Method for preparing antifreeze polysaccharide based on low-temperature induction of highland barley grains

Abstract

The invention provides a method for preparing antifreeze polysaccharide based on low-temperature induction of highland barley grains. The method for preparing the antifreeze polysaccharide based on low-temperature induction of the highland barley grains comprises the following steps: S1, putting the highland barley grains in a low-temperature environment for low-temperature induction; s2, carrying out a low-temperature induction process by adopting a dynamic temperature gradient adjusting method; and S3, collecting the highland barley grains, and extracting the antifreeze polysaccharide by using a water extraction method assisted by an ultrasonic technology. According to the method for preparing the antifreeze polysaccharide based on low-temperature induction of the highland barley grains, it is ensured that the extracted antifreeze polysaccharide has high purity, and the complexity and operation difficulty of the purification process are reduced. In addition, by accurately regulating and controlling the temperature and humidity conditions in the low-temperature induction process, the whole preparation process is simpler, more convenient and more efficient, and the production cost is further reduced.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction. Background Art

[0002] With climate change and increasing environmental pressure, low temperature damage has had a serious impact on the production of crops, especially crops in high-altitude cold regions such as barley. In recent years, researchers have discovered that under low temperature stress, plants will synthesize a special sugar substance - antifreeze polysaccharides. These polysaccharides can effectively enhance the antifreeze ability of plant cells and protect plants from frost damage. Antifreeze polysaccharides play a vital role in the process of plants resisting low temperature stress. Therefore, studying how to induce plants to synthesize antifreeze polysaccharides has become a hot topic in agricultural biotechnology research. Existing research on antifreeze polysaccharides mainly focuses on the low temperature stress response and antifreeze mechanism of different plant varieties.

[0003] However, current technologies still have several shortcomings, particularly the low efficiency of synthesizing antifreeze polysaccharides from highland barley grains under low-temperature induction conditions. For example, after a 24-hour treatment at -5°C, existing methods yield only 2.5 ± 0.3 mg / g dry weight of antifreeze polysaccharides, with a batch-to-batch coefficient of variation as high as 12%, failing to meet the stability requirements for industrial production. Furthermore, the traditional preparation process relies on a phenol-sulfuric acid method for crude separation, followed by three ultrafiltration steps, two ethanol precipitations, and finally a 7–10-day dialysis purification step, ultimately achieving a purity of only 65 ± 8%. The total process takes over 144 hours, which is time-consuming and energy-intensive. Furthermore, ethanol consumption is approximately 500 L per batch, and solvent recovery is only 60%. This results in purification costs as high as 1200 yuan / g, and polysaccharide yields of less than 0.8%. Efficient synthesis and precise control of antifreeze polysaccharides from highland barley grains, particularly optimization of the synthesis pathway under gradient induction conditions from -10°C to 0°C, remain a pressing technical challenge in this field. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a method for preparing antifreeze polysaccharides from highland barley grains based on low-temperature induction, which solves the problem of low efficiency of antifreeze polysaccharide synthesis under low-temperature induction; the existing methods for preparing antifreeze polysaccharides have the problems of high cost and complicated operation.

[0005] To achieve the above objectives, the present invention is implemented by the following technical solutions: a method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction, comprising: S1. placing highland barley grains in a low temperature environment for low temperature induction, wherein the low temperature induction conditions include temperature, induction time and humidity;

[0006] S2. A dynamic temperature gradient adjustment method is used for low-temperature induction process, wherein the dynamic temperature gradient adjustment method gradually reduces the induction temperature and adjusts the time interval so that the barley grains gradually adapt to low temperature stress to promote the synthesis of antifreeze polysaccharides;

[0007] S3. Collect highland barley grains and extract antifreeze polysaccharides using water extraction supplemented by ultrasonic technology;

[0008] S4. Purify the extracted antifreeze polysaccharide using gel filtration chromatography to ensure that the purity of the antifreeze polysaccharide is suitable for application.

[0009] Preferably, the temperature range of the low-temperature induction condition is -2°C to 4°C, the induction time is 16 hours to 48 hours, and the humidity is controlled between 60% and 75%.

[0010] Preferably, the dynamic temperature gradient adjustment method in S2 comprises the following steps:

[0011] S2.1 Set the initial temperature to 4°C and gradually reduce it to 3°C over 4 hours;

[0012] S2.2 Reduce the temperature by 0.5°C to 1°C every 4 hours until the induction temperature drops to -2°C;

[0013] S2.3 The low temperature induction process lasts for 24 hours, and the temperature is gradually adjusted to ensure that the barley grains gradually adapt to the low temperature stress.

[0014] Preferably, the temperature gradient change in the low-temperature induction process follows the following model formula:

[0015]

[0016] Where T(t) is the temperature at time t, T0 is the initial temperature, ΔT is the temperature change amplitude, T t is the total induction time, and t is the current time.

[0017] Preferably, the humidity change model formula of the low temperature induction process is:

[0018]

[0019] Where H(t) is the humidity at time t, H0 is the initial humidity, ΔH is the humidity change amplitude, T t is the total induction time, t is the current time, and the humidity is maintained between 60% and 75% throughout the induction process.

[0020] Preferably, the solvent in the water extraction method is deionized water, and the frequency range of the ultrasonic technology is 30kHz to 60kHz, and the power is 100W to 200W, so as to ensure efficient extraction of antifreeze polysaccharides.

[0021] Preferably, the chromatographic column used in the gel filtration chromatography is Sephadex G-25, PBS buffer is used in the separation process, and the purification process lasts for 2 to 4 hours.

[0022] The present invention provides a method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction. The method has the following beneficial effects:

[0023] This method for preparing antifreeze polysaccharides from highland barley grains by inducing them to low temperatures utilizes a dynamic temperature gradient regulation method to precisely control the adaptation of highland barley grains to low temperatures, promoting the efficient synthesis of antifreeze polysaccharides. By gradually lowering the temperature and adjusting the induction time, the method allows the highland barley grains to gradually adapt to low-temperature stress, significantly improving the synthesis efficiency of antifreeze polysaccharides and addressing the low synthesis efficiency of antifreeze polysaccharides in the prior art. By optimizing induction conditions such as temperature and humidity, the method improves the yield and quality of antifreeze polysaccharides, ensuring highland barley's ability to resist freezing in low-temperature environments.

[0024] The present invention significantly improves the extraction efficiency of antifreeze polysaccharides by using ultrasonic technology to assist water extraction, effectively reducing the amount of solvent required during the extraction process, and alleviating the problems of excessive solvent use and high costs in traditional methods. Combining gel filtration chromatography with the purification of antifreeze polysaccharides ensures that the extracted antifreeze polysaccharides have a high purity and reduces the complexity and operational difficulty of the purification process. Furthermore, by precisely controlling the temperature and humidity conditions during the low-temperature induction process, the present invention makes the entire preparation process simpler and more efficient, further reducing production costs.

[0025] The antifreeze polysaccharide prepared by the present invention has the advantages of being natural, biodegradable, non-toxic and harmless, and can be used as a food-grade natural antifreeze agent. It has broad prospects in the field of frozen food, conforms to the development trend of "green, natural and healthy", and can be used in large-scale industrial frozen ice cream: adding 0.2%-0.5% of the antifreeze polysaccharide of the present invention to the formula can significantly reduce the growth rate of ice crystals and improve the taste. Freeze-thaw cycle ( After 10 times of freezing (10 times), the average diameter of ice crystals in the added group was 30 μm, while that in the control group was 60 μm. Frozen fruits and vegetables: Spraying broccoli with a 1% antifreeze polysaccharide solution and refrigerating it at -18°C for one month increased the rate of intact tissue after refrigeration by approximately 20%, while the rate of juice loss decreased by approximately 15%. Quick-frozen pastries: Adding 1% antifreeze polysaccharide to dough, followed by quick freezing (-40°C, 2 hours) and then re-steaming, reduced the collapse rate of the finished product by 30%, resulting in a softer texture. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a flowchart for implementing the invention. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] Example 1

[0029] like Figure 1 As shown, an embodiment of the present invention provides a method for preparing antifreeze polysaccharides from highland barley grains based on low-temperature induction, comprising: S1. placing highland barley grains in a low-temperature environment for low-temperature induction, the low-temperature induction conditions including temperature, induction time and humidity, the temperature range in the low-temperature induction conditions being -2°C to 4°C, the induction time being 16 hours to 48 hours, and the humidity being controlled between 60% and 75%. 4°C is the threshold temperature for low-temperature signals to activate the CBF transcription factor family, while -2°C is close to the cell membrane phase transition temperature, causing a moderate change in membrane fluidity without causing freezing to destroy the cell structure. By covering this interval, the optimum temperature zones of membrane receptor sensing, secondary messenger cascades and enzymatic reactions can be triggered simultaneously, thereby achieving cumulative amplification of cold shock signals and efficient activation of antifreeze polysaccharide synthesis.

[0030] S2. A dynamic temperature gradient adjustment method is used for the low-temperature induction process. The dynamic temperature gradient adjustment method gradually lowers the induction temperature and adjusts the time interval so that the highland barley grains gradually adapt to the low-temperature stress to promote the synthesis of antifreeze polysaccharides.

[0031] The dynamic temperature gradient adjustment method in S2 includes the following steps:

[0032] S2.1 Set the initial temperature to 4°C and gradually reduce the temperature to 3°C within 4 hours.

[0033] S2.2 lowers the temperature by 0.5°C to 1°C every 4 hours until the induction temperature drops to -2°C. This slow cooling strategy of 0.5–1°C every 4 hours maintains membrane fluidity and the optimal temperature range for enzymatic reactions while providing a continuous and gentle low-temperature signal, allowing the membrane receptor-secondary messenger-transcription factor cascade to accumulate and activate antifreeze gene expression. The slow gradient also avoids metabolic collapse and enzyme inactivation caused by sudden cooling, and seamlessly integrates with existing cold chain equipment and PLC programs, achieving industrial controllability with a balance between temperature control precision and energy consumption, making it the optimal low-temperature induction solution.

[0034] S2.3 The low temperature induction process lasts for 24 hours, and the temperature is gradually adjusted to ensure that the barley grains gradually adapt to the low temperature stress.

[0035] The temperature gradient change in the low temperature induction process follows the following model formula:

[0036]

[0037] Where T(t) is the temperature at time t, T0 is the initial temperature, ΔT is the temperature change amplitude, T t is the total induction time, and t is the current time.

[0038] Corresponding to this step, we set up a static control group (4℃, lasting 24h) to compare the polysaccharide synthesis in the two groups. The results showed that the dynamic gradient group was 75mg / g, while the static group was only 45mg / g, indicating that step-by-step cooling is more conducive to the synthesis of antifreeze polysaccharides. In terms of biological mechanism, the dynamic gradient can make the intracellular cold shock signal (Ca 2+ , ABA) accumulate step by step, enhancing the expression of key synthetic enzymes such as sucrose synthase.

[0039] In order to further verify the effects of dynamic cooling and ultrasonic pretreatment on cold shock signal transduction and polysaccharide synthase system, the present invention, under the conditions of Example 1, 2+ Signal / ABA levels, key transcription factor gene expression, and glycosyltransferase activity were tested. The results showed that:

[0040] Ca 2+ Peak accumulation: intracellular free Ca after treatment 2+ The concentration increased by 2.3 times compared with the control (ultrasound only or cooling only);

[0041] ABA content: The ABA level in leaf tissue increased from the baseline level of 45±5ng·g -1 Increased to 78±6ng·g -1 ;

[0042] Gene expression: The relative expression levels of cold-resistant core transcription factors CBF1, CBF3 and downstream glycosyltransferase gene UGT74F2 increased by 4.5-fold, 3.8-fold and 5.2-fold, respectively;

[0043] Enzyme activity: The activity of the key enzyme for polysaccharide synthesis (UDP-glucose glycosyltransferase) increased from 0.12±0.01 U·mg in the control group to -1 Protein increased to 0.28±0.02U·mg -1 protein.

[0044] The humidity change model formula for the low temperature induced process is:

[0045]

[0046] Where H(t) is the humidity at time t, H0 is the initial humidity, ΔH is the humidity change amplitude, T t is the total induction time, t is the current time, and the humidity is maintained between 60% and 75% throughout the induction process.

[0047] The specific implementation is as follows:

[0048] Induction conditions: temperature range -2°C to 4°C, induction time 16 hours, humidity 60% to 75%.

[0049] Temperature control method:

[0050] The initial temperature was set at 4°C and the temperature was gradually decreased to 3°C over 4 hours.

[0051] The temperature was lowered by 0.5°C to 1°C every 4 hours until the induction temperature dropped to -2°C.

[0052] The low-temperature induction process lasted for 16 hours, and the temperature was gradually adjusted to ensure that the barley grains gradually adapted to the low-temperature stress.

[0053] Implementation effect: Antifreeze polysaccharide synthesis amount: Under 16-hour induction conditions, the antifreeze polysaccharide synthesis amount of highland barley grains is 50 mg / g (calculated on dry weight), which is about 20% higher than that of conventional methods.

[0054] Freeze resistance: In the freeze resistance test at low temperature (-4℃, 24 hours), the freeze damage rate of highland barley grains was 15%, which was significantly lower than that of the conventional method (freeze damage rate was 30%).

[0055] Characteristic parameters of the purified polysaccharide: average molecular weight: 12±1 kDa; PDI≈1.15; monosaccharide composition: glucose 52%, galactose 22%, rhamnose 14%, xylose 12%.

[0056] Polysaccharide structure identification:

[0057] Molecular weight determination: HPSEC-MALLS was used to determine the average molecular weight and polydispersity index of the purified antifreeze polysaccharide. For example, in Example 2, Mw was measured to be ≈15.2 kDa and PDI was ≈1.18.

[0058] Monosaccharide composition analysis: The purified product was hydrolyzed with 2M sulfuric acid (110°C, 2h), and then the monosaccharide composition was quantitatively analyzed by HPLC. The results showed that glucose was 55 mol%, galactose was 20 mol%, rhamnose was 15 mol%, and xylose was 10 mol%.

[0059] Antifreeze activity assay:

[0060] Ice crystal inhibition rate: The IRI at 86 mg / mL was determined using ice crystal microscopy. The experiment showed that after 30 minutes, the ice crystal growth increased from 50 μm to 55 μm only, while the control group (uninduced polysaccharide) increased to 80 μm, with an IRI of 0.31.

[0061] Thermal hysteresis activity: The THA of a 15 mg / mL solution was measured using a nanotitrator and was approximately 0.6°C, which was significantly higher than that of common edible polysaccharides (THA < 0.1°C).

[0062] Biological verification: A -5℃ / 4h freezing test was conducted on rice seedlings. The survival rate of the treated group was ≈85%, while that of the control group was only 45%.

[0063] S3. Collect highland barley grains and extract antifreeze polysaccharides using a water extraction method supplemented by ultrasonic technology. The solvent in the water extraction method is deionized water. The frequency range of the ultrasonic technology is 30kHz to 60kHz, and the power is 100W to 200W to ensure efficient extraction of antifreeze polysaccharides.

[0064] Ultrasonic cavitation can disrupt cell walls and significantly increase cell permeability. Experiments have shown that, given the same extraction time, ultrasound-assisted extraction increases yield by approximately 20%–30% compared to water extraction alone. Furthermore, pre-treatment with low-intensity ultrasound (30kHz, 100W) for 2 hours before the cold induction stage promotes cold shock signaling, ultimately increasing polysaccharide yield by approximately 10%.

[0065] S4. Gel filtration chromatography is used to purify the extracted antifreeze polysaccharide to ensure that the purity of the antifreeze polysaccharide is suitable for application. The chromatographic column used in the gel filtration chromatography is Sephadex G-25, and PBS buffer is used in the separation process. The purification process lasts for 2 to 4 hours. Before purification by Sephadex G-25 gel filtration, the crude polysaccharide content is about 60% (determined by phenol-sulfuric acid method); after purification, the purity is quantitatively measured to be ≥90%, and no protein impurities are detected. Compared with the purity of 70% ± 5% obtained by the prior art multi-step precipitation + dialysis method, the purification efficiency of the present invention is significantly improved, the operation process is reduced by about 50%, and the solvent consumption is reduced by 30%.

[0066] Example 2

[0067] The difference from the first embodiment is that the low temperature induction conditions of this embodiment are different.

[0068] Induction conditions: temperature range -2°C to 4°C, induction time 24 hours, humidity 60% to 75%.

[0069] Temperature control method:

[0070] The initial temperature was set at 4°C and the temperature was gradually decreased to 3°C over 4 hours.

[0071] The temperature was lowered by 0.5°C to 1°C every 4 hours until the induction temperature dropped to -2°C.

[0072] The low-temperature induction process lasts for 24 hours, and the temperature is gradually adjusted to ensure that the barley grains gradually adapt to the low-temperature stress.

[0073] Implementation effect: Antifreeze polysaccharide synthesis amount: Under 24-hour induction conditions, the antifreeze polysaccharide synthesis amount of highland barley grains is 75 mg / g (calculated on dry weight), which is about 50% higher than that of conventional methods.

[0074] Freeze resistance: In the freeze resistance test at low temperature (-4℃, 24 hours), the freeze damage rate of highland barley grains was 8%, which was significantly lower than the conventional method (freeze damage rate was 25%).

[0075] Characteristic parameters of the purified polysaccharide: average molecular weight: 15.2 kDa; PDI≈1.18; monosaccharide composition: glucose 55%, galactose 20%, rhamnose 15%, xylose 10%.

[0076] Example 3

[0077] The difference from the first embodiment is that the low temperature induction conditions of this embodiment are different.

[0078] Induction conditions: temperature range -2°C to 4°C, induction time 48 hours, humidity 60% to 75%.

[0079] Temperature control method:

[0080] The initial temperature was set at 4°C and the temperature was gradually decreased to 3°C over 4 hours.

[0081] The temperature was lowered by 0.5°C to 1°C every 4 hours until the induction temperature dropped to -2°C.

[0082] The low-temperature induction process lasted for 48 hours, and the temperature was gradually adjusted to ensure that the barley grains gradually adapted to the low-temperature stress.

[0083] Implementation effect: Antifreeze polysaccharide synthesis amount: Under 48 hours of induction conditions, the antifreeze polysaccharide synthesis amount of highland barley grains is 100 mg / g (calculated on dry weight), which is about 80% higher than the conventional method.

[0084] Freeze resistance: In the freeze resistance test at low temperature (-4℃, 24 hours), the freeze damage rate of highland barley grains was 5%, which was significantly reduced compared with the conventional method (freeze damage rate was 20%).

[0085] Characteristic parameters of the purified polysaccharide: average molecular weight: 18.5 kDa; PDI≈1.25; monosaccharide composition: glucose 58%, galactose 18%, rhamnose 12%, xylose 12%.

[0086] Example 4

[0087] This embodiment is an application example of frozen dough.

[0088] Materials and conditions:

[0089] Highland barley antifreeze polysaccharide: prepared according to Example 1, with a purity of 85% and a content of 5.8 mg / g.

[0090] Wheat flour: high-gluten flour, protein content 12.5%.

[0091] Water: deionized water.

[0092] Other ingredients: salt 1.8%, yeast 0.5% (both based on flour mass).

[0093] Process flow:

[0094] 1. Take 1000g flour, add 0.5% (w / w) highland barley antifreeze polysaccharide, salt and yeast, and mix well.

[0095] 2. Slowly add 600mL of deionized water and stir at low speed until the dough becomes flocculent.

[0096] 3. Knead the dough at medium speed for 10 minutes and let it rise at room temperature (25℃) for 30 minutes.

[0097] 4. Divide the proofed dough into 50g portions and freeze them in a -18℃ freezer for 24 hours.

[0098] 5. Thaw (place in a 4°C refrigerator for 2 hours), return to room temperature for 10 minutes, and conduct baking texture and water retention tests.

[0099] Test results: Out-of-baker volume retention rate: The volume recovery rate of the frozen and thawed group was 92%, while that of the control group (without antifreeze polysaccharide) was only 78%.

[0100] Texture profile hardness: The dough hardness after thawing (TPA test) was 1.2N, while the control group was 1.8N, with a softer texture.

[0101] Water retention rate: After thawing, the water content remained at 65%, while the control group only maintained 52%.

[0102] Sensory evaluation: After a blind test by 10 people, the overall flavor score of the group containing antifreeze polysaccharides was 8.5 / 10, and the control group was 6.8 / 10.

[0103] Application effect:

[0104] In this example, the prepared highland barley antifreeze polysaccharide was applied to frozen dough, which significantly improved the volume retention, texture softness and water retention after frozen storage, and enhanced the overall flavor, verifying the practical value of the antifreeze polysaccharide in the field of food cold chain.

[0105] Example 5

[0106] Different from Example 1, this example only uses ultrasonic technology to extract polysaccharides.

[0107] Materials and conditions:

[0108] The highland barley grain samples were taken from the same batch, washed, air-dried and set aside; the ultrasonic pretreatment conditions were the same as those in Example 1: ultrasonic power 200 W, frequency 20 kHz, treatment time 30 min, and water bath temperature maintained at 25°C.

[0109] Process flow:

[0110] S1. Place the washed and air-dried highland barley grains in an ultrasonic reactor and add deionized water at a mass ratio of 1:10.

[0111] S2. Start ultrasonic treatment for 30 minutes and collect the supernatant after treatment.

[0112] S3. The supernatant was directly subjected to crude separation: polysaccharides were precipitated by the phenol-sulfuric acid method, and then precipitated once with 70% ethanol.

[0113] S4. Directly perform 24 h dialysis (molecular weight cutoff 3 kDa) without dynamic cooling induction.

[0114] S5. After dialysis, freeze-dry to obtain dry powder antifreeze polysaccharide.

[0115] Test results:

[0116] Polysaccharide yield: 4.1±0.2 mg / g (dry weight).

[0117] Polysaccharide purity: 82±4%.

[0118] Total treatment time: 30 min (ultrasound) + 24 h (dialysis) ≈ 24.5 h.

[0119] Ethanol dosage: 200 L / batch, recovery rate 85%.

[0120] Implementation effect: Compared with Example 1 of the present invention (dynamic cooling + ultrasound), relying solely on ultrasonic wall breaking can shorten part of the process (the total processing time is reduced by about 50%), but the polysaccharide yield (4.1 vs. 7.2 mg / g) and purity (82% vs. 88%) are significantly reduced, and the cost-effectiveness is not as good as the composite process, which verifies the necessity of the synergistic effect of ultrasound and dynamic cooling.

[0121] Example Induction time Temperature range Humidity range Antifreeze polysaccharide synthesis Frost damage rate Example 1 16 hours -2℃ to 4℃ 60% to 75% 50mg / g 15％ Example 2 24 hours -2℃ to 4℃ 60% to 75% 75mg / g 8％ Example 3 48 hours -2℃ to 4℃ 60% to 75% 100mg / g 5％

[0122] Compared with the existing technology, the present invention significantly improves the synthesis efficiency of antifreeze polysaccharides from highland barley grains under low temperature gradient induction conditions. Experimental data show that:

[0123] The polysaccharide yield increased from 2.5±0.3mg / g of the existing technology to 5.8±0.4mg / g, an increase of 132%;

[0124] The purity of polysaccharide was increased from 65±8% of the traditional method to 85±5%.

[0125] The total extraction time was shortened by 66% from the original 144 h (3 steps of ultrafiltration + 2 ethanol precipitations + 7–10 d dialysis) to 48 h (2 steps of ultrafiltration + 1 ethanol precipitation + 24 h dialysis).

[0126] Solvent consumption and costs have been significantly reduced: ethanol usage has been reduced from 500L / batch to 200L / batch, solvent recovery has increased from 60% to 90%, and the unit product purification cost has been reduced from 1,200 yuan / g to 450 yuan / g, resulting in a cost saving of approximately 62%.

[0127] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction, characterized in that: include: S1. The highland barley grains are placed in a low temperature environment for low temperature induction, wherein the low temperature induction conditions include temperature, induction time and humidity; S2. A dynamic temperature gradient adjustment method is used for low-temperature induction process, wherein the dynamic temperature gradient adjustment method gradually reduces the induction temperature and adjusts the time interval so that the barley grains gradually adapt to low temperature stress; S3. Collect highland barley grains and extract antifreeze polysaccharides using water extraction supplemented by ultrasonic technology; S4. Purify the extracted antifreeze polysaccharide using gel filtration chromatography.

2. The method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction according to claim 1, characterized in that: The temperature range of the low-temperature induction condition is -2°C to 4°C, the induction time is 16 hours to 48 hours, and the humidity is controlled between 60% and 75%.

3. The method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction according to claim 1, characterized in that: The dynamic temperature gradient adjustment method in S2 comprises the following steps: S2.1 Set the initial temperature to 4°C and gradually reduce it to 3°C over 4 hours; S2.2 Reduce the temperature by 0.5°C to 1°C every 4 hours until the induction temperature drops to -2°C; S2.3 The low temperature induction process lasts for 24 hours, and the temperature is gradually adjusted.

4. The method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction according to claim 1, characterized in that: The temperature gradient change during the low temperature induction process follows the following model formula: Where T(t) is the temperature at time t, T0 is the initial temperature, ΔT is the temperature change amplitude, T t is the total induction time, and t is the current time.

5. The method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction according to claim 1, characterized in that: The humidity change model formula of the low temperature induced process is: Where H(t) is the humidity at time t, H0 is the initial humidity, ΔH is the humidity change amplitude, T t is the total induction time, t is the current time, and the humidity is maintained between 60% and 75% throughout the induction process.

6. The method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction according to claim 1, characterized in that: The solvent in the water extraction method is deionized water, the frequency range of the ultrasonic technology is 30kHz to 60kHz, and the power is 100W to 200W.

7. The method for preparing antifreeze polysaccharides from highland barley grains based on low temperature induction according to claim 1, characterized in that: The chromatographic column used in the gel filtration chromatography is Sephadex G-25, PBS buffer is used in the separation process, and the purification process lasts for 2 to 4 hours.

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