An agar oligosaccharide with antioxidant function, its preparation method and application

Abstract

This invention provides an agar oligosaccharide with antioxidant function, its preparation method, and its application, belonging to the field of natural polysaccharide degradation technology. This invention utilizes H2O2 / Fe... 2+ The system generates hydroxyl radicals to degrade agar polysaccharides, utilizing H2O2 in Fe... 2+ Catalysis oxidizes and breaks the glycosidic bonds in agar polysaccharides, degrading them into low-molecular-weight fragments to obtain agar oligosaccharides with good antioxidant activity. These fragments are then combined with vitamin C, which reduces the color of the lyophilized and reconstituted agar oligosaccharide solution and imparts stronger antioxidant activity to the degraded agar oligosaccharides. The preparation method provided by this invention has a simple process flow, low equipment requirements, low cost, and a high yield of agar oligosaccharides, reaching over 85%, making it suitable for large-scale industrial application.

Description

Technical Field

[0001] This invention relates to the field of natural polysaccharide degradation technology, and in particular to an agar oligosaccharide with antioxidant function, its preparation method and application. Background Technology

[0002] Agar, scientifically known as agar, is a polysaccharide extracted from seaweeds of the genera *Gracilaria*, *Gelsemium*, and *Gnaphalium* within the red algae phylum. The basic structural unit of agar is the agar disaccharide unit, consisting of a 1,3-linked β-D-galactose and a 1,4-linked 3,6-anhydro-α-L-galactose unit. Agar is a hydrophilic colloid with strong water absorption, high gel strength, and relatively constant viscosity. However, its high viscosity and poor solubility in water limit its widespread application. The structure of agar determines its functional properties. To overcome these limitations, some researchers have conducted structural modification studies, using chemical modifications to improve its application properties. These studies mainly include agar degradation and the introduction of chemical groups for modification.

[0003] Currently, the most common hydrolysis methods for agar are acid hydrolysis and enzymatic hydrolysis. Acid hydrolysis is a traditional method for degrading agar. Acid acts on the α-(1,3) glycosidic bonds of agar, producing ultra-oligosaccharides or agar oligosaccharides with β-D-galactose as the non-reducing end and 3,6-endoether-α-L-galactose as the reducing end. Acid hydrolysis involves harsh reaction conditions, requires sophisticated equipment, produces products with poor uniformity, and presents challenges in product analysis and recovery. Furthermore, the acids used in the process pollute the environment. Enzymatic hydrolysis uses agarases to degrade agar. Based on the different ways agarases cleave glycosidic bonds, they can be divided into α-agarases and β-agarases. Agarases are important seaweed polysaccharide-degrading enzymes that catalyze the breaking of glycosidic bonds in agar. Because enzymes require strict temperature control, enzymatic hydrolysis demands sophisticated equipment and results in a higher impurity content during preparation.

[0004] Fermented yogurt is a product made primarily from fresh milk, with the addition of fermentation starters, sweeteners, and other auxiliary ingredients, through lactic acid fermentation. However, whey separation and uneven texture are common problems affecting yogurt stability during production and storage. To address these issues, stabilizers are added during yogurt production to improve its quality and stability. Currently, most stabilizers on the market are applied in the form of compound polysaccharides in yogurt production; there are relatively few reports on the use of agar oligosaccharides alone as stabilizers in yogurt production.

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

[0006] The purpose of this invention is to provide an agar oligosaccharide with antioxidant function, its preparation method and application. The preparation method provided by this invention is simple to operate, and the obtained agar oligosaccharide has excellent antioxidant function. It can be used alone as a stabilizer in the production process of fermented yogurt to improve the quality and stability of yogurt.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing agar oligosaccharides with antioxidant functions, comprising the following steps:

[0009] (1) Mix agar polysaccharide and water, then heat to dissolve, to obtain an agar polysaccharide solution;

[0010] (2) Add H2O2 solution and ferrous salt solution to the agar polysaccharide solution obtained in step (1) to carry out the degradation reaction and obtain the agar polysaccharide degradation solution;

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

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

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

[0014] Preferably, the concentration of the ferrous 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 ferrous 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 30-50 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] This invention provides an agar oligosaccharide with antioxidant function prepared by the preparation method described in the above technical solution.

[0020] This invention provides the application of agar oligosaccharides with antioxidant function as stabilizers in fermented yogurt, as described in the above technical solution.

[0021] This invention provides a method for preparing agar oligosaccharides with antioxidant functions, comprising the following steps: (1) mixing agar polysaccharide and water, and then heating to dissolve, to obtain an agar polysaccharide solution; (2) adding H2O2 solution and ferrous 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 functions. This invention utilizes H2O2 / Fe 2+ The system generates hydroxyl radicals to degrade agar polysaccharides, utilizing H2O2 in Fe... 2+ Catalysis oxidizes and breaks the glycosidic bonds in agar polysaccharides, degrading them into low-molecular-weight fragments to obtain agar oligosaccharides with good antioxidant activity. These fragments are then combined with vitamin C. The addition of vitamin C reduces the color of the lyophilized and reconstituted agar oligosaccharide solution and imparts stronger antioxidant activity to the degraded agar oligosaccharides. The preparation method provided by this invention has a simple process flow, low equipment requirements, low cost, and a high yield of agar oligosaccharides, reaching over 85%, making it suitable for large-scale industrial application. Results from the examples show that the antioxidant activity of the prepared agar oligosaccharides is significantly improved compared to undegraded agar polysaccharides. Centrifugal sedimentation rate, particle size, and antioxidant activity during storage of yogurt with added agar oligosaccharides are measured. The results indicate that the addition of agar oligosaccharides increases the centrifugal sedimentation rate of fermented yogurt, reduces the particle size, and provides a longer-lasting antioxidant activity. Attached Figure Description

[0022] Figure 1 The images show the actual agar polysaccharide solutions with added H2O2 obtained in Comparative Examples 1-4.

[0023] Figure 2 The images show the actual agar polysaccharide solutions with added H2O2 and FeSO4·7H2O obtained in Comparative Examples 5-7.

[0024] Figure 3 The images show the physical samples of the agar polysaccharide solutions obtained in Comparative Examples 8 and 9, which contained H2O2 and FeSO4·7H2O, as well as those containing H2O2 and vitamin C.

[0025] Figure 4 The image shows the agar oligosaccharide solutions obtained in Comparative Example 10 and Example 1 before vacuum freeze-drying.

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

[0027] Figure 6 A comparison of the ·OH radical scavenging rates of agar oligosaccharides, vitamin C, H2O2 and a mixture of FeSO4·7H2O and vitamin C, and agar solutions;

[0028] Figure 7 A comparison of DPPH scavenging rates of agar oligosaccharides, vitamin C, H2O2 and FeSO4·7H2O mixed with vitamin C, and agar solution;

[0029] Figure 8 The graph shows the reducing power of agar oligosaccharides.

[0030] Figure 9 To compare the actual photos of the yogurt from different angles after 8 hours of fermentation in Application Example 1;

[0031] Figure 10 The images are actual photos of the yogurt from different angles after 12 hours of fermentation, as shown in Application Example 1.

[0032] Figure 11 A comparison graph showing the centrifugation sedimentation rates of yogurt obtained from Application Examples 1-7 and Comparative Application Example 1 on days 2, 4, and 8.

[0033] Figure 12 Comparison chart of yogurt particle size obtained from Application Examples 1-7 and Comparative Application Example 1;

[0034] Figure 13 A comparison chart showing the ·OH radical scavenging rate of yogurt obtained from Application Example 1 and Comparative Application Example 1. Detailed Implementation

[0035] This invention provides a method for preparing agar oligosaccharides with antioxidant functions, comprising the following steps:

[0036] (1) Mix agar polysaccharide and water, then heat to dissolve, to obtain an agar polysaccharide solution;

[0037] (2) Add H2O2 solution and ferrous salt solution to the agar polysaccharide solution obtained in step (1) to carry out a degradation reaction to obtain agar polysaccharide degradation solution;

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

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

[0040] This invention involves mixing agar polysaccharide and water, then heating to dissolve them, to obtain an agar polysaccharide solution.

[0041] In this invention, the agar polysaccharide is preferably agar polysaccharide powder. This invention does not impose any special limitation on the particle size of the agar polysaccharide powder; commercially available agar polysaccharide powder can be used.

[0042] In this invention, the water is preferably deionized water. Using deionized water reduces the impurity content.

[0043] The present invention does not have any special limitations on the specific operation of mixing the agar polysaccharide and water. Any method known to those skilled in the art that can completely mix the agar polysaccharide and water can be used.

[0044] In this invention, the heating and dissolving temperature is preferably ≥90℃. This invention utilizes heating to dissolve agar polysaccharides that are insoluble in cold water in hot water; by controlling the heating temperature, the agar polysaccharides can be completely dissolved in water, thereby forming a homogeneous solution.

[0045] In one embodiment of the present invention, the heating and melting temperature can be 92°C, 93°C, 94°C, 95°C, and 96°C.

[0046] In this invention, the concentration of the agar polysaccharide solution is preferably 2–15 g / L. By controlling the concentration of the agar polysaccharide solution, this invention ensures that the agar polysaccharide is completely dissolved in water, avoiding residue, and also facilitates control over the amount of agar polysaccharide added in relation to the subsequent addition of H2O2 and ferrous salts.

[0047] In one 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 to the agar polysaccharide solution to carry out a degradation reaction, thereby obtaining an agar polysaccharide degradation solution.

[0049] In this invention, the temperature of the agar polysaccharide solution is preferably 50–70°C when H2O2 solution and FeSO4·7H2O solution are added. In this invention, when the temperature of the agar polysaccharide solution does not meet the above requirements, the agar polysaccharide solution is preferably cooled naturally. As one embodiment of this invention, the temperature of the agar polysaccharide solution can be 50°C, 55°C, 60°C, 65°C, and 70°C. By controlling the temperature of the agar polysaccharide solution, this invention allows the reaction to proceed directly under water bath heating conditions after the addition of H2O2 solution and FeSO4·7H2O solution.

[0050] In this invention, the mass concentration of the H2O2 solution is preferably 20-40%; the solvent of the H2O2 solution is preferably deionized water. In this invention, H2O2 can be used in Fe... 2+ Catalysis causes the glycosidic bonds in agar polysaccharides to oxidize and break, thereby degrading agar polysaccharides into low molecular weight fragments.

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

[0052] In this invention, the ferrous salt is preferably FeSO4·7H2O; the concentration of the ferrous salt solution is preferably 4–40 mg / mL; and the solvent of the ferrous salt solution is preferably deionized water. This invention utilizes Fe… 2+ Catalytic H2O2 oxidizes and breaks the glycosidic bonds in agar polysaccharides, thereby degrading agar polysaccharides into low molecular weight fragments.

[0053] In one embodiment of the present invention, the concentration of the ferrous 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 this invention, the preferred method for preparing the ferrous salt solution is as follows: 0.4 g of ferrous salt powder is dissolved in 10 mL of deionized water to obtain a 40 mg / mL ferrous salt solution. Then, deionized water is added to the 40 mg / mL ferrous salt solution for dilution as needed to obtain the desired ferrous salt solution. The preparation method provided by this invention allows for precise control of the concentration of the ferrous salt solution.

[0055] In this invention, the preferred volume ratio of the agar polysaccharide solution, H2O2 solution, and ferrous salt solution is 40:(20-25):(0.5-3), more preferably 40:(20-21):(1-1.8). By controlling the volume ratio of the agar polysaccharide solution, H2O2 solution, and ferrous salt solution, this invention can control the amount of agar polysaccharide, H2O2, and ferrous salt, thereby ensuring that the agar polysaccharide can be completely degraded into agar oligosaccharides under the action of H2O2 and ferrous salt.

[0056] In this invention, the temperature of the degradation reaction is preferably 50–70°C; the duration of the degradation reaction is preferably 3–5 hours; and the degradation reaction is preferably carried out under water bath heating conditions. By controlling the parameters of the degradation reaction, this invention can ensure that agar polysaccharides are completely degraded into agar oligosaccharides.

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

[0058] This invention utilizes H2O2 / Fe 2+ The system generates hydroxyl radicals to degrade agar polysaccharides, utilizing H2O2 in Fe... 2+ Catalysis causes the glycosidic bonds in agar polysaccharides to oxidize and break, thereby degrading agar polysaccharides 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 vitamin C solution to the agar polysaccharide degradation solution, and after drying, obtains agar oligosaccharide with antioxidant function.

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

[0067] In one 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 this invention, the preferred method for preparing the vitamin C solution is to dissolve vitamin C powder in deionized water to obtain a vitamin C solution.

[0069] In this 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 this invention can reduce the color of the agar oligosaccharide solution after lyophilization and reconstitution, and at the same time impart stronger antioxidant activity to the degraded agar oligosaccharides.

[0070] In this invention, the drying method is preferably vacuum freeze-drying. This invention does not impose specific limitations on the specific operation and process parameters of the vacuum freeze-drying; based on the technical knowledge of those skilled in the art, any method that achieves solvent removal is acceptable.

[0071] This invention utilizes H2O2 / Fe 2+ The system generates hydroxyl radicals to degrade agar polysaccharides, utilizing H2O2 in Fe... 2+ Catalysis causes the glycosidic bonds in agar polysaccharides to oxidize and break, thereby degrading agar polysaccharides into low molecular weight fragments, yielding agar oligosaccharides with good antioxidant activity. Then, these are compounded with vitamin C. The addition of vitamin C can reduce the color of the agar oligosaccharide solution after freeze-drying and reconstitution, and at the same time, it can give the degraded agar oligosaccharides stronger antioxidant activity.

[0072] The preparation method provided by this invention has a simple process flow, low equipment requirements, low cost, and a high yield of agar oligosaccharides, reaching over 85%, making it suitable for large-scale industrial application.

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

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

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

[0076] In this invention, the application of the agar oligosaccharide in fermented yogurt preferably includes the following steps:

[0077] 1) Mix milk, white sugar, baking powder, and agar oligosaccharides to obtain a mixture;

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

[0079] The present invention preferably involves mixing milk, white sugar, baking powder and agar oligosaccharides to obtain a mixture.

[0080] The present invention does not have any special limitation on the specific sources of the milk, white sugar and baking powder, and commercially available products known to those skilled in the art can be used.

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

[0082] In this invention, the mass of the agar oligosaccharide is preferably 0.01 to 0.1% of the total mass of milk, white sugar and baking powder, 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 this invention, the fermentation temperature is preferably 43°C. By controlling the fermentation temperature, this invention can further improve the stability of the fermentation process and avoid spoilage or incomplete fermentation.

[0085] This invention significantly improves the texture and stability of fermented yogurt by applying the prepared agar oligosaccharides to it.

[0086] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0087] Comparative Examples 1-4

[0088] (1) Weigh 0.08g, 0.2g, 0.28g and 0.4g of agar polysaccharide powder, add them to 40mL of deionized water respectively, and then heat to 95℃ to dissolve them to obtain agar polysaccharide solutions with concentrations of 2g / L, 5g / L, 7g / L and 10g / L respectively;

[0089] (2) Cool the agar polysaccharide solution obtained in step (1) to 60°C, and then add 3 mL of 30% H2O2 solution 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). Observe the state of the agar solution on the magnetic stirrer. If it solidifies, heat it and add another 3 mL of H2O2. Repeat this step until the agar solution does not solidify. Finally, determine the amount of H2O2 added. The results obtained are comparative examples 1 to 4.

[0090] The actual images of the agar polysaccharide solutions with added H2O2 obtained in Comparative Examples 1-4 are shown below. Figure 1 As shown. Figure 1 In the middle, from left to right, are comparative examples 1 to 4.

[0091] The amounts of H2O2 solution added and the solution states determined in Comparative Examples 1–4 are shown in Table 1.

[0092] Table 1 shows the amount of H2O2 solution added and the solution state determined in Comparative Examples 1-4.

[0093]

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

[0095] Comparative Examples 5-7

[0096] (1) Weigh 0.2g, 0.28g and 0.4g of agar polysaccharide powder, add them to 40mL of deionized water respectively, and then heat to 95℃ to dissolve them to obtain agar polysaccharide solutions with concentrations of 5g / L, 7g / L and 10g / L respectively;

[0097] (2) Cool the agar polysaccharide solution obtained in step (1) to 60°C. Then, under magnetic stirring and water bath heating (60°C), add 3 mL of 30% H2O2 solution and 4 mL of 4 mg / mL FeSO4·7H2O solution to the agar polysaccharide solutions with concentrations of 5 g / L, 7 g / L and 10 g / L obtained in step (1), respectively. Observe the state of the agar solution on the magnetic stirrer. If it solidifies, heat it and add another 3 mL of H2O2 and 2 mL of FeSO4·7H2O solution. Repeat this step until the agar solution does not solidify. Finally, determine the amount of H2O2 solution and FeSO4·7H2O solution to be added. The results obtained are comparative examples 5 to 7.

[0098] The physical images of the agar polysaccharide solutions with added H2O2 and FeSO4·7H2O obtained in Comparative Examples 5-7 are shown below. Figure 2 As shown. Figure 2 In the middle, from left to right are comparative examples 5 to 7.

[0099] The amounts of H2O2 solution and FeSO4·7H2O solution added, as well as the solution states, were determined in Comparative Examples 5–7, as shown in Table 2.

[0100] Table 2 shows the amounts of H2O2 solution and FeSO4·7H2O solution added, as well as the solution states, as determined in Comparative Examples 5–7.

[0101]

[0102]

[0103] From Table 2 and Figure 2 It can be seen that when the amount of H2O2 solution remains constant, the amount of FeSO4·7H2O solution required also remains constant, indicating that Fe 2+ It can catalyze the oxidation and breakage of glycosidic bonds in agar polysaccharides by H2O2.

[0104] Comparative Examples 8-9

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

[0106] (2) Cool one portion of the agar polysaccharide solution obtained in step (1) to 60°C. Then, under magnetic stirring and water bath heating (60°C), add 6 mL of 30% H2O2 solution and 0.6 mL of 4 mg / mL FeSO4·7H2O solution to the 15 g / L agar polysaccharide solution. Observe the state of the agar solution on the magnetic stirrer. If it solidifies, heat it and add 3 mL of H2O2 and 0.4 mL of 4 mg / mL FeSO4·7H2O solution. Repeat this step until the agar solution does not solidify. Finally, determine the amount of H2O2 solution and FeSO4·7H2O solution added. The result is Comparative Example 8.

[0107] (3) Cool another portion of the agar polysaccharide solution obtained in step (1) to 60°C. Then, under magnetic stirring and water bath heating (60°C), add 6 mL of 30% H2O2 solution and 0.6 mL of 40 mg / mL vitamin C solution to the 15 g / L agar polysaccharide solution. Observe the state of the agar solution on the magnetic stirrer. After the first solidification, heat and add 3 mL of H2O2 and 0.4 mL of 40 mg / mL vitamin C solution. After the second solidification, heat and add another 3 mL of H2O2 and 1 mL of 40 mg / mL vitamin C solution. Repeat this step until the agar solution does not solidify. Finally, determine the amount of H2O2 solution and vitamin C solution added. The result is Comparative Example 9.

[0108] Physical images of the agar polysaccharide solutions obtained in Comparative Examples 8 and 9 with added H2O2 and FeSO4·7H2O, and with added H2O2 and vitamin C, are shown below. Figure 3 As shown. Figure 3 In the middle, from left to right are comparative examples 8 to 9.

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

[0110] Table 3 shows the states of the agar polysaccharide solutions obtained from Comparative Examples 8-9, which included H2O2 and FeSO4·7H2O, as well as those with added H2O2 and vitamin C.

[0111]

[0112]

[0113] Comparative Example 10

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

[0115] (2) Cool the agar polysaccharide solution obtained in step (1) to 60°C, and then add 21 mL of H2O2 solution with a mass concentration of 30% and 1.8 mL of FeSO4·7H2O solution with a concentration of 4 mg / mL to the agar polysaccharide solution under magnetic stirring and water bath heating (60°C) for 4 h of degradation reaction to obtain agar polysaccharide degradation solution;

[0116] (3) The agar polysaccharide degradation solution obtained in step (2) is subjected to vacuum freeze drying at -80°C to obtain agar oligosaccharide.

[0117] Example 1

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

[0119] (2) Cool the agar polysaccharide solution obtained in step (1) to 60°C, and then add 21 mL of H2O2 solution with a mass concentration of 30% and 1.8 mL of FeSO4·7H2O solution with a concentration of 4 mg / mL to the agar polysaccharide solution under magnetic stirring and water bath heating (60°C) for 4 h of degradation reaction to obtain agar polysaccharide degradation solution;

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

[0121] The actual images of the agar oligosaccharide solutions obtained in Comparative Example 10 and Example 1 before vacuum freeze-drying are shown below. Figure 4 As shown. Figure 4 In the middle, 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 image below. Figure 5 As shown.

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

[0124] Table 4 shows the state and yield of the agar oligosaccharide solutions obtained in Comparative Example 10 and Example 1 before vacuum freeze-drying.

[0125]

[0126]

[0127] Depend on Figures 4-5 As can be seen from Table 4, the agar oligosaccharides prepared in this invention have better stability.

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

[0129] (1) Determination of OH radical scavenging rate: Hydroxyl radicals were generated using the Fenton reaction: H2O2 + Fe 2+ =·OH + H₂O + Fe 3+Salicylic acid is added to the reaction system. The hydroxyl radicals generated in the Fenton reaction react with the salicylic acid to form 2,3-dihydroxybenzoic acid, which has a specific absorption at 510 nm. Adding an analyte with hydroxyl radical scavenging capabilities to the reaction system reduces the generation of hydroxyl radicals, thereby correspondingly reducing the amount of colored compound formed. The absorbance of the reaction solution containing the analyte is measured at 510 nm using a fixed reaction time method and compared with a blank solution to determine the analyte's hydroxyl radical scavenging effect. 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 and react in a 37°C water bath for 30 min. Measure the absorbance at 510 nm. The results are shown below. Figure 6 As shown;

[0131] The OH scavenging 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 formula for calculating the OH removal rate is shown in Equation I:

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

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

[0137] Experimental results on the ·OH free radical scavenging rate of the agar oligosaccharide prepared in this invention show that its scavenging ability continuously increases with the increase of agar oligosaccharide concentration. When the agar oligosaccharide concentration 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%. This indicates that the degraded agar oligosaccharide prepared in this invention has a significantly 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 effect of the three benzene rings and steric hindrance, preventing the unpaired electrons on the nitrogen atom sandwiched in the middle from playing their proper electron pairing role. It has maximum absorption at a wavelength of 517 nm. In the presence of free radical scavengers, the single electrons of DPPH are captured, causing its color to lighten and the absorbance at the maximum absorption wavelength to decrease, with the decrease showing a linear relationship. The decrease in absorbance level indicates an increase in antioxidant activity, thus evaluating the antioxidant capacity of the test sample. The specific steps are as follows:

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

[0140] The reaction system for DPPH scavenging rate determination is shown in Table 6:

[0141] Table 6. Reaction system for DPPH scavenging rate determination

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

[0143] The formula for calculating DPPH removal rate is shown in Equation II:

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

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

[0146] The experimental results of DPPH scavenging rate of the agar oligosaccharide prepared in this invention show that its scavenging ability continuously increases with the increase of agar oligosaccharide concentration. When the agar oligosaccharide concentration reaches 1.0 mg / mL, the DPPH scavenging rate reaches 69.78%, while when the agar concentration reaches 1.0 mg / mL, the DPPH scavenging rate is 32.73%. This indicates that the DPPH scavenging rate of the degraded agar oligosaccharide has been improved to a certain extent.

[0147] (3) Reducing power determination: Antioxidants (reducing agents) scavenge free radicals by donating electrons through their own reduction action. The stronger the reducing power, the stronger the antioxidant activity. In the experiment, the antioxidant in the sample could reduce the ferric iron of potassium ferricyanide to ferrous iron (potassium ferrocyanide). The ferrous iron (potassium ferrocyanide) further reacted with ferric chloride to generate Prussian blue (Fe4[Fe(CN)6]3) with maximum absorbance at 700 nm. Therefore, the absorbance at 700 nm 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 each of 1 wt% potassium ferricyanide and 0.2 M PBS (pH 6.6), add 1 mL of the sample solution of different concentrations respectively, mix well, incubate at 50°C for 20 min, cool rapidly, then add 2.5 mL of 10% TFA solution, mix well; centrifuge at 4000 rpm / min for 10 min. Take 2.5 mL of the supernatant, then add 2.5 mL of 1 wt% FeCl3 and 2.5 mL of deionized water respectively, mix well, let stand for 10 min, zero the instrument with deionized water, and measure the absorbance at 700 nm. The results are as follows. Figure 8 As shown.

[0149] The reducing power test results of the agar oligosaccharides prepared in this invention show that the reducing power increases with increasing sample concentration.

[0150] Application Example 1

[0151] 1) Mix 50g milk, 5g white sugar and 0.1g baking powder, then add the agar oligosaccharide obtained in Example 1, and stir until completely dissolved to obtain a mixture; the mass of agar oligosaccharide is 0.01% of the total mass of milk, white sugar and baking powder;

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

[0153] Application Examples 2-7

[0154] The mass of agar oligosaccharides 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, while other conditions remained unchanged, thus obtaining Application Examples 2 to 7.

[0155] Comparative Application Example 1

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

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

[0158] Table 7 shows the fermentation time and post-fermentation state of yogurts with different amounts of agar oligosaccharides added in Application Examples 1-7 and Comparative Application Example 1.

[0159] Table 7. Fermentation time and post-fermentation state of yogurt with different amounts of agar oligosaccharides.

[0160]

[0161]

[0162] Comparative application example 1: The actual picture of the yogurt after 8 hours of fermentation is shown below. Figure 9 As shown; the actual picture of the yogurt after 12 hours of fermentation in Application Example 1 is shown below. Figure 10 As shown.

[0163] From Table 7 and Figures 9-10 It can be seen that as the amount of agar oligosaccharides added to yogurt increases, the antioxidant effect of fermented yogurt becomes better and better.

[0164] The stability of fermented yogurt obtained from corresponding use cases 1-7 and comparative application example 1 was tested. The test principle is as follows: the higher the centrifugal sedimentation rate, the more stable the product. This is because yogurt undergoes whey separation under centrifugal force. Yogurt with good stability separates less whey under centrifugal force, retaining more substances in the sediment, thus exhibiting a higher centrifugal sedimentation rate. Conversely, yogurt with poor stability separates more whey under centrifugal force, thus exhibiting a lower centrifugal sedimentation rate. The test method is as follows: 5 mL of yogurt is taken into a centrifuge tube, and the mass m0 of the sample is measured. It is then centrifuged at 3000 r / min for 25 min. After removing the centrifuge tube and allowing it to stand for 10 min, the supernatant is removed, and the mass m of the residue is measured. The centrifugal sedimentation rate (WHC) is calculated using the formula shown in Equation III. The results are as follows. Figure 11 As shown:

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

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

[0167] Depend on Figure 11 It can be seen that when agar oligosaccharides of the present invention are added as a stabilizer to fermented yogurt, the centrifugal sedimentation rate of the yogurt decreases with the increase of its storage days. Yogurt with added agar oligosaccharides has a better centrifugal sedimentation rate than yogurt without added oligosaccharides, especially yogurt with 0.01% added has a centrifugal sedimentation rate as high as 99% in the first 4 days. Therefore, it can be concluded that adding agar oligosaccharides can improve the stability of yogurt, and the optimal addition amount is 0.01%.

[0168] The particle size distribution of fermented yogurt obtained from corresponding use cases 1-7 and comparative application example 1 was tested. The test principle is as follows: the particle size distribution of the product is an important factor affecting the particle sedimentation rate. Under normal circumstances, the sedimentation rate of casein in yogurt is directly proportional to its particle radius. The smaller the radius, the more uniform the distribution, the slower the sedimentation rate, the longer the acid stabilization time, and the better the stability. The test method is as follows: the fermented yogurt was diluted 100 times until the yogurt solution was transparent, and then the particle size of the diluted yogurt was measured using a Malvern laser particle size analyzer. The results are shown below. Figure 12 As shown.

[0169] Depend on Figure 12 Combination Figures 9-10 As can be seen, when the agar oligosaccharide of the present invention is added to fermented yogurt as a stabilizer, it can be observed that yogurt without added agar oligosaccharide has visible particles. This result was also verified by the 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 yogurt.

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

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

[0172] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of agar oligosaccharide with antioxidant function as stabilizer in fermented yogurt, The preparation method of the agar oligosaccharide with antioxidant function comprises the following steps: (1) mixing agar polysaccharide and water, and then heating and dissolving to obtain an agar polysaccharide solution; (2) adding an H2O2 solution and a divalent iron salt solution to the agar polysaccharide solution obtained in step (1) to perform a degradation reaction, thereby obtaining an agar polysaccharide degradation solution; (3) adding a vitamin C solution to the agar polysaccharide degradation solution obtained in step (2), and drying to obtain the agar oligosaccharide with antioxidant function; The concentration of the agar polysaccharide solution in step (1) is 10-15 g / L; The mass concentration of the H2O2 solution in step (2) is 20-40%; the concentration of the divalent iron salt solution in step (2) is 4-40 mg / mL; and the volume ratio of the agar polysaccharide solution, the H2O2 solution and the divalent iron salt solution in step (2) is 40:(20-25):(0.5-3); The temperature of the degradation reaction in step (2) is 50-70°C, and the time of the degradation reaction is 3-5 h; The concentration of the vitamin C solution in step (3) is 30-50 mg / mL; and the volume ratio of the agar polysaccharide solution in step (1) and the vitamin C solution in step (3) is 40:(0.5-1).

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