Pork bone-based condiment and method for preparing the same

Abstract

The present application belongs to the technical field of condiment, and particularly relates to a kind of condiment based on pig bone and a preparation method thereof.The preparation method comprises the following steps: step 1, the pig bone is broken, degreased, and bone fat extract and defatted bone powder are obtained; the defatted bone powder is mixed with water and extracted by two times of hot pressing; step 2, the first extraction liquid and the second extraction liquid are concentrated respectively, and low-temperature flavor concentrate and high-temperature mineral concentrate are obtained; step 3, the second bone residue is enzymatically hydrolyzed, and enzymatic hydrolysis concentrate is obtained by separation and concentration; step 4, the low-temperature flavor concentrate, sodium L-glutamate, high-temperature mineral concentrate and enzymatic hydrolysis concentrate are mixed and subjected to three-stage Maillard reaction.The method realizes high-value utilization of pig bone through the synergistic process of graded hot pressing extraction, three-stage composite enzymolysis, two-phase nanoemulsion and three-stage Maillard reaction.

Description

Technical Field

[0001] This invention belongs to the field of condiment technology, and specifically relates to a condiment based on pork bones and its preparation method. Background Technology

[0002] Pork bones, a major byproduct of livestock and poultry processing, are rich in collagen, various amino acids, minerals, and flavor peptide precursors, making them a high-quality raw material for preparing natural meat-flavored seasonings. With consumers increasingly demanding "clean labels" and natural flavors, extracting flavor substances from pork bones using techniques such as enzymatic hydrolysis and hot-press extraction to prepare nutritious and delicious bone broth seasonings has become an important research direction in the field of intensive food processing.

[0003] Currently, the processes for preparing seasonings using pork bones mainly fall into two categories: one is the traditional boiling and concentration process, which involves boiling crushed pork bones at high temperatures for a long time to dissolve the collagen and fat in the bones, followed by concentration and blending to obtain bone broth seasoning. While this process can achieve a certain meaty flavor, it suffers from problems such as low extraction efficiency, long production cycle, limited flavor, and significant loss of heat-sensitive flavor compounds. The other type of process uses enzymatic hydrolysis technology, which hydrolyzes bone proteins into small peptides and amino acids using proteases to improve the product's nutritional value and umami flavor. However, a single enzymatic hydrolysis process often results in a thin taste, lacking the richness and oily aroma of traditional bone broth.

[0004] How to achieve efficient utilization of all components of pork bones through grading, construct bone seasonings with three-dimensional flavor and long-lasting aroma, and overcome the problems of single product flavor, poor stability and low resource utilization in existing technologies has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides a pork bone-based seasoning and its preparation method. This method utilizes a synergistic process involving graded hot-press extraction, three-stage complex enzymatic hydrolysis, biphasic nanoemulsion, and a three-stage Maillard reaction to achieve high-value utilization of all components of pork bones. The seasoning obtained by this invention is rich in functional peptides, chelated calcium, and characteristic meat aroma substances, exhibiting a rich flavor profile, stable performance, easy storage, high raw material utilization, and an increased variety of flavor compounds.

[0006] The technical solution of the present invention to solve the above problems is as follows: A method for preparing a pork bone-based seasoning includes the following steps: Step 1: Crush pig bones, add lipase, hydrolyze at 45-55℃ and pH 7.0-8.0 for 2-4 hours, centrifuge to obtain bone fat extract and defatted bone powder; mix the defatted bone powder with water for a first hot-press extraction, and separate the solid and liquid to obtain a first extract and a first bone residue; mix the first bone residue with water for a second hot-press extraction, and separate the solid and liquid to obtain a second extract and a second bone residue; the molecular distillation adopts a three-stage molecular distillation process, with a first-stage distillation temperature of 60-70℃ and a vacuum degree of 100-500Pa, a second-stage distillation temperature of 120-140℃ and a vacuum degree of 0.5-2.0Pa, a third-stage distillation temperature of 160-180℃ and a vacuum degree of 0.1-0.5Pa, a scraper rotation speed of 150-250rpm, and a feed rate of 0.6-1.5kg / h; Step 2: The first extract is concentrated using a low-temperature membrane to a solid content of 25-35% to obtain a low-temperature flavor concentrate; the second extract is concentrated using vacuum evaporation to a solid content of 40-50% to obtain a high-temperature mineral concentrate. Step 3: Mix the second bone residue with water and wet-crush it to D90 < 50 μm, add a compound protease for enzymatic hydrolysis, separate and concentrate to obtain an enzymatic hydrolysate with a solid content of 30-40%; Step 4: Mix 30-40 parts of low-temperature flavor concentrate, 2-3 parts of L-glutamate, 20-30 parts of high-temperature mineral concentrate, and 25-35 parts of enzymatic hydrolysis concentrate for a three-stage Maillard reaction: In the first stage, add 3-5 parts of reducing sugar, 5-10 parts of bone lip nanoemulsion a, and 1.5-2.5 parts of sulfur-containing amino acids, and react at 110-115℃ for 25-35 minutes to generate meat flavor precursors; in the second stage, add 2-3 parts of the first spice component and react at 120-125℃ for 40-50 minutes to form a complex flavor; in the third stage, add 3-6 parts of bone lip nanoemulsion b and react at 105-110℃ for 10-20 minutes to achieve flavor anchoring; after cooling, add 0.5-1.0 parts of the second spice component, and then microfilter, homogenize, and spray dry through a 0.2-0.5μm ceramic membrane to obtain the final product.

[0007] Further, in step 1, the first hot-press extraction is carried out at a temperature of 112-118℃, a pressure of 0.15-0.18MPa, and a time of 3.0-3.5h. After flash evaporation and cooling to 75-80℃, solid-liquid separation is obtained to obtain the first extract and the first bone residue. The second extraction is carried out at a temperature of 138-142℃, a pressure of 0.34-0.38MPa, and a time of 2.0-2.2h. Solid-liquid separation is then carried out to obtain the second extract and the second bone residue.

[0008] Further, in step 3, the enzymatic hydrolysis with the addition of complex protease is divided into three stages: the first stage: adding neutral protease at a dosage of 0.6-0.9% of the second bone residue mass, and hydrolyzing for 90-120 min at a temperature of 50-55℃ and a pH of 6.8-7.2; the second stage: maintaining a temperature of 50-55℃, adjusting the pH to 6.0-6.5, adding transglutaminase at a dosage of 0.5-1.0% of the second bone residue mass, and performing a cross-linking reaction for 30-40 min; the third stage: adjusting the pH to 6.5-7.0, adding flavor protease at a dosage of 0.4-0.6% of the second bone residue mass, and continuing enzymatic hydrolysis at 50-55℃ for 60-90 min; finally, maintaining the temperature at 90-95℃ for 10-15 min to inactivate the enzyme.

[0009] Further, the preparation method of the bone fat nanoemulsion a is as follows: bone fat extract, sucrose fatty acid ester, glyceryl monostearate, and deionized water are pre-sheared at 65-70℃ to form a crude emulsion, and then homogenized three times under high pressure at 40-50 MPa to obtain an emulsion with an average particle size of 100-200 nm. In the bone fat nanoemulsion a, the mass ratio of bone fat extract, sucrose fatty acid ester, glyceryl monostearate, and deionized water is 12-17:2-3:0.8-1.2:78-85.

[0010] Further, the preparation method of the bone fat nanoemulsion b is as follows: bone fat extract, sodium octenyl succinate starch, maltodextrin, and deionized water are pre-sheared at 55-60℃, pre-homogenized at 30MPa, and then homogenized four times by microfluidic jet at 80-100MPa to obtain an emulsion with an average particle size of 30-80nm. In the bone fat nanoemulsion b, the mass ratio of bone fat extract, sodium octenyl succinate starch, maltodextrin, and deionized water is 10-15:2-3:1-2:80-90.

[0011] Furthermore, the first spice component is a mixture of onion powder, garlic powder, and ginger powder in a mass ratio of 1.8-2.1:0.9-1.1:1.

[0012] Furthermore, the second spice component is a mixture of white pepper powder, nutmeg powder, and clove powder in a mass ratio of 2.8-3.2:1:0.4-0.6.

[0013] Furthermore, the reducing sugar is a mixture of glucose and xylose in a mass ratio of 1.8-2.1:1, and the sulfur-containing amino acid is a mixture of L-cysteine ​​and L-methionine in a mass ratio of 2.8-3.2:2.

[0014] The present invention has the following beneficial effects: This invention presents a method for preparing pork bone-based seasonings that overcomes the limitations of traditional "one-pot cooking" processes for bone-based seasonings. By combining physical separation, enzymatic hydrolysis, and temperature-controlled Maillard reactions, it maximizes the utilization rate of pork bone resources. This invention successfully extracts components such as fat, flavor peptides, amino acids, minerals, and collagen from fresh pork bones through sequential lipase hydrolysis, two-stage variable-temperature hot-press extraction, wet ultrafine grinding, and targeted enzymatic hydrolysis. In particular, the wet grinding and combined enzymatic hydrolysis of secondary bone residue significantly improves the conversion rate of poorly soluble components such as bone collagen, achieving full utilization of pork bone components and avoiding resource waste. This invention combines different characteristic components (low-temperature flavor concentrate, high-temperature mineral concentrate, and enzymatic hydrolysis concentrate) obtained through graded extraction with bone lipid nanoemulsion, reducing sugars, and sulfur-containing amino acids. Through precise control of a three-stage Maillard reaction, a meat aroma precursor is first generated at low temperature, followed by a complex roasted meat flavor at medium temperature, and finally, flavor anchoring at low temperature. This results in a final seasoning that is not only rich and mellow in meat aroma but also possesses a unique roasted bone aroma and a sweet aftertaste, exhibiting a strong three-dimensional flavor profile, significantly superior to bone broth seasonings prepared using traditional single-process methods. The first extract (rich in heat-sensitive flavor compounds) is concentrated using a low-temperature membrane to maximize the retention of umami substances; the second extract (rich in minerals and collagen) is concentrated using vacuum evaporation, improving concentration efficiency while promoting partial hydrolysis of collagen. The second bone residue undergoes wet pulverization and complex enzymatic hydrolysis, converting waste bone collagen from conventional processes into small peptides and amino acids, increasing product yield and enriching flavor sources.

[0015] In step 4, the first stage generates meat aroma precursors, providing key intermediates for subsequent reactions. Bone lipid nanoemulsion a begins to release during this stage, where fatty acids react with sulfur-containing compounds to form sulfur-containing heterocyclic compounds (such as thiophene and thiazole), establishing the "meat aroma" base and creating a complex meat flavor. The addition of spices upgrades the flavor from a "single meat flavor" to a "complex" flavor, increasing flavor layers. Small-diameter lipid droplets in nanoemulsion b are embedded in the network structure formed by the Maillard reaction products, locking in volatile flavor substances through both physical encapsulation and chemical binding, significantly enhancing the product's aroma longevity. Emulsion b uses sodium octenyl succinate starch and maltodextrin as wall materials, homogenized by 80-100 MPa microfluidic jets, exhibiting high thermal stability and long-term stability. Emulsion a uses sucrose fatty acid esters and glyceryl monostearate as emulsifiers, homogenized under high pressure at 40-50 MPa, exhibiting high interfacial activity but relatively low thermal stability, gradually breaking down and releasing lipids at 120-125 degrees Celsius. Emulsion a is miscible with Maillard aqueous phase, promoting lipid-amino acid interfacial reactions; emulsion b is strongly hydrophilic, forming a glassy matrix after drying, physically encapsulating flavor compounds. In terms of timing, emulsion a first initiates lipid oxidation to generate meat aroma precursors, and then deeply participates in the reaction to form complex flavors; emulsion b subsequently encapsulates and anchors flavor compounds, preventing volatilization loss.

[0016] Furthermore, in step 3, a three-stage enzymatic hydrolysis is performed. The first stage uses a neutral protease to generate a large number of oligopeptides with molecular weights of 1000 to 5000 Da and a characteristic proline-hydroxyproline-glycine tripeptide, achieving a degree of hydrolysis of 15% to 20%. The second stage introduces transglutaminase, catalyzing the formation of ε-(γ-glutamyl)lysine isopeptide bonds, increasing the peptide chain molecular weight to 10000 to 30000 Da, effectively masking bitter peptides and improving thermal stability. The third stage uses a flavor protease at pH 6.5 to 7.0, increasing the degree of hydrolysis to 35% to 40%, with peptides having a molecular weight less than 1000 Da accounting for more than 75%, and converting glutamine to glutamic acid to enhance umami flavor. The three stages work synergistically: efficient hydrolysis by neutral protease, cross-linking modification by transglutaminase, and flavor optimization by flavor protease work together to improve efficiency compared to traditional single-stage enzymatic hydrolysis, significantly reducing bitterness and increasing the content of functional peptides. The three-stage enzymatic hydrolysis cascade design achieves functional synergy: the efficient hydrolysis of neutral protease, the cross-linking modification of transglutaminase, and the flavor optimization of flavor protease work together to give the product good nutritional functionality, processing adaptability, and sensory quality. Compared with the traditional single enzymatic hydrolysis process, the efficiency is increased by more than 40%, the bitterness is significantly reduced, and the content of functional peptides is increased by more than 50%.

[0017] The small peptides and free amino acids produced by the enzymatic hydrolysis in step 3 have much higher reactivity than large protein molecules, allowing the Maillard reaction to be initiated at a lower temperature in step 4, thus reducing the activation energy requirement. Furthermore, the bitter peptides eliminated by flavor proteases in step 3 prevent the formation of bitter heterocyclic compounds at the high temperature in step 4; simultaneously, the stable peptide structure formed by glutamine transaminase cross-linking acts as a "backbone molecule" in step 4, binding volatile flavor substances and slowing down the evaporation rate, thus providing complementary protection to the flavor anchoring of the bone lipid nanoemulsion b in the third stage of step 4. Attached Figure Description

[0018] Figure 1 The results are the combined sensory evaluation results of the samples obtained in Examples 1-3 and Comparative Examples 1-5; Figure 2 The results are the test results of amino acid nitrogen in the samples obtained in Examples 1-3 and Comparative Examples 1-5. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] All of the following raw materials are commercially available. Lipase, powdered water-soluble, enzyme activity 100,000 U / g, Guangzhou Longde Biotechnology Co., Ltd.; Neutral protease, enzyme activity 200,000 U / g, Shanghai Quanyan Biotechnology Co., Ltd.; Flavor protease, enzyme activity 100,000-200,000 U / g, Hebei Jiuxing Chemical Products Co., Ltd.; Transglutaminase, active ingredient content 98%, enzyme activity 10,000 U / g, Henan Ruilunte Biotechnology Co., Ltd.; Onion powder, extract powder, active ingredient content 99%, Hubei Haijia Biotechnology Co., Ltd.; L-Glutamate monosodium glutamate, active ingredient content 99%, Jinan Huijinchuan Chemical Co., Ltd.; Ginger powder, particle size 100 mesh, Cangzhou Dinghao Food Co., Ltd.; Garlic powder, content 99%, Chongqing Tianrun Biological Products Co., Ltd.; Nutmeg powder, active ingredient content 99%, Zhejiang Yicun Biotechnology Co., Ltd.; Clove powder, 80-100 mesh, Fufeng Sinote Biotechnology Co., Ltd.; L-Methionine, content 99%, 80-100 mesh; L-Cysteine, 80-100 mesh, content 99%, all purchased from Jiangsu Caiwei Biotechnology Co., Ltd.

[0021] Example 1 A method for preparing a pork bone-based seasoning includes the following steps: Step 1: 100 portions of fresh pork bones are crushed into 2-5 cm pieces, then further pulverized to 0.4-0.8 mm. Lipase is added, and the mixture is enzymatically hydrolyzed at 50°C and pH 7.0-8.0 for 3 hours. After centrifugation, the upper layer of enzymatically hydrolyzed bone fat is molecularly distilled to obtain a bone fat extract, while the lower layer is dried at 55°C for 2 hours to obtain defatted bone powder. The defatted bone powder is mixed with water at a mass ratio of 1:7 for a first hot-press extraction, followed by solid-liquid separation to obtain a first extract and a first bone residue. The first bone residue is then mixed with water at a mass ratio of 1:5 for a second hot-press extraction, followed by solid-liquid separation to obtain a second extract and a second bone residue. In this process, the molecular distillation employs a three-stage molecular distillation process: first-stage distillation at 65℃ and 300Pa vacuum, second-stage distillation at 130℃ and 1.5Pa vacuum, and third-stage distillation at 170℃ and 0.3Pa vacuum, with a scraper rotation speed of 200rpm and a feed rate of 1kg / h; the first hot-press extraction is performed at 112℃, 0.18MPa, and 3.5h, followed by flash evaporation and cooling to 80℃ to obtain a first extract and a first bone residue; the second extraction is performed at 138℃, 0.38MPa, and 2.2h, resulting in a second extract and a second bone residue. Step 2: The first extract is concentrated to a solid content of 30% by nanofiltration at a temperature of 28°C to obtain a low-temperature flavor concentrate; the second extract is concentrated by vacuum evaporation at a vacuum degree of -0.09MPa and a temperature of 60°C to a solid content of 45% to obtain a high-temperature mineral concentrate. Step 3: The second bone residue and water are mixed at a mass ratio of 1:3 and wet-milled until D90 < 50 μm. A complex protease is added for enzymatic hydrolysis: First stage: Neutral protease is added at a dosage of 0.8% of the second bone residue mass, and enzymatic hydrolysis is performed at 52℃ and pH 7 for 100 min; Second stage: The temperature is maintained at 52℃, the pH is adjusted to 6.3, and transglutaminase is added at a dosage of 0.8% of the second bone residue mass, with a cross-linking reaction for 35 min; Third stage: The pH is adjusted to 7, flavor protease is added at a dosage of 0.5% of the second bone residue mass, and enzymatic hydrolysis continues at 53℃ for 75 min; Finally, the enzyme is inactivated by maintaining the temperature at 92℃ for 13 min, and the enzymatic hydrolysate is separated and concentrated to obtain a concentrated hydrolysate with a solid content of 35%. Step 4: Mix 35 parts of low-temperature flavor concentrate, 2.5 parts of L-glutamate, 25 parts of high-temperature mineral concentrate, and 30 parts of enzymatic hydrolysis concentrate for a three-stage Maillard reaction: In the first stage, add 4 parts of reducing sugar, 8 parts of bone lip nanoemulsion a, and 2 parts of sulfur-containing amino acids, and react at 113℃ for 30 min to generate meat flavor precursors; in the second stage, add 2.5 parts of the first spice component and react at 123℃ for 45 min to form a complex flavor; in the third stage, add 4 parts of bone lip nanoemulsion b and react at 108℃ for 15 min to achieve flavor anchoring; after cooling, add 0.8 parts of the second spice component, and then perform microfiltration, homogenization, and spray drying using a 0.5μm ceramic membrane to obtain the final product.

[0022] The preparation method of the bone fat nanoemulsion a is as follows: bone fat extract, sucrose fatty acid ester, glyceryl monostearate, and deionized water are pre-sheared at 68°C to form a crude emulsion, and then homogenized under high pressure at 45MPa three times to obtain an emulsion with an average particle size of 100-200nm; wherein, the mass ratio of bone fat extract, sucrose fatty acid ester, glyceryl monostearate, and deionized water is 15:2:1:80.

[0023] The preparation method of the bone fat nanoemulsion b is as follows: bone fat extract, sodium octenyl succinate starch, maltodextrin, and deionized water are pre-sheared at 58°C, pre-homogenized at 30 MPa, and then homogenized four times by microfluidic jet at 90 MPa to obtain an emulsion with an average particle size of 30-80 nm. The mass ratio of bone fat extract, sodium octenyl succinate starch, maltodextrin, and deionized water is 12:2.5:1.5:85.

[0024] The first spice component is a mixture of onion powder, garlic powder, and ginger powder in a mass ratio of 2:1:1. The second spice component is a mixture of white pepper powder, nutmeg powder, and clove powder in a mass ratio of 3:1:0.5. The reducing sugar is a mixture of glucose and xylose in a mass ratio of 2:1, and the sulfur-containing amino acid is a mixture of L-cysteine ​​and L-methionine in a mass ratio of 3:2.

[0025] Example 2 A method for preparing a pork bone-based seasoning includes the following steps: Step 1: 100 portions of fresh pork bones are crushed into 2-5 cm pieces, then further pulverized to 0.4-0.8 mm. Lipase is added, and the mixture is enzymatically hydrolyzed at 45°C and pH 7.0-8.0 for 4 hours. After centrifugation, the upper layer of enzymatically hydrolyzed bone fat is subjected to molecular distillation (the molecular distillation process is the same as in Example 1) to obtain bone fat extract. The lower layer is dried at 50°C for 2.5 hours to obtain defatted bone powder. The defatted bone powder is mixed with water at a mass ratio of 1:8 and subjected to a first hot-press extraction. Solid-liquid separation is then performed to obtain... The first extract and the first bone residue were mixed with water at a mass ratio of 1:6 and subjected to a second hot-press extraction. Solid-liquid separation was performed to obtain the second extract and the second bone residue. The first hot-press extraction was carried out at a temperature of 118℃, a pressure of 0.15MPa, and a time of 3.5h. After flash evaporation and cooling to 75℃, solid-liquid separation was performed to obtain the first extract and the first bone residue. The second extraction was carried out at a temperature of 142℃, a pressure of 0.34MPa, and a time of 2.2h. Solid-liquid separation was performed to obtain the second extract and the second bone residue. Step 2: The first extract is concentrated to a solid content of 35% by nanofiltration at a temperature of 35°C to obtain a low-temperature flavor concentrate; the second extract is concentrated by vacuum evaporation at a vacuum degree of -0.085MPa and a temperature of 65°C to a solid content of 50% to obtain a high-temperature mineral concentrate. Step 3: The second bone residue and water are mixed at a mass ratio of 1:3.5 and wet-milled until D90 < 50 μm. A complex protease is added for enzymatic hydrolysis: First stage: Neutral protease is added at 0.9% of the second bone residue mass, and hydrolysis is carried out at 55℃ and pH 6.8 for 90 min; Second stage: The temperature is maintained at 55℃, the pH is adjusted to 6.0, and transglutaminase is added at 1.0% of the second bone residue mass, with a cross-linking reaction for 30 min; Third stage: The pH is adjusted to 7.0, flavor protease is added at 0.4% of the second bone residue mass, and enzymatic hydrolysis continues at 50℃ for 90 min; Finally, the enzyme is inactivated at 95℃ for 10 min, and the solution is separated and concentrated to obtain an enzymatic hydrolysate with a solid content of 40%. Step 4: Mix 30 parts of low-temperature flavor concentrate, 3 parts of L-glutamate monosodium glutamate, 20 parts of high-temperature mineral concentrate, and 35 parts of enzymatic hydrolysis concentrate for a three-stage Maillard reaction: In the first stage, add 3 parts of reducing sugar, 10 parts of bone lip nanoemulsion a, and 1.5 parts of sulfur-containing amino acids, and react at 115℃ for 25 min to generate meat flavor precursors; in the second stage, add 3 parts of the first spice component and react at 120℃ for 50 min to form a complex flavor; in the third stage, add 3 parts of bone lip nanoemulsion b and react at 110℃ for 10 min to achieve flavor anchoring; after cooling, add 1.0 part of the second spice component, and then perform microfiltration, homogenization, and spray drying through a 0.2μm ceramic membrane to obtain the final product.

[0026] The preparation method of the bone fat nanoemulsion a is as follows: bone fat extract, sucrose fatty acid ester, glyceryl monostearate, and deionized water are pre-sheared at 70°C to form a crude emulsion, and then homogenized under high pressure of 50MPa three times to obtain an emulsion with an average particle size of 100-200nm; wherein, the mass ratio of bone fat extract, sucrose fatty acid ester, glyceryl monostearate, and deionized water is 12:3:1.2:78.

[0027] In the preparation method of the bone lipid nanoemulsion b, the mass ratio of bone lipid extract, sodium octenyl succinate starch, maltodextrin, and deionized water is 15:3:1:80, and the rest is the same as in Example 1.

[0028] The first spice component is a mixture of onion powder, garlic powder, and ginger powder in a mass ratio of 2:1:1. The second spice component is a mixture of white pepper powder, nutmeg powder, and clove powder in a mass ratio of 3:1:0.5. The reducing sugar is a mixture of glucose and xylose in a mass ratio of 2:1, and the sulfur-containing amino acid is a mixture of L-cysteine ​​and L-methionine in a mass ratio of 3:2.

[0029] Example 3 A method for preparing a pork bone-based seasoning includes the following steps: Step 1: Crush 100 portions of fresh pork bones into 2-5 cm pieces, then further pulverize to 0.4-0.8 mm. Add lipase and hydrolyze at 45-55°C and pH 7.0-8.0 for 2-4 hours. Centrifuge to separate the enzymatically hydrolyzed bone fat. Perform molecular distillation on the upper layer (molecular distillation process as in Example 1) to obtain bone fat extract. Dry the lower layer at 60°C for 1.5 hours to obtain defatted bone powder. Mix the defatted bone powder with water at a mass ratio of 1:6 and perform a first hot-press extraction to separate the solid and liquid phases. A first extract and a first bone residue were obtained. The first bone residue was mixed with water at a mass ratio of 1:4 and subjected to a second hot-press extraction. Solid-liquid separation was performed to obtain a second extract and a second bone residue. The first hot-press extraction was performed at a temperature of 115℃, a pressure of 0.17MPa, and a time of 3.0h. After flash evaporation and cooling to 78℃, solid-liquid separation was performed to obtain the first extract and the first bone residue. The second extraction was performed at a temperature of 140℃, a pressure of 0.36MPa, and a time of 2.1h. Solid-liquid separation was performed to obtain the second extract and the second bone residue. Step 2: The first extract is concentrated to a solid content of 25% by nanofiltration at a temperature of 20°C to obtain a low-temperature flavor concentrate; the second extract is concentrated by vacuum evaporation at a vacuum degree of -0.095MPa and a temperature of 55°C to a solid content of 40% to obtain a high-temperature mineral concentrate. Step 3: The second bone residue and water are mixed at a mass ratio of 1:4 and wet-milled until D90 < 50 μm. A complex protease is added for enzymatic hydrolysis: First stage: Neutral protease is added at a dosage of 0.6% of the second bone residue mass, and enzymatic hydrolysis is performed at 50℃ and pH 7.2 for 120 min; Second stage: Maintaining the temperature at 50℃, the pH is adjusted to 6.5, and transglutaminase is added at a dosage of 0.5% of the second bone residue mass, with a cross-linking reaction for 40 min; Third stage: The pH is adjusted to 6.5, and flavor protease is added at a dosage of 0.6% of the second bone residue mass, with enzymatic hydrolysis continuing at 55℃ for 60 min; Finally, the enzyme is inactivated by maintaining the temperature at 90℃ for 15 min, and the enzymatic hydrolysate is separated and concentrated to obtain a concentrated enzymatic hydrolysate with a solid content of 30%. Step 4: Mix 40 parts of low-temperature flavor concentrate, 2 parts of L-glutamate, 30 parts of high-temperature mineral concentrate, and 25 parts of enzymatic hydrolysis concentrate for a three-stage Maillard reaction: In the first stage, add 5 parts of reducing sugar, 10 parts of bone lip nanoemulsion a, and 2.5 parts of sulfur-containing amino acids, and react at 110℃ for 35 min to generate meat flavor precursors; in the second stage, add 2 parts of the first spice component and react at 125℃ for 40 min to form a complex flavor; in the third stage, add 6 parts of bone lip nanoemulsion b and react at 105℃ for 20 min to achieve flavor anchoring; after cooling, add 1.0 part of the second spice component, and then perform microfiltration, homogenization, and spray drying using a 0.5μm ceramic membrane to obtain the final product.

[0030] The preparation method of the osteolipin nanoemulsion a is the same as in Example 1.

[0031] In the preparation method of the bone lipid nanoemulsion b, the mass ratio of bone lipid extract, sodium octenyl succinate starch, maltodextrin, and deionized water is 10:2:2:90, and the rest is the same as in Example 1.

[0032] The first spice component is a mixture of onion powder, garlic powder, and ginger powder in a mass ratio of 2:1:1. The second spice component is a mixture of white pepper powder, nutmeg powder, and clove powder in a mass ratio of 3:1:0.5. The reducing sugar is a mixture of glucose and xylose in a mass ratio of 2:1, and the sulfur-containing amino acid is a mixture of L-cysteine ​​and L-methionine in a mass ratio of 3:2.

[0033] Comparative Example 1 100 portions of fresh pork bones were enzymatically degreased. The defatted bone powder was then mixed with water at a mass ratio of 1:6 and extracted for 5 hours at 140°C and 0.35 MPa. Solid-liquid separation was performed to obtain the extract and bone residue. The extract was not graded but directly concentrated under vacuum to a solid content of 35%, and used as a single concentrate for subsequent steps. The remaining steps were the same as in Example 1.

[0034] Comparative Example 2 The three-stage enzymatic hydrolysis process of neutral protease-TGase-flavor protease was eliminated, and a single neutral protease was used instead. Specifically, after wet pulverizing the second bone residue, 1.5% neutral protease (approximately twice the total enzyme amount in Example 1) was added, and enzymatic hydrolysis was carried out at 52°C and pH 7.0 for 4 hours, followed by direct enzyme inactivation and concentration. TGase cross-linking and flavor protease treatment were not performed. The remaining steps were the same as in Example 1.

[0035] Comparative Example 3 Only bone lipid nanoemulsion a was used. In the first stage of step 4, 12 parts of emulsion a were added. In the third stage, emulsion b was not added. The rest was the same as in Example 1.

[0036] Comparative Example 4 Remove bone lipid nanoemulsion a and bone lipid nanoemulsion b. In step 4, add 8 parts of bone lipid extract in the first stage. In the third stage, do not add bone lipid extract or any bone nanoemulsion. The rest is the same as in Example 1.

[0037] Comparative Example 5 The three-stage Maillard reaction is omitted. Step 4 is changed to: A single mixture of the low-temperature flavor concentrate, L-glutamate monosodium glutamate, high-temperature mineral concentrate, enzymatic hydrolysis concentrate, 4 parts reducing sugar, 8 parts bone fat nanoemulsion a, 2 parts sulfur-containing amino acids, and 2.5 parts of the first spice component is mixed and reacted at 120°C for 90 minutes. After cooling, 0.8 parts of the second spice component are added, followed by microfiltration, homogenization, and spray drying. The bone fat nanoemulsion b and the staged addition are omitted. The rest is the same as in Example 1.

[0038] The following performance tests were performed on the samples obtained in Examples 1-3 and Comparative Examples 1-5: 1. Sensory evaluation: 100g of noodles were boiled in water, drained, and then 200mL of 0.6% (w / v) pork bone seasoning solution was added as the soup base. No other seasonings were added. Referring to the sensory evaluation criteria in Table 1, 10 judges were set up for each group, and the average score of the 10 judges was taken as the final score of the group. The results are shown in Table 2.

[0039] 2. For the determination of amino acid nitrogen by formaldehyde titration, please refer to GB 5009.235-2016 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food"; 3. Determine the calcium content according to GB 5009.92-2016 "National Food Safety Standard - Determination of Calcium in Food"; 4. Determine the content of glutamic acid, aspartic acid, glycine, and leucine in the sample according to GB 5009.124-2016 "National Food Safety Standard - Determination of Amino Acids in Food".

[0040] Table 1. Evaluation Criteria score Simulation of bone broth Flavor fullness Texture and taste Overall acceptance 1 point Like MSG water, boneless broth-like Bland as water, lacking any depth or complexity The noodles and soup were completely separated. Unacceptable, dislike 5 points It's like a "flavorful soup," but not like bone broth. It has a savory and salty flavor, but it's rather bland. Soup is soup, noodles are noodles, it's a bit incongruous. Overall acceptable 9 points Just like freshly made bone broth noodles It has a full range of flavors, including umami, richness, and mellowness, with distinct layers of taste. The soup and bread blend together perfectly, with a great "sauce-coating" effect. I really like it and I'm very satisfied.

[0041] Table 2. Sensory Evaluation Results sample Bone broth simulation accuracy (30%) Flavor fullness (25%) Taste balance (25%) Overall acceptance (20%) Overall score Example 1 8.8 8.5 8.6 8.7 8.64 Example 2 8.2 8.0 8.1 8.3 8.14 Example 3 7.5 7.6 7.8 7.6 7.62 Comparative Example 1 3.5 4.0 4.2 4.0 3.90 Comparative Example 2 5.0 5.2 5.5 5.3 5.25 Comparative Example 3 6.5 6.8 6.5 6.6 6.60 Comparative Example 4 2.8 3.2 3.0 3.0 3.00 Comparative Example 5 4.5 4.8 5.0 4.8 4.78

[0042] Table 2. Composition Determination sample Amino acid nitrogen (g / 100g) Calcium (mg / 100g) Glutamic acid (g / 100g) Aspartic acid (g / 100g) Glycine (g / 100g) Example 1 1.35 285 4.82 2.15 3.68 Example 2 1.30 272 4.51 2.03 3.42 Example 3 1.28 268 4.37 1.96 3.35 Comparative Example 1 0.65 186 2.83 1.45 2.10 Comparative Example 2 0.72 258 3.25 1.78 2.65 Comparative Example 3 0.92 280 4.73 2.10 3.55 Comparative Example 4 0.93 278 4.70 2.08 3.52 Comparative Example 5 0.90 275 4.15 1.92 3.20

[0043] From Table 2, Table 3 and Figure 1 , Figure 2 It can be seen that Example 1 achieved a comprehensive score of 8.64, significantly better than all comparative examples, with a bone broth simulation score of 8.8. This demonstrates that the synergistic effect of three-stage molecular distillation, dual-temperature extraction, and three-stage Maillard reaction successfully simulated the complex flavor system of freshly brewed bone broth. Comparative Example 1 scored only 3.90, with single-stage high-temperature extraction leading to a significant loss of volatile flavor substances, resulting in a distorted product with flavor but no bone aroma. Comparative Example 4 scored the lowest (3.00), as the bone fat extract was added directly without nano-emulsification, and the oil-water separation and oxidative flavor completely destroyed the product's texture, verifying the crucial role of the dual nano-emulsion system. The amino acid nitrogen content of Example 1 was 1.35 g / 100 g, higher than that of Comparative Example 1 (0.65 g / 100 g), indicating that the three-stage complex enzymatic hydrolysis (neutral protease-TGase-flavor protease) significantly enhanced the protein hydrolysis. The high content of glutamic acid (4.82g / 100g) and aspartic acid (2.15g / 100g) imparts a strong umami flavor to the product, while glycine (3.68g / 100g) provides a rich and rounded mouthfeel. Comparative Example 2, using a single neutral protease, had an amino acid nitrogen content of only 0.72g / 100g, resulting in incomplete enzymatic hydrolysis and a thin flavor profile. Comparative Examples 3-4 had amino acid contents similar to Example 1, but their sensory scores differed significantly, indicating that the nanoemulsion not only protects fats but also promotes the participation of lipid-soluble precursors in the Maillard reaction, achieving deep transformation and layered construction of flavor compounds.

[0044] The process innovation was validated. Comparative Example 5 had a good amino acid composition but scored 4.78 points. The single-stage high-temperature reaction led to excessive Maillard reaction, producing a burnt and bitter taste, proving the necessity of precise temperature control in stages. Calcium content data also revealed process differences: Comparative Example 1 had the highest calcium dissolution rate (186 mg / 100g) from single-stage high-temperature extraction but the worst flavor. Examples 1-3 controlled the calcium content within the range of 268-285 mg / 100g, achieving a balance between nutrition and flavor. Example 3, due to low-temperature long-term enzymatic hydrolysis, had slightly lower amino acid nitrogen and calcium content than Example 1, but its sensory score of 7.62 points was still good, suggesting that excessive pursuit of hydrolysis degree may lead to the accumulation of bitter peptides.

[0045] In summary, this invention constructs a complete technology chain of "precursor release - flavor transformation - stable anchoring" through multi-technology coupling of graded extraction, compound enzymatic hydrolysis, dual nanoemulsion and staged Maillard reaction. Each process step is indispensable and together support the industrial preparation of high-quality pork bone seasoning.

[0046] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0047] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a pork bone-based seasoning, characterized in that, Includes the following steps: Step 1: Crush the pig bones, add lipase, hydrolyze at 45-55℃ and pH 7.0-8.0 for 2-4 hours, centrifuge to obtain bone fat extract and defatted bone powder; mix the defatted bone powder with water for a first hot-press extraction, and separate the solid and liquid to obtain a first extract and a first bone residue; mix the first bone residue with water for a second hot-press extraction, and separate the solid and liquid to obtain a second extract and a second bone residue. Step 2: The first extract is concentrated using a low-temperature membrane to a solid content of 25-35% to obtain a low-temperature flavor concentrate; the second extract is concentrated using vacuum evaporation to a solid content of 40-50% to obtain a high-temperature mineral concentrate. Step 3: Mix the second bone residue with water and wet-crush it, add a compound protease for enzymatic hydrolysis, and separate and concentrate to obtain the enzymatic hydrolysis concentrate; Step 4: Mix the low-temperature flavor concentrate, L-glutamate monosodium glutamate, high-temperature mineral concentrate, and enzymatic hydrolysis concentrate, and then carry out a three-stage Maillard reaction: In the first stage, add reducing sugar, bone lipid nanoemulsion a, and sulfur-containing amino acids, and react at 110-115℃ for 25-35 min; in the second stage, add the first spice component and react at 120-125℃ for 40-50 min; in the third stage, add bone lipid nanoemulsion b and react at 105-110℃ for 10-20 min; after cooling, add the second spice component, and then microfilter, homogenize, and spray dry to obtain the final product.

2. The method for preparing a pork bone-based seasoning according to claim 1, characterized in that, In step 1, the first hot-press extraction is carried out at a temperature of 112-118℃, a pressure of 0.15-0.18MPa, and a time of 3.0-3.5h. After flash evaporation and cooling to 75-80℃, solid-liquid separation is performed to obtain the first extract and the first bone residue. The second extraction is carried out at a temperature of 138-142℃, a pressure of 0.34-0.38MPa, and a time of 2.0-2.2h. Solid-liquid separation is performed to obtain the second extract and the second bone residue.

3. The method for preparing a pork bone-based seasoning according to claim 1, characterized in that, In step 3, the enzymatic hydrolysis with the addition of complex protease is divided into three stages: the first stage: adding neutral protease at a dosage of 0.6-0.9% of the second bone residue mass, and hydrolyzing for 90-120 minutes at a temperature of 50-55℃ and a pH of 6.8-7.2; the second stage: maintaining a temperature of 50-55℃, adjusting the pH to 6.0-6.5, adding transglutaminase at a dosage of 0.5-1.0% of the second bone residue mass, and performing a cross-linking reaction for 30-40 minutes; the third stage: adjusting the pH to 6.5-7.0, adding flavor protease at a dosage of 0.4-0.6% of the second bone residue mass, and continuing enzymatic hydrolysis at 50-55℃ for 60-90 minutes; finally, maintaining the temperature at 90-95℃ for 10-15 minutes to inactivate the enzyme.

4. The method for preparing a pork bone-based seasoning according to claim 1, characterized in that, In step 4, the preparation method of the bone fat nanoemulsion a is as follows: bone fat extract, sucrose fatty acid ester, glyceryl monostearate and deionized water are pre-sheared at 65-70℃ to form a crude emulsion, and then homogenized under high pressure of 40-50MPa three times to obtain an emulsion with an average particle size of 100-200nm.

5. The method for preparing a pork bone-based seasoning according to claim 1, characterized in that, In step 4, the preparation method of the bone fat nanoemulsion b is as follows: bone fat extract, sodium octenyl succinate starch, maltodextrin, and deionized water are pre-sheared at 55-60℃, pre-homogenized at 30MPa, and then homogenized four times by microfluidic jet at 80-100MPa to obtain an emulsion with an average particle size of 30-80nm.

6. The method for preparing a pork bone-based seasoning according to claim 1, characterized in that, In step 4, the first spice component is a mixture of onion powder, garlic powder, and ginger powder in a mass ratio of 1.8-2.1:0.9-1.1:

1.

7. The method for preparing a pork bone-based seasoning according to claim 1, characterized in that, In step 4, the second spice component is a mixture of white pepper powder, nutmeg powder, and clove powder in a mass ratio of 2.8-3.2:1:0.4-0.

6.

8. The method for preparing a pork bone-based seasoning according to claim 1, characterized in that, In step 4, the reducing sugar is a mixture of glucose and xylose in a mass ratio of 1.8-2.1:1, and the sulfur-containing amino acid is a mixture of L-cysteine ​​and L-methionine in a mass ratio of 2.8-3.2:

2.

9. A seasoning prepared by the method for preparing pork bone-based seasonings as described in any one of claims 1-8.

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