Zero-lactose bovine colostrum lactase enzymolysis method suitable for lactose intolerant people

Abstract

The invention relates to the technical field of lactose hydrolysis, in particular to a zero-lactose bovine colostrum lactase enzymolysis method suitable for lactose intolerant people. A zero-lactose bovine coloctrum lactase enzymolysis method suitable for lactose intolerant people comprises the following steps: adding lactase into cow milk, and carrying out hydrolysis reaction on lactose in bovine coloctrum to enable the hydrolysis rate of the lactose in the bovine coloctrum to be 100%, so as to obtain a zero-lactose emulsion; in the hydrolysis reaction process, the reaction temperature is 45-50 DEG C; the reaction time is 0.5 to 5 hours; the invention discloses a zero-lactose bovine colostrum lactase enzymolysis method suitable for lactose intolerant people, which improves the lactose hydrolysis efficiency by optimizing the lactase addition amount and reaction conditions without high-temperature heating, so that the lactose hydrolysis rate in the lactose hydrolysis process can reach 100% within a short time, and the lactose hydrolysis rate can reach 100% within a short period of time. Meanwhile, active ingredients in the bovine coloctrum are reserved to the maximum extent, and the hydrolyzed cow milk initially meets the market requirements of lactose-free dairy products.

Description

Technical Field

[0001] This application relates to the technical field of lactose hydrolysis, and in particular to a lactose-free enzymatic hydrolysis method for bovine colostrum suitable for lactose-intolerant individuals. Background Technology

[0002] Lactose is the most abundant carbohydrate in cow's milk, accounting for approximately 35% to 40% of the total milk solids. It is composed of one molecule of glucose and one molecule of galactose linked by a β-1,4-glycosidic bond. As a natural disaccharide in mammalian milk, lactose plays an important nutritional and physiological role in infants and young children. However, approximately 65% ​​to 70% of adults worldwide experience lactose intolerance because their intestinal lactase activity naturally declines with age, making them unable to effectively hydrolyze ingested lactose. Typical symptoms include bloating, flatulence, abdominal pain, and diarrhea after consuming dairy products, severely limiting dairy consumption and impacting quality of life.

[0003] With the diversification of the dairy market, consumer demand for dairy products is gradually becoming more personalized and health-oriented, making low-lactose or lactose-free dairy products a growing market trend. Currently, ultrafiltration technology is commonly used to produce low-lactose or lactose-free dairy products, but this process generates lactose as a byproduct. Statistics show that the annual production of lactose-containing liquid whey byproducts reaches approximately 150 to 200 million tons, which not only puts pressure on the environment but also represents a huge waste of resources. Lactose, a dairy byproduct, contains rich nutrients with great potential applications in pharmaceuticals, health products, and functional foods. However, due to the difficulty in recovering and extracting these beneficial components, their application remains somewhat limited.

[0004] Lactase, an enzyme that hydrolyzes lactose into glucose and galactose, is widely found in the small intestine. Through the action of lactase, lactose in dairy products can be effectively broken down into more easily digestible glucose and galactose, thus alleviating the digestive problems caused by lactose intolerance. At the same time, the application of lactase not only helps improve the taste of dairy products but also enhances their nutritional value. For example, lactose-hydrolyzed dairy products are more easily absorbed by the body. Lactose hydrolysis yields glucose, galactose, and lactoligosaccharides; glucose provides rapid energy, while galactose contributes to brain development and immune system function. Therefore, lactose hydrolysis technology plays an important role in modern dairy production, especially in the production of low-lactose or lactose-free dairy products, where lactase plays a crucial role.

[0005] Furthermore, the production cost of lactase is a major obstacle to its widespread application. Although researchers have made some progress in increasing the yield and activity of lactase through genetic engineering, its production cost remains high, especially limiting its application in low-priced dairy products.

[0006] Existing technologies remove lactose from bovine colostrum using lactase hydrolysis, which involves adding lactase to the colostrum to break down lactose into glucose, galactose, and lactooligosaccharides through enzymatic reactions. However, existing processes have significant drawbacks: firstly, to achieve complete hydrolysis, a large amount of lactase is often required, resulting in high production costs; secondly, due to the complex matrix of bovine colostrum, lactase is prone to conformational changes or adsorption by impurities, leading to low hydrolysis efficiency and making it difficult to reduce the lactose content to a level acceptable to lactose-intolerant individuals in a short period of time.

[0007] Therefore, how to improve hydrolysis efficiency while reducing the amount of lactase used by optimizing the production process has become a key issue in industrial applications. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this application provides a lactose-free bovine colostrum lactase hydrolysis method suitable for lactose-intolerant individuals.

[0009] This application proposes an innovative method for lactose hydrolysis that achieves 100% hydrolysis of lactose through enzymatic catalysis without the need for high-temperature heating, while preserving the bioactivity of the components and maximizing the utilization of all remaining components. This innovative technology overcomes the limitations of traditional lactose hydrolysis methods and demonstrates its enormous potential for improving production efficiency, reducing environmental burden, and achieving sustainable development in the dairy industry.

[0010] This application improves the lactase application process by optimizing the amount of lactase added and the reaction conditions, thereby increasing the lactose hydrolysis efficiency. This allows the lactose hydrolysis process to achieve a lactose hydrolysis rate of over 100% in a shorter time, significantly improving lactose decomposition efficiency and meeting the market demand for lactose-free dairy products.

[0011] In a first aspect, this application provides a lactose-free bovine colostrum lactase hydrolysis method suitable for lactose-intolerant individuals, employing the following technical solution: A lactose-free bovine colostrum lactase hydrolysis method suitable for lactose-intolerant individuals includes the following steps: adding lactase to bovine colostrum and hydrolyzing the lactose in the bovine colostrum to achieve a 100% hydrolysis rate of lactose in the bovine colostrum, thereby obtaining a lactose-free emulsion. During the hydrolysis reaction, the reaction temperature is 45-50℃ and the reaction time is 0.5-5 hours.

[0012] Preferably, the pH of the bovine colostrum solution is 5.2-7 during the hydrolysis reaction.

[0013] Preferably, the amount of lactase added is 0.5-2.5% of the lactose content in the cow's milk.

[0014] Preferably, the enzyme activity of the lactase is 43-220 U / g.

[0015] More preferably, the amount of lactase added is 1.5-2.5% of the lactose content in bovine colostrum.

[0016] Preferably, the bovine colostrum is fresh bovine colostrum or defatted bovine colostrum.

[0017] Preferably, the method for preparing the defatted bovine colostrum includes the following steps: centrifuging the bovine colostrum to obtain defatted bovine colostrum; During centrifugal defatting, the current setting of the centrifugal defatting machine is 7.5-8.5A, and the speed is 3000-6000rpm; the temperature of bovine colostrum is controlled to be ≤50℃ during the centrifugal defatting process.

[0018] Preferably, the bovine colostrum used for centrifugation and defatting is frozen bovine colostrum or fresh bovine colostrum.

[0019] Preferably, when the bovine colostrum used for centrifugal defatting is frozen bovine colostrum, the frozen bovine colostrum is pretreated before centrifugal defatting. The steps are as follows: after crushing the frozen bovine colostrum, it is heated to 40-50℃ to obtain pretreated frozen bovine colostrum.

[0020] Secondly, this application provides an application of a lactose-free emulsion obtained by enzymatic hydrolysis of bovine colostrum lactose using the above method in the preparation of food, health products, or pharmaceuticals, employing the following technical solution: The application of the lactose-free emulsion obtained by enzymatic hydrolysis of bovine colostrum lactose using the above method in the preparation of food, health products or pharmaceuticals.

[0021] In summary, this application includes at least one of the following beneficial technical effects: 1. This application discloses a lactose-free bovine colostrum lactase hydrolysis method suitable for lactose-intolerant individuals. Under conditions where high-temperature heating is not required, lactase is used to hydrolyze the lactose in bovine colostrum to 100%, while retaining the effective components in bovine colostrum to the maximum extent. The hydrolyzed bovine colostrum meets the market demand for lactose-free dairy products. 2. This application discloses a lactose-free bovine colostrum lactase hydrolysis method suitable for lactose-intolerant individuals. By optimizing the amount of lactase added and the reaction conditions, the lactose hydrolysis efficiency is improved, enabling the lactose hydrolysis process to achieve a 100% lactose hydrolysis rate in a shorter time. 3. The method of this application can not only ensure the high efficiency of lactose hydrolysis, but also reduce the waste of lactase, thereby effectively reducing the cost of using lactase. Attached Figure Description

[0022] Figure 1This is a graph showing the products and hydrolysis rate changes of the lactose hydrolysis process of this application at different reaction temperatures; wherein, Figure 1 (Left) is a thin-layer chromatography (TLC) chromatogram of the products of the lactose hydrolysis process of this application at different reaction temperatures; Figure 1 (Right) is a graph showing the changes in lactose content and lactose hydrolysis rate at different reaction temperatures in the lactose hydrolysis process of this application; Figure 2 This is a graph showing the products and hydrolysis rate changes of the lactose hydrolysis process of this application under different pH conditions; wherein, Figure 2 (Left) is a thin-layer chromatography (TLC) chromatogram of the products of the lactose hydrolysis process of this application under different pH conditions; Figure 2 (Right) is a graph showing the changes in lactose content and lactose hydrolysis rate under different pH conditions according to the lactose hydrolysis process of this application; Figure 3 The X-ray diffraction (XRD) spectra of the lactose-free emulsions prepared under different pH conditions in this application are shown. Figure 4 The image shows the changes in the infrared absorption spectra of the lactose-free emulsions prepared under different pH conditions in this application. Figure 5 This is a graph showing the products and hydrolysis rate changes of the lactose hydrolysis process of this application at different reaction times; wherein, Figure 5 (Left) is a thin-layer chromatography (TLC) chromatogram of the products of the lactose hydrolysis process of this application at different reaction times; Figure 5 (Right) is a graph showing the changes in lactose content and lactose hydrolysis rate at different reaction times in the lactose hydrolysis process of this application; Figure 6 The graph shows the products and hydrolysis rate changes of the lactose hydrolysis process of this application under different enzyme addition conditions. Figure 6 (Left) is a thin-layer chromatography (TLC) chromatogram of the products of the lactose hydrolysis process of this application under different enzyme addition conditions; Figure 6 (Right) is a graph showing the changes in lactose content and lactose hydrolysis rate under different enzyme addition conditions in the lactose hydrolysis process of this application; Figure 7 The infrared absorption spectrum of the zero-lactose emulsion prepared under different lactase addition amounts in this application is shown. Figure 8 The X-ray diffraction (XRD) spectra of the lactose-free emulsions prepared under different lactase addition amounts in this application are shown. Figure 9 The results of the molecular docking between lactase and lactose in this application are as follows: Figure 9 (A) shows the overall conformation of the lactase-lactose complex; Figure 9 (B) represents the two-dimensional interaction network between lactase and lactose; Figure 9(C) represents the binding site of lactose in the lactase active pocket; Figure 9 (D) represents the three-dimensional interaction between lactase and lactose. Detailed Implementation

[0023] The technical solutions of this application are further illustrated by specific embodiments below. These specific embodiments do not represent a limitation on the scope of protection of this application. Any non-essential modifications and adjustments made by others based on the concept of this application still fall within the scope of protection of this application.

[0024] All raw materials involved in this application are commercially available products, among which, Lactase was purchased from Xi'an Mixianer Biotechnology Co., Ltd.; Food Additive Production License No. SC10661042200511.

[0025] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0026] A lactose-free bovine colostrum lactase hydrolysis method suitable for lactose-intolerant individuals includes the following steps: Step 1: Sources of Bovine Colostrum (a) Skimmed bovine colostrum: (1) When the bovine colostrum is frozen, the frozen bovine colostrum is put into a slicer to slice and crush. After crushing, it is put into a thawing tank for heating and thawing. The temperature of the bovine colostrum after heating and thawing is 40-50℃. The thawed bovine colostrum is then transported to a centrifugal defatting machine for defatting to obtain defatted bovine colostrum. The current setting of the centrifugal defatting machine is 7.5-8.5A, and the speed is 3000-6000rpm; the temperature of bovine colostrum is controlled to be ≤50℃ during the centrifugal defatting process. (2) When the bovine colostrum is fresh, milk is squeezed from the cow and the bovine colostrum is collected. It is then immediately placed in a 4°C refrigerator and then transported to a centrifugal defatting machine for defatting to obtain defatted bovine colostrum. The current setting of the centrifugal defatting machine is 7.5-8.5A, and the speed is 3000-6000rpm; the temperature of bovine colostrum is controlled to be ≤50℃ during the centrifugal defatting process. (ii) Fresh milk colostrum Fresh bovine colostrum was immediately placed in a constant temperature environment of 50°C after collection; Step 2: Hydrolysis of lactose in bovine colostrum Add lactase to bovine colostrum (bovine colostrum is defatted bovine colostrum or fresh bovine colostrum, prepared in step one) and hydrolyze the lactose in the bovine colostrum to achieve a 100% hydrolysis rate of lactose in the bovine colostrum, thus obtaining a lactose-free emulsion. During the hydrolysis reaction, the reaction temperature is 45-50℃.

[0027] Preferably, the reaction time during the hydrolysis process is 0.5-5 hours.

[0028] Preferably, the pH of the solution is 5.2-7 during the hydrolysis reaction.

[0029] Preferably, the amount of lactase added is 0.5-2.5% of the lactose content in the cow's milk.

[0030] Preferably, the enzyme activity of the lactase is 43-220 U / g.

[0031] Furthermore, the amount of lactase added is 1.5-2.5% of the lactose content in bovine colostrum.

[0032] Preferably, when hydrolyzing lactose in bovine colostrum, a mixing device is used to mix the raw materials, and the mixing speed is 100rpm-200rpm.

[0033] Step 3: Application of the lactose-free emulsion obtained after hydrolyzing bovine colostrum lactose. Application of the lactose-free emulsion obtained by enzymatic hydrolysis of bovine colostrum lactose using the above method in the preparation of food, health products or pharmaceuticals.

[0034] (1) Method 1, drying: Bovine colostrum, after lactose hydrolysis, is concentrated by ultrafiltration and then dried; the drying methods include freeze drying, spray drying, and other methods. (2) Method 2: The bovine colostrum after lactose hydrolysis is directly developed into a liquid beverage.

[0035] Example 1: A lactose-free bovine colostrum lactase hydrolysis method suitable for lactose-intolerant individuals, comprising the following steps: Step 1: Preparation of Skimmed Bovine Colostrum Freeze-Dried Powder Milk is milked from cows and the colostrum is collected and immediately placed in a 4°C freezer. The colostrum is then transported to a centrifugal defatting machine for defatting to obtain defatted colostrum. The centrifugal defatting machine is set to a current of 8A and a speed of 4500rpm; the temperature of the bovine colostrum is controlled to be ≤50℃ during the centrifugal defatting process. Skimmed bovine colostrum was dried using a freeze-drying method for 24 hours to obtain skimmed bovine colostrum freeze-dried powder.

[0036] Step 2: Hydrolysis of lactose in bovine colostrum Skimmed bovine colostrum freeze-dried powder (containing 30.6% lactose) was mixed with water to obtain a skimmed emulsion with a mass fraction of 14%. Lactase was added to 1 kg of skim emulsion with a mass fraction of 14% to carry out a hydrolysis reaction, and finally 100% of lactose was hydrolyzed to obtain a lactose-free emulsion.

[0037] The hydrolysis reaction conditions are as follows: reaction temperature is 45℃; reaction time is 4 hours.

[0038] During the hydrolysis reaction, the pH of the bovine colostrum solution was 6.2.

[0039] During the hydrolysis reaction, a mixing device is used to mix the raw materials at a speed of 150 rpm.

[0040] The enzyme activity of the lactase is 219.4 U / g.

[0041] The amount of lactase added is 2.5% of the lactose content in the skim emulsion.

[0042] Examples 2-6:

[0043] The difference between this embodiment and Example 1 is that the reaction conditions for lactase hydrolysis are different, as detailed in the table below: Table 1. Hydrolysis reaction conditions for different lactases

[0044] Performance Test 1: The active ingredients in the lactose-free emulsions prepared using the methods in Examples 1-6 were tested, and the results are as follows: Table 2. Active ingredients in lactose-free emulsions

[0045] As shown in Table 2, the lactase hydrolysis of bovine milk progenitor (i.e., the skimmed emulsion in Example 1) using the methods of Examples 1-6 resulted in a lactose-free emulsion containing protein, Igg, ash, fat, glucose, galactose, and lactoligosaccharides. This indicates that the lactase hydrolysis of bovine milk progenitor (i.e., the skimmed emulsion in Example 1) using the methods of this application does not affect the active ingredients in the lactose-free emulsion.

[0046] Example 7: To investigate the effect of different hydrolysis reaction temperatures on lactose hydrolysis in primary milk (i.e., the skimmed milk in Example 1), the reaction temperature of hydrolysis in step two of Example 1 was adjusted, and the hydrolysis rate of lactose in the skimmed milk and the lactose content in the final product, zero-lactose milk, were measured at 30-60°C.

[0047] In this embodiment, lactase hydrolysis was performed on bovine colostrum (i.e., the skimmed emulsion in Example 1) under different temperature conditions. The fixed conditions were: pH 6.2, lactase activity of 219.4 U / g, lactase addition of 2.5% of the weight of lactose in the skimmed emulsion (on dry weight), and reaction time of 4 hours. The lactose content and degree of hydrolysis of the obtained product were determined by high performance liquid chromatography. The results are as follows: Figure 1 As shown.

[0048] like Figure 1 As shown on the right, as the reaction temperature increased from 30℃ to 45℃, the residual lactose content significantly decreased from 10.86% to 0%, while the corresponding degree of hydrolysis increased from 69.06% to 100%, indicating that increasing the temperature effectively promoted the enzymatic reaction rate. When the temperature rose to 45-50℃, the degree of hydrolysis reached a peak of 100%, and the residual lactose content dropped to 0%, meeting the technical goal of "zero lactose" in this application. Further increasing the temperature to 55℃ and 60℃, the degree of hydrolysis actually decreased to 93.40% and 85.91%, respectively. This is because the enzyme activity was partially deactivated at high temperatures, leading to a decrease in catalytic efficiency. This result confirms that the optimal temperature range for the process in this application is 45℃ to 50℃, within which the optimal balance between high hydrolysis rate and enzyme stability can be achieved.

[0049] Thin-layer chromatography (TLC) chromatograms Figure 1 (Left) This further corroborates the quantitative results above. In the figure, samples 30 to 60 from left to right correspond to treatment groups from 30℃ to 60℃, respectively. The vertically labeled areas from top to bottom represent the monosaccharide region, lactose region, and lactoligosaccharide region. It can be seen that as the temperature increases, the intensity of the lactose band gradually weakens, while the monosaccharide band significantly strengthens. At 45℃-50℃, the lactose spots are almost invisible, while the monosaccharide spots are the most dense, and... Figure 1 The degree of hydrolysis was consistent at 100%. Furthermore, no obvious byproducts or impurities were observed in the temperature group at 50℃ and below, indicating high reaction selectivity and no excessive degradation. In the 60℃ group, although the lactose band was weaker, diffuse tailing appeared in the monosaccharide region, possibly due to Maillard reaction or protein denaturation induced by high temperature, further suggesting that 50℃ is the optimal upper limit temperature.

[0050] Therefore, when the hydrolysis reaction conditions are pH 6.2, lactase activity is 219.4 U / g, lactase addition is 2.5% of the weight of lactose in the skim emulsion (on dry weight), and reaction time is 4 hours, this application can achieve efficient lactose hydrolysis without destroying the milk protein structure by controlling the reaction temperature in the range of 45℃ to 50℃, taking into account efficiency, stability and product quality, and fully adapting to the needs of continuous industrial production.

[0051] Example 8:

[0052] To investigate the effect of different hydrolysis reaction pH on lactose hydrolysis in the initial milk (i.e., the skimmed milk in Example 1), the pH of the hydrolysis in step two of Example 1 was adjusted, and the hydrolysis rate of lactose in the skimmed milk and the lactose content in the final product, the zero-lactose milk, were measured at pH 5.2-7.

[0053] In this embodiment, lactase hydrolysis was performed on bovine colostrum (i.e., the skimmed emulsion in Example 1) under different pH conditions. The fixed conditions were: temperature 45℃, lactase activity 219.4 U / g, lactase addition amount 2.5% of the weight of lactose in the skimmed emulsion (on dry weight), and reaction time 4 hours. The lactose content and degree of hydrolysis of the obtained product were determined by high performance liquid chromatography. The results are as follows: Figure 2 As shown.

[0054] like Figure 2 As shown on the right, the lactose residue was 0% in the pH range of 5.2 to 6.2, corresponding to a degree of hydrolysis of 100%. As the pH increased to above 6.2, the degree of hydrolysis began to decrease significantly. When the pH was 6.4, the degree of hydrolysis dropped to 97.87%, and when the pH was 7.0, it further decreased to 84.39%, indicating that enzyme activity was significantly inhibited in neutral and alkaline environments.

[0055] Thin-layer chromatography (TLC) chromatograms Figure 2 (Left) Further evidence of the above trend: In the figure, samples 5.2 to 7 from left to right correspond to treatment groups with pH values ​​of 5.2 to 7, respectively. The vertically labeled areas from top to bottom represent the monosaccharide region, lactose region, and lactooligosaccharide region. In the pH range of 5.2 to 6.2, the lactose band is the weakest, while the monosaccharide band is the densest, indicating that lactose is efficiently hydrolyzed into glucose and galactose. As the pH increases above 6.4, the lactose band gradually strengthens, while the monosaccharide band becomes lighter, which is completely consistent with the decreasing trend of hydrolysis degree in the curve. It is worth noting that no obvious byproducts or spots were observed in the pH range of 5.2 to 6.0, suggesting high reaction selectivity and no over-degradation.

[0056] The results confirm that the lactase used in this application exhibits excellent catalytic activity and stability under acidic conditions, with its optimal pH ranging from 5.6 to 6.2, significantly different from traditional neutral lactases. By controlling the pH of the reaction system within the range of 5.2 to 6.2, efficient hydrolysis of lactose can be achieved at a mild temperature of 45°C, while avoiding the risk of protein denaturation caused by strong acid or strong base adjustments.

[0057] Performance Test 2:

[0058] 1. Analysis of the crystal structure stability of lactose-free emulsions prepared under different pH conditions X-ray diffraction (XRD) analysis was performed to assess the crystal structure stability of lactose-free emulsions prepared under different pH conditions. Specifically, following the steps in Example 8, lactase hydrolysis was performed on bovine milk progenitor milk (i.e., the skimmed emulsion in Example 1) under different pH conditions (pH 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, and 7) to obtain lactose-free emulsions. The lactose-free emulsions were then freeze-dried for 24 hours to obtain dry powder samples. The obtained dry powder samples were directly subjected to XRD analysis, and the results are as follows: Figure 3 As shown.

[0059] The results showed that all samples exhibited a broadened and diffuse diffraction background, indicating that the milk matrix was mainly composed of amorphous or non-crystalline substances. In the lactose-free emulsion, the XRD patterns of all samples did not change significantly across the pH range of 5.2 to 7.0, indicating that pH adjustment did not induce crystalline transformation, chemical reactions, or new phase formation in lactase or milk components.

[0060] The results confirm that even under a wide pH range and high enzyme addition, the prepared lactose-free emulsion still maintains a physically dispersed state and does not destroy the naturally occurring amorphous structure in milk.

[0061] 2. Molecular structural stability analysis of lactose-free emulsions prepared under different pH conditions Fourier transform infrared spectroscopy (FTIR) analysis was performed to analyze the molecular structure stability of the lactose-free emulsions prepared under different pH conditions. Specifically, following the steps in Example 8, lactase hydrolysis was performed on bovine milk propolis (i.e., the skimmed emulsion in Example 1) under different pH conditions (pH 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, and 7) to obtain lactose-free emulsions. The lactose-free emulsions were then freeze-dried for 24 hours to obtain dry powder samples. The obtained dry powder samples were directly subjected to FTIR detection, and the results are as follows: Figure 4 As shown.

[0062] The results showed that within the pH range of 5.2 to 7.0, the positions and intensities of the protein characteristic peaks in all samples remained highly consistent, with no significant shifts, broadening, or new peak formation, indicating that the secondary structure of the proteins was not disrupted by pH changes or enzyme addition. Simultaneously, the characteristic peaks of lipids and phosphate minerals also showed no significant changes. Furthermore, as the pH increased, some peak shapes underwent slight adjustments, but the overall spectral profile remained stable, with no characteristic peaks of side reactions or degradation products appearing.

[0063] The results confirm that even under a wide pH range and high enzyme addition, the prepared lactose-free emulsion can still maintain the molecular structural integrity of the main functional components.

[0064] Example 9:

[0065] To investigate the effect of different hydrolysis reaction times on lactose hydrolysis in primary milk (i.e., the skimmed milk in Example 1), the hydrolysis reaction time in step two of Example 1 was adjusted, and the hydrolysis rate of lactose in the skimmed milk and the lactose content in the final product, zero-lactose milk, were measured when the reaction time was 0-5 hours.

[0066] In this embodiment, bovine colostrum (i.e., the skimmed emulsion in Example 1) was subjected to lactase hydrolysis under different time conditions. The fixed conditions were: pH 6.2, temperature 45℃, lactase activity of 219.4 U / g, and lactase addition of 2.5% of the weight of lactose in the skimmed emulsion (on a dry weight basis). The lactose content and degree of hydrolysis of the obtained products were determined by high performance liquid chromatography. The results are as follows: Figure 5 As shown.

[0067] like Figure 5 As shown on the right, at the start of the reaction (0 hours), the lactose content was 30.60% and the degree of hydrolysis was 0%. As the reaction proceeded, the residual lactose content continuously decreased, while the degree of hydrolysis steadily increased. After 1 hour of reaction, the degree of hydrolysis reached 56.97%, and the lactose content decreased to 13.17%. After 2 hours of reaction, the degree of hydrolysis increased to 79.89%, and the lactose content was 6.15%. After 4-5 hours of reaction, the degree of hydrolysis reached 100%, and the lactose content decreased to 0%.

[0068] TLC map Figure 5 (Left) Further evidence of the above trend: In the 0-hour sample, the lactose band was the densest, while the monosaccharide band was almost invisible; as the reaction time increased, the lactose band gradually became lighter, while the monosaccharide band significantly strengthened. At 4-5 hours, the lactose spots essentially disappeared, and the monosaccharide region was the densest, perfectly consistent with the high degree of hydrolysis trend in the curve. It is noteworthy that no obvious byproducts or impurities were observed throughout the entire reaction process, indicating high reaction selectivity and no excessive degradation.

[0069] The results confirm that the method of this application can achieve 100% lactose hydrolysis within 4-5 hours under mild conditions (pH 6.2, 45°C).

[0070] Example 10:

[0071] To investigate the effect of different enzyme addition amounts on lactose hydrolysis in bovine milk primordial (i.e., the skimmed milk in Example 1), the enzyme addition amount in step two of Example 1 was adjusted, and the lactose hydrolysis rate in the skimmed milk and the lactose content in the final product, the zero-lactose milk, were measured when the amount of lactase added was 0-2.5% of the weight of lactose in the skimmed milk (on dry weight).

[0072] In this embodiment, lactase hydrolysis was performed on bovine colostrum (i.e., the skimmed milk in Example 1) under different enzyme addition conditions. The fixed conditions were: pH 6.2, temperature 45℃, and reaction time 4 hours. The enzyme addition amount was calculated based on the mass of lactose in the skimmed milk. The lactose content and degree of hydrolysis of the obtained products were determined by high performance liquid chromatography, and the results are as follows: Figure 6 As shown.

[0073] like Figure 6 As shown on the right, in the control group without added enzyme (lactase addition was 0% of the lactose mass in the skim emulsion), the lactose content was 30.60% and the degree of hydrolysis was 0%. With increasing enzyme addition, the residual lactose content continuously decreased, while the degree of hydrolysis steadily increased. When the lactase addition was 0.5% of the lactose mass in the skim emulsion, the degree of hydrolysis reached 47.25%, and the lactose content decreased to 16.14%. When the lactase addition was 1.0% of the lactose mass in the skim emulsion, the degree of hydrolysis increased to 69.99%, and the lactose content was 9.18%. When the lactase addition was 2.5% of the lactose mass in the skim emulsion, the degree of hydrolysis reached 100%, and the lactose content decreased to 0%.

[0074] Thin-layer chromatography (TLC) chromatograms Figure 6 (Left) Further evidence of the above trend: In the figure, sample numbers 0 to 2.5 from left to right correspond to treatment groups with lactase addition levels of 0 to 2.5%, respectively. The vertically labeled areas from top to bottom represent the monosaccharide area, lactose area, and lactoligosaccharide area. In the samples without added enzyme, the lactose band was the densest, while the monosaccharide band was almost invisible. As the enzyme addition level increased, the lactose band gradually became lighter, while the monosaccharide band significantly strengthened. When the lactase addition level was 2.5% of the lactose mass, the lactose spots basically disappeared, and the monosaccharide area was the densest, which is completely consistent with the high degree of hydrolysis trend in the curve. It is worth noting that no obvious byproducts or spots were observed throughout the entire enzyme level range, indicating high reaction selectivity and no over-degradation.

[0075] The results confirm that the method described in this application, by optimizing the amount of enzyme added, can achieve efficient hydrolysis of lactose under mild conditions (pH 6.2, reaction temperature 45℃, and reaction time 4 hours). When the amount of lactase added is 2.5% of the mass of lactose in the skim emulsion, the enzyme activity is 219.4 U / g, and the degree of hydrolysis reaches 100%.

[0076] Performance Test 3:

[0077] 1. Analysis of the effect of different lactase addition amounts on the prepared lactose-free emulsion Fourier transform infrared spectroscopy (FTIR) analysis was performed on the lactose-free emulsions prepared with different amounts of lactase to evaluate the effect of the enzyme itself on the molecular structure of milk components. Specifically, following the steps of Example 10, lactase was added to the primary milk (i.e., the skim milk in Example 1) at proportions of 0%, 0.5% (enzyme activity 43.9 U / g), 1.0% (enzyme activity 86.4 U / g), 1.5% (enzyme activity 130.7 U / g), 2.0% (enzyme activity 175.1 U / g), and 2.5% (enzyme activity 219.4 U / g) of lactose by mass, respectively, to perform lactase hydrolysis treatment and obtain lactose-free emulsions. The lactose-free emulsions were freeze-dried for 24 hours to obtain dry powder samples. The obtained dry powder samples were directly analyzed by FTIR, and the results are as follows. Figure 7 As shown.

[0078] The results showed that, at all enzyme addition levels, the characteristic peak of milk protein (e.g., 3300 cm⁻¹) was the same. -1 NH / OH stretching vibration, 1650 cm -1 The position and intensity of the amide I band remained stable, without significant shift or broadening, indicating that the protein's secondary structure (α-helix, β-sheet) was not disrupted; simultaneously, the lipid CH stretching vibration peak (2900 cm⁻¹) remained intact. -1 ) and phosphate mineral peaks (1050-1100cm) -1 The absorption peaks and shoulder peaks showed no significant changes, indicating that the fat globule membrane and calcium phosphate crystal structure were completely preserved. Furthermore, no new absorption peaks or shoulder peaks appeared in any of the samples, suggesting that the addition of lactase did not trigger any side reactions or structural degradation.

[0079] The results confirm that even at high doses (2.5% w / w lactose), lactase itself does not damage the main functional components of milk and has good biocompatibility and reaction selectivity.

[0080] 2. Analysis of the effect of different lactase addition amounts on the structure of the prepared lactose-free liquid crystal. X-ray diffraction (XRD) analysis was performed on lactose-free emulsions prepared with different amounts of lactase to evaluate the effect of lactase itself on the crystalline state of minerals and proteins in milk. Specifically, following the steps of Example 10, lactase was added to the primary milk (i.e., the skim milk in Example 1) at proportions of 0%, 0.5% (enzyme activity 43.9 U / g), 1.0% (enzyme activity 86.4 U / g), 1.5% (enzyme activity 130.7 U / g), 2.0% (enzyme activity 175.1 U / g), and 2.5% (enzyme activity 219.4 U / g) of lactose by mass, respectively, to obtain lactose-free emulsions. The lactose-free emulsions were then freeze-dried for 24 hours to obtain dry powder samples. The obtained dry powder samples were directly subjected to XRD detection, and the results are as follows: Figure 8 As shown.

[0081] The results showed that the unenzyme-added milk sample exhibited a broad and diffuse diffraction background, indicating that its main components were amorphous or non-crystalline substances. In contrast, the pure lactase sample showed a sharp and high-intensity diffraction peak at approximately 20°, confirming its good crystallinity. As the enzyme addition increased from 0.5% to 2.5%, the intensity of the characteristic peak at 20° in the lactose-free emulsion gradually increased, and the peak shape remained sharp, without any peak shift or new diffraction peaks, indicating that lactase existed in the system in a physically mixed form. Meanwhile, the original broad background of the milk sample did not change significantly with the addition of the enzyme.

[0082] The results confirm that even at high doses, the addition of lactase exists only in a physically dispersed manner and does not trigger the crystallization transformation, aggregation, or phase separation of functional components in milk.

[0083] 3. Analysis of the molecular docking results between lactase and lactose in this application This application systematically studied the interaction mechanism between lactase and the substrate lactose through molecular docking simulation, and the results are as follows: Figure 9 As shown in the figure, this diagram comprehensively reveals the binding pattern of lactose within the lactase active site from four dimensions: overall structure (Figure A), two-dimensional interaction network (Figure B), binding pocket surface (Figure C), and three-dimensional spatial conformation (Figure D).

[0084] The results showed that the lactose molecule was precisely embedded in a recessed active pocket on the surface of lactase, consisting of multiple key amino acid residues. Two-dimensional interaction analysis (Figure B) revealed that lactose formed multiple non-covalent interactions with several amino acids within the active site: including conventional hydrogen bonds with residues such as GLU A:142, GLU A:200, and HIS A:355; π-hydrogen bonds with residues such as TYRA:96 and TYR A:260; and hydrophobic interactions with aromatic residues such as PHE A:264, PHE A:304, and TRP A:806.

[0085] The results confirm that the lactase used in this application has high structural complementarity and specific recognition ability for lactose. This precise binding mode provides a molecular-level structural basis for the subsequent efficient catalytic hydrolysis of lactose into glucose and galactose, and mechanistically explains the fundamental reason why the process in this application can achieve a high hydrolysis rate of 100%.

[0086] This application discloses a lactose-free bovine colostrum lactase hydrolysis method suitable for lactose-intolerant individuals, with the following positive effects: (1) Highly efficient lactose hydrolysis to meet market demand: This application optimizes the application process of lactase, enabling a stable 100% lactose hydrolysis rate, significantly improving the efficiency of lactose hydrolysis. This not only meets the market demand for low-lactose or lactose-free dairy products but also better addresses the digestive issues of lactose-intolerant individuals, enhancing the digestibility and nutritional value of dairy products.

[0087] (2) Reduce the production cost of lactase: This application improves the efficiency of lactase utilization by using a reasonable amount of lactase added and optimized reaction conditions. This not only ensures high efficiency in lactose hydrolysis but also reduces lactase waste, thereby effectively lowering the cost of lactase use. This provides a feasible solution for dairy manufacturers to achieve efficient production under low-cost conditions.

[0088] (3) Optimize resource utilization and promote the recycling of by-products: This technological solution utilizes highly efficient lactose hydrolysis to effectively avoid the lactose byproduct generated during ultrafiltration processing, a method employed in existing technologies to produce low-lactose or lactose-free dairy products. This innovative approach reduces production waste emissions, aligning with the environmental protection and sustainable development requirements of modern dairy production.

[0089] (4) Enhance the nutritional value and taste of dairy products: Lactose-hydrolyzed dairy products are more easily digested and absorbed by the body. The glucose in these products provides rapid energy, while galactose has a positive impact on brain development and immune system function. This not only enhances the nutritional value of dairy products but also improves their taste, making them more suitable for people with lactose intolerance and other consumers.

[0090] (5) Environmental protection and sustainable development: The lactose hydrolysis process used in this application does not require high-temperature heating, thus avoiding the loss of nutrients that may occur during high-temperature processing. This non-heating hydrolysis method not only preserves the nutritional components of dairy products but also reduces energy consumption, meets environmental protection requirements, and promotes the dairy industry towards a greener and more sustainable development direction.

[0091] (6) Flexible product development and diversified applications: The technology presented in this application is not only applicable to lactose-hydrolyzed milk, but also allows for flexible product development based on market demand, such as diversified applications like liquid beverages, milk powder, and functional foods. Different drying methods (such as freeze-drying and spray drying) can be selected according to different product requirements, greatly expanding the market application space for dairy products.

[0092] (7) Enhance the market competitiveness of products: This application effectively enhances the market competitiveness of dairy products by optimizing the lactose hydrolysis process, improving the nutritional value of dairy products, and reducing production costs. With the increasing consumer demand for healthy and personalized dairy products, this technology will help manufacturers stand out in a highly competitive market.

Claims

1. A zero lactose bovine colostrum lactase hydrolysis method suitable for use in a lactose intolerant human population, characterised in that: The method comprises the following steps: adding lactase into the bovine colostrum and performing a hydrolysis reaction on lactose in the bovine colostrum, so that the hydrolysis rate of lactose in the bovine colostrum is 100%, and a zero-lactose emulsion is obtained; In the hydrolysis reaction, the reaction temperature is 45-50℃, and the reaction time is 0.5-5 hours.

2. A zero lactose Bovine Colostrum lactase hydrolysis method suitable for lactose intolerant human population as claimed in claim 1, wherein: In the hydrolysis reaction, the pH of the bovine colostrum solution is 5.2-7.

3. A zero lactose Bovine Colostrum lactase hydrolysis method suitable for lactose intolerant human population as claimed in claim 1, wherein: The lactase is added in an amount of 0.5-2.5% of the lactose content in the bovine colostrum.

4. A zero lactose Bovine Colostrum lactase hydrolysis method suitable for lactose intolerant human population as claimed in claim 3, wherein: The lactase has an enzyme activity of 43-220 U / g.

5. A zero lactose Bovine colostrum lactase hydrolysis method as claimed in claim 3, wherein: The lactase is added in an amount of 1.5-2.5% of the lactose content in the bovine colostrum.

6. A zero lactose Bovine colostrum lactase hydrolysis method suitable for lactose intolerant human population as claimed in claim 1, wherein: The bovine colostrum is fresh bovine colostrum or defatted bovine colostrum.

7. A zero lactose Bovine Colostrum lactase hydrolysis method suitable for lactose intolerant human population as claimed in claim 6, wherein: The defatted bovine colostrum is prepared by the following steps: centrifugal defatting of the bovine colostrum to obtain defatted bovine colostrum. In the centrifugal defatting, the current setting value of the centrifugal defatting machine is 7.5-8.5 A, and the rotation speed is 3000-6000 rpm; and the temperature of the bovine colostrum during the centrifugal defatting process is ≤50℃.

8. A zero lactose Bovine Colostrum lactase hydrolysis method suitable for lactose intolerant human population as claimed in claim 7, wherein: The bovine colostrum used in the centrifugal defatting is frozen bovine colostrum or fresh bovine colostrum.

9. A zero lactose Bovine Colostrum lactase hydrolysis method suitable for lactose intolerant human population as claimed in claim 8, wherein: When the bovine colostrum used in the centrifugal defatting is frozen bovine colostrum, the frozen bovine colostrum is pretreated before the centrifugal defatting, and the pretreatment steps are as follows: the frozen bovine colostrum is crushed and heated to 40-50℃ to obtain pretreated frozen bovine colostrum.

10. Use of the zero-lactose emulsion obtained by the enzymatic hydrolysis of bovine colostrum lactose according to any one of claims 1-9 in the preparation of food, health products or medicines.

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