A cold-soluble agar-zein plant-based stabilizer, and a preparation method and application thereof

Abstract

The application discloses a cold-soluble agar-plant-based prolamine stabilizer, a preparation method and application thereof, and belongs to the technical field of novel food additive preparation. The technical problem of poor cold water solubility and poor emulsification of instant agar in the prior art is solved, and the use range of the instant agar is limited. The preparation method of the cold-soluble agar-plant-based prolamine stabilizer comprises the following steps: carrying out a Maillard reaction on acid-dissolved instant agar and alcohol-soluble prolamine, grafting the prolamine, adding ethanol to the solution, alcohol-sedimenting and dehydrating, and replacing alcohol by supercritical carbon dioxide technology to dry the solution to obtain the cold-soluble agar-plant-based prolamine stabilizer. The cold-soluble agar-plant-based prolamine stabilizer has good cold water solubility and good emulsification, and can replace animal-based colloid to obtain good effect in butter whipping.

Description

Technical Field

[0001] This invention relates to the field of food additive preparation technology, specifically to a cold-soluble agar-alcohol-soluble protein plant-based stabilizer, its preparation method, and its application. Background Technology

[0002] The Maillard reaction, also known as the carbonyl-amine reaction, is a common browning reaction in food processing. It is a reaction between carbonyl compounds (reducing sugars) and amino compounds (amino acids and proteins). Agar is a high-molecular-weight functional polysaccharide derived from marine red algae such as Gracilaria and Agaricus, with galactose as its main component. It possesses excellent sol-gel properties not found in many terrestrial polysaccharides and is widely used in food, medicine, daily chemical industry, and bioengineering.

[0003] Conventional agar requires high temperatures to dissolve; even instant agar cannot dissolve at room temperature or in cold water, significantly limiting its use at room temperature. Furthermore, while agar has good gelling properties, its emulsifying properties are generally poor, greatly restricting its use in whipping butter. In whipping butter, gelatin, although possessing gelling and emulsifying properties, is not easily dissolved at room temperature, the gel melts easily, and the texture is not smooth. Summary of the Invention

[0004] This invention addresses the shortcomings and deficiencies of existing technologies by providing a method and application of a plant-based stabilizer for cold-soluble agar-alcohol protein. This stabilizer is prepared by controlling the concentration, temperature, and alcohol content of the reaction between acid-soluble agar and alcohol-soluble protein, and grafting alcohol-soluble protein onto acid-soluble agar via Maillard reaction. This improves the emulsification performance while altering the dissolution and gelation properties, resulting in a plant-based stabilizer with excellent low-temperature solubility.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The preparation method of the cold-soluble agar-alcohol-soluble protein plant-based stabilizer provided by the present invention includes the following steps:

[0006] The instant agar was acid-hydrolyzed and then dissolved at high temperature with alcohol-soluble protein. The alcohol-soluble protein was grafted onto the acid-hydrolyzed instant agar via Maillard reaction. After the reaction, alcohol precipitation and dehydration were performed. The ethanol was then separated by supercritical carbon dioxide replacement technology to obtain a dry, cold-soluble agar-alcohol-soluble protein plant-based stabilizer.

[0007] Preferably, the agar raw material is derived from one or two of the red algae Gracilaria and Gracilaria; the gliadin is one or two of the wheat gliadin and zein gliadin.

[0008] Preferably, the acid hydrolysis of the rapidly dissolving agar step includes:

[0009] Prepare an aqueous solution of 5%-30% by mass using a certain amount of instant agar. Stir continuously and heat to 50-55℃ to dissolve the agar for 5-10 minutes. Add hydrochloric acid with an acidity of 0.1-0.2 mol / L, at a concentration of 10%-20% of the agar powder added. Keep the reaction at this temperature for 2-4 hours. Then neutralize with sodium hydroxide to neutral. Vacuum filter, concentrate and freeze-dry using hollow fiber nanofiltration to obtain acid-hydrolyzed instant agar.

[0010] The strength range of 5% acid-soluble agar is 100-300 g / cm³. 2 .

[0011] Preferably, the alcohol-soluble protein is first dissolved in alcohol with a concentration of 50%-90%, and the mass ratio of alcohol-soluble protein to alcohol is 0.5-1:1.

[0012] Next, the acid-hydrolyzed agar, alcohol-soluble protein, and purified water were subjected to Maillard reaction at a mass ratio of 2-2.5:1:10 to graft the alcohol-soluble protein onto the oligosaccharide acid-hydrolyzed agar. The reaction temperature was 50℃-90℃, the reaction time was 2h-4h, and the reaction pH was 6-8 to generate the grafted product.

[0013] Preferably, the grafted product is verified by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm that the sugar grafting reaction actually occurred and that a glycoprotein complex was generated.

[0014] Preferably, the method for determining the grafting degree includes the following steps:

[0015] Preparation of OPA reagent: Dissolve 40.0 mg OPA in 1.0 mL methanol, add 2.5 mL of prepared 20% (w / w) sodium dodecyl sulfate (SDS), 25 mL of borax solution (0.1 mol / L), 100 μL of β-mercaptoethanol, and finally dilute to 50 mL with distilled water. Prepare fresh before use.

[0016] The grafting degree was calculated using the free amino content determined by OPA reagent: 200 μL of the test sample was mixed with 4.0 mL of OPA reagent and reacted in a 35°C constant temperature water bath for 2 min. The absorbance value A was measured at 340 nm using a UV spectrophotometer. 200 μL of distilled water was added to the blank group. The formula for calculating the grafting degree is shown below. Each sample was measured three times.

[0017]

[0018] In the formula: A0 is the absorbance value before reaction, and A1 is the absorbance value after saccharification.

[0019] Preferably, the alcohol precipitation and dehydration steps include: precipitating the cold-soluble agar-alcohol-protein complex with 75% ethanol under a 40°C water bath condition; then dehydrating the cold-soluble agar-alcohol-protein complex with 95% ethanol to obtain a cold-soluble agar-alcohol-protein complex with low moisture content; the dehydrated cold-soluble agar-alcohol-protein complex has a water content of less than or equal to 30%.

[0020] Preferably, the grafted cold-soluble agar-alcohol-soluble protein complex is placed in a displacement vessel, and supercritical carbon dioxide is introduced at a temperature of 20℃-25℃ and a pressure of 5.7MPa-6.4MPa for a displacement time of 2h-4h.

[0021] During gas intake, to prevent damage to the grafting of the complex, both the upper and lower inlet valves of the extraction vessel are fully opened. After the pressure stabilizes, the upper inlet valve is closed and the outlet valve is opened to release the gas slowly.

[0022] Ethanol and carbon dioxide are recovered and recycled via a separation vessel;

[0023] The entire process must be carried out slowly to avoid damaging the grafted complex in the reactor. When no ethanol can be observed in the separation reactor, the temperature is kept constant and the gas is slowly released to atmospheric pressure to obtain the dried cold-soluble agar-alcohol-soluble protein complex.

[0024] The cold-soluble agar-alcohol-soluble protein plant-based stabilizer prepared by the above method.

[0025] Application of cold-soluble agar-alcohol-soluble protein plant-based stabilizer in the field of food additive preparation technology.

[0026] This invention provides a cold-soluble agar-alcohol-soluble protein plant-based stabilizer, its preparation method, and its application. It has the following beneficial effects:

[0027] (1) The cold-soluble agar-alcohol-soluble protein plant-based stabilizer of the present invention and its preparation method and application are as follows: the fast-soluble agar is acid-hydrolyzed to obtain more carbonyl ends, the alcohol-soluble protein is dissolved in ethanol, the acid-hydrolyzed fast-soluble agar and the alcohol-soluble protein are dissolved together at high temperature, the alcohol-soluble protein is grafted onto the acid-hydrolyzed fast-soluble agar through Maillard reaction, and then two high-concentration alcohol precipitation and dehydration are performed. The alcohol and the acid-hydrolyzed fast-soluble agar-alcohol-soluble protein complex are separated by supercritical carbon dioxide replacement process to obtain the cold-soluble agar-alcohol-soluble protein plant-based stabilizer with cold water solubility.

[0028] By controlling the concentration, temperature, and alcohol content of the reaction between acid-hydrolyzed fast-dissolving agar and alcohol-soluble protein, alcohol-soluble protein is grafted onto acid-hydrolyzed fast-dissolving agar via the Maillard reaction. This improves emulsifying properties while altering dissolution and gelation properties, resulting in excellent low-temperature rapid solubility. It dissolves at 30°C without gelling, and upon heating above 50°C, it returns to room temperature to form a stable porous gel texture. A low-moisture-content cold-dissolving agar-alcohol-soluble protein complex is obtained through ethanol precipitation and dehydration. The alcohol in the complex is then removed using supercritical carbon dioxide replacement technology, and the alcohol and carbon dioxide are recovered and reused. Finally, a cold-dissolving agar-alcohol-soluble protein plant-based stabilizer with good emulsifying properties and rapid solubility at room temperature is obtained.

[0029] (2) The cold-soluble agar-alcohol-soluble protein plant-based stabilizer of the present invention has good cold water solubility, dissolves at room temperature of 30°C but does not gel, and after heating to above 50°C, it returns to room temperature to form a stable porous gel texture.

[0030] It exhibits excellent emulsifying properties, effectively replacing animal-based colloids in whipping butter. Compared to gelatin, it is stable at room temperature and does not melt, offering a refreshing and non-greasy texture. It can replace gelatin in the preparation of mousse products and gel-based candies, as well as egg whites in baked goods, thus finding wide application in the field of food additive preparation technology. Attached Figure Description

[0031] Figure 1 The bar chart shows the emulsification index (T) results of the emulsification test of the cold-soluble agar-alcohol-soluble protein plant-based stabilizer of the present invention.

[0032] Figure 2 The bar chart shows the emulsification stability (%) results of the cold-soluble agar-alcohol-soluble protein plant-based stabilizer of the present invention. Detailed Implementation

[0033] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0034] Example 1

[0035] A method for preparing a cold-soluble agar-alcohol-soluble protein plant-based stabilizer includes the following steps:

[0036] S1 involves acid hydrolysis of the instant agar. The acid hydrolysis steps for the instant agar include:

[0037] A certain amount of our company's self-produced instant agar (patent number CN 107602729 A) was prepared into a 15% aqueous solution. Under continuous stirring, the solution was heated to 53°C to dissolve the agar for 8 minutes. Hydrochloric acid with an acidity of 0.1 mol / L was added, at a dosage of 10% of the agar powder added. The reaction was maintained at this temperature for 2 hours. The solution was then neutralized with sodium hydroxide. Vacuum filtration was performed, followed by concentration via hollow fiber nanofiltration and freeze-drying to obtain acid-hydrolyzed instant agar. The 5% strength of the acid-hydrolyzed instant agar was 170 g / cm³. 2 The agar raw material is derived from the red algae *Glechoma heliotrope*; the gliadin is wheat gliadin.

[0038] S2 is dissolved at high temperature with alcohol-soluble proteins, and the alcohol-soluble proteins are grafted onto acid-hydrolyzed fast-dissolving agar via Maillard reaction. The steps include the following:

[0039] First, the alcohol-soluble protein is dissolved in 90% alcohol at a mass ratio of 0.5:1. Then, 1 ton of acid-hydrolyzed agar, alcohol-soluble protein, and purified water are added at a mass ratio of 2.5:1:10. The reaction is carried out at 50°C for 4 hours at a pH of 6-8, and the Maillard reaction is performed to graft the alcohol-soluble protein onto the oligosaccharide acid-hydrolyzed agar to generate the grafted product.

[0040] After the reaction was completed, a portion of the grafted product was taken out and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining, Coomassie staining, and Shiff reagent staining confirmed that the sugar grafting reaction actually occurred and a glycoprotein complex was generated. The degree of grafting was then detected.

[0041] The method for determining grafting degree includes the following steps:

[0042] Preparation of OPA reagent: Dissolve 40.0 mg OPA in 1.0 mL methanol, add 2.5 mL of prepared 20% (w / w) sodium dodecyl sulfate (SDS), 25 mL of borax solution (0.1 mol / L), 100 μL of β-mercaptoethanol, and finally dilute to 50 mL with distilled water. Prepare fresh before use.

[0043] The grafting degree was calculated using the free amino content determined by OPA reagent: 200 μL of the test sample was mixed with 4.0 mL of OPA reagent and reacted in a 35°C constant temperature water bath for 2 min. The absorbance value A was measured at 340 nm using a UV spectrophotometer. 200 μL of distilled water was added to the blank group. The formula for calculating the grafting degree is shown below. Each sample was measured three times.

[0044]

[0045] In the formula: A0 is the absorbance value before reaction, and A1 is the absorbance value after saccharification.

[0046] After the S3 reaction, alcohol precipitation and dehydration are performed. Then, the ethanol is separated using supercritical carbon dioxide replacement technology to obtain a dried, cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The specific steps are as follows:

[0047] S3.1 The alcohol precipitation and dehydration steps include: precipitating the cold-soluble agar-alcohol-protein complex with 75% ethanol under a 40°C water bath; then dehydrating the cold-soluble agar-alcohol-protein complex with 95% ethanol to obtain a cold-soluble agar-alcohol-protein complex with low moisture content; the water content of the dehydrated cold-soluble agar-alcohol-protein complex is less than or equal to 30%.

[0048] S3.2 The grafted and dehydrated cold-soluble agar-alcohol-soluble protein complex was placed in a displacement vessel, and supercritical carbon dioxide was introduced at a temperature of 25°C and a pressure of 6.4 MPa. Ethanol and carbon dioxide were recovered and recycled through a separation vessel. The displacement extraction time was 4 hours. When no ethanol was observed in the separation vessel, drying continued. Then the inlet valve was closed, and under constant temperature, the gas was slowly released to atmospheric pressure. After cooling to room temperature, the dried agar-alcohol-soluble protein complex was obtained.

[0049] During gas intake, to prevent damage to the grafting of the complex, both the upper and lower inlet valves of the extraction vessel are fully opened. After the pressure stabilizes, the upper inlet valve is closed and the outlet valve is opened to slowly release the gas.

[0050] The entire process must be carried out slowly to avoid damaging the grafted complex in the reactor. When no ethanol can be observed in the separation reactor, the temperature is kept constant and the gas is slowly released to atmospheric pressure to obtain the dried cold-soluble agar-alcohol-soluble protein complex.

[0051] S4 The cold-soluble agar-alcohol-protein complex obtained from the reaction was subjected to gel strength testing and whipping performance testing in cream application, including foaming rate and foam stability.

[0052] Protein grafting degree 25 Gel strength of cold-soluble agar-alcohol-soluble protein complex after heating <![CDATA[120g / cm 2 ]]> Cold-melted agar-alcohol protein complex whipping cream foaming rate 118％ Cold-melted agar-alcohol protein complex for stable whipped cream foam 80％

[0053] Example 2

[0054] A method for preparing a cold-soluble agar-alcohol-soluble protein plant-based stabilizer includes the following steps:

[0055] S1 involves acid hydrolysis of the instant agar. The acid hydrolysis steps for the instant agar include:

[0056] A certain amount of our company's self-produced instant agar (patent number CN 107602729 A) was prepared into a 20% aqueous solution. Under continuous stirring, the solution was heated to 55°C to dissolve the agar for 10 minutes. Hydrochloric acid with an acidity of 0.1 mol / L was added, at a dosage of 12% of the agar powder added. The reaction was maintained at this temperature for 4 hours. The solution was then neutralized with sodium hydroxide. Vacuum filtration was performed, followed by concentration via hollow fiber nanofiltration and freeze-drying to obtain acid-hydrolyzed instant agar. The 5% strength of the acid-hydrolyzed instant agar was 210 g / cm³. 2 The agar raw material is derived from the red algae *Glechoma heliotrope*; the gliadin is wheat gliadin.

[0057] After the S2 reaction, alcohol precipitation and dehydration are performed. Then, the ethanol is separated using supercritical carbon dioxide replacement technology to obtain a dried, cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The specific steps are as follows:

[0058] First, the alcohol-soluble protein is dissolved in 50% alcohol at a mass ratio of 1:1. Then, 1 ton of acid-hydrolyzed agar, alcohol-soluble protein, and purified water are added at a mass ratio of 2.4:1:10. The reaction is carried out at 60°C for 4 hours at a pH of 8, and the Maillard reaction is performed to graft the alcohol-soluble protein onto the oligosaccharide acid-hydrolyzed agar to generate the grafted product.

[0059] After the reaction was completed, a portion of the grafted product was taken out and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining, Coomassie staining, and Shiff reagent staining confirmed that the sugar grafting reaction actually occurred and a glycoprotein complex was generated. The degree of grafting was then detected.

[0060] After the S3 reaction, alcohol precipitation and dehydration are performed. Then, the ethanol is separated using supercritical carbon dioxide replacement technology to obtain a dried, cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The specific steps are as follows:

[0061] S3.1 The alcohol precipitation and dehydration steps include: precipitating the cold-soluble agar-alcohol-protein complex with 75% ethanol under a 40°C water bath; then dehydrating the cold-soluble agar-alcohol-protein complex with 95% ethanol to obtain a cold-soluble agar-alcohol-protein complex with low moisture content; the water content of the dehydrated cold-soluble agar-alcohol-protein complex is less than or equal to 30%.

[0062] S3.2 The grafted and dehydrated cold-soluble agar-alcohol-soluble protein complex was placed in a displacement vessel, and supercritical carbon dioxide was introduced at a temperature of 20°C and a pressure of 5.7 MPa. Ethanol and carbon dioxide were recovered and recycled through a separation vessel. The displacement extraction time was 4 hours. When no ethanol was observed in the separation vessel, drying continued. Then the inlet valve was closed, and under constant temperature, the gas was slowly released to atmospheric pressure. After cooling to room temperature, the dried agar-alcohol-soluble protein complex was obtained.

[0063] S4 The cold-soluble agar-alcohol-protein complex obtained from the reaction was subjected to gel strength testing and whipping performance testing in cream application, including foaming rate and foam stability.

[0064]

[0065]

[0066] Example 3

[0067] The preparation method of cold-soluble agar-alcohol-soluble protein plant-based stabilizer includes the following steps:

[0068] S1 involves acid hydrolysis of the instant agar. The acid hydrolysis steps for the instant agar include:

[0069] A certain amount of our company's self-produced instant agar (patent number CN 107602729A) was prepared into a 25% aqueous solution. Under continuous stirring, the solution was heated to 55°C to dissolve the agar for 10 minutes. Hydrochloric acid with an acidity of 0.1 mol / L was added, at a dosage of 12% of the agar powder added. The reaction was maintained at this temperature for 4 hours. The solution was then neutralized with sodium hydroxide. Vacuum filtration was performed, followed by concentration via hollow fiber nanofiltration and freeze-drying to obtain acid-hydrolyzed instant agar. The 5% strength of the acid-hydrolyzed instant agar was 250 g / cm³. 2 The agar raw material is derived from the red algae *Glechoma heliotrope*; the gliadin is wheat gliadin.

[0070] After the S2 reaction, alcohol precipitation and dehydration are performed. Then, the ethanol is separated using supercritical carbon dioxide replacement technology to obtain a dried, cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The specific steps are as follows:

[0071] First, the alcohol-soluble protein was dissolved in 90% alcohol at a mass ratio of 0.6:1. Then, 1 ton of acid-hydrolyzed agar, alcohol-soluble protein, and purified water were added at a mass ratio of 2.3:1:10. The reaction was carried out at 70°C for 4 hours at a pH of 6, and the Maillard reaction was performed to graft the alcohol-soluble protein onto the oligosaccharide acid-hydrolyzed agar, generating the grafted product.

[0072] After the reaction was completed, a portion of the grafted product was taken out and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining, Coomassie staining, and Shiff reagent staining confirmed that the sugar grafting reaction actually occurred and a glycoprotein complex was generated. The degree of grafting was then detected.

[0073] After the S3 reaction, alcohol precipitation and dehydration are performed. Then, the ethanol is separated using supercritical carbon dioxide replacement technology to obtain a dried, cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The specific steps are as follows:

[0074] S3.1 The alcohol precipitation and dehydration steps include: precipitating the cold-soluble agar-alcohol-protein complex with 75% ethanol under a 40°C water bath; then dehydrating the cold-soluble agar-alcohol-protein complex with 95% ethanol to obtain a cold-soluble agar-alcohol-protein complex with low moisture content; the water content of the dehydrated cold-soluble agar-alcohol-protein complex is less than or equal to 30%.

[0075] S3.2 The grafted and dehydrated cold-soluble agar-alcohol-soluble protein complex was placed in a displacement vessel, and supercritical carbon dioxide was introduced at a temperature of 23°C and a pressure of 6.1 MPa. Ethanol and carbon dioxide were recovered and recycled through a separation vessel. The displacement extraction time was 4 hours. When no ethanol was observed in the separation vessel, drying continued. Then the inlet valve was closed, and under constant temperature, the gas was slowly released to atmospheric pressure. After cooling to room temperature, the dried agar-alcohol-soluble protein complex was obtained.

[0076] S4 The cold-soluble agar-alcohol-protein complex obtained from the reaction was subjected to gel strength testing and whipping performance testing in cream application, including foaming rate and foam stability.

[0077] Protein grafting degree 42 Gel strength of cold-soluble agar-alcohol-soluble protein complex after heating <![CDATA[120g / cm 2 ]]> Cold-melted agar-alcohol protein complex whipping cream foaming rate 142％ Cold-melted agar-alcohol protein complex for stable whipped cream foam 88％

[0078] Example 4

[0079] The preparation method of cold-soluble agar-alcohol-soluble protein plant-based stabilizer includes the following steps:

[0080] S1 involves acid hydrolysis of the instant agar. The acid hydrolysis steps for the instant agar include:

[0081] A certain amount of our company's self-produced instant agar (patent number CN 107602729A) was prepared into a 28% aqueous solution. Under continuous stirring, the solution was heated to 50°C to dissolve the agar for 10 minutes. Hydrochloric acid with an acidity of 0.1 mol / L was added, at a dosage of 12% of the agar powder added. The reaction was maintained at this temperature for 3 hours. The solution was then neutralized with sodium hydroxide. Vacuum filtration was performed, followed by concentration via hollow fiber nanofiltration and freeze-drying to obtain acid-hydrolyzed instant agar. The 5% strength of the acid-hydrolyzed instant agar was 280 g / cm³. 2 The agar raw material is derived from the red algae *Glechoma heliotrope*; the gliadin is wheat gliadin.

[0082] After the S2 reaction, alcohol precipitation and dehydration are performed. Then, the ethanol is separated using supercritical carbon dioxide replacement technology to obtain a dried, cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The specific steps are as follows:

[0083] First, the alcohol-soluble protein is dissolved in 90% alcohol at a mass ratio of 0.5:1. Then, 1 ton of acid-hydrolyzed agar, alcohol-soluble protein, and purified water are added at a mass ratio of 2.4:1:10. The reaction is carried out at 80℃ for 4 hours at a pH of 7, and the Maillard reaction is performed to graft the alcohol-soluble protein onto the oligosaccharide acid-hydrolyzed agar to generate the grafted product.

[0084] After the reaction was completed, a portion of the grafted product was taken out and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining, Coomassie staining, and Shiff reagent staining confirmed that the sugar grafting reaction actually occurred and a glycoprotein complex was generated. The degree of grafting was then detected.

[0085] After the S3 reaction, alcohol precipitation and dehydration are performed. Then, the ethanol is separated using supercritical carbon dioxide replacement technology to obtain a dried, cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The specific steps are as follows:

[0086] S3.1 The alcohol precipitation and dehydration steps include: precipitating the cold-soluble agar-alcohol-protein complex with 75% ethanol under a 40°C water bath; then dehydrating the cold-soluble agar-alcohol-protein complex with 95% ethanol to obtain a cold-soluble agar-alcohol-protein complex with low moisture content; the water content of the dehydrated cold-soluble agar-alcohol-protein complex is less than or equal to 30%.

[0087] S3.2 The grafted and dehydrated cold-soluble agar-alcohol-soluble protein complex was placed in a displacement vessel, and supercritical carbon dioxide was introduced at a temperature of 22°C and a pressure of 6.0 MPa. Ethanol and carbon dioxide were recovered and recycled through a separation vessel. The displacement extraction time was 4 hours. When no ethanol was observed in the separation vessel, drying continued. Then the inlet valve was closed, and under constant temperature, the gas was slowly released to atmospheric pressure. After cooling to room temperature, the dried agar-alcohol-soluble protein complex was obtained.

[0088] S4 The cold-soluble agar-alcohol-protein complex obtained from the reaction was subjected to gel strength testing and whipping performance testing in cream application, including foaming rate and foam stability.

[0089] Protein grafting degree 45 Gel strength of cold-soluble agar-alcohol-soluble protein complex after heating <![CDATA[100g / cm 2 ]]> Cold-melted agar-alcohol protein complex whipping cream foaming rate 159％ Cold-melted agar-alcohol protein complex for stable whipped cream foam 91％

[0090] Example 5

[0091] The preparation method of cold-soluble agar-alcohol-soluble protein plant-based stabilizer includes the following steps:

[0092] S1 involves acid hydrolysis of the instant agar. The acid hydrolysis steps for the instant agar include:

[0093] A certain amount of our company's self-produced instant agar (patent number CN 107602729 A) was prepared into a 10% aqueous solution. Under continuous stirring, the solution was heated to 55°C to dissolve the agar for 10 minutes. Hydrochloric acid with an acidity of 0.1 mol / L was added, at a dosage of 15% of the agar powder added. The reaction was maintained at this temperature for 3 hours. The solution was then neutralized with sodium hydroxide. Vacuum filtration was performed, followed by concentration via hollow fiber nanofiltration and freeze-drying to obtain acid-hydrolyzed instant agar. The 5% strength of the acid-hydrolyzed instant agar was 190 g / cm³. 2 The agar raw material is derived from the red algae *Glechoma heliotrope*; the gliadin is wheat gliadin.

[0094] After the S2 reaction, alcohol precipitation and dehydration are performed. Then, the ethanol is separated using supercritical carbon dioxide replacement technology to obtain a dried, cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The specific steps are as follows:

[0095] First, the alcohol-soluble protein was dissolved in 90% alcohol at a mass ratio of 0.5:1. Then, 1 ton of acid-hydrolyzed agar, alcohol-soluble protein, and purified water were added at a mass ratio of 2.2:1:10. The reaction was carried out at 90℃ for 2 hours at a pH of 7, and the Maillard reaction was performed to graft the alcohol-soluble protein onto the oligosaccharide acid-hydrolyzed agar, generating a grafted product.

[0096] After the reaction was completed, a portion of the grafted product was taken out and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining, Coomassie staining, and Shiff reagent staining confirmed that the sugar grafting reaction actually occurred and a glycoprotein complex was generated. The degree of grafting was then detected.

[0097] After the S3 reaction, alcohol precipitation and dehydration are performed. Then, the ethanol is separated using supercritical carbon dioxide replacement technology to obtain a dried, cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The specific steps are as follows:

[0098] S3.1 The alcohol precipitation and dehydration steps include: precipitating the cold-soluble agar-alcohol-protein complex with 75% ethanol under a 40°C water bath; then dehydrating the cold-soluble agar-alcohol-protein complex with 95% ethanol to obtain a cold-soluble agar-alcohol-protein complex with low moisture content; the water content of the dehydrated cold-soluble agar-alcohol-protein complex is less than or equal to 30%.

[0099] S3.2 The grafted and dehydrated cold-soluble agar-alcohol-soluble protein complex was placed in a displacement vessel, and supercritical carbon dioxide was introduced at a temperature of 20°C and a pressure of 5.7 MPa. Ethanol and carbon dioxide were recovered and recycled through a separation vessel. The displacement extraction time was 4 hours. When no ethanol was observed in the separation vessel, drying continued. Then the inlet valve was closed, and under constant temperature, the gas was slowly released to atmospheric pressure. After cooling to room temperature, the dried agar-alcohol-soluble protein complex was obtained.

[0100] S4 The cold-soluble agar-alcohol-protein complex obtained from the reaction was subjected to gel strength testing and whipping performance testing in cream application, including foaming rate and foam stability.

[0101] Protein grafting degree 35 Gel strength of cold-soluble agar-alcohol-soluble protein complex after heating <![CDATA[110g / cm 2 ]]> Cold-melted agar-alcohol protein complex whipping cream foaming rate 126％ Cold-melted agar-alcohol protein complex for stable whipped cream foam 86％

[0102] Note: Table 1 below is a collection of experimental data from Examples 1 to 5.

[0103]

[0104] The performance of the cold-soluble agar-alcohol-soluble protein plant-based stabilizer prepared in this invention was tested:

[0105] 1. Solubility

[0106] Test samples: cold-soluble agar-alcohol-soluble protein plant-based stabilizers prepared in Examples 1-5; control group 1 was the grafted product prepared in step S2 of Example 5; control group 2 was the cold-soluble agar-alcohol-soluble protein complex with low moisture content obtained in step S3.1 of Example 5; and the blank group was instant agar.

[0107] Test method: Take 10g of the test sample and dissolve it in 20ml of pure water. Heat the solution to 30℃ and test its solubility and gel properties. After the test is completed, continue heating to 50℃ and then cool it down to room temperature of 25℃ to check its gel properties.

[0108] Testing showed that the cold-soluble agar-alcohol-soluble protein plant-based stabilizers prepared in Examples 1-5 were soluble at 30°C but did not gel. After heating to above 50°C and then returning to room temperature of 25°C, they all formed a stable porous gel texture, fully demonstrating that the cold-soluble agar-alcohol-soluble protein plant-based stabilizers prepared in Examples 1-5 of this invention have good cold water solubility. The preparation method of the cold-soluble agar-alcohol-soluble protein plant-based stabilizer of this invention controls the reaction concentration, temperature, and alcohol content of acid-hydrolyzed fast-dissolving agar and alcohol-soluble protein. Through the Maillard reaction, alcohol-soluble protein is grafted onto the acid-hydrolyzed fast-dissolving agar, improving emulsifying properties while altering dissolution and gelation properties, resulting in good low-temperature rapid solubility. Acid hydrolysis of the fast-dissolving agar provides more terminal carbonyl groups, which is beneficial for increasing the grafting degree of alcohol-soluble protein. The Maillard reaction grafts alcohol-soluble protein onto agarose to improve the functional properties of the polysaccharide complex, thereby enhancing the functional properties of the polysaccharide-protein complex, such as emulsifying properties, thermal stability, solubility, and anti-allergenic properties.

[0109] Control group 1 is the grafted product of Example 5. The difference between control group 1 and the cold-soluble agar-alcohol-soluble protein plant-based stabilizer in Example 5 is that it has not undergone two alcohol precipitations or supercritical carbon dioxide replacement treatment. Its solubility test results are as follows: it can dissolve at 30°C but does not gel. After heating to above 50°C and then returning to room temperature of 25°C, it does not gel.

[0110] Control group 2 is the low-moisture content cold-soluble agar-alcohol-soluble protein complex of Example 5. The difference between control group 2 and the cold-soluble agar-alcohol-soluble protein plant-based stabilizer in Example 5 is that after two alcohol precipitations, it was not subjected to supercritical carbon dioxide replacement treatment. Its solubility test results are as follows: it can dissolve at 30°C, but does not gel. After heating to above 50°C and then returning to room temperature of 25°C, the gelation effect is poor and unstable.

[0111] The above test results fully demonstrate that in the preparation method of the cold-soluble agar-alcohol-soluble protein plant-based stabilizer prepared in this invention, after the Maillard reaction, two alcohol precipitations are performed. In the solution, the agar molecular chains are in a random dispersion state, and they usually entangle with each other to form a double helix structure when cooled. In this invention, ethanol is used to precipitate the solution. After ethanol enters the interstices of the molecular chains, it hinders the formation of double helix chains through steric hindrance. In order to prevent the cold-soluble agar from forming double helix chains again during the drying process as the alcohol evaporates, supercritical carbon dioxide technology is used to replace the alcohol. The alcohol is replaced in the replacement vessel, and at the same time, the formation of double helixes is hindered by the replacement of carbon dioxide during this process. Thus, the cold-soluble agar-alcohol-soluble protein complex is prepared, which gives it good cold water solubility, dissolves at room temperature (30°C) but does not gel, and forms a stable porous gel texture after being heated to above 50°C and returning to room temperature.

[0112] The preparation method of the cold-soluble agar-alcohol-soluble protein plant-based stabilizer prepared by the present invention is essentially to improve the hydrophilic-lipophilic balance of the covalent complex by utilizing the polyhydroxy properties of the biomolecule polysaccharide, improve the thermal stability of the complex by utilizing the random coil structure of the polysaccharide, and improve the interfacial spatial stability of the complex by utilizing the macromolecular nature of the polysaccharide.

[0113] 2. Emulsifying properties

[0114] Test samples: The experimental group consisted of cold-soluble agar-alcohol-soluble protein plant-based stabilizers prepared in Examples 1-5; the control group 1 consisted of animal-based colloidal gelatin; and the blank group consisted of instant-soluble agar.

[0115] Test method:

[0116] Accurately weigh 0.5 g of sample into a 250 mL beaker, add 50 mL of deionized water, heat to dissolve (40 °C) for a certain time, then slowly add 5 mL of soybean salad oil under magnetic stirring, and stir with a high-speed disperser at 20500 r / min for 1 min. Immediately after stirring, take 1 mL of the emulsion into a 100 mL volumetric flask, add 0.1% SDS solution to the mark and shake well, then measure the absorbance of the sample at 500 nm. Additionally, immediately after stirring, take 25 mL of sample into a stoppered test tube and place it in a 30 °C constant temperature water bath for 1 h. After 1 h, remove the test tube and gently shake to mix the sample. Then take 1 mL of sample and dilute it again using the above method, and measure the absorbance of the diluted sample at 500 nm.

[0117] The turbidity of the sample is T, where T = 2.303A500 / I, and I is the optical path length, which is 1 cm in this case. Since the same dilution factor is used, the emulsifying ability (EC) of the emulsifier can be directly expressed by the turbidity (T1) of the sample after stirring.

[0118] Emulsion stability (ES) is expressed as the degree of change in turbidity (T2) after 1 hour, ES = T2 / T1.

[0119] The test results are as follows:

[0120]

[0121]

[0122] The above results indicate that the cold-soluble agar-alcohol-soluble protein plant-based stabilizers prepared in Examples 1-5 of this invention have better emulsifying properties. The emulsifying index (T) and emulsifying stability (%) are close to those of gelatin in the prior art. The blank group, instant agar—untreated, clearly shows that the instant agar without modification by the preparation method of this invention has poor emulsifying properties and cannot replace existing gelatin.

[0123] 3. Homogenization of cream emulsion:

[0124] The following are the methods for determining the whipping performance of the cold-soluble agar-alcohol-soluble protein plant-based stabilizer of the present invention in butter applications, including the foaming rate and foam stability testing methods:

[0125] Add 0.09% cold-soluble agar-alcohol protein plant-based stabilizer to the cream, and adjust the pressure of the high-pressure homogenizer to 20MPa, 30MPa, 40MPa, 50MPa and 60MPa respectively. Add the cream emulsion to the inlet, homogenize once, and collect the cream emulsion at the outlet.

[0126] Cream whipping performance test - foaming rate

[0127] Use a household electric mixer to beat the cream, employing a speed cycle of low (30s), high (180s), and low (30s) until soft peaks form. Stop beating when soft peaks appear. Weigh equal volumes of the cream emulsion before and after beating, and calculate the foaming rate as follows:

[0128]

[0129] In the formula: m1 is the mass (g) of the cream emulsion before whipping, and m2 is the mass (g) of the cream after whipping.

[0130] Cream whipping performance test - foam stability

[0131] A certain amount of whipped butter was placed on a copper wire mesh at room temperature for 2 hours. The weight of the separated water was collected and calculated as follows:

[0132]

[0133] In the formula: m1 is the weight of the collected water extracted from the butter (g), and m2 is the initial weight of the butter (g).

[0134] Tests showed that the cold-soluble agar-alcohol-protein plant-based stabilizers prepared in Examples 1-5 of this invention have foaming rates and foam stability that meet the requirements for whipping butter, and have good effects in the field of whipping butter.

[0135] From the above table and Figure 1 , Figure 2 As shown, the results of emulsification tests—including the emulsification index (T) and emulsification stability (%)—as well as the homogenization, foaming rate, and foam stability of the cream emulsion, indicate that the cold-soluble agar-alcohol protein plant-based stabilizer prepared in Examples 1-5 of this invention has excellent emulsification properties. It can effectively replace animal-based colloids in whipping cream. Compared to gelatin, it is stable at room temperature and does not melt, resulting in a refreshing and non-greasy taste. It can replace gelatin in the preparation of mousse products and gel-based candies, and it can also replace egg whites in the preparation of baked goods, thus finding good applications in the field of food additive preparation technology.

[0136] In summary, the cold-soluble agar-alcohol-soluble protein plant-based stabilizer, its preparation method, and its application of the present invention, by controlling the reaction concentration, temperature, and alcohol content of acid-hydrolyzed fast-dissolving agar and alcohol-soluble protein, grafts alcohol-soluble protein onto acid-hydrolyzed fast-dissolving agar via Maillard reaction, thereby improving emulsifying performance and altering dissolving and gelling properties, giving it excellent low-temperature rapid solubility. It can be widely applied in the field of food additive preparation technology.

[0137] The above are merely embodiments of the present invention. For example, the agar raw material can be derived from one or two of the red algae Gracilaria and Gracilaria; the prolysin can be one or two of the wheat prolysin and zein. All of these can realize the cold-soluble agar-prolysin plant-based stabilizer, preparation method and application of the present invention.

[0138] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a cold-soluble agar-alcohol-soluble protein plant-based stabilizer, characterized in that, The steps include the following: The instant agar was acid-hydrolyzed and dissolved at high temperature with alcohol-soluble protein. The alcohol-soluble protein was grafted onto the acid-hydrolyzed instant agar through Maillard reaction. After the reaction, alcohol precipitation and dehydration were performed. The ethanol was then separated by supercritical carbon dioxide replacement technology to obtain a dry cold-soluble agar-alcohol-soluble protein plant-based stabilizer. The agar raw material is derived from one or two of the red algae Gracilaria and Gracilaria; the prolysin is one or two of the wheat prolysin and zein. Prepare an aqueous solution with a mass fraction of 5%-30% using a certain amount of instant agar. Stir continuously and heat to 50-55℃ to dissolve the agar for 5-10 minutes. Add hydrochloric acid with an acidity of 0.1-0.2 mol / L, using 10%-20% of the amount of agar powder added. Keep the reaction at this temperature for 2-4 hours. Then neutralize with sodium hydroxide to neutral. Vacuum filter, concentrate and freeze-dry using hollow fiber nanofiltration to obtain acid-hydrolyzed instant agar. The strength range of the acid-hydrolyzed rapid-dissolving agar is 100-300 g / cm³. 2 ; First, dissolve the prolactin in 50%-90% alcohol, with the mass ratio of prolactin to alcohol being 0.5-1:

1. Next, the acid-hydrolyzed instant agar, alcohol-soluble protein, and purified water were subjected to Maillard reaction at a mass ratio of 2-2.5:1:10 to graft the alcohol-soluble protein onto the oligosaccharide acid-hydrolyzed instant agar. The reaction temperature was 50℃-90℃, the reaction time was 2h-4h, and the reaction pH was 6-8 to generate the grafted product. The alcohol precipitation and dehydration steps include: precipitating the cold-soluble agar-alcohol-soluble protein complex with 75% ethanol under a 40°C water bath condition; The cold-soluble agar-alcohol-protein complex was then dehydrated with 95% ethanol to obtain a cold-soluble agar-alcohol-protein complex with low moisture content; the water content of the dehydrated cold-soluble agar-alcohol-protein complex was less than or equal to 30%. The grafted and dehydrated cold-soluble agar-alcohol-soluble protein complex was placed in a displacement vessel, and supercritical carbon dioxide was introduced. The temperature was 20℃-25℃, the pressure was 5.7MPa-6.4MPa, and the displacement time was 2h-4h. During gas intake, to prevent damage to the grafting of the complex, both the upper and lower inlet valves of the extraction vessel are fully opened. After the pressure stabilizes, the upper inlet valve is closed and the outlet valve is opened to release the gas slowly. Ethanol and carbon dioxide are recovered and recycled via a separation vessel; The entire process must be carried out slowly to avoid damaging the grafted complex in the vessel. When no ethanol can be observed in the separation vessel, maintain a constant temperature and continue to slowly release the gas to atmospheric pressure to obtain a dry, cold-soluble agar-alcohol-soluble protein complex.

2. The method for preparing a cold-soluble agar-alcohol-soluble protein plant-based stabilizer according to claim 1, characterized in that, The grafted product was confirmed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis to show that the sugar grafting reaction actually occurred and a glycoprotein complex was generated.

3. The method for preparing a cold-soluble agar-alcohol-soluble protein plant-based stabilizer according to claim 1, wherein the method for determining the grafting degree includes the following steps: Preparation of OPA reagent: Dissolve 40.0 mg OPA in 1.0 mL of methanol, add 2.5 mL of 20% w / w sodium dodecyl sulfate, 25 mL of 0.1 mol / L borax solution, 100 μL of β-mercaptoethanol, and finally dilute to 50 mL with distilled water. Prepare fresh before use. The grafting degree was calculated using the free amino content determined by OPA reagent: 200 μL of the test sample was mixed with 4.0 mL of OPA reagent and reacted in a 35℃ constant temperature water bath for 2 min. The absorbance value A was measured at 340 nm using a UV spectrophotometer. 200 μL of distilled water was added to the blank group. The formula for calculating the grafting degree is shown below. Each sample was measured three times. ， In the formula: A0 is the absorbance value before reaction, and A1 is the absorbance value after grafting.

4. A cold-soluble agar-alcohol-soluble protein plant-based stabilizer prepared by the method of any one of claims 1-3.

5. The application of the cold-soluble agar-alcohol-soluble protein plant-based stabilizer as described in claim 4 in the preparation of food additives.

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