Milk-derived taste enhancing peptide as well as preparation method and application thereof

Abstract

The invention provides an enzymolysis preparation method of a milk-derived taste enhancing peptide, which comprises the following steps: carrying out enzymolysis on a liquid dairy product by using flavourzyme and glutaminase, and then carrying out enzyme deactivation to obtain an enzyme-deactivated product; carrying out acidification centrifugation and ultrafiltration on the enzyme-deactivated product, and freeze-drying the separated effluent to obtain freeze-dried powder; and loading the freeze-dried powder, and separating by a glucose gel column. The product disclosed by the invention does not generate bitter taste, and compared with a sample which is not subjected to enzymolysis, the flavor level of an original skimmed milk product is enriched, and the sample subjected to enzymolysis has the mellow taste of caramel and frankincense. The method comprises the following steps: separating milk-derived taste active peptides from milk-derived taste active peptides, predicting delicate flavor, anti-oxidation and anti-inflammatory activity and ACE blood pressure lowering functional activity of separated peptide liquid through a model, performing taste verification on the peptides predicted to have taste activity through Fmoc solid-phase synthesis, and performing sensory verification on the milk-derived taste active peptides LSFD, EDIKQME, IKQMEAE and TEDELQDK after synthesis, so that the milk-derived taste active peptides are obtained for the first time and are subjected to sensory verification.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, in particular to a milk-derived taste-enhancing peptide and a preparation method and application thereof. BACKGROUND

[0002] At present, the dairy industry is still experiencing the pain of the downward cycle of raw milk. According to the data of the Ministry of Agriculture and Rural Affairs, the average price of fresh milk in the main producing areas fell to 3.11 yuan / kg in the second week of February 2025, down 15% from the beginning of 2024. The root cause is that the supply of raw milk is more than the demand, and the consumption demand is far from expected, which makes the price of raw milk continue to be inverted, which has been sustained for 3 years and a half since August 2021. The global dairy market presents the contradictory phenomenon of coexistence of consumption upgrading and overproduction of raw materials. The overproduction rate of raw milk in major producing areas such as the European Union and China is as high as 15%-20%. The traditional dairy processing mode has been unable to digest the excess production capacity, and it is urgent to develop high-value-added products through biotechnology innovation. The development of high-value-added products mainly considers two directions of flavor and health, and according to the Innovations market report, consumers first consider the flavor when choosing products. Flavor is the soul of food, which directly affects the consumption and acceptability of products. There are the following industry problems, according to the 2030 Plan Outline advocated by China to reduce fat, and due to the loss of fat-soluble flavor substances, the flavor of skimmed milk powder is insufficient in layers, and the milk product is not thick, which affects the acceptability of the product. The thick taste of the taste perception brought by fat can be enriched by proteinase to produce fresh and thick milk-derived taste small molecule active peptides, which can enrich the taste of skimmed milk powder and impart certain healthy active ingredients. Existing research produces bitter active peptides through proteinase, which affects the taste and flavor of the product. Suitable enzyme hydrolysis technology is an important prerequisite for natural dairy products to produce taste active peptides with aroma effect, and does not need to add other food additives, which meets the requirements of the advocacy of green and clean labels.

[0003] The future food is not only delicious, but also beneficial to health. There are relevant reports that generate fatty acid compounds by single enzyme complex enzymolysis, provide the taste perception of fat, so as to achieve the purpose of "reducing fat and increasing flavor". Some studies report that milk flavor can be produced by enzymolysis of butter protein, but the enzymolysis condition is single, and the milk flavor substances are not further explored. Some studies on the increase of umami taste of cheese during the reprocessing process show that the fermentation and enzymolysis method can produce high-quality taste peptides, but the fermentation process is uncontrollable. The existing trypsin enzymolysis of butter protein to prepare milk flavor base to improve the overall flavor of milk may have certain application defects in improving the quality of natural liquid milk: (1) the short peptides produced by enzymolysis of butter protein usually have certain bitterness; (2) the fatty acid compounds produced by enzymolysis of butter protein have a certain masking effect on the taste peptides produced by enzymolysis of protein; (3) the enzymolysis of butter protein produces some bitter medium and long chain fatty acids, bitter peptides and amino acids; (4) adding the enzymolysis of butter to dairy products in proportion will change the stable system of milk itself, and then affect the quality of dairy products.

[0004] Therefore, it is necessary to provide a method for improving the overall flavor level of milk by customizing enzymolysis to produce more milk source taste enhancing peptides. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a method for customizing enzymolysis to produce new milk source taste enhancing peptides and a preparation method thereof. The milk source taste enhancing peptides provided by the present application have better natural milk taste, rich flavor and umami flavor, increase the flavor level of skimmed milk, and have antioxidant, anti-inflammatory and / or blood pressure lowering effects through model prediction.

[0006] The enzymolysis preparation method of the milk source taste enhancing peptides provided by the present application comprises the following steps:

[0007] A) Enzymolysis of liquid dairy products by flavor protease and glutamine enzyme, and then enzyme inactivation to obtain an enzyme inactivated product;

[0008] B) Acidification centrifugation, ultrafiltration, and freeze-drying of the separated effluent to obtain a freeze-dried powder;

[0009] C) Sample loading and G-15 glucose gel column separation of the freeze-dried powder.

[0010] D) The application adopts a composite enzymatic hydrolysis method to improve the flavor level of milk, mainly solving the problem that the existing lipase hydrolysis flavor enhancement technology may produce medium and long chain bitter fatty acids to a certain extent. In high-protein milk powder samples, enzymatic hydrolysis can produce healthy and taste-active substances. The existing protein enzymatic hydrolysis method mainly adopts single enzyme hydrolysis or double enzyme hydrolysis (endoenzyme + exoenzyme) combination method. The final product has bitter activity, which affects the sensory enjoyment and acceptance of consumers. The application proposes a combined enzymatic hydrolysis method (comprehensive improvement of skimmed milk powder flavor and production of more umami peptides) to improve the flavor level of milk, so as to make up for the flavor defects caused by the lack of fat.

[0011] The enzymatic preparation method of the milk source taste-enhancing peptide provided by the application first uses flavor protease and glutamine enzyme to hydrolyze skimmed milk products.

[0012] The liquid milk product in the application can be skimmed milk, low-fat milk, skimmed milk powder, low-fat milk powder, skimmed high-protein powder, etc. The fat content of the fat-containing milk is 0% to 3%.

[0013] The flavor protease in the application is 1000L; the added mass of the flavor protease is 1% to 5%; specifically, it can be 1%, 2%, 3%, 4%, or 5%.

[0014] The added amount of the glutamine enzyme is 3% to 8%. Specifically, it can be 3%, 4%, 5%, 6%, 7%, or 8%.

[0015] The flavor enzyme in the application is an exopeptidase that releases amino acids through N-terminal peptide bond hydrolysis. The glutamine enzyme GLU100 is extracted from Bacillus amyloliquefaciens through liquid submerged fermentation, refining, and extraction. It contains glutamic acid and gamma-glutamyl taste peptides, enhances the taste performance of food, improves the overall mouthfeel, enhances the solubility and emulsification of protein, and improves the adverse properties such as protein throat feeling and grit feeling.

[0016] The temperature of the enzymatic hydrolysis reaction in the application is 50℃, and the time is 1h.

[0017] Then, the enzyme is inactivated to obtain an inactivated product; the inactivation is performed at 90℃ for 10min.

[0018] The enzyme inactivation and sterilization can be performed simultaneously.

[0019] The inactivated product is subjected to acidification and centrifugation. The acidification and centrifugation include: adding 0.1M hydrochloric acid to acidify to pH 4.6, and high-speed centrifugation at 10000rmp for 50min.

[0020] After removing the macromolecular protein, the water-soluble small molecule compound is subjected to interception by a 10 kDa ultrafiltration cup, and the separated effluent is freeze-dried; namely, the supernatant obtained by centrifugation of the enzymatic sample is filtered by a 10 kDa ultrafiltration membrane, and the effluent is freeze-dried.

[0021] The freeze-drying parameters in the application are as follows: freezing to about -80 DEG C, vacuumizing to below 20 Pa, dissolving the freeze-dried powder in 5 mL deionized water, and separating by a glucose gel column to obtain the product.

[0022] The glucose gel column is glucose gel G-15.

[0023] The sample loading solvent is 5 mL water for re-dissolving the freeze-dried enzymatic product after ultrafiltration, the eluent is sterilized ultrapure water, the cut-off liquid is collected ( Figure 8 ), and sensory evaluation is performed on it ( Figure 9 ). The separated components with taste activity are selected for secondary mass spectrometry identification, flavor prediction is performed by using the existing machine learning model, the synthesized part is predicted to have umami activity, and the pure peptides with the peptide sequences of LSFD, EDIKQME, IKQMEAE, and TEDELQDK are obtained by primary mass spectrometry identification and purification. The sensory evaluation and verification show that the peptides have certain mellow taste and umami (Table 5).

[0024] The method of the application does not need to add additional buffer solution and maintain the pH of the buffer solution. In addition, the enzymatic stage in the application is based on self-processing to produce enzymatic products, which does not cause energy consumption problems at a certain level. The enzymatic products have good milk flavor and mellow flavor, and can enrich the taste level of milk. After the supernatant is separated by the ultrafiltration cup of the sample with good taste, it is found by glucose gel G-15 analysis that the second component separated by the glucose gel has better milk flavor and mellow flavor, and the first component has certain umami taste. The product obtained by enzymolysis has a more mellow and sweet taste than the existing commercially available sample. Compared with the original product, the product has certain antioxidant and other functional active peptides.

[0025] The separated components are subjected to sensory evaluation, and the peptides identified are subjected to taste activity and functional activity prediction by the UmamiCNN model, the Bitter-RF model, the toxinpred3 model, the in-vitro antioxidant model, the anti-inflammatory peptide PREDAIP model, and the ACE prediction peptide AHTPeptideFusion model.

[0026] The milk-derived taste-enhancing peptide provided by the application is prepared by the preparation method in the above technical solution.

[0027] In some embodiments, the method for preparing the milk-derived taste-enhancing peptide comprises one or more of the polypeptides having the amino acid sequence shown in SEQ ID NO: 1-54.

[0028] The milk-derived taste-enhancing peptide provided by the present application has an amino acid sequence shown in any one of SEQ ID NO: 13, 25, 53 or 54; or an amino acid sequence formed by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in any one of SEQ ID NO: 13, 25, 53 or 54; or an amino acid sequence having not less than 80% identity with the amino acid sequence shown in any one of SEQ ID NO: 13, 25, 53 or 54. The amino acid sequence of the milk-derived taste-enhancing peptide is LSFD, EDIKQME, IKQMEAE or TEDELQDK.

[0029] The milk-derived taste-enhancing peptide described above further comprises one or more of the polypeptides having the amino acid sequence shown in SEQ ID NO: 1-12, 14-24 and 26-52.

[0030] The milk-derived taste-enhancing peptide described above can be prepared by the method described above or obtained by artificial in vitro synthesis according to the peptide sequences of the polypeptides described above respectively or obtained by mixing after synthesis.

[0031] The in vitro synthesis method described by the present application includes one-by-one coupling or segmented coupling. The synthesis method includes liquid phase and / or solid phase synthesis, which is not limited by the present application.

[0032] The present application further provides the use of the milk-derived taste-enhancing peptide described in any one of the above in food additives.

[0033] The milk-derived taste-enhancing peptide described by the present application has a better natural cow milk taste and a rich caramel flavor when added to dairy products, which increases the flavor levels of skimmed milk.

[0034] Compared with traditional enzyme-degraded samples, the product of the present application does not produce bitterness, and compared with non-enzyme-degraded samples, the product of the present application enriches the flavor levels of original skimmed milk products, and the enzyme-degraded sample has a certain caramel sweet and thick sweet flavor.

[0035] The present application provides the use of the milk-derived taste-enhancing peptide described in any one of the above in antioxidation, anti-inflammation or blood pressure reduction.

[0036] The present application identifies the fraction with sensory activity, which has certain sweet and thick milk flavor and antioxidant, anti-inflammatory and blood pressure-lowering functional activity.

[0037] The application carries out FMOC solid-phase synthesis on several peptides with taste activity, and the sensory verification result shows that the LSFD, EDIKQME, IKQMEAE or TEDELQDK peptide has obvious umami and thick taste perception.

[0038] The application provides a product, which comprises the milk-derived taste-enhancing peptide according to any one of the technical solutions.

[0039] The product provided by the application comprises food, health food or medicine, etc.

[0040] The milk-derived taste-enhancing peptide also comprises a common auxiliary material in the field of food, which is not limited in the application and is well known to those skilled in the art.

[0041] The application uses low-fat or skimmed milk as a substrate, and adding compound enzymes helps to improve the reaction efficiency; the reaction condition is mild, so that the enzyme and the substrate can be fully contacted, and a plurality of enzymes are reasonably used and added, so that the release of umami and thick peptides is effectively promoted, the overall flavor of the dairy product is improved, and the taste of skimmed milk is enriched; the whole reaction does not need to add any reagent, and the whole enzymolysis reaction is carried out under the condition of milk source, in addition, the overall taste of skimmed milk is enriched by producing taste-active small peptides, the glucose gel G-15 filtrate component obtained by the best enzymolysis combination has a certain milk flavor and has umami taste activity. Finally, part of the taste activity is verified through solid-phase synthesis and sensory evaluation. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Preparation method and technical route of milk-derived taste umami peptide

[0043] Figure 2 Figure of sensory score of skimmed milk obtained by flavor enzyme enzymolysis ratio in example one

[0044] Figure 3 Figure of sensory score of skimmed milk obtained by flavor enzyme enzymolysis time in example one

[0045] Figure 4 Figure of sensory score of skimmed milk obtained by flavor enzyme enzymolysis temperature in example one

[0046] Figure 5 Figure of sensory score of skimmed milk obtained by flavor enzyme+glutamine enzyme enzymolysis ratio in example two

[0047] Figure 6 Figure of sensory score of skimmed milk obtained by flavor enzyme+glutamine enzyme enzymolysis time in example two

[0048] Figure 7 Figure of sensory score of skimmed milk obtained by flavor enzyme+glutamine enzyme enzymolysis temperature in example two

[0049] Figure 8 Unenzymolysis sample, comparison of Sephadex G-15 separation of flavor enzyme, flavor enzyme + glutamine enzyme under the optimal conditions of example one and three (CKY, FY, FGY represent unenzymolysis sample, flavor enzyme, flavor enzyme + glutamine enzyme after glucose gel separation, respectively)

[0050] Figure 9 Sensory evaluation of flavor enzyme + glutamine enzyme under the optimal conditions of example one and three.

[0051] Figure 10 Primary mass spectrum of purified peptide produced by flavor enzyme + glutamine enzyme. DETAILED DESCRIPTION

[0052] The present application provides a milk-derived taste-enhancing peptide, a preparation method and application thereof. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they all belong to the scope protected by the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0053] It should be understood that the expression "one or more of" includes each object described after the expression and various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0054] The use of the terms "include", "have" or "contain", including their grammatical synonyms, should generally be understood as open and non-limiting, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0055] In this application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B, which can represent the following cases: A exists alone, A and B exist together, and B exists alone. Wherein A, B can be singular or plural.

[0056] In this application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions means any combination of these items, including any combination of single item or multiple items.

[0057] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0058] The use of any and all examples, or exemplary language herein, e.g., "such as" or "including", is intended merely to better illustrate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0059] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are merely intended to convey general information as to the scope of the application. Consistent with the application as claimed, the numerical ranges and parameters should be construed in light of the number of requests in a particular test. Therefore, unless otherwise explicitly provided, it should be understood that all ranges, quantities, values and percentages used herein are modified by "about". Here, "about" generally means within 10%, 5%, 1%, or 0.5% of a particular value or range.

[0060] It should be understood that the size of the sequence number of the above-mentioned processes in various embodiments of the present application does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0061] Some cases are described in the examples and comparative examples of the present application, in which the examples show some implementations of the present application. However, this does not mean that the effect of the present application can only be achieved in these cases.

[0062] In order to further illustrate the present application, the milk-derived taste-enhancing peptide provided by the present application, its preparation method and application are described in detail below in combination with examples.

[0063] Example 1:

[0064] The method flow used is shown in Figure 1 The raw milk used is from lactating Holstein cows in a dairy farm.

[0065] 1. The skimmed milk powder of Holstein cow raw milk spray powder was weighed to 8.475 g and dissolved in 100 mL of water solution.

[0066] 2. In this example, single enzyme hydrolysis was carried out using flavor enzyme, and single factor variance analysis was carried out on the amount of flavor enzyme, hydrolysis temperature and hydrolysis time, and the amount of flavor enzyme for reconstitution was set to 1% to 5%.

[0067] 3. Under the optimal enzymolysis ratio of step 2, the enzymolysis time is optimized, and the enzymolysis time is set to 1 h, 2 h, 3 h, 4 h, and 5 h, respectively.

[0068] 4. Under the optimal enzymolysis ratio and time of steps 2 and 3, the enzymolysis temperature is optimized, and the enzymolysis temperature is set to 40°C, 45°C, 50°C, 55°C, and 60°C, respectively.

[0069] 5. After obtaining the optimal optimized enzymolysis conditions, the enzymolysis product obtained under the optimal conditions is subjected to glucose gel for G-15 separation, and the cut-off liquid is subjected to sensory evaluation. Figure 8 , 9)

[0070] Comparative Example: A same brand of defatted high-protein milk powder: protein content: 59%, fat content: 3%, and carbohydrate content: 18%. Compared with the defatted milk powder obtained in Example One, no enzymolysis process is involved, and other processes are the same as those in the preparation process of the defatted milk powder obtained in Example One.

[0071] Table 1. Taste-active peptides and functionally active peptides significantly up-regulated (compared with peptides identified in the unenzymolysis sample) in the first group of components of the flavor enzyme enzymolysis product in Example One

[0072]

[0073] Example Two:

[0074] 1. The method flow adopted is shown in Figure 1 , and the New Zealand original imported defatted milk powder is used.

[0075] 2. The existing defatted milk powder is weighed at 8.475 g and dissolved in 100 mL of water. The enzyme combination is flavor enzyme + glutamine enzyme, and is added according to different enzymolysis ratios, which are 3:1, 3:3, 3:5, 3:7, and 3:9, respectively. The samples with different ratios of combined enzyme enzymolysis are subjected to sensory evaluation.

[0076] 3. In step 2, the enzymolysis time is optimized, and the enzymolysis time is set to 1 h, 2 h, 3 h, 4 h, and 5 h, respectively.

[0077] 4. Under the optimal enzymolysis ratio and time of steps 2 and 3, the enzymolysis temperature is optimized, and the enzymolysis temperature is set to 40°C, 45°C, 50°C, 55°C, and 60°C, respectively.

[0078] Comparative Example: A commercially available defatted milk powder: protein content: 59%, fat content: 3%, and carbohydrate content: 18%. Compared with the defatted milk powder obtained in Example Two, no enzymolysis process is involved, and the defatted high-protein milk product used comes from the same production line and has the same production date.

[0079] Example Three:

[0080] 1. The method flow chart as shown below was adopted Figure 1 The raw milk used was imported from New Zealand.

[0081] 2. The raw milk collected was centrifuged or flash evaporated and powder was sprayed to adjust the fat content to low-fat milk powder.

[0082] 3. 100 mL of water was used to dissolve 8.475 g of milk powder, 3% flavor enzyme + 5% glutamine enzyme, the enzyme hydrolysis temperature was set to 50°C, after 3h of enzyme hydrolysis, the sample was inactivated at 90°C for 10 min. After inactivation, the enzyme hydrolysate was acidified and centrifuged to obtain the supernatant, which was ultrafiltered through a 10 kDa ultrafiltration membrane, and the effluent was freeze-dried.

[0083] 4. The freeze-dried components were dissolved in 5 mL of deionized water according to the amount of 10 mg. After loading, the sample was separated by glucose gel G-15, G15 is mainly used to separate small molecules with a molecular weight below 1500 Da. Molecules larger than the gel pore size cannot enter the pores inside the gel particles, but can only flow through the gaps between the particles. Therefore, their path is the shortest and they are eluted first from the chromatographic column (first peak), and the component collected after the peak is component one. Molecules smaller than the gel pore size can freely enter the pores of the gel particles. They take the longest path in the column and take more time to be eluted, so they are eluted last (late peak), which is component two; the above cut-off liquid components were collected for identification of peptidomics and comparison of other active ingredients.

[0084] Comparative example: commercially available skim milk powder: protein content: 59%, fat content: 3%, carbohydrate content: 18%. Compared with the skim milk powder obtained in Example Three, no enzyme hydrolysis process was involved, and the skim milk powder with high protein content used came from the same production line and had the same production date.

[0085] Table 2: Taste-active peptides and functional activity prediction significantly up-regulated in the first component separated by glucose gel in Example Three

[0086] Table 2: Taste-active peptides and functional activity prediction significantly up-regulated in the first component separated by glucose gel in Example Three

[0087] Table 3: Taste-active peptide sequences and functional activity prediction significantly up-regulated in the second component separated by glucose gel in Example Three

[0088]

[0089]

[0090] By optimizing the flavor enzyme combined with glutamine enzyme to hydrolyze the sample, acidify, centrifuge, freeze-drying, and pass through the gel column, two cut-off components are obtained, and the second component has a strong thick and milk flavor after sensory evaluation. Figure 9 The two cut-off components are identified by secondary mass spectrometry and the taste and functional activity are predicted to obtain the above taste and functional activity peptides. After the flavor enzyme combined with glutamine enzyme hydrolysis, the second component of the cut-off liquid produces more functional and taste active peptides.

[0091] Experimental Example

[0092] I. Comparison results of sensory evaluation

[0093] 1. Flavor enzyme hydrolysis optimization results obtained in Example 1

[0094] This study was conducted in a professional sensory evaluation laboratory. The sensory evaluation team consisted of 7 systemically trained sensory evaluators. All evaluators have completed a 12-month sensory evaluation training, which strictly follows the selection, training and examination of ISO8586:2012 international standard. The research plan was approved by the scientific research ethics committee of Beijing Technology and Business University, and all participants signed the informed consent form. The evaluation team established the sensory description of milk flavor, rich flavor, and preference for bitterness, and the sensory evaluation standard is shown in Table 4. The sample preparation followed the standard operating procedure: 20 mL of blank and enzyme-hydrolyzed sample was placed in a food-grade evaluation cup without odor (random three-digit code), and after 20 minutes of equilibrium to room temperature, it was presented to the sensory evaluation personnel in random code order.

[0095] Table 4. Sensory evaluation standard

[0096]

[0097] In Example 1, when the flavor enzyme hydrolysis ratio was 3%, the enzyme-hydrolyzed sample had the highest scores for rich flavor, caramel flavor, and preference, and the highest scores for rich flavor, caramel flavor, and preference were obtained when the enzyme hydrolysis time was 60 min. The lowest bitterness score was obtained when the enzyme hydrolysis was at 50°C (3, 4). Figure 2

[0098] 2. Flavor enzyme + glutamine enzyme complex hydrolysis optimization results obtained in Example 2

[0099] In Example 2, the sensory evaluation score of the sample hydrolyzed by 3% flavor enzyme (F) + 5% glutamine enzyme (G) was 7.58 for rich flavor, and the caramel flavor and milk flavor scores under this hydrolysis condition were also high, and there was no significant difference compared with bitterness. The scores for rich flavor, caramel flavor, and preference were higher when the complex enzyme hydrolysis time was 3h, and the bitterness score had no significant difference with the blank group, so the sample produced by the combination of 3% F + 5% G enzyme hydrolysis was selected as the optimal one for sensory evaluation.​Figure 5 , 6, 7).

[0100] II. Comparison of non-volatile differentially expressed peptide components after glucose gel separation from samples obtained by enzymatic hydrolysis

[0101] 1. Separation of flavor enzyme + glutaminase hydrolysate components

[0102] In Example 3, the combination of 3% flavor enzyme and 5% glutaminase was enzymatically hydrolyzed at 50°C for 3 hours. The hydrolyzed sample was then subjected to enzyme inactivation at 90°C for 10 minutes. After enzyme inactivation, the supernatant was collected by centrifugation and ultrafiltered through a 10 kDa ultrafiltration membrane. The resulting eluent was freeze-dried, and 10 mg of the sample was loaded. After separation by glucose gel G-15, the resulting distillate was subjected to sensory evaluation, especially regarding the richness and fullness of the flavor. Components with a rich flavor were selected for freeze-drying.

[0103] 2. Peptide extraction and targeted assay

[0104] Centrifuge 1 mL of sample and collect the supernatant. Take 500 μL and fractionate using a 3 kDa ultrafiltration tube. Add 10 mM DTT sequentially and incubate at 56°C for 60 minutes. DTT is a strong reducing agent that breaks the disulfide bonds (SS bonds) between cysteine ​​residues in proteins or peptide chains, generating free thiol groups (-SH). Add 20 mM IAM and incubate in the dark for 40 minutes for alkylation. The purpose of alkylation is threefold: to stabilize the protein / peptide structure; to improve the quality of mass spectrometry data; and to ensure the reliability and reproducibility of experimental results. Stop the reaction with 10 mM DTT. Finally, desalt using a C18 column and concentrate by vacuum centrifugation.

[0105] Non-targeted peptidomics analysis was performed using a nanofluidic liquid chromatography system (Easy nLC 1200) coupled with orbital trap mass spectrometry (Orbitrap Exploris). TM 480 is a highly efficient and sensitive combination of technologies suitable for deep coverage and high-precision analysis of peptides in complex biological samples. Chromatographic conditions: Acclaim PepMap RSLC C18 column (75 μm × 15 cm), mobile phase A (0.1% formic acid aqueous solution) and B (0.1% formic acid acetonitrile solution), flow rate 300 nL / min, gradient elution program: 4%–8% B (0–2 min), 8%–28% B (2–35 min), 28%–40% B (35–55 min), 40%–95% B (55–56 min), 95% B isocratic elution (56–66 min). Mass spectrometry parameters: positive ion mode, primary scan range m / z 100–1500, resolution 120,000; secondary scan resolution 15,000, collision energy 30 eV, data-dependent acquisition mode. The identified peptides were subjected to molecular weight statistics, differential analysis, and prediction of taste-active peptides.

[0106] 3. Sensory evaluation results of separated components

[0107] The flavor enzyme + glutaminase enzyme hydrolysis combination hydrolysis sample produced relatively smaller molecular weight, and the glucose gel G-15 separation was performed, and the obtained components had certain milk flavor and mouthfeel effect. The flavor enzyme + glutaminase enzyme hydrolysis obtained the effect of umami taste better than the flavor enzyme, and then the glucose gel separation obtained components were selected for further identification. The peptide segments produced by flavor enzyme hydrolysis and the peptide segments produced by flavor enzyme + glutaminase enzyme combined hydrolysis were subjected to differential comparison analysis of peptide segments and CK, and it was found that the flavor enzyme + glutaminase produced more small molecule taste active peptides less than 1000 Da (Tables 2 and 3). The flavor enzyme hydrolysis produced 11 flavor peptides with taste characteristics, and only 3 were less than 1000 Da (Table 1). The first component after the flavor enzyme + glutaminase enzyme hydrolysis and glucose gel separation had 12 peptides predicted to have umami taste characteristics (within 1000 Da), and the second component had more umami active peptides (42). The active small molecule peptides produced by non-hydrolysis, flavor enzyme hydrolysis and flavor enzyme + glutaminase enzyme hydrolysis were subjected to functional activity prediction, and the flavor enzyme combined glutaminase hydrolysis produced more antioxidant active peptides and ACE peptides. In summary, the flavor enzyme combined glutaminase enzyme hydrolysis combination produced more taste active small molecule peptides with functions and tastes.

[0108] 4. The following four peptides were not retrieved in the database and were new milk-derived taste active peptides.

[0109] For the predicted peptides, we synthesized the following four peptides by FMOC solid-phase synthesis, predicted taste peptides, and performed descriptive sensory evaluation (Table 5) on them. LSFD, EDIKQME, IKQMEAE, and TEDELQDK.

[0110] The specific experimental procedures and experimental results are as follows:

[0111] 4.1 Peptide synthesis and preparation

[0112] (1) According to the first amino acid at the C-terminal of the polypeptide sequence, 0.5 mmol of the corresponding Fmoc protected amino acid-Wang Resin was selected and added to the solid-phase reactor. After swelling the resin in DCM for 30 min, it was dried and washed with DMF three times. 20% piperidine in DMF was added, reacted for 5 min, and then 20% piperidine in DMF was added again and reacted for 10 min. In between, DMF was washed once. After the reaction was completed, it was dried, washed with DMF three times, and then dried.

[0113] (2) According to the sequence of the polypeptide from C-terminal to N-terminal, the condensation reaction and Fmoc removal reaction are alternately carried out in the order of 1.5 mmol reaction amount, and all the remaining amino acids of the polypeptide are condensed onto the resin. After the condensation of the last amino acid is completed, 20% hexahydro pyridine DMF solution is added, reacted for 5 min, 20% hexahydro pyridine DMF solution is added again, reacted for 10 min, washed once with DMF in the middle, and then dried by suction, washed with DMF for 3 times.

[0114] (3) 1 mmol of FITC and N-methyl morpholine are added to the reactor, reacted for 5-10 min, and whether the reaction is complete is detected by ninhydrin. After the reaction is complete, the reaction system is washed with DMF and DMC alternately for 3 times, and then washed with methanol to shrink and obtain the dry polypeptide-resin.

[0115] (4) The polypeptide-resin is placed in a round-bottom flask, and the prepared cleavage solution (the reagent formula of the cleavage solution is TFA: benzyl thioether: phenol: triisopropyl silane: water = 82.5:7.5:5:3:2) is slowly added at 0°C and slowly stirred. The reaction is carried out at low temperature for 0.5 h and at room temperature for 2 h. The cleavage solution is obtained by suction filtration, slowly added to anhydrous ethyl ether and stirred, and the crude polypeptide is separated by filtration and washed with ethyl ether for 3 times to obtain the crude peptide.

[0116] (5) The molecular weight of the crude product is detected by mass spectrometry. If correct, the crude product is purified by high performance liquid chromatography, freeze-dried, and then the pure polypeptide is obtained.

[0117] Figure 10 The synthesized peptide is identified by mass spectrometry and purified by liquid chromatography. The liquid chromatography mass spectrometry uses isocratic elution program, Buffer A: 0.1% acetic acid aqueous solution, Buffer B: 0.1% acetic acid acetone solution, and the flow rate is 0.2 mL / min. After the molecular weight of the mass spectrometry is consistent with the measured molecular weight, the preparation is carried out by liquid chromatography purification. The liquid chromatography column uses 4.6*250 mm, kromasil C18-5 chromatography column, solvent A is 0.1% acetic acid acetone solution, solvent B is 0.1% acetic acid aqueous solution, the separation program of the liquid chromatography is 0.01 min: 5% A, 95% B, 25 min: 50% A, 50% B, 30 min: 90% A, 10% B, the flow rate is 1 mL / min, the wavelength is 214 nm, and the injection amount is 20 μL. The above is the detection signal verification and purification preparation of the mass spectrometry of the new milk-derived taste active peptide. After the tastepeptides-meta taste active peptide database detection, the above is the new milk-derived taste active peptide.

[0118] 5. Sensory verification of the milk-derived taste active peptide

[0119] The descriptive sensory evaluation includes acid, sweet, bitter, salty, umami, mouthful, continuity and fullness. The evaluation intensity 7-point scale method is used from 1 (not perceptible) to 7 (strongly perceptible), and the specific sensory evaluation standard is shown in Table 4. The peptide solution of 1 mg / mL is prepared, and the sensory evaluation personnel take 2 mL each time for the descriptive sensory evaluation. The thick feeling refers to the feeling that the sample fills the mouth and continuously enhances, which means richness and fullness.

[0120] Table 5 Descriptive sensory evaluation of synthetic peptides

[0121]

[0122] The verification results of the descriptive sensory evaluation of the synthetic peptides are shown in Table 5, and the four new peptides have obvious umami, mouthful, continuity and fullness. It is proved that through the enzymatic preparation scheme and the separation and purification procedure, the effective milk taste active peptides can be obtained, and the taste of the skimmed milk product is enriched.

[0123] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for the enzymatic hydrolysis preparation of milk-derived taste-enhancing peptides, characterized in that, Includes the following steps: A) Liquid dairy products are hydrolyzed by a combination of flavor protease and glutaminase, and then the enzymes are inactivated to obtain the enzyme-inactivated product. B) After enzyme inactivation, the product is subjected to acidification and centrifugation. The supernatant is collected, ultrafiltered, and the separated effluent is lyophilized to obtain lyophilized powder. C) Load the lyophilized powder onto a glucose gel column for separation, evaluate the taste-active components by sensory evaluation, and freeze-dry to obtain the final product.

2. The preparation method according to claim 1, characterized in that, The flavor protease is 1000L; the addition mass of flavor protease is 1% to 5%; the addition mass of glutaminase is 3% to 8%; the temperature of the enzymatic hydrolysis reaction is 20 to 55℃, and the time is 1 to 5 hours; the enzyme inactivation is carried out at 90℃ for 10 minutes.

3. The preparation method according to claim 1, characterized in that, The acidification centrifugation includes: adding 0.1M hydrochloric acid to acidify the system to pH 4.6, and centrifuging at 10000 rpm for 50 min; The ultrafiltration process includes separation through a 10 kDa ultrafiltration cup, followed by freeze-drying of the separated effluent. The glucose gel column is Sephadex G-15 glucose gel.

4. Milk-derived taste-enhancing peptides, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 4.

5. The milk-derived taste-enhancing peptide according to claim 4, characterized in that, One or more of the polypeptides comprising the amino acid sequences shown in SEQ ID NO:1 to 54.

6. A milk-derived taste-enhancing peptide having an amino acid sequence as shown in any one of SEQ ID NO:13, 25, 53 or 54; or an amino acid sequence formed by substituting, deleting or adding one or more amino acids in an amino acid sequence shown in any one of SEQ ID NO:13, 25, 53 or 54; or an amino acid sequence having at least 80% identity with an amino acid sequence shown in any one of SEQ ID NO:13, 25, 53 or 54.

7. The milk-derived taste-enhancing peptide according to claim 6, characterized in that, It also includes one or more polypeptides with the amino acid sequences shown in SEQ ID NO:1~12, 14~24, 26~52.

8. The use of the milk-derived taste-enhancing peptide according to any one of claims 5 to 7 as a food additive.

9. The use of the milk-derived taste-enhancing peptide according to any one of claims 5 to 7 in anti-oxidation, anti-inflammatory and / or antihypertensive effects.

10. A product characterized in that, Includes the milk-derived taste-enhancing peptides as described in any one of claims 5 to 7.

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