A preparation method of a protease hydrolyzed polypeptide hollow salt with regular shape and improved salt reduction effect

Abstract

This invention discloses a method for preparing hollow salts from proteolytic polypeptides with regular shapes and improved salt reduction effects, relating to the field of condiment technology. The method includes: thermally reacting clam meat proteolytic solution with acetylglucosamine at 90-100℃ for 3-4 hours; ultrafiltration to remove glycopeptide components with a molecular weight ≤1KD; concentrating to a solid content of 60%-80%; and then mixing with saturated brine and spray drying to obtain hollow salts. This invention utilizes Maillard reaction products as shell-forming agents, increasing the hollow volume of the prepared hollow salt microspheres by more than 50% and improving cavitation rate. The salt reduction effect reaches over 40%, with a smooth and regular surface, low bulk density, and fast dissolution rate, making it widely applicable in the condiment and food industries.

Description

Technical Field

[0001] This invention relates to the field of condiment technology, specifically to a method for preparing proteolytic polypeptide hollow salts with regular shapes and improved salt reduction effects. Background Technology

[0002] Excessive sodium intake is a major contributing factor to significant public health crises such as hypertension and cardiovascular disease. The World Health Organization (WHO) recommends that adults consume less than 2 grams of sodium per day (equivalent to 5 grams of salt), but the actual intake of most people worldwide far exceeds this standard. Therefore, the food industry urgently needs to develop innovative technologies that combine efficient sodium reduction with good flavor preservation.

[0003] Currently, hollow salt (also known as hollow core salt) has received widespread attention as an effective salt reduction strategy. By constructing an internal cavity structure, hollow salt reduces the actual salt content while maintaining the particle size. It also enhances the perceived saltiness by increasing the dissolution rate due to the increased specific surface area, thus achieving the effect of reducing salt without reducing flavor.

[0004] In existing technologies, protease-hydrolyzed peptides, used as natural flavoring agents, are mixed with saturated brine and spray-dried to obtain hollow salt powder with a salty and savory flavor and a hollow structure, increasing the cavitation volume by about 30%. However, the cavitation rate is not high, achieving a salt reduction effect of approximately 30%. Nevertheless, untreated protease-hydrolyzed peptides, as auxiliary agents for hollow salt formation, have significant drawbacks: the resulting hollow salt powder particles have rough surfaces, uneven sizes, low hollowness, poor dispersibility, and the salt reduction effect still needs improvement.

[0005] The Maillard reaction is an important and widespread reaction in the food industry, a non-enzymatic browning reaction between the carbonyl group of reducing sugars and the amino group of amino acids or proteins. Maillard reaction products (such as melanoidins) possess good surface activity, film-forming properties, and antioxidant effects. However, no reported technical solutions have been developed for applying Maillard reaction products to the preparation of hollow salts, particularly for improving the surface structure of hollow salts and enhancing salt reduction effects.

[0006] Therefore, how to further improve the surface structure of hollow salt assisted by proteolytic peptides, increase the cavitation rate of hollow salt powder, and enhance the salt reduction effect is a technical problem that urgently needs to be solved in the field of peptide-based hollow salt preparation. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention provides a hollow salt with regular shape, smooth surface, and significant salt reduction effect, and a method for preparing the same. Under a scanning electron microscope (SEM), the hollow salt exhibits a regular spherical structure, low packing density, high cavitation volume, high hollowness, and a stable salt reduction effect of over 40%.

[0008] This invention involves a thermal reaction (Maillard reaction) between clam meat protease hydrolysate and acetylglucosamine to generate a glycopeptide complex with excellent surface activity and shell-forming properties. This glycopeptide complex, used as a shell-forming agent in the preparation of hollow salts, significantly improves the surface structure of the hollow salts, increases cavitation volume, sphericity, and hollowness, thereby substantially enhancing the salt reduction effect. This technical effect is something that those skilled in the art could not easily foresee based on existing technologies.

[0009] This invention provides a method for preparing proteolytically hydrolyzed polypeptide hollow salts with regular shape and improved salt reduction effect, comprising the following steps: (1) Thermal reaction of clam meat hydrolysate: take clam meat homogenate for enzymatic hydrolysis, add 5%~8% of acetylglucosamine by mass of hydrolysate, and carry out thermal reaction at 90~100℃ for 3~4h to obtain glycopeptide thermal reaction solution. (2) Ultrafiltration separation: The glycopeptide thermal reaction solution obtained in step (1) is subjected to ultrafiltration to extract glycopeptide components with a molecular weight ≤1KD; (3) Concentration: The glycopeptide component obtained in step (2) is concentrated by rotary evaporation to achieve a solid content of 60% to 80%; (4) Spray drying: The concentrated glycopeptide solution from step (3) is mixed with saturated saline solution and then spray dried to obtain hollow salt; wherein, the amount of glycopeptide solution added is 5%~10% of the mass of saturated saline solution, and the spray drying conditions are: air inlet temperature 120~150℃, air outlet temperature 80~100℃, and peristaltic pump speed 15~30mL / min.

[0010] In one embodiment of the present invention, in step (1), the enzymes used for enzymatic hydrolysis are neutral protease and flavor protease. Enzymatic hydrolysis is carried out at 45~55℃. After 1~2 hours of enzymatic hydrolysis with neutral protease, flavor protease is added at 45~55℃ for another 1~2 hours of enzymatic hydrolysis. The amount of neutral protease and flavor protease added is 1~2% of the mass of clam meat.

[0011] In one embodiment of the present invention, in step (1), the temperature of the thermal reaction is 95~100℃ and the reaction time is 3.5~4h.

[0012] In one embodiment of the present invention, in step (1), the amount of acetylglucosamine added is 6% to 7% of the mass of the enzymatic hydrolysate.

[0013] In one embodiment of the present invention, in step (2), the glycopeptide component contains Maillard reaction products, the Maillard reaction products comprising glycopeptide compounds of acetylglucosamine and enzymatically hydrolyzed peptides, and the partial amino acid sequence of the glycopeptide compound being one or more of TPIPDS(GlcNAc)S, ET(GlcNAc)ITPIP, S(GlcNAc)GGP, and TT(GlcNAc)EAP.

[0014] In one embodiment of the present invention, in step (3), the solid content after rotary evaporation concentration is 65%~75%.

[0015] In one embodiment of the present invention, in step (4), the spray drying conditions are: inlet temperature 130~140℃, outlet temperature 85~95℃, and peristaltic pump rate 20~25mL / min.

[0016] The present invention also provides hollow salt microspheres prepared by the above method, wherein the hollow salt microspheres have a hollowness of more than 90%, a salt reduction effect of more than 40%, and are shown by SEM to be smooth and regular spherical with a contact angle of less than 30°.

[0017] In one embodiment of the present invention, the bulk density of the hollow salt microspheres is 0.3~0.5 g / mL, and the dissolution time is less than 30 seconds.

[0018] The present invention also provides the application of the above-mentioned hollow salt microspheres in the preparation of seasonings or food.

[0019] Technical principle: The glycopeptide complex prepared by the Maillard reaction in this invention is used as a shell-forming agent, and its mechanism of action is as follows: 1. Improved surface activity: The glycopeptides in the Maillard reaction products have an amphiphilic structure, which can significantly reduce the surface tension of the reaction solution, making it easier for the solution to form a thin film and encapsulate the salt crystal nuclei during spraying.

[0020] 2. Improved shell-forming performance: The glycosyl groups and peptide chains in the glycopeptide complex form a stable network structure, which is rapidly solidified during spray drying to form a strong and smooth shell, effectively maintaining the integrity of the hollow structure.

[0021] 3. Hollow structure optimization: The thermodynamic properties of the glycopeptide complex enable it to form more uniform vapor escape channels during the drying process, thereby constructing hollow microspheres with regular shapes and smooth surfaces.

[0022] 4. Enhanced salt reduction effect: The regular and smooth hollow structure has a larger specific surface area, which can dissolve in saliva more quickly and rapidly activate the taste buds' salty perception, thus achieving the same saltiness perception at a lower sodium content.

[0023] Compared with the prior art, the present invention has the following significant advantages: 1. Significantly increased cavitation volume: The cavitation volume is increased, and the cavitation rate is significantly improved.

[0024] 2. Significant salt reduction effect: The salt reduction effect is consistently above 40%, a significant improvement over existing technologies (approximately 30%). It is also applicable to salt reduction in liquid conditions.

[0025] 3. Improved surface structure: SEM observation shows that the hollow salt is a smooth and regular sphere with significantly reduced surface roughness.

[0026] 4. Faster dissolution rate: Dissolution time is shortened to about 25 seconds, which is faster than existing technologies.

[0027] 5. Enhanced hydrophilicity: The contact angle is less than 30°, indicating that the glycopeptide complex significantly improves the surface wettability of the hollow salt.

[0028] 6. Reduced bulk density: The bulk density is reduced to 0.3~0.5 g / mL, which is beneficial for uniform dispersion in food. Attached Figure Description

[0029] Figure 1 The mass spectrum of LC-MS / MS for Example 1; Figure 2 Comparison of SEM images of Examples 1-5 and Comparative Examples 1-4; Figure 3 This is a comparison of cavitation volume and bulk density in Examples 1-5 and Comparative Examples 1-4; Figure 4 This is a comparison of the solubility of Examples 1-5 and Comparative Examples 1-4; Figure 5 This is a comparison of the sensory salt reduction effects of Examples 1-5 and Comparative Examples 1-4; Figure 6 The contact angles of Examples 1-5 and Comparative Examples 2-4 are compared. Detailed Implementation

[0030] Raw material source: Clams: Purchased from the local market; Acetaminoglycine: Purchased from Nanjing Xingyunlai Trading Co., Ltd.; Salt: Provided by Dalian Salt Chemical Group; The neutral protease and flavor protease were both from Yizhu Biotechnology Co., Ltd. The neutral protease activity was 10,000 u / g, and the flavor protease activity was 8,000 u / g.

[0031] All other reagents were commercially available analytical grade.

[0032] Measurement method: 1. Mass Spectrometry Determination and Screening: To identify the structure of glycosylated peptides with salt-enhancing activity in Maillard reaction products, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to analyze the active components.

[0033] (1) Sample pretreatment The fraction with the best salinizing activity after ultrafiltration fractionation (500-1000 Da) was taken, freeze-dried, and 20 mg of the sample was dissolved in 200 μL of 50 mM NH4HCO3. The sample was transferred to a 10 kDa ultrafiltration tube, centrifuged at 12,000 rpm for 10 min, and the filtrate (<10 kDa fraction) was collected. 200 μL of water was added and the mixture was centrifuged and washed twice. The protein solution was taken, DTT was added to a final concentration of 10 mM, and the mixture was reduced in a water bath at 56 °C for 1 h; then IAM was added to a final concentration of 20 mM, and the mixture was reacted in the dark at room temperature for 40 min; finally, DTT was added to neutralize the unreacted IAM. The reaction solution was desalted using the C18 Stage-Tip: it was activated with 100% acetonitrile, equilibrated with 0.1% trifluoroacetic acid, loaded onto the sample, desalted with 0.1% trifluoroacetic acid, and finally eluted with 50% acetonitrile-0.1% trifluoroacetic acid. The eluent was dried under vacuum at 45 °C.

[0034] (2) Liquid chromatography-mass spectrometry analysis Analysis was performed using a nano-level liquid chromatography system tandem with an Orbitrap mass spectrometer. The chromatographic column was a C18 column (150 μm × 170 mm, 1.9 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid-80% acetonitrile solution, with a flow rate of 600 nL / min. Elution gradient: 0–2 min, 4% B; 2–35 min, 4%–28% B; 35–55 min, 28%–40% B; 55–56 min, 40%–95% B; 56–66 min, 95% B. Mass spectrometry was performed in data-dependent acquisition (DDA) mode. The primary mass spectrometry resolution was 70,000 m / z, with a scan range of 100–1500 m / z. The top 20 most intense precursor ions were selected for secondary fragmentation using high-energy collisional dissociation (HCD) at 28% collision energy, resulting in a secondary mass spectrometry resolution of 17,500 m / z.

[0035] (3) Database retrieval Raw mass spectrometry data were retrieved from the target protein database using MaxQuant software. Search parameters were set to a fixed O-GalNAc glycosylation modification of cysteine / threonine (O-GalNAc, ST). Enzyme digestion was set to non-specific digestion, with a mass deviation of 20 ppm for primary mass spectrometry and 0.02 Da for secondary mass spectrometry.

[0036] 2. Microstructure determination: A Hitachi / Quorum SU8010 / PP3010T scanning electron microscope cryogenic transport system was used. The product was uniformly dispersed on a sample stage coated with conductive adhesive. Excess product was blown away with a bulb syringe. The sample stage was then placed in an ion sputtering instrument for gold sputtering. The operating voltage of the scanning electron microscope was 10kV.

[0037] 3. Cavitation volume and bulk density determination: Place 3.0 g of sample powder in a 10 mL graduated glass cylinder, gently tap it to level the powder surface (without compaction), and read the sample volume. Record this as the cavitation volume.

[0038] Bulk density is calculated using the following formula: Bulk density (g / mL) = Sample mass (g) / Sample volume (mL). Each sample is measured in triplicate, and the results are expressed as the average.

[0039] 4. Solubility Determination: The dissolution rate of different salt particles was determined by dynamic conductivity measurement. Salt particles were placed in a beaker containing 50 mL of deionized water and dissolved using a magnetic stirrer (60 r / min). The dissolution process was monitored in real time using a conductivity meter, and the changes in conductivity were recorded. The mass of the test sample was accurately converted to ensure that the final mass fraction of sodium chloride in the solution was 0.25%.

[0040] 5. Sensory Evaluation of Salt Reduction Effect: This was conducted by a trained sensory evaluation team (10 people, half male and half female). Before the experiment, 20 mg salt samples were aliquoted into 1.5 mL centrifuge tubes and coded with three-digit random numbers. During the evaluation, the evaluators placed all samples on the front of their tongues, let them stand for 30 seconds, and then quantified the peak saltiness intensity (0-10 points), saltiness release rate (0-10 points), saltiness persistence (0-10 points), and graininess intensity (0-10 points). After each evaluation, the evaluators rinsed their mouths with distilled water three times, with a 2-minute interval between each rinse. Each sample was evaluated three times, and the final result was expressed as the average of the three scores.

[0041] 6. Contact Angle Measurement: Using a contact angle measuring instrument and the seated drop method, 1 μL of ultrapure water was dropped onto a solid surface made of salt powder tablets, and the static contact angle was measured. Each sample was measured in parallel 5 times.

[0042] Example 1 A method for preparing proteolytically hydrolyzed peptide hollow salts with regular shape and improved salt reduction effect includes the following steps: (1) Preparation of peptide solution: Take 50 g of clam meat and add 50 g of water to homogenize. Add 1% neutral enzyme at 50℃ for 1 h of enzymatic hydrolysis, then add 2% flavor protease for 2 h of enzymatic hydrolysis to inactivate the enzyme. After enzymatic hydrolysis, add 5% acetylglucosamine by mass of the hydrolysate and carry out thermal reaction at 100℃ for 4 h. After the reaction, pass the reaction solution through a 1 kD molecular weight cutoff membrane for ultrafiltration and collect the filtrate. Concentrate the filtrate by rotary evaporation to achieve a solid content of 70% to obtain a glycopeptide solution.

[0043] (2) Preparation of hollow salt: The glycopeptide solution obtained in step (1) was added to a saturated sodium chloride aqueous solution at a mass fraction of 10% and mixed thoroughly to obtain a mixed solution. The mixed solution was transported by a peristaltic pump at a rate of 20 mL / min and spray-dried under the conditions of an inlet temperature of 130℃ and an outlet temperature of 85℃ to finally obtain the hollow salt product.

[0044] Example 2 The amount of acetylglucosamine added in step (1) of Example 1 was adjusted to 6% of the mass of the enzymatic hydrolysate. Other conditions and steps were the same as in Example 1, and hollow salt products were obtained.

[0045] Example 3 The amount of acetylglucosamine added in step (1) of Example 1 was adjusted to 7% of the mass of the enzymatic hydrolysate. Other conditions and steps were the same as in Example 1, and hollow salt products were obtained.

[0046] Example 4 The thermal reaction temperature in step (1) of Example 1 was adjusted to 90°C and the reaction time was 4 h. Other conditions and steps were the same as in Example 1, and hollow salt products were obtained.

[0047] Example 5 The thermal reaction temperature in step (1) of Example 1 was adjusted to 95°C and the reaction time was 3.5 h. Other conditions and steps were the same as in Example 1, and hollow salt products were obtained.

[0048] Comparative Example 1 (Blank Control) Saturated brine is directly spray-dried (without adding any shell-forming agent) to obtain solid salt powder.

[0049] Comparative Example 2 (only enzyme hydrolysate added, no thermal reaction performed) The amount of clam enzymatic hydrolysate (without thermal reaction) with a solid content of 70% in step (2) of Example 1 was adjusted to 10%, and other conditions and steps were the same as in Example 1, to obtain hollow salt.

[0050] Comparative Example 3 (with added enzymatic hydrolysate and acetylglucosamine, without thermal reaction) In step (1) of Example 1, clam enzymatic hydrolysate with a solid content of 70% and acetylglucosamine are mixed (without thermal reaction), and other conditions and steps are the same as in Example 1 to obtain hollow salt.

[0051] Comparative Example 4 (using glucose instead of acetylglucosamine) In step (1) of Example 1, glucose was used instead of acetylglucosamine for the thermal reaction, while other conditions and steps were the same as in Example 1, resulting in hollow salt.

[0052] Results analysis: Figure 1 The image shows the LC-MS / MS mass spectra of Example 1. The raw mass spectrometry data were retrieved from the target protein database using MaxQuant software. The following glycopeptides were identified: TPIPDS(GlcNAc)S, ET(GlcNAc)ITPIP, S(GlcNAc)GGP, and TT(GlcNAc)EAP.

[0053] Figure 2 The results are obtained from SEM (Series Electron Microscopy) measurements of Examples 1, 2, 3, 4, 5, Comparative Examples 1, 2, 3, and 4. Figure 1 It can be seen that, compared with the dense crystalline structure of traditional table salt (Comparative Example 1), the hollow salt with only enzymatic hydrolysate added (Comparative Example 2) formed a hollow spherical structure through spray drying, but the sphericity was low and the surface was irregular. The hollow salt with added reducing sugar but without thermal reaction (Comparative Example 3) was not much different from Comparative Example 2. The target group (Example 1) that underwent the Maillard reaction produced a product that served as a natural shell-forming agent, optimizing the robustness and shapeability of the microsphere wall, ultimately yielding hollow salt microspheres with the most regular structure, the smoothest surface, and the highest sphericity. No significant changes were observed after the Maillard reaction was performed with glucose (Comparative Example 4).

[0054] Figure 3 This is a comparison chart of the bulk density and expansion volume of Examples 1, 2, 3, 4, 5, Comparative Examples 1, 2, 3, and 4. Example 1 had the lowest bulk density (0.31 g / mL), significantly lower than Comparative Examples 2 (approximately 0.45 g / mL), 3 (approximately 0.46 g / mL), and 4 (approximately 0.39 g / mL). Solid salt (Comparative Example 1) had the highest bulk density (approximately 0.61 g / mL) due to its lack of internal pores and larger mass.

[0055] Figure 4The graph shows the dissolution rates of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4. Example 1 showed the best solubility, dissolving completely in approximately 23 seconds; Comparative Examples 2, 3, and 4 all required approximately 28 seconds to dissolve completely. This corresponds to the bulk density results; particles with lower bulk density disperse more quickly in water and exhibit better solubility.

[0056] Figure 5 The results show the sensory evaluation of saltiness for Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4. The salt reduction effect of Example 1 is greater than that of Comparative Example 2 and Comparative Example 3. Compared with the blank group (Comparative Example 1), the salt reduction effect reaches more than 42%.

[0057] Figure 6 The contact angles of Example 1, Comparative Example 2, and Comparative Example 3 are compared. Example 1 has the smallest contact angle (19°), which is significantly smaller than that of Comparative Example 2 (approximately 38°), Comparative Example 3 (approximately 36°), and Comparative Example 4 (approximately 37°). A lower contact angle means that the Maillard reaction product has better surface activity, which can significantly improve the spreading ability of the solution on the solid surface, allowing the reaction solution to uniformly coat the surface of the brine droplets and form a complete, smooth, thin-walled shell.

[0058] The comparison between the examples and the comparative examples clearly shows that: 1. Hollow salts prepared by adding only protease hydrolysate (Comparative Example 2) or simply mixing reducing sugar and protease hydrolysate (Comparative Example 3) have significantly lower hollowness, surface smoothness and salt reduction effect than the method of the present invention (Example 1).

[0059] 2. Hollow salts prepared using glucose instead of acetylglucosamine (Comparative Example 4) also showed significantly inferior results compared to the present invention, indicating that acetylglucosamine plays a special role in the present invention.

[0060] 3. The glycopeptide complex prepared by Maillard reaction in this invention can be used as a shell-forming agent to significantly improve the surface structure of hollow salts, increase the hollowness and reduce salt content. This technical effect is something that those skilled in the art cannot easily foresee based on existing technology.

[0061] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for producing a hollow salt, characterized by, Clam meat protein hydrolysate was subjected to Maillard reaction with acetylglucosamine to generate a glycopeptide complex, which was then used as a shell-forming agent to prepare hollow salts.

2. The method of claim 1, wherein, Clam meat protein hydrolysate was thermally reacted with acetylglucosamine at 90-100℃ for 3-4 hours. The glycopeptide fraction with a molecular weight ≤1KD was extracted by ultrafiltration, concentrated to a solid content of 60%-80%, and then spray-dried with saturated brine to obtain hollow salt.

3. The method of claim 1, wherein, The method includes the following steps: (1) Thermal reaction of clam meat hydrolysate: take clam meat homogenate for enzymatic hydrolysis to obtain clam meat hydrolysate, add 5%~8% of acetylglucosamine by mass of hydrolysate, and carry out thermal reaction at 90~100℃ for 3~4h to obtain glycopeptide thermal reaction solution. (2) Ultrafiltration separation: The glycopeptide thermal reaction solution obtained in step (1) is subjected to ultrafiltration to extract glycopeptide components with a molecular weight ≤1KD; (3) Concentration: The glycopeptide component obtained in step (2) is concentrated by rotary evaporation to achieve a solid content of 60% to 80%; (4) Spray drying: The concentrated glycopeptide solution from step (3) is mixed with saturated saline solution and then spray dried to obtain hollow salt.

4. The method of claim 3, wherein, In step (1), the enzymes used for enzymatic hydrolysis are neutral protease and flavor protease. Enzymatic hydrolysis is carried out at 45~55℃. Neutral protease hydrolysis is carried out for 1~2 hours, and then flavor protease is added at 45~55℃ for 1~2 hours. The amount of neutral protease and flavor protease added is 1~2% of the clam meat weight.

5. The method of claim 3, wherein, In step (1), the temperature of the thermal reaction is 95~100℃ and the reaction time is 3.5~4h.

6. The method according to claim 3, characterized in that, In step (1), the amount of acetylglucosamine added is 6% to 7% of the mass of the enzymatic hydrolysate.

7. The method according to claim 3, characterized in that, In step (4), the spray drying conditions are: inlet temperature 120~150℃, outlet temperature 80~100℃, and peristaltic pump speed 15~30mL / min.

8. The method according to claim 3, characterized in that, In step (4), the amount of glycopeptide solution added is 5% to 10% of the mass of saturated saline solution.

9. Hollow salt prepared by any one of claims 1 to 8.

10. The use of the hollow salt of claim 9 in the preparation of seasonings or food.

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