Carrier for improving thermal stability of anthocyanin as well as construction method and application of carrier

Abstract

The invention discloses a carrier for improving thermal stability of anthocyanin as well as a construction method and application of the carrier. The vector construction method comprises the following steps: (1) dissolving whey protein in water, centrifuging to remove precipitate, and taking supernate A; (2) heating and stirring the supernatant A in the step (1) at 50 DEG C, adjusting the pH value to 5.0-9.0, then adding neutral protease and papain, maintaining the pH value to 5.0-9.0 in the enzymolysis process, and after the enzymolysis is finished, performing enzyme deactivation on the enzymatic hydrolysate; and (3) centrifuging the enzymatic hydrolysate subjected to enzyme deactivation in the step (2) at the speed of 3000-5000g for 10-30 minutes, taking supernate B, adding glucan, performing freeze-drying, and reacting the freeze-dried powder at the temperature of 60-90 DEG C and the relative humidity of 70-90% for 24-48 hours to obtain the carrier. The carrier construction method is simple, the carrier raw materials are cheap, and the thermal stability of the anthocyanin is improved.

Description

Technical Field

[0001] The invention relates to the field of natural active ingredient loading and health food processing, and in particular to a method for constructing a carrier for improving the thermal stability of anthocyanins. Background Art

[0002] Anthocyanins are a type of water-soluble pigment distributed in plants. They belong to the flavonoid compounds in plant polyphenols. They mainly include six pigments and their derivatives: delphinidin, cyanidin, morning glory pigment, peony pigment, geranium pigment, and malva pigment. As a natural active ingredient, anthocyanins have health benefits such as antioxidant, anti-cancer, antibacterial, eye protection, and prevention of cardiovascular and cerebrovascular diseases. They are widely used in the food, pharmaceutical and health care products industries.

[0003] However, anthocyanins have poor thermal stability and pH stability, and are easily thermally degraded under neutral conditions, resulting in reduced retention during food sterilization, which greatly limits the application of anthocyanins in food. Existing anthocyanin carriers include microcapsules, liposomes, nanoemulsions, and biomacromolecule complexes composed of proteoglycans. Among them, microcapsules have a large loading capacity and are simple to prepare, but most of them have limited improvement in stability; liposomes and nanoemulsions are difficult to prepare and difficult to use in industrialization; proteins and polysaccharides have large molecular weights, few binding sites with anthocyanins, and low encapsulation efficiency. Therefore, developing a simple, inexpensive, large loading capacity, and carrier construction method that significantly improves the thermal stability of anthocyanins is of great significance to broadening the application of anthocyanins in food, health products, and medicines. Summary of the invention

[0004] The present invention aims to provide a carrier for improving the thermal stability of anthocyanins and a construction method and application thereof.

[0005] The present invention provides a method for constructing a vector for improving the thermal stability of anthocyanins, characterized in that it comprises the following steps:

[0006] (1) Dissolve whey protein in water, discard the precipitate by centrifugation, and take the supernatant A;

[0007] (2) heating the supernatant A in step (1) at 35-60° C. with stirring, adjusting the pH to 5.0-9.0, adding neutral protease and papain, maintaining the pH at 5.0-9.0 during the enzymolysis process, and inactivating the enzyme in the enzymolysis solution after the enzymolysis is completed;

[0008] (3) The enzymatic hydrolysate after the enzyme inactivation in step (2) is centrifuged at 3000-5000 g for 10-30 min, the supernatant B is taken, dextran is added, and the freeze-dried powder is placed at 60-90° C. and 70-90% relative humidity for reaction for 24-48 h to obtain a carrier.

[0009] The precipitate discarded by centrifugation in step (1) is insoluble impurities and large protein aggregates; the supernatant A is whey protein dissolved in monomer form. As an example, in step (1), the centrifugation condition is controlled to be centrifuged at a speed of 3000g for 10-30 minutes, and the supernatant A can be separated. The supernatant A can also be separated by using other centrifugation conditions, which are not listed here one by one.

[0010] Furthermore, in step (2), the addition amounts of the neutral protease and papain are 1-4wt% and 2-6wt% of the whey protein, respectively.

[0011] Furthermore, in step (2), the adjustment solution used to maintain pH is 0.1 M hydrochloric acid / sodium hydroxide solution.

[0012] Furthermore, in step (2), the enzymatic hydrolysis process lasts for 1.5-3 hours, and the enzyme inactivation process is to place the enzymatic hydrolysis solution in a 85° C. water bath for 5 minutes to inactivate the enzyme.

[0013] Furthermore, in step (1), the mass ratio of the whey protein to water is 1:20, and the centrifugation condition is centrifugation at a speed of 3000g for 10-30min.

[0014] Furthermore, in step (2), the stirring is performed by magnetic stirring for 30 minutes, and the solution used for adjusting the pH is 0.1 M hydrochloric acid / sodium hydroxide solution.

[0015] Furthermore, in step (3), the amount of glucan added is 40-200 wt % of the whey protein.

[0016] Furthermore, in step (3), the centrifugation condition is 3000-5000g for 10-30min.

[0017] Furthermore, in step (3), magnetic stirring is performed for 30 minutes during the addition of dextran.

[0018] The present invention also provides a carrier for improving the thermal stability of anthocyanins, wherein the carrier is prepared by the aforementioned construction method.

[0019] The present invention also provides a method for loading anthocyanins on the aforementioned carrier, comprising the following steps: adding the carrier and anthocyanins or anthocyanin extracts into water and mixing them evenly, and then drying to obtain a carrier loaded with anthocyanins. It is clearly stated here that "the carrier" refers to a carrier for loading anthocyanins, and "anthocyanins or anthocyanin extracts" refers to the substance loaded into the carrier.

[0020] Furthermore, the mass volume ratio of the carrier to water is (0.5-3) g:100 ml. "The carrier" refers to the carrier used to load anthocyanins.

[0021] Furthermore, the drying is preferably spray drying.

[0022] In the present invention, unless otherwise specified, "the carrier" refers to a carrier for loading anthocyanins. The present invention also provides a food, health product or medicine comprising the above carrier, wherein the carrier is loaded with anthocyanins.

[0023] The anthocyanin extract of the present invention can be derived from blueberry, strawberry, bilberry, black rice, purple cabbage, eggplant and the like.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The preparation method of the present invention can significantly improve the stability of anthocyanins, the prepared carrier has a large anthocyanin loading capacity (not less than 7.5wt%), and is of great significance for expanding the application of anthocyanins in food, health products and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the retention rate of anthocyanins after heating in the examples of the present invention and the comparative examples. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0028] Unless otherwise specified, the same anthocyanin extract, i.e., blueberry anthocyanin extract, was used in the specific examples and comparative examples. The anthocyanin monomer content measured by pH differential method was 25%.

[0029] Example

[0030] A method for constructing a vector for improving the thermal stability of anthocyanins comprises the following steps:

[0031] (1) Accurately weigh 5 g of whey protein, add 20 times the mass of water, centrifuge at 3000 g for 10-30 min, carefully pour out the supernatant and discard the precipitate.

[0032] (2) The supernatant in step (1) was added to a water bath jacket beaker, heated at 35-60°C with magnetic stirring, the pH was adjusted to 5.0-9.0 with 0.1M hydrochloric acid / sodium hydroxide solution, and then 0.05-0.2g of neutral protease and 0.1-0.3g of papain were added, and the pH was maintained at 5.0-9.0 by titration with 0.1M hydrochloric acid / sodium hydroxide solution. The enzymolysis was continued for 1.5-3h. After completion, the enzymolysis solution was placed in an 85°C water bath to inactivate the enzyme for 5min.

[0033] (3) After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate in step (2) is centrifuged at 3000-5000g for 10-30min, the supernatant is carefully poured out, 2-10g of dextran is added, and magnetic stirring is performed for 30min. The freeze-dried powder is placed in a constant temperature and humidity chamber with the temperature set at 60-90°C and the relative humidity set at 70-90%. The glycosylation reaction is continued for 24-48h to obtain the carrier.

[0034] The carrier obtained in step (3) is used to load anthocyanins:

[0035] Dissolve 2 g of the carrier and no more than 0.6 g of anthocyanin extract in 100 ml of water, stir magnetically for 30 min, ultrasonicate at 200-400 W for 5-10 min, spray-dry the sample at 130-170° C. (inlet temperature) / 70-90° C. (outlet temperature) to obtain a carrier loaded with anthocyanins.

[0036] Example 1

[0037] (1) Accurately weigh 5 g of whey protein, add 20 times the mass of water, centrifuge at 3000 g for 20 min, carefully pour out the supernatant and discard the precipitate.

[0038] (2) The supernatant in step (1) was added to a water bath jacket beaker, heated at 50°C with magnetic stirring for 30 min, the pH was adjusted to 6.0 with 0.1M hydrochloric acid / sodium hydroxide solution, and then 0.2g of neutral protease and 0.2g of papain were added, and the pH was maintained at 6.0 by titration with 0.1M hydrochloric acid / sodium hydroxide solution. The enzymolysis was continued for 2h. After completion, the enzymolysis solution was placed in an 85°C water bath to inactivate the enzyme for 5min.

[0039] (3) After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate in step (2) is centrifuged at 3000g for 20 minutes, the supernatant is carefully poured out, 5g of dextran is added, and magnetic stirring is performed for 30 minutes. The freeze-dried powder is placed in a constant temperature and humidity chamber with the temperature set at 80°C and the relative humidity set at 79%. The reaction is continued for 48 hours to obtain the carrier.

[0040] (4) 2 g of the carrier and 0.2 g of anthocyanin extract were dissolved in 100 ml of water, magnetically stirred for 30 min, ultrasonicated at 300 W power for 5 min, and the sample was spray-dried at 150°C (inlet temperature) / 80°C (outlet temperature) to obtain a carrier loaded with anthocyanins.

[0041] Example 2

[0042] (1) Accurately weigh 5g of whey protein, add 20 times the mass of water, centrifuge at 3000g for 20min, carefully pour out the supernatant and discard the precipitate.

[0043] (2) Take the supernatant in step (1) and add it to a water bath jacket beaker. Heat it at 50°C with magnetic stirring for 30 minutes. Adjust the pH to 7.0 with 0.1M hydrochloric acid / sodium hydroxide solution. Then add 0.1g neutral protease and 0.2g papain. Titrate with 0.1M hydrochloric acid / sodium hydroxide solution to maintain pH 7.0. Continue enzymolysis for 2 hours. After completion, place the enzymolysis solution in an 85°C water bath to inactivate the enzyme for 5 minutes.

[0044] (4) After the enzymatic hydrolysis, the enzymatic hydrolyzate in step (3) was centrifuged at 3000g for 20 minutes, the supernatant was carefully poured out, 5g of dextran was added, and magnetic stirring was performed for 30 minutes. The freeze-dried powder was placed in a constant temperature and humidity chamber with the temperature set at 80°C and the relative humidity set at 79%. The reaction was continued for 48 hours to obtain a blueberry anthocyanin carrier.

[0045] (5) Dissolve 2 g of the carrier and 0.6 g of blueberry anthocyanin extract in 100 ml of water, stir magnetically for 30 min, ultrasonicate at 300 W for 5 min, and spray dry the sample at 150°C (inlet temperature) / 80°C (outlet temperature) to obtain a blueberry anthocyanin carrier.

[0046] Comparative Example 1 is an untreated blueberry anthocyanin extract from the same source.

[0047] Comparative Example 2

[0048] (1) Accurately weigh 5g of whey protein, add 20 times the mass of water, centrifuge at 3000g for 20min, carefully pour out the supernatant, discard the precipitate and freeze-dry to obtain freeze-dried whey protein.

[0049] (2) Dissolve 2 g of freeze-dried whey protein and 0.2 g of blueberry anthocyanin extract in 100 ml of water, stir magnetically for 30 min, and ultrasonicate at 300 W for 5 min. Spray dry the sample at 150°C (inlet temperature) / 80°C (outlet temperature) to obtain a blueberry anthocyanin carrier.

[0050] Comparative Example 3

[0051] 2 g of glucan and 0.2 g of blueberry anthocyanin extract were dissolved in 100 ml of water, magnetically stirred for 30 min, ultrasonicated at 300 W power for 5 min, and the sample was spray-dried at 150° C. (inlet temperature) / 80° C. (outlet temperature) to obtain a blueberry anthocyanin carrier.

[0052] Comparative Example 4

[0053] (1) Accurately weigh 5g of whey protein, add 20 times the mass of water, centrifuge at 3000g for 20min, carefully pour out the supernatant and discard the precipitate.

[0054] (2) Take the supernatant in step (1) and add it to a water bath jacket beaker. Heat it at 50°C and stir it magnetically for 30 minutes. Adjust the pH to 7.0 with 0.1M hydrochloric acid / sodium hydroxide solution, then add 5g of dextran and stir it magnetically for 30 minutes. After freeze-drying, place the powder in a constant temperature and humidity chamber with the temperature set at 80°C and the relative humidity set at 79%. The reaction is continued for 48 hours to obtain the carrier.

[0055] (3) Dissolve 2 g of the carrier and 0.2 g of blueberry anthocyanin extract in 100 ml of water, stir magnetically for 30 min, and ultrasonicate at 300 W for 5 min. Spray dry the sample at 150°C (inlet temperature) / 80°C (outlet temperature) to obtain a blueberry anthocyanin carrier.

[0056] The blueberry anthocyanin carriers prepared in the embodiment and the comparative example were respectively used to carry out the following experiments:

[0057] Take 0.1g of anthocyanin extract and a carrier containing an equal amount of anthocyanins (the sampling amount of blueberry anthocyanin carrier in Example 1 and Comparative Examples 2-4 is 1.1g, and the sampling amount of blueberry anthocyanin carrier in Example 2 is 0.43g) and dissolve them in 100ml of 0.05M PBS buffer (pH=7.4), respectively, and place them in a water bath at 80°C for 100min. Take out the solution, cool it to room temperature, and determine the anthocyanin retention rate by pH differential method: take 0.1ml of the solution after heating in a water bath, dissolve it in 0.9ml of pH 1.0 and 4.5 buffer, measure the absorbance at 520nm and 700nm, and calculate the anthocyanin content according to the following formula:

[0058] Anthocyanin content (%, w / w) = (ΔA pH1.0 -ΔA pH4.5 )×MW×DF×V / (ε×L×M t )×100

[0059] Where: ΔA pH1.0 ——The difference in absorbance of the sample at 520nm and 700nm at pH 1.0;

[0060] ΔA pH4.5 ——The difference in absorbance of the sample at 520nm and 700nm at pH 4.5;

[0061] MW——molecular weight of cyanidin-3-glucoside (484.82 g / mol);

[0062] DF – dilution factor;

[0063] V——volume of diluent;

[0064] ε——molar extinction coefficient of cyanidin-3-glucoside, 26900;

[0065] L——optical path, 1cm;

[0066] M t ——Mass of anthocyanins before heating, g.

[0067] The thermal stability of Examples 1-2 and Comparative Examples 1-4 is as follows Figure 1 As shown in the figure, after water bath treatment at 80°C for 100 minutes, the carrier in Example 1 significantly increased the anthocyanin retention rate from 8.35% to 44.79%; the anthocyanin monomer content in the anthocyanin extract added in Example 2 accounted for 7.5wt% of the carrier, and the carrier protection ability decreased slightly, but was still significantly higher than the comparative example, indicating that the effective loading of anthocyanins can still be achieved at this anthocyanin content; Comparative Example 2 with only whey protein added, Comparative Example 3 with only glucan added, and Comparative Example 4 with whey protein and glucan added and grafted by Maillard reaction did not significantly improve the thermal stability of anthocyanins.

[0068] In summary, the present invention proposes a method for constructing a vector that can significantly improve the thermal stability of blueberry anthocyanins. The preparation of the vector is simple and inexpensive, and the thermal stability of blueberry anthocyanins is significantly improved, which is of great significance for ensuring the stability of blueberry anthocyanins added to food during processing and storage. The above are only a few examples of the implementation of the present invention. Obviously, the present invention is not limited to the above embodiments, and there may be many variations. All variations that can be directly derived or associated with the contents disclosed by ordinary technicians in this field should be considered to be within the scope of protection of the present invention.

Claims

1. A method for constructing a vector for improving the thermal stability of anthocyanins, characterized in that: The following steps are involved: (1) Dissolve whey protein in water, discard the precipitate by centrifugation, and take the supernatant A; (2) heating the supernatant A in step (1) at 35-60° C. with stirring, adjusting the pH to 5.0-9.0, adding neutral protease and papain, maintaining the pH at 5.0-9.0 during the enzymolysis process, and inactivating the enzyme in the enzymolysis solution after the enzymolysis is completed; (3) The enzymatic hydrolysate after the enzyme inactivation in step (2) is centrifuged at 3000-5000 g for 10-30 min, the supernatant B is taken, dextran is added, and the freeze-dried powder is placed at 60-90° C. and 70-90% relative humidity for reaction for 24-48 h to obtain a carrier.

2. The construction method according to claim 1, characterized in that: In step (2), the addition amounts of the neutral protease and papain are 1-4wt% and 2-6wt% of the whey protein, respectively.

3. The construction method according to claim 1, characterized in that: In step (2), the adjusting solution used to maintain pH is 0.1 M hydrochloric acid / sodium hydroxide solution.

4. The construction method according to claim 1, characterized in that: In step (2), the enzymatic hydrolysis process lasts for 1.5-3 hours, and the enzyme inactivation process is to place the enzymatic hydrolysis solution in a water bath at 85° C. for 5 minutes to inactivate the enzyme.

5. The construction method according to claim 1, characterized in that: In step (1), the mass ratio of whey protein to water is 1:20, and the centrifugation condition is centrifugation at a speed of 3000g for 10-30min.

6. The construction method according to claim 1, characterized in that: In step (2), the stirring is performed by magnetic stirring for 30 minutes, and the solution used for adjusting the pH is 0.1 M hydrochloric acid / sodium hydroxide solution.

7. The construction method according to claim 1, characterized in that: In step (3), the amount of glucan added is 40-200wt% of the whey protein.

8. A carrier for improving the thermal stability of anthocyanin, characterized in that: The vector is prepared by the construction method described in any one of claims 1 to 7.

9. The method for loading anthocyanins onto a carrier according to claim 8, characterized in that: The following steps are involved: The carrier and anthocyanidin or anthocyanidin extract according to claim 8 are added into water and mixed evenly, and then dried to obtain a carrier loaded with anthocyanidin.

10. A food, health product or medicine comprising the carrier according to claim 8, wherein the carrier is loaded with anthocyanins.

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