Preparation method and application of gel protein based on composite modification of milk concentrate milk

Abstract

The invention provides a preparation method and application of gel protein based on milk concentrated milk composite modification, and belongs to the technical field of dairy product processing. According to the method, the milk concentrated milk is subjected to combined treatment through glutamine transaminase and a magnetic field; and then adding a food-grade coagulator into the treated concentrated milk, and incubating to form the gel protein. Experimental results show that the composite modification strategy significantly accelerates the gelation process, significantly improves the texture characteristics of the gel, increases the hardness by nearly 4 times, increases the chewiness by 5-7 times, and increases the water binding capacity by about 23%; and the whey separation phenomenon in 30-day refrigeration storage is obviously reduced, and the microstructure is more compact and uniform. The method and the modified product thereof are suitable for preparing a high-quality milk-based gel product, and have good industrial application value and innovation potential.

Description

Technical Field

[0001] This invention belongs to the field of dairy processing technology, specifically relating to a method for preparing gel protein based on enzyme-magnetic field treatment and its application. Background Technology

[0002] In the context of dairy product safety, efficient cold gelation modification of milk concentrate (MC) provides a feasible solution for the food industry to achieve the texture and stability of dairy products and desserts without the addition of artificial chemical components. Therefore, exploring efficient methods for modifying milk concentrate and preparing gelling proteins significantly improves its nutritional value and bioavailability, enhances its physicochemical properties such as solubility, gelling ability, water retention, and emulsifying properties, broadens the application range of gelling proteins, and provides a guarantee for the production of green and safe functional dairy-based ingredients.

[0003] Current research on the modification and preparation of gelling proteins mainly focuses on physical modifications such as acidification to prepare gelling soy protein isolate; enzymatic modifications such as glucose oxidase and polysaccharide hydrolase to prepare highly gelling egg white products and soy protein concentrate; and chemical modifications such as epigallocatechin gallate to prepare gelling ginkgo biloba protein. However, there are currently no reports on research and patents regarding the modification of milk concentrate using enzymatic cross-linking technology combined with magnetic field technology for use in food industry production as a substitute for polysaccharide additives, indicating that this field still has broad exploration space and potential innovation opportunities.

[0004] Current technologies still rely on additives to maintain the texture of dairy products, which cannot meet the needs of green and safe food production. Furthermore, there are still significant limitations in terms of gel strength, network uniformity, storage stability, and functional property improvement, making it difficult to meet the comprehensive requirements of high-quality dairy products for improved texture, taste, nutritional value, and shelf life. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method and application for the preparation of gel protein by synergistic modification of milk concentrate. This method, by rationally combining enzyme treatment and physical treatment, can significantly improve the gel performance of the prepared gel protein, forming a denser, more uniform and stable gel network structure, thereby effectively improving the texture properties, water retention and storage stability of the final product.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing gel protein based on a composite modification of milk concentrate involves sequentially treating milk concentrate with transglutaminase (MTGase) and magnetic field (MF), followed by adding a food-grade coagulant and incubation to form gel protein. This invention improves the gel properties, water retention, and storage stability of the gel protein through an MTGase-MF composite modification strategy. The milk concentrate is obtained by ultrafiltration, pasteurization, and frozen storage of low-fat raw milk.

[0008] Furthermore, the process conditions for the transglutaminase treatment are as follows: enzyme addition amount is 0.5-1 U / g, reaction temperature is 45-55℃, reaction time is 2-3 h, and then the enzyme is inactivated by heat treatment.

[0009] Furthermore, the heat treatment temperature is 75-85℃ and the time is 10-15 minutes.

[0010] Furthermore, the process conditions for the magnetic field treatment are as follows: the magnetic field strength is 0.475-0.625 T, and the concentrated milk flows through the magnetic field at a flow rate of 15-25 mL / min.

[0011] Furthermore, the concentrated milk is circulated in the magnetic field 1-2 times.

[0012] Furthermore, the amount of the food-grade coagulant added is 2.5-3.5% w / v of the milk concentrate.

[0013] Furthermore, the incubation temperature is 30-45℃, and the time is 1.5-2.5 h.

[0014] Preferably, the food-grade coagulant includes one or more of glucono-δ-lactone, calcium chloride, and calcium sulfate to achieve stability and coagulation rate control during the protein gelation process, without introducing any chemically synthesized stabilizers.

[0015] An application of the gel protein prepared by the preparation method described above is to apply the prepared gel protein to food ingredients to improve the emulsifying properties, stability, and textural characteristics of the food system, including the enhancement of emulsifying activity and stability.

[0016] Furthermore, the gelling protein is used in emulsion-based gel products.

[0017] Furthermore, the milk-based gel products include yogurt, processed cheese, dairy desserts, or other protein gel foods.

[0018] Further functional additives, including stabilizers, thickeners, or natural flavorings, may be added to the emulsion-based gel product to further enhance the product's texture, taste, and shelf life.

[0019] Microbial transglutaminase (MTGase) can catalyze the formation of ε-(γ-glutamyl)-lysine isopeptide bonds within or between milk protein molecules. These bonds act as "fixation sites" to maintain the stability of the milk protein gel network, limiting the gel network rearrangement that can cause whey separation during storage and achieving effective enzymatic modification.

[0020] In the preparation method of gel protein described in this invention, the synergistic effect of enzyme cross-linking and physical modification promotes intermolecular and intramolecular cross-linking of milk protein in concentrated milk, reduces protein particle size, improves dispersibility and orderly arrangement, and forms a denser, more uniform and stable gel network structure.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention achieves synergistic enhancement of gel protein structure properties at multiple levels through the construction of an MTGase-MF composite modification strategy. Experimental results show that the composite modification significantly accelerates the gelation process of MC, shortening the gel point time from 22 min to 10-11 min; simultaneously, it significantly improves the textural properties of the gel protein, increasing hardness by nearly 4 times, chewiness by 5-7 times, while maintaining excellent elasticity and cohesion. In terms of stability, the water-holding capacity of the composite-modified gel protein is improved by approximately 23%, and whey separation is significantly reduced during 30-day refrigerated storage, indicating superior storage stability. Microstructural analysis (CLSM, SEM, AFM) further confirms that the composite modification promotes the formation of a denser and more uniform protein network, with more uniform fat distribution and significantly enhanced gel structure integrity. In addition, the gel protein prepared by the composite modification of milk concentrate also shows simultaneous improvement in emulsifying activity and stability. In summary, the composite modification method described in this invention not only comprehensively optimizes the gelling properties and functional characteristics of MC gel protein, but also provides reliable technical support for the development of high-quality, long-shelf-life dairy products, and has significant industrial application value. Attached Figure Description

[0023] To more clearly illustrate the present invention, the accompanying drawings will be briefly described below, in which:

[0024] Figure 1These are the screening results of the process for preparing gel protein according to the present invention; wherein, (a) and (b) are the effects of enzyme concentration and reaction time on the viscosity and elastic modulus G' of gel protein, respectively; (c) and (d) are the effects of HPH pressure and number of cycles on the viscosity and elastic modulus G' of gel protein, respectively; (e) and (f) are the effects of ultrasonic power and duration on the viscosity and elastic modulus G' of gel protein, respectively; (g), (h), and (i) are the effects of magnetic field cycle number, flow rate, and magnetic field strength on the viscosity and elastic modulus G' of gel protein, respectively; different letters AE and ad indicate that the results have significant differences, p<0.05;

[0025] Figure 2 The effects of different modification methods of this invention on the structure and properties of gel proteins are shown in Figures (a) and (b), which show the changes in particle size and Zeta potential of gel proteins prepared by different modification methods, respectively; Figures (c) and (d) show the circular dichroism spectroscopy and secondary structure composition of gel proteins prepared by different modification methods, respectively; Figure (e) shows the fluorescence spectra of gel proteins prepared by different modification methods; and (f) shows the emulsifying properties and emulsion stability of gel proteins prepared by different modification methods, with different letters (AD, ac) indicating significant differences, p<0.05.

[0026] Figure 3 Figures (a) and (b) show the gel states of the gel proteins prepared by different modification methods of the present invention after 30 minutes and after 30 days of refrigeration, respectively.

[0027] Figure 4 The effects of different modification methods on the texture of gel proteins prepared according to the present invention are shown in Figure (a), which reflects hardness and elasticity; and Figure (b), which reflects cohesiveness and chewiness.

[0028] Figure 5 These are CLSM images of gel proteins prepared by different modification methods of this invention, with green representing proteins and red representing fats;

[0029] Figure 6 These are SEM images of gel proteins prepared by different modification methods of this invention. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of the invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] The following is the formula information for milk concentrate (MC):

[0032] MC (protein 9.21%, fat 3.59%, lactose 5.15%, total solids 19.64%) was provided by New Hope Group. It is concentrated milk obtained by ultrafiltration, pasteurization, and freezing storage at -20°C of low-fat raw milk (fat 1.5%) using membrane equipment for later use. Specific implementation method one:

[0034] Process screening of gel proteins prepared by MC-based composite modification - Single-factor screening of enzyme concentration: MTGase was added to MC at concentrations of 0.5, 0.75, 1, 1.5, and 2 U / g protein, and the mixture was treated at 45°C for 2 h. Single-factor screening of enzyme reaction time: MTGase was added at a ratio of 0.75 U / g protein, and the mixture was treated at 45°C for 1.5, 2, 2.5, 3, and 4 h, followed by enzyme inactivation by heat treatment (85°C for 10 min).

[0035] Pressure single-factor screening: MC was treated with high-pressure homogenization (HPH) at pressures of 0, 5, 10, 15, 20, and 25 MPa using a high-pressure homogenizer, with 5 cycles per cycle. Cycle number single-factor screening: MC was treated at 10 MPa for 1, 2, 3, 4, 5, 6, and 10 cycles.

[0036] Single-factor screening of ultrasound intensity: MCs were treated with ultrasound at 200, 250, 300, 350, 400, and 500 W for 30 min. Single-factor screening of ultrasound duration: MCs were treated with ultrasound at 250 W for 0, 5, 15, 30, 60, and 120 min.

[0037] Single-factor screening of magnetic field treatment cycle number: MC was circulated 0, 1, 2, 3, 4, 5, and 10 times under an electromagnetic field of 0.7 T (magnetic induction thermal reactor, MIH-10, INDUC Scientific Co., Ltd.). Single-factor screening of flow rate: MC was circulated twice under an electromagnetic field of 0.7 T at flow rates of 15, 20, 25, 30, and 35 mL / min. Single-factor screening of magnetic field strength: Modified MC was obtained by treating MC with electromagnetic fields of 0, 0.25, 0.4, 0.475, 0.55, 0.625, and 0.7 T for 2 cycles.

[0038] MC modified by different methods was mixed with 3% (w / v) gluconate-δ-lactone (GDL), gently stirred for 1 min, and then incubated in a water bath at 30°C for 2 h to prepare MC gel protein. The viscosity (Pa·s) and elastic modulus (G') of the MC gel protein were measured using a rheometer, with each group performed in triplicate. Results are shown below. Figure 1As shown, based on the comprehensive analysis of viscosity and elastic modulus data of MC gel protein, the gel protein prepared by enzyme-magnetic field modified MC has better gel performance. Further experiments used the gel protein prepared by enzyme-high pressure modified MC as a control for comparison.

[0039] Specific Implementation Method Two: Determination of Particle Size and Zeta Potential. Gel proteins prepared from MC modified by different methods were diluted to a protein concentration of 0.3 mg / mL using phosphate buffer (10 mM, pH 7.0). The particle size and zeta potential (Z, mV) of the gel proteins were measured using a Zetasizer Nano ZS90 (Malvern Instruments Co., Ltd., Worcestershire, UK) at 25°C, with measurements repeated three times. Results are as follows... Figure 2 As shown in (a) and (b) in the figure.

[0040] Specific Implementation Method 3: Determination of Circular Dichroism (CD). Gel proteins prepared from MC modified by different methods were diluted to 1 mg / mL with phosphate buffer (10 mmol / L, pH 7.0). The CD spectra in the 190-250 nm range were recorded using a circular dichroism spectrometer with a path length of 1 cm. Each sample was scanned three times, and the average value was taken. The results are as follows: Figure 2 As shown in (c) and (d) in the figure.

[0041] Specific Implementation Method 4: Measurement of Fluorescence Spectroscopy. Gel proteins prepared from MC modified by different methods were diluted to 1 mg / mL with phosphate buffer (10 mmol / L, pH 7.0). The intrinsic fluorescence intensity was measured using a fluorescence spectrometer with an excitation wavelength of 280 nm and an emission wavelength range of 300-400 nm. Each sample was analyzed at least three times, and the results are as follows: Figure 2 As shown in (e) in the diagram.

[0042] Specific Implementation Method 5: Determination of Emulsifying Activity and Stability. 40 mL of gel protein prepared from MC modified by different methods and 10 mL of corn germ oil were placed in 100 mL beakers and emulsified at 2000 rpm for 2 min using an IKA T25 high-speed homogenizer. Immediately, 10 μL of the resulting emulsion was taken and diluted in 5 mL of 0.1 mol / L phosphate buffer (containing 0.1% SDS by weight), and the absorbance was measured at 500 nm using a UV-2300 spectrophotometer. Turbidity (T) was calculated as T = 2.303 A / l, where A is the absorbance at 500 nm and l is the path length of the reaction vessel (1 cm). Emulsifying activity (EAI) was calculated according to equation (1):

[0043] Where T represents the turbidity value, D is the dilution factor, Φ is the mass fraction of corn germ oil in the emulsion, and C is the protein concentration before emulsification (g / mL). The unit of EAI is m. 2 g -1 The emulsion solution was allowed to stand at 4°C for 20 min, and then the absorbance was measured again. The turbidity value was calculated using the method described above. The emulsion stability (ESI) was then calculated according to equation (2):

[0044] Where T is the turbidity value at T=0, ΔT is the change in turbidity over time, and the unit of ESI is min. Each sample should be analyzed at least three times, and the results are as follows: Figure 2 As shown in (f) in the figure.

[0045] Specific Implementation Method Six: Gel Properties Analysis of Gel Proteins. Modified MC prepared by different modification methods was mixed with 3% (w / v) gluconate-δ-lactone GDL, gently stirred for 1 min, and then incubated in a 30°C water bath for 2 h to form a gel. Gel stability was assessed by examining whey separation after storage at 4°C for 30 days. The results are as follows: Figure 3 As shown in the figure; texture analysis was performed using a texture analyzer (TA-XT plus, Stable Micro System, Godalming, UK) to measure hardness, elasticity, chewiness, and cohesion, and the results are as follows. Figure 4 As shown in (a) and (b) in the figure.

[0046] Specific Implementation Method Seven: Protein-Lipid Distribution Analysis of Gel Proteins. Gel proteins prepared using different modification methods were placed on glass slides. Proteins were stained with Fast Green, and lipids with Nile Red, then covered with coverslips. CLSM images were obtained using a Leica TCS SP8 (Leica Microsystems Inc., Heidelberg, Germany) 40x magnifying glass for ordered scanning. The excitation lines for proteins and lipids were 633 nm and 488 nm, respectively. The results are as follows: Figure 5 As shown in the image.

[0047] Specific Implementation Method 8: Microstructure Analysis of Gel Proteins. Gel proteins prepared by different modification methods were fixed in 2.5% glutaraldehyde for 1.5 h, washed three times with 0.1 M phosphate buffer solution for 10 min each time, and then dehydrated in a series of ethanol concentrations (50, 70, 90, and 100%), displaced in a 1:1 mixture of ethanol and tert-butanol for 15 min, and then displaced twice with pure tert-butanol. After freezing at -20℃ for 30 min, the samples were transferred to an ES-2030 (HITACHI) freeze dryer for drying. After drying, a 100-150 Å metal film was coated onto the samples using a sputtering coating machine. Subsequently, the cross-sectional microstructure of the samples was observed using a scanning electron microscope, with at least three images obtained for each sample. The results are as follows: Figure 6 As shown in the image.

[0048] One-way ANOVA was performed using SPSS 25.0. All data are expressed as mean ± standard deviation (SD). Significance of differences between results was determined using Duncan's multiple range test. A difference was considered statistically significant when p < 0.05.

[0049] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing gel protein based on milk concentrate composite modification, characterized in that, The steps are as follows: The milk concentrate is treated with transglutaminase and magnetic field successively, and then food-grade coagulant is added to the treated milk concentrate and incubated to form gel protein.

2. The preparation method according to claim 1, characterized in that: The process conditions for the transglutaminase treatment are as follows: enzyme addition amount is 0.5-1 U / g, reaction temperature is 45-55℃, reaction time is 2-3 h, and then the enzyme is inactivated by heat treatment.

3. The preparation method according to claim 2, characterized in that: The heat treatment is performed at a temperature of 75-85℃ for 10-15 minutes.

4. The preparation method according to claim 1, characterized in that: The process conditions for the magnetic field treatment are as follows: the magnetic field strength is 0.475-0.625 T, and the concentrated milk flows through the magnetic field at a flow rate of 15-25 mL / min.

5. The preparation method according to claim 4, characterized in that: The concentrated milk is circulated in a magnetic field 1-2 times.

6. The preparation method according to claim 1, characterized in that: The amount of the food-grade coagulant added is 2.5-3.5% w / v of the milk concentrate.

7. The preparation method according to claim 1, characterized in that: The incubation temperature is 30-45℃, and the time is 1.5-2.5 h.

8. The preparation method according to claim 1, characterized in that: The food-grade coagulant includes one or more of glucono-δ-lactone, calcium chloride, and calcium sulfate.

9. The application of a gel protein prepared by the preparation method according to any one of claims 1-8, characterized in that: Gel proteins can be used in food ingredients or emulsion-based gel products.

10. The application according to claim 9, characterized in that: The milk-based gel products include yogurt, processed cheese, or dairy desserts.

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