A beta lactoglobulin powder with low allergenic properties and a method for its preparation
By using pH adjustment and covalent modification of oligosaccharides, the problem of balancing the allergenicity and functional properties of β-lactoglobulin in existing technologies has been solved. This method produces β-lactoglobulin powder with low allergenicity, reduces IgE binding capacity, and improves solubility and foaming properties, making it suitable for industrial applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG GONGSHANG UNIVERSITY
- Filing Date
- 2026-03-12
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies cannot improve the functional properties of β-lactoglobulin while reducing its allergenicity, especially its IgE binding capacity.
A method combining pH adjustment (acidity) and covalent modification with oligosaccharides was employed. The pH of the β-lactoglobulin solution was adjusted to acidity, followed by conformational change and then adjustment to neutrality. The solution was then mixed with oligosaccharides, freeze-dried, and subjected to Maillard reaction under mild humid heat conditions. Finally, the mixture was dialyzed and freeze-dried to prepare β-lactoglobulin powder with low allergenicity.
It significantly reduces the allergenicity of β-lactoglobulin while improving its solubility and foaming properties. It is easy to operate, safe, and leaves no residue, making it suitable for industrial production.
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Figure CN122139845A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of food technology, specifically relating to a β-lactoglobulin powder with low allergenicity and its preparation method. Background Technology
[0002] Milk is a nutrient-dense food, rich in protein, lipids, carbohydrates, and various beneficial bacteria. However, milk protein is also a significant food allergen, especially in infants and young children, where it easily triggers immune responses, with an allergy incidence of approximately 2% to 3% in the first year of life. Among the many milk proteins, β-lactoglobulin is the main sensitizing component, accounting for about 10% of the total protein content in milk. β-lactoglobulin belongs to the lipoprotein family, existing as a dimer under neutral conditions and transforming into a monomer under acidic conditions. Its unique structural characteristics endow it with a high-affinity IgE binding epitope, thus exhibiting strong sensitization. Although strict avoidance is currently the main management method for milk allergy, it is often difficult to fully implement in real life and food consumption. Therefore, reducing the sensitization of β-lactoglobulin and expanding the safe application of milk and its products is of significant practical importance.
[0003] Currently, methods for preparing low-allergenic β-lactoglobulin-related products include physical modification methods (such as heat treatment and ultra-high pressure treatment), chemical modification methods (such as glycosylation and acetylation), enzymatic modification methods (such as protease hydrolysis), and combined modification methods (such as ultrasonication + enzymatic hydrolysis). While these methods each have their advantages, they share a common problem: none of these preparation methods can reduce the allergenicity of β-lactoglobulin while improving its functional properties. Summary of the Invention
[0004] To address the problem that existing methods for preparing low-allergenic β-lactoglobulin-related products cannot simultaneously improve the functional properties of β-lactoglobulin while reducing its allergenicity, this invention provides a low-allergenic β-lactoglobulin powder and its preparation method. This preparation method can reduce the allergenicity of β-lactoglobulin while improving its functional properties such as solubility and foaming properties. To achieve the above objectives, this invention adopts the following technical solution.
[0005] The first objective of this invention is to provide a method for preparing β-lactoglobulin powder with low allergenic properties, comprising the following steps: The pH of the β-lactoglobulin solution was adjusted to acidic, stirred until homogeneous, and allowed to undergo conformational change or denaturation at room temperature. The solution was then adjusted to neutral to obtain a pH-changed (acidic) β-lactoglobulin solution.
[0006] Add oligosaccharide solution to the pH-adjusted β-lactoglobulin solution and stir until homogeneous to obtain a β-lactoglobulin-oligosaccharide mixed solution.
[0007] The β-lactoglobulin-oligosaccharide mixed solution was freeze-dried to obtain a mixed powder.
[0008] The mixed powder was subjected to a Maillard reaction at a temperature of 50°C to 60°C and a relative humidity of 70% to 80%. The resulting mixed powder was dissolved, dialyzed, and freeze-dried to obtain the hypoallergenic β-lactoglobulin powder. The hypoallergenic β-lactoglobulin powder is abbreviated as hypoallergenic β-lactoglobulin powder.
[0009] This invention employs a pH-change (acidic) combined with glycosylation to covalently modify β-lactoglobulin, preparing β-lactoglobulin powder with low allergenicity, providing an important theoretical basis for the development of novel high-functionality, low-allergenic protein foods. The preparation method provided by this invention first adjusts the pH of a β-lactoglobulin solution to acidic, stirs it evenly, and allows it to undergo a conformational change or denaturation reaction at room temperature. Then, it is adjusted to neutral to obtain a pH-change (acidic) β-lactoglobulin solution. Next, an oligosaccharide solution is added to the pH-change β-lactoglobulin solution and stirred evenly to obtain a β-lactoglobulin-oligosaccharide mixed solution. The β-lactoglobulin-oligosaccharide mixed solution is then stored at low temperature and freeze-dried to obtain a mixed powder. The mixed powder is then subjected to a Maillard reaction at 50°C–60°C and 70%–80% relative humidity. Finally, the mixed powder after the Maillard reaction is dissolved, dialyzed, and freeze-dried to obtain β-lactoglobulin powder with low allergenicity. This invention solves the problem of balancing functional properties and allergenicity in existing technologies by using a synergistic mechanism of pH-change pretreatment and mild solid-state glycosylation reaction. First, the β-lactoglobulin solution is switched between acidic and neutral environments to induce moderate conformational unfolding of the protein, exposing internal glycosylation sites and antigenic epitopes, laying the foundation for subsequent reactions. Then, it is mixed with an oligosaccharide solution, freeze-dried to prepare a mixed powder, and subjected to a solid-state Maillard reaction under mild conditions of 50°C–60°C and 70%–80% relative humidity. These conditions allow for precise control of the degree of glycosylation, avoiding excessive protein denaturation and aggregation caused by prolonged high temperatures. This allows the oligosaccharide chains to selectively modify antigenic epitopes through covalent bonds, generating a steric hindrance effect that reduces IgE binding capacity. Simultaneously, the introduced glycans significantly improve functional properties such as solubility, thermal stability, and emulsifying properties. This "structure regulation first, then site-specific modification" strategy achieves simultaneous improvement in functional properties while reducing allergenicity, overcoming the shortcomings of traditional processes where harsh conditions make it difficult to balance these two aspects.
[0010] Preferably, the acidity refers to a pH of 1 to 3.
[0011] Preferably, the pH of the β-lactoglobulin solution is adjusted to acidity by adjusting the solution system with 1 mol / L HCl.
[0012] Preferably, the condition for adjusting to neutral is: adjusting the pH of the solution system to 7.0 with 1 mol / L NaOH.
[0013] Preferably, the β-lactoglobulin solution is obtained by dissolving β-lactoglobulin in phosphate buffer.
[0014] Preferably, the pH of the phosphate buffer solution is 7.2 to 7.6, and the concentration is 0.01 mol / L to 0.02 mol / L.
[0015] Preferably, the mass ratio of the pH-changing β-lactoglobulin to oligosaccharides is 1:1 to 3.
[0016] Preferably, the concentration of the xylooligosaccharide solution is 100 mg / mL and the volume is 1 mL.
[0017] Preferably, the concentration of the β-lactoglobulin solution is 10 mg / mL and the volume is 10 mL.
[0018] Preferably, the oligosaccharide comprises xylooligosaccharides with a molecular weight of 300kDa to 1050kDa.
[0019] Preferably, the time for the conformational change or denaturation reaction to occur at room temperature is 0.5h to 1.5h.
[0020] Preferably, the β-lactoglobulin-oligosaccharide mixed solution is frozen in a 60 mm culture dish.
[0021] Preferably, the mixed powder after the reaction is dissolved using deionized water.
[0022] Preferably, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 4kDa to 10kDa at room temperature for 20 to 28 hours.
[0023] Preferably, the freeze-drying temperature is -75℃ to -85℃, and the time is 42h to 54h.
[0024] A second objective of this invention is to provide a β-lactoglobulin powder with low allergenicity prepared by the aforementioned preparation method.
[0025] Preferably, the sensitization of the β-lactoglobulin powder is reduced by more than 50% compared to natural β-lactoglobulin, while maintaining its solubility.
[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a method for preparing β-lactoglobulin powder with low allergenicity. The preparation method includes pH adjustment (acidic), oligosaccharide complexation, freeze-drying pretreatment, mild humid heat reaction, and purification and drying steps: First, the β-lactoglobulin solution is adjusted to acidic conditions at room temperature and then returned to neutral. It is then complexed with oligosaccharides and freeze-dried to obtain a mixed powder. After Maillard reaction at 50°C~60°C and 70%~80% relative humidity, the β-lactoglobulin powder with low allergenicity is obtained through dissolution, dialysis, and freeze-drying. pH adjustment (acidic) can reconstruct the protein conformation, fully exposing the sensitizing antigenic epitopes and glycosylation sites; oligosaccharides stably bind to the antigenic epitopes under mild humid heat conditions, completely blocking antibody recognition sites; the entire process involves no chemical reagents and mild reaction conditions, avoiding residual risks while preserving the original solubility and foaming properties of the protein; the process steps are clear, parameters are easily controlled, and no complex equipment is required. This method has the advantages of thorough low allergenicity, complete product function, safety and no residue, and strong industrial feasibility. It effectively solves the problem that existing methods for preparing low-allergenic β-lactoglobulin-related products cannot reduce their allergenicity while improving the functional properties of β-lactoglobulin.
[0027] Specifically, the preparation method provided by this invention first adjusts the pH of the β-lactoglobulin solution to acidic, stirs it evenly, and allows it to undergo a conformational change or denaturation reaction at room temperature. Then, it is adjusted to neutral to obtain a pH-changed (acidic) β-lactoglobulin solution. Next, an oligosaccharide solution is added to the pH-changed β-lactoglobulin solution and stirred evenly to obtain a β-lactoglobulin-oligosaccharide mixed solution. The β-lactoglobulin-oligosaccharide mixed solution is then stored at low temperature and freeze-dried to obtain a mixed powder. The mixed powder is then subjected to a Maillard reaction at 50°C–60°C and 70%–80% relative humidity. Finally, the mixed powder after the Maillard reaction is dissolved, dialyzed, and freeze-dried to obtain a β-lactoglobulin powder with low allergenic properties. This invention solves the problem of balancing functional properties and allergenicity in existing technologies by using a synergistic mechanism of pH-change pretreatment and mild solid-state glycosylation reaction. First, the β-lactoglobulin solution is switched between acidic and neutral environments to induce moderate conformational unfolding of the protein, exposing internal glycosylation sites and antigenic epitopes, laying the foundation for subsequent reactions. Then, it is mixed with an oligosaccharide solution, freeze-dried to prepare a mixed powder, and subjected to a solid-state Maillard reaction under mild conditions of 50°C–60°C and 70%–80% relative humidity. These conditions allow for precise control of the degree of glycosylation, avoiding excessive protein denaturation and aggregation caused by prolonged high temperatures. This allows the oligosaccharide chains to selectively modify antigenic epitopes through covalent bonds, generating a steric hindrance effect that reduces IgE binding capacity. Simultaneously, the introduced glycans significantly improve functional properties such as solubility, thermal stability, and emulsifying properties. This "structure regulation first, then site-specific modification" strategy achieves simultaneous improvement in functional properties while reducing allergenicity, overcoming the shortcomings of traditional processes where harsh conditions make it difficult to balance these two aspects.
[0028] 2. The preparation method provided by this invention can effectively reduce the allergenicity of β-lactoglobulin while improving its functional properties. Specifically, it can significantly reduce the IgE binding capacity of β-lactoglobulin and enhance its solubility and foaming properties. Furthermore, this preparation method is simple to operate, has a rapid onset of action, and provides significant improvement. Xylooligosaccharides are highly safe and widely available. This work provides a promising strategy for developing dairy products with low allergenicity and high functionality. Attached Figure Description
[0029] Figure 1 The graph shows the results of measuring the binding capacity of β-lactoglobulin (powder) with IgE in the serum of patients with milk allergy in Examples 1-3 and Comparative Example 1 of this invention.
[0030] Figure 2 The results are the determination results of the solubility of β-lactoglobulin (powder) in Examples 1-3 and Comparative Example 1 of this invention.
[0031] Figure 3The graph shows the results of the foaming properties determination of β-lactoglobulin (powder) in Examples 1-3 and Comparative Example 1 of this invention. Detailed Implementation
[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.
[0033] The test materials used in this invention are all common commercial products and can be purchased on the market.
[0034] The room temperature in this invention is 25°C.
[0035] According to embodiments of the present invention, a method for preparing β-lactoglobulin powder with low allergenicity is provided, comprising the following steps (preferred conditions): (1) Dissolve β-lactoglobulin in phosphate buffer (pH 7.4, 0.01 mol / L) to obtain a β-lactoglobulin solution; adjust the pH of the β-lactoglobulin solution to acidic, stir evenly and react at room temperature for 1 h, then adjust to neutral to obtain a pH-changed (acidic) β-lactoglobulin solution. According to a specific embodiment of the present invention, the specific operation is as follows:
[0036] β-lactoglobulin was prepared into a homogeneous protein solution with a concentration of 10 mg / mL using phosphate buffer (pH 7.4, 0.01 mol / L). The pH of the β-lactoglobulin solution was then adjusted to 2.0 using 1 mol / L HCl, and the reaction was carried out in the air for 1 hour. Subsequently, the pH of the solution was adjusted to 7.0 using 1 mol / L NaOH. This yielded the pH-adjusted (acidic) β-lactoglobulin solution.
[0037] (2) Add oligosaccharide solution to the pH-adjusted (acidic) β-lactoglobulin solution, stir until homogeneous, and obtain a β-lactoglobulin-oligosaccharide mixed solution. According to a specific embodiment of the present invention, the specific operation is as follows:
[0038] Add 1 mL of xylooligosaccharide solution with a concentration of 100 mg / mL to the pH-adjusted (acidic) β-lactoglobulin solution, stir well to obtain a β-lactoglobulin-oligosaccharide mixed solution.
[0039] The method for preparing the xylooligosaccharide solution is as follows: dissolve 500 mg of xylooligosaccharide in 5 mL of phosphate buffer (pH 7.4, 0.01 mol / L).
[0040] (3) The obtained β-lactoglobulin-oligosaccharide mixed solution is cooled at -20°C and freeze-dried to obtain a mixed powder. According to a specific embodiment of the present invention, the specific operation is as follows:
[0041] The obtained β-lactoglobulin-oligosaccharide mixed solution was placed in a 60 mm petri dish and cooled at -20 °C. After freeze-drying, a mixed powder was obtained.
[0042] The freeze-drying process was carried out at a temperature of -80℃ for 48 hours.
[0043] (4) The mixed powder is reacted at a temperature of 55°C ± 5°C and a relative humidity of 75% ± 5% for 1 hour. After dissolving in deionized water, dialyzing, and freeze-drying, low-allergenic β-lactoglobulin powder is obtained. According to a specific embodiment of the present invention, the specific operation is as follows:
[0044] The mixed powder was subjected to a Maillard reaction at 55°C and 74.41% relative humidity for 1 hour. After the reaction, β-lactoglobulin was dissolved in deionized water. Unreacted xylooligosaccharides were then removed using a dialysis bag with a molecular weight cutoff of 7 kDa, and the retentate was collected. Finally, the collected retentate was freeze-dried to obtain β-lactoglobulin powder with low allergenic properties. This low-allergenic β-lactoglobulin powder is simply referred to as low-allergenic β-lactoglobulin powder.
[0045] The dialysis conditions are as follows: the dialysis temperature is room temperature, the dialysis time is 24 hours, and the dialysis solution is changed every 2 hours.
[0046] In summary, the present invention, through the above-described preparation method, utilizes the alteration effect of pH change pretreatment (acidity) on the structure of β-lactoglobulin to expose more glycosylation reaction regions and increase the degree of glycosylation. This, combined with the synergistic effect of xylooligosaccharide glycosylation modification on the masking of sensitizing linear epitopes and the disruption of conformational epitopes, significantly reduces the sensitization of β-lactoglobulin and improves its functional properties such as solubility and foaming properties.
[0047] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.
[0048] Example 1 A method for preparing a β-lactoglobulin powder with low allergenicity includes the following steps: (1) Prepare a homogeneous protein solution of 10 mg / mL with 10 mL of phosphate buffer (pH 7.4, 0.01 mol / L), i.e., β-lactoglobulin solution. Adjust the pH of the β-lactoglobulin solution to 2.0 with 1 mol / L HCl and expose the reaction to air for 1 h. Then adjust the pH of the solution system to 7.0 with 1 mol / L NaOH. The resulting pH-adjusted (acidic) β-lactoglobulin solution is obtained.
[0049] (2) The pH-changed (acidic) β-lactoglobulin solution prepared in step (1) was subjected to a dialysis bag with a molecular weight cutoff of 7 kDa to remove excess salt, and the retentate in the dialysis bag was collected. Finally, the collected retentate was freeze-dried to obtain pH-changed β-lactoglobulin powder.
[0050] The dialysis conditions were as follows: the dialysis temperature was room temperature, the dialysis time was 24 hours, and the dialysis fluid was changed every 2 hours.
[0051] The freeze-drying temperature was -80℃ and the time was 48 hours.
[0052] Example 2 A method for preparing a β-lactoglobulin powder with low allergenicity includes the following steps: (1) Prepare 10 mL of homogeneous protein solution with a concentration of 10 mg / mL using phosphate buffer (pH 7.4, 0.01 mol / L), which is called β-lactoglobulin solution.
[0053] (2) Add 1 mL of xylooligosaccharide solution with a concentration of 100 mg / mL to the β-lactoglobulin solution prepared in step (1), stir evenly, and obtain a β-lactoglobulin-xylooligosaccharide mixed solution.
[0054] The method for preparing the xylooligosaccharide solution is as follows: dissolve 500 mg of xylooligosaccharide in 5 mL of phosphate buffer (pH 7.4, 0.01 mol / L).
[0055] Xylooligosaccharides were purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a molecular weight of 300 kDa to 1050 kDa.
[0056] (3) The β-lactoglobulin-xylooligosaccharide mixed solution prepared in step (2) was placed in a 60 mm petri dish and cooled at -20 °C. After freeze-drying, the mixed powder was obtained.
[0057] The freeze-drying conditions were -80℃ for 48 hours.
[0058] (4) The mixed powder prepared in step (3) was placed at a temperature of 55°C and a relative humidity of 74.41% for 1 hour. After the reaction was completed, β-lactoglobulin was dissolved in deionized water. Unreacted xylooligosaccharides were then removed using a dialysis bag with a molecular weight cutoff of 7 kDa, and the retentate in the dialysis bag was collected. Finally, the collected retentate was freeze-dried to obtain hypoallergenic β-lactoglobulin powder.
[0059] The dialysis conditions were as follows: the dialysis temperature was room temperature, the dialysis time was 24 hours, and the dialysis fluid was changed every 2 hours.
[0060] Example 3 A method for preparing a β-lactoglobulin powder with low allergenicity includes the following steps: (1) Prepare a homogeneous protein solution of 10 mg / mL with 10 mL of phosphate buffer (pH 7.4, 0.01 mol / L), i.e., β-lactoglobulin solution. Adjust the pH of the β-lactoglobulin solution to 2.0 with 1 mol / L HCl, and expose the reaction to air for 1 h. Then adjust the pH of the solution system to 7.0 with 1 mol / L NaOH. The resulting pH-adjusted (acidic) β-lactoglobulin solution is obtained.
[0061] (2) Add 1 mL of xylooligosaccharide solution with a concentration of 100 mg / mL to the pH-changed (acidic) β-lactoglobulin solution prepared in step (1), stir evenly, and obtain a β-lactoglobulin-xylooligosaccharide mixed solution.
[0062] The method for preparing the xylooligosaccharide solution is as follows: dissolve 500 mg of xylooligosaccharide in 5 mL of phosphate buffer (pH 7.4, 0.01 mol / L).
[0063] Xylooligosaccharides were purchased from Shanghai Yuanye Biotechnology Co., Ltd., with molecular weights ranging from 300 kDa to 1050 kDa.
[0064] (3) The β-lactoglobulin-xylooligosaccharide mixed solution prepared in step (2) was placed in a 60 mm petri dish and cooled at -20 °C. After freeze-drying, the mixed powder was obtained.
[0065] The freeze-drying process was carried out at a temperature of -80℃ for 48 hours.
[0066] (4) The mixed powder prepared in step (3) was placed at a temperature of 55°C and a relative humidity of 74.41% for 1 hour. After the reaction was completed, β-lactoglobulin was dissolved in deionized water. Unreacted xylooligosaccharides were then removed using a dialysis bag with a molecular weight cutoff of 7 kDa, and the retentate in the dialysis bag was collected. Finally, the collected retentate was freeze-dried to obtain β-lactoglobulin powder.
[0067] The dialysis conditions were as follows: the dialysis temperature was room temperature, the dialysis time was 24 hours, and the dialysis fluid was changed every 2 hours.
[0068] Comparative Example 1 A method for preparing β-lactoglobulin powder includes the following steps: (1) Prepare 10 mL of homogeneous protein solution with a concentration of 10 mg / mL using phosphate buffer (pH 7.4, 0.01 mol / L), which is called β-lactoglobulin solution.
[0069] (2) The β-lactoglobulin solution prepared in step (1) was dialyzed using a dialysis bag with a molecular weight cutoff of 7 kDa, and the retention solution in the dialysis bag was collected. Finally, the collected retention solution was freeze-dried to obtain β-lactoglobulin powder.
[0070] The dialysis conditions were as follows: the dialysis temperature was room temperature, the dialysis time was 24 hours, and the dialysis fluid was changed every 2 hours.
[0071] The freeze-drying temperature was -80℃ and the time was 48 hours.
[0072] Test case The protein samples (β-lactoglobulin powder) prepared in Examples 1-3 and Comparative Example 1 were used as test subjects to test the efficacy of β-lactoglobulin.
[0073] 1. IgE binding capacity test—Indirect enzyme-linked immunosorbent assay (ELISA) Protein samples (β-lactoglobulin powder) prepared in Examples 1-3 and Comparative Example 1 at a concentration of 50 μg / mL were added to a high-affinity 96-well ELISA plate, 0.1 mL per well. After overnight incubation at 4°C, the plate was washed 5 times with washing buffer (250 μL / well each time), incubated at room temperature for 5 min, and then blotted dry. 200 μL of blocking buffer containing 5% (w / v) bovine serum albumin was added to each well, and the plate was incubated at 37°C for 2 h, followed by 5 washes. Then, 100 μL of a 1:50 diluted serum from milk-allergic patients was added to each well, and the plate was incubated at 37°C for 1 h, followed by 5 washes. Next, 200 μL of a 1:5000 diluted biotin-labeled goat anti-human IgE antibody was added to each well, and the plate was incubated at 37°C for 2 h, followed by 5 washes. 100 μL of TMB chromogenic solution was added to each well, and the plate was incubated at 37°C in the dark for 30 min. Finally, 50 μL of 2 mol / L TMB chromogenic buffer was added to each well. Using H2SO4 as a stop solution, the absorbance (OD) of each well was measured at 450 nm using an ELISA reader. 450nm The results are expressed as a percentage of IgE binding capacity relative to the untreated control example 1.
[0074] Overnight stays refer to stays of 12 hours or more.
[0075] Test results as follows Figure 1 As shown. By Figure 1 It can be seen that the IgE binding capacity of the β-lactoglobulin powder prepared in Examples 1-3 is significantly lower than that of Comparative Example 1 (untreated). P <0.05). Among them, Example 3 (pH change (acidity)-glycosylation co-modification) showed the lowest IgE binding capacity, which decreased by 24.81%~40.02% compared with Comparative Example 1.
[0076] In summary, Example 3 showed the best desensitization effect, proving that the combined pH change (acidity) and glycosylation modification treatment of the present invention can effectively reduce the binding ability of β-lactoglobulin to specific IgE, and the effect is better than using pH change (acidity) or glycosylation modification alone.
[0077] 2. Solubility test 20 mg of the protein samples (β-lactoglobulin powder) prepared in Examples 1-3 and Comparative Example 1 were dissolved in 10 mL of deionized water and stirred at room temperature for 2 h to ensure complete protein hydration. The solution was then centrifuged at 6000 × g for 15 min at 25°C. The protein content in the supernatant was determined using a BCA protein quantification kit (Nanjing Jiancheng Biotechnology Institute, China). Solubility is expressed as the percentage of soluble protein concentration in the supernatant relative to the total protein concentration.
[0078] Test results as follows Figure 2 As shown. By Figure 2 It can be seen that the solubility of the β-lactoglobulin powder prepared in Examples 1 and 3 is higher than that of Comparative Example 1 (untreated). P <0.05), the solubility increased by 14.08%~22.94% and 14.17%~20.52%, respectively. This further demonstrates that Example 3 (pH change (acidity)-glycosylation co-modification) has low sensitization and high solubility.
[0079] 3. Foaming test 10 mL of protein samples (β-lactoglobulin powder) prepared in Examples 1-3 and Comparative Example 1 at a concentration of 1.5 mg / mL were transferred to a graduated cylinder and homogenized twice at 10,000 rpm for 30 s each time. Foaming ability was measured immediately after homogenization, while foam stability was measured after the foam had stood at room temperature for 20 min. These values were calculated using the following formula:
[0080] ; In the above formula, the unit of foaming ability is % %. h 0 This refers to the height of the solution before homogenization, in cm. h1 This refers to the height of the solution and air bubbles after homogenization, measured in cm. ; In the above formula, the unit for foam stability is %; h 0 This refers to the height of the solution before homogenization, in cm. h 2 This refers to the height of the solution and bubbles after homogenization and standing for 20 minutes, measured in cm.
[0081] Test results as follows Figure 3 As shown. By Figure 3 It can be seen that the foaming ability and foam stability of the β-lactoglobulin powder prepared in Examples 1-3 are significantly higher than those of Comparative Example 1 (untreated). P <0.05). Example 3 (pH change (acidity)-glycosylation combined modification) showed the highest FC and FS, increasing by 158.82%~182.81% and 219.57%~237.78% compared to Comparative Example 1. This indicates that Example 3 has good foaming properties. This demonstrates that the pH change (acidity)-glycosylation combined modification of the present invention can effectively improve the functional properties of flavo-β-lactoglobulin, and the effect is superior to using pH change (acidity) or glycosylation modification alone.
[0082] All data in the examples were statistically analyzed using IBM SPSS Statistics 27.0 software; experimental graphs were plotted using Prism 10 software. It should be noted that different letters indicate statistically significant differences between different samples. P <0.05).
[0083] This invention utilizes a strategy of pH-change (acidic) modification, glycosylation modification, and combined treatment to significantly reduce the binding ability of β-lactoglobulin to specific IgE, while simultaneously improving its functional properties (solubility and foaming properties) while reducing sensitization. The combined pH-change (acidic) glycosylation modification (Example 3) showed the best desensitization effect and optimal functional properties. The core mechanism is as follows: β-lactoglobulin unfolds its protein structure in strong acid and is preserved for a period of time, then refolds into a more flexible "molten globular state" after neutralization. This alters the secondary and tertiary structures of β-lactoglobulin, increasing its chance of binding to sugars; while glycosylation modification masks the linear sensitizing epitopes of β-lactoglobulin through covalent bonding, simultaneously disrupting its conformational sensitizing epitopes and introducing hydrophilic groups to change the protein's functional properties. Through synergistic effects, the desensitization effect is significantly superior to that of pH-change (acidic treatment) alone, which only changes the protein state but cannot disrupt the sensitizing epitopes, and to glycosylation alone, which has limited reaction targets and low modification efficiency. Meanwhile, this treatment process has technical advantages such as mild reaction conditions, simple operation, rapid onset of action, and strong stability, making it easy to achieve large-scale industrial application. It not only effectively breaks through the technical bottleneck of poor desensitization effect of existing single modification technology, but also provides practical technical support for improving the functional characteristics of β-lactoglobulin and expanding its application in the food industry.
[0084] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.
[0085] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.
Claims
1. A method for preparing a β-lactoglobulin powder with low allergenicity, characterized in that, Includes the following steps: The pH of the β-lactoglobulin solution was adjusted to acidic, stirred evenly, and allowed to undergo conformational change or denaturation at room temperature. The pH was then adjusted to neutral to obtain a pH-adjusted β-lactoglobulin solution. Add oligosaccharide solution to the pH-adjusted β-lactoglobulin solution and stir until homogeneous to obtain a β-lactoglobulin-oligosaccharide mixed solution; The β-lactoglobulin-oligosaccharide mixed solution was freeze-dried to obtain a mixed powder; The mixed powder was subjected to Maillard reaction at a temperature of 50°C to 60°C and a relative humidity of 70% to 80%. The mixed powder after Maillard reaction was dissolved, dialyzed, and freeze-dried to obtain the β-lactoglobulin powder with low allergenicity.
2. The preparation method according to claim 1, characterized in that, The acidity refers to a pH value of 1 to 3.
3. The preparation method according to claim 1, characterized in that, The β-lactoglobulin solution is obtained by dissolving β-lactoglobulin in phosphate buffer.
4. The preparation method according to claim 3, characterized in that, The phosphate buffer solution has a pH of 7.2 to 7.6 and a concentration of 0.01 mol / L to 0.02 mol / L.
5. The preparation method according to claim 1, characterized in that, The mass ratio of β-lactoglobulin to oligosaccharides in the pH-changing process is 1:1~3.
6. The preparation method according to claim 5, characterized in that, The oligosaccharides include xylooligosaccharides with a molecular weight of 300kDa to 1050kDa.
7. The preparation method according to claim 1, characterized in that, The time for the conformational change or denaturation reaction to occur at room temperature is 0.5 h to 1.5 h.
8. The preparation method according to claim 1, characterized in that, The dialysis is performed at room temperature for 20-28 hours using a dialysis bag with a molecular weight cutoff of 4kDa to 10kDa.
9. A β-lactoglobulin powder with low allergenicity prepared by the preparation method according to any one of claims 1 to 8.