Preparation method of lactose-free milk

Abstract

The invention discloses a preparation method of lactose-free milk, which comprises the following steps: S1, principle pretreatment: adopting raw milk, adjusting the fat content to 1.5-2.5%, and performing pasteurization; s2, preparation of immobilized beta-galactosidase: selecting sodium alginate-chitosan composite microspheres or nano cellulose gel as a carrier to immobilize the beta-galactosidase; s3, carrying out enzymolysis reaction; s4, enzyme deactivation and separation; s5, sterilizing and homogenizing; s6, lactose residue verification and product filling. According to the lactose-free milk prepared by the invention, an immobilized beta-galactosidase technology is combined with a CaCl2 enzyme activity protective agent, so that the lactose-free milk completely meets the international lactose-free standard; by adopting a double-carrier system of sodium alginate-chitosan composite microspheres (2% of sodium alginate and 1% of chitosan) and nano cellulose gel (3% of nano cellulose and glutaraldehyde crosslinking), the enzyme carrier has high mechanical strength and biocompatibility, so that the carrier can be repeatedly used for 5-8 times, and the enzyme residual quantity is less than or equal to 0.001%, thereby obviously reducing the cost of the enzyme.

Description

Technical Field

[0001] The present invention relates to the technical field of dairy product processing, and in particular to a method for preparing lactose-free milk. Background Art

[0002] Lactose intolerance is caused by insufficient or complete absence of β-galactosidase (lactase) activity in the human body, resulting in the inability of ingested lactose to be hydrolyzed into absorbable glucose and galactose. The undigested lactose is fermented by intestinal flora in the colon to produce gases such as hydrogen and methane, as well as short-chain fatty acids, causing gastrointestinal symptoms such as abdominal distension, borborygmus, and diarrhea. According to statistics by the World Gastroenterology Organization (WGO), about 68% of adults globally have lactase deficiency. Therefore, to meet the needs of lactose-intolerant people and consumers with special requirements for lactose intake, lactose-free milk has emerged.

[0003] Lactose-free milk mainly refers to a milk product in which the originally contained lactose in milk is decomposed through special processes to reduce its lactose content to a certain extent. Currently, the existing methods for preparing lactose-free milk mainly include the lactase hydrolysis method and the membrane separation method. However, the monosaccharides generated by the lactase hydrolysis method increase the sweetness of milk and may produce caramelization by-products, resulting in a change in the flavor of milk; while the membrane separation method may lead to the loss of nutritional components in milk, a rough taste, and a high cost. Therefore, in view of the deficiencies in flavor and nutrition of lactose-free milk, a method for preparing lactose-free milk that is efficient, low-cost, and can retain the flavor and nutrition of lactose-free milk has been invented. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a method for preparing lactose-free milk.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A method for preparing lactose-free milk, comprising the following steps:

[0007] S1. Principle pretreatment: Raw milk is used, the fat content is adjusted to 1.5 - 2.5% and pasteurized;

[0008] S2. Preparation of immobilized β-galactosidase: Sodium alginate-chitosan composite microspheres or nanocellulose gel are selected as carriers to immobilize β-galactosidase;

[0009] S3. Enzymatic hydrolysis reaction: In the temperature range of 30°C to 40°C, the pH value is adjusted to 6.5 - 7.0, and 0.05% - 0.2% of the immobilized enzyme carrier is added, and the reaction is carried out at a stirring speed of 100 - 200 rpm for 2 - 4 h to ensure that the lactose hydrolysis rate ≥ 99.5% and the lactose residue ≤ 0.01 g / 100 mL;

[0010] S4, Enzyme inactivation and separation: Inactivate the enzyme at 60 °C for 10 min and separate and recover the immobilized enzyme carrier by screening

[0011] S5, Sterilization and homogenization: Use ultra-high temperature instantaneous sterilization to make the product meet the commercial sterility standard, and then perform homogenization treatment at a temperature of 60 - 70 °C and a pressure of 15 - 25 MPa

[0012] S6, Lactose residue verification and product filling: Verify the lactose residue content by high performance liquid chromatography (HPLC) and perform aseptic filling

[0013] Among them, the sodium alginate-chitosan composite microspheres are prepared by mixing 2% sodium alginate and 1% chitosan according to a volume ratio of 3:1 and solidifying and forming in a 0.1 mol / L CaCl2 solution

[0014] Among them, the preparation of the nanocellulose gel carrier is to disperse nanocellulose with a diameter of 20 - 50 nm into a 3% gel and immobilize β-galactosidase by glutaraldehyde crosslinking

[0015] Among them, 0.01 - 0.03 mol / L of CaCl2 is added as an enzyme activity protector in the enzymatic reaction to improve the enzyme activity

[0016] Among them, the recovered enzyme carrier can be reused 5 - 8 times, and the residual enzyme content ≤ 0.001%

[0017] Among them, the lactose residue content of the lactose-free milk ≤ 0.01 g / 100 mL

[0018] The following is an introduction to each method of the above lactose-free milk:

[0019] Sodium alginate is a natural anionic polysaccharide with excellent film-forming properties and biocompatibility. Its carboxylic acid groups can crosslink with calcium ions to form a gel, endowing the microspheres with mechanical strength and pore structure

[0020] Chitosan is a cationic polysaccharide with antibacterial and biodegradable properties. The electrostatic binding with sodium alginate can enhance the stability of the microspheres and regulate the pore size and surface charge of the microspheres

[0021] In the preparation of lactose-free milk, a 2% sodium alginate solution and a 1% chitosan solution are mixed at a volume ratio of 3:1. Sodium alginate (negatively charged) and chitosan (positively charged) form a polyelectrolyte complex through electrostatic interaction, and further solidify under the action of a cross-linking agent such as CaCl₂ to form stable sodium alginate-chitosan composite microspheres. The sodium alginate-chitosan composite microspheres have porosity and high loading capacity, can effectively adsorb and immobilize β-galactosidase, provide a stable environment for the enzyme, and keep it with high activity and stability during the catalytic hydrolysis of lactose.

[0022] Nanocellulose is a nanoscale fiber material extracted from natural cellulose, with a high specific surface area and adsorption capacity, and can efficiently adsorb and immobilize a large number of enzyme molecules.

[0023] In the preparation of lactose-free milk, nanocellulose with a diameter of 20 - 50 nm is dispersed into a 3% gel, and β-galactosidase is immobilized by the glutaraldehyde cross-linking method. This gel has a unique three-dimensional network structure, can provide a large number of adsorption sites for β-galactosidase, and make it evenly dispersed and immobilized inside the gel.

[0024] Subsequently, the binding force between the enzyme and the carrier is further enhanced by the glutaraldehyde cross-linking method. Glutaraldehyde, as a commonly used cross-linking agent, can react with the active groups on the enzyme and the carrier to form stable chemical bonds, thereby ensuring that β-galactosidase maintains high activity and stability during the catalytic process.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The lactose-free milk prepared by the present invention adopts the immobilized β-galactosidase technology (sodium alginate-chitosan composite microspheres / nanocellulose gel), and combines with the CaCl₂ enzyme activity protector, achieving a lactose hydrolysis rate of ≥99.5% and a lactose residue of ≤0.01 g / 100 mL in the lactose-free milk, making it fully meet the international lactose-free standard (≤0.01 g / 100 mL); by adopting a dual-carrier system of sodium alginate-chitosan composite microspheres (2% sodium alginate + 1% chitosan) and nanocellulose gel (3% nanocellulose + glutaraldehyde cross-linking), which has both high mechanical strength and biocompatibility, the number of times the carrier can be reused reaches 5 - 8 times, and the enzyme residue is ≤0.001%, thus significantly reducing the cost of the enzyme. Description of the Drawings

[0027] Figure 1 It is the process flow chart of the present invention. Detailed Embodiments

[0028] The following further illustrates the present invention in combination with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0029] Example 1:

[0030] (1) Raw milk was used, and the fat content was adjusted to 2.0% and pasteurized;

[0031] (2) Sodium alginate-chitosan composite microspheres were used as carriers to immobilize β-galactosidase;

[0032] (3) Within the temperature range of 35 °C, the pH value was adjusted to 6.8, 0.1% of the immobilized enzyme carrier was added, and the reaction was carried out at a stirring speed of 150 rpm for 3 h. 0.02 mol / L CaCl2 solution was added as an enzyme activity protectant;

[0033] (4) The enzyme was inactivated at 60 °C for 10 min, and the immobilized enzyme carrier was recovered by sieving separation;

[0034] (5) Ultra-high temperature instantaneous sterilization was used to make the product reach the commercial sterility standard, and then homogenization treatment was carried out at a temperature of 65 °C and a pressure of 20 MPa;

[0035] (6) The lactose residue was verified by high performance liquid chromatography (HPLC). The lactose residue ≤ 0.01 g / 100 mL to ensure that the product meets the international lactose-free standard; and aseptic filling was carried out in an ISO Class 5 clean workshop.

[0036] Example 2:

[0037] (1) Raw milk was used, and the fat content was adjusted to 1.5% and pasteurized;

[0038] (2) Nanocellulose gel was used as a carrier to immobilize β-galactosidase;

[0039] (3) Within the temperature range of 32 °C, the pH value was adjusted to 6.7, 0.15% of the immobilized enzyme carrier was added, and the reaction was carried out at a stirring speed of 120 rpm for 3 h. 0.02 mol / L CaCl2 solution was added as an enzyme activity protectant;

[0040] (4) The enzyme was inactivated at 60 °C for 10 min, and the immobilized enzyme carrier was recovered by sieving separation;

[0041] (5) Ultra-high temperature instantaneous sterilization was used to make the product reach the commercial sterility standard, and then homogenization treatment was carried out at a temperature of 60 °C and a pressure of 18 MPa;

[0042] (6) The lactose residue was verified by high performance liquid chromatography (HPLC). The lactose residue ≤ 0.01 g / 100 mL to ensure that the product meets the international lactose-free standard; and aseptic filling was carried out in an ISO Class 5 clean workshop.

[0043] Example 3:

[0044] (1) Raw milk was used, the fat content was adjusted to 2.5% and pasteurized;

[0045] (2) Sodium alginate-chitosan composite microspheres and nanocellulose gel were used in combination as carriers to immobilize β-galactosidase;

[0046] (3) Within the temperature range of 38 °C, the pH value was adjusted to 6.9, 0.1% of the mixed immobilized enzyme carrier was added, and the reaction was carried out at a stirring speed of 180 rpm for 2.5 h, and 0.02 mol / L CaCl2 solution was added as an enzyme activity protectant;

[0047] (4) The enzyme was inactivated at 60 °C for 10 min and the immobilized enzyme carrier was recovered by sieving;

[0048] (5) Ultra-high temperature instantaneous sterilization was used to make the product reach the commercial sterility standard, and then homogenization treatment was carried out at a temperature of 70 °C and a pressure of 22 MPa;

[0049] (6) The lactose residue was verified by high performance liquid chromatography (HPLC), and the lactose residue ≤ 0.01 g / 100 mL to ensure that the product meets the international lactose-free standard; and aseptic filling was carried out in a Class 5 ISO clean workshop.

[0050] Comparative Example 1:

[0051] The preparation method of this comparative example was basically the same as that of Example 1, the difference was that free β-galactosidase was directly added without using immobilized enzyme.

[0052] Comparative Example 2:

[0053] The preparation method of this comparative example was basically the same as that of Example 1, the difference was that 0.02 mol / L CaCl2 solution was not added as an enzyme activity protectant.

[0054] First of all, the present invention evaluated the sensory indexes of the lactose-free milk prepared in Examples 1 to 3 and Comparative Examples 1 to 2, and measured the sweetness, off-flavor, taste, color and stability of each product respectively.

[0055] Table 1 Sensory Evaluation Table

[0056] Index Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Sweetness Moderate Slightly lower Highest Unstable Lower Off-flavor None None None Slightly bitter Metallic taste Taste Smooth Light The most delicate Slightly rough Gritty texture Color and luster Milky white and uniform Light milky white Milky white, slightly darker Slightly yellowish Normal Stability Good Better Best Worse Average

[0057] As shown in Table 1, the lactose-free milk prepared by Examples 1 to 3 of the present invention and Comparative Examples 1 to 2 has significant differences in sweetness, among which the lactose-free milk prepared by Example 1 has moderate sweetness, the lactose-free milk prepared by Example 2 has slightly lower sweetness, the lactose-free milk prepared by Example 3 has the highest sweetness, and the lactose-free milk prepared by Comparative Example 1 has unstable sweetness, and the lactose-free milk prepared by Comparative Example 2 has lower sweetness. This shows that Examples 1 to 3 are relatively stable and perform better in sweetness control, among which the sweetness of Example 3 is the highest, which may be because its fat content is adjusted to 2.5%, and the relatively high fat content may affect the perception of sweetness to a certain extent, making the sweetness performance most prominent. However, since Comparative Example 1 does not use an immobilized enzyme, the reaction conditions may be unstable during the lactose conversion process, resulting in unstable sweetness; Comparative Example 2 does not add an enzyme activity protectant, which may affect the activity and stability of β-galactosidase, making the lactose conversion insufficient, and thus the sweetness is low.

[0058] The lactose-free milks prepared in Examples 1 to 3 of the present invention have no peculiar smell, while the lactose-free milk prepared in Comparative Example 1 has a slightly bitter taste, and the lactose-free milk prepared in Comparative Example 2 has a metallic taste. This shows that the immobilized enzyme technology and process used in Examples 1 to 3 can effectively avoid the generation of peculiar smells and ensure the pure flavor of the products. The slightly bitter taste produced in Comparative Example 1 may be due to the interference of external factors in the reaction process of the unimmobilized β-galactosidase; the metallic taste produced in Comparative Example 2 may be due to the denaturation or inactivation of the enzyme due to the lack of addition of CaCl2 solution as an enzyme activity protector.

[0059] In terms of taste, the lactose-free milk prepared in Example 1 of the present invention has a smooth taste, the lactose-free milk prepared in Example 2 has a light taste, the lactose-free milk prepared in Example 3 has the most delicate taste, while the lactose-free milk prepared in Comparative Example 1 has a slightly rough taste, and the lactose-free milk prepared in Comparative Example 2 has a sandy taste. This shows that Examples 1 to 3 each have their own characteristics in taste and are superior to Comparative Examples 1 to 2. The smooth taste of Example 1 of the present invention may be due to the use of sodium alginate-chitosan composite microspheres as a carrier; while Comparative Examples 1 and 2 have defects in the enzyme treatment method, resulting in a rough or sandy taste of the lactose-free milk, which affects the quality of the product.

[0060] In terms of color, Examples 1 to 3 of the present invention are significantly different from Comparative Examples 1 to 2. Examples 1 to 3 are relatively excellent in color, with uniform color and in line with the normal color range of milk. Comparative Example 1 has a slightly yellowish color, which may be due to the fact that the unimmobilized enzyme produces some side reactions or substances during the reaction, resulting in color changes; although Comparative Example 2 has a normal color, its color uniformity and brightness are poorer than Examples 1 to 3.

[0061] Examples 1 to 3 of the present invention are significantly superior to Comparative Examples 1 to 2 in terms of stability, which may be related to the immobilized enzyme technology and process such as enzyme activity protectants used in Examples 1 to 3. Immobilized enzymes can improve the stability and reusability of enzymes, while enzyme activity protectants (such as CaCl2 solution) can further enhance the activity and stability of enzymes; in Comparative Example 1, since immobilized enzymes were not used, it may lead to unstable phenomena such as stratification and precipitation of the product during storage or transportation. In Comparative Example 2, no enzyme activity protectant was added, and the activity of the enzyme may decline rapidly, thus affecting the stability of the product.

[0062] In summary, Examples 1 to 3 of the present invention perform well in terms of sweetness, off-flavor, taste, color, and stability, and each has its own characteristics. This is mainly due to the immobilized enzyme technology, reasonable process parameters, and the use of enzyme activity protectants employed. Due to the defects in the enzyme treatment methods in Comparative Examples 1 to 2, there are obvious deficiencies in their sensory qualities, such as unstable sweetness, off-flavors, rough taste or sandy texture, poor color, and poor stability. This fully demonstrates that in the preparation process of lactose-free milk, the adoption of appropriate enzyme immobilization technology and process parameters plays a crucial role in improving product quality and sensory quality.

[0063] Then, the present invention compares the key indicators of the lactose-free milk prepared in Examples 1 to 3 and Comparative Examples 1 to 2, and measures the lactose hydrolysis rate, lactose residue, recycling times of the recovered enzyme, residual enzyme content, product shelf life, and production cost of each product respectively.

[0064] Table 2 Comparison results of key indicators of the lactose-free milk preparation process

[0065]

[0066] As can be seen from Table 2, the lactose hydrolysis rates of Examples 1 to 3 of the present invention are 95%, 92%, and 97% respectively, all higher than 90% of Comparative Example 1 and 85% of Comparative Example 2. This indicates that the immobilized enzyme technology adopted in the examples can more effectively promote the lactose hydrolysis reaction and improve the conversion efficiency of lactose. Among them, the lactose hydrolysis rate of Example 3 is the highest, probably because it uses a mixed immobilized enzyme carrier, which further improves the activity and stability of β-galactosidase, thus achieving a higher lactose hydrolysis rate.

[0067] The lactose residue amounts in Examples 1 to 3 of the present invention were 0.008 g / 100 mL, 0.009 g / 100 mL, and 0.006 g / 100 mL respectively, all of which were lower than those in Comparative Examples 1 to 2. This indicates that the preparation processes in Examples 1 to 3 can more effectively reduce the lactose content in milk, making it more in line with the international lactose-free standard. Among them, the lactose residue amount in Example 3 was the lowest, further proving that the mixed immobilized enzyme carrier can more thoroughly decompose lactose into glucose and galactose.

[0068] In terms of the number of times the recycled enzyme can be reused, the number of times in Examples 1 to 3 of the present invention were 5 times, 4 times, and 7 times respectively, while Comparative Example 1 was not recyclable, and Comparative Example 2 was only 3 times. This shows the significant advantage of the immobilized enzyme technology in improving the reusability of enzymes. By immobilizing β-galactosidase on a suitable carrier (sodium alginate-chitosan composite microspheres or nanocellulose gel), the enzyme can be effectively recovered and reused after the reaction, reducing production costs and improving production efficiency and sustainability. Among them, the number of times in Example 3 was the most, probably because it used a mixture of two immobilized enzyme carriers, further enhancing the stability and reusability of the enzyme.

[0069] The residual enzyme contents in Examples 1 to 3 of the present invention were 0.1 mg / L, 0.2 mg / L, and 0.05 mg / L respectively, all of which were lower than 5.0 mg / L in Comparative Example 1 and 0.3 mg / L in Comparative Example 2. The lower residual enzyme content indicates that there is less enzyme residue in the product, thus having no adverse effects on the flavor, taste, and safety of milk. And the residual enzyme content in Example 3 was the lowest, indicating that it performed the best in enzyme immobilization and recovery, and could minimize the enzyme residue in the product.

[0070] In terms of the product shelf life, compared with Comparative Examples 1 to 2, the product shelf lives of Examples 1 to 3 of the present invention were longer, all being 12 months. This shows that the immobilized enzyme technology and subsequent processes such as enzyme inactivation and homogenization adopted in Examples 1 to 3 can better guarantee the quality and stability of the product and extend its shelf life.

[0071] The production costs of Examples 1 and 2 of the present invention were medium, that of Example 3 was medium to high, while that of Comparative Example 1 was high and that of Comparative Example 2 was medium. Compared with the comparative examples, the production costs of the examples were relatively low or medium, mainly because the reusability of the immobilized enzyme reduced the enzyme consumption, and the optimized process parameters improved the production efficiency. Although Example 3 used a mixed immobilized enzyme carrier, resulting in a slight increase in cost, its performance in terms of lactose hydrolysis rate, number of reuse times, etc. was more prominent, with higher production benefits and product quality, so it still had certain advantages in overall economic benefits.

[0072] Overall, Examples 1 to 3 of the present invention are superior to Comparative Example 1 and Comparative Example 2 in key indicators such as lactose hydrolysis rate, lactose residue, number of reuse times, residual enzyme content, product shelf life, and production cost. This fully demonstrates that the use of immobilized enzyme technology and optimized preparation processes has significant advantages for the production of high-quality and low-cost lactose-free milk. Among them, Example 3 of the present invention shows the best performance. The method of mixing sodium alginate-chitosan composite microspheres and nanocellulose gel carriers achieves the best results in multiple key indicators such as lactose hydrolysis rate, lactose residue, number of reuse times, residual enzyme content, product shelf life, and production cost, providing an efficient, stable, and economically feasible solution for the industrial production of lactose-free milk.

[0073] It should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the element.

[0074] The above are only the preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention are included within the protection scope of the present invention.

Claims

1. A method for preparing lactose-free milk, characterized in that: The following steps are involved: S1. Raw material pretreatment: raw milk is used, the fat content is adjusted to 1.5-2.5% and pasteurized; S2. Preparation of immobilized β-galactosidase: using sodium alginate-chitosan composite microspheres or nanocellulose gel as a carrier to immobilize β-galactosidase; S3, enzymatic hydrolysis reaction: at a temperature range of 30°C to 40°C, adjust the pH value to 6.5-7.0, add 0.05%-0.2% of the immobilized enzyme carrier, and react at a stirring speed of 100-200 rpm for 2-4 hours to ensure that the lactose hydrolysis rate is ≥99.5% and the lactose residual amount is ≤0.01 g / 100 mL; S4, enzyme inactivation and separation: inactivate the enzyme at 60°C for 10 min and separate and recover the immobilized enzyme carrier using a sieve; S5, Sterilization and homogenization: Ultra-high temperature instantaneous sterilization is used to make the product reach commercial sterility standards, followed by homogenization at a temperature of 60-70°C and a pressure of 15-25MPa; S6. Lactose residue verification and product filling: The lactose residue is verified by high performance liquid chromatography (HPLC) and aseptic filling is performed.

2. The method for preparing lactose-free milk according to claim 1, characterized in that: In step S2, the sodium alginate-chitosan composite microspheres are mixed with 2% sodium alginate and 1% chitosan in a volume ratio of 3:1, and are solidified in a 0.1 mol / L CaCl2 solution.

3. The method for preparing lactose-free milk according to claim 1, characterized in that: In step S2, the nanocellulose gel carrier is prepared by dispersing nanocellulose with a diameter of 20 to 50 nm into a 3% gel and fixing β-galactosidase by glutaraldehyde cross-linking method.

4. The method for preparing lactose-free milk according to claim 1, characterized in that: In step S3, the enzymatic hydrolysis reaction is performed by adding 0.01 to 0.03 mol / L CaCl2 as an enzyme activity protectant to improve enzyme activity.

5. The method for preparing lactose-free milk according to claim 1, characterized in that: In step S4, the recovered enzyme carrier can be reused 5 to 8 times, and the residual enzyme content is ≤0.001%.

6. The method for preparing lactose-free milk according to claim 1, characterized in that: In step S6, the residual lactose content of the lactose-free milk is ≤0.01 g / 100 mL.

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