A method for preparing highly active ACE inhibitory peptides based on a combined microbial fermentation-freeze-drying technique and its application.

Abstract

This application relates to the fields of food biotechnology and functional peptide preparation technology, and in particular to a method for preparing highly active ACE inhibitory peptides based on a combined microbial fermentation-freeze-drying technology and its application. The method involves solid-state fermentation using *Aspergillus oryzae* or *Bacillus subtilis*, or a mixture thereof, combined with a five-stage gradient temperature freeze-drying process. This method utilizes a multi-enzyme system of microorganisms to synergistically hydrolyze protein substrates and protects the peptide structure through low-temperature gradient dehydration. The resulting ACE inhibitory peptide has an IC₅₀ ≤ 0.8 mg / mL, a yield ≥ 18%, and significantly improved thermal and storage stability, making it suitable for the development of blood pressure-lowering functional foods, health foods, and pharmaceuticals.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology and functional peptide preparation technology, specifically a method for preparing highly active ACE inhibitory peptides based on a combined microbial fermentation-freeze-drying technology and its application. Background Technology

[0002] Hypertension is one of the major risk factors for cardiovascular and cerebrovascular diseases. ACE inhibitory peptides are short peptides released from food proteins that effectively lower blood pressure by inhibiting angiotensin-converting enzyme (ACE) activity. They have advantages such as high safety and few toxic side effects, and are widely used in the development of functional foods, health products, and drugs. Currently, the industrial preparation of ACE inhibitory peptides mainly uses direct enzymatic hydrolysis combined with spray drying. Although this method is simple to operate, it has limitations in terms of the limited types of peptides and low activity (IC50). 50 (Mostly in the range of 1.5–2.5 mg / mL), high-temperature drying can easily lead to the inactivation of heat-sensitive peptides, moisture absorption and clumping of products, and poor storage stability.

[0003] In recent years, microbial fermentation has attracted attention due to its ability to produce various endogenous proteases, promote the release of complex peptides, and enhance bioactivity. Some studies have attempted to combine fermentation with subsequent separation and purification processes to improve the yield and activity of ACE inhibitory peptides. However, current technologies still predominantly employ spray drying in the drying stage, failing to effectively address the structural damage of heat-sensitive peptides at high temperatures. While freeze-drying can better preserve the active structure of peptides, its high cost when used alone for drying enzymatic hydrolysis or fermentation products, and its limited effect on further enhancing peptide activity, make an integrated process that synergistically leverages the advantages of microbial fermentation and freeze-drying to achieve the efficient preparation of highly active and stable ACE inhibitory peptides.

[0004] Among the existing patents retrieved, the following two technical solutions are most similar to the present invention, "A method for preparing highly active ACE inhibitory peptides based on a combined microbial fermentation-freeze-drying technology and its application": Patent CN108048520B discloses a method for preparing ACE inhibitory peptides from shellfish meat. This method involves fermenting the shellfish meat with Bacillus subtilis and Aspergillus niger, and using magnetic carbon microspheres to enhance the activity and purity of the peptides. The final product is obtained through purification using a magnetic silica adsorption column. This method introduces a microbial fermentation step, which helps to enrich the types of peptides and improve their activity. However, it does not employ freeze-drying, relying instead on adsorption column purification. This lack of protection measures for heat-sensitive peptides during the drying stage may limit the stability of the obtained product during long-term storage, and the overall process is complex, making it unsuitable for large-scale production.

[0005] Patent CN113832207B discloses a method for preparing ACE-inhibiting peptides from edible fungi. This method involves directly enzymatically hydrolyzing the pulverized fruiting bodies of the edible fungi, or first subjecting them to natural fermentation followed by enzymatic hydrolysis, ultimately obtaining peptide powder containing ACE-inhibiting peptides. This approach combines fermentation and enzymatic hydrolysis, improving raw material utilization and peptide extraction efficiency. However, it does not explicitly state the use of freeze-drying as the drying method, and it fails to synergistically optimize the fermentation-drying process parameters. This makes it difficult to fully utilize the structural preservation advantages of the fermented active peptides under low-temperature drying conditions, limiting the activity level and stability of the final product.

[0006] In summary, although existing technologies have made improvements in raw material selection, enzymatic hydrolysis methods, or the introduction of fermentation, they still suffer from limitations in enzymatic hydrolysis alone, the coupling of microbial fermentation and freeze-drying processes, the protection of the activity of heat-sensitive peptides throughout the process, and the systematic improvement of product stability. A highly efficient, stable, and industrially applicable high-activity ACE inhibitory peptide preparation technology system has not yet been formed. Summary of the Invention

[0007] This invention provides a method for preparing highly active ACE inhibitory peptides based on a combined microbial fermentation-freeze-drying technology and its application. The aim is to achieve full protection and enhanced activity of the thermosensitive ACE inhibitory peptide structure by releasing the synergistic effect of multiple enzyme systems during solid-state fermentation using specific protease-producing strains, combined with a freeze-drying process with a gradient temperature program.

[0008] In a first aspect, the present invention provides a method for preparing highly active ACE inhibitory peptides, comprising the following steps: Step 1: Strain screening and activation. Microbial strains with high protease secretion capacity were selected and cultured on slant agar at 30°C for 48 hours to obtain activated strains. The microbial strains were Aspergillus oryzae CICC 40186 or Bacillus subtilis CICC 10027. Step 2: Inoculate the activated microbial strain at an inoculum rate of 5% to 15% into a solid-state fermentation substrate with a moisture content of 45% to 55%. Perform solid-state fermentation at a temperature of 25°C to 37°C, a pH of 6.0 to 7.2, and a relative humidity of 60% to 80% for 48 to 72 hours. The solid-state fermentation substrate is one or more of defatted soybean meal, whey protein powder, fish protein powder, or wheat gluten powder. Step 3: Add deionized water to the fermented solid material at a material-to-liquid ratio of 1:8 to 1:12. Stir and extract at 45°C to 55°C for 1.5 to 2.5 hours, then centrifuge at 7000 to 9000 rpm for 10 to 20 minutes and collect the supernatant. Ultrafilter the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 3,000 Daltons to obtain an ACE inhibitory peptide extract with a molecular weight of less than 3,000 Daltons. Step 4: Place the ACE inhibitory peptide extract in a freeze dryer and pre-freeze at -45℃ to -40℃ for 3 to 5 hours. Then, dry it under a vacuum of no more than 10 Pascals using a five-stage gradient temperature program: the first stage maintains -40℃ to -35℃ for 4 to 6 hours; the second stage increases the temperature to -30℃ to -25℃ for 4 to 6 hours; the third stage increases the temperature to -20℃ to -15℃ for 4 to 6 hours; the fourth stage increases the temperature to -10℃ to -5℃ for 4 to 6 hours; and the fifth stage increases the temperature to 20℃ to 30℃ for 2 to 4 hours. The total drying time is controlled between 20 and 28 hours to obtain highly active ACE inhibitory peptide powder.

[0009] According to the present invention, during solid-state fermentation, microorganisms not only secrete various endogenous proteases such as alkaline proteases, neutral proteases, and acidic proteases to hydrolyze substrate proteins at multiple sites and along multiple pathways, generating a rich and diverse short peptide spectrum, but also metabolize and produce organic acids, extracellular polysaccharides, and small molecule peptide cofactors. These cofactors coexist with ACE inhibitory peptides in the fermentation system, forming a microenvironment with natural protective effects. In the subsequent freeze-drying process, a gradient temperature program is used to avoid the mechanical damage to the peptide chain conformation caused by ice crystal recrystallization, allowing the peptides to be dehydrated and solidified in a non-thermal manner under low temperature and low pressure conditions, thus fully preserving their spatial structure and biological activity. This process combination produces a synergistic effect that is not simply additive, resulting in a final product that is significantly superior to existing technologies in terms of activity, stability, yield, and peptide composition.

[0010] In some embodiments, in step 1, the slant culture medium is potato dextrose agar or beef extract peptone agar.

[0011] In some embodiments, in step 2, the solid-state fermentation substrate is pulverized through an 80-100 mesh sieve before inoculation and the initial pH is adjusted to 6.5-7.0.

[0012] In some implementations, in step 2, the material is turned over every 12 hours during solid-state fermentation to ensure oxygen supply and temperature uniformity.

[0013] In some embodiments, in step 3, the extraction process uses a constant temperature water bath oscillator with an oscillation frequency of 120 to 180 revolutions per minute.

[0014] In some embodiments, in step 3, the precipitate obtained after centrifugation can be extracted once more, and the supernatants from both extractions can be combined for ultrafiltration.

[0015] In some embodiments, in step 3, the ultrafiltration operation is performed at a pressure of 0.1 MPa to 0.3 MPa and a circulation flow rate of 2 to 5 liters per minute.

[0016] In some embodiments, in step 4, the extract is dispensed into stainless steel trays before freeze-drying, and the liquid layer thickness is controlled between 0.8 cm and 1.2 cm.

[0017] In some implementations, in step 4, the temperature change rate at each stage of the gradient heating is controlled at 1°C to 2°C per hour to avoid sudden temperature changes that could lead to local collapse or structural damage.

[0018] In some embodiments, the moisture content of the highly active ACE inhibitory peptide powder is controlled at 2.5% to 3.5%, and the product is light yellow to light brown, loose and porous, without clumping.

[0019] In some embodiments, the proportion of short peptides with a molecular weight in the range of 500 Daltons to 2000 Daltons in the ACE inhibitory peptide obtained by the method is not less than 85%, and the total content of dipeptides and tripeptides exceeds 60%.

[0020] Secondly, the present invention provides a highly active ACE inhibitory peptide product, prepared by the method described in any embodiment of the first aspect, wherein its ACE inhibitory activity is IC50. 50 The value is not higher than 0.8 mg / mL, the peptide yield is not less than 18% based on protein substrate, and the activity retention rate is not less than 90% after accelerated storage at 40℃ for 30 days.

[0021] Thirdly, the present invention provides the application of the highly active ACE inhibitory peptide in the preparation of blood pressure-lowering functional foods, health foods, or pharmaceuticals.

[0022] In some embodiments, the highly active ACE inhibitory peptide is added as an active ingredient to solid beverages, compressed candies, capsules or protein powder in an amount of 0.1% to 5% (by mass).

[0023] In some embodiments, the highly active ACE inhibitory peptide is used in combination with other antihypertensive ingredients such as γ-aminobutyric acid, tea polyphenols, or potassium salts to enhance the synergistic antihypertensive effect.

[0024] In some embodiments, the stability of the highly active ACE inhibitory peptide in the food matrix is ​​further improved by adding maltodextrin, trehalose, or β-cyclodextrin as a carrier, with the mass ratio of carrier to peptide powder being 1:1 to 4:1.

[0025] In some embodiments, the highly active ACE inhibitory peptide powder is sealed and stored at room temperature and protected from light for 12 months, and the activity retention rate is not less than 85%.

[0026] In some embodiments, the extracellular polysaccharide content produced by the microbial strain during solid-state fermentation is 0.8% to 1.5% (on a dry basis). This polysaccharide forms a hydrogen bond network with the peptide, which helps maintain the secondary structure of the peptide during freeze-drying.

[0027] In some embodiments, the solid-state fermentation substrate contains a protein content of not less than 45% (on a dry basis), an ash content of not more than 8%, and a fat residue of not more than 2%.

[0028] In some embodiments, the retentate obtained from ultrafiltration (the portion with a molecular weight greater than 3,000 Daltons) can be reused in the next batch of solid-state fermentation substrate as a nitrogen source supplement, thereby improving the utilization rate of raw materials.

[0029] In some embodiments, the cold trap temperature is maintained at -50°C to -45°C during the freeze-drying process to ensure efficient water vapor capture and prevent moisture reabsorption.

[0030] In some embodiments, the method does not require the addition of any exogenous proteases, and relies entirely on the enzyme system secreted by the microorganisms themselves to complete the protein hydrolysis, thereby reducing production costs and avoiding the risk of exogenous enzyme residues.

[0031] In some implementations, the fifth stage of the gradient heating process is set to an end temperature of 25°C, so that the product reaches room temperature before leaving the warehouse, thus avoiding moisture absorption.

[0032] In some embodiments, the proportion of hydrophobic amino acids (such as alanine, valine, leucine, isoleucine, phenylalanine, and proline) in the amino acid composition of the highly active ACE inhibitory peptide is not less than 40%, and this compositional characteristic is closely related to its high ACE inhibitory activity.

[0033] In some embodiments, the method is applicable to a variety of plant or animal-derived protein substrates, including but not limited to soy protein isolate, whey protein concentrate, collagen hydrolysate, and zein, and has broad raw material adaptability.

[0034] In some embodiments, during the solid-state fermentation process, the degree of protein hydrolysis reaches 15% to 20% at 48 hours, 22% to 28% at 60 hours, and tends to stabilize at 72 hours, indicating that the synergistic effect of the multi-enzyme system is time-dependent.

[0035] In some embodiments, the freeze-dried product has a specific surface area of ​​30 to 50 square meters per gram and a porosity of 60% to 75%, which is beneficial for rapid dissolution and dispersion in subsequent food systems.

[0036] In some implementations, the entire process of the method can be completed in a closed bioreactor system, reducing the risk of contamination by other microorganisms and complying with GMP production standards.

[0037] In some embodiments, the highly active ACE inhibitory peptide retains no less than 80% of its ACE inhibitory activity after being digested in vitro using simulated gastrointestinal fluid, indicating that it has good digestive stability.

[0038] In some embodiments, the organic acids produced during the microbial fermentation process mainly include lactic acid, acetic acid and citric acid, with a total acidity of 0.3% to 0.7% (calculated as lactic acid). This weakly acidic environment helps to inhibit the growth of other microorganisms and promote the release of specific peptides.

[0039] In some embodiments, the product obtained by the method has no Maillard reaction products detected, a color difference value L* greater than 85, a* less than 2, b* less than 10, a pure white color, and excellent sensory quality.

[0040] In some implementations, the energy consumption of the freeze-drying process is reduced by 15% to 25% compared to traditional constant-temperature freeze-drying by optimizing the gradient heating program and vacuum control strategy, thereby improving the economic efficiency of the process.

[0041] In some embodiments, the particle size distribution D50 of the highly active ACE inhibitory peptide powder is 30 to 60 micrometers, exhibiting good flowability and suitability for automated filling.

[0042] In some implementations, the method can be combined with online near-infrared monitoring technology to monitor the degree of protein hydrolysis in the fermentation process in real time, thereby achieving intelligent process control.

[0043] In summary, this invention, through the coupling of microbial solid-state fermentation and gradient temperature freeze-drying processes, not only solves the problems of single peptide fragments, low activity, thermal inactivation, and poor stability in the traditional enzymatic hydrolysis-spray drying method, but also achieves a comprehensive breakthrough in the activity, yield, composition, and storage performance of ACE inhibitory peptides through the synergistic effect of endogenous cofactors and low-temperature structural protection. This provides a brand-new technical path for the green, efficient, and large-scale preparation of highly active bioactive peptides.

[0044] Beneficial effects of the present invention Compared with the closest existing technology, the unexpected technical effects of this invention are mainly reflected in: 1. Significantly enhanced activity: The obtained ACE inhibitory peptide IC 50 ≤0.8 mg / mL, a reduction of 40-50% compared to traditional methods (usually 1.5-2.5 mg / mL); 2. Excellent stability: Accelerated storage test (40℃, 30 days) shows an activity retention rate of ≥90%, which is much higher than that of spray-dried products (usually ≤70%). 3. High yield: Peptide yield (based on protein substrate) ≥18%, higher than traditional methods (approximately 12-15%). 4. Optimized peptide composition: The proportion of short peptides with a molecular weight of 500-2000 Da is high, of which dipeptides and tripeptides account for more than 60%, making them easier to absorb and more active. 5. Green and economical process: Fermentation does not require the addition of exogenous enzymes, and the energy consumption of freeze drying can be controlled through optimized procedures, making the overall cost controllable. Detailed Implementation

[0045] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0046] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0047] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0048] As described in the background section above, the preparation of ACE inhibitory peptides currently relies heavily on exogenous protease hydrolysis combined with spray drying, which suffers from problems such as limited peptide composition, significant loss of thermosensitive activity, poor product stability, and high production costs. To address these issues, this invention couples microbial solid-state fermentation with gradient-temperature freeze-drying to construct an endogenous multi-enzyme synergistic hydrolysis system. This system achieves non-thermal dehydration and solidification of peptides under low-temperature and low-pressure conditions, thereby obtaining ACE inhibitory peptide products with high activity, high stability, and high yield.

[0049] In a first aspect, the present invention provides a method for preparing highly active ACE inhibitory peptides based on a combined microbial fermentation-freeze-drying technology, comprising the following steps: Step 1: Strain screening and activation. Microbial strains with high protease secretion capacity were selected and cultured on slant agar at 30°C for 48 hours to obtain activated strains. The microbial strains were Aspergillus oryzae CICC 40186 or Bacillus subtilis CICC 10027. Step 2: Inoculate the activated microbial strain at an inoculum rate of 5% to 15% into a solid-state fermentation substrate with a moisture content of 45% to 55%. Perform solid-state fermentation at a temperature of 25°C to 37°C, a pH of 6.0 to 7.2, and a relative humidity of 60% to 80% for 48 to 72 hours. The solid-state fermentation substrate is one or more of defatted soybean meal, whey protein powder, fish protein powder, or wheat gluten powder. Step 3: Add deionized water to the fermented solid material at a material-to-liquid ratio of 1:8 to 1:12. Stir and extract at 45°C to 55°C for 1.5 to 2.5 hours, then centrifuge at 7000 to 9000 rpm for 10 to 20 minutes and collect the supernatant. Ultrafilter the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 3,000 Daltons to obtain an ACE inhibitory peptide extract with a molecular weight of less than 3,000 Daltons. Step 4: Place the ACE inhibitory peptide extract in a freeze dryer and pre-freeze at -45℃ to -40℃ for 3 to 5 hours. Then, dry it under a vacuum of no more than 10 Pascals using a five-stage gradient temperature program: the first stage maintains -40℃ to -35℃ for 4 to 6 hours; the second stage increases the temperature to -30℃ to -25℃ for 4 to 6 hours; the third stage increases the temperature to -20℃ to -15℃ for 4 to 6 hours; the fourth stage increases the temperature to -10℃ to -5℃ for 4 to 6 hours; and the fifth stage increases the temperature to 20℃ to 30℃ for 2 to 4 hours. The total drying time is controlled between 20 and 28 hours to obtain highly active ACE inhibitory peptide powder.

[0050] According to the present invention, during solid-state fermentation, microorganisms not only secrete various endogenous proteases such as alkaline proteases, neutral proteases, and acidic proteases to hydrolyze substrate proteins at multiple sites and along multiple pathways, generating a rich and diverse short peptide spectrum, but also metabolize and produce organic acids, extracellular polysaccharides, and small molecule peptide cofactors. These cofactors coexist with ACE inhibitory peptides in the fermentation system, forming a microenvironment with natural protective effects. In the subsequent freeze-drying process, a gradient temperature program is used to avoid the mechanical damage to the peptide chain conformation caused by ice crystal recrystallization, allowing the peptides to be dehydrated and solidified in a non-thermal manner under low temperature and low pressure conditions, thus fully preserving their spatial structure and biological activity. This process combination produces a synergistic effect that is not simply additive, resulting in a final product that is significantly superior to existing technologies in terms of activity, stability, yield, and peptide composition.

[0051] In some embodiments, in step 1, the slant culture medium is potato dextrose agar medium or beef extract peptone agar medium. Specifically, the potato dextrose agar medium is prepared by adding 200g of potato juice, 20g of glucose, and 20g of agar to water to a final volume of 1000ml; the beef extract peptone agar medium is prepared by adding 5g of beef extract, 10g of peptone, 5g of sodium chloride, and 20g of agar to water to a final volume of 1000ml, with the pH adjusted to 7.0.

[0052] In some embodiments, in step 2, the solid-state fermentation substrate is pulverized through an 80-100 mesh sieve before inoculation, and the initial pH is adjusted to 6.5-7.0. pH adjustment can be performed using a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrochloric acid solution.

[0053] In some embodiments, during step 2, the material is turned over every 12 hours during solid-state fermentation to ensure oxygen supply and temperature uniformity. The turning operation can be performed on a clean workbench using a sterile stainless steel shovel, and each turning session should be limited to no more than 3 minutes to avoid contamination by other microorganisms.

[0054] In some embodiments, in step 3, the extraction process uses a constant temperature water bath oscillator with an oscillation frequency of 120 to 180 revolutions per minute.

[0055] In some embodiments, in step 3, the precipitate obtained after centrifugation can be extracted once more, and the supernatants from both extractions are combined for ultrafiltration. The conditions for the second extraction are the same as those for the first.

[0056] In some embodiments, in step 3, the ultrafiltration operation is performed at a pressure of 0.1 MPa to 0.3 MPa, with a circulation flow rate of 2 to 5 liters per minute. The ultrafiltration equipment may employ hollow fiber ultrafiltration membrane modules made of polyethersulfone with a molecular weight cutoff of 3 kilodaltons and an effective membrane area of ​​0.5 square meters.

[0057] In some embodiments, in step 4, the extract is dispensed into stainless steel trays before freeze-drying, with the liquid layer thickness controlled between 0.8 cm and 1.2 cm. The stainless steel trays are 300 mm × 200 mm × 30 mm in size and made of 304 stainless steel.

[0058] In some embodiments, in step 4, the temperature change rate at each stage of the gradient heating is controlled at 1°C to 2°C per hour to avoid sudden temperature changes that could lead to local collapse or structural damage. This heating rate is controlled by a built-in program in the freeze dryer.

[0059] In some embodiments, the moisture content of the highly active ACE inhibitory peptide powder is controlled between 2.5% and 3.5%, and the product is a light yellow to light brown loose porous product without clumping. The moisture content is determined using a halogen moisture analyzer.

[0060] In some embodiments, the proportion of short peptides with a molecular weight in the range of 500 Daltons to 2000 Daltons in the ACE-inhibiting peptides obtained by the method is not less than 85%, of which the total content of dipeptides and tripeptides exceeds 60%. The molecular weight distribution is determined by high performance liquid chromatography-mass spectrometry (HPLC-MS) using a ZORBAX 300SB-C18 column and a gradient elution of 0.1% formic acid aqueous solution and acetonitrile.

[0061] Secondly, the present invention provides a highly active ACE inhibitory peptide product, prepared by the method described in any embodiment of the first aspect, wherein its ACE inhibitory activity is IC50. 50 The value is not higher than 0.8 mg / mL, the peptide yield is not less than 18% based on protein substrate, and the activity retention rate is not less than 90% after accelerated storage at 40℃ for 30 days.

[0062] Thirdly, the present invention provides the application of the highly active ACE inhibitory peptide in the preparation of blood pressure-lowering functional foods, health foods, or pharmaceuticals.

[0063] In some embodiments, the highly active ACE inhibitory peptide is added as an active ingredient to solid beverages, compressed candies, capsules or protein powder in an amount of 0.1% to 5% (by mass).

[0064] In some embodiments, the highly active ACE inhibitory peptide is used in combination with other antihypertensive ingredients such as γ-aminobutyric acid, tea polyphenols, or potassium salts to enhance the synergistic antihypertensive effect.

[0065] In some embodiments, the stability of the highly active ACE inhibitory peptide in the food matrix is ​​further improved by adding maltodextrin, trehalose, or β-cyclodextrin as a carrier, with the mass ratio of carrier to peptide powder being 1:1 to 4:1.

[0066] In some embodiments, the highly active ACE inhibitory peptide powder is sealed and stored at room temperature and protected from light for 12 months, and the activity retention rate is not less than 85%.

[0067] In some embodiments, the extracellular polysaccharide content produced by the microbial strain during solid-state fermentation is 0.8% to 1.5% (on a dry basis). This polysaccharide forms a hydrogen bond network with the peptides, which helps maintain the secondary structure of the peptides during freeze-drying. The extracellular polysaccharide content is determined using the phenol-sulfuric acid method.

[0068] In some embodiments, the solid-state fermentation substrate contains a protein content of not less than 45% (on a dry basis), an ash content of not more than 8%, and a fat residue of not more than 2%. The protein content is determined by the Kjeldahl method, the ash content is determined by the muffle furnace ignition method at 550°C, and the fat residue is determined by the Soxhlet extraction method.

[0069] In some embodiments, the retentate obtained from ultrafiltration (the portion with a molecular weight greater than 3,000 Daltons) can be reused in the next batch of solid-state fermentation substrate as a nitrogen source supplement, thereby improving the utilization rate of raw materials.

[0070] In some embodiments, the cold trap temperature is maintained at -50°C to -45°C during the freeze-drying process to ensure efficient water vapor capture and prevent moisture reabsorption.

[0071] In some embodiments, the method does not require the addition of any exogenous proteases, and relies entirely on the enzyme system secreted by the microorganisms themselves to complete the protein hydrolysis, thereby reducing production costs and avoiding the risk of exogenous enzyme residues.

[0072] In some implementations, the fifth stage of the gradient heating process is set to an end temperature of 25°C, so that the product reaches room temperature before leaving the warehouse, thus avoiding moisture absorption.

[0073] In some embodiments, the amino acid composition of the highly active ACE inhibitory peptide contains at least 40% hydrophobic amino acids (such as alanine, valine, leucine, isoleucine, phenylalanine, and proline), a compositional characteristic closely related to its high ACE inhibitory activity. The amino acid composition is determined using an automated amino acid analyzer.

[0074] In some embodiments, the method is applicable to a variety of plant or animal-derived protein substrates, including but not limited to soy protein isolate, whey protein concentrate, collagen hydrolysate, and zein, and has broad raw material adaptability.

[0075] In some embodiments, during the solid-state fermentation process, the degree of protein hydrolysis reaches 15% to 20% after 48 hours, 22% to 28% after 60 hours, and tends to stabilize after 72 hours, indicating that the synergistic effect of the multi-enzyme system is time-dependent. The degree of protein hydrolysis is determined by formaldehyde titration.

[0076] In some embodiments, the freeze-dried product has a specific surface area of ​​30 to 50 square meters per gram and a porosity of 60% to 75%, which is beneficial for rapid dissolution and dispersion in food systems. The specific surface area is determined using the BET nitrogen adsorption method, and the porosity is determined using the mercury porosimetry method.

[0077] In some embodiments, the entire process of the method can be completed in a closed bioreactor system, reducing the risk of contamination by other microorganisms and complying with GMP production standards. The closed bioreactor system includes a solid-state fermenter, an extraction tank, an ultrafiltration unit, and a freeze dryer; each unit is connected by pipelines to achieve closed-loop material transport.

[0078] In some embodiments, after digestion with simulated gastrointestinal fluid in vitro, the ACE inhibitory activity retention rate of the highly active ACE inhibitory peptide is not less than 80%, indicating that it has good digestive stability. Simulated gastrointestinal fluid digestion is performed in accordance with GB / T 22250-2008 standard.

[0079] In some embodiments, the organic acids produced during the microbial fermentation process mainly include lactic acid, acetic acid, and citric acid, with a total acidity of 0.3% to 0.7% (calculated as lactic acid). This weakly acidic environment helps inhibit the growth of other microorganisms and promotes the release of specific peptides. The types and contents of organic acids are determined by high-performance liquid chromatography (HPLC) using an Aminex HPX-87H column and a 5 mmol / L sulfuric acid solution as the mobile phase.

[0080] In some embodiments, the product obtained by the method shows no Maillard reaction products, a color difference value L* greater than 85, a* less than 2, b* less than 10, a pure white color, and excellent sensory quality. The color difference value is measured using a colorimeter.

[0081] In some implementations, the freeze-drying energy consumption is reduced by 15% to 25% compared to traditional isothermal freeze-drying by optimizing the gradient heating program and vacuum control strategy, thus improving the process economy. Energy consumption data is recorded through an electricity meter.

[0082] In some embodiments, the highly active ACE inhibitory peptide powder has a particle size distribution D50 of 30 to 60 micrometers, exhibiting good flowability and suitability for automated filling. The particle size distribution is determined using a laser particle size analyzer.

[0083] In some embodiments, the method can be combined with online near-infrared monitoring technology to monitor the degree of protein hydrolysis during fermentation in real time, thereby achieving intelligent process control. The near-infrared probe is installed on the side wall of the solid-state fermenter, and the data is collected and fed back through a PLC system to adjust fermentation parameters.

[0084] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially. Example

[0085] Step 1: Take Aspergillus oryzae CICC 40186 strain and incubate it on potato dextrose agar slant at 30°C for 48 hours to obtain activated strain; Step 2: Grind the defatted soybean meal through an 80-mesh sieve, adjust the initial pH value to 6.8, add water to adjust the moisture content to 50%, put it into a solid fermentation tank, inoculate with activated bacteria at a 10% inoculation rate, and ferment for 60 hours at 32℃ and 70% relative humidity, turning the material every 12 hours during the fermentation. Step 3: Add deionized water to the fermented material at a material-to-liquid ratio of 1:10. Extract by shaking at 50°C and 150 rpm for 2 hours, then centrifuge at 8000 rpm for 15 minutes and collect the supernatant. Repeat the extraction once with the precipitate and combine the supernatants. Pass the combined liquid through a 3,000 Dalton ultrafiltration membrane at a pressure of 0.2 MPa and a circulation flow rate of 3 liters per minute, and collect the permeate. Step 4: Dispense the permeate into a stainless steel tray with a liquid layer thickness of 1.0 cm, place it in a freeze dryer, and pre-freeze at -45°C for 4 hours; then dry it under a vacuum of 8 Pascals according to a five-stage gradient temperature program: the first stage is drying at -40°C for 5 hours, the second stage is drying at -30°C for 5 hours, the third stage is drying at -20°C for 5 hours, the fourth stage is drying at -10°C for 5 hours, and the fifth stage is drying at 25°C for 4 hours, for a total drying time of 24 hours, to obtain ACE inhibitory peptide powder.

[0086] Example 2: Bacillus subtilis CICC 10027 was used to replace Aspergillus oryzae CICC 40186, and the rest was the same as in Example 1.

[0087] Comparative Example 1: Except for the following two points, the other conditions are the same as in Example 1: (1) Instead of microbial fermentation, alkaline protease (enzyme-to-base ratio 2%) was directly added and enzymatically hydrolyzed at 50℃ for 6 h; (2) The drying method was changed to spray drying (inlet air temperature 180℃, outlet air temperature 85℃).

[0088] Comparative Example 2: The fermentation steps were the same as in Example 1, but the drying was carried out under the same spray drying conditions as in Comparative Example 1.

[0089] Comparative Example 3: The enzymatic hydrolysis steps were the same as in Comparative Example 1, but the drying was performed under the same freeze-drying conditions as in Example 1. Comparative Example 4: Lactobacillus helveticus CICC 6064 was used instead of the strain used in this application, and the rest was the same as in Example 1.

[0090] The key indicators of the polypeptide products obtained in the above examples and comparative examples were measured respectively, and the results are shown in Table 1.

[0091] Table 1 Comparison of Technical Effects As shown in the table above, the present invention (Examples 1 and 2) is significantly superior to the respective comparative examples in terms of ACE inhibitory activity, yield, stability, and short peptide content. It is particularly noteworthy that: Comparative Example 1 (Conventional Method), the present invention IC 50 The yield was reduced by approximately 60%, the activity retention rate was increased by approximately 40%, and the short peptide content was increased by approximately 27%, achieving a comprehensive improvement. In Comparative Examples 2 and 3, the effects of this invention are superior to single-technology improvements (modifying fermentation only or drying only), demonstrating a synergistic effect between microbial fermentation and freeze-drying, an effect that cannot be expected by simple additive methods. Comparative Example 4 used *Lactobacillus helveticus* CICC 6064 instead of the strain used in this application, resulting in a significant reduction in the technical effectiveness.

[0092] The synergistic effect may stem from the fact that the fermentation produces a richer polypeptide spectrum, accompanied by natural protective components (such as microbial polysaccharides), which are then freeze-dried to solidify without damage, together forming a highly active and stable microenvironment.

[0093] As shown in the table above, the product obtained in the embodiments of the present invention is significantly superior to the comparative example in terms of peptide yield, ACE inhibitory activity, low molecular weight peptide content, and storage stability. The present invention effectively solves the above-mentioned problems through a combination of synergistic hydrolysis by microbial multi-enzyme systems and gradient temperature freeze-drying.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing highly active ACE inhibitory peptides based on a combined microbial fermentation-freeze-drying technology, characterized in that, Includes the following steps: Step 1 is strain screening and activation; Step 2 is solid-state fermentation; Step 3 is extraction and separation; Step 4 is freeze drying.

2. The method according to claim 1, characterized in that, Step 1: Strain screening and activation. Microbial strains with high protease secretion capacity are selected and cultured on slant agar at 30°C for 48 hours to obtain activated strains. The microbial strains are *Aspergillus oryzae* CICC 40186 or *Bacillus subtilis* CICC. 10027 or its mixed strains; Step 2: Inoculate the activated strain at an inoculum of 5% to 15% into a solid fermentation substrate with a moisture content of 45% to 55%, and carry out solid fermentation at a temperature of 25℃ to 37℃, a pH of 6.0 to 7.2, and a relative humidity of 60% to 80% for 48 to 72 hours; the solid fermentation substrate is one or more of defatted soybean meal, whey protein powder, fish protein powder, or wheat gluten powder; Step 3: Add deionized water to the fermented solid material at a material-to-liquid ratio of 1:8 to 1:12, stir and extract at 45℃ to 55℃ for 1.5 to 2.5 hours, then centrifuge at 7000 rpm to 9000 rpm for 10 to 20 minutes, and collect the supernatant; pass the supernatant through a molecular weight cutoff of 3... Ultrafiltration is performed using a 1,000 Dalton ultrafiltration membrane to obtain an ACE inhibitory peptide extract with a molecular weight of less than 3,000 Daltons; Step 4: The ACE inhibitory peptide extract is placed in a freeze dryer and pre-frozen at -45°C to -40°C for 3 to 5 hours, and then dried under a vacuum of no more than 10 Pascals using a five-stage gradient temperature program: the first stage maintains -40°C to -35°C for 4 to 6 hours, the second stage increases the temperature to -30°C to -25°C for 4 to 6 hours, the third stage increases the temperature to -20°C to -15°C for 4 to 6 hours, the fourth stage increases the temperature to -10°C to -5°C for 4 to 6 hours, and the fifth stage increases the temperature to 20°C to 30°C for 2 to 4 hours. The total drying time is controlled between 20 and 28 hours to obtain highly active ACE inhibitory peptide powder.

3. The method according to claim 1, characterized in that, In step 1, the slant culture medium is potato dextrose agar medium or beef extract peptone agar medium.

4. The method according to claim 1, characterized in that, In step 2, the solid-state fermentation substrate is pulverized through an 80-100 mesh sieve before inoculation and the initial pH value is adjusted to 6.5-7.

0. During the solid-state fermentation process, the substrate is turned over once every 12 hours.

5. The method according to claim 1, characterized in that, In step 3, the extraction process uses a constant temperature water bath oscillator with an oscillation frequency of 120 to 180 revolutions per minute.

6. The method according to claim 1, characterized in that, In step 3, the precipitate obtained after centrifugation is extracted once more, and the supernatants from both extractions are combined for ultrafiltration.

7. The method according to claim 1, characterized in that, In step 3, the ultrafiltration operation is carried out at a pressure of 0.1 MPa to 0.3 MPa and a circulation flow rate of 2 to 5 liters per minute.

8. The method according to claim 1, characterized in that, In step 4, before freeze-drying, the extract is dispensed into stainless steel trays, and the liquid layer thickness is controlled between 0.8 cm and 1.2 cm.

9. The method according to claim 1, characterized in that, In step 4, the temperature change rate at each stage of the gradient heating is controlled at 1°C to 2°C per hour.

10. A highly active ACE inhibitory peptide product, characterized in that, Prepared by the method according to any one of claims 1 to 9.