Use of Candida in improving the effectiveness of antifreeze proteins

Abstract

The present invention relates to the field of plant growth regulators and provides the use of Candida to enhance the effects of antifreeze proteins. The antifreeze proteins have the effect of enhancing the cold tolerance of crops. The Candida is Candida XHZG06-95A3, which is taxonomically categorized as Candida palmioleophila and has the accession number CGMCC No. 29215. The amino acid sequence of the antifreeze protein is set forth in SEQ ID NO: 1. In the present invention, the use of Candida XHZG06-95A3 and / or its fermentation supernatant can enhance the effects of the antifreeze proteins, thereby further improving the cold tolerance of crops.

Description

[Technical Field]

[0001] The present invention relates to the field of plant growth regulators, and in particular to the use of Candida in enhancing the effectiveness of antifreeze proteins. [Background technology]

[0002] Low temperature is one of the most significant factors that adversely affect plant growth, development, and harvest quality. When plants are exposed to low temperature stress, they undergo physiological changes such as damage to cell membrane structure, decreased antioxidant enzyme activity, and impaired photosynthesis. Macroscopically, these changes manifest as delayed germination and growth, wilting, yellowing of leaves, tissue softening, reduced fruit set, seedling weakness, and even seedling death.

[0003] Crop antifreeze agents are a type of exogenous plant growth regulator that stabilizes cell membranes and induces a series of physiological and biochemical reactions in crops, thereby enhancing their cold tolerance. They are typically applied by soaking seeds or spraying them on plants. Currently reported crop antifreeze agents include salicylic acid, abscisic acid, fulvic acid, ascorbic acid, potassium dihydrogen phosphate, paclobutrazol, chitosan, and locust bean gum. However, the effectiveness of a single antifreeze agent in improving crop cold tolerance is limited. Therefore, the current mainstream of research and development in crop antifreeze agents is to design an antifreeze formulation scheme based on the crop's cold tolerance and the mechanism of action of the antifreeze agent.

[0004] Patent Document 1 discloses an antifreeze protein and its manufacturing method and application. This patent claims that an antifreeze protein was discovered in the BSI20414 strain of Marinomonas arctica. This antifreeze protein protects cells in low-temperature environments by inhibiting ice crystal growth and recrystallization, and can be used as a protective agent during cryopreservation of cells. However, when the antifreeze protein in this patent is used as an antifreeze agent for crops, although it can improve the cold tolerance of crops to some extent, the effect is not good. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Chinese Patent Application Publication No. 112876545 Summary of the Invention [Problem to be solved by the invention]

[0006] To solve the technical problem that antifreeze proteins used as crop antifreeze agents are not effective in improving the cold resistance of crops, the present invention provides the use of Candida to improve the effect of antifreeze proteins. In the present invention, the use of Candida XHZG06-95A3 and / or its fermentation supernatant can enhance the effect of antifreeze proteins and further improve the cold resistance of crops. [Means for solving the problem]

[0007] The technical solutions of the present invention are as follows:

[0008] In the use of Candida XHZG06-95A3 and / or its fermentation supernatant to improve the effect of an antifreeze protein, the effect of the antifreeze protein is to enhance the cold resistance of crops by the antifreeze protein, and the Candida is Candida XHZG06-95A3, which is taxonomically categorized as Candida palmioleophila and has the accession number CGMCC No. 29215. The amino acid sequence of the antifreeze protein is set forth in SEQ ID NO: 1.

[0009] The present invention uses the antifreeze protein shown in the amino acid sequence SEQ ID NO: 1 to inhibit the growth of small ice crystals when they form in crops, and at the same time, to make the ice crystals formed in the crop spherical, thereby preventing them from perforating the cells. By using the above method, the antifreeze protein can improve the cold tolerance of crops.

[0010] Based on this, the present invention proposes that by combining Candida XHZG06-95A3 and / or its fermentation supernatant with a specific antifreeze protein, a synergistic effect can be achieved, and Candida XHZG06-95A3 and / or its fermentation supernatant can enhance the effect of the antifreeze protein in improving the cold tolerance of crops. Furthermore, this synergistic effect is strain-specific, and not all Candida strains can achieve the effect of improving the cold tolerance of crops in combination with an antifreeze protein as in the present invention. The mechanism of this synergistic effect is speculated to be as follows: Candida XHZG06-95A3 can secrete a specific glycoprotein substance that can form a stable complex with an antifreeze protein with a specific structure. Furthermore, this strain may produce inhibitory metabolites (such as specific lipid substances) that weaken the active structure of the antifreeze protein.

[0011] The deposit information for Candida XHZG06-95A3 is as follows: Depositary: Center of Ordinary Microorganisms, China Committee for the Preservation and Management of Microorganisms Address of depository institution: No. 3, Courtyard, No. 1, Beichen West Road, Chaoyang District, Beijing Deposit number: CGMCC No.29215 Deposit date: December 4, 2023 Classification: Candida palmioleophila - This is a species of fungus in the genus Candida. Currently, there is no unified Chinese translation for this species.

[0012] Previous research by the present inventors has reported that Candida XHZG06-95A3 has the ability to improve acidic soil, but the mechanism by which it improves acidic soil is different from the mechanism by which it improves the cold tolerance of crops. Previous research has not reported that this strain has the ability to improve plant cold tolerance and enhance the effectiveness of antifreeze proteins. Other existing technologies have not reported the existence of Candida strains that can enhance the effectiveness of antifreeze proteins and improve the cold tolerance of crops.

[0013] Preferably, the Candida XHZG06-95A3 and / or its fermentation supernatant and the antifreeze protein are prepared into an agent for improving environmental stress resistance.

[0014] Preferably, the agent for improving environmental stress resistance further comprises one or more of glycerol, trehalose, and a phosphate buffer solution.

[0015] Preferably, the mass ratio of the fermentation supernatant to the antifreeze protein is 1 to 2:1, the fermentation supernatant is produced by fermenting Candida XHZG06-95A3 in a yeast extract peptone glucose liquid medium supplemented with glycerol, and the polysaccharide content is 0.10 to 0.15 g / mL.

[0016] Furthermore, the amount of glycerol added to the yeast extract peptone glucose liquid medium is 4 to 7 wt %.

[0017] Preferably, the method for preparing the agent for improving environmental stress tolerance includes a step of mixing Candida XHZG06-95A3 and / or a fermentation broth supernatant thereof with an antifreeze protein to prepare the agent for improving environmental stress tolerance.

[0018] Preferably, the method for preparing the agent for improving environmental stress tolerance comprises the step of: 600 The method includes a step of obtaining an environmental stress tolerance improver by mixing a Candida XHZG06-95A3 bacterial suspension having a mass / volume ratio of 0.5 to 1.5, an antifreeze protein, and a dispersion medium, wherein the volume ratio of the Candida XHZG06-95A3 bacterial suspension to the environmental stress tolerance improver is 1:1.5 to 2.5, and the mass / volume ratio of the antifreeze protein to the Candida XHZG06-95A3 bacterial suspension is 0.08 to 0.15 g:50 mL.

[0019] Preferably, the method for preparing the environmental stress resistance improver includes the steps of mixing the fermentation supernatant of Candida XHZG06-95A3 with an antifreeze protein, adding a dispersant until the solid content reaches 10 to 15 wt %, adding an emulsifier to homogenize and emulsify the mixture to obtain the environmental stress resistance improver.

[0020] Furthermore, the dispersion medium is a phosphate (PBS) buffer solution.

[0021] Preferably, the method for preparing the fermentation supernatant comprises inoculating Candida XHZG06-95A3 into a yeast extract peptone glucose liquid medium supplemented with glycerol, and then culturing the Candida XHZG06-95A3 at an OD 600 The method includes a step of fermenting and culturing the culture medium until the pH reaches 1.8 to 2.2, recovering the supernatant by centrifugation, and concentrating the supernatant until the polysaccharide content reaches 0.10 to 0.15 g / mL.

[0022] Preferably, improving the cold tolerance of the crop comprises reducing intracellular electrolyte exudation, lowering the degree of cell membrane lipid peroxidation, and increasing superoxide dismutase activity under low temperature stress, thereby improving the survival rate of the crop, and the crop is tea or wheat. [Effects of the Invention]

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention combines Candida XHZG06-95A3 and / or its fermentation supernatant with a specific antifreeze protein, which acts synergistically to significantly improve the cold tolerance of crops. However, existing technology has not yet found a Candida strain that enhances the effect of the antifreeze protein and improves the cold tolerance of crops. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will be further described below in conjunction with examples.

[0026] The use of Candida XHZG06-95A3 and / or its fermentation supernatant in improving the effect of an antifreeze protein is characterized in that the effect of the antifreeze protein is to increase the cold resistance of crops through the antifreeze protein, and the Candida is Candida XHZG06-95A3, which is taxonomically categorized as Candida palmioleophila and has the deposit number CGMCC No. 29215, and the amino acid sequence of the antifreeze protein is set forth in SEQ ID NO: 1.

[0027] In some specific embodiments, the Candida XHZG06-95A3 and / or its fermentation supernatant and the antifreeze protein are prepared into an agent for improving environmental stress resistance.

[0028] In some specific embodiments, the agent for improving environmental stress resistance further comprises one or more of glycerol, trehalose, and phosphate buffer.

[0029] In some specific embodiments, the mass ratio of the fermentation broth supernatant to the antifreeze protein is 1-2:1.

[0030] In some specific embodiments, the fermentation supernatant is produced by fermenting Candida XHZG06-95A3 in a yeast extract peptone glucose liquid medium supplemented with glycerol, and has a polysaccharide content of 0.10 to 0.15 g / mL, wherein optionally or preferably, the amount of glycerol added to the yeast extract peptone glucose liquid medium is 4 to 7 wt%.

[0031] In some specific embodiments, the method for preparing the environmental stress resistance improver includes mixing Candida XHZG06-95A3 and / or a fermentation broth supernatant thereof with an antifreeze protein to prepare the environmental stress resistance improver.

[0032] In some specific embodiments, the method for preparing the agent for improving environmental stress tolerance comprises: 600The method includes a step of obtaining an environmental stress tolerance improver by mixing a Candida XHZG06-95A3 bacterial suspension having a pH of 0.5 to 1.5, an antifreeze protein, and a dispersion medium, wherein the volume ratio of the Candida XHZG06-95A3 bacterial suspension to the environmental stress tolerance improver is 1:1.5 to 2.5, and the mass-volume ratio of the antifreeze protein to the Candida XHZG06-95A3 bacterial suspension is 0.08 to 0.15 g:50 mL. Optionally or preferably, the dispersion medium is a phosphate buffer solution.

[0033] In some other specific embodiments, the method for preparing the agent for improving environmental stress tolerance includes the steps of mixing the fermentation broth supernatant of Candida XHZG06-95A3 with an antifreeze protein, adding a dispersant until the solid content reaches 10 to 15 wt %, and adding an emulsifier to homogenize and emulsify the mixture to obtain the agent for improving environmental stress tolerance, where the dispersant is optionally or preferably a phosphate buffer solution.

[0034] In some specific embodiments, the method for preparing the fermentation supernatant comprises inoculating Candida XHZG06-95A3 into a yeast extract peptone glucose liquid medium supplemented with glycerol, and 600 The method includes fermenting and culturing the yeast extract peptone glucose liquid medium until the pH reaches 1.8 to 2.2, recovering the supernatant by centrifugation, and concentrating the supernatant until the polysaccharide content reaches 0.10 to 0.15 g / mL. Optionally or preferably, the amount of glycerol added to the yeast extract peptone glucose liquid medium is 4 to 7 wt %.

[0035] In some specific embodiments, improving the cold tolerance of the crop comprises reducing intracellular electrolyte exudation, decreasing the degree of cell membrane lipid peroxidation, and increasing superoxide dismutase activity under low temperature stress, thereby improving crop survival rate, hi some specific embodiments, the crop is tea or wheat.

[0036] The present invention will be described below through specific examples. These examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. It should be understood that all modifications and advantages that a person skilled in the art may conceive without departing from the spirit and scope of the present invention are included in the present invention, and the scope of protection of the present invention is within the scope of the appended claims and their equivalents.

[0037] In the following Examples and Comparative Examples, unless otherwise specified, the Candida XHZG06-95A strain used in the present invention is CGMCC No. 29215, and the antifreeze protein used in the present invention is a protein having the amino acid sequence shown in SEQ ID NO: 1. The specific sequence is as follows: DSATHHDYNYDQWHQFGSGNDQIVIDNDVEKWLDAGDGDNSIYVGDDVNRNNSAGIKTGSGDDDIFVKDNVDSTIQTGGGNDRVQIGYDLGNGYHSAINLGDGDNNLIIKNDVNNYSTV HSGSGDDVVSIGDDDVRYDADIQLGDGNDRLTIGDKIEKEVSINLGSGNDVLVVGGKVSDEAWVDGGSGSDSVWFESYSRSDYNSDKDGIKSRFANFENFKFSDGTVIGNASAFDGSSVSG.

[0038] Example 1: Effect of an environmental stress tolerance enhancer containing Candida XHZG06-95A on improving cold tolerance in crops

[0039] 1.1 Preparation of environmental stress tolerance enhancer 1.1.1 Raw material ingredients (1) Candida XHZG06-95A3 bacterial solution: OD 600 =1.0, usage amount 50mL. (2) Antifreeze protein dispersion: concentration 0.5 mg / mL, amount used 20 mL. (3) Supplementary materials: 5% v / v glycerol, 0.05 g / mL trehalose, phosphate (PBS) buffer solution with a pH of 7.0. 1.1.2 Preparation process of environmental stress tolerance improver (1) Cultivation of strains: Candida XHZG06-95A3 was inoculated into YPD liquid medium (yeast extract 1%, peptone 2%, glucose 2%) and cultured at 25°C and 180 rpm for 48 hours with shaking. After that, the medium was centrifuged at 5000 × g for 10 minutes to collect the bacterial cells. 600 The bacterial cells were resuspended in PBS buffer so that the pH became 1.0, to obtain a bacterial suspension. (2) Mixing preparation: Bacterial suspension (50mL, OD 600 Mix the antifreeze protein dispersion (20 mL, 0.5 mg / mL), glycerol (5 mL), and trehalose (5 g) in a PBS buffer (pH 7.0) to a final volume of 100 mL. Stir at 200 rpm at 4°C for 30 minutes to obtain an environmental stress resistance enhancer, which is then aseptically packaged.

[0040] 1.2 Measurement of the effect of improving crop cold tolerance 1.2.1 Experimental design Three experimental groups and one control group were set up, and the treatment solutions used in each group were as follows: (1) Experimental group 1: The environmental stress tolerance improver prepared in this example. (2) Experimental Group 2: Antifreeze protein dilution. The antifreeze protein dispersion (20 mL, 0.5 mg / mL) was prepared as follows: The antifreeze protein dispersion (20 mL, 0.5 mg / mL) was diluted to 100 mL with PBS buffer (pH = 7.0) and stirred at 200 rpm at 4 °C for 30 minutes. (3) Experimental Group 3: Candida XHZG06-95A3 solution. The preparation method was as follows: Candida XHZG06-95A3 was inoculated into YPD liquid medium (yeast extract 1%, peptone 2%, glucose 2%) and cultured at 25°C and 180 rpm for 48 hours with shaking. After that, the medium was centrifuged at 5000 × g for 10 minutes to collect the bacterial cells. The OD 600 The bacterial cells were resuspended in PBS buffer until OD = 1.0 to obtain a bacterial suspension. 600 = 1.0) was adjusted to 100 mL with PBS buffer (pH = 7.0) and stirred at 4°C at 200 rpm for 30 minutes. (4) Control group: supplementary material solution. The preparation method was as follows: Glycerol (5 mL) and trehalose (5 g) were mixed, and the resulting solution was adjusted to 100 mL with PBS buffer (pH = 7.0), and the mixture was stirred at 200 rpm at 4 °C for 30 minutes. The treatment conditions were as follows: The tea leaves were sprayed with the treatment solution of each group until they were completely wet, and then subjected to low-temperature stress at -20°C for 24 hours. After returning to room temperature (25°C), physiological indices such as relative conductivity, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and plant survival rate were measured. 1.2.2 Experimental results The measurement results of physiological indices for each group are shown in Table 1. In Table 1, "black triangles" indicate significant differences (p<0.05) between Group 1 and Groups 2 and 3. The data are the mean ± standard deviation of three repeated experiments. [Table 1] As can be seen from the experimental results in Table 1, (1) Compared with the control group, the crops in experimental groups 2 and 3 had lower relative conductivity and MDA content, and higher SOD activity and plant survival rate after low temperature stress, indicating that both the antifreeze protein of the present invention and Candida XHZG06-95A3 can improve the cold tolerance of crops to some extent. (2) Compared with Experimental Groups 2 and 3, the crops in Experimental Group 1 had lower relative conductivity and MDA content, and higher SOD activity and plant survival rate after low-temperature stress. This indicates that the antifreeze protein of the present invention and Candida XHZG06-95A3 exert a synergistic effect, and that combining the two can further improve the cold tolerance of crops.

[0041] Example 2: Effect of an environmental stress tolerance enhancer containing Candida XHZG06-95A fermentation supernatant on improving cold tolerance in crops

[0042] 2.1 Preparation of environmental stress tolerance enhancer Candida XHZG06-95A3 was inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol, and incubated at 30°C and 180 rpm until OD 600 The fermentation culture was performed until the pH reached 2.0, then centrifuged at 8000 × g for 15 minutes. The supernatant was collected and concentrated until the polysaccharide content reached 0.1 g / mL, yielding a fermentation supernatant. The fermentation supernatant and antifreeze protein were mixed at specific mass ratios (the mass ratios used for groups A to E were 3:1, 2:1, 1:1, 1:2, and 1:3, respectively) and diluted with PBS buffer to prepare a mixed solution with a solids concentration of 15 wt%. 0.1 wt% Tween-80 was added to the mixture, which was then homogenized and emulsified at 40 °C and 5000 rpm for 20 minutes to obtain the environmental stress tolerance improvers for groups A to E. Candida XHZG06-95A3 was inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol, and incubated at 30°C and 180 rpm until OD 600 The resulting culture was fermented until the pH reached 2.0, then centrifuged at 8000 x g for 15 minutes. The supernatant was collected and concentrated to a polysaccharide content of 0.1 g / mL to obtain a fermentation supernatant. The fermentation supernatant was diluted with PBS buffer to a solids concentration of 15 wt%, and 0.1 wt% Tween-80 was added. The mixture was homogenized at 40°C and 5000 rpm for 20 minutes to obtain the F group environmental stress tolerance enhancer. The antifreeze protein was mixed with PBS buffer to obtain an antifreeze protein dispersion with a solids concentration of 15 wt%, to which 0.1 wt% Tween-80 was added, and the mixture was homogenized at 40°C and 5000 rpm for 20 minutes to emulsify, thereby obtaining a group G environmental stress resistance improver.

[0043] 2.2 In vitro antifreeze activity measurement Each group of environmental stress resistance improvers was diluted to a solids concentration of 1 wt% and stored in a freezer at -20°C. The freezing point of the solution was measured using differential scanning calorimetry (DSC) and the freezing point depression value (ΔTf) was calculated. The ΔTf values ​​for each group are shown in Table 2. The data in Table 2 are the mean ± standard deviation of three replicate experiments. [Table 2]

[0044] 2.3 Measurement of the effect of improving crop cold tolerance: Winter wheat seedlings (at the three-leaf stage) were sprayed with 5 mL of each environmental stress tolerance enhancer per plant, while the control group was sprayed with 5 mL of PBS buffer per plant. After 24 hours of cold stress at -8°C, the plants were allowed to grow again for 7 days, and physiological indicators such as electrolyte permeability, MDA content, SOD activity, and plant survival rate were measured. The results are shown in Table 3. The data in Table 3 are the mean ± standard deviation of three replicate experiments. [Table 3]

[0045] 2.4 Analysis of experimental results As can be seen from the experimental results in Tables 2 and 3, (1) Compared with groups F and G, the freezing points of the solutions in groups A to E were significantly higher. After the crops were subjected to low-temperature stress, the electrolyte permeability and MDA content decreased, while the SOD activity and plant survival rate were higher. This indicates that in this invention, by adding antifreeze protein to the fermentation supernatant of Candida XHZG06-95A3, the two can synergistically enhance the cold tolerance of crops. (2) Compared with groups A and D-F, the freezing points of the solutions in groups B and C were significantly higher. After the crops were subjected to cold stress, electrolyte permeability and MDA content decreased, while SOD activity and plant survival rates were higher. This indicates that the ratio of Candida albicans fermentation supernatant to antifreeze protein affected the synergistic effect of the two. When the mass ratio of the two was 1-2:1, the crop's cold tolerance was improved, with the optimal mass ratio being 2:1.

[0046] Example 3: Storage stability of an environmental stress resistance improving agent containing Candida XHZG06-95A fermentation supernatant

[0047] 2.1 Preparation of environmental stress tolerance enhancer Candida XHZG06-95A3 was inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol, and incubated at 30°C and 180 rpm until OD 600The mixture was fermented until the pH reached 2.0, then centrifuged at 8000 x g for 15 minutes. The supernatant was collected and concentrated until the polysaccharide content reached 0.1 g / mL, yielding a fermentation supernatant. The fermentation supernatant and antifreeze protein were mixed at a mass ratio of 2:1 and diluted with PBS buffer to prepare a mixed solution with a solids concentration of 15 wt%. 0.1 wt% Tween-80 was added to the mixture, which was then homogenized and emulsified at 40°C and 5000 rpm for 20 minutes to obtain an environmental stress tolerance improver.

[0048] 2.2 Measurement of the effect of improving crop cold tolerance The environmental stress tolerance enhancers were divided into two groups, Group B1 and Group B2. The effects of Group B1 on improving crop cold tolerance were immediately measured, while Group B2 was stored at 4°C for 30 days before being measured for its effects on improving crop cold tolerance. The measurement method was as follows: 5 mL of each environmental stress tolerance enhancer per plant was sprayed onto the leaves of winter wheat seedlings (at the three-leaf stage). 5 mL of PBS buffer per plant was sprayed on the control plant. After 24 hours of cold stress at -8°C, the plants were allowed to grow for 7 days, and physiological indicators such as electrolyte permeability, MDA content, SOD activity, and plant survival rate were measured. The results are shown in Table 4. The data in Table 4 are the mean ± standard deviation of three replicate experiments. [Table 4] As can be seen from the experimental results in Table 4, after being subjected to low temperature stress, the electrolyte permeability, MDA content, SOD activity, and plant survival rate of group B2 were close to those of group B1. This indicates that the environmental stress tolerance improver of the present invention has good storage stability and can maintain its ability to improve the cold tolerance of crops even after long-term storage.

[0049] Example 4: Effect of Candida and antifreeze protein selection on environmental stress tolerance enhancers

[0050] 4.1 Preparation of environmental stress tolerance enhancer 4.1.1 Experimental materials In this example, Candida utilis ACCC 20060 was used as a control strain. This strain was purchased from the China Agricultural Microorganism Species Preservation and Management Center (abbreviated as "ACCC 20060"). 4.1.2 Preparation process of environmental stress tolerance improver Candida XHZG06-95A3 was inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol, and incubated at 30°C and 180 rpm until OD 600 The mixture was fermented until the pH reached 2.0, then centrifuged at 8000 x g for 15 minutes. The supernatant was collected and concentrated until the polysaccharide content reached 0.1 g / mL, yielding a fermentation supernatant. The fermentation supernatant and antifreeze protein were mixed at a mass ratio of 2:1 and diluted with PBS buffer to prepare a mixed solution with a solids concentration of 15 wt%. 0.1 wt% Tween-80 was added to the mixture, which was then homogenized and emulsified at 40°C and 5000 rpm for 20 minutes to obtain the environmental stress tolerance improver of Group B. Candida XHZG06-95A3 was inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol, and incubated at 30°C and 180 rpm until OD 600 The resulting mixture was fermented until the pH reached 2.0, then centrifuged at 8000 x g for 15 minutes. The supernatant was collected and concentrated to a polysaccharide content of 0.1 g / mL to obtain a fermentation supernatant. The fermentation supernatant was diluted with PBS buffer to a solids concentration of 15 wt%, and 0.1 wt% Tween-80 was added. The mixture was homogenized at 40°C and 5000 rpm for 20 minutes to obtain the F group environmental stress tolerance enhancer. The antifreeze protein was mixed with PBS buffer to obtain an antifreeze protein dispersion with a solids concentration of 15 wt%, to which 0.1 wt% Tween-80 was added, and the mixture was homogenized and emulsified at 40°C and a rotation speed of 5000 rpm for 20 minutes to obtain a group G environmental stress resistance improver. Candida utilis ACCC 20060 was inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol, and incubated at 30°C and 180 rpm until OD 600The mixture was fermented until the pH reached 2.0, then centrifuged at 8000 x g for 15 minutes. The supernatant was collected and concentrated until the polysaccharide content reached 0.1 g / mL, yielding a fermentation supernatant. The fermentation supernatant and antifreeze protein were mixed at a mass ratio of 2:1 and diluted with PBS buffer to prepare a mixed solution with a solids concentration of 15 wt%. 0.1 wt% Tween-80 was added to the mixture, which was then homogenized and emulsified at 40°C and 5000 rpm for 20 minutes to obtain the environmental stress tolerance improver for Group H. Candida utilis ACCC 20060 was inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol, and the OD was measured at 30°C and 180 rpm. 600 The resulting mixture was fermented until the pH reached 2.0, then centrifuged at 8000 x g for 15 minutes. The supernatant was collected and concentrated to a polysaccharide content of 0.1 g / mL to obtain a fermentation supernatant. The fermentation supernatant was diluted with PBS buffer to a solids concentration of 15 wt%, and 0.1 wt% Tween-80 was added. The mixture was homogenized at 40°C and 5000 rpm for 20 minutes to obtain Group I environmental stress tolerance enhancers.

[0051] 4.2 Measurement of the effect of improving crop cold tolerance Winter wheat seedlings (at the three-leaf stage) were sprayed with 5 mL of each environmental stress tolerance enhancer per plant, while the control group was sprayed with 5 mL of PBS buffer per plant. After 24 hours of cold stress at -8°C, the plants were allowed to grow again for 7 days, and physiological indicators such as electrolyte permeability, MDA content, SOD activity, and plant survival rate were measured. The results are shown in Table 5. The data in Table 5 are the mean ± standard deviation of three replicate experiments. [Table 5] As can be seen from the experimental results in Table 5, after the crops in Group I were subjected to low-temperature stress, the electrolyte permeability, MDA content, SOD activity, and plant survival rate were similar to those of Group F. However, the electrolyte permeability and MDA content of Group H were significantly higher than those of Group B, and the SOD activity and plant survival rate were significantly lower than those of Group B. This indicates that the synergistic effect between Candida and antifreeze proteins is strain-specific, and the selection of the Candida strain influences the expression of this synergistic effect. However, not all Candida strains can achieve the synergistic effect of improving the cold tolerance of crops in the present invention when combined with antifreeze proteins. Based on this, it is speculated that the synergistic effect between Candida XHZG06-95A3 and antifreeze proteins in the present invention is due to the fact that this strain can secrete specific glycoprotein substances that can form stable complexes with antifreeze proteins with specific structures, and that this strain may also produce inhibitory metabolites (e.g., specific lipid substances) that weaken the active structure of the antifreeze proteins.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional in the art and available from ordinary commercial sources. The methods used in the present invention are conventional in the art unless otherwise specified.

[0053] The above description is only a preferred embodiment of the present invention, and does not limit the present invention. Any simple modification, change, or equivalent variation of the above embodiment based on the technical essence of the present invention remains within the protection scope of the technical solution of the present invention.

[0054] (Addendum) (Appendix 1) Use of Candida XHZG06-95A3 and / or a fermentation broth supernatant thereof in improving the effect of an antifreeze protein, wherein the effect of the antifreeze protein is to improve the cold resistance of crops by the antifreeze protein, and the Candida XHZG06-95A3 belongs to the taxonomic genus Candida palmioleophila and has the deposit number CGMCC No. 29215, and the amino acid sequence of the antifreeze protein is set forth in SEQ ID NO: 1.

[0055] (Appendix 2) Use of the Candida and / or fermentation supernatant thereof according to Appendix 1, characterized in that the Candida XHZG06-95A3 and / or fermentation supernatant thereof and the antifreeze protein are prepared into an environmental stress resistance improver.

[0056] (Appendix 3) The use of Candida and / or its fermentation supernatant according to Appendix 2, characterized in that the agent for improving environmental stress resistance further comprises one or more of glycerol, trehalose, and phosphate buffer.

[0057] (Appendix 4) Use of Candida and / or its fermentation supernatant according to Appendix 2, characterized in that the mass ratio of the fermentation supernatant to antifreeze proteins is 1 to 2:1, the fermentation supernatant is produced by fermenting Candida XHZG06-95A3 in a yeast extract peptone glucose liquid medium supplemented with glycerol, and the polysaccharide content is 0.10 to 0.15 g / mL.

[0058] (Appendix 5) The use of Candida and / or its fermentation supernatant described in Appendix 2, characterized in that the method for preparing the environmental stress resistance improver includes a step of mixing the Candida XHZG06-95A3 and / or its fermentation supernatant with an antifreeze protein to prepare an environmental stress resistance improver.

[0059] (Appendix 6) Appendix 5. Use of Candida and / or a fermentation broth supernatant thereof according to Appendix 5, wherein the method for preparing the environmental stress tolerance improver comprises a step of mixing a Candida XHZG06-95A3 bacterial suspension having an OD600 of 0.5 to 1.5, an antifreeze protein, and a dispersing medium to obtain the environmental stress tolerance improver, wherein the volume ratio of the Candida XHZG06-95A3 bacterial suspension to the environmental stress tolerance improver is 1:1.5 to 2.5, and the mass-volume ratio of the antifreeze protein to the Candida XHZG06-95A3 bacterial suspension is 0.08 to 0.15 g:50 mL.

[0060] (Appendix 7) The use of Candida and / or its fermentation supernatant according to Appendix 5, characterized in that the method for preparing the agent for improving environmental stress resistance includes the steps of mixing the fermentation supernatant of Candida XHZG06-95A3 with an antifreeze protein, adding a dispersant until the solid content reaches 10 to 15 wt%, adding an emulsifier to homogenize and emulsify the mixture to obtain the agent for improving environmental stress resistance.

[0061] (Appendix 8) The use of Candida and / or its fermentation supernatant according to Appendix 5 or 7, characterized in that the method for preparing the fermentation supernatant comprises the steps of inoculating Candida XHZG06-95A3 into a yeast extract peptone glucose liquid medium supplemented with glycerol, culturing the strain by fermentation until the OD600 reaches 1.8 to 2.2, collecting the supernatant by centrifugation, and concentrating it until the polysaccharide content reaches 0.10 to 0.15 g / mL.

[0062] (Appendix 9) The use of Candida and / or its fermentation supernatant described in Appendix 1, wherein improving the cold tolerance of the crop comprises reducing intracellular electrolyte exudation, reducing the degree of cell membrane lipid peroxidation, increasing superoxide dismutase activity, and improving the crop survival rate under low temperature stress, wherein the crop is tea or wheat.

Claims

1. Use of Candida XHZG06-95A3 and / or a fermentation broth supernatant thereof in improving the effect of an antifreeze protein, wherein the effect of the antifreeze protein is to improve the cold tolerance of crops by the antifreeze protein, and the Candida XHZG06-95A3 belongs to the taxonomic genus Candida palmioleophila and has the deposit number CGMCC No. 29215, and the amino acid sequence of the antifreeze protein is set forth in SEQ ID NO:

1.

2. The use of Candida and / or its fermentation supernatant according to claim 1, characterized in that the Candida XHZG06-95A3 and / or its fermentation supernatant and the antifreeze protein are prepared into an environmental stress resistance improver.

3. The use of Candida and / or its fermentation supernatant according to claim 2, wherein the agent for improving environmental stress resistance further comprises one or more of glycerol, trehalose, and phosphate buffer.

4. The use of Candida and / or its fermentation supernatant according to claim 2, characterized in that the mass ratio of the fermentation supernatant to antifreeze proteins is 1 to 2:1, the fermentation supernatant is produced by fermenting Candida XHZG06-95A3 in a yeast extract peptone glucose liquid medium supplemented with glycerol, and the polysaccharide content is 0.10 to 0.15 g / mL.

5. The use of Candida and / or a fermentation broth supernatant thereof according to claim 2, characterized in that the method for preparing the environmental stress resistance improver comprises a step of mixing the Candida XHZG06-95A3 and / or a fermentation broth supernatant thereof with an antifreeze protein to prepare the environmental stress resistance improver.

6. The method for preparing the environmental stress resistance improver comprises: ６００ 6. The use of Candida and / or a fermentation broth supernatant thereof according to claim 5, comprising a step of obtaining an environmental stress resistance improver by mixing a Candida XHZG06-95A3 bacterial suspension having a molecular weight of 0.5 to 1.5, an antifreeze protein, and a dispersion medium, wherein the volume ratio of the Candida XHZG06-95A3 bacterial suspension to the environmental stress resistance improver is 1:1.5 to 2.5, and the mass-volume ratio of the antifreeze protein to the Candida XHZG06-95A3 bacterial suspension is 0.08 to 0.15 g:50 mL.

7. The use of Candida and / or its fermentation broth supernatant according to claim 5, characterized in that the method for preparing the agent for improving environmental stress resistance comprises the steps of mixing the fermentation broth supernatant of Candida XHZG06-95A3 with an antifreeze protein, adding a dispersant until the solid content reaches 10 to 15 wt %, and adding an emulsifier to homogenize and emulsify the mixture to obtain the agent for improving environmental stress resistance.

8. The method for preparing the fermentation supernatant was as follows: Candida XHZG06-95A3 was inoculated into a yeast extract peptone glucose liquid medium containing glycerol, and the OD ６００ Use of Candida and / or its fermentation supernatant according to claim 5 or 7, characterized in that it comprises the steps of fermenting and culturing the Candida and / or its fermentation supernatant until the polysaccharide content reaches 1.8 to 2.2, collecting the supernatant by centrifugation, and concentrating it until the polysaccharide content reaches 0.10 to 0.15 g / mL.

9. The use of Candida and / or its fermentation supernatant according to claim 1, wherein improving the cold tolerance of the crop comprises reducing intracellular electrolyte exudation, reducing the degree of cell membrane lipid peroxidation, increasing superoxide dismutase activity, and improving the survival rate of the crop under low temperature stress, wherein the crop is tea or wheat.

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