Casein hydrolysate as well as preparation method and application thereof

Abstract

The invention discloses a casein hydrolysate as well as a preparation method and application thereof. The present invention provides a casein hydrolysate in which the content of a protein component having a molecular weight between 196 Da and 500 Da is 40% by mass or more among the protein components of the casein hydrolysate. The casein hydrolysate provided by the invention has lasting milk fat fragrance.

Description

Technical Field

[0001] This invention belongs to the field of protease hydrolysis technology, specifically, it relates to a casein hydrolysate, its preparation method, and its application. Background Technology

[0002] Protein supplements are commonly used for protein fortification (e.g., in sports drinks), as well as in other dietary drinks, dry blended beverages, nutrition bars, infant formula, and adult milk powder.

[0003] Generally speaking, pre-digested, partially hydrolyzed milk-based proteins are more easily absorbed than unhydrolyzed proteins, which is why protein hydrolysates are considered to have nutritional benefits. However, hydrolyzed casein often has a slightly bitter or astringent taste, which makes it unpalatable to most people when used in foods such as beverages or adult milk powder.

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

[0005] Reference 2 relates to a low-bitterness casein hydrolysate and its preparation method. The provided preparation method includes: performing three enzymatic hydrolysis treatments on casein to obtain the casein hydrolysate; the enzyme used in the first hydrolysis is trypsin, the enzyme used in the second hydrolysis is a flavor protease, and the enzyme used in the third hydrolysis is TG enzyme; by mass, the amount of trypsin is 0.4–2.2% of the casein; the amount of flavor protease is 0.08–0.5% of the casein; and the amount of TG enzyme is 0.4–2.2% of the casein. By optimizing the enzymatic hydrolysis conditions for casein, the bitterness of the obtained hydrolysate is significantly reduced, and a casein hydrolysate with virtually no bitterness is obtained without adding exogenous non-enzymatic substances.

[0006] Existing technologies, such as those cited in references 1-2, employ prolonged enzymatic hydrolysis and multiple enzymatic treatments. While these techniques can reduce the bitterness of casein hydrolysates to some extent, the flavor of the current products needs improvement. Further research and improvements are needed to enhance the flavor of casein hydrolysates during the production process.

[0007] Therefore, there is an urgent need in the field for casein hydrolysates with good flavor.

[0008] References

[0009] Reference 1: CN116268174A

[0010] Reference 2: CN119234918A Summary of the Invention

[0011] The problem the invention aims to solve

[0012] Deep hydrolysis results in more protein breakdown, but it also increases the exposure of bitter substances. For example, references 1-2 employ prolonged enzymatic hydrolysis and multiple enzymatic treatments. These techniques can reduce the bitterness of casein hydrolysates to some extent, but the flavor of the products still needs improvement.

[0013] Further research and improvement of the production process are needed to enhance the flavor of casein hydrolysates and meet market demands. Therefore, this invention primarily provides a casein hydrolysis method and the resulting hydrolysate. This method, by controlling the composition of the casein solution and the use of specific enzymes, can suppress undesirable flavors, reduce bitterness, and enhance the milky aroma of the product.

[0014] Solution for solving the problem

[0015] In a first aspect of the present invention, a casein hydrolysate is provided, wherein the content of the protein component with a molecular weight between 196 Da and 500 Da is 40% by mass or more, preferably between 50% and 60% by mass, and more preferably between 52% and 54% by mass.

[0016] In one embodiment, the casein hydrolysate has a milk fat aroma.

[0017] In a preferred embodiment, the casein hydrolysate achieves a combined score of 84-87 points for milk fat aroma and lingering fragrance.

[0018] In one implementation, the sensory evaluation method involves five evaluators scoring the casein hydrolysate sample in three aspects: taste (1-25 points), aroma intensity (1-50 points), and aroma duration (1-25 points). The overall score is calculated by adding the scores of the three indicators together.

[0019] In one embodiment, the average molecular weight of the casein hydrolysate does not exceed 1500 Da.

[0020] In a preferred embodiment, the average molecular weight of the casein hydrolysate is not less than 850 Da.

[0021] In a more preferred embodiment, the casein hydrolysate has an average molecular weight of 950-980 Da.

[0022] In one embodiment, the protein component of the casein hydrolysate contains at least 17% by mass of a protein component with a molecular weight between 500 Da and 1000 Da.

[0023] In a preferred embodiment, the content of protein components with a molecular weight between 500 Da and 1000 Da in the casein hydrolysate is between 18% and 25% by mass.

[0024] In a more preferred embodiment, the content of protein components with a molecular weight between 500 Da and 1000 Da in the casein hydrolysate is between 19% and 23% by mass.

[0025] In one embodiment, the degree of hydrolysis of the casein hydrolysate is not less than 10%, preferably 11%-15%.

[0026] In a second aspect of the present invention, a method for preparing the casein hydrolysate described in the first aspect of the present invention is provided, wherein the preparation method comprises:

[0027] a) Add metalloproteinases and bacterial endopeptidases simultaneously or sequentially to an aqueous solution containing casein; and

[0028] b) Incubate to hydrolyze the casein.

[0029] In a preferred embodiment, the metalloproteinase and bacterial endopeptidase are added simultaneously.

[0030] In a more preferred embodiment, the metalloproteinase and bacterial endopeptidase are added in the form of a protease composition.

[0031] In one embodiment, the metalloproteinase in the preparation method is a metalloproteinase derived from Bacillus amyloliquefaciens.

[0032] In one embodiment, the bacterial endopeptidase in the preparation method is a bacterial endopeptidase derived from Nocardia strain NRRL 18262.

[0033] In one embodiment, the metalloproteinase in the preparation method is selected from the group consisting of:

[0034] i) a polypeptide comprising an amino acid sequence having at least 60% identity with the polypeptide shown in SEQ ID NO:1; and

[0035] ii) A variant of the polypeptide shown in SEQ ID NO:1 that includes substitutions, deletions, and / or insertions of one or more amino acids.

[0036] In one embodiment, the bacterial endopeptidase in the preparation method is selected from the group consisting of:

[0037] i) a polypeptide comprising an amino acid sequence having at least 60% identity with the polypeptide shown in SEQ ID NO:2; and

[0038] ii) A variant of the polypeptide shown in SEQ ID NO:2 that includes substitutions, deletions, and / or insertions of one or more amino acids.

[0039] In one embodiment, in the preparation method, in step a), the amount of metalloproteinase added is 0.0001-3 AU-N / g casein.

[0040] In a preferred embodiment, the amount of metalloproteinase added is 0.001-2 AU-N / g casein.

[0041] In a more preferred embodiment, the amount of metalloproteinase added is 0.05-1 AU-N / g casein.

[0042] In one or even a more preferred embodiment, the amount of metalloproteinase added is 0.01-0.1 AU-N / g casein.

[0043] In one embodiment, the amount of bacterial endopeptidase added is 0.0001-3 KPROT / g casein.

[0044] In a preferred embodiment, the amount of bacterial endopeptidase added is 0.001-2 KPROT / g casein.

[0045] In a more preferred embodiment, the amount of bacterial endopeptidase added is 0.05-1 KPROT / g casein.

[0046] In one or even a more preferred embodiment, the amount of bacterial endopeptidase added is 0.01-0.1 KPROT / g casein.

[0047] In one embodiment, in the preparation method, in step b), hydrolysis is carried out between 45°C and 70°C, preferably between 50°C and 60°C; and / or

[0048] In step b), hydrolysis is carried out at a pH value in the range of 5 to 11, preferably in the range of 6 to 10, and more preferably in the range of 7 to 9; and / or

[0049] In step b), the hydrolysis time is between 5 minutes and 6 hours, preferably between 20 minutes and 3 hours, and more preferably between 30 and 60 minutes.

[0050] In one embodiment, the casein in the preparation method is casein obtained by membrane separation of milk.

[0051] In a preferred embodiment, the milk is fresh milk.

[0052] In a third aspect of the invention, a product is provided, wherein the product comprises the casein hydrolysate of the first aspect of the invention or the casein hydrolysate prepared by the preparation method of the second aspect of the invention, and optionally, the product comprises any one or more of the following components: plant product components, animal meat product components, animal dairy product components, functional additives, and any acceptable excipients.

[0053] In a fourth aspect of the invention, the use of the casein hydrolysate described in the first aspect of the invention or the casein hydrolysate prepared by the preparation method described in the second aspect of the invention in food or food preparation is provided.

[0054] The effects of the invention

[0055] The casein hydrolysate prepared by this invention has an excellent molecular weight distribution, good hydrolysis effect, rich taste, natural milk fat aroma, and long-lasting fragrance.

[0056] The preparation of casein hydrolysate of the present invention does not require additional processes such as membrane filtration or chromatography. It can be prepared solely through protease combination hydrolysis and precise hydrolysis control technology, which allows casein to undergo full hydrolysis under suitable conditions, balancing flavor and degree of hydrolysis, further reducing production costs and meeting market demand.

[0057] The casein hydrolysate prepared by this invention through specific processes and methods has a unique molecular weight and outstanding aroma, and can be applied to various foods for people of different ages. Attached Figure Description

[0058] Figure 1 This displays the high-performance liquid chromatography (HPLC) chromatogram of the hydrolysis products of casein solution by metalloproteinases.

[0059] Figure 2This displays the high-performance liquid chromatography (HPLC) chromatogram of the hydrolysis products of casein solution by bacterial endopeptidase.

[0060] Figure 3 This displays high-performance liquid chromatography (HPLC) chromatograms of the hydrolysis products of casein solution by metalloproteinases and bacterial endopeptidases. Detailed Implementation

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0065] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," or "implementation" 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.

[0066] 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.

[0067] In this invention, “Da” is used to represent the unit of molecular weight, “Dalton”.

[0068] 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.

[0069] In this invention, the term "infant" is used to refer to the human group aged 0 to 6 months.

[0070] In this invention, the term "older infant" refers to the human group aged 6 to 12 months.

[0071] In this invention, the term "infant" is used to refer to the human group aged 12 to 36 months.

[0072] In this invention, the term "infant" refers to the human group under the age of 3 years.

[0073] 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.

[0074] In this manual, the term "adult" refers to a person who is 18 years of age or older.

[0075] In this manual, the term "teenager" refers to people aged 7-40.

[0076] In this manual, the term "middle-aged person" refers to a person aged 41-65.

[0077] 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.

[0078] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0079] In this specification, the correlation between two amino acid sequences is described by the parameter “Sequence Identity”. For the purposes of this invention, the sequence identity between two amino acid sequences is determined using the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277, preferably version 6.6.0 or later) as implemented in the Needle program. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Niedle output labeled “Longest Identity” (obtained using the non-simplification option) is used as the identity percentage and calculated as follows:

[0080] (identical residues × 100) / (alignment length - total number of vacancies in alignment).

[0081] In this specification, the three-letter and single-letter codes for amino acids are as described in J. biol. chem, 243, p3558 (1968).

[0082] According to this disclosure, an amino acid "addition" refers to the addition of an amino acid to the C-terminus or N-terminus of an amino acid sequence. According to this disclosure, an amino acid "deletion" refers to the deletion of one, two, or three or more amino acids from an amino acid sequence. According to this disclosure, an amino acid "insertion" refers to the insertion of an amino acid residue at an appropriate position in an amino acid sequence; the inserted amino acid residues may be all or partly adjacent to each other, or none of the inserted amino acids may be adjacent to each other. According to this disclosure, an amino acid "substitution" refers to the replacement of an amino acid residue at a certain position in an amino acid sequence by another amino acid residue; wherein, "substitution" can be a conserved amino acid substitution.

[0083] According to this disclosure, "conservative modification," "conservative substitution," or "conservative replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), such that it can be frequently altered without changing the protein's biological activity. Those skilled in the art will appreciate that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, the substitution of structurally or functionally similar amino acids is unlikely to impair biological activity.

[0084] <First Aspect>

[0085] A first aspect of the present invention provides a casein hydrolysate. That is, the casein hydrolysate is derived from the degradation (hydrolysis) of casein to form a degradation product (hydrolysate), and therefore, the casein hydrolysate contains at least a portion of the degraded casein component, i.e., a composition comprising a variety of proteins of different molecular weights.

[0086] In some embodiments, the protein components of the casein hydrolysate contain at least 40% by mass, preferably between 50% and 60% by mass, and more preferably between 52% and 54% by mass, of the protein components with a molecular weight between 196 Da and 500 Da, based on dry weight.

[0087] In some embodiments, the content of protein components with a molecular weight between 500 Da and 1000 Da in the casein hydrolysate is 17% by mass or more, preferably between 18% and 25% by mass, and more preferably between 19% and 23% by mass, wherein the content refers to the percentage of protein components with a molecular weight between 500 Da and 1000 Da, based on dry weight, in the total protein content of the casein hydrolysate.

[0088] In some embodiments, the average molecular weight of the casein hydrolysate of the present invention does not exceed 1500 Da.

[0089] In some preferred embodiments, the average molecular weight of the casein hydrolysate is not more than 1200 Da, more preferably not more than 1000 Da.

[0090] In some further preferred embodiments, the average molecular weight of the casein hydrolysate is not less than 850 Da.

[0091] In some further preferred embodiments, the casein hydrolysate has an average molecular weight of 950-980 Da, and most preferably, the casein hydrolysate has an average molecular weight of 967.82 Da.

[0092] In some embodiments, the degree of hydrolysis (DH) of the casein hydrolysate is not less than 10%, preferably, the degree of hydrolysis of the casein hydrolysate is between 11% and 15%, and more preferably, the degree of hydrolysis of the casein hydrolysate is 13%.

[0093] In this specification, the degree of hydrolysis (DH) represents the extent to which the protein obtained by the method is hydrolyzed. In the context of this invention, the degree of hydrolysis (DH) is defined as follows:

[0094] DH = (Number of cleaved peptide bonds / Total number of peptide bonds) × 100%

[0095] Those skilled in the art can measure DH using methods described in Adler-Nissen, J., 1986, Enzymatic Hydrolysis of Food Proteins, Chapter 5, pp. 122-124.

[0096] In some preferred embodiments, the present invention uses high-performance liquid chromatography (HPLC) to evaluate the characteristics of the hydrolysis products. Specifically, the method can be found in the People's Republic of China National Standard GB / T22729-2008. After precipitating the protein with trichloroacetic acid, o-phthalaldehyde (OPA) and fluorenyl chloroformate (FMOC-Cl) are used as derivatization reagents for primary and secondary amino acids, respectively. The total amount of free amino acids in the casein hydrolysate is determined using pre-column automated derivatization reversed-phase HPLC.

[0097] In some implementations, the hydrolysis products are evaluated through sensory assessment.

[0098] In some preferred embodiments, sensory evaluation members evaluate the taste, aroma intensity, and aroma retention time of the casein hydrolysate obtained by enzymatic hydrolysis, and give sensory evaluation scores according to specific scoring criteria.

[0099] In some specific embodiments, the casein hydrolysate provided by the present invention has a milk fat aroma.

[0100] In some embodiments, the casein hydrolysate provided by the present invention has a comprehensive score of 80 or above for milk fat aroma and lingering fragrance, and preferably a comprehensive score between 84 and 87 for milk fat aroma and lingering fragrance.

[0101] According to the present invention, the flavor of casein hydrolysates is correlated with the degree of hydrolysis, but not linearly. In the early stages of hydrolysis, bitterness intensifies with increasing degree of hydrolysis; however, after reaching the peak of bitterness, the bitterness decreases as the degree of hydrolysis continues to rise. Therefore, the hydrolysates provided by the present invention further balance the relationship between flavor and degree of hydrolysis in order to achieve good flavor and control costs.

[0102] In some preferred embodiments, the casein hydrolysate of the present invention is a casein hydrolysate.

[0103] <Second aspect>

[0104] A second aspect of the present invention provides a method for preparing the casein hydrolysate according to the first aspect of the present invention, the method comprising:

[0105] a) Add metalloproteinases and bacterial endopeptidases simultaneously or sequentially to an aqueous solution containing casein;

[0106] b) Incubate to hydrolyze the casein.

[0107] The casein hydrolysate prepared by the method provided in this invention has a rich taste, prominent aroma, and long-lasting fragrance.

[0108] (Casein)

[0109] In the context of this invention, casein is the main protein in milk, constituting 75-80% of all proteins in milk and cheese. Soluble forms of casein can be coagulated by acid and / or by rennet.

[0110] Casein can be acidic casein or nonfat milk solids. Casein can be used in the form of liquid concentrate or powder.

[0111] In the method of the present invention, the casein material is diluted or reconstituted into an aqueous solution or suspension, preferably containing about 2-35% by weight, more preferably about 5-30% by weight of casein.

[0112] In some exemplary embodiments, the casein content in the diluted or reconstituted casein material in an aqueous solution or suspension is 8.87% by weight, and its average molecular weight is 9271.60 Da.

[0113] In some preferred embodiments, the casein is derived from milk.

[0114] In some preferred embodiments, the casein may be casein obtained by membrane separation of milk; preferably, the milk is fresh milk, not dried milk that has been hydrolyzed.

[0115] The casein used in the method of the present invention may be, for example, in the form of sodium caseinate, potassium caseinate, or calcium caseinate.

[0116] In some specific embodiments, the casein material may be a casein liquid.

[0117] In some embodiments, the casein liquid, which is the casein material, can be obtained by separating it from the casein raw material (e.g., milk) through a membrane separation method.

[0118] 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 as a casein material. 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 as a casein material can be a casein solution prepared using a ceramic membrane or a spiral wound membrane via a membrane separation method.

[0119] In some preferred embodiments, the casein liquid used as the casein material in this invention has a dry matter content of 10.0-12.8 g / 100 g, a protein content (on a dry basis) of 84.0-86.9 g / 100 g, a casein content (as a percentage of protein) of 82.0-84.5 g / 100 g, and a fat content (on a dry basis) of 1.0-5.0 g / 100 g. Casein liquids meeting these specifications facilitate the preparation of the casein hydrolysate of this invention through hydrolysis, thereby enhancing the taste, aroma, and lingering flavor of the casein hydrolysate.

[0120] (metalloproteinases)

[0121] The term "metalloproteinase," as used herein, refers to a protease having one or more metal ions at its binding / active site. Metalloproteinases are proteolytic enzymes whose activity absolutely requires a metal ion. Metalloproteinases can be classified into several subtypes, primarily including matrix metalloproteinases (MMPs), integrin-dissociating metalloproteinases (ADAMs), and ADAMs with thromboplastin motifs (ADAMTS).

[0122] In some specific embodiments, the active site of the matrix metalloproteinase may contain at least one metal ion, such as calcium ions or zinc ions.

[0123] In some specific embodiments, the metalloproteinases described in this invention are zinc-dependent.

[0124] In some specific embodiments, the metalloproteinase is derived from microorganisms.

[0125] In some specific embodiments, the metalloproteinase is a metalloproteinase (Uniprot: P06832) from Bacillus amyloliquefaciens, which is simply referred to as metalloproteinase in the embodiments of the present invention.

[0126] Metalloproteinases, such as Bacillus amyloliquefaciens metalloproteinase, are commercially available from Novozymes (Bagswell, Denmark) and are commercially available neutral metalloproteinases. TM It can be used to ensure the uniformity of the dough and the texture of the bread.

[0127] In some specific embodiments, examples of the metalloproteinase are:

[0128] (i) A metalloproteinase (Uniprot: P06832) from *Bacillus amyloliquefaciens*, the sequence of which is shown in SEQ ID NO: 1. It is a commercially available neutral metalloproteinase, Neutrase. TM It can be used to ensure the uniformity of dough and the texture of bread, and is commercially available from Novozymes (Bagswell, Denmark);

[0129] (ii) metalloproteinases having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the metalloproteinases of (i);

[0130] (iii)(i) or (ii) metalloproteinases exhibiting protease activity mutants, variants or fragments.

[0131] In some preferred embodiments, the metalloproteinase is selected from the group consisting of:

[0132] (i) a polypeptide comprising an amino acid sequence having at least 60% identity with SEQ ID NO:1; and

[0133] (ii) A variant of SEQ ID NO:1 containing one or more amino acid substitutions, deletions, and / or insertions.

[0134] In some preferred embodiments, the metalloproteinase contains an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1.

[0135] >SEQ ID NO:1 (Metalloproteinase)

[0136] AATTGTGTTLKGKTVSLNISSESGKYVLRDLSKPTGTQIITYDLQNREYNLPGTLVSSTTNQFTTSSQRAAVDAHYNLGKVYDYFYQKFNRNSYDNKGGKIVSSVHYGSRYNNAAWIGDQMIYGDGDGSFFSPLSGSMDVTAHEMTHGVT QETANLNYENQPGALNESFSDVFGYFNDTEDWDIGEDITVSQPALRSLSNPTKYGQPDNFKNYKNLPNTDAGDYGGVHTNSGIPNKAAYNTITKIGVNKAEQIYYRALTVYLTPSSTFKDAKAALIQSARDLYGSQDAASVEAAWNAVGL

[0137] (bacterial endopeptidase)

[0138] According to the bacterial endopeptidases of the present invention, the term "bacterial" or "bacterial" indicates that the endopeptidase is derived from or originates from bacteria, or is derived from bacterial analogs, fragments, variants, mutants, or synthetic endopeptidases. It can be produced or expressed in the original wild-type bacterial strain, in another microbial strain, or in plants; that is, the term covers the expression of wild-type, naturally occurring endopeptidases, as well as the expression of recombinant endopeptidases, genetically engineered endopeptidases, or synthetic endopeptidases in any host.

[0139] In some embodiments, the bacterial endopeptidase is derived from actinomycetes.

[0140] In some specific embodiments, the bacterial endopeptidase is derived from Nocardiopsis.

[0141] In some specific embodiments, the bacterial endopeptidase is a bacterial endopeptidase derived from Nocardia strain NRRL 18262, which is disclosed in WO01 / 58276.

[0142] Examples of bacterial endopeptidases used according to the present invention are:

[0143] (i) A bacterial endopeptidase derived from Nocardia species NRRL 18262, disclosed in WO01 / 58276, the sequence of which is shown in SEQ ID NO:2;

[0144] (ii) Bacterial endopeptidases of (i) having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid identity;

[0145] (iii)(i) or (ii) Bacterial endopeptidases showing endopeptidase activity mutants, variants or fragments.

[0146] In a preferred embodiment, the bacterial endopeptidase is selected from the group consisting of:

[0147] (i) a polypeptide comprising an amino acid sequence having at least 60% identity with the polypeptide shown in SEQ ID NO:2; and

[0148] (ii) A variant of the polypeptide shown in SEQ ID NO:2 that includes substitutions, deletions, and / or insertions of one or more amino acids.

[0149] In some preferred embodiments, the bacterial endopeptidase contains an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:2.

[0150] In some preferred embodiments, the metalloproteinases or bacterial endopeptidases used in the methods of the present invention are purified to contain at least 20%, preferably at least 30%, at least 40%, or at least 50% (w / w) of the said metalloproteinase or bacterial endopeptidase in the total protein. The amount of metalloproteinase or bacterial endopeptidase can be calculated by dividing the prepared activity measurement by the specific activity (activity / mg EP) of the said metalloproteinase or bacterial endopeptidase, or can be quantified by SDS-PAGE or any other method known in the art. The amount of total protein can be determined, for example, by amino acid analysis.

[0151] >SEQ ID NO:2 (Bacterial Endopeptidase)

[0152] ADIIGGLAYTMGGRCSVGFAATNAAGQPGFVTAGHCGRVGTQVTIGNGRGVFEQSVFPGNDAAFVRGTSNFTLTNLVSRYNTGGYATVAGHNQAPIGSSVCRSGSTTGWHCGTIQARGQSVSYPEGTVTNMTRTTVCAEPGDSGGSYISGTQAQGVTSGGSGNCRTGGTTFYQEVTPMVNSWGVRLRT

[0153] In one specific embodiment, the metalloproteinase or bacterial endopeptidase used in this invention has an optimal pH value (optimal pH) close to neutral.

[0154] The term "pH activity optimum near neutral" can mean that the enzyme has an optimal pH of 5-11, preferably 6-10, more preferably 7-9, and most preferably at an optimum pH of about 8.

[0155] (hydrolysis)

[0156] After dispersing casein material in water to form a casein solution, such as an aqueous solution containing casein, the pH and / or temperature of the casein solution can be adjusted to optimize the hydrolysis reaction, particularly ensuring that the functions of the metalloproteinases and bacterial endopeptidases used in the hydrolysis reaction are close to their optimal activity levels. The pH of the casein solution can be adjusted and monitored according to methods known in the art. The pH of the casein solution can be adjusted to about 5 to about 11. In one embodiment, the pH of the casein solution can be adjusted to about 6 to about 10. In a preferred embodiment, the pH of the casein solution can be adjusted to about 7 to about 9, for example, pH at 7.5, 7.6, 7.7, 7.8, 7.9, 8, etc. The pH of the casein solution can be maintained at the above levels during the hydrolysis reaction, or it can be allowed to decrease as the hydrolysis reaction proceeds.

[0157] In this invention, the pH of the casein solution can be adjusted by adding a pH alkaline adjuster. In some embodiments of this invention, the pH alkaline adjuster is selected from food-grade sodium bicarbonate, calcium hydroxide, potassium hydroxide, sodium hydroxide, or any combination thereof, preferably a sodium hydroxide solution (e.g., an aqueous solution) with a concentration of 5-30% by mass, and more preferably a 20% sodium hydroxide solution.

[0158] In this invention, the pH of the casein solution can be adjusted by adding a pH acidity adjuster. In some embodiments of this invention, the pH acidity adjuster is selected from any combination of food-grade hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, malic acid, etc., preferably a food-grade hydrochloric acid solution (e.g., an aqueous solution) with a concentration of 5-30% by mass, and more preferably a food-grade hydrochloric acid solution with a concentration of 20%.

[0159] During the hydrolysis reaction, the temperature of the casein solution is preferably adjusted and maintained at about 45°C to about 70°C, more preferably about 50°C to about 60°C, according to methods known in the art. In a preferred embodiment, the temperature of the casein solution can be adjusted and maintained at about 50°C to about 60°C, for example, 55°C, 58°C, 59°C, 60°C, etc., during the hydrolysis reaction.

[0160] The hydrolysis reaction is typically initiated by simultaneously or sequentially adding the metalloproteinase and bacterial endopeptidase to a casein solution. Alternatively, the enzymes may be dispersed separately or simultaneously in water, and the protein material (e.g., casein material) may be slowly added while stirring. The latter method is advantageous when preparing a concentrated casein solution to avoid excessive viscosity. In one embodiment, if the metalloproteinase and bacterial endopeptidase do not function at the same pH and / or temperature, the protein material (e.g., casein material) may be incubated first with the metalloproteinase hydrolase, followed by optionally adjusting the pH and / or temperature and subsequently incubating with the bacterial endopeptidase hydrolase. In a further embodiment, if the metalloproteinase and bacterial endopeptidase do not function at the same pH and / or temperature, the protein material (e.g., casein material) may be incubated first with the bacterial endopeptidase, followed by optionally adjusting the pH and / or temperature and subsequently incubating with the metalloproteinase. Alternatively, in a further embodiment, the protein material (e.g., casein material) may be incubated together with the metalloproteinase and bacterial endopeptidase at the same pH and / or temperature. In a preferred embodiment, the metalloproteinase and bacterial endopeptidase are added simultaneously. In a more preferred embodiment, the metalloproteinase and bacterial endopeptidase are added simultaneously in the form of a protease composition.

[0161] The amount of metalloproteinases and bacterial endopeptidases added to a protein substance can and will vary depending on the source of the protein substance, the required degree of hydrolysis, and the duration of the hydrolysis reaction.

[0162] In one embodiment of the present invention, the amount of metalloproteinase added is 0.0001-3 AU-N / g casein, preferably 0.001-2 AU-N / g casein, more preferably 0.05-1 AU-N / g casein, and even more preferably 0.01-0.1 AU-N / g casein.

[0163] The activity AU-N of metalloproteinases was obtained using Konelab analysis. The principle is that dimethyl casein (DMC) is hydrolyzed by the protease into small polypeptide fragments, and the released amino acid clusters form a colored complex with trinitrobenzenesulfonic acid (TNBS). This reaction can be monitored in situ, and enzyme activity is determined by calculating the change in absorbance per unit time. The reaction conditions were: temperature 50℃, pH 7.1, wavelength 405nm, reaction time 6 min, measurement time 2 min, substrate concentration 0.25%, and enzyme concentration 0.0090-0.0448 AU-N / L. The enzyme activity of the sample was calculated relative to a standard curve.

[0164] AU-N is defined relative to the Novozymes standard enzyme and has the same unit as the standard: Anson unit (N standard). Specific methods can be obtained from Novozymes (Baggswell, Denmark) upon request.

[0165] In one embodiment of the present invention, the amount of bacterial endopeptidase added is 0.0001-3 KPROT / g casein, preferably 0.001-2 KPROT / g casein, more preferably 0.05-1 KPROT / g casein, and even more preferably 0.01-0.1 KPROT / g casein.

[0166] The principle behind the bacterial endopeptidase activity PROT is that bacterial endopeptidase hydrolyzes the substrate N-succinyl-alanine-alanine-proline-phenylalanine p-nitroaniline. The release of yellow p-nitroaniline (pNA) leads to an increase in absorbance at 405 nm, and this increase is proportional to the enzyme activity.

[0167] The reaction conditions were: temperature 37℃, pH 9.0, wavelength 405nm, reaction time 180 seconds before measurement, kinetic detection interval 198 seconds (12 measurements, 18-second intervals), substrate concentration 0.62 mg / mL, enzyme concentration [6.6; 26] mPROT / mL. Enzyme activity was calculated relative to a standard curve. Specific methods can be obtained from Novozymes (Baguswell, Denmark) upon request. The bacterial endopeptidase activity KPROT was 1000 times that of the bacterial endopeptidase activity PROT.

[0168] As those skilled in the art will understand, the duration of a hydrolysis reaction can and will vary. Generally, the duration of a hydrolysis reaction can range from a few minutes to many hours, for example, from 5 minutes to 6 hours, preferably from 20 minutes to 3 hours, and more preferably from 30 to 60 minutes.

[0169] Preferably, casein hydrolysate having a degree of hydrolysis (DH) of about 11% to about 15%, more preferably about 13%, is treated with metalloproteinases and bacterial endopeptidases.

[0170] In a preferred embodiment, before step a), the casein solution is preheated to 50-60°C, for example, 55°C, 58°C, 59°C, or 60°C, to ensure uniform heating of the solution and to promote the subsequent reaction.

[0171] (Post-processing steps)

[0172] After step b) incubation to hydrolyze the casein, the process further includes a post-treatment step of the hydrolysate, which includes pH adjustment and / or enzyme inactivation.

[0173] pH adjustment treatment

[0174] In some embodiments of the present invention, after step b) of the present invention is completed, the pH of the reaction solution (also referred to as hydrolysate in this specification) in step b) can be adjusted to 7-8, preferably 7.5-8.

[0175] In some embodiments, a food-grade sodium hydroxide solution (e.g., an aqueous solution) with a concentration of 5-30% by mass is used, more preferably a food-grade sodium hydroxide solution with a concentration of 20%.

[0176] Enzyme inactivation treatment

[0177] In a preferred embodiment, after completing step b), the enzyme can be inactivated (enzyme inactivation treatment). This inactivation can be carried out by any inhibition method in the art, for example, by heating to at least 75°C, such as 95-100°C.

[0178] 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 duration is less than 8 minutes, for example 6 minutes, 5 minutes, or 4 minutes.

[0179] In some optional embodiments, the enzyme hydrolysate after enzyme inactivation can be appropriately concentrated and / or dried.

[0180] There are no particular limitations on the concentration and drying methods of this invention; methods commonly used by those skilled in the art can be employed.

[0181] <Third aspect>

[0182] In a third aspect of the present invention, a product is provided, the product comprising or using the casein hydrolysate described in the first aspect or containing the casein hydrolysate prepared by the preparation method described in the second aspect.

[0183] In addition to the essential components described above, the product of this invention can also be obtained by processing the casein hydrolysate as described in the first aspect above, depending on the needs of the final product, including other components, such as:

[0184] Plant-based ingredients include fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, rubber, grape, pear, cherry, blueberry, blackberry, blackcurrant, cranberry, raspberry, melon, amla, and bilberry, or their extracts; fruits and vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; grains such as rice (indica, japonica, glutinous rice), cereals (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow millet, buckwheat, soybean, broad bean, pea, mung bean, red bean, and kidney bean, or their extracts; nuts such as walnut, pistachio, cashew, hazelnut, almond, apricot kernel, pine nut, peanut, sunflower seed, chestnut, macadamia nut, and ginkgo, or their extracts; coffee or its extracts; and some medicinal and edible herbal medicines or their extracts.

[0185] Animal meat product ingredients, including meat products from pigs, cattle, sheep, aquatic products, or poultry.

[0186] Animal dairy products include fresh milk from cows, sheep, etc., as well as processed dairy products such as milk powder, whey protein, or cheese.

[0187] Functional additives include vitamins (one or more of vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, biotin, L-carnitine, and lutein); starch; modified starch; amino acids (L-lysine-L-glutamic acid, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, L-leucine, casein phosphopeptide, etc.); dietary fiber (inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, raffinose, polydextrose, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, or other similar ingredients). Soy fiber, etc.); trace element supplements (which may include metal ion salts of organic acids, such as calcium citrate, L-calcium lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate, and magnesium gluconate, etc.); fat supplements (such as saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured lipids, OPL structured lipids, LPL structured lipids, DHA, EPA, ARA, phospholipids, etc.); nucleotide supplements; human milk oligosaccharides (such as 2'-FL, 3-FL, DFL, LNFPI, LNFPII, LNT, LNnT, 3'-SL, 6'-SL, DSLNT, etc.), etc.

[0188] Any acceptable excipients, including but not limited to solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, fillers, disintegrants, lubricants, anti-caking agents, flavoring agents, sweeteners, food flavorings, food colorings, etc.

[0189] The present invention does not specifically limit the type of the above-mentioned products.

[0190] The casein hydrolysate provided by this invention has a persistent milk fat aroma and can be used in food products. Non-limiting examples of such food products include nutrition bars, dry mixed drinks, sports drinks, energy drinks, and adult milk powder. The casein hydrolysate provided by this invention can also be used in clinical nutrition.

[0191] In some embodiments, the product is food and / or health food.

[0192] For different product categories, the present invention does not particularly limit the specific form of the product. For example, it can be in the form of solid, liquid or semi-solid.

[0193] The present invention does not specifically limit the target population of the product. The food of the present invention can be food suitable for infants, teenagers, adults and / or middle-aged and elderly people.

[0194] In some embodiments, the products described in this invention may be infant formula, baby food, children's formula, children's snacks, formula milk powder for pregnant women, adult milk powder, milk powder for middle-aged and elderly people, or nutritional or dietary supplements.

[0195] In other embodiments, the products of the present invention may be beverages (carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages, milk beverages, alcoholic beverages, instant coffee, dietary beverages, grain powder, nut powder or lotus root powder, etc.), candies (gel candies, hard candies, compressed candies, etc.), milk and dairy products (fresh milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc. derived from fresh cow (sheep) milk), pasta products (noodles, instant noodles, steamed buns, dumplings or wontons, as well as baked foods such as bread, cakes or biscuits), and instant food products, etc.

[0196] There are no special restrictions on beverages; they can include fruit drinks, vegetable drinks, milk tea drinks, tea drinks, milk, yogurt, dry mixed drinks, sports drinks, and energy drinks.

[0197] In addition to the casein hydrolysate of the present invention, dry mixed beverages may also include flavored blends commonly used by those skilled in the art, such as coffee, tea, and oatmeal.

[0198] For sports drinks and energy drinks, in addition to including the casein hydrolysate of the present invention, they mainly contain various functional vitamins and other functional ingredients, such as white sugar, cyclamate, acesulfame potassium, taurine, potassium sorbate, lysine, inositol, vitamin PP, vitamin B6, vitamin B12, citric acid, etc.

[0199] In some specific embodiments, the casein hydrolysate obtained by the method of the present invention can also be used in clinical nutritional supplements, such as protein supplements and protein powder.

[0200] For reconstituteable foods, typical examples are reconstituteable milk powder products, such as infant formula, adult milk powder, and milk powder for middle-aged and elderly people.

[0201] All references to documents cited in this article are incorporated herein by reference in their entirety.

[0202] The present invention is further described through the following embodiments, which should not be regarded as limiting the scope of the invention.

[0203] Examples and Comparative Examples

[0204] The embodiments and comparative examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0205] Comparative Example 1: Hydrolysis of casein solution by metalloproteinases

[0206] This embodiment describes a process for treating casein using metalloproteinases via enzymatic hydrolysis.

[0207] (I) Materials

[0208] Casein solution: First, use membrane-separated casein solution for preparation. The casein solution should have a dry matter content of 10.0-12.8 g / 100g, a protein content (dry basis) of 84.0-86.9 g / 100g, a casein content (as a percentage of protein) of 82.0-84.5 g / 100g, and a fat content (dry basis) of 1.0-5.0 g / 100g. It can be obtained from Heilongjiang Feihe Dairy Co., Ltd. (Qiqihar City, Heilongjiang Province, China). Prepare a casein solution before use, with a casein content of 8.87% and an average molecular weight of 9271.60 Da.

[0209] Enzyme preparation: metalloproteinase, derived from Bacillus amyloliquefaciens, as shown in SEQ ID NO:1 of this application, and also disclosed in WO2007 / 044993.

[0210] (II) Steps

[0211] 1. Preheat casein solution

[0212] Preheat 500g of casein solution to 60°C, ensuring uniform heating to promote subsequent reactions.

[0213] 2. Adjust the pH value

[0214] Use a pH meter to measure the pH of the solution and adjust it to 7.8. This step can be achieved by adding a 20% hydrochloric acid or sodium hydroxide solution to ensure optimal enzyme activity.

[0215] 3. Add enzyme preparations

[0216] Add 2.2175 AU-N of metalloproteinase to the solution, which is equivalent to 0.05 AU-N / g casein.

[0217] 4. Enzymatic hydrolysis reaction

[0218] The hydrolysis reaction was carried out at 55°C for 40 minutes. During this process, stirring was maintained to ensure uniform enzyme distribution and complete reaction.

[0219] 5. Adjust the pH to 7.8.

[0220] After hydrolysis, the pH of the solution was adjusted to 7.8 using a pH adjuster to facilitate subsequent processing.

[0221] 6. Termination of reaction

[0222] The solution was heated to 95°C and held for 5 minutes to terminate enzyme activity and prevent further hydrolysis.

[0223] 7. Sampling and Analysis

[0224] Samples were taken from the reaction solution for molecular weight and degree of hydrolysis determination (results shown in Table 1). The characteristics of the hydrolysis products were evaluated using high-performance liquid chromatography (HPLC) according to the People's Republic of China National Standard GB / T 22729-2008 (results shown in Table 1). Figure 1 ).

[0225] Comparative Example 2: Hydrolysis of casein solution by bacterial endopeptidase

[0226] This embodiment describes a process for treating casein using bacterial endopeptidase via enzymatic hydrolysis.

[0227] (I) Materials

[0228] Casein solution: First, use membrane-separated casein solution for preparation. The casein solution should have a dry matter content of 10.0-12.8 g / 100g, a protein content (dry basis) of 84.0-86.9 g / 100g, a casein content (as a percentage of protein) of 82.0-84.5 g / 100g, and a fat content (dry basis) of 1.0-5.0 g / 100g. It can be obtained from Heilongjiang Feihe Dairy Co., Ltd. (Qiqihar City, Heilongjiang Province, China). Prepare a casein solution before use, with a casein content of 8.87% and an average molecular weight of 9271.60 Da.

[0229] Enzyme preparation: bacterial endopeptidase, derived from Nocardia species NRRL 18262, disclosed in WO01 / 58276, the sequence of which is shown in SEQ ID NO:2 of this application.

[0230] (II) Steps

[0231] 1. Preheat casein solution

[0232] Preheat 500g of casein solution to 60°C, ensuring uniform heating to promote subsequent reactions.

[0233] 2. Adjust the pH value

[0234] Use a pH meter to measure the pH of the solution and adjust it to 7.8. This step can be achieved by adding 20% ​​hydrochloric acid or sodium hydroxide solution to ensure optimal enzyme activity.

[0235] 3. Add enzyme preparations

[0236] Add 2.661 KPROT of bacterial endopeptidase to the solution, which is equivalent to 0.06 KPROT / g casein.

[0237] 4. Enzymatic hydrolysis reaction

[0238] The hydrolysis reaction was carried out at 55°C for 40 minutes. During this process, stirring was maintained to ensure uniform enzyme distribution and complete reaction.

[0239] 5. Adjust the pH to 7.8.

[0240] After hydrolysis, the pH of the solution was adjusted to 7.8 using a pH adjuster to facilitate subsequent processing.

[0241] 6. Termination of reaction

[0242] The solution was heated to 95°C and held for 5 minutes to terminate enzyme activity and prevent further hydrolysis.

[0243] 7. Sampling and Analysis

[0244] Samples were taken from the reaction solution for molecular weight and degree of hydrolysis determination (results are shown in Table 1). The characteristics of the hydrolysis products were evaluated using high-performance liquid chromatography (HPLC) according to the People's Republic of China National Standard GB / T 22729-2008 (results are shown in Table 1). Figure 2 ).

[0245] Example 1: Hydrolysis of casein solution by metalloproteinases and bacterial endopeptidases

[0246] This embodiment describes a process for treating casein using metalloproteinases and bacterial endopeptidases via enzymatic hydrolysis to improve the aroma and persistence of casein hydrolysate.

[0247] (I) Materials

[0248] Casein solution: First, use membrane-separated casein solution for preparation. The casein solution should have a dry matter content of 10.0-12.8 g / 100g, a protein content (dry basis) of 84.0-86.9 g / 100g, a casein content (as a percentage of protein) of 82.0-84.5 g / 100g, and a fat content (dry basis) of 1.0-5.0 g / 100g. It can be obtained from Heilongjiang Feihe Dairy Co., Ltd. (Qiqihar City, Heilongjiang Province, China). Prepare a casein solution before use, with a casein content of 8.87% and an average molecular weight of 9271.60 Da.

[0249] Enzyme preparations:

[0250] A combination of a metalloproteinase and a bacterial endopeptidase, wherein the metalloproteinase is derived from Bacillus amyloliquefaciens, as shown in SEQ ID NO:1, disclosed in WO2007 / 044993;

[0251] Bacterial endopeptidase, derived from Nocardia species NRRL 18262, disclosed in WO01 / 58276, has the sequence shown in SEQ ID NO:2 of this application.

[0252] (II) Steps

[0253] 1. Preheat casein solution

[0254] Preheat 500g of casein solution to 60°C, ensuring uniform heating to promote subsequent reactions.

[0255] 2. Adjust the pH value

[0256] Use a pH meter to measure the pH of the solution and adjust it to 7.8. This step can be achieved by adding 20% ​​hydrochloric acid or sodium hydroxide solution to ensure optimal enzyme activity.

[0257] 3. Add enzyme preparations

[0258] 1.774 AU-N metalloproteinase was added to the solution, equivalent to 0.04 AU-N / g casein; and 2.661 KPROT of bacterial endopeptidase was added simultaneously, equivalent to 0.06 KPROT / g casein.

[0259] 4. Enzymatic hydrolysis reaction

[0260] The hydrolysis reaction was carried out at 55°C for 40 minutes. During this process, stirring was maintained to ensure uniform enzyme distribution and complete reaction.

[0261] 5. Adjust the pH to 7.8.

[0262] After hydrolysis, the pH of the solution was adjusted to 7.8 using a pH adjuster to facilitate subsequent processing.

[0263] 6. Termination of reaction

[0264] The solution was heated to 95°C and held for 5 minutes to terminate enzyme activity and prevent further hydrolysis.

[0265] 7. Sampling and Analysis

[0266] Samples were taken from the reaction solution for molecular weight and degree of hydrolysis determination (results are shown in Tables 1 and 2). The characteristics of the hydrolysis products were evaluated using high-performance liquid chromatography (HPLC) according to the People's Republic of China National Standard GB / T 22729-2008 (results are shown in Tables 1 and 2). Figure 3 ).

[0267] Test Example 1: Determination of molecular weight and degree of hydrolysis of casein hydrolysate

[0268] Samples of casein hydrolysates from Comparative Examples 1, 2, and 3 were taken, and their molecular weight and degree of hydrolysis were determined (results are shown in Tables 1 and 2). The detection method followed the People's Republic of China National Standard GB / T 22729-2008, using high-performance liquid chromatography (HPLC) to analyze the hydrolysis products and evaluate their characteristics. Figure 1-3 More specifically, the data were processed by gel permeation chromatography (GPC) using a Waters 2695 instrument with a UV detector 2489 (commercially available from Waters Corporation, Milford, Massachusetts, USA) and a GE Healthcare Superdex™ Peptide 10 / 300GL column; the average molecular weight was calculated using the software program within the instrument.

[0269] Table 1: Molecular weight distribution

[0270] Molecular weight distribution data Comparative Example 1 Comparative Example 2 Example 1 >10000Da 8.93％ 43.58％ 2.29％ 10000-5000Da 6.29％ 17.88％ 2.26％ 5000-3000Da 7.55％ 9.48％ 2.4％ 3000-2000Da 4.82％ 8.68％ 1.86％ 2000-1000Da 17.09％ 11.97％ 12.43％ 1000-500Da 16.47％ 4.14％ 21.01％ 500-196Da 35.63％ 4.06％ 53.4％ <196Da 3.22％ 0.22％ 4.35％ Average molecular weight (Da) 2195.85 5860.91 967.82

[0271] Table 2: Degree of hydrolysis

[0272] sample Comparative Example 1 Comparative Example 2 Example 1 DH% 9.7 6.1 13.0

[0273] As can be seen from Table 1, by simultaneously treating casein with metalloproteinases and bacterial endopeptidases, the average molecular weight of the casein hydrolysate of the present invention is less than 1000 Da. The content of protein components with molecular weight of 1000-500 Da in the casein hydrolysate is more than 17% by mass, and the content of protein components with molecular weight of 500-196 Da in the casein hydrolysate is more than 36% by mass.

[0274] As can be seen from Table 2, by simultaneously treating casein with metalloproteinases and bacterial endopeptidases, the degree of hydrolysis exceeds 10%. This invention can prepare casein hydrolysates with a degree of hydrolysis exceeding 10% simply by using protease combination hydrolysis and precise hydrolysis control technology.

[0275] Test Example 2: Sensory evaluation of casein hydrolysate

[0276] Five sensory evaluation members evaluated the casein hydrolysates obtained from the enzymatic hydrolysis of Comparative Example 1, Comparative Example 2, and Example 1, as well as the control (without added enzymes), in terms of taste, aroma intensity, and aroma retention time. The specific scoring criteria are shown in Table 3, and the sensory evaluation form is shown in Table 4.

[0277] Table 3: Scoring Criteria

[0278]

[0279] Table 4: Sensory Evaluation Form

[0280]

[0281] As can be seen from Tables 3 and 4, the simultaneous treatment of casein with metalloproteinases and bacterial endopeptidases achieves a synergistic effect that surpasses that of a single enzyme, resulting in significant improvements in three sensory dimensions: taste, aroma, and longevity. The combined score for milk fat aroma and longevity reaches 84-87 points, and the casein hydrolysate surprisingly possesses a long-lasting milk fat aroma.

[0282] 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.

[0283] 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 hydrolysate, characterized in that, In the protein components of the casein hydrolysate, the content of protein components with a molecular weight between 196 Da and 500 Da is more than 40% by mass, preferably between 50% and 60% by mass, and more preferably between 52% and 54% by mass.

2. The casein hydrolysate according to claim 1, characterized in that, The average molecular weight of the casein hydrolysate does not exceed 1500 Da. Preferably, the average molecular weight of the casein hydrolysate is not less than 850 Da. More preferably, the casein hydrolysate has an average molecular weight of 950-980 Da.

3. The casein hydrolysate according to claim 1 or 2, characterized in that, In the protein components of the casein hydrolysate, the content of protein components with a molecular weight between 500 Da and 1000 Da is 17% by mass or more, preferably between 18% and 25% by mass, and more preferably between 19% and 23% by mass.

4. The casein hydrolysate according to any one of claims 1 to 3, characterized in that, The degree of hydrolysis of the casein hydrolysate is not less than 10%, preferably 11%-15%.

5. A method for preparing the casein hydrolysate as described in any one of claims 1 to 4, characterized in that, The preparation method includes: a) Add metalloproteinases and bacterial endopeptidases simultaneously or sequentially to an aqueous solution containing casein; and b) Incubate to hydrolyze the casein; Preferably, the metalloproteinase and bacterial endopeptidase are added simultaneously; more preferably, they are added in the form of a protease composition.

6. The preparation method according to claim 5, characterized in that, The metalloproteinase is a metalloproteinase derived from Bacillus amyloliquefaciens; and / or, The bacterial endopeptidase is derived from the Nocardia strain NRRL 18262.

7. The preparation method according to claim 5 or 6, wherein the metalloproteinase is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence having at least 60% identity with the polypeptide shown in SEQ ID NO:1; and ii) A variant of the polypeptide shown in SEQ ID NO:1 that includes substitutions, deletions, and / or insertions of one or more amino acids; And / or the bacterial endopeptidases mentioned therein are selected from the group consisting of: i) a polypeptide comprising an amino acid sequence having at least 60% identity with the polypeptide shown in SEQ ID NO:2; and ii) A variant of the polypeptide shown in SEQ ID NO:2 that includes substitutions, deletions, and / or insertions of one or more amino acids.

8. The preparation method according to any one of claims 5 to 7, characterized in that, In step a), the amount of metalloproteinase added is 0.0001-3 AU-N / g casein. Preferably, the amount of metalloproteinase added is 0.001-2 AU-N / g casein. More preferably, the amount of metalloproteinase added is 0.05-1 AU-N / g casein. Even more preferred is 0.01-0.1 AU-N / g casein; and / or The amount of bacterial endopeptidase added is 0.0001-3 KPROT / g casein. Preferably, the amount of bacterial endopeptidase added is 0.001-2 KPROT / g casein. More preferably, the amount of bacterial endopeptidase added is 0.05-1 KPROT / g casein. Even more preferred is 0.01-0.1 KPROT / g casein.

9. The preparation method according to any one of claims 5 to 8, characterized in that, In step b), hydrolysis is carried out at a temperature between 45°C and 70°C, preferably between 50°C and 60°C; and / or In step b), hydrolysis is carried out at a pH value in the range of 5 to 11, preferably in the range of 6 to 10, and more preferably in the range of 7 to 9; and / or In step b), the hydrolysis time is between 5 minutes and 6 hours, preferably between 20 minutes and 3 hours, and more preferably between 30 and 60 minutes.

10. The preparation method according to any one of claims 5 to 9, characterized in that, The casein is casein obtained by membrane separation of milk; preferably, the milk is fresh milk.

11. A product characterized in that, The product includes casein hydrolysate as described in any one of claims 1 to 4 or casein hydrolysate prepared by the preparation method as described in any one of claims 5 to 10, and optionally, the product includes any one or more of the following components: plant product components, animal meat product components, animal dairy product components, functional additives, and any acceptable excipients.

12. Use of the casein hydrolysate as described in any one of claims 1 to 4 or the casein hydrolysate prepared by the preparation method as described in any one of claims 5 to 10 in food or food preparation.

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