A processing method for improving the stability of characteristic aroma of fermented milk based on heat-induced casein conformation rearrangement

Abstract

The present application relates to a kind of processing methods based on heat-induced casein conformation rearrangement to improve the characteristic aroma stability of fermented milk, comprising the following steps: S1, the fermented milk base of fermentation completion is broken and is smoothed treatment;S2, the temperature of the fermented milk base after S1 step processing is adjusted to 25-45 ℃, after adding characteristic ketone flavoring material, constant temperature incubation is carried out;S3, the system after incubation of S2 step is sterilized, after rapid cooling, cold storage is carried out.Compared with prior art, the present application discards the traditional 4 ℃ cold flavoring process, utilizes the "thermal induction conformation rearrangement" mechanism of 25-45 ℃, breaks the conventional, and significantly improves the shelf-life aroma retention rate.

Description

Technical Field

[0001] This invention belongs to the field of food technology and relates to a processing method for improving the stability of characteristic aromas in fermented milk based on heat-induced casein conformational rearrangement. Background Technology

[0002] Dimethylglyoxal (DMGE) and acetoin are the core volatile compounds that give yogurt its characteristic "buttery" and "milky" aroma. However, because DMGE and acetoin are low-boiling-point flavor compounds, they are highly volatile or degraded during fermented milk processing (especially heat treatment) and subsequent shelf-life storage, resulting in a loss of flavor and quality in the later stages of storage.

[0003] Currently, the industry commonly uses a "cold flavoring" process to reduce aroma volatilization, which involves adding flavorings and blending them at a low temperature of around 4°C after fermentation. However, at low temperatures, the core protein (casein) in the fermented milk base has a compact structure, with hydrophobic regions encapsulated within micelles. This causes flavor molecules to remain free in the aqueous phase and unable to form stable bonds with the protein, making them highly susceptible to release during storage.

[0004] Therefore, there is an urgent need to develop a processing method that can improve the stability of the characteristic aroma of fermented milk. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies, such as the easy loss of characteristic aromas of fermented milk during shelf life and the low aroma retention rate of traditional low-temperature flavoring processes, and to provide a processing method based on heat-induced casein conformational rearrangement to improve the stability of characteristic aromas of fermented milk.

[0006] The objective of this invention can be achieved through the following technical solutions: This method breaks away from the traditional technical bias of "low-temperature aroma preservation" by inducing the conformational unfolding of endogenous casein in a specific medium temperature range (25-40℃), exposing binding sites, thereby achieving a strong physical and chemical anchoring of target flavor substances.

[0007] One of the technical solutions of the present invention is to provide a processing method for improving the stability of characteristic aromas in fermented milk based on heat-induced casein conformational rearrangement, comprising the following steps: S1. Demulsify and smooth the fermented milk base material after fermentation; S2. Adjust the temperature of the fermented milk base material after step S1 to 25-45℃, add characteristic ketone flavor substances and then incubate at a constant temperature. S3. Sterilize the system after incubation in step S2, and then refrigerate it after rapid cooling.

[0008] In some specific implementations, in step S1, the pH of the fermented milk base is 4.3-4.6.

[0009] In some specific implementations, step S2 involves adjusting the temperature of the fermented milk base to 37°C.

[0010] In some specific embodiments, in step S2, the characteristic ketone flavor compound is selected from dimethylglyoxal and / or acetoin.

[0011] In some specific embodiments, in step S2, the amount of the characteristic ketone flavor substance added is such that its final concentration is 40~180 mg / L.

[0012] In some specific embodiments, the incubation process conditions for step S2 are: stirring at 100-150 rpm for 30-50 minutes.

[0013] In some specific embodiments, the incubation process conditions for step S2 are: stirring at 100-150 rpm for 40 minutes.

[0014] In some specific implementations, step S3 involves sterilization using pasteurization at a temperature of 75-85°C for 15-30 seconds.

[0015] In some specific implementations, the process conditions for rapid cooling in step S3 are: rapid cooling to 4°C at a rate greater than 5°C / min.

[0016] The second technical solution of the present invention is to provide a fermented dairy product prepared by the processing method described in one of the above technical solutions.

[0017] Compared with the prior art, the present invention has the following advantages: (1) This invention abandons the traditional 4℃ cold flavoring process and utilizes the "thermal induced conformational rearrangement" mechanism at 25-45℃ to break with convention and significantly improve the aroma retention rate during shelf life. The fermented milk treated by this invention was tested at 4℃ for 14 days, and the absolute retention concentrations of dimethylglyoxal and acetoin were increased by about 24% and 22% respectively compared with the traditional low temperature process.

[0018] (2) The number of binding sites (n value) of casein and acetoin / butanedione in the fermented milk processed by the present invention at 37℃ (310K) increased significantly from <1 at room temperature to between 1.10 and 1.21, and the binding constant (Ka) was significantly improved. This provides reliable support in terms of microscopic mechanism. The processing method of the present invention accurately utilizes this thermodynamic law and realizes the successful transformation of microscopic mechanism into macroscopic process.

[0019] (3) This invention makes full use of the endogenous casein in fermented milk as a natural “flavor microcapsule” without the need to add any artificial thickeners or encapsulation carriers, which is in line with the development trend of food clean labeling and does not increase the cost of raw materials. Attached Figure Description

[0020] Figure 1 The concentrations of dimethylglyoxal and acetoin in the samples prepared for the examples and comparative examples after storage at 4°C for 14 days are shown.

[0021] Figure 2 The images show the UV-Vis absorption spectra of casein before and after binding with dimethyl ethyl ketone (A) and acetoin (B), respectively.

[0022] Figure 3 The images show the microstructure of the casein-butanedione-acetoin composite system at different processing temperatures, as compared by scanning electron microscopy.

[0023] Figure 4 The graph shows the molecular dynamics simulation parameters of the binding system between each casein subunit and aroma molecule. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0026] Unless otherwise specified, the materials and processes used in the following embodiments or examples are conventional materials and processes employed in the art to achieve the corresponding functions.

[0027] Example 1: Take a yogurt base with a fermentation endpoint of pH 4.5. After fermentation, gently stir the yogurt base to break up the gel structure (demulsification), and then perform moderate homogenization (smoothing) to obtain a fermented milk base with a uniform texture and fine consistency. Then, control the temperature of the fermented milk base system at 25℃. Prepare 200mM acetoin stock solution and dimethyl ethyl ketone stock solution separately. Add 1mL of dimethyl ethyl ketone stock solution and 1mL of acetoin stock solution to 99mL of fermented milk to make their final concentrations 180 mg / L, respectively. Incubate at a constant temperature for 40 minutes with continuous gentle stirring (around 120 rpm). Then, perform the same pasteurization, and after pasteurization, quickly cool to 4℃ for storage.

[0028] Example 2: Yogurt base material from the same batch as in Example 1, with a fermentation endpoint of pH 4.5, was demulsified and smoothed, and the system temperature was controlled at 37°C. 200 mM acetoin and dimethyl ethyl ketone (DMQ) stock solutions were prepared separately. 1 mL of DMQ and 1 mL of acetoin stock solution were added to 99 mL of fermented milk to achieve final concentrations of 180 mg / L, respectively. The mixture was incubated at a constant temperature for 40 minutes with continuous gentle stirring (approximately 120 rpm). Subsequently, the same pasteurization process was performed, followed by rapid cooling to 4°C for storage.

[0029] Example 3: Yogurt base material from the same batch as in Example 1, with a fermentation endpoint of pH 4.5, was demulsified and smoothed, and the system temperature was controlled at 45°C. 200 mM acetoin and dimethyl ethyl ketone stock solutions were prepared separately. 1 mL of dimethyl ethyl ketone stock solution and 1 mL of acetoin stock solution were added to 99 mL of fermented milk to achieve a final concentration of 180 mg / L, respectively. The mixture was incubated at a constant temperature for 40 minutes with continuous gentle stirring (approximately 120 rpm). Subsequently, the same pasteurization process was performed, followed by rapid cooling to 4°C for storage.

[0030] Comparative Example 1: Yogurt base material from the same batch as in Example 1, with a fermentation endpoint of pH 4.5, was demulsified and smoothed, and the system temperature was controlled at 4°C. 200 mM acetoin and dimethyl ethyl ketone stock solutions were prepared separately. 1 mL of dimethyl ethyl ketone stock solution and 1 mL of acetoin stock solution were added to 99 mL of fermented milk to achieve a final concentration of 180 mg / L, respectively. The mixture was incubated at a constant temperature for 40 minutes with continuous gentle stirring (approximately 120 rpm). Subsequently, the same pasteurization process was performed, followed by rapid cooling to 4°C for storage.

[0031] After storing the samples prepared in the above examples and test examples at 4°C for 14 days, the headspace aroma concentration was determined by gas chromatography-mass spectrometry (GC-MS).

[0032] The results are as follows Figure 1 As shown, in Comparative Example 1, the concentration of dimethylglyoxal (DMG) was 63.7 mg / L and the concentration of acetoin was 24.6 mg / L; in Example 1, the concentration of DMG was 85.2 mg / L and the concentration of acetoin was 46.8 mg / L; in Example 2, the concentration of DMG was 106.3 mg / L and the concentration of acetoin was 64.4 mg / L; and in Example 3, the concentration of DMG was 87.4 mg / L and the concentration of acetoin was 52.1 mg / L.

[0033] It is evident that the heating processing technology of the present invention can significantly improve the aroma retention rate during shelf life. In particular, after constant temperature incubation at 37°C, the absolute retention concentrations of dimethylglyoxal and acetoin are increased by approximately 24% and 22% respectively compared to the traditional low temperature (4°C) process.

[0034] Since the core protein in the fermented milk of this invention is casein, casein was directly used to further explore the mechanism of action of the processing method of this invention, including the following tests: (1) Samples were prepared by mixing 0.5 mg / mL casein with dimethyl ethyl ketone and acetoin respectively (the final concentrations of aroma substances in the system were 0, 10, 20, 30, 40, and 50 mM, respectively). The mixtures were incubated at 37°C for 30 minutes to ensure sufficient interaction. The absorption peak positions and intensity changes in the range of 250-500 nm were recorded using a UV-Vis spectrophotometer. The results are as follows: Figure 2 As shown, A is the casein and dimethylglyoxal system, and B is the casein and acetoin system, where 0 mM represents the casein control alone.

[0035] The results are as follows Figure 2 As shown in Figure A, the maximum absorption wavelength of the pure casein system is 295 nm (λmax = 0.179). The maximum absorption wavelength of the complex system after adding 10 mM acetoin casein is 294 nm (λmax = 0.239). The maximum absorption wavelength of the complex system after adding 20 mM acetoin casein is 294 nm (λmax = 0.284). The maximum absorption wavelength of the complex system after adding 30 mM acetoin casein is 294 nm (λmax = 0.367). The maximum absorption wavelength of the complex system after adding 40 mM acetoin casein is 294 nm (λmax = 0.367). The maximum absorption wavelength of the complex system after adding 50 mM acetoin casein is 294 nm (λmax = 0.399).

[0036] like Figure 2 As shown in Figure B, the maximum absorption wavelength of the complex system after adding 10 mM dimethylglyoxal casein is 298 nm (λmax = 0.348), the maximum absorption wavelength of the complex system after adding 20 mM dimethylglyoxal casein is 300 nm (λmax = 0.426), the maximum absorption wavelength of the complex system after adding 30 mM dimethylglyoxal casein is 300 nm (λmax = 0.464), the maximum absorption wavelength of the complex system after adding 40 mM dimethylglyoxal casein is 302 nm (λmax = 0.565), and the maximum absorption wavelength of the complex system after adding 50 mM dimethylglyoxal casein is 302 nm (λmax = 0.614), demonstrating the spectral changes between different systems.

[0037] The results showed that, compared with the pure casein system, the addition of acetoin and dimethyl ethyl ketone caused slight changes in the maximum absorption wavelength of the composite system. Although the peak shift was small, the change was reproducible in repeated experiments. Combined with the changes in absorbance intensity, this indicates that the interaction between aroma substances and casein may affect the local structural environment of the protein.

[0038] (2) At 4℃ and 37℃ respectively, aroma compounds (butanedione and acetoin) were added to 0.5 mg / mL casein to a final concentration of 1 mM and incubated for 30 minutes to ensure sufficient interaction. Then, routine pasteurization was performed, followed by cooling to 4℃. The composite system was freeze-dried and its microstructure was observed using scanning electron microscopy. The results are as follows: Figure 3 As shown, A is scented at 37℃, making it denser; B is scented at 4℃, making it coarser.

[0039] The results showed that, compared with low-temperature fragrance addition at 4℃ ( Figure 3 Compared to B), the composite system under thermally induced conditions at 37℃ ( Figure 3 A) The surface exhibits a large number of irregular granular structures aggregated, resulting in a denser overall morphology. This directly demonstrates that moderate heating promotes the structural rearrangement and micro-region exposure of casein, thereby more firmly encapsulating aroma molecules.

[0040] (3) Based on the structure of casein α, β, and κ subunits, a semi-flexible molecular docking method was used to predict the binding sites and binding modes of each aroma molecule, calculate the binding energy, and screen the optimal conformation. Based on the docking results, the temperature was adjusted to 37℃ during the temperature control stage to conduct molecular dynamics simulations, analyze the structural stability, conformational changes, and changes in the protein flexible region of the system, in order to further verify the interaction mechanism.

[0041] The results are as follows Figure 4 The results showed that the RMSD curves of all complex systems tended to plateau after 50 ns, and the N-terminal region of β-casein exhibited higher RMSF fluctuations. This further confirms from a theoretical perspective that, at 37°C, β-casein can form a highly stable complex conformation with diacetyl and acetoin through adaptive adjustment of its highly flexible region.

[0042] (4) The concentration of casein in the test sample was 0.5 mg / mL, and the final concentrations of aroma substances were 0, 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM and 0.05 mM respectively. Phosphate buffer was used as the solvent to adjust the pH to 7.4. Three representative temperatures of 298K, 303K and 310K were selected to simulate the temperature change of food from storage to entering the human mouth. The excitation wavelength was 280nm, and the fluorescence spectrum was measured at 300-450nm. In the binding model of small molecules attached to proteins, the binding constant Ka and the binding point n can be obtained by the modified Stern-Volmer equation.

[0043] The results are shown in Table 1. From 298K to 310K (approximately 37℃), the number of binding sites (n value) of casein with acetoin / butanedione increased significantly from <1 at room temperature to between 1.10 and 1.21, and the binding constant (Ka) was significantly improved.

[0044] Table 1 The parameters are labeled as follows: K sv / 10 3 M -1 Stern-Volmer Quenching Constant; K q / 10 11 M -1 S -1 Bimolecular quenching rate constant. K a / 10 3 M -1 Association / Binding Constant; n: Binding Site Number. ΔH / (kJ·mol -1 ): Enthralpy Change; ΔS / (J·mol -1 ·K -1 Entropy change; ΔG / (kJ·mol -1 Gibbs Free Energy Change.

[0045] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A processing method for improving the stability of characteristic aromas in fermented milk based on heat-induced casein conformational rearrangement, characterized in that, Includes the following steps: S1. Demulsify and smooth the fermented milk base material after fermentation; S2. Adjust the temperature of the fermented milk base material after step S1 to 25-45℃, add characteristic ketone flavor substances and then incubate at a constant temperature. S3. Sterilize the system after incubation in step S2, and then refrigerate it after rapid cooling.

2. The processing method according to claim 1, characterized in that, In step S1, the pH of the fermented milk base is 4.3-4.

6.

3. The processing method according to claim 1, characterized in that, Step S2: Adjust the temperature of the fermented milk base to 37°C.

4. The processing method according to claim 1, characterized in that, In step S2, the characteristic ketone flavor compounds are selected from dimethylglyoxal and / or acetoin.

5. The processing method according to claim 1, characterized in that, In step S2, the amount of the characteristic ketone flavor substance added is such that its final concentration is 40~180 mg / L.

6. The processing method according to claim 1, characterized in that, In step S2, the incubation conditions are: stirring at 100-150 rpm for 30-50 minutes.

7. The processing method according to claim 6, characterized in that, In step S2, the incubation conditions are: stirring at 100-150 rpm for 40 minutes.

8. The processing method according to claim 1, characterized in that, Step S3 involves sterilization using pasteurization at a temperature of 75-85℃ for 15-30 seconds.

9. The processing method according to claim 1, characterized in that, In step S3, the rapid cooling process conditions are: rapid cooling to 4°C at a rate greater than 5°C / min.

10. A fermented dairy product, characterized in that, It is prepared by any of the processing methods described in claims 1 to 9.

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