Agar oligosaccharide with antioxidant function as well as preparation method and application thereof

Abstract

The invention provides agar oligosaccharide with an antioxidant function as well as a preparation method and application of the agar oligosaccharide, and belongs to the technical field of degradation of natural polysaccharides. According to the method, a H2O2 / Fe < 2 + > system is adopted to generate hydroxyl free radicals to degrade agar polysaccharide, glycosidic bonds in the agar polysaccharide are oxidized and broken by H2O2 under the catalysis of Fe < 2 + >, so that the agar polysaccharide is degraded into low-molecular fragments, agar oligosaccharide with relatively good antioxidant activity is obtained, then the agar oligosaccharide is compounded with vitamin C, and the antioxidant activity of the agar oligosaccharide is improved. The color of the freeze-dried and redissolved agar oligosaccharide solution can be reduced, and meanwhile, the degraded agar oligosaccharide can be endowed with stronger antioxidant activity. The preparation method provided by the invention is simple in process flow, low in equipment requirement, low in cost and high in agar oligosaccharide yield which can reach 85% or above, and is suitable for industrial large-scale popularization.

Description

Technical Field

[0001] The invention relates to the technical field of natural polysaccharide degradation, and in particular to agar oligosaccharide with antioxidant function and a preparation method and application thereof. Background Art

[0002] Agar, scientifically known as agar, also known as agar, jelly powder, agar essence and agar, is a seaweed polysaccharide extracted from seaweeds such as Gracilaria, Gelidium and Glechoma. The basic structural unit of agar is agar disaccharide unit, namely 1,3-linked β-D-galactose and 1,4-linked 3,6-anhydroendoether-α-L-galactose units. Agar is a hydrophilic colloid with strong water absorption, high gel strength and relatively constant viscosity, but agar has high viscosity and poor solubility in water, which limits its wide application. The structure of agar determines its functional properties. In order to break through the limitations of agar in application, some scholars have conducted research on structural modification to achieve the needs of improving its application properties through chemical modification. The content mainly includes the degradation of agar and the introduction of chemical group modification.

[0003] At present, the most commonly used hydrolysis methods for agar hydrolysis are acid hydrolysis and enzymatic hydrolysis. Acid hydrolysis is a traditional method for degrading agar. Acid can act on the α-(1,3) glycosidic bond of agar, and the generated product is an ultra-oligosaccharide or agar oligosaccharide with β-D-galactose as the non-reducing end and 3,6-endo-α-L-galactose as the reducing end. The reaction conditions of the acid hydrolysis method are drastic, and the production equipment is required to be high. The uniformity of the obtained product is poor, and there are problems such as product analysis and recovery difficulties. At the same time, the acid substances used in the production process will pollute the environment. Enzymatic hydrolysis is a method of degrading agar using agarase. According to the different ways in which agarase cleaves glycosidic bonds, it can be divided into α-agarase and β-agarase. Agarase is an important class of seaweed polysaccharide degrading enzymes that can catalyze the cleavage of glycosidic bonds in agar. Since the enzyme has strict requirements for temperature control, the enzymatic hydrolysis method has high requirements for equipment, and the impurity content is high during the preparation process.

[0004] Fermented yogurt is a product obtained by lactic acid fermentation with fresh milk as the main raw material, adding fermentation bacteria, sweeteners and other auxiliary materials. However, whey precipitation and uneven texture are problems that affect the stability of yogurt during the production and storage of yogurt. In order to solve the above problems, some stabilizers are added during the production of yogurt to improve the quality and stability of yogurt. At present, most of the stabilizers on the market are applied to the production process of yogurt in the form of compound polysaccharides, and there are few reports on the addition of only agar oligosaccharides as stabilizers in the production process of yogurt.

[0005] Therefore, providing an agar-oligosaccharide with a simple preparation method and antioxidant function so that it can be used as a stabilizer in the yogurt production process has become a technical problem to be urgently solved in the field. Summary of the invention

[0006] The object of the present invention is to provide an agar oligosaccharide with antioxidant function and a preparation method and application thereof. The preparation method provided by the present invention has a simple operation process, and the obtained agar oligosaccharide has excellent antioxidant function and can be applied alone as a stabilizer in the production process of fermented yogurt to improve the quality and stability of yogurt.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing agaro-oligosaccharide with antioxidant function, comprising the following steps:

[0009] (1) mixing agar polysaccharide and water, and then heating and dissolving them to obtain an agar polysaccharide solution;

[0010] (2) adding H2O2 solution and divalent iron salt solution to the agar polysaccharide solution obtained in step (1) to carry out a degradation reaction to obtain an agar polysaccharide degradation solution;

[0011] (3) Adding vitamin C solution to the agar polysaccharide degradation solution obtained in step (2), and drying to obtain agar oligosaccharides with antioxidant function.

[0012] Preferably, the concentration of the agar polysaccharide solution in step (1) is 2 to 15 g / L.

[0013] Preferably, the mass concentration of the H2O2 solution in step (2) is 20-40%.

[0014] Preferably, the concentration of the divalent iron salt solution in step (2) is 4 to 40 mg / mL.

[0015] Preferably, in step (2), the volume ratio of agar polysaccharide solution, H2O2 solution and divalent iron salt solution is 40:(20-25):(0.5-3).

[0016] Preferably, the temperature of the degradation reaction in step (2) is 50-70° C., and the time of the degradation reaction is 3-5 hours.

[0017] Preferably, the concentration of the vitamin C solution in step (3) is 30 to 50 mg / mL.

[0018] Preferably, the volume ratio of the agar polysaccharide solution in step (1) to the vitamin C solution in step (3) is 40:(0.5-2).

[0019] The present invention provides agaro-oligosaccharides with antioxidant function prepared by the preparation method described in the above technical scheme.

[0020] The present invention provides the use of the agar-oligosaccharide with antioxidant function described in the above technical solution as a stabilizer in fermented yogurt.

[0021] The present invention provides a method for preparing agar oligosaccharides with antioxidant function, comprising the following steps: (1) mixing agar polysaccharides and water, and then heating and dissolving them to obtain an agar polysaccharide solution; (2) adding H2O2 solution and divalent iron salt solution to the agar polysaccharide solution obtained in step (1) to carry out degradation reaction to obtain an agar polysaccharide degradation solution; (3) adding vitamin C solution to the agar polysaccharide degradation solution obtained in step (2), and drying to obtain agar oligosaccharides with antioxidant function. The present invention adopts H2O2 / Fe 2+ The system produces hydroxyl radicals to degrade agar polysaccharide, and H2O2 is used in Fe 2+ Under the catalysis of, the glycosidic bonds in agar polysaccharide are oxidatively broken, so that agar polysaccharide is degraded into low molecular weight fragments, and agar oligosaccharide with good antioxidant activity is obtained, which is then compounded with vitamin C. Adding vitamin C can reduce the color of the agar oligosaccharide solution after freeze-drying and re-dissolving, and at the same time can give the degraded agar oligosaccharide stronger antioxidant activity. The preparation method provided by the present invention has a simple process flow, low requirements for equipment, low cost, and a high yield of agar oligosaccharide, which can reach more than 85%, and is suitable for large-scale industrial promotion. The results of the embodiment show that the prepared agar oligosaccharide is subjected to antioxidant activity determination, and the results show that the antioxidant activity of the degraded agar oligosaccharide is significantly improved compared with that of the undegraded agar polysaccharide; the yogurt with added agar oligosaccharide is subjected to centrifugal sedimentation rate, particle size determination, and antioxidant activity determination during storage, and the results show that the addition of agar oligosaccharide increases the centrifugal sedimentation rate of fermented yogurt, reduces the particle size of yogurt, and has a longer antioxidant activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a physical picture of the agar polysaccharide solution with added H2O2 obtained in Comparative Examples 1 to 4;

[0023] Figure 2 The actual picture of the agar polysaccharide solution with added H2O2 and FeSO4·7H2O obtained in Comparative Examples 5 to 7;

[0024] Figure 3 The actual pictures of the agar polysaccharide solutions obtained in Comparative Examples 8 to 9 with the addition of H2O2 and FeSO4·7H2O and the addition of H2O2 and vitamin C;

[0025] Figure 4 The actual pictures are of the agar oligosaccharide solutions before vacuum freeze drying obtained in Comparative Example 10 and Example 1;

[0026] Figure 5 This is a physical picture of the vacuum freeze-dried agar-oligosaccharide obtained in Example 1;

[0027] Figure 6 This is a comparison chart of ·OH radical scavenging rates of agar oligosaccharide, vitamin C, H2O2, FeSO4·7H2O and vitamin C mixed solution, and agar solution;

[0028] Figure 7 It is a comparison chart of DPPH scavenging rate of agar oligosaccharide, vitamin C, H2O2, FeSO4·7H2O and vitamin C mixture, and agar solution;

[0029] Figure 8 This is a graph showing the reduction power of agar oligosaccharides;

[0030] Fig. 9 For comparison, the actual pictures of yogurt at different angles after 8h fermentation in Application Example 1 are shown;

[0031] Fig.10 This is a physical picture of yogurt at different angles after 12 hours of fermentation in Application Example 1;

[0032] Fig.11 A comparison chart of the centrifugal sedimentation rates of the yogurt obtained in Application Examples 1 to 7 and Comparative Application Example 1 on the 2nd, 4th and 8th days;

[0033] Fig.12 A comparison chart of yogurt particle sizes obtained in Application Examples 1 to 7 and Comparative Application Example 1;

[0034] Fig.13 This is a comparison chart of the OH radical scavenging rates of the yogurts obtained in Application Example 1 and Comparative Application Example 1. DETAILED DESCRIPTION

[0035] The present invention provides a method for preparing agaro-oligosaccharide with antioxidant function, comprising the following steps:

[0036] (1) mixing agar polysaccharide and water, and then heating and dissolving them to obtain an agar polysaccharide solution;

[0037] (2) adding H2O2 solution and divalent iron salt solution to the agar polysaccharide solution obtained in step (1) to carry out degradation reaction to obtain an agar polysaccharide degradation solution;

[0038] (3) Adding vitamin C solution to the agar polysaccharide degradation solution obtained in step (2), and drying to obtain agar oligosaccharides with antioxidant function.

[0039] In the present invention, unless otherwise specified, the raw materials used are commercially available products well known to those skilled in the art or products prepared by methods well known to those skilled in the art.

[0040] The invention mixes agar polysaccharide and water, and then heats and dissolves them to obtain an agar polysaccharide solution.

[0041] In the present invention, the agar polysaccharide is preferably agar polysaccharide powder. The present invention has no particular limitation on the particle size of the agar polysaccharide powder, and commercially available agar polysaccharide powder can be used.

[0042] In the present invention, the water is preferably deionized water. The use of deionized water in the present invention can reduce the impurity content.

[0043] The present invention has no particular limitation on the specific operation of mixing the agar polysaccharide and water, and any method known to those skilled in the art that can completely mix the agar polysaccharide and water can be used.

[0044] In the present invention, the temperature of the heating dissolution is preferably ≥ 90° C. The present invention can dissolve agar polysaccharide insoluble in cold water in hot water by heating; and can completely dissolve agar polysaccharide in water by controlling the heating temperature, thereby forming a uniform solution.

[0045] As an embodiment of the present invention, the temperature of heating and dissolving may be 92°C, 93°C, 94°C, 95°C and 96°C.

[0046] In the present invention, the concentration of the agar polysaccharide solution is preferably 2 to 15 g / L. By controlling the concentration of the agar polysaccharide solution, the present invention can ensure that the agar polysaccharide is completely dissolved in water to avoid residue, and is also beneficial for controlling the dosage relationship between the agar polysaccharide and the subsequently added H2O2 and divalent iron salt.

[0047] As an embodiment of the present invention, the concentration of the agar polysaccharide solution can be 2 g / L, 5 g / L, 7 g / L, 10 g / L, 12 g / L and 15 g / L.

[0048] After obtaining the agar polysaccharide solution, the present invention adds H2O2 solution and FeSO4.7H2O solution into the agar polysaccharide solution to carry out degradation reaction, thereby obtaining the agar polysaccharide degradation solution.

[0049] In the present invention, the temperature of the agaropolysaccharide solution is preferably 50-70°C when the H2O2 solution and the FeSO4·7H2O solution are added. In the present invention, when the temperature of the agaropolysaccharide solution does not meet the above requirements, the present invention preferably cools the agaropolysaccharide solution naturally. As an embodiment of the present invention, the temperature of the agaropolysaccharide solution can be 50°C, 55°C, 60°C, 65°C and 70°C. By controlling the temperature of the agaropolysaccharide solution, the present invention can react directly under the condition of water bath heating after adding the H2O2 solution and the FeSO4·7H2O solution.

[0050] In the present invention, the mass concentration of the H2O2 solution is preferably 20-40%; the solvent of the H2O2 solution is preferably deionized water. 2+ Under the catalysis of , the glycosidic bonds in agaropolysaccharide are oxidatively broken, thereby degrading agaropolysaccharide into low molecular weight fragments.

[0051] As an embodiment of the present invention, the mass concentration of the H2O2 solution can be 20%, 25%, 30%, 35% and 40%.

[0052] In the present invention, the divalent iron salt is preferably FeSO4·7H2O; the concentration of the divalent iron salt solution is preferably 4-40 mg / mL; and the solvent of the divalent iron salt solution is preferably deionized water. 2+ Catalyze H2O2 to oxidatively break the glycosidic bonds in agaropolysaccharide, thereby degrading agaropolysaccharide into low molecular weight fragments.

[0053] As an embodiment of the present invention, the concentration of the divalent iron salt solution can be 4 mg / mL, 8 mg / mL, 12 mg / mL, 16 mg / mL, 20 mg / mL, 24 mg / mL, 28 mg / mL, 32 mg / mL, 36 mg / mL and 40 mg / mL.

[0054] In the present invention, the preparation method of the ferrous iron salt solution is preferably: dissolving 0.4 g of ferrous iron salt powder in 10 mL of deionized water to obtain a 40 mg / mL ferrous iron salt solution, and then adding deionized water to the 40 mg / mL ferrous iron salt solution as needed to dilute it to obtain the desired ferrous iron salt solution. The preparation method provided by the present invention can accurately control the concentration of the ferrous iron salt solution.

[0055] In the present invention, the volume ratio of the agar polysaccharide solution, the H2O2 solution and the divalent iron salt solution is preferably 40: (20-25): (0.5-3), and more preferably 40: (20-21): (1-1.8). The present invention can control the dosage relationship of agar polysaccharide, H2O2 and divalent iron salt by controlling the volume ratio of the agar polysaccharide solution, the H2O2 solution and the divalent iron salt solution, thereby ensuring that the agar polysaccharide can be completely degraded into agaro-oligosaccharides under the action of H2O2 and divalent iron salt.

[0056] In the present invention, the temperature of the degradation reaction is preferably 50-70°C; the time of the degradation reaction is preferably 3-5 hours; and the degradation reaction is preferably carried out under the condition of water bath heating. The present invention can ensure that agar polysaccharide is completely degraded into agar oligosaccharides by controlling the parameters of the degradation reaction.

[0057] As an embodiment of the present invention, the temperature of the water bath heating can be 50°C, 55°C, 60°C, 65°C and 70°C; the time of the degradation reaction can be 3h, 4h and 5h.

[0058] The present invention adopts H2O2 / Fe 2+ The system produces hydroxyl radicals to degrade agar polysaccharide, and H2O2 is used in Fe 2+ Under the catalysis of , the glycosidic bonds in agar polysaccharide are oxidatively broken, thereby degrading agar polysaccharide into low molecular weight fragments. The degradation mechanism is as follows:

[0059] Initiation process: Fe 2+ +H2O2→Fe 3+ + OH+OH

[0060] Growth process:

[0061]

[0062] Termination process: OH+Fe 2+ →Fe 3+ +OH

[0063]

[0064]

[0065] After obtaining the agar polysaccharide degradation solution, the present invention adds a vitamin C solution to the agar polysaccharide degradation solution, and obtains agar oligosaccharides with antioxidant function after drying.

[0066] In the present invention, the concentration of the vitamin C solution is preferably 30 to 50 mg / mL.

[0067] As an embodiment of the present invention, the concentration of the vitamin C solution can be 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL and 50 mg / mL.

[0068] In the present invention, the method for preparing the vitamin C solution is preferably: dissolving vitamin C powder in deionized water to obtain the vitamin C solution.

[0069] In the present invention, the volume ratio of the agar polysaccharide solution to the vitamin C solution is preferably 40: (0.5-2), more preferably 40: 1. The addition of vitamin C in the present invention can reduce the color of the agar oligosaccharide solution after freeze-drying and reconstitution, and can also give the degraded agar oligosaccharide stronger antioxidant activity.

[0070] In the present invention, the drying method is preferably vacuum freeze drying. The present invention has no special restrictions on the specific operation and process parameters of the vacuum freeze drying, which can be determined according to the technical common sense of those skilled in the art as long as the solvent can be removed.

[0071] The present invention adopts H2O2 / Fe 2+ The system produces hydroxyl radicals to degrade agar polysaccharide, and H2O2 is used in Fe 2+ Under the catalysis of , the glycosidic bonds in agar polysaccharide are oxidatively broken, thereby degrading agar polysaccharide into low molecular weight fragments to obtain agar oligosaccharides with good antioxidant activity, which are then compounded with vitamin C. The addition of vitamin C can reduce the color of the agar oligosaccharide solution after freeze-drying and re-dissolving, and at the same time can give the degraded agar oligosaccharides stronger antioxidant activity.

[0072] The preparation method provided by the invention has simple process flow, low requirements on equipment, low cost, and high yield of agaro-oligosaccharide, which can reach more than 85%, and is suitable for large-scale industrial promotion.

[0073] The present invention also provides agar oligosaccharides with antioxidant function prepared by the preparation method described in the above technical scheme.

[0074] The agar oligosaccharide provided by the invention has good solubility and good antioxidant activity.

[0075] The present invention also provides the use of the agar-oligosaccharide with antioxidant function described in the above technical solution in fermented yogurt.

[0076] In the present invention, the use of agar-oligosaccharide in fermented yogurt preferably comprises the following steps:

[0077] 1) mixing milk, white sugar, baking powder and agar-oligosaccharide to obtain a mixture;

[0078] 2) fermenting the mixture obtained in step 1) to obtain fermented yogurt.

[0079] The present invention preferably mixes milk, white granulated sugar, baking powder and agar-oligosaccharide to obtain a mixture.

[0080] The present invention has no particular limitation on the specific sources of the milk, white sugar and baking powder, and commercially available products well known to those skilled in the art may be used.

[0081] In the present invention, the mass ratio of the milk, white sugar and baking powder is preferably 50:(3-6):(0.01-0.2), and more preferably 50:5:0.1.

[0082] In the present invention, the mass of the agar-oligosaccharide is preferably 0.01-0.1% of the total mass of milk, white sugar and baking powder, and more preferably 0.01%, 0.02%, 0.03%, 0.04%, 0.06%, 0.08% or 0.1%.

[0083] After obtaining the mixture, the present invention preferably ferments the mixture to obtain fermented yogurt.

[0084] In the present invention, the fermentation temperature is preferably 43° C. By controlling the fermentation temperature, the present invention can further improve the stability of the fermentation process and avoid deterioration or incomplete fermentation.

[0085] The invention can significantly improve the product texture and product stability of the fermented yogurt by applying the prepared agar-oligosaccharide to the fermented yogurt.

[0086] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0087] Comparative Examples 1 to 4

[0088] (1) Weighing 0.08 g, 0.2 g, 0.28 g and 0.4 g of agar polysaccharide powder, respectively, added them into 40 mL of deionized water, and then heated to 95° C. for dissolution to obtain agar polysaccharide solutions with concentrations of 2 g / L, 5 g / L, 7 g / L and 10 g / L, respectively;

[0089] (2) The agar polysaccharide solution obtained in step (1) was cooled to 60° C., and then 3 mL of a 30% H2O2 solution was added to the agar polysaccharide solutions with concentrations of 2 g / L, 5 g / L, 7 g / L and 10 g / L obtained in step (1) under magnetic stirring and water bath heating (60° C.), and the state of the agar solution on the magnetic stirrer was observed. If it solidified, 3 mL of H2O2 was added after heating. This step was repeated until the agar solution did not solidify. Finally, the amount of H2O2 added was determined. The results obtained are Comparative Examples 1 to 4, respectively.

[0090] The actual pictures of the agar polysaccharide solutions with added H2O2 obtained in Comparative Examples 1 to 4 are as follows Figure 1 shown. Figure 1 , from left to right are comparative examples 1 to 4.

[0091] The amount of H2O2 solution added and the solution state determined in Comparative Examples 1 to 4 are shown in Table 1:

[0092] Table 1 Comparative Examples 1 to 4 Determination of the amount of H2O2 solution added and the state of the solution

[0093]

[0094] From Table 1 and Figure 1 It can be seen that as the agar polysaccharide concentration in the agar polysaccharide solution increases, the amount of H2O2 solution required will also increase. By observing the state of the agar polysaccharide solution, the optimal amount of H2O2 solution can be determined.

[0095] Comparative Examples 5 to 7

[0096] (1) Weighing 0.2 g, 0.28 g, and 0.4 g of agar polysaccharide powder, respectively, added them into 40 mL of deionized water, and then heated to 95° C. for dissolution to obtain agar polysaccharide solutions with concentrations of 5 g / L, 7 g / L, and 10 g / L, respectively;

[0097] (2) The agar polysaccharide solution obtained in step (1) was cooled to 60°C, and then 3 mL of a 30% mass concentration of H2O2 solution and 4 mL of a 4 mg / mL FeSO4·7H2O solution were added to the agar polysaccharide solutions of 5 g / L, 7 g / L and 10 g / L obtained in step (1) respectively under magnetic stirring and water bath heating (60°C). The state of the agar solution on the magnetic stirrer was observed. If it solidified, 3 mL of H2O2 and 2 mL of FeSO4·7H2O solution were added after heating. This step was repeated until the agar solution did not solidify. Finally, the amounts of H2O2 solution and FeSO4·7H2O solution to be added were determined. The results obtained were respectively Comparative Examples 5 to 7.

[0098] The actual pictures of the agar polysaccharide solutions with added H2O2 and FeSO4·7H2O obtained in Comparative Examples 5 to 7 are as follows Figure 2 shown. Figure 2 , from left to right are comparative examples 5 to 7.

[0099] Comparative Examples 5 to 7 determine the amount of H2O2 solution and FeSO4·7H2O solution added and the solution state as shown in Table 2:

[0100] Table 2 Comparative Examples 5 to 7 Determine the amount of H2O2 solution and FeSO4·7H2O solution added and the solution state

[0101]

[0102]

[0103] From Table 2 and Figure 2 It can be seen that when the amount of H2O2 solution remains unchanged, the amount of FeSO4·7H2O solution required also remains unchanged, indicating that Fe 2+ It can catalyze H2O2 to oxidatively break the glycosidic bonds in agaropolysaccharide.

[0104] Comparative Examples 8 to 9

[0105] (1) Weigh two portions of 0.6 g agar polysaccharide powder, add them into 40 mL of deionized water respectively, and then heat to 95° C. to dissolve, to obtain two portions of agar polysaccharide solutions with a concentration of 15 g / L;

[0106] (2) A portion of the agar polysaccharide solution obtained in step (1) was cooled to 60° C., and then 6 mL of a 30% mass concentration of H2O2 solution and 0.6 mL of a 4 mg / mL FeSO4·7H2O solution were added to the agar polysaccharide solution with a concentration of 15 g / L under magnetic stirring and water bath heating (60° C.), and the state of the agar solution on the magnetic stirrer was observed. If it solidified, 3 mL of H2O2 and 0.4 mL of a 4 mg / mL FeSO4·7H2O solution were added after heating. This step was repeated until the agar solution did not solidify. Finally, the amounts of the H2O2 solution and the FeSO4·7H2O solution to be added were determined, and the result obtained was Comparative Example 8;

[0107] (3) Another portion of the agar polysaccharide solution obtained in step (1) was cooled to 60° C., and then 6 mL of a 30% mass concentration of H2O2 solution and 0.6 mL of a 40 mg / mL vitamin C solution were added to the agar polysaccharide solution with a concentration of 15 g / L under magnetic stirring and water bath heating (60° C.), and the state of the agar solution on the magnetic stirrer was observed. After the first solidification, 3 mL of H2O2 and 0.4 mL of a 40 mg / mL vitamin C solution were added after heating. After the second solidification, 3 mL of H2O2 and 1 mL of a 40 mg / mL vitamin C solution were added after heating. This step was repeated until the agar solution did not solidify. Finally, the amounts of H2O2 solution and vitamin C solution to be added were determined. The result obtained was Comparative Example 9.

[0108] The actual pictures of the agar polysaccharide solutions obtained in Comparative Examples 8 to 9 with the addition of H2O2 and FeSO4·7H2O and the addition of H2O2 and vitamin C are as follows: Figure 3 shown. Figure 3 In the figure, from left to right are comparative examples 8 to 9.

[0109] The states of the agar polysaccharide solutions obtained in Comparative Examples 8 to 9 with the addition of H2O2 and FeSO4·7H2O and the addition of H2O2 and vitamin C are shown in Table 3:

[0110] Table 3 The state of agar polysaccharide solution with addition of H2O2 and FeSO4·7H2O and H2O2 and vitamin C obtained in Comparative Examples 8 to 9

[0111]

[0112]

[0113] Comparative Example 10

[0114] (1) Weigh 0.6 g of agar polysaccharide powder, add it to 40 mL of deionized water, and then heat it to 95° C. to dissolve it, to obtain an agar polysaccharide solution with a concentration of 15 g / L;

[0115] (2) cooling the agar polysaccharide solution obtained in step (1) to 60° C., then adding 21 mL of a 30% H2O2 solution and 1.8 mL of a 4 mg / mL FeSO4·7H2O solution to the agar polysaccharide solution under magnetic stirring and water bath heating (60° C.), and performing a degradation reaction for 4 hours to obtain an agar polysaccharide degradation solution;

[0116] (3) The agaropolysaccharide degradation solution obtained in step (2) is subjected to vacuum freeze drying at a temperature of -80°C to obtain agaro-oligosaccharides.

[0117] Example 1

[0118] (1) Weigh 0.6 g of agar polysaccharide powder, add it to 40 mL of deionized water, and then heat it to 95° C. to dissolve it, to obtain an agar polysaccharide solution with a concentration of 15 g / L;

[0119] (2) cooling the agar polysaccharide solution obtained in step (1) to 60° C., then adding 21 mL of a 30% H2O2 solution and 1.8 mL of a 4 mg / mL FeSO4·7H2O solution to the agar polysaccharide solution under magnetic stirring and water bath heating (60° C.), and performing a degradation reaction for 4 hours to obtain an agar polysaccharide degradation solution;

[0120] (3) Add 1 mL of a 40 mg / mL vitamin C solution to the agar polysaccharide degradation solution obtained in step (2), mix well, and then perform vacuum freeze drying at a temperature of -80°C to obtain agar oligosaccharides with antioxidant function.

[0121] The actual pictures of the agar oligosaccharide solutions before vacuum freeze drying obtained in Comparative Example 10 and Example 1 are as follows: Figure 4 shown. Figure 4 , from left to right are Example 1 and Comparative Example 10.

[0122] The vacuum freeze-dried agar oligosaccharide obtained in Example 1 is shown in the figure below: Figure 5 shown.

[0123] The state and yield of the agar oligosaccharide solution before vacuum freeze drying obtained in Comparative Example 10 and Example 1 are shown in Table 4:

[0124] Table 4 State and yield of agar oligosaccharide solution before vacuum freeze drying obtained in Comparative Example 10 and Example 1

[0125]

[0126]

[0127] Depend on Figures 4-5 As can be seen from Table 4, the agarooligosaccharide prepared by the present invention has better stability.

[0128] The antioxidant activity of the agar-oligosaccharide prepared in Example 1 was tested, including (1) OH free radical scavenging rate determination; (2) DPPH scavenging rate determination; and (3) reducing power determination.

[0129] (1) Determination of OH radical scavenging rate: Hydroxyl radicals are generated by Fenton reaction: H2O2+Fe 2+ = OH + H2O + Fe 3+Salicylic acid is added to the reaction system, and the hydroxyl radicals generated by the Fenton reaction react with salicylic acid to generate 2,3-dihydroxybenzoic acid with special absorption at 510nm. If a test substance with the function of scavenging hydroxyl radicals is added to the reaction system, the generated hydroxyl radicals will be reduced, thereby reducing the amount of colored compounds generated accordingly. Using the fixed reaction time method, the absorbance of the reaction solution containing the test substance is measured at 510nm and compared with the blank solution to determine the scavenging effect of the test substance on hydroxyl radicals. The specific steps are as follows:

[0130] according to Figure 6 Take 1 mL of sample solution, add 1 mL of 9 mM salicylic acid-ethanol solution, then add 1 mL of 3 mM FeSO4 solution, and finally add 1 mL of 8.8 mM H2O2 solution to start the reaction, mix well, react in a 37°C water bath for 30 minutes, and measure the absorbance at 510 nm. The results are as follows Figure 6 As shown;

[0131] The OH removal rate determination system is shown in Table 5:

[0132] Table 5 OH scavenging rate determination system

[0133] <![CDATA[A0]]> <![CDATA[A i ]]> <![CDATA[A j ]]> Sample solution - 1mL 1mL Deionized water 1mL - 1mL 9mM salicylic acid-ethanol solution 1mL 1mL 1mL <![CDATA[3 mM FeSO4 solution]]> 1mL 1mL 1mL <![CDATA[8.8 mM H2O2 solution]]> 1mL 1mL -

[0134] The calculation formula of OH removal rate is shown in formula I:

[0135] Clearance % = [1-(A i -A j ) / A0]×100% Formula I

[0136] In formula I, A0 is the absorbance of blank control; A i A is the absorbance after adding the sample; j is the background absorbance of the sample solution.

[0137] The experimental results of the ·OH free radical scavenging rate of the agar-oligosaccharides prepared by the present invention show that as the concentration of agar-oligosaccharides increases, its scavenging ability continues to increase. When the concentration of agar-oligosaccharides reaches 1.0 mg / mL, the ·OH scavenging rate reaches 92.37%, while when the agar concentration reaches 1.0 mg / mL, the ·OH scavenging rate is only 17.82%, which indicates that the degraded agar-oligosaccharides prepared by the present invention have greatly improved ·OH scavenging ability.

[0138] (2) DPPH scavenging rate determination: DPPH is a very stable nitrogen-centered free radical. Its stability mainly comes from the resonance stabilization of the three benzene rings and the spatial barrier, which prevents the unpaired electrons on the nitrogen atom in the middle from playing their due electron pairing role. It has maximum absorption at a wavelength of 517nm. In the presence of free radical scavengers, the single electron of DPPH is captured and its color becomes lighter. The absorbance value at the wavelength of maximum light absorption decreases, and the degree of decrease is linear. The decrease in the absorbance level indicates an increase in antioxidant activity, thereby evaluating the antioxidant capacity of the test sample. The specific steps are as follows:

[0139] Prepare 0.002% DPPH solution: weigh 5 mg DPPH, dissolve it in anhydrous ethanol and transfer it to a 250 mL volumetric flask, adjust the volume, and store it in a brown bottle at 4°C; take 2 mL of sample solution, add 2 mL DPPH solution and 2 mL sample solvent, mix well, react in the dark for 30 minutes, and measure the absorbance at 517 nm. The results are as follows: Figure 7 shown.

[0140] The DPPH clearance determination reaction system is shown in Table 6:

[0141] Table 6 DPPH clearance determination reaction system

[0142] <![CDATA[A0]]> <![CDATA[A i ]]> <![CDATA[A j ]]> DPPH 2mL 2mL - sample - 2mL 2mL Sample solvent 2mL - 2mL

[0143] The calculation formula of DPPH clearance rate is shown in Formula II:

[0144] DPPH·scavenging rate %=[1-(A i -A j ) / A0]×100%Formula II

[0145] In formula II, A0 is the absorbance of DPPH and sample solution; A i A is the absorbance after adding the sample; j is the background absorbance of the sample solution.

[0146] The experimental results of DPPH clearance rate of agar oligosaccharides prepared by the present invention show that as the concentration of agar oligosaccharides increases, the clearance ability thereof is continuously enhanced. When the concentration of agar oligosaccharides reaches 1.0 mg / mL, the DPPH clearance rate reaches 69.78%, and when the concentration of agar reaches 1.0 mg / mL, the DPPH clearance rate is 32.73%, which indicates that the DPPH clearance rate of the degraded agar oligosaccharides is improved to a certain extent.

[0147] (3) Determination of reducing power: Antioxidants (reducing agents) remove free radicals by giving away electrons through their own reducing action. The stronger the reducing power, the stronger the antioxidant property. In the experiment, the antioxidant in the sample can reduce the trivalent iron of potassium ferrocyanide to divalent iron (potassium ferrocyanide). The divalent iron (potassium ferrocyanide) further reacts with ferric chloride to generate Prussian blue (Fe4[Fe(CN)6]3) with maximum absorbance at 700nm. Therefore, measuring the level at 700nm can indirectly reflect the reducing power of the antioxidant. The greater the absorbance, the stronger the reducing power. The specific steps are as follows:

[0148] Take 2.5 mL of 1 wt% potassium ferricyanide and 2.5 mL of PBS with a concentration of 0.2 M and a pH of 6.6, add 1 mL of sample solution of different concentrations, mix well, place in a 50°C water bath for 20 minutes, cool quickly, add 2.5 mL of 10% TFA solution, mix well, centrifuge at 4000 rpm / min for 10 minutes. Take 2.5 mL of supernatant, add 2.5 mL of 1 wt% FeCl3 and 2.5 mL of deionized water, mix well, let stand for 10 minutes, adjust to zero with deionized water, and measure the absorbance at 700 nm. The results are as follows: Figure 8 shown.

[0149] The reducing power test results of the agar oligosaccharide prepared by the present invention show that as the sample concentration increases, its reducing power becomes stronger.

[0150] Application Example 1

[0151] 1) 50 g of milk, 5 g of white sugar and 0.1 g of baking powder were mixed, and then the agar-oligosaccharide obtained in Example 1 was added and stirred until completely dissolved to obtain a mixture; the mass of the agar-oligosaccharide was 0.01% of the total mass of the milk, white sugar and baking powder;

[0152] 2) fermenting the mixture obtained in step 1) at 43° C. to obtain fermented yogurt.

[0153] Application Examples 2 to 7

[0154] The mass of agar-oligosaccharide in Application Example 1 was adjusted to 0.02%, 0.03%, 0.04%, 0.06%, 0.08% and 0.1% of the total mass of milk, white sugar and baking powder, respectively, and other conditions remained unchanged, and Application Examples 2 to 7 were obtained in turn.

[0155] Comparative application example 1

[0156] 1) Mix 50 g of milk, 5 g of white sugar and 0.1 g of baking powder, and stir until completely dissolved to obtain a mixture;

[0157] 2) fermenting the mixture obtained in step 1) at 43° C. to obtain fermented yogurt.

[0158] The fermentation time of yogurt with different agar oligosaccharide addition amounts in Application Examples 1 to 7 and Comparative Application Example 1 and the state after fermentation are shown in Table 7:

[0159] Table 7 Fermentation time of yogurt with different agar oligosaccharide additions and its post-fermentation state

[0160]

[0161]

[0162] Comparative Application Example 1: The actual picture of yogurt after 8 hours of fermentation is as follows Fig. 9 As shown in the figure; the actual picture of yogurt after 12h fermentation in Application Example 1 is as follows Fig.10 shown.

[0163] From Table 7 and Figures 9-10 It can be seen that with the increase of the amount of agar-oligosaccharide in yogurt, the antioxidant effect of fermented yogurt becomes better and better.

[0164] The stability of the fermented yogurt obtained from Examples 1 to 7 and Comparative Application Example 1 was tested. The testing principle is: the greater the centrifugal sedimentation rate, the more stable the product. This is because the yogurt undergoes centrifugal separation under the action of centrifugal force, separating the whey in the yogurt. Yogurt with good stability separates less whey under the action of centrifugal force, and more substances are retained in the sediment, so it shows a higher centrifugal sedimentation rate; on the contrary, yogurt with poor stability separates more whey under the action of centrifugal force, so its centrifugal sedimentation rate is low. The testing method is: take 5 mL of yogurt in a centrifuge tube, and after measuring the mass m0 of the sample, put it into a centrifuge and centrifuge it at 3000r / min for 25 minutes, take out the centrifuge tube, let it stand for 10 minutes, remove the supernatant, measure the mass m of the residue, and calculate the centrifugal sedimentation rate (WHC). The calculation formula is shown in Formula III, and the results are shown in Fig.11 As shown:

[0165] WHC (%) = m / m0 × 100% Formula III

[0166] In formula III, m is the mass of the residue; m0 is the mass of the sample.

[0167] Depend on Fig.11 It can be seen that the agar oligosaccharide of the present invention is added to the fermented yogurt as a stabilizer, and the centrifugal sedimentation rate of the yogurt decreases as the number of days it is placed increases. The yogurt with added agar oligosaccharide has a better centrifugal sedimentation rate than the yogurt without oligosaccharide, especially the yogurt with 0.01% added has a centrifugal sedimentation rate of up to 99% in the first 4 days. It can be seen that the addition of agar oligosaccharide can improve the stability of yogurt, and the optimal addition amount is 0.01%.

[0168] The particle size distribution of the fermented yogurt obtained in Examples 1 to 7 and Comparative Application Example 1 was tested. The testing principle is: the particle size distribution of the product is an important factor affecting the particle sedimentation rate. Under normal circumstances, the sinking speed of casein in yogurt is proportional to its particle radius. The smaller the radius, the more uniform the distribution, the slower the sinking speed, the longer the acid stability time, and the better the stability. The testing method is: after diluting the fermented yogurt 100 times until the yogurt solution is transparent, the particle size of the diluted yogurt is measured using a Malvern laser particle size analyzer. The results are as follows: Fig.12 shown.

[0169] Depend on Fig.12 Combination Figures 9-10 It can be seen that the agar-oligosaccharide of the present invention is added to the fermented yogurt as a stabilizer. It can be seen from visual observation that the yogurt without the addition of agar-oligosaccharide has particles visible to the naked eye. This result is also verified after detection by Malvern laser particle size analyzer, indicating that the addition of the agar-oligosaccharide prepared by the present invention can significantly reduce the particle size of the yogurt.

[0170] The antioxidant activity of the fermented yogurt obtained in Example 1 and Comparative Application Example 1 was tested. The test method was as follows: 5 g of yogurt was accurately weighed and diluted with anhydrous ethanol at a material-liquid ratio of 1:9. After mixing evenly, the mixture was centrifuged at 4000 r / min for 10 min. The supernatant was taken for later use. The ·OH scavenging ability of the yogurt was measured to obtain the antioxidant activity of the fermented yogurt. The results are shown in FIG. Fig.13 shown.

[0171] Depend on Fig.13 It can be seen that the agar oligosaccharide of the present invention is added as a stabilizer to fermented yogurt, and the antioxidant activity of the yogurt without adding agar oligosaccharide is similar to that of the yogurt with 0.01% agar oligosaccharide added (·OH clearance rate is about 38%). On the fourth day of storage, the yogurt without adding agar oligosaccharide no longer has antioxidant activity, while the yogurt with 0.01% agar oligosaccharide added has an ·OH clearance rate of 20.51%, and until the eighth day of storage, its ·OH clearance rate is 5.76%. The experimental results show that adding the agar oligosaccharide provided by the present invention can increase the antioxidant days of yogurt and improve the stability of its product.

[0172] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing agar oligosaccharide with antioxidant function, comprising the following steps: (1) mixing agar polysaccharide and water, and then heating and dissolving them to obtain an agar polysaccharide solution; (2) adding H2O2 solution and divalent iron salt solution to the agar polysaccharide solution obtained in step (1) to carry out a degradation reaction to obtain an agar polysaccharide degradation solution; (3) Adding vitamin C solution to the agar polysaccharide degradation solution obtained in step (2), and drying to obtain agar oligosaccharides with antioxidant function.

2. The preparation method according to claim 1, characterized in that: The concentration of the agar polysaccharide solution in step (1) is 2 to 15 g / L.

3. The preparation method according to claim 1, characterized in that: The mass concentration of the H2O2 solution in step (2) is 20-40%.

4. The preparation method according to claim 1, characterized in that: The concentration of the divalent iron salt solution in step (2) is 4 to 40 mg / mL.

5. The preparation method according to claim 1, characterized in that: In the step (2), the volume ratio of agar polysaccharide solution, H2O2 solution and divalent iron salt solution is 40: (20-25): (0.5-3).

6. The preparation method according to claim 1, characterized in that: The temperature of the degradation reaction in step (2) is 50-70° C., and the time of the degradation reaction is 3-5 hours.

7. The preparation method according to claim 1, characterized in that: The concentration of the vitamin C solution in step (3) is 30 to 50 mg / mL.

8. The preparation method according to claim 1 or 7, characterized in that: The volume ratio of the agar polysaccharide solution in step (1) to the vitamin C solution in step (3) is 40:(0.5-2).

9. Agaro-oligosaccharide with antioxidant function prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the agar-oligosaccharide with antioxidant function according to claim 9 as a stabilizer in fermented yogurt.

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