipoic acid solution and Chlamydomonas reinhardtii solution and stir to form a film-forming solution; and performing film-forming treatment on the film-forming solution to prepare a Chlamydomonas reinhardtii-based film.

[0008] The embodiment of the present invention also provides a Chlamydomonas reinhardtii-based film prepared by the aforementioned preparation method.

[0009] The embodiments of the present invention also provide uses of Chlamydomonas reinhardtii, the aforementioned composition for preserving fruits and vegetables, or the Chlamydomonas reinhardtii-based film in the field of fruit and vegetable preservation.

[0010] The embodiment of the present invention further provides a method for preserving fruits and vegetables, which comprises: Dissolve carboxymethyl chitosan and sodium alginate in water, then add lipoic acid solution and Chlamydomonas reinhardtii solution and stir to form a film-forming solution; Also, placing fruits or vegetables in the film-forming solution, taking them out, drying them, and then storing them.

[0011] Compared with the prior art, the present invention has the following beneficial effects: (1) In the present invention, by using the edible microalga Chlamydomonas reinhardtii in the field of fruit preservation, the prepared Chlamydomonas reinhardtii-based film can effectively regulate the gas environment around fruits and vegetables, reduce their respiration rate, thereby delaying the aging and deterioration of fruits and vegetables, and ultimately achieving a good preservation effect; (2) This invention not only fills the gap in the application of microalgae in the field of fruit and vegetable preservation, but also may provide a new, green, environmentally friendly and efficient solution to the preservation problems that have long plagued the fruit and vegetable industry. It has important theoretical research value and practical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0013] Figure 1 is an infrared spectrum of carboxymethyl chitosan, sodium alginate and thin film CS in a typical embodiment of the present invention; Figure 2 is an infrared spectrum of lipoic acid, thin film CS and thin film CSA in a typical embodiment of the present invention; Figure 3 In a typical embodiment of the present invention, carboxymethyl chitosan, sodium alginate and film CS are 1 H NMR spectrum; Figure 4 In a typical embodiment of the present invention, lipoic acid, thin film CS and thin film CSA are 1 H NMR spectrum; Figure 5 Graphs showing water vapor transmission rates of films prepared in Example 1 and Comparative Examples 1-3 of the present invention; Figure 6 2. It is a graph showing the E. coli inhibition rate of the films and their components in Example 1 and Comparative Examples 1-3 of the present invention; Figure 7 Graph showing the Staphylococcus aureus inhibition rates of the films and their components prepared in Example 1 and Comparative Examples 1-3 of the present invention; Figure 8 Graph showing the gray mold inhibition rates of the films and their components prepared in Example 1 and Comparative Examples 1-3 of the present invention; Figure 9 This is a graph showing the weight loss rate of the films prepared in Example 1 and Comparative Examples 1-3 of the present invention when used to preserve blueberries; Figure 10 This is a graph of the respiration rate of the films prepared in Example 1 of the present invention and Comparative Examples 1-3 when used to preserve blueberries; Figure 11 This is a graph showing the hardness of the films prepared in Example 1 and Comparative Examples 1-3 of the present invention when used to preserve blueberries; Figure 12 The color difference values of the films prepared in Example 1 and Comparative Examples 1-3 of the present invention when applied to blueberry preservation are shown in FIG. △E picture; Figure 13 This is a graph showing the total soluble solids content of the films prepared in Example 1 and Comparative Examples 1-3 of the present invention when used to preserve blueberries. DETAILED DESCRIPTION

[0014] In view of the shortcomings of the prior art, the inventors of this case, after long-term research and extensive practice, have proposed the technical solution of the present invention. To facilitate understanding of this application, this application will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to facilitate a more thorough and comprehensive understanding of the disclosure of the present invention.

[0015] Specifically, as one aspect of the technical solution of the present invention, a composition for preserving fruits and vegetables comprises: Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate (SA); wherein the mass ratio of Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate is 0.1-0.3:0.25-0.5:15:10-30.

[0016] As another aspect of the technical solution of the present invention, it relates to a method for preparing a Chlamydomonas reinhardtii-based film, which comprises: Dissolve carboxymethyl chitosan and sodium alginate in water, then add lipoic acid solution and Chlamydomonas reinhardtii solution and stir to form a film-forming solution; and performing film-forming treatment on the film-forming solution to prepare a Chlamydomonas reinhardtii-based film.

[0017] In some embodiments, the preparation method specifically comprises: Dissolving carboxymethyl chitosan and sodium alginate in water and stirring at 40-50°C for 5 h to form a carboxymethyl chitosan / sodium alginate solution, then adding lipoic acid solution and stirring at room temperature for 30 min, then adding Chlamydomonas reinhardtii solution and stirring at room temperature (RT) for 30 min to form the film-forming solution; and placing the mold containing the film-forming solution in a calcium chloride solution for film-forming treatment to prepare a Chlamydomonas reinhardtii-based film.

[0018] Furthermore, the lipoic acid solution includes lipoic acid and water, and the concentration of the lipoic acid solution is 0.25-0.5 g / L.

[0019] Furthermore, the concentration of Chlamydomonas reinhardtii in the Chlamydomonas reinhardtii liquid is 0.1-0.3 g / L.

[0020] Furthermore, the mass ratio of Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate in the film-forming solution is 0.1-0.3:0.25-0.5:15:10-30.

[0021] Furthermore, the concentration of the calcium chloride solution is 2.5-5 wt%.

[0022] The carboxymethyl chitosan-sodium alginate-lipoic acid-microalgae hydrogel film of the present invention forms a dense protective layer, which acts like a solid barrier, effectively blocking the penetration of external water vapor. Due to the high water content of blueberry fruit, the intrusion of water vapor easily causes an increase in the surface humidity of the fruit, thereby accelerating the decay of the fruit. At the same time, the film can also significantly delay the oxidative aging process of blueberries. Carboxymethyl chitosan and sodium alginate themselves have good film-forming properties, and the two are interwoven to form a stable membrane structure skeleton. Lipoic acid has a strong antioxidant capacity and is evenly distributed in the membrane structure. When free radicals in the external environment attempt to attack the blueberry fruit, lipoic acid can quickly react with them, capture and neutralize them, thereby reducing the damage of free radicals to blueberry cells, maintaining the normal physiological function of the cells, and thus delaying the oxidative aging of the fruit.

[0023] Microalgae play a key and unique role in slowing blueberry aging. Working synergistically with carboxymethyl chitosan, sodium alginate, and lipoic acid, microalgae possess a unique respiratory metabolic mechanism. In the dark, hydrogel film system, the microalgae compete with the blueberries for oxygen in the surrounding environment. As a fresh fruit, blueberries undergo respiration during storage, and the intensity of this respiration is closely related to the rate of aging. When the microalgae consume some of the oxygen, the oxygen concentration surrounding the blueberries decreases, and their respiration rate also decreases. This lower respiration rate slows the blueberry's metabolism and reduces energy consumption, effectively delaying the fruit's ripening and aging process. Furthermore, the biocompatibility of carboxymethyl chitosan and sodium alginate provides the microalgae with a stable and suitable microenvironment, ensuring their normal physiological activities and enabling them to continue competing with the blueberries for oxygen.

[0024] In terms of antimicrobial activity, carboxymethyl chitosan, lipoic acid, and microalgae exhibit a close synergistic effect. Carboxymethyl chitosan, with its positive charge, interacts with the negatively charged surface of microbial cell membranes, disrupting their structural integrity and causing leakage of cellular contents, thereby inhibiting their growth and reproduction. Lipoic acid, on the other hand, inhibits microorganisms by altering the redox potential within microbial cells and disrupting their normal metabolic pathways. Microalgae secrete bioactive substances, such as antimicrobial peptides and polysaccharides, which enhance the destructive effects of carboxymethyl chitosan and lipoic acid on microbial cell membranes. Furthermore, these substances, along with carboxymethyl chitosan and lipoic acid, act on key microbial metabolic enzymes, further disrupting their normal physiological functions. These three ingredients complement and reinforce each other, significantly enhancing the antimicrobial capacity of the composite film and further reducing microbial contamination of blueberry fruit.

[0025] In summary, the carboxymethyl chitosan-sodium alginate-lipoic acid-microalgae hydrogel film achieves the stability of blueberry quality and nutritional enhancement through the complex and exquisite synergistic effect among its components, providing an efficient and safe solution for blueberry preservation.

[0026] As another aspect of the technical solution of the present invention, it also relates to a Chlamydomonas reinhardtii-based film prepared by the aforementioned preparation method.

[0027] As another aspect of the technical solution of the present invention, it also relates to the use of Chlamydomonas reinhardtii, the aforementioned composition for preserving fruits and vegetables, or the Chlamydomonas reinhardtii-based film in the field of fruit and vegetable preservation.

[0028] As another aspect of the technical solution of the present invention, it also relates to a method for preserving fruits and vegetables, which comprises: Dissolve carboxymethyl chitosan and sodium alginate in water, then add lipoic acid solution and Chlamydomonas reinhardtii solution and stir to form a film-forming solution; Also, placing fruits or vegetables in the film-forming solution, taking them out, drying them, and then storing them.

[0029] This invention innovatively focuses on the edible microalga Chlamydomonas reinhardtii (C. r.), a key player in fruit preservation. C. r. only respires in the dark. By constructing a C. r.-based preservation system, the gaseous environment surrounding fruits and vegetables is effectively regulated, reducing their respiration rate, thereby slowing aging and deterioration, ultimately achieving excellent preservation results. This provides a novel, environmentally friendly solution for the application of microalgae in fruit and vegetable preservation, with significant theoretical and practical value.

[0030] In some embodiments, the method for preserving fruits and vegetables specifically comprises: Dissolving carboxymethyl chitosan and sodium alginate in water and stirring at 40-50°C for 5 h to form a carboxymethyl chitosan / sodium alginate solution, then adding lipoic acid solution and stirring at room temperature for 30 min, then adding Chlamydomonas reinhardtii solution and stirring at room temperature (RT) for 30 min to form the film-forming solution; Also, soak the fruits or vegetables in the film-forming solution for 5 to 7 minutes, take them out, dry them, and then store them.

[0031] Furthermore, the lipoic acid solution includes lipoic acid and water, and the concentration of the lipoic acid solution is 0.25-0.5 g / L.

[0032] Furthermore, the concentration of Chlamydomonas reinhardtii in the Chlamydomonas reinhardtii liquid is 0.1-0.3 g / L.

[0033] Furthermore, the mass ratio of Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate in the film-forming solution is 0.1-0.3:0.25-0.5:15:10-30.

[0034] Furthermore, the storage environment temperature is room temperature (RT) and the humidity is 60-80%.

[0035] The present invention is further illustrated by the following examples. The present invention can be better understood according to the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the present invention and should not, and do not, limit the present invention as described in detail in the claims.

[0036] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment and testing methods used in the following examples are all well known in the art.

[0037] Blueberries (Vaccinium spp.) of uniform size and free of any damage or infection were purchased from a fruit shop in Hefei, China. Carboxymethyl chitosan (CAS: 83512-85-0, Mw = 100,000-200,000, degree of carboxylation ≥ 80%), lipoic acid (CAS: 62-46-4), and sodium alginate (CAS: 9005-38-3, Mw = 200,000, M / G = 0.8) were purchased from MacLean Biochemical Co., Ltd. (Shanghai, China). Anhydrous calcium chloride (CAS: 10043-52-4), sodium hydroxide (CAS: 1310-73-2), and anhydrous ethanol (CAS: 64-17-5) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0038] Example 1 First, a mixture of 15 g / L carboxymethyl chitosan (CMCS) and 20 g / L sodium alginate (SA) was dissolved in deionized water and stirred at 50°C for 5 hours until completely dissolved, forming a CMCS / SA solution. Subsequently, 0.5 g / L lipoic acid (ALA) was added and stirred at room temperature for 30 minutes. A concentrated live Chlamydomonas reinhardtii solution (algae concentration 0.2 g / L) was then added and stirred for 30 minutes until homogeneous, forming a CSA-Cr solution (CSA-Cr solution). The mass ratio of C. reinhardtii, ALA, CMCS, and SA in the solution was 0.2:0.5:15:20). A 10.0 mL aliquot of the CSA-Cr solution was then poured into a polytetrafluoroethylene mold. The mold containing the CSA-Cr solution was then immersed in a 2.5–5% (weight percent) anhydrous calcium chloride solution to form a CSA-Cr membrane.

[0039] Comparative Example 1 First, a mixture of 15 g / L CMCS and 20 g / L SA was dissolved in deionized water and stirred at 50°C for 5 hours until completely dissolved, forming a CMCS / SA solution (CS solution, with a CMCS:SA mass ratio of 15:20). A 10.0 mL aliquot of this solution was then poured into a polytetrafluoroethylene mold. The mold containing the CMCS / SA solution was then immersed in a 2.5–5 wt% anhydrous calcium chloride solution to form a CS membrane (CMCS-SA). CMCS membranes were prepared similarly without the addition of SA.

[0040] Comparative Example 2 First, a mixture of 15 g / L CMCS and 20 g / L SA was dissolved in deionized water and stirred at 50°C for 5 hours until completely dissolved. Subsequently, 0.5 g / L ALA was added and stirred at room temperature for 30 minutes to form a CSA solution (the mass ratio of ALA, CMCS, and SA in the solution was 0.5:15:20). A 10.0 mL aliquot of the CSA solution was then poured into a polytetrafluoroethylene mold. The mold containing the CSA solution was then immersed in a 2.5–5 wt% anhydrous calcium chloride solution to form a CSA membrane (CMCS-SA-ALA).

[0041] Comparative Example 3 First, a mixture of 15 g / L CMCS and 20 g / L SA was dissolved in deionized water and stirred at 50°C for 5 hours until completely dissolved to form a CMCS / SA solution. Subsequently, concentrated live Chlamydomonas reinhardtii solution (algae solution concentration of 0.2 g / L) was added and stirred for 30 min until mixed to form a CS-Cr solution (the mass ratio of Chlamydomonas reinhardtii, CMCS, and SA in the solution was 0.2:15:20). Then, a 10.0 mL aliquot of the CS-Cr solution was poured into a polytetrafluoroethylene mold, and the mold containing the CS-Cr solution was immersed in a 2.5-5% (weight percentage) anhydrous calcium chloride solution to form a CS-Cr membrane.

[0042] Performance Characterization (1) Structural characterization: The CMCS, SA, CMCS-SA (CS), and CMCS-SA-ALA (CSA) films were characterized by Fourier transform infrared spectroscopy, e.g. Figure 1-Figure 2 As shown, sodium alginate has a wavelength of 1600 cm -1 and 1407 cm -1 The peaks at 1024 cm represent the symmetric and asymmetric stretching vibrations of the COO- group. -1 The characteristic C-OH band is represented by carboxymethyl chitosan at 1589 cm -1 and 1427 cm-1 The peaks correspond to the carboxyl anions (COO - ) asymmetric and symmetric stretching vibrations. 1600 cm in SA -1 and 1589cm in CMCS -1 The peak at 1595 cm -1 The reason for the shift is that the carboxyl group COOH is ionized to -COO-. -1 The peak is blue-shifted to 1060 cm -1 , 1024 cm in SA -1 The peak red-shifted to 1060 cm -1 , which indicates that Ca in CMCS-SA 2+ Physically cross-linked by hydrogen bonds, the -NH 3+ The shift of characteristic peaks also indicates that SA and Ca 2+ It may be cross-linked by forming eggshell structure and gel polymerization. The characteristic absorption peak of disulfide bond in infrared spectrum mainly appears at 500 cm -1 to 600 cm -1 At the same time, CMCS, SA, CMCS-SA (CS), CMCS-SA-ALA (CSA) films were characterized by NMR, such as Figure 3-Figure 4 As shown, it can be seen that ALA was successfully added into the CS composite film.

[0043] (2) Water vapor transmission rate: According to GB / T 1037-2021, the water vapor transmission rate (WVTR) was measured. The film was placed on a vent cup filled with 20 mL of distilled water and sealed. The mass was recorded every 2 hours and the WVTR was calculated based on the mass change. Each test was performed in triplicate. Δm (g) is time t The mass change at , A (m²) is the permeable area of the membrane, WVTR ( ) represents the water vapor transmission rate.

[0044] Water vapor transmission rate (WVTR) is the ability of a substance to transport water to the surrounding environment or atmosphere and is a key parameter for films used as packaging materials. Studies have shown that the chemical and structural characteristics of the polymer matrix and the degree of hydrophobic interactions in the film network can greatly affect WVTR. Figure 5 The results showed that compared with the CMCS group, the CS group, CS-Cr group and CSA-Cr group all showed excellent water vapor transmission rate performance, which was significantly higher than that of the CMCS group.

[0045] (3) Antibacterial properties test: The antibacterial assay evaluated representative Gram-positive and Gram-negative bacteria, namely Staphylococcus aureus and Escherichia coli. Bacterial colonies were incubated at 37°C for 14-16 hours and then diluted to 10 7 The concentration of CFU / mL was determined. For the experimental groups, 100 μL of sample suspension (0.5 mg / mL ALA solution, CS solution, CSA solution, CS-Cr solution, and CSA-Cr solution, respectively) and diluted bacterial suspension were added to 5 mL sterile EP tubes. For the control group, the diluted bacterial suspension was added to 100 μL of PBS solution and incubated at 37°C for 3 hours. This resulted in the ALA, CS, CSA, CS-Cr, and CSA-Cr groups, as well as the control group. Subsequently, the bacterial culture was diluted 100-fold. 50 μL of the bacterial culture was spread on an agar plate and incubated overnight at 37°C. Each experiment was performed in triplicate to obtain accurate results. The inhibition rate against Staphylococcus aureus and Escherichia coli was calculated using the following formula.

[0046] in, N 0 represents the initial bacterial colony count, N 1 represents the number of bacterial colonies after adding the material.

[0047] Antifungal experiments tested the inhibitory effect against Botrytis cinerea using the inhibition zone method. Sterile filter paper discs were immersed in ALA, CS, CSA, CS-Cr, or CSA-Cr solutions and allowed to fully absorb. Botrytis cinerea was then inoculated into a Petri dish. The filter paper disc was placed in the center of the dish and gently pressed to ensure proper contact. Sterile water was used as a control, resulting in the control, ALA, CS, CSA, CS-Cr, and CSA-Cr groups. Each experiment consisted of three replicates.

[0048] In in vitro experiments, Figure 6-Figure 8 The results showed that compared with the control group, the CS, ALA, CSA, CS-Cr, and CSA-Cr groups all had a certain antibacterial effect against Staphylococcus aureus, Escherichia coli, and Botrytis cinerea. The CSA and CSA-Cr groups showed significantly higher antibacterial effects than the other three groups. Overall, it was found that CS film and ALA possess certain antibacterial properties on their own, and their combination achieved a synergistic effect, resulting in a highly effective antibacterial effect.

[0049] (3) Freshness preservation performance test Freshly picked blueberries of similar maturity, uniform size, and free of rot and shrunkenness were randomly grouped and immersed in a 200 μL / L sodium hypochlorite solution for 2 minutes, rinsed with purified water, and air-dried at ambient temperature. Five groups of fruit were immersed in ALA, CS, CSA, CS-Cr, and CSA-Cr solutions for 5–7 minutes, respectively. Another group was immersed in deionized water as a control. The fruit was then dried at room temperature (RT) and bagged for storage at a humidity of 60–80% for 6 days.

[0050] ①Weight loss rate test: The weight loss of fruit during storage is determined gravimetrically. The weight loss is calculated as follows: in, w 0 (g) is the initial weight of the fruit, w 1 (g) is the weight of the fruit at different storage days.

[0051] like Figure 9 As shown in the data, under storage conditions of 25 ℃, the weight loss rate of the CSA-Cr group remained at a low level, and the weight loss rate of the CSA-Cr group on the 6th day was 10.9%, which was significantly lower than the 39.8% of naked blueberries. The hydrogel coating with the addition of this concentration of Chlamydomonas reinhardtii achieved the purpose of maintaining the freshness of blueberries.

[0052] ②Respiratory rate test: To measure the respiration rate of blueberries, add 0.4 M NaOH to a Petri dish at the bottom of a glass desiccator, place the septum and blueberries inside, and seal the dish for a while. Then, remove 10.0 mL of NaOH and place it in an Erlenmeyer flask. Add 2.5 mL of BaCl2 and 1 drop of phenolphthalein reagent, and titrate with 0.3 M oxalic acid, recording the amount of oxalic acid consumed. Also, take 10.0 mL of freshly prepared sodium hydroxide as a blank control. The respiration rate of the strawberries is calculated as follows: in, V 1 is the volume of oxalic acid consumed by the control group, V 0 is the volume of oxalic acid consumed by the experimental group, C is the molar concentration of oxalic acid, M is the weight of the fruit, h is the storage time of the fruit, and 44 is the molar mass of CO2.

[0053] In the present invention, Chlamydomonas reinhardtii (Cr) and ALA are added to the film to prevent fungal decay and preserve the freshness of post-harvest fruits. In the early stages of storage, the microalgae compete with blueberries for oxygen, thereby reducing the respiration of the blueberries and playing a certain role in preserving freshness. In the early stages of storage, the system significantly reduces the respiratory metabolic rate through the dynamic oxygen competition mechanism between Chlamydomonas reinhardtii and blueberry fruits. At the same time, ALA slows down the electron transfer of the respiratory chain by regulating the activity of cytochrome oxidase. The dual effects keep the respiration intensity of blueberries at a low level. Therefore, Figure 10 It can be seen that the respiration rate of the CSA-Cr group has been at a lower level, so its preservation effect is better.

[0054] ③Hardness test: Fruit pulp firmness was measured using a TA.XT2i texture analyzer (Stable Micro Systems, Guildford, UK) using a 6 mm diameter probe (P / 6) penetrating 10 mm of pulp at a speed of 5 mm / s. Three biological replicates were performed, each with 20 blueberry samples. Total soluble solids (TSS) in homogenized litchi juice during storage was determined using a digital handheld refractometer (AtagoPAL-1, Japan). Each measurement was performed in triplicate.

[0055] At the experimental temperature, the hardness of the coated blueberries of the present invention is significantly higher than that of the bare blueberries. Figure 11 As shown, this indicates increased firmness. Combined with the above results, the high firmness of the coated blueberries is attributed to the high water retention rate in the plant cells, which leads to high cell elasticity. Furthermore, firmness is an indicator of blueberry ripening, further confirming that the CSA-Cr group can effectively delay blueberry ripening.

[0056] ④Color difference test: Determination of blueberry 、 and , color difference ΔE ΔE is calculated by the following formula: ; Where: is the chromaticity value of blueberry during measurement; 、 、 is the chromaticity value of the initial blueberry.

[0057] The color of fruits and vegetables reflects the maturity, freshness and nutritional value of the fruit trees to a certain extent. Figure 12 The results showed that the total color difference of the coating group during the entire storage period was △E All of them were lower than the control group, among which the CSA-Cr group was the most significant, maintaining the freshness of the blueberry fruit.

[0058] ⑤Total soluble solids test: During storage, the total soluble solids of the homogenized blueberry juice were determined by a digital handheld refractometer (AtagoPAL-1, Japan) in triplicate.

[0059] Studies have shown that a reduction in soluble sugars in blueberry fruit can lead to a shortened shelf life. Figure 13 As shown, the total soluble solids content of blueberries initially increased and then decreased during storage. This phenomenon may be related to the breakdown of carbohydrates, which leads to the production of total soluble solids, and the consumption of soluble solids due to respiration. Throughout the storage period, the total soluble solids content of the coating groups was lower than that of the control group, with the CSA-Cr group showing the most significant decrease, maintaining the freshness of the blueberries.

[0060] Example 2 A mixture of 15 g / L CMCS and 10 g / L SA was dissolved in deionized water and stirred at 40°C for 5 hours until completely dissolved, forming a CMCS / SA solution. Subsequently, 0.25 g / L ALA was added and stirred at room temperature for 30 minutes. A concentrated live Chlamydomonas reinhardtii solution (algae concentration 0.1 g / L) was then added and stirred for 30 minutes until thoroughly mixed, forming a CSA-Cr solution (CSA-Cr solution with a mass ratio of C. reinhardtii, ALA, CMCS, and SA of 0.1:0.25:15:10). A 10.0 mL aliquot of the CSA-Cr solution was then poured into a polytetrafluoroethylene mold. The mold containing the CSA-Cr solution was then immersed in a 2.5–5% (weight percent) anhydrous calcium chloride solution to form a CSA-Cr membrane.

[0061] Example 3 A mixture of 15 g / L CMCS and 30 g / L SA was dissolved in deionized water and stirred at 45°C for 5 hours until completely dissolved, forming a CMCS / SA solution. Subsequently, 0.3 g / L ALA was added and stirred at room temperature for 30 minutes. A concentrated live Chlamydomonas reinhardtii solution (algae concentration 0.3 g / L) was then added and stirred for 30 minutes until thoroughly mixed, forming a CSA-Cr solution (CSA-Cr solution with a mass ratio of C. reinhardtii, ALA, CMCS, and SA of 0.3:0.5:15:30). A 10.0 mL aliquot of the CSA-Cr solution was then poured into a polytetrafluoroethylene mold. The mold containing the CSA-Cr solution was then immersed in a 2.5–5% (weight percent) anhydrous calcium chloride solution to form a CSA-Cr membrane.

[0062] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments using other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0063] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A composition for preserving fruits and vegetables, characterized in that: include: Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate; wherein the mass ratio of Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate is 0.1-0.3:0.25-0.5:15:10-30.

2. A method for preparing a Chlamydomonas reinhardtii-based film, characterized in that: include: Dissolve carboxymethyl chitosan and sodium alginate in water, then add lipoic acid solution and Chlamydomonas reinhardtii solution and stir to form a film-forming solution; and performing film-forming treatment on the film-forming solution to prepare a Chlamydomonas reinhardtii-based film.

3. The preparation method according to claim 2, characterized in that Specifically include: Dissolving carboxymethyl chitosan and sodium alginate in water and stirring at 40-50°C for 5 h to form a carboxymethyl chitosan / sodium alginate solution, then adding lipoic acid solution and stirring at room temperature for 30 min, then adding Chlamydomonas reinhardtii solution and stirring at room temperature for 30 min to form the film-forming solution; and placing the mold containing the film-forming solution in a calcium chloride solution for film-forming treatment to prepare a Chlamydomonas reinhardtii-based film.

4. The preparation method according to claim 3, wherein: The lipoic acid solution comprises lipoic acid and water, and the concentration of the lipoic acid solution is 0.25-0.5 g / L; and / or, the concentration of Chlamydomonas reinhardtii in the Chlamydomonas reinhardtii solution is 0.1-0.3 g / L; and / or, the mass ratio of Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate in the film-forming solution is 0.1-0.3:0.25-0.5:15:10-30; and / or, the concentration of the calcium chloride solution is 2.5-5 wt%.

5. A Chlamydomonas reinhardtii-based film prepared by the preparation method according to any one of claims 2 to 4.

6. Use of Chlamydomonas reinhardtii, the composition for preserving fruits and vegetables according to claim 1, or the Chlamydomonas reinhardtii-based film according to claim 5 in the field of fruit and vegetable preservation.

7. A method for preserving fruits and vegetables, characterized in that: include: Dissolve carboxymethyl chitosan and sodium alginate in water, then add lipoic acid solution and Chlamydomonas reinhardtii solution and stir to form a film-forming solution; Also, placing fruits or vegetables in the film-forming solution, taking them out, drying them, and then storing them.

8. The method for preserving fruits and vegetables according to claim 7, characterized in that: Specifically include: Dissolving carboxymethyl chitosan and sodium alginate in water and stirring at 40-50°C for 5 h to form a carboxymethyl chitosan / sodium alginate solution, then adding lipoic acid solution and stirring at room temperature for 30 min, then adding Chlamydomonas reinhardtii solution and stirring at room temperature for 30 min to form the film-forming solution; Also, soak the fruits or vegetables in the film-forming solution for 5 to 7 minutes, take them out, dry them, and then store them.

9. The method for preserving fruits and vegetables according to claim 7, characterized in that: The lipoic acid solution comprises lipoic acid and water, and the concentration of the lipoic acid solution is 0.25-0.5 g / L; and / or the concentration of Chlamydomonas reinhardtii in the Chlamydomonas reinhardtii solution is 0.1-0.3 g / L; and / or the mass ratio of Chlamydomonas reinhardtii, lipoic acid, carboxymethyl chitosan and sodium alginate in the film-forming solution is 0.1-0.3:0.25-0.5:15:10-30.

10. The method for preserving fruits and vegetables according to claim 7, characterized in that: The storage environment temperature is room temperature and the humidity is 60-80%.

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