Method for stabilizing anthocyanin in liquid dairy product

Abstract

The invention discloses a method for stabilizing anthocyanin in a liquid dairy product. Aiming at the problems that anthocyanin is poor in stability and easy to degrade and fade, fiber easy-aggregation peptide is prepared from cow milk protein through enzymolysis, polypeptide is induced to be assembled by utilizing a high-pressure microjet technology to form amyloid fiber with a crossed-beta-folded structure, and the amyloid fiber is used for encapsulating anthocyanin and improving the stability of the anthocyanin. After stabilization treatment, the thermal degradation half-life period of the anthocyanin is increased from 29.2 min to 52.9 min. Compared with the prior art, the stabilization method can effectively improve the stability of anthocyanin in the liquid milk product, and the prepared milk product is long in shelf life, resistant to heat processing and stable in color generation.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a method for stabilizing anthocyanins in liquid milk products. Background Art

[0002] Anthocyanins are a class of water-soluble natural pigments widely distributed in plants. They appear red, purple, blue and other colors depending on pH, metal ions, etc., and have a variety of health benefits including antioxidant, anti-aging, anti-cancer, anti-diabetic, eye protection and neuroprotective effects. They are one of the most commonly used natural pigments in food production. A large number of dairy products on the market, including flavored milk drinks, flavored fermented milk, etc., add anthocyanin-rich pulp and jam to the products to provide the products with bright colors, rich taste and flavor. In dairy products targeting infants and the elderly, extracts of medicinal and edible plants rich in anthocyanins are also added to give the products antioxidant, anti-aging, eye protection and other health benefits.

[0003] However, anthocyanins are easily degraded under conditions such as heating, light, oxygen and metal ions, which greatly limits the product's processing technology, shelf life and health benefits. Although anthocyanins can improve stability by forming complexes with macromolecules such as proteins and polysaccharides, the structural characteristics of proteins from different sources (such as amino acid composition, surface hydrophobicity and charge distribution) significantly affect their binding ability. Although whey protein and casein naturally present in dairy products can interact with anthocyanins, their binding affinity is low, making it difficult to effectively protect anthocyanin molecules from damage by external factors.

[0004] At present, common methods for stabilizing anthocyanins include adding auxiliary colorants, microencapsulation, chemical modification, etc. Among them, chemical modification requires the introduction of strong oxidants such as thionyl chloride, which causes environmental pollution and the product is difficult to use in food production. Microencapsulation is often used in the production of solid powders, and has limited protective effects on anthocyanins in liquid milk systems. In addition, microcapsules may interact with proteins and fat globules in dairy products, causing unpredictable changes. The addition of phenolic acid auxiliary colorants also has limited effects on the stability of anthocyanins, and often changes the pH and taste of food, induces milk protein aggregation and precipitation, and destroys the physical stability of dairy products.

[0005] In response to the above problems, some studies have attempted to improve the stability of anthocyanins through amyloid fibers. For example, CN117770448A discloses a method for preparing anthocyanin protein complexes, which forms amyloid fibers by acidifying and heating soybean globulin, and then prepares the complexes by ultra-high pressure treatment. However, this method relies on the acidification and heating steps to prepare amyloid fibers. Acidification and long-term heating greatly destroy the nutritional value of protein, and the acidification process introduces safety hazards to the food processing process, which puts higher requirements on processing equipment.

[0006] Therefore, developing an innovative method that can fully avoid destructive processing (such as high-temperature acidification) while efficiently and harmlessly improving the stability of anthocyanins has become one of the key issues that need to be overcome in the application of anthocyanins in dairy production. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the present invention aims to provide a method for stabilizing anthocyanins in liquid milk products. The treatment method is simple, environmentally friendly, does not introduce inedible ingredients and does not damage the milk system, and the stability of anthocyanins in the obtained product is significantly improved, which is suitable for application in the dairy product processing process.

[0008] Dairy products contain natural whey protein and casein. If the protein peptides in dairy products can be induced to assemble into amyloid fibers, a natural and harmless anthocyanin stabilizing carrier can be obtained, thereby improving the stability of anthocyanins in dairy products.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] In a first aspect of the present invention, a method for stabilizing anthocyanins in a liquid milk product is provided, comprising the following steps:

[0011] (1) mixing liquid milk product raw materials with protease and adjusting the temperature for enzymatic hydrolysis;

[0012] (2) subjecting the liquid dairy product raw material after enzymatic hydrolysis in step (1) to high-pressure microfluidization treatment;

[0013] (3) mixing the liquid milk product raw material treated by high-pressure microfluidization in step (2) with anthocyanins and sterilizing the mixture to obtain a liquid milk product containing anthocyanins;

[0014] The amount of protease added in step (1) is 1-100u / g protein, the enzymatic hydrolysis conditions are 30-65°C, and the enzymatic hydrolysis time is 3-60min.

[0015] The above 1-100u / g protein refers to a protease having an enzyme activity content of 1-100u per gram of protein.

[0016] As an example, the liquid milk product raw material is one or more of whole milk, skim milk, modulated milk, and fermented milk. The preparation and acceptance of the liquid milk product raw material are carried out according to conventional methods in the art, and the present invention does not specifically limit or elaborate on it. For example, the liquid milk product raw material in step (1) is accepted whole milk, skim milk obtained after centrifugation, milk beverage after raw milk is modulated, and yogurt obtained by fermenting raw milk.

[0017] After the step (1) of the present invention, fiber aggregation-prone peptides can be prepared from dairy protein.

[0018] In the above method, the liquid dairy product raw material in step (1) has no special restrictions on its composition, but the average particle size must be below 5 μm, preferably below 2.5 μm, to avoid affecting the high-pressure microfluidization effect.

[0019] For example, the protease in step (1) is one or more of pepsin, acid protease, papain, neutral protease, trypsin, and alkaline protease.

[0020] In the above method, the amount of protease added in step (1) is preferably 50-80u / g protein.

[0021] In the above method, the enzymatic hydrolysis conditions in step (1) are preferably 40-60° C.; and the enzymatic hydrolysis time is preferably 5-40 min.

[0022] In the above method, the pressure of the high-pressure microjet in step (2) is 150-240 MPa, preferably 180-210 MPa, the maintenance time is 10-18 s, preferably 12-15 s, and the number of repetitions is 2-5 times. After step (2) of the present invention, the fiber aggregation-prone peptide can be assembled into amyloid fibers rich in cross-β-folded structure.

[0023] In the above method, the anthocyanins in step (3) are derived from one or more anthocyanin-rich vegetables or fruit pulps, or extracts thereof.

[0024] In the above method, the amount of anthocyanin added in step (3) is 20-500 mg / g protein, preferably 50-300 mg / g protein, measured by pH differential method.

[0025] In the above method, the sterilization temperature in step (3) is 125-135° C., preferably 128-134° C., and the time is 3-10 seconds, preferably 4-7 seconds. After step (3) of the present invention, a stable liquid milk product containing anthocyanins can be obtained.

[0026] In a second aspect of the present invention, a liquid milk product containing anthocyanins is provided, wherein the milk product is prepared by the method according to the first aspect of the present invention.

[0027] In the above-mentioned liquid milk product containing anthocyanins, the content of anthocyanins is 20-500 mg / g protein, preferably 50-300 mg / g protein. The anthocyanin content is calculated by pH differential method. This method belongs to the commonly used anthocyanin content detection method in this field, and the present invention is not specifically limited and elaborated here.

[0028] In the above-mentioned liquid milk product containing anthocyanins, the thermal degradation half-life of anthocyanins at 90°C is longer than 45 minutes, preferably longer than 50 minutes.

[0029] The method for stabilizing anthocyanins in liquid milk products disclosed in the present invention has the following advantages:

[0030] (1) The present invention uses restricted enzymatic hydrolysis to convert proteins naturally contained in dairy products into fiber-aggregation-prone peptides, and uses high-pressure microfluidization technology to induce the polypeptides to assemble into protein fibers rich in cross-β-folding. The cross-β-folding structure of the fibers is used to stabilize anthocyanins, thereby significantly improving the thermal stability and storage stability of anthocyanins. The resulting dairy products have a long shelf life, stable anthocyanin color, and no negative impact on the stability and taste of the dairy product system.

[0031] (2) The method for stabilizing anthocyanins in liquid milk products of the present invention utilizes proteins naturally present in dairy products as carrier materials for stabilizing anthocyanins, and does not introduce any inedible reagents. It is environmentally friendly, low-cost, and has no potential safety hazards.

[0032] (3) The present invention uses enzymatic hydrolysis combined with high-pressure microfluidics technology to achieve protein modification, which efficiently and harmlessly improves the stability of anthocyanins, realizes dynamic and gentle processing, and avoids destructive processing such as strong acid and high temperature treatment to prevent the loss of nutritional components of dairy products; it realizes production process coupling to the greatest extent, saves costs, reduces operating procedures, and is suitable for industrial production.

[0033] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The secondary structure composition of proteins in anthocyanin-containing milk products prepared by different treatments is shown;

[0035] Figure 2 The fluorescence intensity of Thioflavin T (ThT) staining of anthocyanin-containing milk products prepared by different treatments is shown;

[0036] Figure 3 The degradation kinetics of anthocyanins in anthocyanin-containing milk products prepared by different treatments under heating at 90°C are shown;

[0037] Figure 4 The graph shows the change in the retention rate of anthocyanins in anthocyanin-containing dairy products prepared by different treatments at 25°C in a dark environment with storage time (the anthocyanin content before sterilization is 100%). DETAILED DESCRIPTION

[0038] The following is a description of the preferred embodiments of the present invention with reference to the accompanying drawings to make the technical content clearer and easier to understand. The detection methods mentioned in the experiment are in accordance with conventional methods in the field. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text. Unless otherwise specified, the percentages mentioned in the following examples and comparative examples refer to mass percentages.

[0039] Example 1

[0040] The flavored milk containing blueberry anthocyanins was prepared by the following method:

[0041] Raw materials: inspected whole milk, protein content of 3.2%, average particle size of 1.57±0.08μm; blueberry anthocyanin extract, anthocyanin content of 25% (measured by pH differential method); trypsin, enzyme activity of 250u / mg; neutral protease, enzyme activity of 50u / mg.

[0042] Preparation method:

[0043] (1) adding protease (including trypsin and neutral protease) to whole milk and stirring and mixing, wherein the amount of trypsin added is 6.4 mg / L (50 u / g protein), and the amount of neutral protease added is 6.4 mg / L (10 u / g protein), and the temperature of the whole milk is adjusted to 45° C. for enzymolysis, and the enzymolysis time is 15 min;

[0044] (2) subjecting the enzymatically hydrolyzed whole milk obtained in step (1) to high-pressure microfluidization treatment, wherein the microfluidization pressure is 180 MPa, the maintenance time is 12 s, and the number of repetitions is 4 times;

[0045] (3) The whole milk treated with high-pressure microfluidization obtained in step (2) is stirred and mixed with a blueberry anthocyanin extract, wherein the amount of blueberry anthocyanin extract added is 12.8 mg / L (equivalent to 100 mg anthocyanin / g protein, measured by pH differential method), and the mixed milk product is sterilized at a temperature of 132° C. for 4 seconds to obtain a flavored milk containing blueberry anthocyanins.

[0046] Example 2

[0047] The raw materials are the same as those in Example 1. The difference between the preparation method and Example 1 is that: the amount of trypsin added is 80u / g protein, no neutral protease is added, the enzymatic hydrolysis temperature is 30°C, and the enzymatic hydrolysis time is 60min; the microjet pressure is 150MPa, the holding time is 18s, and the number of repetitions is 5 times; the amount of blueberry anthocyanin extract added is 20mg anthocyanin / g protein, the sterilization temperature is 135°C, and the sterilization time is 3s.

[0048] Example 3

[0049] The raw materials are the same as those in Example 1. The difference between the preparation method and Example 1 is that: the amount of trypsin added is 30u / g protein, the amount of neutral protease added is 20u / g protein, the enzymatic hydrolysis temperature is 65°C, and the enzymatic hydrolysis time is 3min; the microjet pressure is 240MPa, the holding time is 10s, and the number of repetitions is 2 times; the amount of blueberry anthocyanin extract added is 500mg anthocyanin / g protein, the sterilization temperature is 125°C, and the sterilization time is 10s.

[0050] Comparative Example 1

[0051] In the process of preparing flavored modulated milk containing blueberry anthocyanins in Comparative Example 1, enzymatic hydrolysis and high-pressure microfluidization treatment were not performed, and the raw materials were the same whole milk and blueberry anthocyanin extract as those described in Experimental Example 1.

[0052] Preparation method:

[0053] Add blueberry anthocyanidin extract to whole milk and stir to mix, wherein the amount of blueberry anthocyanidin extract added is 12.8 mg / L (equivalent to 100 mg anthocyanidin / g protein, calculated by pH differential method), and sterilize the mixed milk product at a sterilization temperature of 132° C. for 4 seconds to obtain flavored modulated milk containing blueberry anthocyanidins.

[0054] Comparative Example 2

[0055] In Comparative Example 2, in the process of preparing flavored modulated milk containing blueberry anthocyanins, only enzymatic hydrolysis was performed without high-pressure microfluidization treatment, and the raw materials were the same as those in Experimental Example 1.

[0056] Preparation method:

[0057] (1) adding protease (including trypsin and neutral protease) to whole milk and stirring and mixing, wherein the amount of trypsin added is 6.4 mg / L (50 u / g protein), and the amount of neutral protease added is 6.4 mg / L (10 u / g protein), and the temperature of the whole milk is adjusted to 45° C. for enzymolysis, and the enzymolysis time is 15 min;

[0058] (2) The enzymatically hydrolyzed whole milk obtained in step (1) is stirred and mixed with a blueberry anthocyanin extract, wherein the amount of blueberry anthocyanin extract added is 12.8 mg / L (equivalent to 100 mg anthocyanin / g protein, measured by pH differential method), and the mixed milk product is sterilized at a temperature of 132° C. for a sterilization time of 4 seconds to obtain a flavored modulated milk containing blueberry anthocyanins.

[0059] Comparative Example 3

[0060] In Comparative Example 3, in the process of preparing flavored reconstituted milk containing blueberry anthocyanins, only high-pressure microfluidization treatment was performed without enzymatic hydrolysis, and the raw materials were the same whole milk and blueberry anthocyanin extract as described in Experimental Example 1.

[0061] (1) Whole milk was treated with high-pressure microfluidization, with a microfluidization pressure of 180 MPa, a maintenance time of 12 s, and a repetition frequency of 4 times;

[0062] (2) The whole milk treated with high-pressure microfluidization obtained in step (1) is stirred and mixed with a blueberry anthocyanin extract, wherein the amount of blueberry anthocyanin extract added is 12.8 mg / L (equivalent to 100 mg anthocyanin / g protein, measured by pH differential method), and the mixed milk product is sterilized at a sterilization temperature of 132° C. for a sterilization time of 4 seconds to obtain a flavored modulated milk containing blueberry anthocyanins.

[0063] Comparative Example 4

[0064] In Comparative Example 4, enzymatic hydrolysis was combined with high-pressure microfluidization process parameters in the process of preparing flavored modulated milk containing blueberry anthocyanins, but the enzymatic hydrolysis process was not optimized and the raw materials were the same as those in Experimental Example 1.

[0065] Preparation method:

[0066] (1) adding protease (including trypsin and neutral protease) to whole milk and stirring and mixing, wherein the amount of trypsin added is 6.4 mg / L (50 u / g protein), and the amount of neutral protease added is 12.8 mg / L (20 u / g protein), adjusting the temperature of the whole milk to 45° C. for enzymolysis, and the enzymolysis time is 100 min;

[0067] (2) subjecting the enzymatically hydrolyzed whole milk obtained in step (1) to high-pressure microfluidization treatment, wherein the microfluidization pressure is 180 MPa, the maintenance time is 12 s, and the number of repetitions is 4 times;

[0068] (3) The whole milk treated with high-pressure microfluidization obtained in step (2) is stirred and mixed with a blueberry anthocyanin extract, wherein the amount of blueberry anthocyanin extract added is 12.8 mg / L (equivalent to 100 mg anthocyanin / g protein, measured by pH differential method), and the mixed milk product is sterilized at a temperature of 132° C. for 4 seconds to obtain a flavored milk containing blueberry anthocyanins.

[0069] Comparative Example 5

[0070] In Comparative Example 5, enzymatic hydrolysis was combined with high-pressure microfluidization process parameters in the process of preparing flavored modulated milk containing blueberry anthocyanins, but the high-pressure microfluidization process was not optimized, and the raw materials were the same as those in Experimental Example 1.

[0071] Preparation method:

[0072] (1) adding protease (including trypsin and neutral protease) to whole milk and stirring and mixing, wherein the amount of trypsin added is 6.4 mg / L (50 u / g protein), and the amount of neutral protease added is 6.4 mg / L (10 u / g protein), and the temperature of the whole milk is adjusted to 45° C. for enzymolysis, and the enzymolysis time is 15 min;

[0073] (2) subjecting the enzymatically hydrolyzed whole milk obtained in step (1) to high-pressure microfluidization treatment, wherein the microfluidization pressure is 60 MPa, the maintenance time is 12 s, and the number of repetitions is 2 times;

[0074] (3) The whole milk treated with high-pressure microfluidization obtained in step (2) is stirred and mixed with a blueberry anthocyanin extract, wherein the amount of blueberry anthocyanin extract added is 12.8 mg / L (equivalent to 100 mg anthocyanin / g protein, measured by pH differential method), and the mixed milk product is sterilized at a temperature of 132° C. for 4 seconds to obtain a flavored milk containing blueberry anthocyanins.

[0075] First, the protein structure of the processed flavored milk was analyzed to determine the generation of protein fibers. The secondary structures of the proteins in Experimental Example 1 and Comparative Examples 1-5 were measured by circular dichroism spectroscopy. The results are as follows: Figure 1 As shown, in Experimental Example 1, the α-helix of the protein was converted to β-fold after restricted enzymatic hydrolysis and high-pressure microfluidization treatment, indicating that the rigidity of the protein was enhanced. Similar secondary structure transformation also occurred in Comparative Example 3, indicating that high-pressure microfluidization promoted protein modification. Thioflavin T (ThT) is a hydrophobic dye that can specifically bind to fibers. After binding, the fluorescence at an excitation wavelength of 440nm and an emission wavelength of 480nm is significantly enhanced. Figure 2 As shown, the fluorescence of Experimental Example 1 and Comparative Example 5 is significantly enhanced, indicating that the proteins of Experimental Example 1 and Comparative Example 5 assemble to form amyloid fibers, and the production of fiber aggregation-prone peptides by restricted enzymatic hydrolysis and high-pressure microfluidization treatment are indispensable for the assembly of protein fibers. The ThT fluorescence intensity of Comparative Example 4 did not increase, indicating that the protein peptides after excessive enzymatic hydrolysis could not form amyloid fibers. The ThT fluorescence intensity of Comparative Example 5 is significantly lower than that of Experimental Example 1, indicating that the high-pressure microfluidization conditions affect the assembly of amyloid fibers.

[0076] Secondly, the stabilization effect of anthocyanins in flavored milk was evaluated. Experimental Example 1 and Comparative Examples 1-5 were heated at 90°C. The relationship between the logarithm of the anthocyanin retention rate and the heating time is shown in the following figure: Figure 3As shown, compared with comparative examples 1-5, the degradation of anthocyanins in experimental example 1 was significantly slowed down. The relationship between anthocyanin retention rate and heating time was fitted according to the first-order reaction kinetics, and the thermal degradation half-lives of experimental example 1 and comparative examples 1-5 were 52.9min, 29.2min, 34.5min, 31.5min, 38.7min, and 36.2min, respectively. This shows that the thermal stability of anthocyanins is significantly improved. The storage stability at room temperature is shown in FIG. Figure 4 As shown, the anthocyanin retention rates of Experimental Example 1 and Comparative Examples 1-5 after sterilization were 88.1±1.1%, 79.7±1.7%, 83.4±1.3%, 82.1±0.4%, 84.2±0.8%, and 85.0±1.1%, respectively. After 35 days of storage, the anthocyanin retention rates dropped to 55.6±1.4%, 20.2±1.3%, 25.5±1.3%, 22.9±1.3%, 26.6±1.3%, and 28.8±1.3%. The above results show that the method for stabilizing anthocyanins in liquid milk products proposed in the present invention can significantly improve the thermal stability and storage stability of anthocyanins, and that restricted enzymolysis and high-pressure microfluidization are both necessary steps for stabilizing anthocyanins. As in Example 1, compared with the treatment processes of only enzymatic hydrolysis, unoptimized enzymatic hydrolysis process, only high-pressure microfluidization or unoptimized enzymatic hydrolysis process, the anthocyanins treated in Examples 2-3 of the present invention have significantly improved thermal stability after heating and storage stability at room temperature.

[0077] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

[0078] Based on the protective effect of the characteristic cross-β-folding structure in amyloid fibers on anthocyanins, the inventor combined restricted enzymolysis and high-pressure microfluidization technology to decompose the naturally existing proteins in dairy products to release fiber-prone peptides, and assemble to form amyloid fibers, thereby protecting the anthocyanins in dairy products and significantly enhancing the thermal stability and storage stability of anthocyanins without introducing any organic reagents, small molecule auxiliary colorants, and macromolecular microcapsule wall materials. Compared with the current anthocyanin stabilization method, the present invention is specially designed for dairy products, and significantly improves the stability of anthocyanins without introducing any food risks and ingredients that destroy the flavor; and it is simple to operate, can be highly coupled with the dairy production line, saves costs to the greatest extent, and is convenient for large-scale promotion, paving the way for the use of anthocyanins as natural pigments and functional ingredients in dairy products.

Claims

1. A method for stabilizing anthocyanins in liquid milk products, characterized in that: The following steps are involved: (1) mixing liquid milk product raw materials with protease and adjusting the temperature for enzymatic hydrolysis; (2) subjecting the liquid dairy product raw material after enzymatic hydrolysis in step (1) to high-pressure microfluidization treatment; (3) mixing the liquid milk product raw material treated by high-pressure microfluidization in step (2) with anthocyanins and sterilizing the mixture to obtain a liquid milk product containing anthocyanins; The amount of protease added in step (1) is 1-100u / g protein; the enzymatic hydrolysis conditions in step (1) are 30-65°C, and the enzymatic hydrolysis time is 3-60min; The pressure used for the high-pressure microfluidization in step (2) is 150-240 MPa.

2. The method according to claim 1, characterized in that The liquid milk product raw material in step (1) is one or more of whole milk, skimmed milk, modified milk and fermented milk.

3. The method according to claim 1, characterized in that The average particle size of the liquid dairy product raw material in step (1) is less than 5 μm.

4. The method according to claim 1, characterized in that The protease in step (1) is one or more of pepsin, acid protease, papain, neutral protease, trypsin and alkaline protease.

5. The method according to claim 1, characterized in that The amount of protease added in step (1) is 50-80u / g protein.

6. The method according to claim 1, characterized in that The enzymatic hydrolysis conditions in step (1) are 40-60° C. and the enzymatic hydrolysis time is 5-40 min.

7. The method according to claim 1, characterized in that The pressure used for the high-pressure microjet in step (2) is 180-210 MPa, the maintenance time is 10-18 s, and the number of repetitions is 2-5 times.

8. The method according to claim 1, characterized in that Calculated by pH differential method, the amount of anthocyanin added in step (3) is 20-500 mg / g protein.

9. The method according to claim 1, characterized in that The sterilization temperature in step (3) is 125-135° C. and the time is 3-10 seconds.

10. Anthocyanin-containing liquid milk product obtained by the method according to any one of the preceding claims.

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