Modified casein-polyphenol compound as well as preparation method and application thereof

Abstract

The invention provides a modified casein-polyphenol compound as well as a preparation method and application thereof, and belongs to the technical field of protein processing. The invention provides the application of the small red garlic polyphenol in casein modification for the first time, and further provides a preparation method of a modified casein-small red garlic polyphenol compound, and the preparation method comprises the following steps: preparing a casein culture solution, inoculating casein casei for fermentation, and sterilizing after fermentation to obtain a fermentation solution; mixing the small red garlic polyphenol with the fermentation liquor for reaction, and drying a reaction product to obtain the modified casein-small red garlic polyphenol covalent complex. The stability, the oxidation resistance, the solubility, the emulsifying capacity, the emulsifying stability, the foamability and the foaming stability of the casein can be obviously improved, and the method has a wide application prospect in the field of protein processing.

Description

Technical Field

[0001] The invention belongs to the technical field of protein processing, and in particular relates to a modified casein-polyphenol complex and a preparation method and application thereof. Background Art

[0002] As the main protein component in dairy products, casein is widely used in food, medicine and cosmetics due to its good nutritional value and functional properties (such as emulsification and gelation). However, natural casein has functional limitations, such as poor thermal stability, low solubility, and weak antioxidant capacity, which restrict its application in complex processing environments.

[0003] To improve its performance, researchers often use physical, chemical or enzymatic modification methods, but traditional methods have significant defects. For example, chemical modification (such as acid treatment, cross-linking agent modification) may introduce toxic reagent residues, affecting product safety; although enzymatic modification conditions are mild, the cost is high and the reaction efficiency is limited by enzyme activity and substrate specificity; physical modification (such as ultrasound, high-pressure homogenization) is environmentally friendly, but the improvement of functional properties is limited and it is difficult to achieve stable binding at the molecular level.

[0004] In recent years, polyphenol-protein covalent compound technology has attracted much attention due to its green and efficient characteristics. Polyphenols can bind to proteins through oxidative cross-linking or non-covalent interactions to enhance their antioxidant, thermal stability and emulsifying properties. Studies have reported the compounding of tea polyphenols, grape seed polyphenols, etc. with casein, but these polyphenols are widely available and have low specificity, and the compounding process mostly relies on strong oxidative conditions (such as alkaline pH or high temperature), which can easily destroy the natural structure of proteins and lead to functional loss.

[0005] As a traditional medicinal plant, the fresh bulb of Eleutherine bulbosa is rich in unique phenolic compounds (such as naphthols and flavonoids) with strong antioxidant activity and biocompatibility. However, the current research on Eleutherine bulbosa polyphenols is mostly focused on extraction technology and pharmacological activity, and its application in protein modification has not been reported. In addition, the existing casein-polyphenol composite technology mostly directly mixes polyphenols with natural casein without combining microbial fermentation strategies, making it difficult to achieve deep modification of protein molecular structure and functional synergy.

[0006] Therefore, developing a green and efficient casein modification method that combines microbial fermentation with specific polyphenol compounding to break through the limitations of traditional technology has become a key issue that needs to be urgently addressed in this field. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a modified casein-polyphenol complex, its preparation method and application, which can improve the stability, antioxidant capacity, solubility, emulsifying ability, emulsifying stability, foaming ability and foaming stability of casein.

[0008] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0009] Application of polyphenols from Lycoris radiata var. radiata in casein modification.

[0010] The present invention also provides a preparation method of a modified casein-polyphenol complex, which includes the following steps: preparing a casein culture solution, inoculating Lactobacillus casei subsp. casei for fermentation, and sterilizing the fermented product to obtain a fermentation broth; mixing polyphenols from Lycoris radiata var. radiata with the fermentation broth for reaction, and drying the reaction product to obtain a modified casein-polyphenols from Lycoris radiata var. radiata covalent complex.

[0011] Preferably, by mass-volume concentration, the casein content in the casein culture solution is 5% - 12%, and the glucose content is 1% - 4%.

[0012] Preferably, during the fermentation, the viable count of Lactobacillus casei subsp. casei is 1×10 7 ~1×10 9 cfu / mL, the temperature is 30 - 37 °C, and the time is 10 - 20 h.

[0013] Preferably, the preparation method of the polyphenols from Lycoris radiata var. radiata includes: using freeze-dried fresh bulbs of Lycoris radiata var. radiata as raw materials, extracting with 20% - 50% ethanol as the extractant, and centrifuging to obtain the extract; evaporating the solvent from the extract to obtain a crude polyphenol extract; dissolving the crude polyphenol extract in deionized water and purifying it by column chromatography to obtain the polyphenols from Lycoris radiata var. radiata.

[0014] Preferably, during the extraction, the material-liquid ratio of freeze-dried fresh bulbs of Lycoris radiata var. radiata to ethanol is 1 g:10 - 25 mL, stirring and extracting for 2 - 5 h, and repeating the extraction 3 - 5 times.

[0015] Preferably, the solvent evaporation is carried out by rotary evaporation at 50 °C; the column chromatography purification is purification by an AB-8 macroporous adsorption resin column.

[0016] Preferably, the mass ratio of the polyphenols from Lycoris radiata var. radiata to the fermentation broth is 1:5 - 15, reacting in a sealed brown container at 4 °C for 8 - 15 h, and the reaction pH is 8.0 - 9.0.

[0017] The present invention also provides the modified casein-polyphenols from Lycoris radiata var. radiata covalent complex prepared by the above preparation method and its application in food processing.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention first applies polyphenols from Rhodophiala bulbocodioides var. minor to the field of casein functional modification, and constructs a casein-polyphenol composite system through fermentation synergistic polyphenol covalent coupling technology. The modified casein-polyphenols from Rhodophiala bulbocodioides var. minor covalent complex of the present invention exhibits unique structure-function synergistic effects, including improved stability, enhanced emulsifying property, and enhanced antioxidant capacity, and can be widely applied to the fields of plant-based protein beverages, low-fat dairy products, and functional embedding carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the instability index of the modified casein-polyphenols from Rhodophiala bulbocodioides var. minor covalent complex of Examples 1 to 4 and Comparative Examples 1 to 4;

[0021] Figure 2 It is the particle size of the modified casein-polyphenols from Rhodophiala bulbocodioides var. minor covalent complex of Examples 1 to 4 and Comparative Examples 1 to 4;

[0022] Figure 3 It is the solubility of the modified casein-polyphenols from Rhodophiala bulbocodioides var. minor covalent complex of Examples 1 to 4 and Comparative Examples 1 to 4. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides an application of polyphenols from Rhodophiala bulbocodioides var. minor in casein modification.

[0024] The present invention also provides a preparation method of a modified casein-polyphenol complex, comprising the following steps:

[0025] (1) Prepare a casein culture solution, inoculate Lactobacillus casei subsp. paracasei and carry out fermentation, and sterilize the fermentation broth after fermentation to obtain a fermentation liquid. The protease produced by Lactobacillus casei subsp. paracasei can degrade casein, expose the hydrophobic amino acid side chains in the protein, and increase the number of static charges of the protein molecules, thereby improving the functional properties of the protein.

[0026] In the present invention, preferably by mass-volume concentration (w / v), the casein content in the casein culture solution is 5% to 12%, and more preferably 8%; the glucose content is 1% to 4%, and more preferably 2%.

[0027] In the present invention, preferably the preparation method of the casein culture solution is as follows: Weigh a certain amount of casein and mix it evenly with deionized water to prepare a 10% to 16% (w / v) casein solution, and mix it with a 4% to 8% (w / v) glucose solution at a ratio of 1 to 3:1 to obtain a casein culture solution. More preferably, a 12% (w / v) casein solution is prepared and mixed with a 6% (w / v) glucose solution at a ratio of 2:1 to obtain a casein culture solution. More preferably, the casein solution and the glucose solution are sterilized at 115 to 121 °C for 15 to 20 min at natural pH, and then prepared under sterile conditions.

[0028] In the present invention, preferably during fermentation, the viable count of Lactobacillus paracasei is 1×10 7 ~1×10 9 cfu / mL, more preferably 1×10 8 cfu / mL; the preferred fermentation temperature is 30 - 37°C, more preferably 35°C; the preferred fermentation time is 10 - 20 h, more preferably 15 h. As an implementable embodiment, the Lactobacillus paracasei of the present invention is Lactobacillus paracasei CICC 6108, purchased from the China Center for Industrial Culture Collection.

[0029] In the present invention, preferably the fermentation broth is heated at 60 - 65°C for 2 - 5 min to kill Lactobacillus paracasei.

[0030] In the present invention, preferably the pH of the fermentation broth is adjusted to 8.0 - 9.0 with 1 mol / L NaOH solution for use in reacting with polyphenols from small red garlic.

[0031] (2) Extraction of polyphenols from small red garlic;

[0032] In the present invention, preferably the preparation method of polyphenols from small red garlic includes: using freeze-dried fresh bulbs of small red garlic as raw materials, extracting with 20% - 50% (v / v) ethanol as the extraction solution, and centrifuging to obtain the extraction solution; evaporating the solvent from the extraction solution to obtain a crude polyphenol extract; dissolving the crude polyphenol extract in deionized water and purifying by column chromatography to obtain polyphenols from small red garlic. The main phenolic compounds in the polyphenols from small red garlic of the present invention are pyrogallol, and the three ortho-phenolic hydroxyl structures can form a strong hydrogen bond network. With phenolic compounds such as taxifolin and hesperetin, they are more likely to undergo a Schiff base reaction with the lysine residues of casein under alkaline conditions, enhancing the covalent binding force; the catechol structures of rosmarinic acid and salvianolic acid D have redox activity and can form dynamic disulfide bonds between casein molecules, enhancing the emulsifying stability of casein. These phenolic compounds communicate to achieve the transformation of casein from a linear structure to a three-dimensional network structure, forming a more stable composite system.

[0033] In the present invention, more preferably, the fresh bulbs of small red garlic after maturity are collected, cut into small pieces, and freeze-dried to make freeze-dried products.

[0034] In the present invention, more preferably, the extraction is carried out with 35% (v / v) ethanol as the extraction solution.

[0035] In the present invention, during extraction, it is further preferred that the material-liquid ratio of freeze-dried fresh bulbs of Rhizoma Curcumae Longae to ethanol is 1 g: 10 - 25 mL, more preferably 1 g: 20 mL; it is further preferred to stir and extract for 2 - 5 h, more preferably for 3.5 h; it is further preferred to repeat extraction 3 - 5 times, more preferably 4 times, and then mix the extraction solutions. It is further preferred to centrifuge at 3000 - 6000×g for 8 - 15 min, more preferably at 5000×g for 10 min.

[0036] In the present invention, during evaporation of the solvent, it is further preferred to use rotary evaporation at 50 °C, and during column purification, it is preferred to use an AB-8 macroporous adsorption resin column (16×300 mm) for purification.

[0037] In the present invention, it is further preferred to lyophilize the obtained polyphenols from Rhizoma Curcumae Longae and store them at -20 °C for later use.

[0038] (3) Mix and react the fermentation broth in step (1) with the polyphenols from Rhizoma Curcumae Longae in step (2), and dry the reaction product to obtain a modified casein-polyphenols from Rhizoma Curcumae Longae covalent complex.

[0039] In the present invention, it is preferred that the mass ratio of polyphenols from Rhizoma Curcumae Longae to the fermentation broth is 1:5 - 15, more preferably 1:10. React at 4 °C for 8 - 15 h in a sealed brown container, more preferably for 10 h, to prepare a modified casein-polyphenols from Rhizoma Curcumae Longae covalent complex solution.

[0040] In the present invention, it is preferred to lyophilize the modified casein-polyphenols from Rhizoma Curcumae Longae covalent complex solution. More preferably, the lyophilization parameters are a temperature of -54 °C and a vacuum of 0.08 mbar to obtain the modified casein-polyphenols from Rhizoma Curcumae Longae covalent complex.

[0041] The present invention also provides the modified casein-polyphenols from Rhizoma Curcumae Longae covalent complex prepared by the above preparation method and its application in food processing, preferably including the preparation of plant-based protein beverages, low-fat dairy products, and functional embedding carriers.

[0042] The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] A method for preparing a modified casein-polyphenols from Rhizoma Curcumae Longae covalent complex comprises the following steps:

[0045] (1) Preparation of casein culture medium:

[0046] Weigh casein and mix it with deionized water to prepare a 14% (w / v) casein solution. Sterilize it at 118 °C for 18 min at natural pH, and set it aside after cooling; weigh glucose and mix it with deionized water to prepare a 5% (w / v) glucose solution. Sterilize it at 118 °C for 18 min at natural pH, and set it aside after cooling; mix the casein solution and the glucose solution at a ratio of 2:1 (v / v) to obtain a casein culture medium (casein concentration 9.3%, glucose concentration 1.7%).

[0047] (2) Fermentation of casein culture medium:

[0048] Inoculate Lactobacillus paracasei CICC6108 with a viable count of 5×10 8 cfu / mL into the casein culture medium; ferment at 33 °C for 12 h, and heat at 62 °C for 4 min to sterilize after fermentation to obtain a fermentation broth.

[0049] (3) Extraction of polyphenols from Lycoris radiata var. radiata:

[0050] Collect the mature fresh bulbs of Lycoris radiata var. radiata, freeze-dry them as raw materials, add 40% (v / v) ethanol according to the solid-liquid ratio of 1 g:15 mL, stir and extract for 4 h, centrifuge at 4000×g for 12 min to obtain the supernatant, repeat 4 times, and combine the extracts; remove ethanol by rotary evaporation at 50 °C to obtain a crude polyphenol extract; dissolve the crude polyphenol extract with deionized water, purify it through an AB-8 macroporous adsorption resin column, and store it at -20 °C after freeze-drying.

[0051] (4) Composite reaction:

[0052] Mix the fermentation broth and polyphenols from Lycoris radiata var. radiata at a mass ratio of 8:1, store them in a sealed brown container, and react at 4 °C for 12 h; after the reaction, freeze-dry (-54 °C, 0.08 mbar) to obtain a modified casein-polyphenols covalent complex from Lycoris radiata var. radiata.

[0053] Example 2

[0054] A preparation method of a modified casein-polyphenols covalent complex from Lycoris radiata var. radiata is as follows:

[0055] (1) Preparation of casein culture medium:

[0056] Weigh casein and mix it with deionized water to prepare a 10% (w / v) casein solution. Sterilize it at 115 °C for 20 min at natural pH, and set it aside after cooling; weigh glucose and mix it with deionized water to prepare a 4% (w / v) glucose solution. Sterilize it at 115 °C for 20 min at natural pH, and set it aside after cooling; mix the casein solution and the glucose solution at a ratio of 1:1 (v / v) to obtain a casein culture medium (casein concentration 5%, glucose concentration 2%).

[0057] (2) Fermentation of casein culture medium:

[0058] Inoculate Lactobacillus paracasei CICC6108 into the casein culture medium, with the viable cell count being 1×10 7 cfu / mL; ferment at 30 °C for 20 h, and after fermentation, heat at 60 °C for 5 min for sterilization to obtain the fermentation broth.

[0059] (3) Extraction of polyphenols from Lycoris radiata var. radiata:

[0060] Collect the mature fresh bulbs of Lycoris radiata var. radiata, freeze-dry them as raw materials, add 20% (v / v) ethanol according to the solid-liquid ratio of 1 g:10 mL, stir and extract for 5 h, centrifuge at 3000×g for 15 min to obtain the supernatant, repeat 3 times, and combine the extracts; remove ethanol by rotary evaporation at 50 °C to obtain the crude polyphenol extract; dissolve the crude polyphenol extract with deionized water, purify it through an AB-8 macroporous adsorption resin column, and store it at -20 °C after freeze-drying.

[0061] (4) Composite reaction:

[0062] Mix the fermentation broth and polyphenols from Lycoris radiata var. radiata at a mass ratio of 5:1, store them in a sealed brown container, and react at 4 °C for 15 h; after the reaction, perform freeze-drying (-54 °C, 0.08 mbar) to obtain the modified casein-polyphenols from Lycoris radiata var. radiata covalent complex.

[0063] Example 3

[0064] A preparation method of a modified casein-polyphenols from Lycoris radiata var. radiata covalent complex, the steps are as follows:

[0065] (1) Preparation of casein culture medium:

[0066] Weigh casein and mix it with deionized water to prepare a 16% (w / v) casein solution, sterilize it at 121 °C for 15 min at natural pH, and set aside after cooling; weigh glucose and mix it with deionized water to prepare an 8% (w / v) glucose solution, sterilize it at 121 °C for 15 min at natural pH, and set aside after cooling; mix the casein solution and the glucose solution at a ratio of 3:1 (v / v) to obtain the casein culture medium (casein concentration 12%, glucose concentration 2%).

[0067] (2) Fermentation of casein culture medium:

[0068] Inoculate Lactobacillus paracasei CICC6108 into the casein culture medium, with the viable cell count being 1×10 9cfu / mL; Ferment at 37 °C for 10 h, and after the fermentation is completed, heat at 65 °C for 2 min to sterilize and obtain the fermentation broth.

[0069] (3) Extraction of polyphenols from small red garlic

[0070] Collect the mature fresh bulbs of small red garlic, freeze-dry them as raw materials, add 50% (v / v) ethanol according to the solid-liquid ratio of 1 g:25 mL, stir and extract for 2 h, centrifuge at 6000×g for 8 min to obtain the supernatant, repeat 5 times, and combine the extracts; Rotate and evaporate at 50 °C to remove ethanol to obtain the crude polyphenol extract; The crude polyphenol extract is dissolved in deionized water, purified by passing through an AB-8 macroporous adsorption resin column, and stored at -20 °C after freeze-drying.

[0071] (4) Composite reaction

[0072] Mix the fermentation broth and small red garlic polyphenols at a mass ratio of 15:1, store in a sealed brown container, and react at 4 °C for 8 h; After the reaction is completed, freeze-dry (-54 °C, 0.08 mbar) to obtain the modified casein-small red garlic polyphenol covalent complex.

[0073] Example 4

[0074] A preparation method of a modified casein-small red garlic polyphenol covalent complex, the steps are as follows:

[0075] (1) Preparation of casein culture solution

[0076] Weigh casein and mix it with deionized water to prepare a 12% (w / v) casein solution, sterilize at 121 °C for 15 min at natural pH, and cool for later use; Weigh glucose and mix it with deionized water to prepare a 6% (w / v) glucose solution, sterilize at 121 °C for 15 min at natural pH, and cool for later use; Mix the casein solution and the glucose solution at a ratio of 2:1 (v / v) to obtain the casein culture solution (casein concentration 8%, glucose concentration 2%).

[0077] (2) Fermentation of casein culture solution

[0078] Inoculate Lactobacillus paracasei CICC6108 into the casein culture solution, and the viable cell count is 1×10 8 cfu / mL. Ferment at 35 °C for 15 h, and after the fermentation is completed, heat at 65 °C for 3 min to sterilize and obtain the fermentation broth.

[0079] (3) Extraction of polyphenols from small red garlic

[0080] The mature fresh bulbs of small red garlic were collected and freeze-dried as raw materials. Ethanol with a volume fraction of 35% (v / v) was added at a solid-liquid ratio of 1 g:20 mL, and the mixture was stirred and extracted for 3.5 h. After centrifugation at 5000×g for 10 min, the supernatant was taken, and the extraction was repeated 4 times. The extracts were combined; ethanol was removed by rotary evaporation at 50 °C to obtain a crude polyphenol extract; the crude polyphenol extract was dissolved in deionized water and purified by passing through an AB-8 macroporous adsorption resin column, and then freeze-dried and stored at -20 °C.

[0081] (4) Composite reaction:

[0082] The fermentation broth and small red garlic polyphenols were mixed at a mass ratio of 10:1, stored in a sealed brown container, and reacted at 4 °C for 10 h; after the reaction, freeze-drying (-54 °C, 0.08 mbar) was carried out to obtain a modified casein-small red garlic polyphenol covalent complex.

[0083] Comparative Example 1

[0084] The preparation method was the same as that of Example 1, except that casein was subjected to high-pressure modification (50 MPa high-pressure microfluidics).

[0085] Comparative Example 2

[0086] The preparation method was the same as that of Example 1, except that casein was not subjected to fermentation treatment.

[0087] Comparative Example 3

[0088] The preparation method was the same as that of Example 1, except that small red garlic polyphenols were replaced with tea polyphenols (purchased from the market).

[0089] Comparative Example 4

[0090] The preparation method was the same as that of Example 1, except that small red garlic polyphenols were replaced with grape seed polyphenols (purchased from the market).

[0091] Test Example 1

[0092] The purified small red garlic polyphenols (100 mg) in Example 1 were mixed evenly with 0.5 mL of 80% (v / v) methanol solution. After centrifugation at 15000×g for 20 min (4 °C), the supernatant was collected and diluted with LC-MS grade water until the methanol concentration reached 53%. The diluted sample was passed through a 0.22 μm filter membrane, and phenolic compounds were determined by UPLC / MS / MS.

[0093] Test results: The total phenolic content of the purified Polygonum minus Roxb. Hance polyphenols in the present invention reaches 358 mg / g of the sample. As determined by UPLC-MS / MS, the results of the phenolic compound composition are shown in Table 1. Among them, the main phenolic compound is pyrogallol, and the three ortho-phenolic hydroxyl structures can form a strong hydrogen bond network. With phenolic compounds such as flavonoids from Larix sibirica Ledeb. and hesperetin, they are more likely to undergo Schiff base reaction with lysine residues of casein under alkaline conditions, enhancing the covalent binding force. The catechol structures of rosmarinic acid and salvianolic acid D have redox activity and can form dynamic disulfide bonds between casein molecules, enhancing the emulsifying stability of casein. These phenolic compounds together achieve the transformation of casein from a linear structure to a three-dimensional network structure, forming a more stable composite system.

[0094] Table 1 Relative contents of phenolic compounds in Polygonum minus Roxb. Hance polyphenols

[0095] name Relative content (%) name Relative content (%) Pyrogallol 56.45 Kaempferol 0.47 Siberian larch flavonoids 10.95 m-Cresol 0.39 Hesperidin 8.05 Eriochoride 0.34 Luteolin 4.52 Isoferulic acid 0.33 gallic acid 2.99 Caffeic acid 0.28 Catechol 2.60 4-Methylcatechol 0.25 Sinapic acid 2.60 Salvianolic acid D 0.24 Rosmarinic acid 2.10 Syringic acid 0.23 Isozepin 1.31 4-Ethylphenol 0.20 Catechins 1.04 Isorhamnetin 0.19 Sakura 0.88 Protocatechuic acid 0.16 Iris flavone 0.77 Puerarin 0.15 6-Methylflavone 0.56 o-Phenylphenol 0.14 Dihydroquercetin 0.54 Zingerone 0.11 Hydroxygenkandrin 0.48 Catechins 0.11 Ferulic acid 0.47 Olive Oil 0.11

[0096] Test Example 2

[0097] Take the covalent complexes prepared in Examples 1 to 4 and Comparative Examples 1 to 4 and measure their instability indices.

[0098] Mix different modified casein-polyphenol covalent complexes with 20 mmol / L phosphate buffer solution at pH 7.0 in a ratio of 1:100, and use a stability analyzer to measure the instability index of the complexes. Slowly add the complex solution to the bottom of a standard cuvette, cover the cuvette, and place it horizontally in the instrument. Set the centrifugal force at 3500 rpm, and record the separation characteristics of the sample every 20 s. Use sepview6 software to calculate the instability index.

[0099] Test results: As Figure 1 shown, the instability indices of the modified casein-Polygonum minus Roxb. Hance polyphenol covalent complexes prepared in Examples 1 to 4 are significantly lower than those in Comparative Examples 1 to 4. Among them, the instability index of Example 4 is the lowest, indicating the best stability, followed by Example 1. The instability indices of Comparative Examples 1, 3, and 4 are relatively high, and the instability index of Comparative Example 2 is the highest, indicating that the traditional physical modification method and other polyphenol reactions have limited effects on improving the stability of casein.

[0100] Test Example 3

[0101] Take the covalent complexes prepared in Examples 1 to 4 and Comparative Examples 1 to 4 and measure their antioxidant properties, including DPPH radical scavenging ability (spectrophotometry) and ABTS radical scavenging ability (spectrophotometry).

[0102] Test results: As can be seen from Table 2, the DPPH and ABTS free radical scavenging abilities of the modified casein - polyphenol covalent complexes of Examples 1 - 4 are higher than those of Comparative Examples 1 - 4. Among them, Example 4 has the strongest antioxidant ability (DPPH scavenging rate is 84.12%, ABTS scavenging rate is 82.27%), indicating that the covalent binding of polyphenol from Allium cepa var. proliferum Regel to casein significantly improves the antioxidant performance of the complex. While Comparative Examples 1 - 4 reflect the deficiencies of traditional physical modification methods and other polyphenol reactions.

[0103] Table 2 Antioxidant ability of modified casein - polyphenol covalent complexes

[0104] Group DPPH free radical scavenging ability (%) ABTS free radical scavenging ability (%) Example 1 <![CDATA[75.80±1.53 b > <![CDATA[78.06±0.58 b > Example 2 <![CDATA[62.79±3.60 d > <![CDATA[59.47±0.17 d > Example 3 <![CDATA[70.36±2.14 c > <![CDATA[70.18±3.44 c > Example 4 <![CDATA[84.12±0.28 a > <![CDATA[82.27±1.26 a > Comparative Example 1 <![CDATA[50.18±1.87 f > <![CDATA[53.37±2.01 e > Comparative Example 2 <![CDATA[47.15±3.06 f > <![CDATA[48.66±0.70 f > Comparative Example 3 <![CDATA[56.44±0.78 e > <![CDATA[60.19±1.46 d > Comparative Example 4 <![CDATA[60.25±2.33 d > <![CDATA[69.47±2.36 c >

[0105] Note: Different letters in the same column of the table represent significant differences, P < 0.05. The same below.

[0106] Test Example 4

[0107] Take the covalent complexes prepared in Examples 1 - 4 and Comparative Examples 1 - 4 to measure their particle sizes

[0108] Dilute the freeze - dried samples of different covalent complexes to 1 mg / mL, and detect the average particle size of the samples at 25 °C on a Zetasizer Nano S90 dynamic light scattering analyzer.

[0109] Test results: As Figure 2 shown, the average particle sizes of the covalent complexes of Examples 1 - 4 are 190.14 nm, 255.00 nm, 220.19 nm, and 141.77 nm respectively; the average particle sizes of the covalent complexes of Comparative Examples 1 - 4 are 262.99 nm, 295.32 nm, 275.31 nm, and 258.67 nm respectively. This further verifies the limitations of the modification methods in Comparative Examples 1, 3, and 4.

[0110] Test Example 5

[0111] Take the covalent complexes prepared in Examples 1 - 4 and Comparative Examples 1 - 4 to measure their solubility

[0112] Weigh appropriate amounts of freeze - dried powder samples of different covalent complexes and dissolve them in phosphate buffer (pH 7.0) to prepare a protein solution with a mass concentration of 3 mg / mL. Take 2 mL of the protein solution, centrifuge at 6500×g for 10 min, successively absorb the supernatant and the corresponding stock solution, mix them with 4 mL of biuret solution, then incubate in a water bath at 30 °C for 30 min. Using 1 mL of phosphate buffer as a control, measure the absorbance of the test sample at a wavelength of 540 nm. Using bovine serum albumin as a standard curve, the solubility is expressed as the percentage of the protein mass concentration (mg / mL) in the supernatant to the original protein mass concentration (mg / mL).

[0113] Test results: As Figure 3 shown, the solubility of the modified casein - Polygonum minus polyphenol covalent complexes in Examples 1 - 4 was significantly higher than that in Comparative Examples 1 - 4. Among them, the solubility of Example 4 was the highest, indicating that its dispersibility and solubility in aqueous solution were the best, followed by Example 1, while the solubility of Comparative Examples 1 - 4 was relatively low, indicating that fermentation treatment and the combination of Polygonum minus polyphenol played an important role in improving the solubility of casein.

[0114] Test Example 6

[0115] Take the covalent complexes prepared in Examples 1 - 4 and Comparative Examples 1 - 4 and measure their emulsifying ability and emulsifying stability.

[0116] Prepare protein solutions with a volume fraction of 1% respectively. Mix the solutions with soybean oil at a water:oil ratio of 3:1. After stirring with a homogenizer at a speed of 20000 r / min for 2 min, take 20 μL samples at 0 min and 10 min respectively, and dilute them to 10 mL with a 1% sodium dodecyl sulfate dilute solution, and measure the absorbance at 500 nm. The following formulas are used to calculate the emulsifying ability (EAI) and emulsifying stability (ESI) of the emulsion.

[0117]

[0118] Test results: As can be seen from Table 3, the emulsifying ability (EAI) and emulsifying stability (ESI) of the modified casein - Polygonum minus polyphenol covalent complexes in Examples 1 - 4 were better than those in Comparative Examples 1 - 4. Among them, the emulsifying ability and emulsifying stability of Example 4 were the highest (EAI was 6.11 m 2 / g, ESI was 38.28%), indicating that the covalent binding of Polygonum minus polyphenol and casein significantly improved the emulsifying performance of the complex. While the emulsifying ability and emulsifying stability of Comparative Examples 1 and 2 were relatively low, indicating that non - fermentation modification and traditional physical modification methods had limited effects on improving the emulsifying performance of casein.

[0119] Table 3 Emulsifying ability and emulsifying stability of modified casein - Polygonum minus polyphenol covalent complexes

[0120] Group <![CDATA[EAI(m 2 / g)]]> ESI(%) Example 1 <![CDATA[6.06±0.25 a > <![CDATA[35.23±1.05 b > Example 2 <![CDATA[5.57±0.07 b > <![CDATA[30.49±1.00 c > Example 3 <![CDATA[5.89±0.11 a > <![CDATA[31.24±2.47 c > Example 4 <![CDATA[6.11±0.10 a > <![CDATA[38.28±1.23 a > Comparative Example 1 <![CDATA[5.21±0.22 c > <![CDATA[28.44±0.08 c > Comparative Example 2 <![CDATA[5.00±0.17 c > <![CDATA[25.60±0.17 d > Comparative Example 3 <![CDATA[5.20±0.03 c > <![CDATA[30.05±1.10 c > Comparative Example 4 <![CDATA[5.48±0.13 b > <![CDATA[27.56±2.23 c >

[0121] Test Example 7

[0122] Take the covalent complexes prepared in Examples 1 - 4 and Comparative Examples 1 - 4 and measure their foaming properties.

[0123] Prepare 20 mL of protein solution with a volume fraction of 1% respectively. After complete dissolution, adjust the pH of the solution to 7.0. Use a high-speed disperser to homogenize for 2 min at a rotation speed of 10,000 r / min. Transfer the sample to a graduated cylinder and record the total volume after 25 s. Record the liquid volume after standing for 30 min. Calculate the foaming property (%) and foam stability (%).

[0124] Test results: As can be seen from Table 4, the foaming property and foaming stability of the modified casein - polyphenol covalent complex of Examples 1 - 4 are higher than those of Comparative Examples 1 - 4. Among them, the foaming property and foaming stability of Examples 1 and 4 are the best, indicating that the covalent binding of polyphenol with casein significantly improves the foaming performance of the complex, while the foaming property and foaming stability of Comparative Example 2 are the lowest.

[0125] Table 4 Foaming property and foaming stability of the modified casein - polyphenol covalent complex

[0126]

[0127]

[0128] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of garlic polyphenols in casein modification.

2. A method for preparing a modified casein-polyphenol complex, characterized in that: The following steps are involved: A casein culture solution is prepared, and Lactobacillus casei is inoculated for fermentation, and the fermentation solution is sterilized after fermentation to obtain a fermentation solution; garlic polyphenol is mixed with the fermentation solution for reaction, and the reaction product is dried to obtain a modified casein-garlic polyphenol covalent complex.

3. The preparation method according to claim 2, characterized in that: According to mass volume concentration, the casein content in the casein culture solution is 5% to 12%, and the glucose content is 1% to 4%.

4. The preparation method according to claim 2, characterized in that: During the fermentation, the number of viable Lactobacillus casei was 1×10 7 ~1×10 9 cfu / mL, temperature is 30-37℃, and time is 10-20h.

5. The preparation method according to claim 2, characterized in that: The preparation method of garlic polyphenols comprises: using freeze-dried fresh garlic bulbs as raw materials, extracting with 20% to 50% ethanol as an extracting solution, and centrifuging to obtain an extracting solution; evaporating the solvent from the extracting solution to obtain a crude polyphenol extract; dissolving the crude polyphenol extract in deionized water, and purifying it through a column to obtain garlic polyphenols.

6. The preparation method according to claim 5, characterized in that: During the extraction, the solid-liquid ratio of freeze-dried fresh bulbs of small red garlic to ethanol is 1 g: 10-25 mL, the extraction is stirred for 2-5 hours, and the extraction is repeated 3-5 times.

7. The preparation method according to claim 5, characterized in that: The evaporation solvent is rotary evaporated at 50° C.; the column purification is AB-8 macroporous adsorption resin column purification.

8. The preparation method according to claim 2, characterized in that: The mass ratio of the garlic polyphenol to the fermentation liquid is 1:5-15, and the reaction is carried out in a sealed brown container at 4° C. for 8-15 hours, and the reaction pH is 8.0-9.

0.

9. The modified casein-garlic polyphenol covalent complex obtained by the preparation method according to any one of claims 2 to 8.

10. Use of the modified casein-garlic polyphenol covalent complex according to claim 9 in food processing.

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