Casein peptide composition containing multifunctional peptide and preparation method thereof

Abstract

The invention discloses a casein peptide composition containing multifunctional peptide and a preparation method of the casein peptide composition. The present invention provides a casein peptide composition containing a multifunctional peptide, characterized in that the casein peptide composition is obtained by enzymatic hydrolysis of a casein liquid, in the casein peptide composition, the relative strength of total functional peptides is 25% or more, the relative strength of casein phosphopeptides is 9% or more, and the relative strength of the casein phosphopeptides is 9% or more. The relative strength of the functional peptides except the casein phosphopeptides is above 11%, and the enzyme in the enzymolysis at least comprises serine protease.

Description

Technical Field

[0001] This invention relates to a protein composition containing multifunctional peptides and its preparation method, belonging to the technical field of nutritional compositions. Background Technology

[0002] In recent years, my country's dairy deep 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 deep processing industry. In my country, due to 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. Casein peptides from hydrolyzed milk protein contain various essential amino acids, effectively supplementing protein, promoting tissue and cell growth and development, and promoting bone health. Furthermore, casein peptides from hydrolyzed milk protein contain various functional peptides that can regulate and enhance immune function.

[0003] Casein peptide products are designed to meet the needs of people of all ages, promoting the healthy growth of infants and adolescents and enhancing the health benefits for middle-aged and elderly individuals. Hydrolyzed milk protein products contain over 20 functional peptides, including casein phosphopeptides (CPP), ACE-inhibiting peptides, antimicrobial peptides, antioxidant peptides, bone anabolic peptides, and calcium absorption-promoting peptides. Casein phosphopeptides (CPP) effectively promote calcium absorption, prevent mineral loss, and have anti-caries effects.

[0004] However, casein is highly unstable during hydrolysis and readily forms casein micelles. Furthermore, in large-scale industrial production, current technologies commonly employ prolonged deep hydrolysis to increase the content of functional peptides, followed by purification of small molecule peptides using membrane filtration and chromatography. This may lead to a reduction in functional peptides such as phosphopeptides, thus affecting their efficacy.

[0005] Reference 1 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 2 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.

[0007] For example, the existing technologies cited in references 1 and 2 generally employ prolonged enzymatic hydrolysis followed by purification using methods such as membrane filtration and chromatography. Currently, the functional peptide content and production cost of the products both need improvement. Further research and improvements are needed to increase the functional peptide content and reduce production costs in large-scale industrial production to meet application requirements. Furthermore, references 1 and 2 do not disclose the efficacy of their prepared products in intestinal protection and in assisting to improve enteritis symptoms.

[0008] References

[0009] Reference 1: CN104046673A

[0010] Reference 2: CN105385738A Summary of the Invention

[0011] The problem the invention aims to solve

[0012] In existing technologies, casein hydrolysis is highly unstable and readily forms casein micelles. In large-scale industrial production, current techniques typically employ prolonged, deep hydrolysis followed by purification using methods such as membrane filtration and chromatography to increase the content of functional peptides.

[0013] For example, reference 2 uses a centrifugation-assisted multi-stage membrane separation system to purify yak casein phosphopeptides. This process is also cumbersome and time-consuming, but the membrane separation process results in nutrient loss. Similarly, reference 1 uses a long-duration deep hydrolysis method, which is complex, inefficient, and only slightly increases the content of functional peptides.

[0014] Furthermore, the efficacy of the products obtained from the aforementioned cited literature in improving or alleviating intestinal discomfort symptoms and / or protecting the intestines is unknown.

[0015] In addition, for casein hydrolysates, it is more ideal to have a greater variety of total peptides and a higher proportion of protein phosphopeptides (CPPs) in the functional peptides. However, in the above case, it usually means that the proportion of total functional peptides in the hydrolysate is reduced.

[0016] Therefore, in view of the above problems, the present invention provides a casein peptide composition prepared by water enzymatic method, in which, under the condition of using a specific enzyme, not only is the proportion of CPP maintained at a relatively high level in the enzymatic hydrolysate, but the proportion of total functional peptides is also maintained at a high level. Furthermore, it has been verified that the composition has the effect of improving or relieving intestinal discomfort symptoms and / or protecting the intestine.

[0017] Solution for solving the problem

[0018] [1]. A casein peptide composition containing multifunctional peptides, wherein the casein peptide composition is obtained by enzymatic hydrolysis of casein solution, wherein the casein peptide composition has a relative strength of more than 25% of total functional peptides, a relative strength of more than 9% of casein phosphopeptides, a relative strength of more than 11% of functional peptides other than casein phosphopeptides, and the enzyme in the enzymatic hydrolysis includes at least serine protease.

[0019] [2]. The casein peptide composition according to [1], wherein the casein liquid is obtained by membrane separation of the milk raw material.

[0020] [3]. The casein peptide composition according to [1] or [2], wherein the relative strength of the total functional peptides in the casein peptide composition is 25% to 45%; and the relative strength of the functional peptides other than casein phosphopeptides is 15% to 30%.

[0021] [4]. The casein peptide composition according to [1] or [2], wherein the total number of peptide types in the casein peptide composition is 1350 or more.

[0022] [5]. A method for preparing a casein peptide composition as described in any one of [1] to [4], wherein the method comprises: an enzymatic hydrolysis step, wherein the amount of serine protease added is 0.0001-0.015% by mass based on the mass of the casein liquid, and the serine protease includes Bacillus licheniformis serine protease.

[0023] [6]. According to the preparation method described in [5], the enzymatic hydrolysis step further uses flavor proteases, including Aspergillus oryzae flavor protease and Aspergillus niger flavor protease.

[0024] [7]. According to the preparation method described in [5] or [6], wherein, in the enzymatic hydrolysis step, based on the mass of the 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.

[0025] [8]. The preparation method according to any one of [5] to [7], wherein the membrane separation method uses a ceramic membrane and / or a spiral wound membrane; preferably, after obtaining casein liquid from casein raw material by membrane separation method, it is pasteurized.

[0026] [9]. The preparation method according to any one of [5] to [8], wherein, in the enzymatic hydrolysis step, the hydrolysis time is less than 60 min; the pH of the casein solution is adjusted to 7.5-8 before enzymatic hydrolysis; and the hydrolysis temperature does not exceed 62°C.

[0027]

[10] . Use of the casein peptide composition according to any one of [1] to [4] or the casein peptide composition prepared according to any one of [5] to [9] in the preparation of food and / or health products for improving gut health.

[0028]

[11] . According to the use described in

[10] , the improvement of gut health includes at least one of regulating gut microbiota, assisting in the improvement of enteritis discomfort, and protecting the gut.

[0029] The effects of the invention

[0030] In some embodiments, the casein peptide composition prepared by the present invention maintains a high CPP content while having a higher overall content of functional peptides. The casein peptide composition of the present invention does not require additional processes such as membrane filtration or chromatography. It can be prepared with a high content of functional peptides simply by using compound protease directed enzymatic hydrolysis and precise enzymatic hydrolysis control technology. The product of the present invention contains more than 20 functional peptides, such as casein phosphopeptide (CPP), ACE inhibitory peptide, antimicrobial peptide, antioxidant peptide, bone anabolic peptide, and calcium absorption promoting peptide. The relative strength of functional peptides in the sample is greater than 25%, while the relative strength of functional peptides in commercially available products is less than 25%.

[0031] In some embodiments, the casein peptide compositions prepared according to the present invention also have the effect of improving or relieving intestinal discomfort symptoms and / or protecting the intestines. Attached Figure Description

[0032] Figure 1 This is a schematic spectrum of the mass spectrometry detection results of the sample in Example 4.

[0033] Figure 2 This is a schematic spectrum of the mass spectrometry detection results of the sample in Example 1.

[0034] Figure 3 This is a schematic spectrum of the mass spectrometry detection results of the sample in Example 2.

[0035] Figure 4 This is a schematic spectrum of the mass spectrometry detection results of the sample in Example 3.

[0036] Figure 5 The image shows a schematic spectrum of the mass spectrometry results for sample 1 in Comparative Example 1.

[0037] Figure 6 This is a schematic spectrum of the mass spectrometry detection results of the sample in Example 5.

[0038] Figure 7 The image shows a schematic spectrum of the mass spectrometry results for sample 2 in Comparative Example 2.

[0039] Figure 8 This is a schematic diagram showing the total number of peptides in the sample.

[0040] Figure 9 This is a schematic diagram showing the distribution of peptide types in the sample.

[0041] Figure 10 This is a schematic diagram showing the comparison of the percentage content of phosphopeptides and functional peptides in the sample.

[0042] Figure 11 This is a schematic diagram showing the number of phosphopeptides identified in the sample.

[0043] Figure 12 This is a schematic diagram showing the statistical results of body weight in the aged mice group.

[0044] The data volume in the figure is "n=8"; Figure 12 In the modeling process, A represents the results of seven weight measurements. Figure 12 B in the figure represents the result of the last weight statistical analysis.

[0045] Figure 13 This is a schematic diagram of the physiological observation scoring results.

[0046] Figure 14 This is a diagram illustrating the DAI scoring results.

[0047] Figure 15 This is a schematic diagram showing the statistical results of intestinal tissue sampling data.

[0048] The data volume in the figure is "n=8"; Figure 15 A in the table represents the statistical results of small intestine length. Figure 15 In the diagram, B represents the statistical results of colon length; and C represents the statistical results of colon weight.

[0049] Figure 16 This is a schematic diagram of the statistical results of organ ratio.

[0050] Note: The data size is "n=8"

[0051] Figure 17 This is a schematic diagram of the CD45 flow cytometry results in the gut.

[0052] Figure 17 In the diagram, A represents the results of the blank group; Figure 17 B in the diagram represents the combined group results; Figure 17 C in the diagram represents the results of the positive control group; Figure 17 D in the diagram represents the results of the casein hydrolysate group; Figure 17 E in the diagram represents the results of the model group.

[0053] Figure 18 This is a schematic diagram of the flow cytometry results for E-cadherin in the gut.

[0054] Figure 18 In the diagram, A represents the results of the blank group; Figure 18 B in the diagram represents the combined group results; Figure 18 C in the diagram represents the results of the positive control group; Figure 18 D in the diagram represents the results of the casein hydrolysate group; Figure 18 E in the diagram represents the results of the model group.

[0055] Figure 19 This is a schematic diagram of the statistical results of flow cytometry detection.

[0056] Figure 19 In the diagram, A represents the results of CD45 single staining. Figure 19 B in the diagram represents a single staining of E-cadherin.

[0057] Figure 20 This is a schematic diagram of the results of flow cytometry double staining of CD24 and LGR5 in the intestine.

[0058] Figure 20 In the diagram, A represents the results of the blank group; Figure 20 B in the diagram represents the combined group results; Figure 20 C in the diagram represents the results of the positive control group; Figure 20 D in the diagram represents the results of the casein hydrolysate group; Figure 20 E in the diagram represents the results of the model group.

[0059] Figure 21 This is a schematic diagram of the statistical results of CD24 and LGR5 flow cytometry detection.

[0060] Figure 22 This is a schematic diagram of the PEPT1 immunofluorescence staining results in the ileum.

[0061] The data volume in the figure is "n=4"; Figure 22 In the diagram, A represents the results of the blank group; Figure 22 B in the diagram represents the combined group results; Figure 22 C in the diagram represents the results of the positive control group; Figure 22 D in the diagram represents the results of the casein hydrolysate group; Figure 22 E in the figure represents a schematic diagram of the model group results. The first row in the figure shows PEPT1 staining, the second row shows DAPI staining, and the third row shows the merged image.

[0062] Figure 23 This is a schematic diagram of the results of PEPT1 immunofluorescence staining in the colon.

[0063] The data volume in the figure is "n=4"; Figure 23 In the diagram, A represents the results of the blank group; Figure 23 B in the diagram represents the combined group results; Figure 23 C in the diagram represents the results of the positive control group; Figure 23 D in the diagram represents the results of the casein hydrolysate group; Figure 23 E in the figure represents a schematic diagram of the model group results. The first row in the figure shows PEPT1 staining, the second row shows DAPI staining, and the third row shows the merged image.

[0064] Figure 24 This is a schematic diagram of the PEPT1 immunofluorescence staining results in the jejunum.

[0065] The data volume in the figure is "n=4"; Figure 24 In the diagram, A represents the results of the blank group; Figure 24 B in the diagram represents the combined group results; Figure 24 C in the diagram represents the results of the positive control group; Figure 24 D in the diagram represents the results of the casein hydrolysate group; Figure 24 E in the figure represents a schematic diagram of the model group results. The first row in the figure shows PEPT1 staining, the second row shows DAPI staining, and the third row shows the merged image.

[0066] Figure 25 This is a schematic diagram of the PEPT1 immunofluorescence staining results analysis.

[0067] The data volume in the figure is "n=4"; Figure 25 In this context, A represents the statistical results of ileal immunofluorescence. Figure 25 B in the figure represents the statistical results of colonic immunofluorescence. Figure 25 C in the figure represents the statistical results of jejunal immunofluorescence.

[0068] Figure 26 This is a schematic diagram of the results of the Alpha diversity analysis.

[0069] Figure 27 The image shows a schematic diagram of the β diversity analysis results.

[0070] Figure 28 Venn diagram showing the differences in microbial communities between groups.

[0071] Figure 29 This is a clustered stacked graph.

[0072] Figure 30 A bubble chart for analyzing significant differences. Detailed Implementation

[0073] 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.

[0074] 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.

[0075] 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.

[0076] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0077] 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.

[0078] 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.

[0079] In this invention, “Da” is used to represent the unit of molecular weight, “Dalton”.

[0080] 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.

[0081] In this invention, the term "infant" is used to refer to the human group aged 0 to 6 months.

[0082] In this invention, the term "older infant" refers to the human group aged 6 to 12 months.

[0083] In this invention, the term "infant" is used to refer to the human group aged 12 to 36 months.

[0084] In this invention, the term "infant" refers to the human group under the age of 3 years.

[0085] 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.

[0086] In this manual, the term "adult" refers to a person who is 18 years of age or older.

[0087] In this manual, the term "teenager" refers to people aged 7-40.

[0088] In this manual, the term "middle-aged person" refers to a person aged 41-65.

[0089] 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.

[0090] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0091] All unit names used in this manual are international standard unit names.

[0092] In this specification, "LGR5" refers to leucine-rich repeat-containing G-protein coupled receptor 5.

[0093] In this instruction manual, "CD45" represents leukocyte common antigen (LCA).

[0094] In this specification, "PEPT1" refers to oligopeptide transporter 1 (PEPT1).

[0095] In this specification, "CD24" refers to a highly glycosylated glycosylphosphatidylinositol (GPI)-anchored membrane protein (Cluster of differentiation 24, CD24).

[0096] <First Aspect>

[0097] A first aspect of the present invention provides 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, i.e., a composition comprising multiple peptides (peptide fragments).

[0098] The casein peptide composition of the present invention, while ensuring excellent CPP content, also has significantly improved relative strength of functional peptides, thereby having better functions and effects, such as helping to improve or alleviate symptoms of enteritis and / or intestinal protection.

[0099] In some embodiments, the relative strength of the total functional peptides in the casein peptide composition of the present invention is 25% or more, preferably 30% or more, more preferably 35% or more, for example 25% to 45%.

[0100] The relative intensity of total functional peptides refers to the signal intensity of the target sample / the signal intensity of the reference sample in the casein peptide composition detected by liquid chromatography-mass spectrometry × 100%.

[0101] Based on the structure of peptides, this invention retrieves peptide efficacy data and classifies all peptides into three categories: ordinary peptides, phosphorylated peptides, and other functional peptides.

[0102] The strength and content of the aforementioned peptides are directly proportional. This invention adopts the principle of normalization, using the relative strength of phosphorylated peptides and other functional peptides (i.e., total functional peptides) in the casein peptide composition to reflect the relative content.

[0103] Total functional peptides refer to the collection of peptides in existing peptide libraries that are labeled with functions such as casein phosphopeptide (CPP), ACE inhibitory peptides, antimicrobial peptides, antioxidant peptides, bone anabolic peptides, and calcium absorption-promoting peptides. Each of these functional peptides has been recognized as being associated with a certain function. For example, in some specific implementations, peptide libraries can be used to identify these functional peptides:

[0104] >sp|P02662|CASA1_BOVIN Alpha-S1-casein OS=Bos taurus OX=9913GN=CSN1S1 PE=1SV=2

[0105] >sp|P02663|CASA2_BOVIN Alpha-S2-casein OS=Bos taurus OX=9913GN=CSN1S2 PE=1SV=2

[0106] >sp|P02666|CASB_BOVIN Beta-casein OS=Bos taurus OX=9913GN=CSN2 PE=1SV=2

[0107] >sp|P02668|CASK_BOVIN Kappa-casein OS=Bos taurus OX=9913GN=CSN3 PE=1SV=1

[0108] Other functional peptides or functional peptides other than casein phosphopeptide refer to the collection of functional peptides other than casein phosphopeptide among the aforementioned functional peptides.

[0109] In the casein peptide composition of the present invention, the relative strength of the casein phosphopeptide functional peptide is 9% or more, preferably 10% or more, and more preferably 11% or more. The strength of the casein phosphopeptide of the present invention can be maintained at a level equivalent to that of casein phosphopeptides in existing CPP products.

[0110] Furthermore, in the casein peptide composition of the present invention, the relative strength of functional peptides other than casein phosphopeptides is 11% or more, preferably 15% or more, more preferably 20% or more, or even 25% or more, for example 11 to 30%.

[0111] Furthermore, in some preferred embodiments, the total number of peptide types in the casein peptide composition is 1350 or more, preferably 1500 or more, and more preferably 1700 or more. In the technical solution of the present invention, a greater variety of peptide types facilitates the realization of more functionalities. In other embodiments, by adjusting the enzymatic hydrolysis conditions, the total number of peptides in the casein peptide composition can reach up to 2000, for example, 1950 or 1900.

[0112] The total number of peptides mentioned in this invention refers to the total number of different peptide (peptide segments) contained in the casein peptide composition, that is, the total number of peptides (peptide segments) with different amino acid sequences. These peptide segments can be the functional peptide segments mentioned above, or non-functional peptide segments. Under the specific composition of the above-mentioned peptides, the casein peptide composition based on hydrolyzed milk protein has a richer variety of protein peptide functional peptide segments, which can achieve better functions and effects, such as the effects of protecting the intestines and / or helping to improve the discomfort of enteritis.

[0113] In some preferred embodiments, the functional peptides in the casein peptide composition include casein phosphopeptides, ACE inhibitory peptides, antioxidant peptides, immunomodulatory peptides, antimicrobial peptides, tumor suppressor peptides, dipeptidyl peptidase-IV inhibitory peptides, opioid peptides, and functional peptides that promote bone synthesis and metabolism.

[0114] <Second aspect>

[0115] A second aspect of the present invention provides a method for preparing the casein peptide composition described in the first aspect of the present invention. The casein peptide composition of the present invention is obtained by hydrolyzing a raw protein solution in the presence of a specific enzyme.

[0116] In some specific embodiments, the preparation method of the present invention includes an enzymatic hydrolysis step, wherein a casein solution containing a specific protease is used to enzymatically hydrolyze the casein solution.

[0117] In principle, there are no particular restrictions on the source and composition of the casein solution. From the perspective of facilitating subsequent enzymatic hydrolysis, the casein solution of the present invention can be a casein solution obtained by separating the whole protein components through a membrane.

[0118] In some preferred embodiments of the present invention, each 100g of the casein liquid contains 5-15g of dry matter; each 100g of the casein liquid contains 75-100g of protein, and each 100g of the protein contains 75-100g of casein.

[0119] The protease of the present invention necessarily includes at least a serine protease, and in some other embodiments, the protease may further include other flavor proteases. The casein peptide composition prepared by the method for preparing the hydrolyzed milk protein-based casein peptide composition provided by the present invention has a higher content of functional peptides and can achieve a better enzymatic hydrolysis effect.

[0120] (Casein solution)

[0121] 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.

[0122] 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.

[0123] In some embodiments, the casein solution can be obtained from casein raw materials (e.g., milk) via membrane separation. Casein solution obtained from casein raw materials (e.g., milk) via membrane separation can be combined with subsequent enzymatic hydrolysis using complex proteases to achieve better enzymatic hydrolysis results, leading to better intestinal protection or assistance in improving enteritis discomfort. Conversely, casein solutions prepared by enzymatic hydrolysis or acid hydrolysis may affect the mechanism of subsequent combined enzymatic hydrolysis with complex proteases, impacting the hydrolysis effect, functional peptide content, and other effects.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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).

[0128] 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.

[0129] In some specific implementation schemes, the pasteurized material is cooled to 4-6°C and temporarily stored in an enzymatic hydrolysis tank.

[0130] 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.

[0131] In some embodiments, the casein solution contains 0-15g of fat per 100g of dry matter.

[0132] 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.

[0133] 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.

[0134] Casein solutions that meet the above criteria are advantageous for obtaining the functional components of the casein composition of the present invention through hydrolysis.

[0135] (Protein)

[0136] In the hydrolyzed casein solution of the present invention, the protease used must include a serine protease, and in some preferred embodiments, from the perspective of further increasing the content of functional peptides, the protease can be other proteases, such as the complex protease that can be used in the present invention, which includes a serine protease and a flavor protease.

[0137] serine protease

[0138] 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.

[0139] In some specific embodiments, the serine protease includes chymotrypsin-like protease.

[0140] In some specific implementations, the serine protease is an endopeptidase.

[0141] In some specific implementations, the serine protease is derived from microorganisms.

[0142] 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.

[0143] 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.

[0144] Regarding the dosage of serine protease, in some preferred embodiments, the amount of serine protease added is 0.0001-0.015% by mass of the casein solution.

[0145] Flavor proteases

[0146] In this invention, flavor protease, or simply flavor enzyme, is derived from microorganisms such as Aspergillus. It can not only cleave peptide bonds between amino acids from within the 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 the peptide chain, like an exonuclease, to release specific amino acids.

[0147] 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).

[0148] Aspergillus oryzae flavor protease is commercially available to those skilled in the art, for example, from Novo Neogene (…). Food-grade flavor enzymes, such as those sold by Nanning Pangbo Biotechnology Co., Ltd. (500MG), are included, but are not limited to these.

[0149] 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.

[0150] 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.

[0151] In this invention, the combined use of the aforementioned complex proteases is beneficial for obtaining a casein peptide composition with a higher content of functional peptides. In particular, when combined with a casein solution that meets the above-mentioned specifications, even better enzymatic hydrolysis results are achieved.

[0152] In some specific embodiments, the amount of serine protease, especially Bacillus licheniformis serine protease, added is 0.0001-0.015% by weight of the casein solution, preferably 0.0005-0.005% by weight, more preferably 0.001-0.003% by weight, and even more preferably 0.001215-0.001836% by weight, for example: 0.001215%, 0.001836%, 0.001377%.

[0153] The addition of the above-mentioned Bacillus licheniformis serine protease can further increase the content of functional peptides in the obtained casein peptide composition.

[0154] 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.

[0155] 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%.

[0156] The addition of the aforementioned complex protease ensures optimal enzymatic hydrolysis without wasting enzymes or affecting subsequent enzyme inactivation processes, thus preserving the final casein peptide composition's composition and nutritional value. This results in a higher content of functional peptides in the obtained casein peptide composition, leading to superior intestinal protection and improved relief of enteritis symptoms. Especially when combined with casein solution meeting the above criteria, even better enzymatic hydrolysis results are achieved.

[0157] 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.

[0158] (Enzymatic hydrolysis)

[0159] 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 protease used.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] In some specific implementations, the enzymatic hydrolysis time for casein solution is less than 60 minutes. At this hydrolysis time, the combined use of the aforementioned proteases, especially complex proteases, results in a casein peptide composition with superior functional peptide content and better intestinal protection or auxiliary improvement of enteritis discomfort. Especially when combined with casein solution that meets the above-mentioned indicators, even better enzymatic hydrolysis results are obtained.

[0164] From the perspective of further increasing the content of functional peptides, 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.

[0165] 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.

[0166] 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.

[0167] (Post-processing steps)

[0168] Following the step of enzymatic hydrolysis of casein solution, 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.

[0169] Enzyme inactivation treatment

[0170] 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.

[0171] 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.

[0172] Concentration

[0173] The enzyme hydrolysate after enzyme inactivation can be appropriately concentrated.

[0174] 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.

[0175] 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.

[0176] Drying process

[0177] The product after the above concentration process can be dried.

[0178] 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.

[0179] The casein peptide composition based on hydrolyzed milk protein provided by this invention uses a complex protease-directed enzymatic hydrolysis and precise enzymatic hydrolysis control technology to rationally control the content of functional peptides in the casein peptide composition.

[0180] The casein peptide composition based on hydrolyzed milk protein provided by this invention requires no additional processes such as membrane filtration or chromatography. It can produce a product with a high content of functional peptides solely through (compound) protease-directed enzymatic hydrolysis and precise enzymatic hydrolysis control technology. The product contains more than 20 functional peptides, including casein phosphopeptides (CPP), ACE-inhibiting peptides, antimicrobial peptides, antioxidant peptides, bone anabolic peptides, and calcium absorption-promoting peptides. Furthermore, the resulting casein peptide composition has a higher content of functional peptides and exhibits superior intestinal protection or auxiliary improvement of enteritis discomfort.

[0181] <Third aspect>

[0182] A third aspect of the present invention provides a product and its use. The product comprises the casein peptide composition described in the first aspect or a product prepared according to the method described in the second aspect. The present invention does not particularly limit the specific form of the product for different product categories; for example, it may be in powder or liquid form.

[0183] In some implementations, the product is a food product, which is a nutritional food, functional food, or health food.

[0184] In some preferred embodiments, the food product is milk and dairy products. Examples include liquid milk, milk powder, fermented milk, cheese, or condensed milk.

[0185] In some specific implementations, the product is used to improve gut health.

[0186] (Improving gut health)

[0187] The casein peptide composition provided by the present invention helps improve gut health. Therefore, the present invention also provides the use of the casein peptide composition prepared by the preparation method according to the first aspect of the present invention or the casein peptide composition according to the second aspect of the present invention in the preparation of food or health products for improving gut health.

[0188] In some implementations, the improvement of gut health is for non-therapeutic purposes.

[0189] In some implementations, the improvement of gut health includes at least one of regulating gut microbiota, assisting in the improvement of enteritis discomfort, and protecting the gut.

[0190] Protect the gut

[0191] The present invention has found that the casein peptide composition prepared by the above-described preparation method can enhance the intestinal barrier function and has a significant protective effect on the intestine.

[0192] This invention provides the use of a casein peptide composition prepared by the method described in this invention in the preparation of products for protecting the gut.

[0193] In some implementations, the gut protection is for non-therapeutic purposes.

[0194] In some specific implementations, the protection of the gut includes at least one of the following: increasing the number of goblet cells in the ileum, jejunum, and colon; increasing the proportion of Ki-67 and LGR5 positive cells in the ileum, jejunum, and colon; increasing the proportion of CD24+ and LGR5+ positive cells in the gut; and increasing the proportion of E-cadherin positive cells in the gut.

[0195] In some more specific embodiments, as demonstrated in the test examples, the casein peptide composition of the present invention increases the number of goblet cells in the ileum, jejunum, and colon, thereby protecting the intestine and enhancing barrier function.

[0196] In some more specific embodiments, as demonstrated in the test examples, the casein peptide composition of the present invention increases the proportion of Ki-67 and LGR5 positive cells in the ileum, jejunum, and colon, indicating that the casein peptide composition has a protective effect on the intestine.

[0197] In some more specific embodiments, as demonstrated in the test examples, the casein peptide composition of the present invention increases the proportion of CD24+ and LGR5+ positive cells in the gut, causing stem cells to re-proliferate, thereby activating the gut repair function.

[0198] In some more specific embodiments, as demonstrated in the test examples, the casein peptide compositions of the present invention increase the proportion of E-cadherin-positive cells in the gut, promote stem cell differentiation into epithelial cells, and enhance the barrier function of epithelial cells.

[0199] The present invention also found that when subjects using the casein peptide composition provided by the present invention take antibiotics, the protective effect on the intestines is further enhanced.

[0200] In this specification, the term "object" can refer to an individual exhibiting enteritis-related discomfort symptoms or a need for intestinal function protection. For example, when an object experiences increased frequency of diarrhea, abdominal pain, or mucosal damage, intestinal protection may be required. It should be noted that different objects have different subjective perceptions of intestinal discomfort symptoms. As long as their intestinal condition produces clear discomfort symptoms, they can be considered as the applicable objects of the product described in this invention. Furthermore, the use of the product includes current intestinal protection for individuals with pre-existing discomfort symptoms, as well as the auxiliary relief of discomfort symptoms for groups currently experiencing intestinal discomfort symptoms. It also includes physiological states requiring special intestinal care (such as daily intestinal protection).

[0201] In some embodiments, the antibiotics include quinolone antibacterial agents whose basic structure contains a 4-quinolone, such as norfloxacin, levofloxacin, ciprofloxacin, and moxifloxacin.

[0202] In some preferred embodiments, the quinolone antibacterial drug is levofloxacin.

[0203] Therefore, the present invention also provides the use of the casein peptide composition prepared according to the above-described preparation method of the present invention, or the casein peptide composition described above combined with an antibiotic, in the preparation of products for protecting the intestine.

[0204] Helps improve the discomfort symptoms of enteritis

[0205] The present invention has found that the casein peptide composition provided by the present invention has a significant effect on helping to improve the discomfort of enteritis.

[0206] The present invention provides the use of the casein peptide composition prepared by the preparation method described above according to the present invention in the preparation of products for the adjunctive improvement of enteritis discomfort for non-therapeutic purposes.

[0207] In some implementation schemes, aiding in the improvement of enteritis discomfort includes aiding in the improvement of discomfort and symptoms caused by enteritis, specifically including improving weight loss; improving rectal bleeding; increasing the length and weight of the small intestine and colon; reducing inflammatory factors in the intestine, weakening the infiltration of inflammatory cells, and alleviating fibroblast proliferation; reducing DAI score; reducing organ ratio and inhibiting spleen inflammation; increasing villus length and crypt depth in the ileum, jejunum, and colon; and reducing at least one of the following: the proportion of CD45 positive cells in the intestine.

[0208] The present invention also found that when subjects using the casein peptide composition provided by the present invention take anti-infective drugs (e.g., antibiotics), the effect of assisting in the improvement of enteritis discomfort is further enhanced.

[0209] In some more specific embodiments, as demonstrated in the test examples, the casein peptide composition of the present invention can alleviate intestinal mucosal damage, fibrosis or ulceration, and increase the length of the small intestine, indicating that the casein peptide composition of the present invention can help improve enteritis discomfort.

[0210] In some more specific embodiments, as demonstrated in the test examples, the casein peptide compositions of the present invention can reduce inflammation in the ileum, jejunum, and colon, thereby helping to improve enteritis discomfort.

[0211] In some more specific embodiments, as demonstrated in the test examples, the casein peptide composition of the present invention can reduce the degree of inflammatory cell infiltration and decrease the expression of CD45 inflammatory factor in the intestine, thus having the effect of helping to improve the discomfort of enteritis.

[0212] In some more specific embodiments, as demonstrated in the test examples, the casein peptide compositions of the present invention increase the transport proteins PEPT1 and SGLT1 in the small intestine, promote intestinal transport capacity, and help improve enteritis discomfort.

[0213] The present invention also found that when subjects using the casein peptide composition provided by the present invention take antibiotics, the effect of aiding in the improvement of enteritis discomfort can be enhanced.

[0214] In some implementations, the enteritis includes enteritis caused by infection with one or more bacteria.

[0215] In some specific implementations, the enteritis includes enteritis caused by Salmonella infection.

[0216] In addition, this casein peptide composition can be applied to a variety of target groups (such as adolescents, young adults, and the elderly) to meet the needs of different age groups for intestinal protection or to help improve the discomfort of enteritis. Furthermore, the casein peptide composition can also be used in combination with levofloxacin for intestinal protection or to help improve enteritis-related diseases.

[0217] Helps improve gut microbiota

[0218] The present invention also found that the casein peptide composition provided by the present invention helps to regulate the intestinal flora. Therefore, the present invention also provides the use of the casein peptide composition prepared by the preparation method according to the first aspect of the present invention or the casein peptide composition according to the second aspect of the present invention in the preparation of food or health products for regulating the intestinal flora.

[0219] In some embodiments, the casein peptide composition is used for non-therapeutic purposes to regulate the gut microbiota.

[0220] In some embodiments, the casein peptide composition can regulate intestinal flora imbalance and increase the richness of intestinal flora.

[0221] In some specific embodiments, the casein peptide composition can increase the enrichment of beneficial bacteria such as Firmicutes and Bacteroidetes in the intestine. More specifically, the casein peptide composition can increase the enrichment of beneficial bacteria such as Firmicutes and Bacteroidetes in the intestine during enteritis.

[0222] In some specific embodiments, the casein peptide composition can increase the enrichment level of Streptococcus, Enterococcus, and Lactococcus in the intestine. More specifically, the casein peptide composition can increase the enrichment level of beneficial bacteria of Streptococcus, Enterococcus, and Lactococcus in the intestine during enteritis.

[0223] In some specific embodiments, the casein peptide composition can increase microbial species diversity.

[0224] In some specific embodiments, the casein peptide composition provided by the present invention can be used in combination with antibiotics to regulate the intestinal flora in an enteritis state.

[0225] Example

[0226] 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.

[0227] 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);

[0228] The flavor proteases used in the following examples and comparative examples include enzyme preparation BD, specifically:

[0229] Enzyme preparation B: Aspergillus oryzae aminopeptidase, which is from Novo Nordisk. 500MG);

[0230] Enzyme preparation C: Aspergillus niger flavored protease, which is from Sternzym Food Ingredients (Suzhou) Co., Ltd. (Sternzym FP23290);

[0231] Enzyme preparation D: Aspergillus oryzae flavored protease, which is from Nanning Pangbo Biotechnology Co., Ltd.

[0232] Example 1

[0233] 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).

[0234] 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;

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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%;

[0239] 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.

[0240] 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.

[0241] 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;

[0242] 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.

[0243] 7. After enzyme inactivation is complete, the enzyme hydrolysate is cooled to 4°C and transferred to a storage tank for later use.

[0244] 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.

[0245] 9. The concentrated enzymatic hydrolysate can be processed into the final product using a freeze-drying or spray-drying system.

[0246] Example 2

[0247] 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.

[0248] 2. Accurately weigh the four enzyme preparations (AD) and add them to the enzymatic hydrolysis tank, then add RO water to fully dissolve them;

[0249] The enzyme preparation AD is:

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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;

[0257] 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.

[0258] 7. After enzyme inactivation is complete, the enzyme hydrolysate is cooled to 6°C and transferred to a storage tank for later use.

[0259] 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.

[0260] 9. The concentrated enzymatic hydrolysate can be processed into the final product using a freeze-drying or spray-drying system.

[0261] Example 3

[0262] 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.

[0263] 2. Accurately weigh the four enzyme preparations (AD) and add them to the enzymatic hydrolysis tank, then add RO water to fully dissolve them;

[0264] The enzyme preparation AD is:

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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;

[0272] 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.

[0273] 7. After enzyme inactivation is complete, the enzyme hydrolysate is cooled to 5°C and transferred to a storage tank for later use.

[0274] 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.

[0275] 9. The concentrated enzymatic hydrolysate can be processed into the final product using a freeze-drying or spray-drying system.

[0276] Example 4

[0277] 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.

[0278] Other conditions are the same as in Example 1.

[0279] Example 5

[0280] 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.

[0281] Comparative Example

[0282] Comparative Example 1

[0283] 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.

[0284] Comparative Example 2

[0285] In Example 1, step 2, the amount of serine protease added was 0.02145%, and other conditions were the same as in Example 1.

[0286] Test case

[0287] Test Example 1

[0288] 1. Functional peptide differential analysis

[0289] Liquid chromatography conditions: EASY nLC 1200 nanoliter liquid chromatograph, reverse-phase C18 liquid chromatography column (150μm×150mm, 1.9μm)

[0290] 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.

[0291] The raw mass spectrometry data were retrieved from the relevant database using Proteome Discoverer 2.2 software to obtain peptide identification and modification results.

[0292] Peptide spectrum library:

[0293] >sp|P02662|CASA1_BOVIN Alpha-S1-casein OS=Bos taurus OX=9913GN=CSN1S1 PE=1SV=2

[0294] >sp|P02663|CASA2_BOVIN Alpha-S2-casein OS=Bos taurus OX=9913GN=CSN1S2 PE=1SV=2

[0295] >sp|P02666|CASB_BOVIN Beta-casein OS=Bos taurus OX=9913GN=CSN2 PE=1SV=2

[0296] >sp|P02668|CASK_BOVIN Kappa-casein OS=Bos taurus OX=9913GN=CSN3 PE=1SV=1

[0297] Table 1: Results of HPLC-MS / MS

[0298]

[0299] Relative intensity = (Signal intensity of target sample / Signal intensity of reference sample) × 100%.

[0300] Functional peptides refer to polypeptides in the aforementioned peptide library that are labeled with functions such as casein phosphopeptide (CPP), ACE-inhibiting peptides, antimicrobial peptides, antioxidant peptides, bone anabolic peptides, and calcium absorption-promoting peptides. This includes casein phosphopeptide and other functional peptides mentioned below.

[0301] Casein phosphopeptide count refers to the number of functional peptides labeled as casein phosphopeptides in the aforementioned peptide library among the total peptides in the tested sample. Other functional peptide count refers to the number of functional peptides other than casein phosphopeptides labeled in the aforementioned peptide library among the total peptides in the tested sample.

[0302] Casein phosphopeptide percentage refers to the ratio of the number of casein phosphopeptides to the total number of peptides. Other functional peptide percentage refers to the ratio of the number of other functional peptides to the total number of peptides.

[0303] The relative intensity of casein phosphopeptides in the sample is calculated as (signal intensity of casein phosphopeptides / signal intensity of the reference sample) × 100%. The relative intensity of other functional peptides in the sample is calculated as (signal intensity of other functional peptides / signal intensity of the reference sample) × 100%. The relative intensity of functional peptides in the sample is calculated as (signal intensity of functional peptides / signal intensity of the reference sample) × 100%. Normalization was used to represent the relative content of phosphorylated peptides and other functional peptides in the casein peptide composition.

[0304] In this experiment, the reference sample is the product of Comparative Example 1.

[0305] Detection using liquid chromatography-mass spectrometry and database analysis revealed that the products in Examples 1-5 contained functional peptides such as casein phosphopeptide (CPP), ACE inhibitory peptide, antimicrobial peptide, antioxidant peptide, bone anabolic peptide, bone growth peptide, antimicrobial peptide, tumor suppressor peptide, dipeptidyl peptidase-IV inhibitory peptide, and opioid peptide. The relative intensity of the functional peptides in the samples was greater than 35% (Table 1). Figure 1-7 The relative intensities of functional peptides in the samples of both the commercially available product in Comparative Example 1 and Comparative Example 2 were less than 23%.

[0306] 2. Casein phosphopeptide content analysis

[0307] The samples were tested according to Appendix A of the National Food Safety Standard GB 31617-2014, which specifies the detection method for casein phosphopeptides used as a food fortifier.

[0308] Table 2: Results of Casein Phosphopeptide Content Detection

[0309]

[0310]

[0311] Table 2 shows that the test results in this embodiment do not require additional processes such as membrane filtration or chromatography. Casein phosphopeptides can be prepared simply by using the directional enzymatic hydrolysis of compound protease and precise enzymatic hydrolysis control technology.

[0312] Examples 1-5 have the advantage of higher functional peptide content. No additional processes such as membrane filtration and chromatography are required. Products with high functional peptide content can be prepared simply by directional enzymatic hydrolysis of compound protease and precise enzymatic hydrolysis control technology. The casein phosphopeptide (CPP) content (w / %) in the products of Examples 1-5 is 24-30% on a dry basis. The products prepared by the enzymatic hydrolysis method of the present invention have abundant casein phosphopeptide.

[0313] 3. Comparison of the total number of peptides produced during hydrolysis

[0314] Casein is a complete protein, rich in the eight essential amino acids required by the human body, which can provide essential amino acids for organisms and thus promote their development and growth (Reference: Yang Fan, Su Deliang, Song Li. Optimization of alkaline protease hydrolysis process of casein [J]. Food Industry, 2024, 45(08):35-39.). Casein is also an important source of bioactive peptides. Casein has a loose and soft structure, and different functional peptides are produced after enzymatic hydrolysis in different cases. The number of peptides generated after hydrolysis in Examples 1-5 and Comparative Examples 1-2 is shown in Table 1 and 2 after peptidomics identification. Figure 8 As shown.

[0315] The results showed that the total number of peptide types generated in Examples 1-5 was more than 1350, and the total number of peptide types generated in Example 4 was the highest at 1796. Furthermore, the total number of peptide types generated in Examples 1-5 was higher than that in Comparative Example 1 (1304 peptide types) and Comparative Example 2 (1217 peptide types). The total number of peptide types generated in Comparative Examples 1-2 was less than 1350. The more peptide types there are, the more types of functional peptides will be generated.

[0316] 4. Types and content distribution of peptides during enzymatic hydrolysis.

[0317] Database comparison identified 25 functional peptides, including casein phosphopeptides, ACE inhibitory peptides, antioxidant peptides, immunomodulatory peptides, antimicrobial peptides, tumor suppressor peptides, dipeptidyl peptidase-IV inhibitory peptides, opioid peptides, and peptides that promote bone synthesis and metabolism. In addition, many blank peptides not previously identified in the database were also identified. The peptide distribution is as follows: Figure 9 As shown, other functional peptides and casein phosphopeptides account for, for example, Figure 10 As shown.

[0318] Both the examples and comparative examples contain a large number of unidentified blank peptides. The deeper the red color, the stronger the relative intensity of the identified peptide and the higher its content. The casein phosphopeptides in Examples 1-5 and Comparative Example 1 are a deeper red, indicating a higher abundance of casein phosphopeptides in these groups. However, Examples 1-5 are even richer in ACE-related peptides such as ACE inhibitory peptides, ACE inhibitory / bone metabolism promoting peptides, ACE inhibitory / bone metabolism promoting peptides, ACE inhibitory / antioxidant / antithrombotic peptides, as well as antimicrobial peptides, antioxidant peptides, dipeptidyl peptidase-IV inhibitory peptides, and immunomodulatory peptides. This suggests that, in addition to phosphopeptides, the examples are rich in a wider range of nutritional peptides. Figure 10 It can also be seen that the casein phosphopeptide content in Example 4 is the most abundant, indicating that this example provides a preferred solution for preparing a high content of casein phosphopeptide fragments.

[0319] 5. Types and content distribution of peptides during enzymatic hydrolysis.

[0320] Casein phosphopeptides are β-, α- s1 - and α s2 - A series of bioactive peptides rich in phosphoserine acyl groups were obtained from casein through protease hydrolysis and subsequent separation and purification (References: Liu Guo. Preparation, identification, calcium absorption-promoting activity and mechanism of casein phosphopeptides [D]. South China Agricultural University, 2018.; Shi Yanan, Zhang Jiayan, Huang Aixiang. Casein hydrolysis sites of Moringa chymoses and their casein phosphopeptides and casein glycomacropeptides [J]. Food Science, 2021, 42(10):104-110.). The number of phosphopeptide fragments identified in the samples is as follows: Figure 11As shown, the number of repetitive peptides is relatively uniform among the example groups, with significant differences in the number of peptides, indicating that the results of each sample are relatively reliable. The composite enzymatic hydrolysis technology provided by this invention offers a feasible technical solution for the enzymatic hydrolysis process of casein to prepare functional peptides with high content of casein phosphopeptides and ACE inhibitory peptides in a short time.

[0321] In summary, the sample from the example differs significantly from the sample from the comparative example. The combined enzymatic hydrolysis technology of the example provides a feasible technical solution for the enzymatic hydrolysis process of casein to prepare functional peptides with high content of casein phosphopeptides and ACE inhibitory peptides in a short time, and provides a better option for functional food processing.

[0322] Test Example 2 Analysis of the symptom relief effect of casein peptide composition on aged mice with enteritis

[0323] I. Experimental Materials and Instruments

[0324] 1. Laboratory animals

[0325] The 50 aged male C57BL / 6J mice used in this experiment, weighing 15-35g, were all provided by Beijing Vital River Laboratory Animal Co., Ltd.

[0326] 2. Experimental apparatus (refer to Table 3)

[0327] Table 3: Experimental Instruments

[0328]

[0329] Other equipment: surgical scissors, hemostats, forceps, glass dishes, centrifuge tubes, etc.

[0330] 3. Experimental reagents (refer to Table 4)

[0331] Table 4: Experimental Reagents

[0332]

[0333] 4. Reagent preparation

[0334] (1) Preparation of PBS buffer (refer to Table 5)

[0335] Table 5: PBS buffer preparation

[0336]

[0337] (2) 4% paraformaldehyde formulation (refer to Table 6)

[0338] Table 6: 4% Paraformaldehyde Configuration

[0339]

[0340]

[0341] (3) Antibody dilution ratio (refer to Table 7)

[0342] Table 7: Antibody Dilution Ratio

[0343]

[0344] II. Experimental Methods (I) Feeding of Experimental Animals

[0345] 1. Animal husbandry

[0346] The indoor environment is controlled by an independent air conditioning fan system, with good ventilation, room temperature of 22℃-24℃, and relative humidity of 28%-50%. The laboratory environment is kept clean and hygienic, and mice have free access to food and water.

[0347] 2. Animal condition observation

[0348] (1) Weight monitoring: Weight was monitored at the following time points throughout the experiment:

[0349] The initial weighing is performed once, once a day during the modeling period, and once before execution.

[0350] (2) Physiological observation: During the entire experiment, the observations included the smoothness of the mice's fur, the consistency of their stool, and their activity level, as shown in Table 8.

[0351] Table 8: Evaluation Criteria for Physiological Observation in Mice

[0352]

[0353] (3) DAI (Disease Activity Index) score: During the entire experiment, the observations included whether the mice had bloody stools, changes in weight, and loose stools, as shown in Table 9.

[0354] Table 9: Mouse DAI Scoring Criteria

[0355]

[0356] 3. Animal grouping

[0357] (1) Control group: fed with ordinary feed, free access to food and water, and without any treatment;

[0358] (2) Model group: fed with normal feed, and a mouse enteritis model was constructed using Salmonella;

[0359] (3) Positive control group: fed with normal feed, and levofloxacin solution (0.4mg / ml, 50μl) was administered by gavage at the same time as modeling;

[0360] (4) Feeding with a diet containing casein peptide composition (containing 40% casein peptide composition by weight) and using Salmonella to construct an enteritis model;

[0361] (5) Combined group: fed with feed containing casein peptide composition (containing 40% casein peptide composition by weight), and levofloxacin (0.4 mg / ml, 50 μL) was administered by gavage at the same time as modeling.

[0362] 4. Animal-based materials

[0363] On day 28 of the experiment, all mice were harvested 24 hours after modeling was completed. The specific procedures were as follows:

[0364] (1) Small intestine: After the small intestine is removed, its length is measured first, and then it is divided into colon, jejunum and ileum according to its location;

[0365] (2) Colon: After the colon was removed, its length and weight were measured first, and then it was divided into two halves. One half of the tissue was frozen in liquid nitrogen, and the other half was fixed with 4% paraformaldehyde for subsequent experiments.

[0366] (3) Ileum: After the ileum was removed, it was divided into two halves. One half of the tissue was frozen in liquid nitrogen, and the other half was fixed with 4% paraformaldehyde for subsequent experiments.

[0367] (4) Jejunum: After removing the jejunum, it was divided into two halves. One half of the tissue was frozen in liquid nitrogen, and the other half was fixed with 4% paraformaldehyde for subsequent experiments.

[0368] (5) Cecal contents: After removing the cecum, collect the cecal contents and freeze them in liquid nitrogen for subsequent experiments;

[0369] (6) Spleen: After removing the spleen, weigh its wet weight;

[0370] Serum: After blood was collected from the eyeball, the serum was centrifuged and frozen in liquid nitrogen for subsequent experiments.

[0371] (II) Cell surface antibody staining (flow cytometry)

[0372] 1. Resuspend the cells in 98 μL of 1×PBS, add 2 μL of LGR5, incubate at 4°C for 30 min, centrifuge at 300g for 5 min, and discard the supernatant;

[0373] 2. Wash cells with 1×PBS, centrifuge at 300g for 5 min, and discard the supernatant;

[0374] 3. Resuspend cells in 100 μL 1×PBS, add 0.1 μL Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM Incubate at 488°C in the dark for 30 minutes, then centrifuge at 300g for 5 minutes and discard the supernatant.

[0375] 4. Add other antibodies according to the proportions specified in the antibody instructions (refer to Table 10), and incubate at 4°C in the dark for 30-60 minutes;

[0376] Table 10: Proportions of Other Antibodies

[0377]

[0378] 5. Add 1 mL of 1×PBS, centrifuge at 200-300 g for 5 min, discard the supernatant, resuspend the cells in 200 μL of 1×PBS, filter through a 200-mesh sieve, and then load the solution into the machine.

[0379] (III) Dehydration and embedding

[0380] 1. Dehydration: overnight with 75% ethanol, 30 min with 80% ethanol, 30 min with 85% ethanol, 30 min with 90% ethanol, 30 min with 95% ethanol, and 30 min each with 100% ethanol I and II.

[0381] 2. Transparent: Xylene I for 20 min, Xylene II for 10 min.

[0382] 3. Wax impregnation: Paraffin I, II, and III, for 30 min, 60 min, and 90 min respectively;

[0383] 4. The dehydrated samples were then embedded in paraffin.

[0384] (iv) IF staining (immunofluorescence staining)

[0385] 1. Preparation of IF reagent (refer to Table 11)

[0386] Table 11: Reagent Preparation

[0387]

[0388] PBST buffer: Weigh 0.46g sodium dihydrogen phosphate, 2.3g sodium dihydrogen phosphate dodecahydrate, and 6.95g sodium chloride, add distilled water to a final volume of 1000ml, and store at room temperature;

[0389] 2% PBST buffer: Add 2 ml of Tween-20 to the PBS buffer prepared above, mix well, and store at room temperature;

[0390] The membrane breaking solution is prepared in a ratio of methanol to H2O2 of 9:1.

[0391] Antigen repair solution: Take 4.75ml of antigen repair solution, add 500ml of distilled water, heat to boiling and then use;

[0392] DAPI working solution: Take 1 μL of DAPI stock solution, add 2 mL of PBS phosphate buffer, mix well, and store at -20°C.

[0393] Note: The final reagent volume depends on the number of experimental samples.

[0394] 2. Experimental Procedure

[0395] 1. Sectioning: Before sectioning, set the temperature of the biological tissue curing oven to 60℃ and the thickness to 4μm. After removing the sections with an adhesive slide, heat the sections for 30 minutes.

[0396] 2. Dewaxing: Place the sections in a basket and immerse them in xylene I, II, and III in sequence for 5 minutes each;

[0397] 3. Hydration: Immerse the sections in anhydrous ethanol I and II sequentially for 5 min each; add an appropriate amount of permeabilizing solution to the sections and allow them to permeate at room temperature for 10 min.

[0398] After membrane rupture, the membrane was rinsed in the following reagents: 95% ethanol for 2 min, 90% ethanol for 2 min, 85% ethanol for 2 min, 80% ethanol for 2 min, 75% ethanol for 2 min, and hydrated 3 times for 5 min each time.

[0399] 4. Antigen retrieval: After boiling the antigen retrieval solution in a microwave oven, place the slide in the solution and cool it to room temperature before proceeding with subsequent operations.

[0400] 5. Blocking: After antigen retrieval, the slides were washed three times with PBST for 5 minutes each time, and then 100 μL of BSA blocking solution was added to each slide. The slides were then incubated at room temperature for 30 minutes.

[0401] 6. Incubate primary antibody: Add 100 μL of diluted primary antibody to each slice, seal with sealing film, and place in a 4°C refrigerator overnight.

[0402] 7. Incubation of secondary antibody: The next day, take out the slides incubated with primary antibody, wash with PBST 3 times for 5 min each time, then add 100 μL of diluted secondary antibody, seal with sealing film, and incubate in a 37℃ oven for 1 h.

[0403] 8. Nuclear staining: After incubating with the secondary antibody, add 100 μL of DAPI working solution, seal with sealing film, and incubate at room temperature for 10 min;

[0404] 9. Mounting: After staining with DAPI, wash the slide twice with PBST for 5 minutes each time, then wash once with pure water for 5 minutes. Finally, mount the slide with an anti-fluorescence quencher.

[0405] 10. Image acquisition using a fluorescence microscope.

[0406] (V) Statistical Analysis

[0407] One-way ANOVA was performed on the experimental data using statistical software such as ImageJ-win64 and Prism 10.0. All experimental results are expressed as mean ± standard error (x ± s). "*" indicates p < 0.05; "**" indicates p < 0.01; "***" indicates p < 0.001; "****" indicates p < 0.0001.

[0408] III. Experimental Results

[0409] 1. Weight measurement results

[0410] From the start to the end of the experiment, body weight was measured 7 times. Before modeling, the average body weight of mice in each group was not significantly different. Starting from the 3rd day after modeling, the body weight of some experimental groups began to change, with the model group showing the most significant decrease and the fastest weight loss during the experiment. The positive control group, casein group, and combined group had higher body weights than the model group. The line graph shows that the combined group mice steadily increased in body weight, similar to the trend of the blank group, while the positive control group and casein group showed a decreasing trend in body weight, but the decrease was less than that of the model group. Overall, the body weight of older mice increased more slowly, and if there were disease factors, there would be a rapid loss of body weight.

[0411] Analysis of the final body weight showed that, compared with the control group, the body weight of mice in the model group was significantly lower (p<0.001). Compared with the model group, the body weight of mice in the positive control group, casein group, and combined group was significantly higher (p<0.001). This indicates that the weight loss of mice was controlled to varying degrees after treatment with the experimental substance. Furthermore, the body weight of mice in the combined group maintained an upward trend before and after modeling, showing no significant difference from the control group. The final body weight of the combined group was significantly higher than that of the positive control group and the casein group (p<0.001). This suggests that the combined treatment with levofloxacin and casein was more effective in improving enteritis than treatment with the experimental substance or the positive control alone. Figure 12 And Table 12.

[0412] Table 12: Statistical Results of Material Sampling Data

[0413] Last weight / g Organ ratio / % Small intestine length / cm Colon length / cm Blank group 27.2±0.1701 1.05±0.16 39.21±0.60 7.60±0.19 Model group 23.3875±0.1903**** 1.87±0.04**** 20.55±0.36**** 4.95±0.13**** Positive control group 25.4875±0.163* 1.27±0.05*** 35.31±0.28**** 6.99±0.15**** Chelate group 25.1375±0.0778**** 1.59±0.05*** 33.00±0.73**** 6.25±0.11* Joint Group 27.05±0.1376**** 1.07±0.06**** 36.96±0.17**** 7.46±0.24****

[0414] Note: Data is represented as “x±s,n=8”, “*” indicates p<0.05; “**” indicates p<0.01; “***” indicates p<0.001; “****” indicates p<0.0001.

[0415] 2. Physiological observation and DAI score results

[0416] Physiological observation and DAI scoring can be used to observe the health status of experimental animals and the severity of discomfort in mice with enteritis, which facilitates the assessment of discomfort from multiple dimensions. The effect of the test substance treatment on improving the discomfort of enteritis can be evaluated by the score differences between groups.

[0417] According to the statistical data of this test case, compared with the blank group, the physiological observation and DAI scores of mice in the model group gradually increased with the increase of modeling time. In the middle and late stages of modeling, they showed obvious kyphosis, severe skin bulging, very rough fur, diarrhea and severe bloody stools, significantly reduced activity, and some mice died during the modeling process. The physiological observation and DAI scores of mice in the positive control group, casein group, and combined group were all lower than those in the model group throughout the modeling period. In the late stage of modeling, the casein group had rough fur, liquid stools, and obvious bloody stools, and some mice also died. The combined group mice showed no significant difference from the blank group in the early stage, with smooth fur, normal stools, no bloody stools, and relatively active overall. In the last two measurements, the stools became softer, with slight bloody stools, and the mice were in good spirits. This indicates that the combined treatment of levofloxacin and casein can effectively alleviate the symptoms of enteritis in aged mice. Figure 13 , Figure 14 And Table 13.

[0418] Table 13: Physiological Observations and DAI Scores

[0419]

[0420]

[0421] 3. Statistical Results of Material Collection

[0422] (1) Statistical results of intestinal sampling data

[0423] In the enteritis model, changes in intestinal length are one of the key indicators for assessing the severity of enteritis and improving the effectiveness of discomfort symptoms. In this test case, the length of the small intestine, colon, and weight of aged mice were measured to evaluate the modeling and experimental treatment effects.

[0424] According to the statistical data of this test case, compared with the blank group, the small intestine length of mice in the model group was significantly shortened (p<0.001), indicating that intestinal inflammation may have caused intestinal mucosal damage, fibrosis, or ulceration, thereby triggering intestinal contraction. Compared with the model group, the small intestine length of the positive control group, the caseinate group, and the combined group was significantly increased (p<0.001), indicating that the treatment with the test substance can reduce intestinal inflammation and stimulate intestinal regeneration. Figure 15 And Table 13.

[0425] According to the statistical data of this test case, compared with the blank group, the colon length of mice in the model group was significantly shortened (p<0.001), indicating that intestinal inflammation may have caused mucosal damage, fibrosis, or ulceration of the colon, thereby triggering intestinal contraction and resulting in overall shortening of the small intestine. Compared with the model group, the colon length of mice in the positive control group, casein group, and combined group was significantly increased (p<0.001), indicating that the treatment with the test substance can alleviate intestinal inflammation and stimulate the regenerative function of the colon. Among them, the combined group of mice had the highest colon length, and the effect of improving enteritis discomfort was more obvious. The results are shown in […]. Figure 15 And Table 13.

[0426] According to the statistical data of this test case, compared with the blank group, the colon weight of mice in the model group was also significantly reduced (p<0.01), indicating that intestinal inflammation may have led to water loss, cell necrosis or shedding in the colon, resulting in tissue reduction. Compared with the model group, the colon weight of the positive control group, the caseinate group, and the combined group increased, but only the combined group showed a significant difference (p<0.05), indicating that the combined group could more effectively alleviate this symptom. The specific effects of the experimental treatment on improving enteritis need to be evaluated in conjunction with other data. The results are shown in [link to results]. Figure 15 And Table 13.

[0427] (2) Statistical results of organ ratio

[0428] The organ ratio can reflect the functional state of organs, pathological changes, and the effects of experimental treatment on the mouse body. In this test case, the organ ratio of the spleen was calculated to assess the degree of organ damage, and the modeling and experimental treatment effects were judged accordingly.

[0429] After spleen removal, organ ratios were calculated. Statistical results showed that, compared to the control group, the spleen organ ratio in the model group was significantly increased (p<0.001), indicating that the spleen may have enlarged or proliferated under the influence of in vivo inflammatory responses, leading to increased weight. Compared to the model group, the organ ratios in the positive control group, casein group, and combined group were significantly decreased (p<0.01), indicating that spleen inflammation was inhibited to varying degrees after treatment with the experimental substances, resulting in significant weight reduction (p<0.01). Levofloxacin combined with casein treatment showed better improvement in enteritis. Figure 16 And Table 13.

[0430] Formula for calculating organ ratio:

[0431] Organ ratio = (Organ weight / Body weight on the day of sampling) × 100%

[0432] 4. Flow cytometry results

[0433] Flow cytometry primarily analyzes the expression levels and quantities of specific molecular markers on or within cells. In this test case, CD45 was selected as an immune cell marker. By detecting its proportion in intestinal cells, the immune system response and inflammation status can be assessed.

[0434] According to the flow cytometry results of this test, compared with the blank group, the proportion of CD45-positive cells in the intestine of the model group was significantly increased (p<0.001), indicating that abnormal aggregation of immune cells and obvious inflammatory response existed in the intestine of mice with enteritis. Compared with the model group, the positive rate of CD45 in the intestine of the positive control group, casein group, and combined group was significantly decreased (p<0.01). Among them, the inflammatory factors in the combined group were significantly lower than those in the positive control group and casein group. Since older mice have lower immunity than younger mice, the inflammatory factors in their bodies were also higher than normal after treatment. However, the above results indicate that all three treatments can play an anti-inflammatory role. In comparison, the treatment of levofloxacin combined with casein has a better effect on improving enteritis. Figure 17 and Figure 20 .

[0435] The E-cadherin index selected in this test case is a key cell adhesion molecule that participates in maintaining the polarity of epithelial cells and the integrity of tissue structure. By detecting its proportion in intestinal cells, the extent of epithelial cell damage in intestinal tissue can be determined.

[0436] According to the flow cytometry results of this test, compared with the blank group, the proportion of E-cadherin-positive cells in the intestine of the model group was significantly reduced (p<0.001), indicating that the adhesion of intestinal cells in mice with enteritis was reduced, leading to severe epithelial cell detachment and damage. Compared with the model group, the positive rate of E-cadherin in the intestine of the positive control group, the casein group, and the combined group was significantly increased (p<0.01), with the combined group being significantly higher than the positive control group and the casein group. Since older mice have weaker recovery ability than younger mice, cell function is more difficult to restore to normal levels after treatment with the experimental substances. However, the above results indicate that all three treatments can improve cell adhesion, thereby maintaining the integrity of epithelial cells and enhancing the barrier function of epithelial cells. The combination of levofloxacin and casein showed better efficacy in improving enteritis. Figure 18 and Figure 19 .

[0437] According to the flow cytometry results of this test, compared with the blank group, the proportion of CD24+ and LGR5+ positive cells in the intestine of the model group was significantly decreased (p<0.001), indicating that the number of stem cells in mice with enteritis was reduced and their differentiation capacity was weakened. Compared with the model group, the positive rates of CD24+ and LGR5+ in the intestine of the positive control group, the casein group, and the combined group were all significantly increased (p<0.001), with the combined group being significantly higher than the positive control group and the casein group. This indicates that the treatment with the test substance can restore stem cell function, promote cell differentiation capacity, and thus activate the intestinal repair function. Compared with the three treatment groups, the treatment with levofloxacin combined with casein showed better improvement in enteritis. Figure 20 and Figure 21 .

[0438] 5. Immunofluorescence assay results

[0439] Immunofluorescence staining uses fluorescently labeled antibodies to detect the expression location and level of specific proteins or antigens. It is mainly used to analyze the expression of specific molecular markers in cells or tissues. In this test example, PEPT1 (peptide transporter 1) and SGLT1 (sodium-glucose cotransporter 1) are important transport proteins in the small intestine, marking different functions of intestinal epithelial cells.

[0440] According to the IF staining results of this test case, compared with the blank group, the expression of PEPT1 and SGLT1 in the ileum of the model group was significantly reduced (p<0.001), indicating that the epithelial cells in the ileum of mice with enteritis were severely damaged. Compared with the model group, the positive rates of PEPT1 and SGLT1 in the intestines of the positive control group, the casein group, and the combined group were increased (p<0.001), with the expression in the combined group being higher than that in the positive control group and the casein group, indicating that the casein had a certain repair effect on the ileal epithelial cells. The combined treatment of casein and levofloxacin had a better effect on improving enteritis. Figure 22 and Figure 25 .

[0441] According to the IF staining results of this test case, compared with the blank group, the expression of PEPT1 and SGLT1 in the jejunum of the model group was significantly reduced (p<0.001), indicating that the epithelial cells in the jejunum of mice with enteritis were severely damaged. Compared with the model group, the positive rates of PEPT1 and SGLT1 in the intestines of the positive control group, the casein group, and the combined group were increased (p<0.001), with the expression in the combined group being higher than that in the positive control group and the casein group, indicating that the casein had a certain repair effect on the epithelial cells of the jejunum. The combined effect of casein and levofloxacin was even better. Figure 23 and Figure 25 .

[0442] According to the IF staining results of this test case, compared with the blank group, the expression of Ki-67 and LGR5 in the colon of the model group was significantly reduced (p<0.001), indicating that the epithelial cells in the colon of mice with enteritis were severely damaged. Compared with the model group, the positive rates of PEPT1 and SGLT1 in the intestines of the positive control group, casein group, and combined group were increased (p<0.001), with the expression in the combined group being higher than that in the positive control group and casein group, indicating that casein had a certain repair effect on the epithelial cells of the colon. The combined treatment of casein and levofloxacin had a better effect on improving enteritis. Figure 24 and Figure 25 .

[0443] Test Example 5 Analysis of the effects of casein peptide composition on gut microbiota in aged mice

[0444] I. Experimental Materials

[0445] 1. Laboratory animals

[0446] The 50 aged male C57BL / 6J mice used in this experiment, weighing 15-35g, were all provided by Beijing Vital River Laboratory Animal Co., Ltd.

[0447] II. Experimental Methods

[0448] 1. Animal husbandry

[0449] The indoor environment is controlled by an independent air conditioning fan system, with good ventilation, room temperature of 22℃-24℃, and relative humidity of 28%-50%. The laboratory environment is kept clean and hygienic, and mice have free access to food and water.

[0450] 2. Animal grouping

[0451] (1) Control group: fed with ordinary feed, free access to food and water, and without any treatment;

[0452] (2) Model group: fed with normal feed, and a mouse enteritis model was constructed using Salmonella;

[0453] (3) Positive control group: fed with normal feed, and levofloxacin solution (0.4 mg / ml, 50 μL) was administered by gavage at the same time as modeling;

[0454] (4) Casein group: fed with a diet containing casein peptide composition (containing 40% by weight of the casein peptide composition prepared in Example 1), and an enteritis model was constructed using Salmonella;

[0455] (5) Combined group: fed with feed containing casein peptide composition (containing 40% by weight of casein peptide composition prepared in Example 1), and levofloxacin (0.4 mg / ml, 50 μL) was administered by gavage at the same time as modeling.

[0456] 3. Animal-based materials

[0457] On day 28 of the experiment, 24 hours after the modeling of all mice was completed, samples were taken. The sampling steps were as follows: First, the mice were anesthetized and their whole bodies were disinfected with 75% alcohol. Blood was taken from the eyeballs first. Then, the abdominal cavity was opened and the colon, ileum, jejunum and spleen were taken for subsequent experiments. Half of the colon, jejunum and ileum were frozen in a -80℃ freezer and the other half were fixed in 4% paraformaldehyde.

[0458] 4.16 sDNA Sequencing Experiment Analysis

[0459] After sequencing, the raw data is obtained. The paired-end data are then spliced ​​using overlap, followed by quality control and chimera filtering to obtain high-quality clean data. DADA2 (Divisive Amplicon Denoising Algorithm) improves data accuracy and species resolution by obtaining representative sequences with single-base precision through steps such as dereplication (equivalent to clustering with 100% similarity). The core of DADA2 is noise reduction, followed by the construction of OTU (Operational Taxonomic Units) tables using ASVs (Amplicon Sequence Variants) to obtain the final feature table and feature sequences. Further diversity analysis, species classification annotation, and differential analysis are then performed.

[0460] III. Experimental Results

[0461] (I) Sequencing Analysis Results

[0462] 1. Valid data statistics

[0463] After performing dual-end splicing, quality control, and chimera filtering on the raw offline data, high-quality data statistics were performed, and the results are shown in Table 14 below:

[0464] Table 14: Statistical Table of Valid Data

[0465]

[0466]

[0467] The meanings of each column in Table 14 above are shown in Table 15 below:

[0468] Table 15: Meaning of Each Column

[0469] Term significance Sample Sequencing sample name Raw_Reads The raw sequencing data is presented in units of four lines, counting the number of sequencing sequences in each file. Raw_Bases The number of sequencing sequences multiplied by the length of the sequencing sequence, expressed in units of M. Valid_Tags After preprocessing, the number of assembled sequencing sequences in each file is counted in units of four lines. Valid_Bases After preprocessing, the number of sequencing sequences is multiplied by the length of the sequencing sequence, and expressed in units of M. Valid% The ratio of valid data to raw data, expressed as a percentage. Q20％ The proportion of valid data with a quality ≥ Q20 Q30％ The proportion of valid data with a quality ≥ Q30 GC% GC content in valid data

[0470] Tables 14 and 15 above show the sequencing data used. This sequencing data underwent processing and statistical analysis, and quality control (QC) was performed. The data shows that the quality of the sequencing data was mainly distributed above Q20 (≥95%) and Q30 (≥90%). QC can eliminate systematic errors and ensure data reliability. The distribution of sequencing base content was also statistically analyzed to detect any AT / GC separation. Overall, the sequencing data quality is high, the library construction process is complete, and there is no obvious contamination in the samples, allowing for subsequent result analysis.

[0471] 2. Alpha diversity analysis

[0472] Alpha diversity refers to the diversity within a specific habitat (or sample). It can be assessed using a series of alpha diversity indices to obtain information such as species richness and diversity within an environmental community. The results of the violin plot show that... Figure 26 The microbial species diversity of mice in the model group was significantly lower than that in the blank group (p<0.01), while the microbial species richness of mice in the combined group was significantly higher than that in the model group and the casein group (p<0.01), and the richness index was higher than that in the positive control group, but the difference was not significant. Compared with the positive control group, the casein peptide composition in the combined group can increase the microbial species diversity to a certain extent, and there is no significant difference between the combined group and the blank group.

[0473] 3. β-diversity analysis

[0474] Beta diversity refers to the species differences between different environmental communities (samples). PCoA analysis is used to analyze beta diversity. Figure 27 Phylogenetic distance can be observed to distinguish the blank group, combined group, positive control group, casein peptide group, and model group. The separation effect shows that the model group has a significant separation trend from the blank group, positive control group, and combined group, indicating that there is a significant difference in the structure and distribution of the microbial community of mice in the model group, while there is no significant difference from the casein peptide group. Compared with other groups, the distribution of the microbial community in the combined group is significantly similar to that of the blank group. Compared with the positive control group, the casein peptide composition in the combined group can increase the separation trend of phylogenetic distance, and the casein peptide composition increases the richness of the intestinal flora of mice.

[0475] 4. Microbial community difference analysis

[0476] To further analyze the differences in microbial communities among groups, Venn analysis was performed. Based on the obtained eigenvalue abundance table, the number of shared features for each sample / group was calculated, and the results were plotted using Venn diagrams. Figure 28This test case visually presents the number of shared and unique features across samples / groups. The expression abundance of each feature in each group was statistically analyzed. If the expression of a feature is non-zero in all three groups, then that feature is shared by all three groups. Similarly, if a feature exists only in one sample / group, then that feature is unique to that sample / group. The results showed that there were 166 shared species among the five groups. The number of unique species in the blank group, combined group, positive control group, casein group, and model group were 353, 236, 265, 203, and 139, respectively. The model group had the lowest species specificity. The casein group showed an increase in the number of unique species compared to the model group, indicating that the casein composition in the casein group had a regulatory effect on the gut microbiota.

[0477] 5. Species composition analysis

[0478] (1) Cluster diagram

[0479] While bar charts of species composition provide a visual overview of species abundance across different samples or groups, they don't directly reveal the similarity between specific samples. To study the differences and similarities between samples in greater detail, cluster analysis was performed based on the species composition distances within the bar charts. This analysis was implemented using the R language's vegan package, employing the Bray-Curtis distance (a commonly used distance metric in hierarchical clustering, primarily used to describe the similarity between samples; the distance is a key criterion for sample classification). The analysis selected the top 30 abundant species, including enrichment of microorganisms such as Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, Verrucous Microbes, Dethiobacteria, Fusobacteria, and Campylobacteria. Bray-Curtis distance was also used for sample clustering. Figure 29 The results showed that the microbial composition of the combined group mice was most similar to that of the control group, with the closest species composition. The positive control group was the next closest. Compared to the positive control group, the casein peptide composition in the combined group helped to make the microbial composition of the mice similar to that of the control group. The casein peptide group showed the highest similarity to the model group. The combined group mice exhibited an enrichment of beneficial bacteria such as Firmicutes and Bacteroidetes in their intestines. Compared to the positive control group, the combined group showed a significant increase in Firmicutes and a significant decrease in harmful bacteria such as Proteobacteria. The casein peptide composition in the combined group could enrich beneficial bacteria in the intestines, and the ratio of Firmicutes to Bacteroidetes was higher than in other experimental groups. The model group mice showed significant enrichment of Proteobacteria in their intestines, with the ratio lower in other treatment groups than in the model group, and the lowest ratio in the combined group.

[0480] (2) Analysis of significant differences

[0481] 16S rDNA sequencing analysis was used to study the composition of microbial communities in specific environments. Fisher's exact test (suitable for comparing differences between samples without biological replicates), Mann-Whitney U test (suitable for comparing differences between two groups of samples with biological replicates), and Kruskal-Wallis test (suitable for comparing multiple groups of samples with biological replicates) were employed to test for species differences.

[0482] Figure 30 The bubble charts shown depict microorganisms with significant differences at each level. The p-value obtained from the statistical tests above was used to determine whether there were significant differences between different groups for the species; in this test case, p < 0.05 was defined as a significantly different species. Species annotation information for the corresponding phylum is also displayed. The results showed that harmful genera such as Salmonella and Shigella were significantly enriched in the model group, while their enrichment levels in the intestine were significantly reduced under the combined effects of casein hydrolysate and antibiotics. Furthermore, the combined group showed enrichment of beneficial bacteria such as Streptococcus, Lactobacillus, Enterococcus, and Bifidobacterium, with enrichment levels higher than other treatment groups. The average enrichment levels of Streptococcus, Enterococcus, and Lactococcus in the combined group were higher than the positive control group. The casein peptide composition in the combined group could enrich beneficial bacteria in the intestine.

[0483] 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.

[0484] 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 casein peptide composition containing multifunctional peptides, characterized in that, The casein peptide composition was obtained by enzymatic hydrolysis of casein solution. In the casein peptide composition, the relative strength of total functional peptides is above 25%, the relative strength of casein phosphopeptides is above 9%, and the relative strength of functional peptides other than casein phosphopeptides is above 11%. Furthermore, the enzymes used in the enzymatic hydrolysis include at least serine proteases.

2. The casein peptide composition according to claim 1, characterized in that, The casein liquid is obtained from milk raw materials through membrane separation.

3. The casein peptide composition according to claim 1 or 2, characterized in that, In the casein peptide composition, the relative strength of the total functional peptides is between 25% and 45%. The relative strength of functional peptides other than casein phosphopeptides is between 15% and 30%.

4. The casein peptide composition according to claim 1 or 2, characterized in that, The total number of peptide types in the casein peptide composition is more than 1350.

5. A method for preparing the casein peptide composition according to any one of claims 1 to 4, characterized in that, The method includes: In the enzymatic hydrolysis step, the amount of serine protease added is 0.0001-0.015% by mass, based on the mass of the casein solution. The serine protease includes Bacillus licheniformis serine protease.

6. The preparation method according to claim 5, characterized in that, The enzymatic hydrolysis step also uses flavor proteases, including Aspergillus oryzae flavor protease and Aspergillus niger flavor protease.

7. The preparation method according to claim 5 or 6, characterized in that, In the enzymatic hydrolysis step, the mass of the casein solution is as follows: 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.

8. The preparation method according to any one of claims 5 to 7, characterized in that, The membrane separation method employs ceramic membranes and / or spiral wound membranes; Preferably, after obtaining casein liquid from casein raw material by membrane separation, it is pasteurized.

9. The preparation method according to any one of claims 5 to 8, 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℃.

10. The use of the casein peptide composition according to any one of claims 1 to 4 or the casein peptide composition prepared by the preparation method according to any one of claims 5 to 9 in the preparation of food and / or health products for improving gut health.

11. The use according to claim 10, characterized in that, The improvement of gut health includes at least one of the following: regulating gut microbiota, helping to improve enteritis discomfort, and protecting the gut.

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