Method for improving antifreeze activity of wheat bran araboxylan based on ion-induced spatial conformation rearrangement and application

Abstract

The invention particularly relates to a method for improving antifreeze activity of wheat bran araboxylan based on ion-induced spatial conformation rearrangement and application, and belongs to the technical field of food processing. The method is characterized in that wheat bran is used as a raw material, wheat bran AX with anti-freezing activity is obtained through urea alkali extraction and alcohol precipitation, ice shell separation and column chromatography, polysaccharide is subjected to conformation modification through Hofmeyer series ions, the anti-freezing activity of AX is remarkably improved, the ice crystal growth size inhibition effect is improved by 20%-86%, and the yeast survival rate is improved by 8%-47%. According to the method, polysaccharide spatial conformation rearrangement is induced through ions, the anti-freezing activity of AX is improved, and the method can be widely applied to frozen food preservation and biological sample low-temperature storage and has the advantages of being environmentally friendly, safe and low in cost.

Description

I. Technical Field

[0001] This invention relates to a method and its application for improving the antifreeze activity of wheat bran arabinoxylan based on ion-induced spatial conformational rearrangement, belonging to the field of food processing technology. II. Background Technology

[0002] my country is a major wheat producer, and wheat bran, a major byproduct of wheat processing, boasts advantages such as high yield, low cost, and health benefits. However, it is primarily used as animal feed, resulting in low utilization and economic value. Arabicxylan (AX) is a crucial component of wheat bran's dietary fiber, accounting for approximately 20%–26%. AX mainly consists of β-1,4-linked D-pyranoyl residues as its backbone, with arabinose groups (Araf) linked to the xylose backbone via O-2, O-3, or O-2,3 bonds. Related studies have shown that inducing conformational evolution in polysaccharides may enhance their antifreeze properties. In ion-specificity studies, Hofmeister investigated a series of ions that specifically affect protein precipitation, termed the Hofmeister series ions. Different types of salts produce ion-specific effects, thereby influencing the structure and functional properties of biomolecules. Simultaneously, the Hofmeister series ions may also further enhance antifreeze properties by affecting the water molecule network.

[0003] Patent (Publication No. CN118460658A, Publication Date August 9, 2024) discloses "A method for preparing yak hide antifreeze peptides." The yak hide antifreeze peptides prepared in this invention have good antifreeze activity, but the yield is low and production is severely limited by raw materials. Patent (Publication No. CN111548428A, Publication Date August 18, 2020) discloses "A method for extracting enoki mushroom antifreeze polysaccharides." This invention prepares enoki mushroom antifreeze polysaccharides with high antifreeze activity by obtaining enoki mushroom mycelium through cold induction treatment, but the raw material cost is high and the functional characteristics are relatively simple.

[0004] This invention relates to a method and its application for enhancing the antifreeze activity of wheat bran arabinoxylan based on ion-induced spatial conformational rearrangement, which has significant advantages such as low cost, greenness, and safety. In the frozen food industry, it can effectively improve product quality and preservation effect, and extend shelf life; in the biomedical field, it can be used for the low-temperature preservation of cells and tissues. Simultaneously, this technology promotes the efficient utilization of wheat bran resources in the frozen food industry, realizing the high-value transformation of agricultural by-products. III. Summary of the Invention

[0005] Technical issues

[0006] To address the insufficient antifreeze properties of natural arabinoxylan (AX), this invention provides a method and application for enhancing the antifreeze activity of wheat bran arabinoxylan based on ion-induced spatial conformational rearrangement. By inducing ion-induced antifreeze AX conformational rearrangement in wheat bran, its antifreeze properties are further improved, enabling the development of highly efficient natural antifreeze agents suitable for the food and biopharmaceutical fields.

[0007] Technical solution

[0008] The technical solution of this invention is summarized as follows: Wheat bran AX with antifreeze activity is prepared from wheat bran, and conformational modification with Hofmeister series ions is performed to enhance the antifreeze activity of wheat bran AX. Specific steps include:

[0009] (1) Preparation of wheat bran AX: Wheat bran was subjected to enzyme inactivation, urea alkaline extraction, fractional alcohol precipitation (20%–60%), ice shell separation, and freeze drying to obtain wheat bran antifreeze polysaccharide crude sugar; a wheat bran AX solution with a mass-volume concentration of 0.5%–1% was prepared, and the solution was initially purified using a DEAE-52 cellulose ion exchange column and freeze-dried; a pre-purified polysaccharide solution with a mass-volume concentration of 0.5%–0.8% was prepared, and the solution was purified a second time using a Sephadex G-100 molecular sieve gel column. The main peak was collected by tracking the elution curve using the phenol-sulfuric acid method, and the solution was concentrated and freeze-dried to obtain antifreeze wheat bran AX; it is preferred to use an AX aqueous solution with an alcohol precipitation concentration of 50% and a mass-volume concentration of 1% for initial purification and a pre-purified polysaccharide solution with a mass-volume concentration of 0.5% for secondary purification. Under these conditions, the yield and antifreeze activity of antifreeze AX were the highest.

[0010] (2) Ion conformation modification: Prepare salt solutions (Na2SO4, NaCl, NaNO3, KCl, MgCl2) containing specific Hofmeister series ions at concentrations of 0.05M to 0.25M. Add wheat bran antifreeze AX to a mass-volume concentration of 0.1% to 1%. Obtain antifreeze AX with different molecular conformations by ion-induced spatial conformational rearrangement. Drop the conformationally modified AX solution onto a pre-cooled glass slide to form thin ice crystals. Observe the ice crystal size at -8℃ using a polarizing microscope equipped with a freezing stage, and calculate the average particle area ratio (MGA%). Preferably, 0.25M solutions containing Hofmeister series anions (SO4, NaCl, NaNO3, KCl, MgCl2) are used. 2- Cl - NO3 - When wheat bran antifreeze AX is added to a salt solution of 1% by mass volume, the antifreeze activity is the highest.

[0011] (3) Cryoprotection of yeast: Yeast cells were dispersed in an equal volume of sterile physiological saline in liquid culture medium. AX with different conformations before and after ion modification was added to a concentration of 0.05%–0.2% (w / v). The cells were frozen at -20°C for 1 hour, then thawed at 30°C for 30 minutes as one cycle, with 1–3 cycles performed. After centrifugation and washing, the frozen-thawed yeast cells were plated and incubated at 30°C for 36–48 hours. The total number of yeast colonies was then determined. The optimal concentration of AX with ion-induced conformational rearrangement in the total system was 0.2%, which provided the best cryoprotection effect for yeast.

[0012] Beneficial effects

[0013] (1) This invention improves the ice crystal growth size inhibition effect by 20-86% by ion-induced AX conformation rearrangement of wheat bran, thus significantly enhancing its antifreeze activity;

[0014] (2) In this invention, AX with ion conformation modification is applied to the cryoprotection of yeast cells, which increases the yeast survival rate by 8% to 47%, and can be developed into a highly efficient natural cryoprotectant suitable for the food and biopharmaceutical fields.

[0015] (3) This invention uses wheat bran, a by-product of wheat processing, as raw material, which is green and safe, and realizes the high-value utilization of wheat bran resources. IV. Detailed Implementation

[0016] Example 1: Wheat bran was subjected to enzyme inactivation, urea alkaline extraction, fractionated alcohol precipitation (50% concentration), ice shell separation, and freeze-drying to obtain wheat bran antifreeze polysaccharide crude sugar. A 1% (w / v) wheat bran AX solution was prepared, preliminarily purified using a DEAE-52 cellulose ion exchange column, and then freeze-dried. A 0.5% (w / v) preliminarily purified polysaccharide solution was prepared, and further purified using a Sephadex G-100 molecular sieve gel column. The main peak was collected by tracking the elution curve using the phenol-sulfuric acid method. After concentration and freeze-drying, antifreeze wheat bran AX was obtained. A 0.25M solution containing specific Hofmeister series anions, SO42-, was prepared. 2- Cl - NO3 -A solution of Na₂SO₄, NaCl, and NaNO₃ salts (dissolved in 0.01M, pH 7 phosphate buffer) was prepared by adding wheat bran antifreeze AX to a concentration of 1% by ion-induced conformational rearrangement of the wheat bran antifreeze AX, thus obtaining antifreeze AX with different molecular conformations. The conformationally modified AX solutions were dropped onto a pre-cooled glass slide to form thin ice crystals. The ice crystal size at -8℃ was observed using a polarizing microscope equipped with a freezing stage, and the average particle area ratio (MGA%) was calculated. Compared with wheat bran antifreeze AX, Na₂SO₄, NaCl, and NaNO₃ improved the inhibition effect on ice crystal size by 86%, 68%, and 60%, respectively. Yeast cells were dispersed in an equal volume of sterile physiological saline in liquid culture medium, and different conformations of AX before and after ion modification were added to a concentration of 0.2% by ion-induced rearrangement. After freezing at -20℃ for 1 hour, the cells were thawed in a 30℃ incubator for 30 minutes as one cycle, and one cycle was performed. Yeast cells after freeze-thaw treatment were obtained by centrifugation and washing, plated, and incubated at 30°C for 36–48 h. The total number of yeast colonies was then determined. Compared with the untreated freeze-thawed AX group, Na₂SO₄, NaCl, and NaNO₃ increased yeast survival rates by 43%, 47%, and 39%, respectively.

[0017] Example 2: The method for preparing antifreeze wheat bran AX is the same as in Example 1, except that the concentration of alcohol precipitation is 60%, and a wheat bran AX solution with a mass-volume concentration of 0.8% is prepared for preliminary purification; a preliminary purified polysaccharide solution with a mass-volume concentration of 0.6% is prepared for secondary purification; and a 0.05M solution containing specific Hofmeister series anions, SO42-, is prepared. 2- Cl - NO3 - A solution of Na₂SO₄, NaCl, and NaNO₃ salts (dissolved in 0.01M, pH 7 phosphate buffer) was prepared by adding wheat bran antifreeze AX to a concentration of 0.5% by ion-induced conformational rearrangement of the wheat bran antifreeze AX, thus obtaining antifreeze AX with different molecular conformations. The conformationally modified AX solutions were dropped onto a pre-cooled glass slide to form thin ice crystals. The ice crystal size at -8°C was observed using a polarizing microscope equipped with a freezing stage, and the average particle area ratio (MGA%) was calculated. Compared with wheat bran antifreeze AX, Na₂SO₄, NaCl, and NaNO₃ showed improved inhibition of ice crystal size by 65%, 48%, and 43%, respectively. The method for determining the cryoprotective effect on yeast cells was the same as in Example 1, except that AX with different conformations before and after ion modification was added to a concentration of 0.1%, and two freeze-thaw cycles were performed to determine the total number of yeast colonies formed. Compared with the untreated antifreeze AX group, Na2SO4, NaCl and NaNO3 increased yeast survival rate by 20%, 28% and 23%, respectively.

[0018] Example 3: The method for preparing antifreeze wheat bran AX is the same as in Example 1, except that the concentration of alcohol precipitation is 40%, and a wheat bran AX solution with a mass-volume concentration of 0.5% is prepared for preliminary purification; a preliminary purified polysaccharide solution with a mass-volume concentration of 0.8% is prepared for secondary purification; and a solution containing specific Hofmeister series cations K is prepared at a concentration of 0.05M. + Na + Mg 2+ KCl, NaCl, and MgCl2 salt solutions (dissolved in 0.01M, pH=7 phosphate buffer) were used to add wheat bran antifreeze AX to a concentration of 0.1% by volume. Different molecular conformations of antifreeze AX were prepared by ion-induced conformational rearrangement. The conformationally modified AX solutions were dropped onto pre-cooled glass slides to form thin ice crystals. The ice crystal size at -8°C was observed using a polarizing microscope equipped with a freezing stage, and the average particle area ratio (MGA%) was calculated. Compared with wheat bran antifreeze AX, KCl, NaCl, and MgCl2 improved the inhibition effect on ice crystal size by 32%, 20%, and 27%, respectively. The method for determining the cryoprotective effect on yeast cells was the same as in Example 1, except that different conformations of AX before and after ion modification were added to a concentration of 0.05% by volume, and three freeze-thaw cycles were performed. The total number of yeast colonies was measured. Compared with the untreated antifreeze AX group, KCl, NaCl, and MgCl2 increased yeast survival rate by 8%, 10%, and 13%, respectively.

[0019] The embodiments of the present invention have been described in detail above, but these are merely examples for ease of understanding and should not be considered as limiting the scope of the invention. Similarly, anyone skilled in the art can make various possible equivalent changes and substitutions based on the technical solutions and preferred embodiments described in this invention, but all such changes and substitutions should fall within the protection scope of the claims of this invention.

Claims

1. A method and application for improving the antifreeze activity of wheat bran arabinoxylan based on ion-induced spatial conformational rearrangement, belonging to the field of food processing technology. Its characteristics are... Wheat bran was used as raw material, and after urea alkaline extraction, alcohol precipitation, ice shell separation, and column chromatography, wheat bran arabinoxylan with antifreeze activity was obtained. The Hofmeister series ions (SO4) were then used for further analysis. 2- Cl - NO3 - K + Na + Mg 2+ Inducing polysaccharide spatial conformational rearrangement to enhance the antifreeze activity of AX, specifically including the following steps: (1) Preparation of wheat bran AX: Wheat bran was subjected to enzyme inactivation, urea alkaline extraction, fractional alcohol precipitation (20% to 60%), ice shell separation, and freeze drying to obtain wheat bran antifreeze polysaccharide crude sugar; an AX solution with a mass-volume concentration of 0.5% to 1% was prepared, and the solution was initially purified by DEAE-52 cellulose ion exchange column and freeze-dried; a pre-purified polysaccharide solution with a mass-volume concentration of 0.5% to 0.8% was prepared, and the solution was purified a second time by Sephadex G-100 molecular sieve gel column and freeze-dried to obtain wheat bran antifreeze AX; (2) Ion conformation modification: Prepare salt solutions (Na2SO4, NaCl, NaNO3, KCl, MgCl2) containing specific Hofmeister series ions at a concentration of 0.05M to 0.25M, add wheat bran antifreeze AX to a mass-volume concentration of 0.1% to 1%, and obtain antifreeze AX with different molecular conformations by ion-induced spatial conformation rearrangement of wheat bran antifreeze AX; (3) Cryoprotection of yeast: Yeast cells were dispersed in sterile physiological saline of equal volume in liquid culture medium, and AX solutions with different conformations before and after ion modification were added. The mass-volume concentration of AX in the system was 0.05% to 0.2%. After freezing at -20℃ for 1 h, the cells were thawed in a constant temperature incubator at 30℃ for 30 min as one cycle, and 1 to 3 cycles were performed. After centrifugation and washing, the frozen and thawed yeast cells were obtained, plated, and cultured at 30℃ for 36 to 48 h. The total number of yeast colonies was then determined.

2. The method and application for improving the antifreeze activity of wheat bran arabinoxylan based on ion-induced spatial conformational rearrangement as described in claim 1, characterized in that... Use SO4 2- Cl - NO3 - K + Na + Mg 2+ Hofmeister series ions enhance the antifreeze activity of wheat bran antifreeze AX, increasing the ice crystal growth size inhibition effect by 20%–86% and yeast survival rate by 8%–47%. This technology further enhances the antifreeze activity of natural polysaccharides and can be widely used in frozen food preservation and low-temperature storage of biological samples, offering advantages of being green, safe, and low-cost.