Preparation method of casein peptide composition based on lactoalbumin hydrolysate

Abstract

The invention discloses a preparation method of a casein peptide composition based on lactoalbumin hydrolysate, and provides the preparation method of the casein peptide composition based on the lactoalbumin hydrolysate, which comprises the following steps: an enzymolysis step: carrying out enzymolysis on a casein solution by adopting compound protease; wherein the compound protease comprises serine protease and flavourzyme; wherein in every 100 g of the casein liquid, the content of dry substances is 5-15 g; the serine protease is added into the casein liquid, the serine protease is added into the casein liquid, 75-100 g of protein is contained in every 100 g of dry matter of the casein liquid, 75-100 g of casein is contained in every 100 g of protein, and the adding amount of the serine protease is 0.0001-0.015% by mass according to the mass of the casein liquid. The product obtained by the preparation method disclosed by the invention is weaker in bad volatile smell, can also retain more nutritional ingredients, and can also obtain good brewing property.

Description

Technical Field

[0001] This invention belongs to the field of proteolytic technology, and more specifically, relates to a method for preparing a casein peptide composition based on hydrolyzed milk protein. Background Technology

[0002] In recent years, my country's dairy processing industry has developed rapidly. Due to the vast market for infant formula and the huge consumption of whey protein, casein has become an important byproduct of the dairy processing industry. In my country, influenced by long-standing dietary culture, the cheese market, the primary consumer of casein, is still in its early stages of development. Therefore, many researchers and companies have turned their attention to the field of casein peptides. Currently, the most mature casein peptide products on the market are mainly casein phosphopeptides used in infant formula, along with smaller quantities of casein ACE inhibitory peptides and casein sleep peptides.

[0003] However, casein is highly unstable during hydrolysis, easily forming casein micelles. Casein hydrolysis also exposes a large amount of hydrophobic amino acids, resulting in an extremely bitter taste and producing numerous undesirable volatile gases such as 1-octen-3-one, (Z)-2-hexanal, 1-octanal, and heptanal. Furthermore, in large-scale industrial production, current technologies commonly employ prolonged deep hydrolysis to increase the phosphopeptide content of casein, followed by purification of small peptides using membrane filtration and chromatography.

[0004] Reference 1 discloses a low-bitterness casein hydrolysate, its preparation method, and its application. The preparation method involves adding casein to water and dispersing it evenly to obtain a casein dispersion; adjusting the pH of the casein dispersion to 6.0-9.0, adding an appropriate amount of protease for restrictive enzymatic hydrolysis; inactivating the enzyme, centrifuging, and drying the supernatant to obtain the casein hydrolysate.

[0005] Reference 2 discloses an industrial manufacturing method for low-allergenic casein peptide powder containing CPPs and its application. The method uses food-grade casein as raw material and employs more than 8 kinds of compound enzymes to perform combined enzymatic hydrolysis in two groups to obtain casein peptide powder. The CPPs content of the casein peptide powder is more than 15%.

[0006] Reference 3 discloses a non-bitter casein phosphopeptide cyclodextrin inclusion complex and its preparation method. This method mainly employs an encapsulation process to encapsulate the exposed bitter amino acids in casein phosphopeptides. The raw materials are composed of the following ingredients in a weight ratio: casein phosphopeptide: cyclodextrin = 1:0.1-30. Through the inclusion effect of cyclodextrin, a supramolecular inclusion complex is formed, shielding the bitter groups and producing a non-bitter casein phosphopeptide inclusion complex.

[0007] Reference 4 discloses a method for preparing yak casein phosphopeptides. The method involves dissolving yak casein in water, then enzymatically hydrolyzing the yak casein solution using a two-step enzymatic hydrolysis method. After the enzymatic hydrolysis is completed, the hydrolysate is purified using a centrifuge in conjunction with a multi-stage membrane separation system to obtain a yak casein phosphopeptide solution. Finally, the yak casein phosphopeptide solution is rapidly dried by spray drying to obtain yak casein phosphopeptides.

[0008] For example, existing technologies cited in references 1-4 generally employ prolonged enzymatic hydrolysis followed by purification of small molecule peptides using methods such as membrane filtration and chromatography. The application of these technologies can reduce the generation of bitterness and unpleasant odors to some extent, and currently, the solubility of the product and the production cost of the process both need improvement. Further research and improvement are needed to enhance the bitter taste and unpleasant volatile odors during large-scale industrial production; optimize reconstitution performance; and reduce production costs to meet application requirements.

[0009] References

[0010] Reference 1: CN116268174A

[0011] Reference 2: CN104046673A

[0012] Reference 3: CN101147800A

[0013] Reference 4: CN105385738A Summary of the Invention

[0014] The problem the invention aims to solve

[0015] In existing technologies, casein hydrolysis is highly unstable and readily forms casein micelles. Casein hydrolysis exposes a large amount of hydrophobic amino acids, resulting in an extremely bitter taste and producing numerous undesirable volatile gases such as 1-octen-3-one, (Z)-2-hexanal, 1-octanal, and heptanal. Furthermore, in large-scale industrial production, existing technologies commonly employ prolonged, deep hydrolysis to increase the phosphopeptide content of casein, followed by purification of small peptides using membrane filtration and chromatography.

[0016] Therefore, deep hydrolysis also presents some challenges. For instance, it leads to more protein cleavage, increasing the exposure of bitter end groups or bitter substances. For example, reference 1 attempted to reduce bitterness, but its process is cumbersome, requiring centrifugation after enzymatic hydrolysis, collection of the supernatant, and further physical screening of the product. Reference 4 uses a centrifugation-assisted multi-stage membrane separation system to purify yak casein phosphopeptides, but this process is also cumbersome, with long enzymatic hydrolysis times, and membrane separation increases bitter components. Reference 2 employs prolonged deep hydrolysis, which is complex and inefficient. Reference 3 uses an encapsulation process to encapsulate exposed bitter amino acids in casein phosphopeptides, attempting to reduce bitterness. Furthermore, while separation methods exist to remove bitterness, these also lead to nutrient loss.

[0017] In addition, deep hydrolysis may result in poor solubility and severe agglomeration of the final product.

[0018] Therefore, the present invention mainly provides a casein hydrolysis method and the obtained hydrolysate. The method, by controlling the composition of the casein solution and the use of specific enzymes, can not only suppress undesirable flavors of the product, but also obtain good reconstitution properties.

[0019] Solution for solving the problem

[0020] [1]. A method for preparing a casein peptide composition based on hydrolyzed milk protein, the method comprising: an enzymatic hydrolysis step, wherein a complex protease is used to hydrolyze casein solution; wherein the complex protease comprises a serine protease and a flavor protease; wherein the dry matter content is 5-15g per 100g of casein solution; the casein solution contains 75-100g of protein per 100g of dry matter, and each 100g of protein contains 75-100g of casein, and the amount of serine protease added is 0.0001-0.015g by mass based on the mass of casein solution.

[0021] [2]. According to the preparation method described in [1], the flavor protease includes Aspergillus oryzae flavor protease and Aspergillus niger flavor protease; the serine protease includes Bacillus licheniformis serine protease.

[0022] [3]. According to the preparation method described in [2], based on the mass of the casein liquid: the amount of Bacillus licheniformis serine protease added is 0.0001-0.015% by mass; the amount of Aspergillus oryzae flavor protease added is 0.001-0.03% by mass; and / or the amount of Aspergillus niger flavor protease added is 0.0001-0.01% by mass.

[0023] [4]. According to any one of the preparation methods described in [1] to [3], each 100g of dry matter of the casein solution contains 0-15g of fat.

[0024] [5]. According to any one of [1] to [4], the casein liquid is obtained by separating it from the casein raw material by a membrane separation method; optionally, the membrane separation method uses a ceramic membrane or a spiral wound membrane; preferably, after obtaining the casein liquid from the casein raw material by the membrane separation method, it is pasteurized.

[0025] [6]. According to any one of [1] to [5], in the enzymatic hydrolysis step, the hydrolysis time is less than 60 min; before hydrolyzing the casein solution, the pH of the casein solution is adjusted to 7.5-8; and the hydrolysis temperature does not exceed 62°C.

[0026] [7]. According to any one of [1] to [6], before the end of the enzymatic hydrolysis step, the pH of the reaction solution in the enzymatic hydrolysis step is adjusted to 7-7.5.

[0027] [8]. The preparation method according to any one of [1] to [7] further includes a post-processing step, wherein the post-processing step includes at least one of enzyme inactivation treatment, concentration treatment and drying treatment.

[0028] [9]. A casein peptide composition, which is a composition prepared by any one of the preparation methods described in [1] to [8].

[0029]

[10] . According to the casein peptide composition of [9], the total number of peptides in the casein peptide composition is such that the sum of peptides with a hydrophobic amino acid at the C-terminus, peptides with a basic amino acid at the N-terminus, and peptides with a hydrophobic amino acid at the C-terminus and a basic amino acid at the N-terminus is 85% or less.

[0030]

[11] . Use of a casein peptide composition prepared according to any one of the preparation methods described in [1] to [8] or a casein peptide composition described in [9] or

[10] in the preparation of food or health products.

[0031]

[12] . A formulated dairy product, the formulated dairy product comprising:

[0032] (A) A casein peptide composition prepared according to any one of the preparation methods described in [1] to [8], or a casein peptide composition according to [9] or

[10] , and one or more of the following additional components: (B) a functional protein component, (C) a carbohydrate component, (D) a fat component, (E) a mineral salt component, and (F) other nutritional supplement components.

[0033] The effects of the invention

[0034] 1. Less bitterness: This invention eliminates the need for additional processes such as encapsulation and ultrafiltration. It utilizes a complex protease-directed enzymatic hydrolysis and precise enzymatic control technology to convert some bitter L-type aromatic amino acids, basic amino acids, and branched-chain amino acids into corresponding amino acid derivatives. This minimizes the number of protein peptides with hydrophobic C-termini and basic N-termini, thus reducing the product's bitterness. The total bitter amino acid content in this invention's product is less than 80%, while the total bitter amino acid content in commercially available products is greater than 90%. The acidic bitterness of this invention's product is significantly lower than that of commercially available products.

[0035] 2. The unpleasant volatile odor is weaker. This invention further reduces the generation of fishy and odorous compounds such as 1-octen-3-one, (Z)-2-hexanal, (E)-2-decenal, 2,4-heptadienal, 1-octen-3-ol, hexanol, 1-octanal, heptanal, n-pentadienal, and n-pentanal by using a compound protease-directed enzymatic hydrolysis technology. Compared with existing products, the product of this invention has a significant effect in reducing unpleasant volatile odor.

[0036] 3. This invention does not require additional processes such as membrane filtration or chromatography. It can prepare products with high content of casein phosphopeptide (CPP) simply by using compound protease-directed enzymatic hydrolysis and precise enzymatic hydrolysis control technology.

[0037] 4. Better dissolving properties: The product of this invention has better solubility, with a wettable wetting time of less than 32 seconds. After dissolving and standing, there is no sediment or obvious insoluble particles visible to the naked eye. In contrast, the wettable wetting time of existing commercially available products is greater than 60 seconds, and after dissolving and standing, there is obvious sediment, visible insoluble particles, and white spots sticking to the wall. The dissolving effect of the product of this invention is significantly better than that of existing commercially available products. Attached Figure Description

[0038] Figure 1 The figure shows a schematic spectrum of the three-dimensional GC-IMS results for volatile components. The three axes in the figure represent the relative migration time (X-axis), retention time (Y-axis), and signal peak intensity (Z-axis), respectively.

[0039] Figure 2 The image shows a top-down view of the two-dimensional GC-IMS results for volatile components. The red vertical line at 1.0 on the horizontal axis represents the RIP peak (reacting ion peak, normalized). The vertical axis represents the retention time (s) of the gas chromatography, and the horizontal axis represents the relative migration time (normalized).

[0040] Figure 3 This is a schematic spectral representation of the GC-IMS difference results for volatile components.

[0041] Figure 4This is a schematic diagram of the fingerprint spectrum results for volatile components.

[0042] Figure 1-18 shows volatile substances that were detected but not identified in the GC-IMS database.

[0043] Figure 5 This is a schematic diagram of the PCA score results for volatile components. Detailed Implementation

[0044] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0045] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0046] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0047] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0048] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0049] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0050] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, while specific related values ​​have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by "about". Here, "about" generally means that the actual value is within ±3%, ±2%, ±1%, or ±0.5% of a specific value or range.

[0051] In this invention, the term "infant" is used to refer to the human group aged 0 to 6 months.

[0052] In this invention, the term "older infant" refers to the human group aged 6 to 12 months.

[0053] In this invention, the term "infant" is used to refer to the human group aged 12 to 36 months.

[0054] In this invention, the term "infant" refers to the human group under the age of 3 years.

[0055] In this invention, the term "children" refers to a group of human beings who are older than 3 years and younger than 12 years and are in the growth and development stage.

[0056] In this manual, the term "adult" refers to a person who is 18 years of age or older.

[0057] In this manual, the term "teenager" refers to people aged 7-40.

[0058] In this manual, the term "middle-aged person" refers to a person aged 41-65.

[0059] In this manual, the term "elderly person" or "senior citizen" refers to a person aged 65 or older. In this manual, the numerical range indicated by "above" or "below" refers to a range that includes the stated number.

[0060] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0061] All unit names used in this manual are international standard unit names.

[0062] First aspect

[0063] The first aspect of the present invention provides a method for preparing a casein peptide composition based on hydrolyzed milk protein, comprising: an enzymatic hydrolysis step, wherein a casein solution is hydrolyzed using a complex protease; wherein the complex protease comprises a serine protease and a flavor protease; wherein the dry matter content is 5-15g per 100g of the casein solution; the casein solution contains 75-100g of protein per 100g of dry matter, and each 100g of the protein contains 75-100g of casein; and the amount of serine protease added is 0.0001-0.015g by mass of the casein solution.

[0064] The casein peptide composition prepared by the method for preparing casein peptide composition based on hydrolyzed milk protein provided by the present invention has a milder bitterness, a weaker unpleasant volatile odor, and better reconstitution properties, thus achieving a better enzymatic hydrolysis effect.

[0065] (Casein solution)

[0066] In this specification, casein is a phosphorus- and calcium-bound protein that is sensitive to acid and precipitates at low pH levels. Casein is the main protein in the milk of mammals, including cows, sheep, and humans, and is also known as casein, casein gluten, or lactose.

[0067] There are no particular restrictions on the source of casein in this invention. For example, it can be casein derived from various animal milks, such as cow's milk, sheep's milk, horse's milk, camel's milk, etc.

[0068] In some embodiments, the casein solution can be obtained by membrane separation from casein raw materials (e.g., milk). Casein solution obtained by membrane separation can be combined with subsequent enzymatic hydrolysis using complex proteases to achieve better enzymatic hydrolysis results. Conversely, casein solutions prepared by enzymatic hydrolysis or acid hydrolysis can affect the subsequent combined action mechanism with complex protease hydrolysis, impacting the hydrolysis effect and failing to achieve the effects of the present invention, such as less bitterness, weaker unpleasant volatile odors, and better reconstitution.

[0069] In this invention, the membrane separation method refers to the use of a filter membrane with a suitable pore size to retain and separate protein components of different molecular weights, thereby obtaining the casein solution of this invention. In some specific embodiments, the membrane separation method uses a ceramic membrane or a spiral wound membrane for membrane separation; that is, the casein solution of this invention can be a casein solution prepared using a ceramic membrane or a spiral wound membrane via a membrane separation method.

[0070] In some specific implementations, the membrane separation method is disclosed in the literature: Li Zhibin. Study on membrane separation preparation of active whey protein and casein micelles from bovine milk [D]. Jiangnan University, 2021. DOI:10.27169 / d.cnki.gwqgu.2021.001167.

[0071] In some embodiments, the casein raw material for preparing the casein liquid of the present invention can be milk, dairy products or any other raw material (sample) containing casein, such as a food sample.

[0072] In some preferred embodiments, the casein solution is also pasteurized (e.g., pasteurized by a pasteurization system) before the enzymatic hydrolysis step (e.g., after being separated from casein raw materials (e.g., milk) by membrane separation methods and before enzymatic hydrolysis).

[0073] In some specific implementations, the pasteurization treatment is carried out at a sterilization temperature of 75-85°C and a sterilization holding time of 15-30 seconds.

[0074] In some specific implementation schemes, the pasteurized material is cooled to 4-6°C and temporarily stored in an enzymatic hydrolysis tank.

[0075] In this invention, pasteurization not only eliminates the interference of the original active protease in the casein solution obtained by membrane separation, but also allows the casein to undergo moderate and slight denaturation, causing the protein spatial structure to unfold, which is beneficial to improving the subsequent enzymatic hydrolysis effect.

[0076] In some embodiments, the casein solution contains 0-15g of fat per 100g of dry matter.

[0077] In some preferred embodiments, the casein solution of the present invention has a dry matter content of 8-14 g / 100 g, a protein content (on a dry basis) of 80-90 g / 100 g, a casein content (accounting for 75-88 g / 100 g of protein), and a fat content (on a dry basis) of 0-10 g / 100 g.

[0078] In some preferred embodiments, the casein solution of the present invention has a dry matter content of 9-13 g / 100g, a protein content (on a dry basis) of 83-89 g / 100g, a casein content (as a percentage of protein) of 81-87 g / 100g, and a fat content (on a dry basis) of 0-4 g / 100g. For example, the casein solution has a dry matter content of 10.0 g / 100g, a protein content (on a dry basis) of 84.0 g / 100g, a casein content (as a percentage of protein) of 82.0 g / 100g, and a fat content (on a dry basis) of 3.6 g / 100g; or a casein solution has a dry matter content of 12.8 g / 100g, a protein content (on a dry basis) of 9-13 g / 100g, a protein content (on a dry basis) of 83-89 g / 100g, a casein content (as a percentage of protein) of 81-87 g / 100g, and a fat content (on a dry basis) of 0-4 g / 100g. The casein liquid has a dry matter content of 86.9g / 100g, with casein (as a percentage of protein) at 84.5g / 100g and fat (on a dry basis) at 1.0g / 100g; the casein liquid has a dry matter content of 12.3g / 100g, with protein (on a dry basis) at 85.2g / 100g, casein (as a percentage of protein) at 83.9g / 100g and fat (on a dry basis) at 2.4g / 100g; and the casein liquid has a dry matter content of 12.5g / 100g, with protein (on a dry basis) at 87.2g / 100g, casein (as a percentage of protein) at 85.8g / 100g and fat (on a dry basis) at 0.296g / 100g.

[0079] Casein solutions that meet the above criteria are more suitable for use with complex proteases for hydrolysis, resulting in better hydrolysis effects.

[0080] (Complex protease)

[0081] In some embodiments, the complex protease of the present invention includes serine proteases and flavor proteases.

[0082] serine protease

[0083] In this invention, serine proteases are a family of proteases whose function is to break peptide bonds in large proteins, reducing them to smaller proteins. Serine proteases are widely found in eukaryotes and prokaryotes. Based on their structure, serine proteases are divided into two main categories: chymotrypsin-like proteases or subtilisin-like proteases.

[0084] In some specific embodiments, the serine protease includes chymotrypsin-like protease.

[0085] In some specific implementations, the serine protease is an endopeptidase.

[0086] In some specific implementations, the serine protease is derived from microorganisms.

[0087] In some more specific embodiments, the serine protease is a serine protease derived from Bacillus licheniformis, also referred to herein as Bacillus licheniformis serine protease.

[0088] Serine proteases, such as Bacillus licheniformis serine protease, are commercially available to those skilled in the art. For example, Novogene (…). CTL 300BG), but not limited to this. Regarding the amount of serine protease used, in some preferred embodiments, the amount of serine protease added is 0.0001-0.015% by mass of the casein solution.

[0089] Flavor proteases

[0090] In this invention, flavor protease, or simply flavor enzyme, refers to a complex enzyme derived from microorganisms such as Aspergillus. It can not only cleave peptide bonds between amino acids within a peptide chain, like an endonuclease, to generate smaller peptide segments and amino acids, but also cleave amino acids from the N-terminus or C-terminus of a peptide chain, like an exonuclease, releasing specific amino acids.

[0091] In some embodiments, the flavor protease includes a flavor protease derived from Aspergillus oryzae (also referred to as Aspergillus oryzae flavor protease in this invention) and / or a flavor protease derived from Aspergillus niger (also referred to as Aspergillus niger flavor protease in this invention).

[0092] Aspergillus oryzae flavor protease is commercially available to those skilled in the art, for example, from Novo Neogene (…). Food-grade flavor enzymes such as 500mg and those sold by Nanning Pangbo Biotechnology Co., Ltd., but not limited to these.

[0093] Similarly, those skilled in the art can commercially obtain Aspergillus niger flavor protease, for example, from Sternzym Food Ingredients (Suzhou) Co., Ltd. (Sternzym FP 23290), but are not limited thereto.

[0094] In some specific embodiments, the complex protease of the present invention includes: Bacillus licheniformis serine protease, Aspergillus oryzae flavored protease, and Aspergillus niger flavored protease.

[0095] In this invention, the combined use of the aforementioned complex proteases results in a casein peptide composition with a milder bitterness, a weaker unpleasant volatile odor, and better reconstitution properties. Especially when combined with casein liquid that meets the above-mentioned specifications, even better enzymatic hydrolysis results are achieved.

[0096] In this invention, the casein solution is hydrolyzed using the aforementioned complex protease, resulting in a peptide distribution in the obtained casein peptide composition that is less bitter, has a weaker unpleasant volatile odor, and is better for reconstitution:

[0097] The proportion of peptides with a C-terminus hydrophobic amino acid, an N-terminus basic amino acid, and a C-terminus hydrophobic amino acid and an N-terminus basic amino acid, based on the total number of peptides in the casein peptide composition, is 85% or less.

[0098] In some specific embodiments, the amount of serine protease, especially Bacillus licheniformis serine protease, added based on the weight of the casein solution is 0.0001-0.015% by mass, preferably 0.0005-0.005% by mass, more preferably 0.001-0.003% by mass, and even more preferably 0.001215-0.001836% by mass, for example: 0.001215%, 0.001836%, 0.001377%.

[0099] The addition of the above-mentioned Bacillus licheniformis serine protease can further result in a casein peptide composition with a lower content of bitter amino acids and a weaker unpleasant volatile odor.

[0100] In some specific embodiments, the amount of Aspergillus niger flavor protease added, based on the weight of the casein solution, is 0.0001-0.01% by mass, preferably 0.0005-0.005% by mass, more preferably 0.001-0.003% by mass, and even more preferably 0.001096-0.001164% by mass, for example: 0.001096% by mass, 0.001164% by mass, and 0.001148% by mass.

[0101] In some specific embodiments, the amount of Aspergillus oryzae flavor protease added is 0.001-0.05% by weight of casein solution; preferably 0.01-0.05% by weight, more preferably 0.02-0.03% by weight, for example: 0.021212%, 0.026006%, 0.025698%.

[0102] The addition of the aforementioned complex protease ensures that enzymatic hydrolysis reaches the appropriate level without wasting enzyme or requiring enzyme inactivation in subsequent steps, or affecting the composition and nutritional components of the final casein peptide combination. Furthermore, it results in a milder bitterness, weaker unpleasant volatile odor, and better reconstitution of the obtained casein peptide composition. Especially when combined with casein solution that meets the above specifications, even better enzymatic hydrolysis results can be achieved.

[0103] Furthermore, in some embodiments of the present invention, there are no particular limitations on the method of adding various enzymes in the enzymatic hydrolysis of the complex protease. They can be added together at the beginning of the enzymatic hydrolysis reaction or added in batches to make the various enzymes adapt to the system temperature and pH value.

[0104] (Enzymatic hydrolysis)

[0105] Regarding the conditions for enzymatic hydrolysis of casein solution in this invention, the temperature and time of the enzymatic hydrolysis reaction, as well as the pH of the reaction system, can be determined based on the optimal pH and optimal temperature of the complex protease used.

[0106] In some preferred embodiments, the pH of the casein solution needs to be adjusted to 7.5-8 during the enzymatic hydrolysis step, preferably to 7.7-7.9.

[0107] In this invention, the pH of the casein solution can be adjusted by adding a pH adjuster. In some embodiments of this invention, the pH adjuster is selected from food-grade sodium bicarbonate, calcium hydroxide, potassium hydroxide, sodium hydroxide, or any combination thereof, preferably a sodium hydroxide or potassium hydroxide solution (e.g., an aqueous solution) with a concentration of 5-10% by mass, and more preferably an 8% sodium hydroxide or potassium hydroxide solution.

[0108] In some preferred embodiments, the enzymatic hydrolysis temperature of casein solution does not exceed 62°C, and more preferably, the temperature is 58-62°C, for example, 58°C, 60°C, or 62°C.

[0109] In some specific implementations, the enzymatic hydrolysis time for casein solution is less than 60 minutes. At this hydrolysis time, combined with the aforementioned complex protease, the resulting casein peptide composition has a milder bitterness, a weaker unpleasant volatile odor, and better reconstitution properties. Especially when used with casein solution that meets the above specifications, even better enzymatic hydrolysis results are achieved.

[0110] From the perspective of further reducing bitterness, in some preferred embodiments, when enzymatically hydrolyzing casein solution, the enzymatic hydrolysis reaction time is less than 60 min, preferably 40-50 min, more preferably 43-47 min, such as 43 min, 45 min, and 47 min.

[0111] The above-mentioned time, pH and / or temperature for enzymatic hydrolysis, used alone or in combination, further enhance the effect of enzymatic hydrolysis.

[0112] Adjust the pH of the enzyme hydrolysate

[0113] In some embodiments of the present invention, 2-10 minutes, preferably 5-8 minutes, before the end of the enzymatic hydrolysis of casein solution, the pH of the reaction solution (also referred to as the hydrolysate in this specification) in the enzymatic hydrolysis step can be adjusted to 7-7.5, preferably 7.2-7.4.

[0114] In some implementations, the pH is adjusted using a 5-10% sodium hydroxide or potassium hydroxide solution, for example, an 8% sodium hydroxide or potassium hydroxide solution.

[0115] (Post-processing steps)

[0116] Following the step of enzymatic hydrolysis of casein solution by complex protease, the method further includes a post-treatment step of the hydrolysate, which includes at least one of enzyme inactivation treatment, concentration treatment, and drying treatment.

[0117] Enzyme inactivation treatment

[0118] In some embodiments of the present invention, for the enzyme inactivation treatment of the present invention, after the enzymatic hydrolysis process is completed, the enzymatic hydrolysate is heated from the enzymatic hydrolysis temperature to the enzyme inactivation temperature of 90-100°C, and the heating time is less than 6 minutes.

[0119] In some preferred embodiments of the present invention, for the enzyme inactivation treatment of the present invention, the enzymatic hydrolysate is heated from the enzymatic hydrolysis temperature to the enzyme inactivation temperature of 90-95°C, more preferably 90°C, 91°C, or 95°C; the heating time is less than 6 minutes, for example 6 minutes, 5 minutes, or 4 minutes.

[0120] Concentration

[0121] The enzyme hydrolysate after enzyme inactivation can be appropriately concentrated.

[0122] There are no particular limitations on the concentration method of the present invention. Evaporation concentration, membrane separation concentration, freeze concentration, extraction concentration, adsorption concentration, dialysis, precipitation, adsorption and other methods can be used. In some preferred embodiments, concentration can be carried out through an RO system or a falling film evaporation concentration system.

[0123] In some preferred embodiments, the dry matter content of the concentrated product of the present invention is 20-30g / 100g, more preferably 22-25g / 100g.

[0124] Drying process

[0125] The product after the above concentration process can be dried.

[0126] There are no particular limitations on the drying method of the present invention. Vacuum drying, freeze drying, airflow drying, microwave drying, spray drying, etc. can be used. In some preferred embodiments, freeze drying or spray drying systems can be selected.

[0127] The method for preparing casein peptide compositions based on hydrolyzed milk protein provided by this invention employs a complex protease-directed enzymatic hydrolysis and precise enzymatic hydrolysis control technology to cause some bitter-tasting L-type aromatic amino acids, basic amino acids, and branched-chain amino acids to form corresponding amino acid derivatives. This minimizes the number of protein peptides with hydrophobic amino acids at the C-terminus and basic amino acids at the N-terminus, thereby reducing the bitterness of the product. Simultaneously, it further reduces the generation of fishy and off-flavor compounds such as 1-octen-3-one, (Z)-2-hexanal, (E)-2-decenal, 2,4-heptadienal, 1-octen-3-ol, hexanol, 1-octanal, heptanal, n-pentadimer, and n-pentanal.

[0128] The method for preparing casein peptide compositions based on hydrolyzed milk protein provided by this invention does not require additional processes such as membrane filtration or chromatography. It can prepare products with reduced bitter amino acid content simply by using directional enzymatic hydrolysis of compound protease and precise enzymatic hydrolysis control technology. In addition, the products also have good solubility, with a wettable time of less than 32 seconds. After mixing and standing, there is no precipitation or obvious insoluble particles visible to the naked eye.

[0129] Second aspect

[0130] A second aspect of the present invention provides a casein peptide composition based on hydrolyzed milk protein, which is obtained according to the above-described method for preparing a casein peptide composition based on hydrolyzed milk protein. That is, the casein peptide composition is derived from degradation products (enzymatic hydrolysates) formed by at least partial degradation (enzymatic hydrolysis) of casein, and therefore, the casein peptide composition contains at least a portion of degraded casein components.

[0131] In some embodiments of the present invention, the proportion of peptides with a C-terminus hydrophobic amino acid, peptides with an N-terminus basic amino acid, and peptides with both a C-terminus hydrophobic amino acid and an N-terminus basic amino acid, based on the total number of peptides in the casein peptide composition based on hydrolyzed milk protein, is 85% or less, preferably 80% or less, and more preferably 75% or less.

[0132] In some embodiments of the present invention, the proportion of peptides with an N-terminus basic amino acid is 36% or less, based on the total number of peptides in the casein peptide composition based on hydrolyzed milk protein.

[0133] In some embodiments of the invention, the acid bitterness value of the casein peptide composition based on hydrolyzed milk protein is below 11, preferably below 10. In some specific embodiments, the acid bitterness value is detected using an electronic tongue, for example, using an Insent SA402B electronic tongue.

[0134] At the aforementioned peptide ratios, the casein peptide composition based on hydrolyzed milk protein exhibits superior taste and flavor with lower bitterness content.

[0135] Third aspect

[0136] In a third aspect, the present invention provides a formulated dairy product and a method for preparing the same. The formulated dairy product of the present invention can be in solid, liquid, or semi-solid form; preferably, the dairy product of the present invention can be in powder form.

[0137] In some specific implementations, the formula dairy product is formula milk powder, such as formula milk powder that can be used by infants, adults, and the elderly.

[0138] Specifically, the dairy products of the present invention include:

[0139] (A) A casein peptide composition based on hydrolyzed milk protein prepared according to the preparation method described in the first aspect or a casein peptide composition based on hydrolyzed milk protein prepared according to the second aspect, and one or more of the following additional components (i.e., these components are not derived from the casein peptide composition based on hydrolyzed milk protein):

[0140] (B) Functional protein components, (C) Carbohydrate components, (D) Fats and oils, (E) Mineral salts, and (F) Other nutritional supplement components.

[0141] (Functional protein components)

[0142] There are generally no special restrictions on the functional protein ingredients that can be used; for example, various whey proteins, immunoglobulins, lactoferrin, etc. There are also no special restrictions on the amount of these ingredients added, as long as they comply with laws and regulations.

[0143] (Carbohydrate composition)

[0144] There are no particular restrictions on the type or source of carbohydrates added to the formula dairy products of this invention; those carbohydrates commonly used in formula dairy products in this field can be used.

[0145] In some specific embodiments, the carbohydrate component described in this invention mainly refers to sugars. These sugars are typically a general term for polyhydroxy aldehydes or polyhydroxy ketones and their condensation polymers and certain derivatives, generally composed of carbon, hydrogen, and oxygen. All sugars can be written with the empirical molecular formula: Cn(H₂O). n .

[0146] In this invention, the carbohydrates typically include monosaccharides, disaccharides, polysaccharides, or oligosaccharides.

[0147] Monosaccharides can mainly include glucose, fructose, etc.

[0148] Disaccharides, polysaccharides, or oligosaccharides can include: sucrose, lactose, fructotriose, fructotetraose, fructopentose, fructooligosaccharides, glucosamine, (maltodextrin), and various forms of human milk oligosaccharides.

[0149] In some specific implementations, the carbohydrates may be added in the form of dietary fiber. Examples of dietary fiber substances include one or more of the following: inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, maltodextrin, resistant dextrin, and soybean fiber.

[0150] Furthermore, there is no particular limitation on the total carbohydrate content in the formula dairy products of the present invention, and it can be formulated with reference to the carbohydrate content commonly used in infant formula milk powder products for the middle-aged and elderly in the art.

[0151] (Oil and fat components)

[0152] There are no particular restrictions on the types of oils added to the formula dairy products of this invention. Fatty acid glycerides commonly used in infant formula and formula milk powder products for the middle-aged and elderly can be used.

[0153] These glycerides can be obtained from plant extraction or through artificial synthesis (transesterification). Transesterification can be achieved by esterification of glycerol with fatty acids in the presence of a catalyst, or by transesterification of triglycerides and fatty acids from various existing sources in the presence of a (specific) catalyst (enzyme). For plant oils extracted from plants, the desired fatty acid glycerides can be obtained by mixing one or more of the following plant oils: rapeseed oil, soybean oil, sunflower oil, olive oil, sesame oil, corn oil, flaxseed oil, and camellia seed oil.

[0154] The fatty acid glycerides described in this invention are primarily triglycerides. Examples of such glycerides include OPL, OPO, MLCT, OOL, OPP, OLO, OLL, LPL, LPLn, OPLn, LPCa, and OPCa.

[0155] The term "MLCT structured mixed ester" refers to a structurally mixed ester whose main components are medium- to long-chain fatty acid triglycerides. "MLCT" stands for Middle to Long Chain Triglycerides, a structural ester whose glycerol backbone contains both medium-chain fatty acids (M) and long-chain fatty acids (L). Medium-chain fatty acids are those containing 6 to 12 carbon atoms, while long-chain fatty acids are those with more than 12 carbon atoms. MLCTs possess the characteristics of both medium- and long-chain fatty acids, offering the advantages of supplementing essential fatty acids, providing rapid energy, and not causing fat accumulation. Common MLCTs include OPLa, LPLa, OPCa, OLaO, OLaL, OMLa, SLaL, and SLaO.

[0156] (Mineral salt composition)

[0157] The mineral salts that can be added to the dairy products formulated in this invention are mainly used to introduce micronutrients, including iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon, tin, etc.

[0158] Furthermore, regarding the content of mineral salts in formula dairy products, as long as it complies with the provisions of laws and regulations, it is acceptable.

[0159] (Other nutritional supplement ingredients)

[0160] There are no particular restrictions on other nutritional supplement ingredients that can be added to the dairy products formulated according to the present invention, and they can be prepared in accordance with existing methods in the art.

[0161] In some specific implementations, these supplement ingredients include one or more selected from vitamins, probiotics, and unsaturated fatty acids.

[0162] Examples of vitamins include one or more of the following: vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, and biotin.

[0163] For probiotics, for example, Bifidobacteria probiotics that are beneficial to the gut.

[0164] Examples of unsaturated fatty acids include arachidonic acid and docosahexaenoic acid.

[0165] Furthermore, there are no particular restrictions on the source of each of the components (B) to (F) above; for example, they can be introduced by mixing with animal milk or other nutritional additives.

[0166] (use)

[0167] The casein peptide composition based on hydrolyzed milk protein obtained by the present invention can be used to prepare various types of food, including infant food, milk powder, and health products.

[0168] Example

[0169] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0170] The serine protease used in the following examples and comparative examples is enzyme preparation A: Bacillus licheniformis serine protease, which is from Novo Nordisk. CTL 300BG);

[0171] The flavor proteases used in the following examples and comparative examples include enzyme preparation BD, specifically:

[0172] Enzyme preparation B: Aspergillus oryzae aminopeptidase, which is from Novo Nordisk. 500MG);

[0173] Enzyme preparation C: Aspergillus niger flavored protease, which is from Sternzym Food Ingredients (Suzhou) Co., Ltd. (Sternzym FP23290);

[0174] Enzyme preparation D: Aspergillus oryzae flavored protease, which is from Nanning Pangbo Biotechnology Co., Ltd.

[0175] Example 1

[0176] 1. First, a casein solution separated by membrane separation was selected for use. The casein solution had a dry matter content of 10.0 g / 100 g, a protein content (on a dry basis) of 84.0 g / 100 g, a casein content (as a percentage of protein) of 82.0 g / 100 g, and a fat content (on a dry basis) of 3.6 g / 100 g. (For the method of membrane separation of protein, please refer to: Li Zhibin. Study on membrane separation preparation of active whey protein and casein micelles from bovine milk [D]. Jiangnan University, 2021. DOI:10.27169 / d.cnki.gwqgu.2021.001167).

[0177] 2. Accurately weigh the four enzyme preparations (AD) and add them to the enzymatic hydrolysis tank, then add reverse osmosis (RO) water to fully dissolve them;

[0178] The enzyme preparation AD is: Enzyme preparation A is Bacillus licheniformis serine protease, with an enzyme activity of 300 KPROT / g, and the addition amount (enzyme preparation / casein solution) is 0.001215% by mass.

[0179] Enzyme B is Aspergillus oryzae flavored protease I with an enzyme activity of 500 LAPU / g and an addition amount (enzyme preparation / casein solution) of 0.02013% by mass.

[0180] Enzyme C is a Aspergillus niger flavored protease with an enzyme activity of 450 U / g and an addition amount (enzyme preparation / casein liquid) of 0.001096% by mass.

[0181] Enzyme preparation D is Aspergillus oryzae flavored protease II, with an enzyme activity of 100,000 U / g, and the addition amount (enzyme preparation / casein liquid) mass ratio is 0.001082%;

[0182] 3. In step 1, the casein solution is poured into an enzymatic hydrolysis tank and heated to the enzymatic hydrolysis temperature of 58°C. During the heating process, the stirring paddle inside the tank is continuously stirring.

[0183] 4. Adjust the pH of the casein solution to 7.7 using an 8% sodium hydroxide or potassium hydroxide solution, and the enzymatic hydrolysis time is 43 minutes.

[0184] 5. Five minutes before the end of the enzymatic hydrolysis process in step 4, adjust the pH to 7.2 again using an 8% sodium hydroxide or potassium hydroxide solution;

[0185] 6. After the enzymatic hydrolysis process is completed, the enzymatic hydrolysate is heated from the enzymatic hydrolysis temperature of 58℃ to the enzyme inactivation temperature of 90℃, and the heating time (i.e., the time taken to heat from 58℃ to the enzyme inactivation temperature of 90℃) is 6 minutes.

[0186] 7. After enzyme inactivation is complete, the enzyme hydrolysate is cooled to 4°C and transferred to a storage tank for later use.

[0187] 8. The enzyme hydrolysate after enzyme inactivation can be concentrated to a dry matter content of 22g / 100g by an RO system or a falling film evaporation and concentration system.

[0188] 9. The concentrated enzymatic hydrolysate can be processed into the final product using a freeze-drying or spray-drying system.

[0189] Example 2

[0190] 1. First, use membrane-separated casein solution for later use. The casein solution has a dry matter content of 12.8g / 100g, a protein content (on a dry basis) of 86.9g / 100g, a casein content (accounting for 84.5g / 100g of protein), and a fat content (on a dry basis) of 1.0g / 100g.

[0191] 2. Accurately weigh the four enzyme preparations (AD) and add them to the enzymatic hydrolysis tank, then add RO water to fully dissolve them;

[0192] The enzyme preparation AD is:

[0193] Enzyme A is Bacillus licheniformis serine protease with an enzyme activity of 300 KPROT / g and an addition amount (enzyme preparation / casein solution) of 0.001836% by mass.

[0194] Enzyme B is Aspergillus oryzae flavored protease I with an enzyme activity of 500 LAPU / g and an addition amount (enzyme preparation / casein solution) of 0.02485% by mass.

[0195] Enzyme preparation C is a Aspergillus niger flavored protease with an enzyme activity of 450 units / g and an addition amount (enzyme preparation / casein liquid) of 0.001164% by mass.

[0196] Enzyme preparation D is Aspergillus oryzae flavored protease II, with an enzyme activity of 100,000 U / g, and the addition amount (enzyme preparation / casein liquid) is 0.001156% by mass.

[0197] 3. In step 1, the casein solution is poured into an enzymatic hydrolysis tank and heated to the enzymatic hydrolysis temperature of 62°C. During the heating process, the stirring paddle inside the tank is continuously stirring.

[0198] 4. Adjust the pH of the casein solution to 7.9 using an 8% sodium hydroxide or potassium hydroxide solution, and the enzymatic hydrolysis time is 47 minutes.

[0199] 5. Five minutes before the end of the enzymatic hydrolysis process in step 4, adjust the pH to 7.4 again using an 8% sodium hydroxide or potassium hydroxide solution;

[0200] 6. After the enzymatic hydrolysis process is completed, the enzymatic hydrolysate is heated from the enzymatic hydrolysis temperature of 62℃ to the enzyme inactivation temperature of 95℃ over a period of 6 minutes.

[0201] 7. After enzyme inactivation is complete, the enzyme hydrolysate is cooled to 6°C and transferred to a storage tank for later use.

[0202] 8. The enzyme hydrolysate after enzyme inactivation can be concentrated to a dry matter content of 25g / 100g by an RO system or a falling film evaporation and concentration system.

[0203] 9. The concentrated enzymatic hydrolysate can be processed into the final product using a freeze-drying or spray-drying system.

[0204] Example 3

[0205] 1. First, use membrane-separated casein solution for later use. The casein solution has a dry matter content of 12.3g / 100g, a protein content (on a dry basis) of 85.2g / 100g, a casein content (accounting for 83.9g / 100g of protein), and a fat content (on a dry basis) of 2.4g / 100g.

[0206] 2. Accurately weigh the four enzyme preparations (AD) and add them to the enzymatic hydrolysis tank, then add RO water to fully dissolve them;

[0207] The enzyme preparation AD is:

[0208] Enzyme A is Bacillus licheniformis serine protease with an enzyme activity of 300 KPROT / g and an addition amount (enzyme preparation / casein solution) of 0.001377% by mass.

[0209] Enzyme B is Aspergillus oryzae flavored protease I with an enzyme activity of 500 LAPU / g and an addition amount (enzyme preparation / casein solution) of 0.02455% by mass.

[0210] Enzyme C is a Aspergillus niger flavored protease with an enzyme activity of 450 units / g and an addition amount (enzyme preparation / casein liquid) of 0.001148% by mass.

[0211] Enzyme preparation D is Aspergillus oryzae flavored protease II, with an enzyme activity of 100,000 U / g, and the addition amount (enzyme preparation / casein liquid) is 0.001148% by mass.

[0212] 3. In step 1, the casein solution is poured into an enzymatic hydrolysis tank and heated to 60°C for enzymatic hydrolysis. During the heating process, the stirring paddle inside the tank is continuously stirring.

[0213] 4. Adjust the pH of the casein solution to 7.8 using an 8% sodium hydroxide or potassium hydroxide solution, and the enzymatic hydrolysis time is 45 minutes.

[0214] 5. Five minutes before the end of the enzymatic hydrolysis process in step 4, adjust the pH to 7.3 again using an 8% sodium hydroxide or potassium hydroxide solution;

[0215] 6. After the enzymatic hydrolysis process is completed, the enzymatic hydrolysate is heated from the enzymatic hydrolysis temperature of 60℃ to the enzyme inactivation temperature of 90℃, and the heating time is 6 minutes.

[0216] 7. After enzyme inactivation is complete, the enzyme hydrolysate is cooled to 5°C and transferred to a storage tank for later use.

[0217] 8. The enzyme hydrolysate after enzyme inactivation can be concentrated to a dry matter content of 23g / 100g by an RO system or a falling film evaporation and concentration system.

[0218] 9. The concentrated enzymatic hydrolysate can be processed into the final product using a freeze-drying or spray-drying system.

[0219] Example 4

[0220] First, a membrane-separated casein solution is selected for use. The casein solution has a dry matter content of 12.5g / 100g, a protein content (on a dry basis) of 87.2g / 100g, a casein content (as a percentage of protein) of 85.8g / 100g, and a fat content (on a dry basis) of 0.296g / 100g. It is then pasteurized using a pasteurization system at a temperature of 75-85℃ for 15-30 seconds. After pasteurization, the material is cooled to 4-6℃ and temporarily stored in an enzymatic hydrolysis tank.

[0221] Other conditions are the same as in Example 1.

[0222] Example 5

[0223] In Example 1, the enzymatic hydrolysis time in step 4 was adjusted to 60 min, while other conditions remained the same as in Example 1.

[0224] Comparative Example

[0225] Comparative Example 1

[0226] Commercially available hydrolyzed casein products are obtained by using a single alkaline protease at 50-60 degrees Celsius, pH 7.3-7.5, and enzymatic hydrolysis for 90-120 minutes.

[0227] Comparative Example 2

[0228] In Example 1, step 2, the amount of Bacillus licheniformis serine protease added was 0.02145%, and other conditions were the same as in Example 1.

[0229] Test case

[0230] Test Example 1

[0231] 1. Liquid chromatography-mass spectrometry analysis and detection

[0232] Liquid chromatography conditions: EASY nLC 1200 nanoliter liquid chromatograph, reverse-phase C18 liquid chromatography column (150μm×150mm, 1.9μm)

[0233] Mass spectrometry conditions: Orbitrap Fusion Lumos high-resolution mass spectrometer, electrospray ionization source (ESI+), collision gas: argon, resolution: 60000, mass scan range: 400-1200 m / z, mass spectrometry data type: profile.

[0234] Table 1: Results of Liquid Chromatography-Mass Spectrometry Analysis

[0235]

[0236] The results of the analysis by liquid chromatography-mass spectrometry are shown in Table 1. It was found that the total proportion of bitter amino acids in each example was less than 85%, especially in examples 1-4 which were less than 79%. The total proportion of bitter amino acids in each comparative example was greater than 85%, with comparative example 1 having a total proportion of bitter amino acids greater than 90% in commercially available products, and comparative example 2 also reaching as high as 89%.

[0237] Among them, bitter amino acid-N indicates a peptide with a basic amino acid at the N-terminus (and a non-hydrophobic amino acid at the C-terminus);

[0238] Bitter amino acid-C indicates a peptide with a hydrophobic amino acid at the C-terminus (and no basic amino acid at the N-terminus);

[0239] Bitter amino acids -N&C represent peptides with a hydrophobic amino acid at the C-terminus and a basic amino acid at the N-terminus.

[0240] 2. Electronic tongue analysis

[0241] Using the Insent SA402B electronic tongue, this device employs an artificial lipid membrane sensor with wide-range selectivity to simulate the taste perception mechanism of living organisms. By testing the changes in membrane potential generated by the electrostatic or hydrophobic interactions between various taste substances and the artificial lipid membrane, it can evaluate acidic bitterness and acidic bitter aftertaste (Aftertaste-B) without the need for any statistical analysis or modeling.

[0242] Table 2: Evaluation results of electronic tongue on bitter and acidic bitter aftertastes

[0243]

[0244] The electronic tongue-specific artificial lipid membrane sensor detected that the acidic bitterness of each embodiment was significantly lower than that of each comparative example (Table 2). Among them, the average acidic bitterness of commercially available comparative example 1 reached 13.23, with a significant bitterness.

[0245] It can be seen that, compared with Comparative Examples 1-2, Examples 1-5 have the advantage of a milder bitterness. No additional processes such as encapsulation or ultrafiltration are required; only targeted enzymatic hydrolysis by a compound protease and precise enzymatic hydrolysis control technology are used to convert some bitter L-type aromatic amino acids, basic amino acids, and branched-chain amino acids into corresponding amino acid derivatives. This minimizes the number of protein peptides with hydrophobic C-terminals and basic N-terminals, thus reducing the bitterness of the product. Liquid chromatography-mass spectrometry analysis revealed that the total proportion of bitter amino acids in each example was less than 85%, especially in Examples 1-4, which was less than 80%. In contrast, the total proportion of bitter amino acids in the commercially available product of Comparative Example 1 was greater than 90%, and in Comparative Example 2, it was as high as 89%. Detection using an electronic tongue-specific artificial lipid membrane sensor showed that the acidic bitterness in each example was significantly lower than that of the commercially available product of Comparative Example 1.

[0246] Test Example 2 Difference analysis of volatile organic compounds in casein peptide solutions

[0247] use Gas phase ion mobility spectrometry, GAS (Germany, Dortmund); CTC-PAL 3 static headspace automated sampler, CTC Analytics AG (Switzerland, Zwingen); VOCal data processing software (0.4.03), GAS (Germany, Dortmund).

[0248] Headspace injection conditions: Incubation temperature: 60℃; incubation for 15 min; injection volume: 500 μL; splitless injection; incubation speed: 500 r / min; injection needle temperature: 85℃.

[0249] The sample numbers for the injections are shown in Table 3.

[0250] GC conditions: Column temperature: 60℃; Carrier gas: High-purity nitrogen (purity ≥99.999%); Programmed pressure: Initial flow rate 2.0 mL / min, held for 2 min, linearly increased to 10.0 mL / min within 8 min, and linearly increased to 100.0 mL / min within 10 min; Run time: 20 min; Injector temperature: 80℃.

[0251] IMS conditions: Ionization source: tritium source (3H); migration tube length: 53 mm; electric field strength: 500 V / cm; migration tube temperature: 45 °C; drift gas: high-purity nitrogen (purity ≥99.999%); flow rate: 150 mL / min; positive ion mode.

[0252] Data Processing: A mixed standard of six ketones was analyzed, and calibration curves for retention time and retention index were established. The retention index of the target analyte was then calculated based on its retention time. Qualitative analysis of the target analyte was performed using the GC retention index (NIST 2020) database and the IMS migration time database built into the VOCal software for retrieval and comparison. The Reporter, Gallery Plot, and Dynamic PCA plugins in the VOCal data processing software were used to generate three-dimensional spectra, two-dimensional spectra, difference spectra, fingerprint spectra, and PCA plots of volatile components for comparison of volatile organic compounds between samples.

[0253] Table 3: Sample Information Table

[0254] Sample number illustrate FH02 Example 1 FH03 Example 2 FH04 Example 3 FH01 Example 4 FH06 Example 5 FH05 Comparative Example 1 FH07 Comparative Example 2

[0255] Figure 1 The image shows a three-dimensional spectrum of GC-IMS, with the three axes representing relative migration time (X-axis), retention time (Y-axis), and signal peak intensity (Z-axis), respectively. Figure 2 This is a two-dimensional GC-IMS spectrum (top view). The entire graph has a blue background. The red vertical line at 1.0 on the horizontal axis represents the RIP peak (reacting ion peak, normalized). The vertical axis represents the gas chromatographic retention time (s), and the horizontal axis represents the relative migration time (normalized). Each point on either side of the RIP peak represents a volatile organic compound. The color represents the peak intensity, ranging from blue to red, with darker colors indicating greater peak intensity.

[0256] To further visually compare the differences in volatile components among the samples, the spectrum of sample FH01 was selected as a reference, and the spectra of other samples were subtracted from the reference to obtain a comparison chart of the differences between the samples, as shown below. Figure 3 As shown. If the volatile organic compound content in the target sample and the reference is the same, the background after subtraction is white, while red indicates that the concentration of the substance in the target sample is higher than that in the reference, and blue indicates that the concentration of the substance in the target sample is lower than that in the reference.

[0257] from Figure 1-3 It can be clearly seen that the differences in volatile organic compounds among the various embodiments are small, while the differences in volatile organic compounds between the comparative examples and between the comparative examples and the embodiments are relatively significant.

[0258] Further comparisons were made of volatile substances in seven different samples, and fingerprint analysis was performed on all volatile substances. Figure 4 Each row in the graph represents all the signal peaks selected from a single sample, and each column in the graph represents the signal peaks of the same volatile organic compound in different samples. Figure 4 The data reveals complete volatile organic compound (VOC) information for each sample and the differences in VOCs between samples. Analysis showed that Comparative Example 1, a commercially available product, contained more hexanol, 1-octaneal, heptanal, n-pentanedimer, n-pentanal, amines, sulfides, and other odorous compounds, while the products in the examples mainly contained 2-butanone, triethylamine, propional, 3-methylbutanal, 1-penten-3-ol, acetoin, 1-penten-3-one, 2-methyl-2-propanol, 3-methyl-2-butenal, ethyl acetate, 2-methyl-1-propanol, 3-pentyl-2-one, and 2-hexanal, demonstrating a significant reduction in unpleasant volatile odors.

[0259] Depend on Figure 5 As shown in the gallery plot, PCA spectral analysis can distinguish all seven samples. Similar samples indicate small differences, while large differences indicate significant component variations. Comparative analysis revealed a total of 72 peaks across the seven samples, identifying 38 substances, including aldehydes, alcohols, esters, amines, and sulfides. The volatile compounds in the various examples (especially FH01, FH02, FH03, and FH04) showed relatively similar differences, while the comparative examples (FH05 and FH07) exhibited larger differences.

[0260] Based on the above results, it can be seen that the differences in volatile substances among the samples (especially FH01, FH02, FH03, and FH04) are relatively similar, while the differences compared to the comparative examples (FH05 and FH07) are significant. In particular, compared to comparative examples 1-2, examples 1-4 have the advantage of weaker unpleasant volatile odors. The use of a compound protease-directed enzymatic hydrolysis technique further reduces the generation of fishy and off-odor compounds such as 1-octen-3-one, (Z)-2-hexanal, (E)-2-decenal, 2,4-heptadienal, 1-octen-3-ol, hexanol, 1-octanal, heptanal, n-pentadienal, and n-pentanal. Gas phase ion mobility spectrometry analysis in a flavor analyzer revealed that, compared to the existing product in Comparative Example 1, the products in Examples 1-5, especially Examples 1-4, showed a significant reduction in unpleasant volatile odors.

[0261] Test Example 3 Casein phosphopeptide content analysis

[0262] The samples were tested according to Appendix A of the National Food Safety Standard GB 31617-2014, which specifies the detection method for casein phosphopeptide as a food fortifier.

[0263] Table 4: Results of Casein Phosphopeptide Content Detection

[0264] Sample number Casein phosphopeptide content (on a dry basis, w) / % Example 1 24.2 Example 2 24.8 Example 3 25.9 Example 4 29.8 Example 5 27.9

[0265] Table 4 shows that this embodiment does not require additional processes such as membrane filtration or chromatography. It can prepare casein phosphopeptides (CPP) with a high content and equivalent quantity to commercially available products simply by using compound protease-directed enzymatic hydrolysis and precise enzymatic hydrolysis control technology.

[0266] Examples 1-5 have the advantage of higher CPP content. No additional processes such as membrane filtration and chromatography are required. Products with high CPP content can be prepared simply by the compound protease directional enzymatic hydrolysis and precise enzymatic hydrolysis control technology described in this invention. The casein phosphopeptide (CPP) content (on a dry basis, w / %) in the products of Examples 1-5 is 24-30%, and the products prepared by this invention have abundant casein phosphopeptide.

[0267] Test Example 4 Impulse-adjustment evaluation

[0268] First, measure 300mL of 55℃ distilled water into a 500mL beaker. Place a suction plate and a stainless steel funnel above the beaker. Then, pour the weighed 45g sample into the stainless steel cylinder with the suction plate, ensuring the surface of the milk powder is level. Gently and continuously remove the bottom plate (complete this process within approximately 2 seconds). Start a stopwatch as you remove the bottom plate. Stop timing once all the product is submerged in the water. Record the time taken; this is the wettable immersion time.

[0269] Table 5: Results of Impatience Evaluation

[0270]

[0271] The products of each embodiment have good solubility, with a wettable wetting time of less than 32s. After mixing and standing, there is no precipitation or obvious insoluble particles visible to the naked eye (Table 5). In contrast, the existing product of the commercially available comparative example 1 has a wettable wetting time of more than 60s, and after mixing and standing, there is obvious precipitation, visible insoluble particles, and white spots sticking to the wall. The solubility of the products of each embodiment is significantly better than that of the existing commercially available products.

[0272] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0273] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a casein peptide composition based on hydrolyzed milk protein, characterized in that, The method includes: The enzymatic hydrolysis step involves using a complex protease to hydrolyze the casein solution. The complex protease includes serine protease and flavor protease; Of which, each 100g of the casein solution contains 5-15g of dry matter; each 100g of the casein solution contains 75-100g of protein, and each 100g of the protein contains 75-100g of casein. The amount of serine protease added is 0.0001-0.015% by mass of the casein solution.

2. The preparation method according to claim 1, characterized in that, The flavor proteases include Aspergillus oryzae flavor protease and Aspergillus niger flavor protease; The serine protease includes Bacillus licheniformis serine protease.

3. The preparation method according to claim 2, characterized in that, Based on the mass of casein solution: The amount of Bacillus licheniformis serine protease added is 0.0001-0.015% by mass; The amount of Aspergillus oryzae flavor protease added is 0.001-0.03% by mass; and / or The amount of Aspergillus niger flavor protease added is 0.0001-0.01% by mass.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The casein solution contains 0-15g of fat per 100g of dry matter.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The casein liquid is obtained by separating it from casein raw material using a membrane separation method; Optionally, the membrane separation method uses a ceramic membrane or a spiral wound membrane; Preferably, after obtaining casein liquid from casein raw material by membrane separation, it is pasteurized.

6. The preparation method according to any one of claims 1 to 5, characterized in that, In the enzymatic hydrolysis step, Enzymatic hydrolysis time should be less than 60 minutes; Before enzymatically hydrolyzing the casein solution, adjust the pH of the casein solution to 7.5-8; The enzymatic hydrolysis temperature should not exceed 62℃.

7. The preparation method according to any one of claims 1 to 6, characterized in that, Before the end of the enzymatic hydrolysis step, adjust the pH of the reaction solution in the enzymatic hydrolysis step to 7-7.

5.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The method further includes a post-processing step, wherein the post-processing step includes at least one of enzyme inactivation treatment, concentration treatment, and drying treatment.

9. A casein peptide composition, characterized in that, It is a composition prepared by the preparation method according to any one of claims 1 to 8.

10. The casein peptide composition according to claim 9, characterized in that, The total number of peptides in the casein peptide composition, calculated by weight, is less than 85% for peptides with a hydrophobic amino acid at the C-terminus, peptides with a basic amino acid at the N-terminus, and peptides with both a hydrophobic amino acid at the C-terminus and a basic amino acid at the N-terminus.

11. The use of the casein peptide composition prepared by the preparation method according to any one of claims 1 to 8, or the casein peptide composition according to claim 9 or 10, in the preparation of food or health products.

12. A formulated dairy product, characterized in that, The formulated dairy products include: (A) A casein peptide composition prepared by the method according to any one of claims 1 to 8, or a casein peptide composition according to claim 9 or 10, and one or more of the following additional components: (B) Functional protein components, (C) Carbohydrate components, (D) Fats and oils, (E) Mineral salts, and (F) Other nutritional supplement components.

Images