Preparation method of buffalo milk active polypeptide

Abstract

The invention discloses a buffalo milk active polypeptide preparation method, and relates to the technical field of dairy product processing, and the buffalo milk active polypeptide preparation method comprises the following steps: preparing buffalo cheese protein into a substrate solution; performing high-pressure homogenization and mild heat treatment on the substrate solution; carrying out two-stage enzymolysis on the pretreated substrate solution, wherein the first-stage enzymolysis adopts an enzyme combination mainly comprising incision enzyme, and the hydrolysis degree is controlled to be 10-22%; in the second-stage enzymolysis, flavourzyme and aminopeptidase or a compound enzyme preparation containing flavourzyme and aminopeptidase are added into the reaction system, and enzymolysis is carried out until the total hydrolysis degree reaches 28%-40%; separating the enzymatic hydrolysate by adopting an ultrafiltration membrane and a nanofiltration membrane, and collecting permeate with the molecular weight of less than 2000Da; and carrying out post-treatment on the permeate to obtain the buffalo milk active polypeptide. According to the method, the bitter taste of the product can be remarkably reduced, and specific sequences with high-value biological activities such as ACE inhibition and oxidation resistance can be better reserved and enriched.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of dairy processing, and particularly relates to a preparation method of active polypeptide of buffalo milk. BACKGROUND

[0002] Milk-derived bioactive peptides are short peptide fragments with multiple physiological functions released after enzymatic hydrolysis, fermentation or gastrointestinal digestion of milk proteins, which have been widely studied and applied in functional foods, health products and other fields. These peptides often exhibit angiotensin-converting enzyme (ACE) inhibition, antioxidant, antibacterial, immunomodulatory and other biological activities. As the main component of milk protein, beta-casein is an important precursor source for producing high-activity peptides.

[0003] At present, most of the preparation researches of milk-derived active polypeptides use ordinary cow milk (mainly Holstein and other breeds), whey protein or soybean protein as raw materials, and there are few reports on special preparation processes for buffalo milk. Buffalo milk has unique advantages in protein composition: the content of beta-casein is usually higher than that of ordinary cow milk, and most of them are A2 type beta-casein or A2 type with high proportion. The A2 type beta-casein has proline at the 67th amino acid, while the A1 type commonly found in ordinary cow milk has histidine. This key difference leads to the easy release of beta-casomorphin-7 (BCM-7) in the digestion or enzymatic hydrolysis process of A1 type, which is a controversial bioactive peptide with potential inflammatory risk and digestive discomfort, while A2 type has a natural low-risk advantage due to the structural difference at this site.

[0004] However, when the existing conventional enzymatic hydrolysis process for ordinary cow milk is directly applied to buffalo milk, the following main technical problems often occur: First, a large amount of bitter peptides are produced, resulting in poor sensory quality of the enzymatic hydrolysate and obvious bitterness, which seriously limits its application in food; Second, due to the more compact casein micelle structure of buffalo milk and lower accessibility of enzyme cleavage sites, the hydrolysis efficiency is not high under conventional enzymatic hydrolysis conditions, making it difficult to obtain a high proportion of small molecule active peptides; Third, it is difficult to effectively control the enzyme cleavage site, which easily leads to excessive cutting in the specific region of beta-casein, potentially increasing the risk of some adverse peptide segments, and cannot be targeted to enrich high-value active sequences according to the characteristics of A2 type sequence; Fourth, the existing debittering techniques mostly rely on additional physical and chemical methods or multi-step enzymatic hydrolysis combined with repeated enzyme inactivation operations, resulting in complex process route, significant cost increase, large loss of active ingredients, and difficulty in realizing continuous industrial production.

[0005] Therefore, there is an urgent need to develop a preparation method specifically for the characteristics of high A2 type β-casein in buffalo milk, in order to simultaneously solve technical problems such as high bitterness, potential risks of BCM-7, low proportion of small molecules, insufficient enrichment of activity, and complex processes, so as to achieve industrial production with low bitterness, low risk, high activity, high yield, and simple process. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing bioactive peptides from buffalo milk, the specific technical solution of which is as follows: A method for preparing bioactive peptides from buffalo milk includes the following steps: S1. Prepare a protein substrate solution with a mass concentration of 8-12% by buffalo casein; S2. The protein substrate solution is subjected to physical pretreatment, the physical pretreatment including high-pressure homogenization and mild heat treatment; S3. The substrate solution after physical pretreatment was subjected to the following enzymatic hydrolysis: a) Use an enzyme combination mainly composed of endonucleases to carry out mild enzymatic hydrolysis under conditions of pH 7.5~8.5 and temperature 45~60℃, and control the degree of hydrolysis to 10%~22%; b) Add flavor protease and aminopeptidase, or a complex enzyme preparation containing flavor protease and aminopeptidase, to the reaction system, and continue enzymatic hydrolysis at pH 6.5-7.8 and temperature 45-60℃ until the total degree of hydrolysis reaches 28%-40%; S4. Use ultrafiltration and nanofiltration membranes to continuously or cyclically separate the enzymatic hydrolysate and collect the permeate with a molecular weight of less than 2000 Da. S5. The permeate is post-processed to obtain buffalo milk active peptides, wherein the post-processing includes concentration and drying.

[0007] Preferably, in step S1, the buffalo casein is obtained from buffalo milk after defatting, precipitation, washing, and neutralization. The precipitation is acid precipitation or enzyme precipitation. The buffalo casein contains ≥36% β-casein, and A2 type β-casein accounts for ≥85% of the β-casein.

[0008] Preferably, in step S2: The high-pressure homogenization process is performed at a pressure of 40-65 MPa, and the process is repeated 2-4 times. The mild heat treatment specifically includes heating the substrate solution to 68~72°C and holding it at that temperature for 8~15 minutes.

[0009] Preferably, in step S3, the enzyme combination mainly composed of endonuclease includes alkaline protease and chymotrypsin, and the total amount of enzyme added is 0.5% to 1.5% of the substrate protein mass, and the activity ratio of alkaline protease to chymotrypsin is (5 to 10):1.

[0010] Preferably, in step S3, the total amount of flavor protease and aminopeptidase added is 1.0% to 2.5% of the substrate protein mass, and the activity ratio of flavor protease to aminopeptidase is (2 to 6):1.

[0011] Preferably, in step S4: The ultrafiltration membrane has a molecular weight cutoff of 5-8 kDa, an operating pressure of 0.10-0.35 MPa, and an operating temperature of 48-56°C. The nanofiltration membrane has a molecular weight cutoff of 1.5~3kDa.

[0012] Preferably, step S5 specifically includes the following sub-steps: S51. The permeate is concentrated under low temperature vacuum, with the concentration temperature controlled at 40~55℃ and the vacuum degree at -0.08~-0.095MPa, until the solid content is 20%~35%, to obtain the concentrated solution. S53. The concentrate is subjected to spray drying or centrifugal atomization drying, wherein the inlet air temperature of the spray drying is 155~175℃, the outlet air temperature is 85~92℃, and the atomization pressure is 0.15~0.25MPa.

[0013] Preferably, after step S51, the following steps are further included: S52. The concentrated liquid is subjected to a fine debittering treatment, wherein the fine debittering treatment employs macroporous adsorption resin for adsorption-desorption, specifically including: a) Adjust the pH of the concentrate to 6.0~6.8, control the temperature at 38~45℃, add macroporous adsorption resin at a feed:resin volume ratio of 1:(10~18), and adsorb for 1.5~3.5 hours, wherein the macroporous adsorption resin is selected from at least one of XAD-16N, HP-20, DA-201-C, and AB-8. b) After adsorption is complete, wash away the unadsorbed impurities with water, and then desorb using ethanol with a volume fraction of 40%~60%, collect the desorbed liquid, and use the desorbed liquid for drying in step S53.

[0014] Preferably, in step S3, the compound enzyme preparation comprises flavor protease, aminopeptidase and carboxypeptidase, wherein the activity of carboxypeptidase accounts for 5% to 30% of the total activity of the compound enzyme preparation.

[0015] Preferably, the compound enzyme preparation is prepared by the following steps: a) Dissolve flavor protease, aminopeptidase and carboxypeptidase in phosphate buffer solution at pH 6.0-7.0 to obtain three enzyme solutions; b) Mix the three enzyme solutions in a ratio of (3~6):(1~3):1 for the activity of flavor protease: aminopeptidase: carboxypeptidase to obtain an enzyme mixture. c) Mix the enzyme mixture with a sodium alginate solution with a mass concentration of 1.5% to 3.0% at a volume ratio of 1:(1 to 3) to obtain a mixture; d) The mixture is added dropwise or squeezed into a calcium chloride solution with a mass concentration of 0.05~0.3 mol / L to form calcium alginate gel beads; e) Immerse the formed calcium alginate gel beads in a chitosan solution with a mass concentration of 0.1%~0.5% and coat them for 0.5~2 hours. The pH of the chitosan solution is adjusted to 5.5~6.5. f) After coating, wash the gel beads with distilled water or phosphate buffer until neutral, filter or dry at low temperature to obtain the composite enzyme preparation.

[0016] Compared with the prior art, the technical solution of the present invention has the following significant advantages: 1. By employing a gentle, targeted enzymatic cleavage and integrated debittering strategy tailored to the characteristics of A2 type β-casein in buffalo milk, the bitterness of the product is significantly reduced, resulting in a substantial improvement in sensory quality and making it suitable for a wider range of food systems; 2. Effectively controls excessive cutting in specific areas, resulting in a significant reduction or even undetectable level of BCM-7 content, thus enhancing safety; 3. Increase the proportion and yield of small molecule bioactive peptides (especially those with a molecular weight of less than 2000 Da), while better preserving and enriching specific sequences with high-value biological activities such as ACE inhibition and antioxidation; 4. The process route is relatively simple, avoiding the complexity, increased cost and activity loss caused by traditional multi-step enzyme inactivation or additional debittering units, which is more conducive to continuous industrial scale-up production. Detailed Implementation

[0017] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below. The description in this part is only exemplary and explanatory, and should not be used to limit the scope of protection of the present invention in any way.

[0018] This embodiment provides a method for preparing bioactive peptides from buffalo milk, comprising the following steps: S1. Prepare a protein substrate solution with a mass concentration of 8-12% by buffalo casein.

[0019] S2. Physical pretreatment of the protein substrate solution, including high-pressure homogenization and mild heat treatment.

[0020] S3. The substrate solution after physical pretreatment was subjected to the following enzymatic hydrolysis: a) Use an enzyme combination mainly composed of endonucleases to carry out mild enzymatic hydrolysis under the conditions of pH 7.5~8.5 and temperature 45~60℃, and control the degree of hydrolysis to 10%~22%.

[0021] b) Add flavor protease and aminopeptidase, or a complex enzyme preparation containing flavor protease and aminopeptidase, to the reaction system, and continue enzymatic hydrolysis at pH 6.5-7.8 and temperature 45-60℃ until the total degree of hydrolysis reaches 28%-40%.

[0022] S4. Use ultrafiltration and nanofiltration membranes to continuously or cyclically separate the enzymatic hydrolysate and collect the permeate with a molecular weight of less than 2000 Da.

[0023] S5. The permeate is post-processed to obtain buffalo milk active peptides. The post-processing includes concentration and drying.

[0024] Specifically, the core of the method described in this embodiment lies in adopting a two-step enzymatic hydrolysis strategy of "mild directional internal hydrolysis + integrated external hydrolysis debittering", combined with physical pretreatment and membrane separation integrated operation, to achieve precise and controllable hydrolysis of buffalo casein.

[0025] First, physical pretreatment significantly improves the dense structure of buffalo casein micelles, enhances the accessibility of enzyme cleavage sites, and creates favorable conditions for subsequent enzymatic hydrolysis.

[0026] Then, in the first stage of enzymatic hydrolysis, an enzyme combination mainly composed of endonucleases is used for limited hydrolysis under mild conditions. By controlling the degree of hydrolysis, the hydrophilic regions of the protein are preferentially opened, while the precursor regions of β-casein that are prone to BCM-7 production are effectively protected, avoiding the release of undesirable peptides caused by excessive cleavage.

[0027] Based on this, without inactivating enzymes, flavor proteases and aminopeptidases (or their complex enzyme preparations) with predominantly exonuclease activity are directly added to the reaction system to continue deep enzymatic hydrolysis. This "non-enzyme-inactivating, integrated addition" design fully utilizes the residual endonuclease activity from the previous stage, creating a synergistic effect with the subsequently added exonuclease: the endonuclease produces more medium- and long peptides as substrates, while the exonuclease sequentially removes hydrophobic amino acids (especially those causing bitterness such as Leu, Val, Phe, Ile, and Trp) from both ends of the peptide chain, thereby significantly reducing the accumulation of bitter peptides, while further reducing molecular weight and enhancing umami flavor.

[0028] The entire enzymatic hydrolysis process is combined with ultrafiltration-nanofiltration membrane separation to achieve simultaneous separation of small molecule permeate, avoiding secondary bitterness caused by excessive substrate accumulation and improving the yield and purity of small molecule bioactive peptides. Finally, a gentle post-processing step maximizes the preservation of bioactivity.

[0029] In the technical solution of this embodiment, the second stage of enzymatic hydrolysis in step S3 particularly emphasizes the method of adding enzyme directly without inactivating it. This design not only avoids the loss of activity and the prolongation of time caused by repeated heating to inactivate enzyme in traditional multi-step enzymatic hydrolysis, but also enables the enzyme system to play a synergistic role in the reaction system, forming a continuous and dynamic enzymatic hydrolysis environment.

[0030] Furthermore, to enhance the debittering effect and address the characteristic of milk proteins containing a large number of proline (Pro) residues, a strategy of using a combination of broad-spectrum aminopeptidase and proline-specific aminopeptidase can be adopted. Proline-specific aminopeptidase has a highly efficient cleavage ability against the Pro-X bond, which helps to more thoroughly remove bitter peptides containing Pro, while reducing damage to other active sequences.

[0031] In addition, under membrane reactor operation mode, the exonuclease activity in the system can be maintained by combining cyclic enzymatic hydrolysis with timed small-volume addition of flavor protease, preventing flux reduction and incomplete hydrolysis caused by enzyme inactivation or membrane fouling, thereby achieving higher continuous production efficiency and small molecule peptide yield.

[0032] Compared with the prior art, the technical solution of this embodiment has the following significant advantages: 1. By employing a gentle, targeted enzymatic cleavage and integrated debittering strategy tailored to the characteristics of A2 type β-casein in buffalo milk, the bitterness of the product is significantly reduced, resulting in a substantial improvement in sensory quality and making it suitable for a wider range of food systems; 2. Effectively controls excessive cutting in specific areas, resulting in a significant reduction or even undetectable level of BCM-7 content, thus enhancing safety; 3. Increase the proportion and yield of small molecule bioactive peptides (especially those with a molecular weight of less than 2000 Da), while better preserving and enriching specific sequences with high-value biological activities such as ACE inhibition and antioxidation; 4. The process route is relatively simple, avoiding the complexity, increased cost and activity loss caused by traditional multi-step enzyme inactivation or additional debittering units, which is more conducive to continuous industrial scale-up production.

[0033] Further, in step S1, buffalo casein is obtained from buffalo milk through defatting, precipitation, washing, and neutralization. The precipitation is either acid precipitation or enzyme precipitation. The proportion of β-casein in buffalo casein is ≥36%, and the proportion of A2 type β-casein is ≥85% of the total β-casein.

[0034] Specifically, A2 type β-casein, because its 67th amino acid is proline (instead of histidine in A1 type), has peptide bonds near this site that are naturally resistant to most proteases during enzymatic hydrolysis, thus greatly reducing the likelihood of β-casein-7 (BCM-7) release. Meanwhile, buffalo milk itself has a high β-casein content, providing a richer sequence source for obtaining high-yield, highly active small molecule peptides.

[0035] Casein is preferably obtained from locally sourced Guangxi buffalo milk (Mora hybrid or local breed) through defatting, acid / enzyme precipitation, washing, and neutralization. The A2 type ratio can be verified by genotyping, mass spectrometry, or high-performance liquid chromatography. In actual production, BCM-7 achieves better control when the A2 type ratio of the raw material is ≥90% or even close to 100%.

[0036] Using buffalo casein with a high A2 ratio as the starting material is the most fundamental guarantee for achieving "extremely low BCM-7 content" in this implementation method. At the same time, it provides a material basis for the subsequent targeted enrichment of highly active peptides unique to the A2 type sequence, significantly improving the safety and functional value of the product.

[0037] Furthermore, in step S2: The high-pressure homogenization process is carried out at a pressure of 40~65 MPa, and the process is repeated 2~4 times.

[0038] The mild heat treatment specifically includes heating the substrate solution to 68~72℃ and holding it at that temperature for 8~15 minutes.

[0039] Specifically, buffalo casein micelles have a denser structure, larger particle size, and tighter binding than those of regular milk, making it difficult for enzyme molecules to effectively access their internal cleavage sites. High-pressure homogenization disrupts the micelle aggregates through intense shearing, cavitation, and impact, significantly increasing the protein's specific surface area and enzyme accessibility. Subsequent mild heat treatment at 68–72°C further loosens the micelle structure and moderately unfolds the peptide chains without causing significant protein denaturation or producing additional bitterness, creating optimal substrate conditions for subsequent mild enzymatic hydrolysis.

[0040] In some implementations, low-intensity sonication can be used as a pretreatment supplement to further improve enzyme digestion efficiency without introducing side reactions caused by excessive energy.

[0041] Beneficially, physical pretreatment significantly improves the hydrolysis efficiency of buffalo casein compared to untreated or conventional milk processing (typically by 15% to 30% or more), thereby significantly increasing the yield of small molecule bioactive peptides and laying the foundation for precise control of the degree of hydrolysis and avoiding over-cutting.

[0042] Furthermore, in step S3, the enzyme combination mainly composed of endonucleases includes alkaline protease and chymotrypsin, with a total enzyme amount of 0.5% to 1.5% of the substrate protein mass, and the activity ratio of alkaline protease to chymotrypsin is (5 to 10):1.

[0043] Specifically, its core principle is to achieve "mild and targeted enzymatic cleavage": alkaline proteases (Alcalases) have broad-spectrum endonuclease capabilities, but prefer to cleave in hydrophilic regions (near acidic / basic amino acids); a small amount of chymotrypsin is specific to aromatic amino acids (Phe, Tyr, Trp). When the two are used in a high ratio, they can preferentially open the more hydrophilic exposed regions in the protein molecule, while cutting less of the more hydrophobic, more compact, and BCM-7 precursor sequence (near positions 67-72) regions in β-casein. Thus, while achieving a certain degree of hydrolysis, it effectively protects key sensitive regions from excessive enzymatic cleavage.

[0044] By strictly controlling the degree of hydrolysis at this stage to a low level, and combining it with an appropriate enzyme dosage ratio and reaction time, "sequence selectivity" regulation of the enzyme cleavage site can be achieved, that is, preferentially producing precursor peptides with beneficial active sequences while keeping the BCM-7 precursor region as intact as possible.

[0045] This targeted enzymatic digestion strategy is a key technical step in achieving extremely low BCM-7 content in the final product. At the same time, it provides a medium-to-long peptide substrate with a suitable chain length for the second-stage exonuclease, thus avoiding the excessive production of bitter peptides.

[0046] Further, in step S3, the total amount of flavor protease and aminopeptidase added is 1.0% to 2.5% of the substrate protein mass, and the activity ratio of flavor protease to aminopeptidase is (2 to 6):1.

[0047] Specifically, the flavor protease and aminopeptidase added in the second stage of step S3 work by using exonuclease to remove hydrophobic amino acid residues (especially bitter amino acids such as Leu, Val, Ile, Phe, Tyr, and Trp) one by one from the N-terminus (aminopeptidase) and C-terminus (some flavor proteases have exonuclease activity) of the peptide chain. This further degrades the potentially bitter long peptides produced in the first stage into short peptides with smaller molecular weight and more neutral or slightly umami taste, thus achieving efficient enzymatic debittering.

[0048] To further enhance the removal of bitter peptides caused by proline (Pro) residues that frequently appear in milk proteins, a certain proportion of proline-specific aminopeptidase (Prolyl aminopeptidase or X-Prolyl dipeptidyl aminopeptidase) can be introduced into the aminopeptidase component. This enzyme has a high efficiency in cleaving the Pro-X bond, which helps to more thoroughly eliminate the bitter structure containing proline.

[0049] Through this integrated external debittering process, the bitterness score of the product can be stably controlled at an extremely low level, while significantly enhancing the umami flavor, improving the overall sensory quality, and making the product more acceptable to consumers.

[0050] Furthermore, in step S4: The ultrafiltration membrane has a molecular weight cutoff of 5~8kDa, an operating pressure of 0.10~0.35MPa, and an operating temperature of 48~56℃.

[0051] The molecular weight cutoff of nanofiltration membranes is 1.5~3kDa.

[0052] Specifically, by utilizing differences in molecular weight cutoff, simultaneous separation during enzymatic hydrolysis can be achieved—the ultrafiltration membrane (5-8 kDa) allows small-molecule bioactive peptides to pass through in a timely manner, while retaining large-molecule substrate proteins and large-fragment peptides back into the reaction system for continued enzymatic hydrolysis; secondary nanofiltration further concentrates and collects the target bioactive peptide fraction <2000-3000 Da. This process, carried out simultaneously with enzymatic hydrolysis, is a typical membrane reactor model, which can significantly improve the yield of small-molecule peptides and prevent the secondary generation of bitterness caused by excessive substrate accumulation.

[0053] In the cyclic operation of the membrane reactor, a small amount of flavor protease can be added to the system periodically according to the decrease in membrane flux to compensate for the natural decline in exonuclease activity, maintain a continuous high hydrolysis rate and membrane flux stability, and achieve a longer continuous operation time.

[0054] This integrated membrane separation method ensures that the proportion of small molecule active peptides with a molecular weight of <2000Da remains stable at a high level (usually ≥85%~92%), while shortening the overall process time, improving production efficiency, and preserving the active ingredients to the greatest extent.

[0055] Furthermore, step S5 specifically includes the following sub-steps: S51. The permeate is concentrated under low temperature vacuum, with the concentration temperature controlled at 40~55℃ and the vacuum degree at -0.08~-0.095MPa, until the solid content is 20%~35%, to obtain the concentrated solution.

[0056] S53. The concentrate is spray-dried or centrifugally atomized and dried. The inlet air temperature for spray drying is 155~175℃, the outlet air temperature is 85~92℃, and the atomization pressure is 0.15~0.25MPa.

[0057] Specifically, the principle of this post-processing (low-temperature vacuum concentration + low-temperature spray drying) is as follows: bioactive peptides, especially short peptides, are extremely sensitive to high temperatures and are prone to Maillard reactions, oxidation, or structural damage. Low-temperature vacuum concentration is used to lower the boiling point and remove moisture at a lower temperature; spray drying uses a lower combination of inlet / outlet air temperatures and completes drying in a short time, achieving rapid moisture removal while maximizing the protection of biological activity.

[0058] Depending on the end use of the product, spray drying or centrifugal atomization drying can be selected. If necessary, a small amount of protective agent (such as maltodextrin) can be added to further improve the activity retention rate.

[0059] The post-processing technology ensures that the final product maintains high biological activity (with minimal loss of functional activities such as ACE inhibition and anti-oxidation), and the finished product has good stability, making it suitable for long-term storage and commercial applications.

[0060] Furthermore, the following steps are included after step S51: S52. The concentrate is subjected to a fine debittering treatment, which employs adsorption-desorption using macroporous adsorption resin, specifically including: a) Adjust the pH of the concentrate to 6.0~6.8 and control the temperature at 38~45℃. Add macroporous adsorption resin at a volume ratio of 1:(10~18) of liquid to resin and adsorb for 1.5~3.5 hours. The macroporous adsorption resin is selected from at least one of XAD-16N, HP-20, DA-201-C, and AB-8.

[0061] b) After adsorption is complete, wash away the unadsorbed impurities with water, and then desorb using ethanol with a volume fraction of 40%~60%. Collect the desorbed liquid and use it for drying in step S53.

[0062] Specifically, macroporous adsorption resin debittering is an optional step. The principle is to utilize the hydrophobic adsorption properties of macroporous resin to selectively adsorb bitter peptides with strong hydrophobicity (especially short peptides containing aromatic amino acids), while most hydrophilic active peptides remain in the liquid phase, thereby further reducing the already low bitterness level if necessary.

[0063] This step is only used for specific products with extremely high sensory requirements (such as high-end functional beverages). Regular products can omit this step to simplify the process and reduce costs.

[0064] Beneficially, this step provides a backup technical means for the demand for extremely low bitterness, allowing the bitterness score of the product to be further reduced to an even better level, while the ethanol desorption process is mature and easy to operate industrially.

[0065] Furthermore, in step S3, the compound enzyme preparation contains flavor protease, aminopeptidase and carboxypeptidase, wherein the activity of carboxypeptidase accounts for 5% to 30% of the total activity of the compound enzyme preparation.

[0066] Specifically, carboxypeptidase can be further introduced into the compound enzyme preparation used in the second stage of enzymatic hydrolysis, with its activity accounting for 5% to 30% of the total activity of the compound enzyme preparation.

[0067] Carboxypeptidase removes amino acid residues one by one from the C-terminus of the peptide chain, forming a complementary exoclease action with aminopeptidase (which removes residues from the N-terminus). It is particularly good at removing bitter amino acids with strong hydrophobicity at the C-terminus (such as Leu, Ile, Val, Phe, Tyr, Trp, etc.).

[0068] Flavor proteases possess both endo- and exo-cleavage activities. The synergistic action of these three enzymes enables a more comprehensive and efficient "double-ended" degradation of bitter peptides. Simultaneously, they convert most of the hydrophobic residues that contribute to bitterness into free amino acids or very short peptides, thereby further reducing the overall bitterness intensity and enhancing umami and richness.

[0069] The proportion of carboxypeptidase introduced (5%~30%) has been optimized and balanced: if the proportion is too low, the C-terminal debittering will be insufficient, and if the proportion is too high, it may over-hydrolyze certain highly active sequences with specific C-terminal structures.

[0070] In practical applications, the proportion of carboxypeptidase can be flexibly adjusted within this range according to the sensory requirements and functional activity needs of the target product, forming product variations with different flavor gradients.

[0071] Beneficially, the introduction of carboxypeptidase significantly enhances the debittering ability of the complex exonuclease system. Under the same enzymatic hydrolysis time and total enzyme dosage, the bitterness score can be further reduced. At the same time, it has a more targeted removal effect on bitter peptides enriched with certain C-terminal hydrophobic amino acids, resulting in better overall sensory quality and higher product acceptance.

[0072] Furthermore, the complex enzyme preparation is prepared through the following steps: a) Dissolve flavor protease, aminopeptidase and carboxypeptidase in phosphate buffer solution at pH 6.0-7.0 to obtain three enzyme solutions.

[0073] b) Mix the three enzyme solutions in a ratio of (3~6):(1~3):1 for the activity of flavor protease: aminopeptidase: carboxypeptidase to obtain an enzyme mixture.

[0074] c) Mix the enzyme mixture with a sodium alginate solution with a mass concentration of 1.5% to 3.0% at a volume ratio of 1:(1 to 3) to obtain a mixture.

[0075] d) The mixture is added dropwise or squeezed into a calcium chloride solution with a mass concentration of 0.05~0.3 mol / L to form calcium alginate gel beads.

[0076] e) Immerse the formed calcium alginate gel beads in a chitosan solution with a mass concentration of 0.1%~0.5% and coat them for 0.5~2 hours. Adjust the pH of the chitosan solution to 5.5~6.5.

[0077] f) After coating, wash the gel beads with distilled water or phosphate buffer until neutral, filter or dry at low temperature to obtain the complex enzyme preparation.

[0078] The core principle of this preparation step is as follows: 1. First, use sodium alginate and Ca... 2+ Rapid ionic cross-linking forms a porous calcium alginate gel bead framework, which physically embeds three enzymes—flavor protease, aminopeptidase, and carboxypeptidase—into the framework, achieving preliminary enzyme immobilization. 2. Then, an electrostatic coating is applied to the surface of the gel beads with chitosan (cationic polysaccharide) to form a dense "core-shell" structure, which further improves mechanical strength, reduces enzyme leakage, and imparts certain pH responsiveness and antibacterial properties; 3. The immobilized enzyme beads can fully contact the substrate under mild conditions (without high-temperature enzyme inactivation). After enzymatic hydrolysis, solid-liquid separation can be achieved by simple filtration / centrifugation. The enzyme beads can be reused or added continuously.

[0079] This immobilization method essentially transforms free enzymes into recyclable "solid catalysts," significantly improving the enzyme's thermal stability, operational stability, and pH tolerance, while avoiding the problem of enzyme loss along with the product in traditional liquid enzymatic hydrolysis.

[0080] The preparation parameters of immobilized enzyme beads (such as sodium alginate concentration, CaCl2 concentration, chitosan coating time and concentration, enzyme solution to carrier volume ratio, etc.) can be fine-tuned according to the actual production equipment and the target enzyme activity recovery rate.

[0081] In industrial scale-up, enzyme beads with uniform particle size can be prepared by dripping, extrusion or vibratory jetting, and the packed bed or fluidized bed operation mode in a continuous flow membrane reactor can be realized to further improve the continuity of production.

[0082] In addition, this immobilization technology is also applicable to the preparation of low-bitter bioactive peptides from other milk proteins or plant proteins, demonstrating a certain degree of versatility.

[0083] Compared with free enzyme systems, the use of this immobilized complex enzyme preparation offers several advantages: 1. The reusability of enzymes is significantly improved (usually it can be reused 5 to 15 times or more, depending on the strength of the enzyme beads and operating conditions), greatly reducing the enzyme consumption per unit product and the production cost; 2. It avoids the activity loss and energy consumption caused by repeated high-temperature enzyme inactivation in traditional processes; 3. After enzymatic hydrolysis, the enzyme and product are automatically separated, simplifying downstream purification steps and reducing the risk of microbial contamination; 4. Immobilized enzymes exhibit better thermal and storage stability, facilitating long-term preservation and transportation on an industrial scale; 5. It performs particularly well in continuous operation of membrane reactors, maintaining stable flux and enzyme activity for longer periods, making it suitable for large-scale, continuous production.

[0084] Specific embodiments are provided below. These embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way.

[0085] Example 1 Step 1: Preparation of substrate solution Weigh 200 g of buffalo casein, add 1.8 L of pure water, stir well, and prepare a protein substrate solution with a mass concentration of 10.0%. Adjust the pH to 7.0 at room temperature using 1 mol / L NaOH.

[0086] Step 2: Physical Pretreatment The substrate solution was placed in a high-pressure homogenizer and homogenized twice at a pressure of 55 MPa. After each cycle, the solution was cooled to <40°C.

[0087] The sample was then transferred to a constant-temperature water bath and heated to 72.0°C, held for 12 minutes with gentle stirring (100 rpm). After treatment, the sample was rapidly cooled to 53°C.

[0088] Step 3: First Stage Enzymatic Digestion Adjust the pH of the solution to 7.9 (using 1 mol / L NaOH) and stabilize the temperature at 53.0℃.

[0089] Add 2.00 g of Alcalase and 0.24 g of chymotrypsin sequentially to start the reaction.

[0090] Reaction conditions: pH 7.9±0.1 (maintained by automatic addition of 1 mol / L NaOH), temperature 53.0±0.5℃, stirring speed 150 rpm.

[0091] Reaction time: 2.0 h.

[0092] The degree of hydrolysis was measured to be 16.8% at the end of the reaction (OPA method).

[0093] Step 4: Second Stage Enzymatic Digestion Flavorzyme 3.20 g and aminopeptidase 0.80 g were added directly to the reaction system.

[0094] The reaction continued, and the pH naturally drifted to around 7.2. The temperature was maintained at 53.0℃, and the stirring speed was 150 rpm.

[0095] Reaction time: 4.2 h.

[0096] The final degree of total hydrolysis was 34.2% (OPA method).

[0097] Step 5: Integrated separation using membrane reactor The enzymatic hydrolysate was transferred to an ultrafiltration membrane reactor (6 kDa cutoff), operating at a pressure of 0.18 MPa, a temperature of 52.5 °C, and a circulation rate of approximately 1.2 L / min.

[0098] During the 2.5 h cyclic enzymatic hydrolysis, 0.10 g of Flavorzyme was added every 45 min to maintain flux and enzyme activity.

[0099] Approximately 1.45 L of permeate was collected.

[0100] The permeate is further passed through a nanofiltration membrane (2.5 kDa cutoff) and concentrated to a solids content of approximately 28%, yielding approximately 320 mL of concentrate.

[0101] Step Six: Post-processing The concentrate was placed in a rotary evaporator and concentrated under vacuum at 40°C and -0.090 MPa until the solid content was 32.5%.

[0102] The concentrate was spray-dried with an inlet air temperature of 168°C, an outlet air temperature of 88°C, an atomization pressure of 0.20 MPa, and a feed rate of 8 mL / min.

[0103] A pale yellow powder product was obtained: approximately 48.7 g.

[0104] Step 7: Data Testing 1) Bitterness rating: A sensory evaluation panel of 12 trained evaluators with a 10-point scale was used. The samples were prepared as a 2.0% aqueous solution, and the evaluation was conducted blindly with 3 replicates and the average value was taken.

[0105] 2) BCM-7 content: LC-MS / MS method, C18 column, MRM mode, internal standard method for quantification, detection limit 0.05 μg / g.

[0106] 3) Molecular weight distribution (<2000 Da proportion): SEC-HPLC (TSKgel G2000SWXL column, mobile phase 0.1 mol / L phosphate buffer pH 7.0, detection wavelength 220 nm), the percentage of transmitted peak area was calculated using standard peptides of known molecular weight.

[0107] 4) ACE inhibitory activity (IC50): The amount of hippuric acid produced at 228 nm was determined by HHL substrate method and spectrophotometry, with 3 replicates.

[0108] 5) DPPH free radical scavenging rate: Classical DPPH method, sample concentration 1.0 mg / mL, measured at 517 nm, with Trolox as standard.

[0109] The test data is shown in Table 1 below:

[0110] The above data show that the buffalo milk active peptide powder prepared according to the process of this embodiment has extremely low bitterness, extremely low BCM-7 risk, high proportion of small molecule active peptides, and excellent ACE inhibition and antioxidant activity.

[0111] Example 2 Step 1: Preparation of substrate solution Weigh 200 g of buffalo casein, add 1.8 L of pure water, stir well, and prepare a protein substrate solution with a mass concentration of 10.0%. Adjust the pH to 7.0 at room temperature using 1 mol / L NaOH.

[0112] Step 2: Physical Pretreatment The substrate solution was placed in a high-pressure homogenizer and homogenized twice at a pressure of 55 MPa. After each cycle, the solution was cooled to <40°C.

[0113] The sample was then transferred to a constant-temperature water bath and heated to 72.0°C, held for 12 minutes with gentle stirring (100 rpm). After treatment, the sample was rapidly cooled to 53°C.

[0114] Step 3: First Stage Enzymatic Digestion Adjust the pH of the solution to 7.9 (using 1 mol / L NaOH) and stabilize the temperature at 53.0℃.

[0115] Add 2.20 g of Alcalase and 0.16 g of chymotrypsin sequentially to start the reaction.

[0116] Reaction conditions: pH 7.9±0.1 (maintained by automatic addition of 1 mol / L NaOH), temperature 53.0±0.5℃, stirring speed 150 rpm.

[0117] Reaction time: 2.0 h.

[0118] The degree of hydrolysis was measured to be 15.9% at the end of the reaction (OPA method).

[0119] Step 4: Second Stage Enzymatic Digestion Flavorzyme 3.20 g and aminopeptidase 0.80 g were added directly to the reaction system.

[0120] The reaction continued, and the pH naturally drifted to around 7.1. The temperature was maintained at 53.0℃, and the stirring speed was 150 rpm.

[0121] Reaction time: 4.2 h.

[0122] The final degree of total hydrolysis was 33.7% (OPA method).

[0123] Step 5: Integrated separation using membrane reactor The enzymatic hydrolysate was transferred to an ultrafiltration membrane reactor (6 kDa cutoff), operating at a pressure of 0.18 MPa, a temperature of 52.5 °C, and a circulation rate of approximately 1.2 L / min.

[0124] During the 2.5 h cyclic enzymatic hydrolysis, 0.10 g of Flavorzyme was added every 45 min to maintain flux and enzyme activity.

[0125] Approximately 1.42 L of permeate was collected.

[0126] The permeate is further passed through a nanofiltration membrane (2.5 kDa cutoff) and concentrated to a solids content of approximately 28%, yielding approximately 315 mL of concentrate.

[0127] Step Six: Post-processing The concentrate was placed in a rotary evaporator and concentrated under vacuum at 40°C and -0.090 MPa until the solid content was 32.1%.

[0128] The concentrate was spray-dried with an inlet air temperature of 168°C, an outlet air temperature of 88°C, an atomization pressure of 0.20 MPa, and a feed rate of 8 mL / min.

[0129] A pale yellow powder product was obtained: approximately 47.9 g.

[0130] Step 7: Data Testing The testing method is the same as in Example 1, and the test data is shown in Table 2 below:

[0131] The above data shows that even after reducing the amount of chymotrypsin, BCM-7 was still not detected in this embodiment, the proportion of small molecules remained at a high level, and although the bitterness and activity index were slightly inferior to those of Example 1, the overall performance was still far superior to the existing conventional process, and the enzyme cost was significantly reduced, making it more economical.

[0132] Example 3 Step 1: Preparation of substrate solution Weigh 200 g of buffalo casein, add 1.8 L of pure water, stir well, and prepare a protein substrate solution with a mass concentration of 10.0%. Adjust the pH to 7.0 at room temperature using 1 mol / L NaOH.

[0133] Step 2: Physical Pretreatment The substrate solution was placed in a high-pressure homogenizer and homogenized twice at a pressure of 55 MPa. After each cycle, the solution was cooled to <40°C.

[0134] The sample was then transferred to a constant-temperature water bath and heated to 72.0°C, held for 12 minutes with gentle stirring (100 rpm). After treatment, the sample was rapidly cooled to 53°C.

[0135] Step 3: First Stage Enzymatic Digestion Adjust the pH of the solution to 7.9 (using 1 mol / L NaOH) and stabilize the temperature at 53.0℃.

[0136] Add 2.00 g of Alcalase and 0.24 g of chymotrypsin sequentially to start the reaction.

[0137] Reaction conditions: pH 7.9±0.1 (maintained by automatic addition of 1 mol / L NaOH), temperature 53.0±0.5℃, stirring speed 150 rpm.

[0138] Reaction time: 2.0 h.

[0139] The degree of hydrolysis was measured to be 16.7% at the end of the reaction (OPA method).

[0140] Step 4: Second Stage Enzymatic Digestion Add 2.80 g of Flavorzyme, 0.70 g of aminopeptidase, and 0.50 g of carboxypeptidase directly to the reaction system.

[0141] The reaction continued, and the pH naturally drifted to around 7.0. The temperature was maintained at 53.0℃, and the stirring speed was 150 rpm.

[0142] Reaction time: 4.2 h.

[0143] The final degree of total hydrolysis was 35.1% (OPA method).

[0144] Step 5: Integrated separation using membrane reactor The enzymatic hydrolysate was transferred to an ultrafiltration membrane reactor (6 kDa cutoff), operating at a pressure of 0.18 MPa, a temperature of 52.5 °C, and a circulation rate of approximately 1.2 L / min.

[0145] During the 2.5 h cyclic enzymatic hydrolysis, 0.10 g of Flavorzyme was added every 45 min to maintain flux and enzyme activity.

[0146] Approximately 1.48 L of permeate was collected.

[0147] The permeate is further passed through a nanofiltration membrane (2.5 kDa cutoff) and concentrated to a solids content of approximately 28%, yielding approximately 325 mL of concentrate.

[0148] Step Six: Post-processing The concentrate was placed in a rotary evaporator and concentrated under vacuum at 40°C and -0.090 MPa until the solid content was 32.8%.

[0149] The concentrate was spray-dried with an inlet air temperature of 168°C, an outlet air temperature of 88°C, an atomization pressure of 0.20 MPa, and a feed rate of 8 mL / min.

[0150] A pale yellow powder product was obtained: approximately 49.2 g.

[0151] Step 7: Data Testing The testing method is the same as in Example 1, and the test data is shown in Table 3 below:

[0152] The data above show that by introducing carboxypeptidase in the second stage, this embodiment further enhances the removal effect of C-terminal hydrophobic bitter amino acids. Compared with Example 1, the bitterness score is reduced to 1.9, the antioxidant activity and ACE inhibitory activity are slightly improved, and the proportion of small molecules is slightly increased. This proves that the introduction of carboxypeptidase can significantly optimize sensory quality while maintaining extremely low BCM-7 risk and high functional activity.

[0153] Example 4 Step 1: Preparation of composite enzyme beads 1) Preparation of enzyme solution Weigh out 2.80 g of flavor protease, 0.70 g of aminopeptidase, and 0.50 g of carboxypeptidase, respectively, and add an appropriate amount of 0.05 mol / L phosphate buffer at pH 6.5 to each. Dissolve them thoroughly and bring the volume up to 15 mL of flavor protease solution, 8 mL of aminopeptidase solution, and 5 mL of carboxypeptidase solution.

[0154] 2) Vitality ratio calibration and mixing Take a small amount (about 0.1~0.2 mL) of each of the three enzyme solutions and determine the actual enzyme activity using the standard substrate method.

[0155] Based on the measured activity, the volumes used were adjusted as follows: 15.0 mL of flavor protease solution, 7.2 mL of aminopeptidase solution, and 4.8 mL of carboxypeptidase solution.

[0156] Combine the three solutions to obtain an enzyme mixture with a total volume of approximately 27 mL.

[0157] 3) Sodium alginate mixture Slowly add 27 mL of the enzyme mixture to 80 mL of 2.0% sodium alginate solution and mix thoroughly for 15 min with magnetic stirring (300 rpm) to avoid generating bubbles.

[0158] 4) Drop-on curing The mixture was added dropwise to 120 mL of 0.2 mol / L CaCl2 solution using a peristaltic pump at a rate of approximately 1.0 mL / min.

[0159] After the addition is complete, continue stirring and solidify for 30 minutes to form gel beads with a diameter of approximately 2.2~3.0 mm.

[0160] 5) Chitosan coating Discard the CaCl2 solution and wash the gel beads three times with distilled water until neutral.

[0161] The washed gel beads were transferred to 40 mL of 0.3% chitosan solution (pH 6.0) and coated for 1.0 h using a constant temperature shaker (30℃, 80 rpm).

[0162] 6) Washing and drying Wash the coated gel beads five times with distilled water until neutral, then filter dry through a 200-mesh sieve.

[0163] The enzyme beads were dried at a low temperature and in a well-ventilated place at 4°C until they reached a constant weight, yielding approximately 32 g of immobilized complex enzyme beads. The enzyme beads were then sealed and stored at 4°C for later use.

[0164] Step 2: Preparation of substrate solution Weigh 200 g of buffalo casein, add 1.8 L of pure water, stir well, and prepare a protein substrate solution with a mass concentration of 10.0%. Adjust the pH to 7.0 at room temperature using 1 mol / L NaOH.

[0165] Step 3: Physical Pretreatment The substrate solution was placed in a high-pressure homogenizer and homogenized twice at a pressure of 55 MPa. After each cycle, the solution was cooled to <40°C.

[0166] The sample was then transferred to a constant-temperature water bath and heated to 72.0°C, held for 12 minutes with gentle stirring (100 rpm). After treatment, the sample was rapidly cooled to 53°C.

[0167] Step 4: First Stage Enzymatic Digestion Adjust the pH of the solution to 7.9 (using 1 mol / L NaOH) and stabilize the temperature at 53.0℃.

[0168] Add 2.00 g of Alcalase and 0.24 g of chymotrypsin sequentially to start the reaction.

[0169] Reaction conditions: pH 7.9±0.1, temperature 53.0±0.5℃, stirring speed 150 rpm.

[0170] Reaction time: 2.0 h.

[0171] The degree of hydrolysis at the end of the reaction was 16.5% (OPA method).

[0172] Step 5: Second Stage Enzymatic Digestion Add 32 g of the immobilized complex enzyme beads prepared in step one directly to the reaction system (washed to neutral with phosphate buffer before use).

[0173] The reaction continued, and the pH naturally drifted to around 7.0. The temperature was 53.0℃, and the stirring speed was 120 rpm.

[0174] Reaction time: 4.5 h.

[0175] The final degree of total hydrolysis was 34.8% (OPA method).

[0176] Step Six: Integrated Separation of Membrane Reactors The enzymatic hydrolysate was transferred to an ultrafiltration membrane reactor (6 kDa cutoff), operating at a pressure of 0.18 MPa, a temperature of 52.5 °C, and a circulation rate of approximately 1.2 L / min.

[0177] During the 2.5-hour cyclic enzymatic hydrolysis, the system was gently shaken every 50 minutes.

[0178] After completion, the enzyme beads were filtered through a 200-mesh sieve to recover the enzyme beads (recovery rate 98.5%), and approximately 1.46 L of permeate was collected.

[0179] The permeate is passed through a nanofiltration membrane (2.5 kDa cutoff) and concentrated to a solids content of approximately 28%, yielding approximately 322 mL of concentrate.

[0180] Step 7: Post-processing The concentrate was placed in a rotary evaporator and concentrated under vacuum at 40°C and -0.090 MPa to a solid content of 32.4%.

[0181] Spray drying: inlet air temperature 168℃, outlet air temperature 88℃, atomization pressure 0.20 MPa, feed rate 8 mL / min.

[0182] A pale yellow powder product was obtained: approximately 48.9 g.

[0183] Step 8: Data Testing The testing method is the same as in Example 1, and the test data is shown in Table 4 below:

[0184] The above data shows that the use of immobilized composite enzyme beads achieves high enzyme recovery and reuse while maintaining enzymatic hydrolysis effects comparable to those of free enzyme systems, providing favorable conditions for continuous and industrial applications of the process.

[0185] Example 5 Step 1: Preparation of substrate solution Weigh 200 g of buffalo casein, add 1.8 L of pure water, stir well, and prepare a protein substrate solution with a mass concentration of 10.0%. Adjust the pH to 7.0 at room temperature using 1 mol / L NaOH.

[0186] Step 2: Physical Pretreatment The substrate solution was placed in a high-pressure homogenizer and homogenized three times at a pressure of 65 MPa, and cooled to <40°C after each cycle.

[0187] After processing, cool directly to 53°C.

[0188] Step 3: First Stage Enzymatic Digestion Adjust the pH of the solution to 7.9 (using 1 mol / L NaOH) and stabilize the temperature at 53.0℃.

[0189] Add 2.00 g of Alcalase and 0.24 g of chymotrypsin sequentially to start the reaction.

[0190] Reaction conditions: pH 7.9±0.1 (maintained by automatic addition of 1 mol / L NaOH), temperature 53.0±0.5℃, stirring speed 150 rpm.

[0191] Reaction time: 1.5 h.

[0192] The degree of hydrolysis was measured to be 11.2% at the end of the reaction (OPA method).

[0193] Step 4: Second Stage Enzymatic Digestion Flavorzyme 3.20 g and aminopeptidase 0.80 g were added directly to the reaction system.

[0194] The reaction continued, and the pH naturally drifted to around 7.1. The temperature was maintained at 53.0℃, and the stirring speed was 150 rpm.

[0195] Reaction time: 4.0 h.

[0196] The final degree of total hydrolysis was 28.6% (OPA method).

[0197] Step 5: Integrated separation using membrane reactor The enzymatic hydrolysate was transferred to an ultrafiltration membrane reactor (6 kDa cutoff), operating at a pressure of 0.18 MPa, a temperature of 52.5 °C, and a circulation rate of approximately 1.2 L / min.

[0198] During the 2.5 h cyclic enzymatic hydrolysis, 0.10 g of Flavorzyme was added every 45 min to maintain flux and enzyme activity.

[0199] Approximately 1.38 L of permeate was collected.

[0200] The permeate is further passed through a nanofiltration membrane (2.5 kDa cutoff) and concentrated to a solids content of approximately 28%, yielding approximately 305 mL of concentrate.

[0201] Step Six: Post-processing The concentrate was placed in a rotary evaporator and concentrated under vacuum at 40°C and -0.090 MPa until the solid content was 31.8%.

[0202] The concentrate was spray-dried with an inlet air temperature of 168°C, an outlet air temperature of 88°C, an atomization pressure of 0.20 MPa, and a feed rate of 8 mL / min.

[0203] A pale yellow powder product was obtained: approximately 45.2 g.

[0204] Step 7: Data Testing The testing method is the same as in Example 1, and the test data is shown in Table 5 below:

[0205] The above data shows that, in this embodiment, even with the removal of mild heat treatment, reliance solely on high-pressure homogenization, and control of the degree of hydrolysis close to the lower limit of the range, BCM-7 was still not detected. Although the bitterness score and functional activity decreased slightly, all indicators still met the basic target range. The results indicate that, with simplified pretreatment conditions and the degree of hydrolysis in the first stage close to the lower limit of the range, the process of this embodiment can still achieve effective control of BCM-7 and a certain level of small molecule peptide yield and functional activity, demonstrating good adaptability to changes in process parameters.

[0206] Comparative Example 1 Step 1: Preparation of substrate solution Weigh 200 g of buffalo casein, add 1.8 L of pure water, stir well, and prepare a protein substrate solution with a mass concentration of 10.0%. Adjust the pH to 8.0 at room temperature using 1 mol / L NaOH.

[0207] Step 2: Enzymatic hydrolysis Adjust the pH of the solution to 8.0 (using 1 mol / L NaOH) and stabilize the temperature at 55.0℃.

[0208] Add 4.40 g of Alcalase to start the reaction.

[0209] Reaction conditions: pH 8.0±0.1 (maintained by automatic addition of 1 mol / L NaOH), temperature 55.0±0.5℃, stirring speed 150 rpm.

[0210] Reaction time: 6.0 h.

[0211] The total degree of hydrolysis was measured to be 32.1% at the end of the reaction (OPA method).

[0212] Step 3: Enzyme inactivation After the reaction was complete, the enzyme hydrolysate was heated to 90°C and kept at that temperature for 10 minutes to inactivate the enzyme. It was then rapidly cooled to room temperature.

[0213] Step 4: Separation and Concentration The enzyme-inactivated hydrolysate was filtered once through an ultrafiltration membrane (6 kDa cutoff), and approximately 1.35 L of permeate was collected.

[0214] The permeate was further concentrated using a nanofiltration membrane (2.5 kDa cutoff) to a solids content of approximately 28%, yielding approximately 300 mL of concentrate.

[0215] Step 5: Post-processing The concentrate was placed in a rotary evaporator and concentrated under vacuum at 40°C and -0.090 MPa to a solid content of 32.0%.

[0216] The concentrate was spray-dried with an inlet air temperature of 168°C, an outlet air temperature of 88°C, an atomization pressure of 0.20 MPa, and a feed rate of 8 mL / min.

[0217] A pale yellow powder product was obtained: approximately 46.1 g.

[0218] Step Six: Data Testing The testing method is the same as in Example 1, and the test data is shown in Table 6 below:

[0219] The data above show that Comparative Example 1, which uses a conventional single enzymatic hydrolysis process, has a significantly higher bitterness, a significantly detected BCM-7 content (4.2 μg / g), a lower proportion of small molecules (78.5%), and a significantly lower functional activity compared to the Example.

[0220] Comparative Example 2 Step 1: Preparation of substrate solution Weigh 200 g of buffalo casein, add 1.8 L of pure water, stir well, and prepare a protein substrate solution with a mass concentration of 10.0%. Adjust the pH to 7.0 at room temperature using 1 mol / L NaOH.

[0221] Step 2: Physical Pretreatment The substrate solution was placed in a high-pressure homogenizer and homogenized twice at a pressure of 55 MPa. After each cycle, the solution was cooled to <40°C.

[0222] The sample was then transferred to a constant-temperature water bath and heated to 72.0°C, held for 12 minutes with gentle stirring (100 rpm). After treatment, the sample was rapidly cooled to 53°C.

[0223] Step 3: First Stage Enzymatic Digestion Adjust the pH of the solution to 7.9 (using 1 mol / L NaOH) and stabilize the temperature at 53.0℃.

[0224] Add 2.00 g of Alcalase and 0.24 g of chymotrypsin sequentially to start the reaction.

[0225] Reaction conditions: pH 7.9±0.1 (maintained by automatic addition of 1 mol / L NaOH), temperature 53.0±0.5℃, stirring speed 150 rpm.

[0226] Reaction time: 2.0 h.

[0227] The degree of hydrolysis was measured to be 16.4% at the end of the reaction (OPA method).

[0228] Step 4: First-stage enzyme inactivation The enzyme hydrolysate was heated to 90.0℃ and kept at that temperature for 10 minutes to inactivate the enzyme at high temperature.

[0229] It is then rapidly cooled to room temperature, with gentle stirring during the process to avoid localized overheating.

[0230] Step 5: Second Stage Enzymatic Digestion The enzyme hydrolysate after enzyme inactivation was reheated to 53.0℃ and the pH was adjusted to 7.2.

[0231] Add 3.20 g of Flavorzyme and 0.80 g of aminopeptidase to initiate the second-stage reaction.

[0232] Reaction conditions: pH naturally drifted to around 7.0, temperature 53.0±0.5℃, stirring speed 150 rpm.

[0233] Reaction time: 4.2 h.

[0234] The final degree of total hydrolysis was 33.9% (OPA method).

[0235] Step Six: Separation and Concentration The second-stage enzymatic hydrolysate was heated to 90°C and kept at that temperature for 5 minutes to inactivate the enzyme.

[0236] After cooling, the solution was filtered once through an ultrafiltration membrane (6 kDa cutoff), and approximately 1.40 L of permeate was collected.

[0237] The permeate was further concentrated using a nanofiltration membrane (2.5 kDa cutoff) to a solids content of approximately 28%, yielding approximately 310 mL of concentrate.

[0238] Step 7: Post-processing The concentrate was placed in a rotary evaporator and concentrated under vacuum at 40°C and -0.090 MPa until the solid content was 32.2%.

[0239] The concentrate was spray-dried with an inlet air temperature of 168°C, an outlet air temperature of 88°C, an atomization pressure of 0.20 MPa, and a feed rate of 8 mL / min.

[0240] A pale yellow powder product was obtained: approximately 47.3 g.

[0241] Step 8: Data Testing The testing method is the same as in Example 1, and the test data is shown in Table 7 below:

[0242] The above data show that although the bitterness of Comparative Example 2, which uses the traditional two-step enzymatic hydrolysis + intermediate high-temperature enzyme inactivation method, is reduced compared to Comparative Example 1, the product still has a noticeable bitterness due to enzyme inactivation, insufficient exonuclease activity in the second stage, and thermal damage to the active ingredients caused by repeated heating. The content of BCM-7 can be detected (1.8 μg / g), and the proportion of small molecules and functional activity are significantly lower than those of the examples in this embodiment.

[0243] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for preparing bioactive polypeptides from buffalo milk, characterized in that, Includes the following steps: S1. Prepare a protein substrate solution with a mass concentration of 8-12% by buffalo casein; S2. The protein substrate solution is subjected to physical pretreatment, the physical pretreatment including high-pressure homogenization and mild heat treatment; S3. The substrate solution after physical pretreatment was subjected to the following enzymatic hydrolysis: a) Use an enzyme combination mainly composed of endonucleases to carry out mild enzymatic hydrolysis under conditions of pH 7.5~8.5 and temperature 45~60℃, and control the degree of hydrolysis to 10%~22%; b) Add flavor protease and aminopeptidase, or a complex enzyme preparation containing flavor protease and aminopeptidase, to the reaction system, and continue enzymatic hydrolysis at pH 6.5-7.8 and temperature 45-60℃ until the total degree of hydrolysis reaches 28%-40%; S4. Use ultrafiltration and nanofiltration membranes to continuously or cyclically separate the enzymatic hydrolysate and collect the permeate with a molecular weight of less than 2000 Da. S5. The permeate is post-processed to obtain buffalo milk active peptides, wherein the post-processing includes concentration and drying.

2. The method for preparing buffalo milk active polypeptides according to claim 1, characterized in that, In step S1, the buffalo casein is obtained from buffalo milk through defatting, precipitation, washing, and neutralization. The precipitation is either acid precipitation or enzyme precipitation. The buffalo casein contains ≥36% β-casein, and A2 type β-casein accounts for ≥85% of the β-casein.

3. The method for preparing buffalo milk active polypeptides according to claim 1, characterized in that, In step S2: The high-pressure homogenization process is performed at a pressure of 40-65 MPa, and the process is repeated 2-4 times. The mild heat treatment specifically includes heating the substrate solution to 68~72°C and holding it at that temperature for 8~15 minutes.

4. The method for preparing buffalo milk active polypeptides according to claim 1, characterized in that, In step S3, the enzyme combination mainly composed of endonuclease includes alkaline protease and chymotrypsin, and the total amount of enzyme added is 0.5% to 1.5% of the substrate protein mass. The activity ratio of alkaline protease to chymotrypsin is (5 to 10):

1.

5. The method for preparing buffalo milk active polypeptides according to claim 1, characterized in that, In step S3, the total amount of flavor protease and aminopeptidase added is 1.0% to 2.5% of the substrate protein mass, and the activity ratio of flavor protease to aminopeptidase is (2 to 6):

1.

6. The method for preparing buffalo milk active polypeptides according to claim 1, characterized in that, In step S4: The ultrafiltration membrane has a molecular weight cutoff of 5-8 kDa, an operating pressure of 0.10-0.35 MPa, and an operating temperature of 48-56°C. The nanofiltration membrane has a molecular weight cutoff of 1.5~3kDa.

7. The method for preparing buffalo milk active polypeptides according to claim 1, characterized in that, Step S5 specifically includes the following sub-steps: S51. The permeate is concentrated under low temperature vacuum, with the concentration temperature controlled at 40~55℃ and the vacuum degree at -0.08~-0.095MPa, until the solid content is 20%~35%, to obtain the concentrated solution. S53. The concentrate is subjected to spray drying or centrifugal atomization drying, wherein the inlet air temperature of the spray drying is 155~175℃, the outlet air temperature is 85~92℃, and the atomization pressure is 0.15~0.25MPa.

8. The method for preparing buffalo milk active polypeptides according to claim 7, characterized in that, Step S51 is followed by the following steps: S52. The concentrated liquid is subjected to a fine debittering treatment, wherein the fine debittering treatment employs macroporous adsorption resin for adsorption-desorption, specifically including: a) Adjust the pH of the concentrate to 6.0~6.8, control the temperature at 38~45℃, add macroporous adsorption resin at a feed:resin volume ratio of 1:(10~18), and adsorb for 1.5~3.5 hours, wherein the macroporous adsorption resin is selected from at least one of XAD-16N, HP-20, DA-201-C, and AB-8. b) After adsorption is complete, wash away unadsorbed impurities with water, and then desorb using 40%~60% ethanol by volume. Collect the desorbed liquid, which is used for drying in step S53.

9. The method for preparing the bioactive polypeptide from buffalo milk according to any one of claims 1 to 8, characterized in that, In step S3, the compound enzyme preparation contains flavor protease, aminopeptidase and carboxypeptidase, wherein the activity of carboxypeptidase accounts for 5% to 30% of the total activity of the compound enzyme preparation.

10. The method for preparing buffalo milk bioactive polypeptides according to claim 9, characterized in that, The complex enzyme preparation is prepared through the following steps: a) Dissolve flavor protease, aminopeptidase and carboxypeptidase in phosphate buffer solution at pH 6.0-7.0 to obtain three enzyme solutions; b) Mix the three enzyme solutions in a ratio of (3~6):(1~3):1 for the activity of flavor protease: aminopeptidase: carboxypeptidase to obtain an enzyme mixture. c) Mix the enzyme mixture with a sodium alginate solution with a mass concentration of 1.5% to 3.0% at a volume ratio of 1:(1 to 3) to obtain a mixture; d) The mixture is added dropwise or squeezed into a calcium chloride solution with a mass concentration of 0.05~0.3 mol / L to form calcium alginate gel beads; e) Immerse the formed calcium alginate gel beads in a chitosan solution with a mass concentration of 0.1%~0.5% and coat them for 0.5~2 hours. The pH of the chitosan solution is adjusted to 5.5~6.

5. f) After coating, wash the gel beads with distilled water or phosphate buffer until neutral, filter or dry at low temperature to obtain the composite enzyme preparation.

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