CN116789793A

CN116789793A - Milk protein composition and preparation method and application thereof

Info

Publication number: CN116789793A
Application number: CN202310551053.1A
Authority: CN
Inventors: 骆滨; 廖丽; 王松伟; 苏世源; 屠均亮; 张改改; 吴蓉; 许昱
Original assignee: Shanghai Changjin Biotechnology Co ltd
Current assignee: Shanghai Changjin Biotechnology Co ltd
Priority date: 2022-05-17
Filing date: 2023-05-16
Publication date: 2023-09-22

Abstract

The application discloses a milk protein composition, a preparation method and application thereof, wherein the milk protein composition comprises one or more recombinant milk proteins and milk protein peptides, and the milk protein peptides are produced by degradation of the recombinant milk proteins when expressed in host cells. Compared with natural milk protein, the milk protein composition provided by the application has lower sensitization; the milk protein composition of the present application exhibits Angiotensin Converting Enzyme (ACE) inhibitory activity; the composition has the solubility and in-vitro digestibility obviously superior to those of natural milk proteins, has the effects of promoting growth, supplementing iron and inhibiting bacteria, and improves the flavor of the product.

Description

Milk protein composition and preparation method and application thereof

The present application claims priority from chinese patent application (application number 2022105409758, title: a milk protein composition, and methods for making and using the same) having a filing date of 2022, 05, and 19, which is incorporated herein by reference in its entirety, and which is adapted for unified alignment of specific expression forms.

Technical Field

The application belongs to the field of dairy product processing, and particularly relates to a milk protein composition, a preparation method and application thereof.

Background

Cow milk is a common source of nutrition and contains abundant proteins, fats, lactose, minerals, vitamins and growth factors, such as immunoglobulins and lactoferrin, etc. There are two main types of cow milk proteins: whey protein (20%) and casein (80%), wherein the whey protein is classified into alpha-lactalbumin, beta-lactoglobulin; casein exists mainly in the form of micelles, consisting of αs1-casein, αs2-casein, β -casein and κ -casein (4:1:4:1). Casein contains almost all essential amino acids and is the most nutritionally valuable protein for young children. Second, casein can increase calcium and phosphorus uptake by newborn young animals. Casein releases a variety of bioactive peptides upon action of gastrointestinal proteases in newborn young animals. These active peptides have regulatory effects on digestion, nutrition, immunity, etc. of young animals. Kappa-casein may also stimulate the growth of beneficial colonies in the gastrointestinal tract of newborn young animals. Beta-lactoglobulin is a high-quality protein in cow milk, has good amino acid proportion and high branched chain amino acid content, and has the specific ability of lipid carrier family (lipocalin family) to bind hydrophobic ligand, for example, can be combined with fat-soluble nutrients such as beta-carotene, retinol, unsaturated fatty acid, vitamin E and the like, and can be used as a carrier or solvent of the fat-soluble nutrients, so that the intake of grease is reduced. Whey protein has better solubility than casein and contains essential amino acids in higher quality. The whey protein has high content of sulfur-containing amino acid, the substance is an important component substance for biosynthesis of glutathione, and the glutathione is a tripeptide related to antioxidation, anticancer and immunity improvement of an organism. Whey proteins are also the highest in content of the natural sources of branched chain amino acids that are thought to stimulate muscle protein synthesis.

However, cow's milk is also one of the eight major allergens published by the united nations grain and agriculture organization, and its major allergens include casein, bovine serum albumin, beta-lactoglobulin, alpha-lactalbumin, etc., about 2% -6% of infants are allergic to cow's milk, and the number of allergic persons in adults is 0.1% -0.5%. Among cow's milk allergic patients, 82% are allergic to beta-lactoglobulin (beta-Lg), mainly because the human milk does not contain beta-Lg, so beta-Lg is the first foreign protein encountered by infants, and is not easily digested by chymotrypsin and pepsin, and can maintain its immune characteristics higher after digestion and absorption by human body, so beta-Lg is considered to be the most important allergen in cow's milk. Most mammalian whey contains lactoglobulin, but it is not contained in the whey of rodents, rabbits and humans, and this is one of the causes of lactoglobulin as a major allergen in cow milk.

Based on the above problems, there is a need to develop a milk protein or composition having the same or similar protein composition as the natural milk protein, while having lower sensitization.

Disclosure of Invention

The application aims to provide a milk protein composition, a preparation method and application thereof, wherein the milk protein composition has a protein composition similar to natural milk protein and at least has lower sensitization.

The application provides the following technical scheme for solving the technical problems:

in a first aspect the present application provides a milk protein composition comprising one or more recombinantly expressed milk proteins, and a milk protein peptide, wherein the milk protein peptide is produced by degradation of the recombinantly expressed milk protein when expressed in a host cell.

In a second aspect the present application provides a method of preparing a milk protein composition according to the first aspect of the application, comprising expressing the one or more recombinantly expressed milk proteins in a host cell and recovering the milk protein composition from the host cell.

In a third aspect the present application provides a food or feed composition comprising the milk protein composition of the first aspect of the application.

The beneficial effects of the application include:

(1) The present application surprisingly achieves a milk protein composition comprising milk protein peptides, which has a lower allergenicity compared to natural milk proteins, by the exogenous expression of milk proteins in host cells.

(2) The milk protein composition provided by the application contains milk protein peptide generated by milk protein degradation, has smaller fragments, and has significantly better solubility and in-vitro digestibility than natural milk protein, thereby having similar or better nutritional value as the natural milk protein.

(3) More unexpectedly, the milk protein has a plurality of functional peptide fragments, such as peptide fragments with Angiotensin Converting Enzyme (ACE) inhibitory activity, in milk protein peptides generated by degradation of the milk protein when expressed in host cells, which provide the milk protein composition with blood pressure reducing effect and further comprise beneficial effects of promoting growth, supplementing iron, inhibiting bacteria and the like.

(4) The milk protein composition is obtained by a heterogeneous expression mode, and can eliminate the problems of environmental pollution, animal welfare and the like caused by natural milk production.

(5) The exogenously expressed milk protein composition contains less unhealthy components such as lactose, saturated fat, cholesterol, etc., and can be added into food instead of natural milk protein to improve food properties, enrich food composition, and improve product flavor.

Drawings

FIG. 1 is a SDS-PAGE result of fermentation supernatants of Kluyveromyces marxianus expression strains of example 1;

FIG. 2A is a physical view of the milk protein composition obtained in example 2;

FIG. 2B is a SDS-PAGE result of the Kluyveromyces marxianus milk protein composition of example 2;

FIG. 3 is a graph showing SDS-PAGE results of milk protein compositions in the supernatant of Kluyveromyces marxianus fermentation broth at various times in example 3;

FIG. 4 is a graph showing SDS-PAGE results of milk protein compositions in the supernatant of Kluyveromyces marxianus fermentation broth at various times in example 4;

FIG. 5 is a diagram showing SDS-PAGE results of milk protein compositions prepared from different Kluyveromyces marxianus strains of example 5;

FIG. 6 is a SDS-PAGE result of a milk protein composition obtained by fermentation of Kluyveromyces lactis of example 6;

FIG. 7 is a diagram showing SDS-PAGE results of the milk protein composition obtained in the comparative example;

FIG. 8 is a high performance liquid chromatogram of a beta-lactoglobulin standard;

FIG. 9 is a high performance liquid chromatogram of the fermentation sample obtained in example 2.

Detailed Description

The terms and descriptions used herein are merely used to describe particular embodiments and are not intended to limit the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

Definition of the definition

In the present application, the terms "a" and "an" and "the" and similar referents refer to the singular and the plural, unless the context clearly dictates otherwise.

In the present application, the terms "about" and "similar to" refer to an acceptable error range for a particular value as determined by one of ordinary skill in the art, which may depend in part on the manner in which the value is measured or determined, or on the limitations of the measurement system.

"milk protein" in the present application refers to a protein found in mammalian-produced milk or a protein having a sequence that is at least 50% (e.g., at least 50%, at least 55%, 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 98%, at least 99%, 100%) identical to the sequence of a protein found in mammalian-produced milk. Non-limiting examples of milk proteins include beta-lactoglobulin, alpha-lactalbumin, kappa-casein, beta-casein, lactoferrin, alpha S1-casein, alpha S2-casein and osteopontin, additional milk proteins being known in the art.

"peptide" refers to a polymeric form of amino acids, i.e., a polymer of two or more amino acids joined by peptide bonds. The term "milk protein peptide" in the present application refers in particular to a combination of peptides of different molecular weights resulting from the degradation of one or more milk proteins, either from the degradation of one milk protein or from the degradation of a plurality of milk proteins.

The term "recombinant" is a term known in the art. When referring to a nucleic acid (e.g., a gene), the term can be used, for example, to describe a nucleic acid that has been isolated from its naturally occurring environment, a nucleic acid that is no longer associated with all or a portion of the nucleic acid that is contiguous or in proximity to nature, a nucleic acid that is operably linked to a nucleic acid that is not linked to nature, or a nucleic acid that is not occurring in nature. When "recombinant" is used to describe a protein, it may refer to a protein produced in a cell of a different species or type than, for example, the type or class of cell in which the protein is produced in nature.

"host cell" refers to a particular recipient cell and the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In the present application, the "host cell" particularly refers to a cell for the heterologous expression of the recombinant milk protein.

In the present application, "degradation" has its general meaning, and refers to a process in which a protein is degraded into a polypeptide and an amino acid by the action of a protein degrading enzyme, and in the present application, particularly, refers to a process in which a milk protein expressed in a host cell is degraded into a mixture of polypeptides having different molecular weights (i.e., milk protein peptides of the present application) by the action of a protein degrading enzyme specific to the host cell.

"filamentous fungus" refers to organisms from the phylum Eumycota (Eumycota) and Oomycota (Oomycota) in filamentous form. Filamentous fungi differ from yeasts in that their hyphae elongate during vegetative growth.

"Yeast" refers to organisms of the order Saccharomyces. The vegetative growth by yeasts is by budding/blebbing of a single cell and carbon catabolism may be fermentative.

"cell culture" means that the cells proliferate under appropriate conditions. Non-limiting examples of suitable conditions include a suitable medium (e.g., a medium having a suitable nutrient content [ e.g., a suitable carbon content, a suitable nitrogen content, a suitable phosphorus content ], a suitable supplement content, a suitable trace metal content, a suitable pH), a suitable temperature, a suitable feed rate, a suitable pressure, a suitable oxygenation level, a suitable culture duration, a suitable culture volume (i.e., a culture medium volume comprising recombinant host cells), and a suitable culture vessel.

"expression" refers to the process by which a cell converts genetic information stored in a DNA sequence, through transcription and translation, into a protein molecule having biological activity.

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Illustratively, one type of vector is a "plasmid," which generally refers to a circular double-stranded DNA loop that can be ligated into an additional DNA segment (exogenous gene), and may also include a linear double-stranded molecule, such as that obtained from amplification by Polymerase Chain Reaction (PCR) or treatment of a circular plasmid with a restriction enzyme. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., vectors having an origin of replication that is functional in the host cell). Other vectors may be introduced into a host cell and integrated into the host cell's genome and thus replicated together with the host genome. In addition, certain preferred vectors are capable of directing the expression of the foreign genes to which they are linked. In the present application, the vector to which the foreign gene is linked is referred to as a "recombinant expression vector" (or simply as an "expression vector").

In a first aspect the present application provides a milk protein composition comprising one or more recombinantly expressed milk proteins, and a milk protein peptide, wherein the milk protein peptide is produced by degradation of the recombinantly expressed milk protein when expressed in a host cell. The inventors have unexpectedly found in the study that when the recombinantly expressed milk protein is expressed in a host cell, degradation occurs, resulting in the corresponding milk protein peptide, and that the milk protein composition of the application comprising the milk protein peptide has a lower allergenicity compared to the natural milk protein.

In some embodiments of the application, the recombinant expressed milk protein is selected from at least one of recombinant expressed β -lactoglobulin, recombinant expressed α -lactalbumin, recombinant expressed kappa-casein, recombinant expressed β -casein, recombinant expressed lactoferrin, recombinant expressed αs1-casein, recombinant expressed αs2-casein, and recombinant expressed osteopontin, and the recombinant expressed milk protein may be one, or a combination of two or more.

In some embodiments of the application, the recombinantly expressed lactoprotein is a recombinantly expressed β -lactoglobulin, which upon expression in a host cell degrades to produce a lactoprotein peptide, which may also be referred to as a lactoglobulin peptide, in which case the milk protein composition comprises the recombinantly expressed β -lactoglobulin and the lactoglobulin peptide.

In some embodiments of the application, the milk protein composition comprises recombinant expressed β -lactoglobulin and lactoglobulin peptides; the content of the recombinant expressed beta-lactoglobulin and lactoglobulin peptide is 10-100% based on the total weight of the milk protein composition; preferably 55-95%.

In some embodiments of the application, the content of said recombinantly expressed β -lactoglobulin is from 0 to 95% based on the total mass of said recombinantly expressed β -lactoglobulin and said lactoglobulin peptide; the content of the lactoglobulin peptide is 5% -100%; preferably, the content of the recombinant expressed beta-lactoglobulin is 10% -90%; the content of the lactoglobulin peptide is 10% -90%; preferably, the content of the recombinant expressed beta-lactoglobulin is 20% -80%; the content of the lactoglobulin peptide is 20% -80%; preferably, the content of the recombinant expressed beta-lactoglobulin is 30% -70%; the content of the lactoglobulin peptide is 30% -70%; preferably, the content of the recombinant expressed beta-lactoglobulin is 40% -60%; the content of the lactoglobulin peptide is 40% -60%. It will be appreciated that the milk protein compositions of the present application may also comprise only milk protein peptides (e.g. lactoglobulin peptides) resulting from the degradation of the recombinantly expressed milk protein (e.g. recombinantly expressed β -lactoglobulin) in a host cell.

In some embodiments of the application, the recombinant expressed milk protein further comprises other recombinant expressed milk proteins, which may be at least one of recombinant expressed alpha-lactalbumin, recombinant expressed kappa-casein, recombinant expressed beta-casein, recombinant expressed lactoferrin, recombinant expressed alpha S1-casein, recombinant expressed alpha S2-casein, and recombinant expressed osteopontin. For example, it may be: the recombinant expression beta-lactoglobulin and the recombinant expression alpha-lactalbumin, the recombinant expression beta-lactoglobulin and the recombinant expression kappa-casein, the recombinant expression beta-lactoglobulin and the recombinant expression beta-casein, the recombinant expression beta-lactoglobulin and the recombinant expression lactoferrin, the recombinant expression beta-lactoglobulin and the recombinant expression alpha S1-casein, the recombinant expression beta-lactoglobulin and the recombinant expression alpha S2-casein, the recombinant expression beta-lactoglobulin and the recombinant expression osteopontin, or other recombinant expression lactoproteins are also included on the basis of the combination of the two recombinant expression lactoproteins, for example, the combination of the recombinant expression beta-lactoglobulin, the recombinant expression lactoferrin and the recombinant expression alpha-lactalbumin, the combination of the recombinant expression beta-lactoglobulin, the recombinant expression lactoferrin and the recombinant expression kappa-casein, the combination of the recombinant expression beta-lactoglobulin, the recombinant expression lactoferrin and the recombinant expression beta-casein, the combination of the recombinant expression beta-lactoglobulin, the recombinant expression beta-lactoferricin and the recombinant expression alpha 1-casein, and the like can be used.

In some embodiments of the application, the other recombinantly expressed milk protein may be expressed directly in the host cell to obtain the milk protein composition; may also be added to the milk protein composition by means of exogenous addition.

In some embodiments of the application, the milk protein peptide may further comprise other milk protein peptides produced by degradation of the other recombinantly expressed milk proteins when expressed in the host cell.

In some embodiments of the application, the further milk protein peptide may be obtained by degradation upon expression of the further recombinantly expressed milk protein in the host cell, or may be added to the milk protein composition by means of exogenous addition.

In some embodiments of the application, the milk protein composition comprises 5 wt.% or more, 10 wt.% or more, 20 wt.% or more, 30 wt.% or more, 40 wt.% or more, 50 wt.% or more, 60 wt.% or more, 70 wt.% or more, 80 wt.% or more, 90 wt.% or more, or 95 wt.% or more of the milk protein peptide.

In some embodiments of the application, the milk protein composition comprises 90 wt.% or less, 80 wt.% or less, 70 wt.% or less, 60 wt.% or less, 50 wt.% or less, 40 wt.% or less, 30 wt.% or less, 20 wt.% or less, 10 wt.% or 5 wt.% or less of the milk protein peptide.

In some embodiments of the application, the milk protein composition, wherein the weight ratio of the recombinantly expressed milk protein to the milk protein peptide is 100:1 to 1:100; preferably 10:1-1:10; more preferably 5:1-1:5, a step of; and more preferably 3:2 to 2:3.

In some embodiments of the application, the content of the recombinantly expressed milk protein is from 0 to 95% and the content of the milk protein peptide is from 5% to 100% based on the total mass of the milk protein composition; preferably, the content of the recombinant expressed milk protein is 10% -90%; the content of the milk protein peptide is 10% -90%; preferably, the content of the recombinant expressed milk protein is

20% -80%, wherein the content of the milk protein peptide is 20% -80%; preferably, the content of the recombinant expressed milk protein is 30% -70%, and the content of the milk protein peptide is 30% -70%; preferably, the content of the recombinant expressed milk protein is 40% -60%, and the content of the milk protein peptide is 40% -60%.

In some embodiments of the application, the β -lactoglobulin may be bovine, ovine, caprine, buffalo, camel, equine, donkey, marmoset, guinea pig, squirrel, bear, cynomolgus monkey, gorilla, chimpanzee, north american wild goat (mountain goat), monkey, ape, cat, dog, gerbil (wallby), rat, mouse, elephant, negative mouse, rabbit, whale, baboon, gibbon, gorilla, mountain (mangrill), pig, wolf, fox, lion, tiger, needle-mole or ragon. Beta-lactoglobulin is the major allergen in mammalian milk (e.g., bovine milk), and the inventors have found that inclusion of lactoglobulin peptides resulting from the degradation of beta-lactoglobulin in the milk protein compositions of the present application significantly reduces the sensitization of beta-lactoglobulin.

In some embodiments of the application, the kappa-casein may be bovine, human, sheep, goat, buffalo, camel, horse, donkey, marmoset, guinea pig, squirrel, bear, macaque, gorilla, north american wild goats (mountain goat), monkey, ape, cat, dog, sand bag mouse (walleby), rat, mouse, elephant, negative mouse, rabbit, whale, baboon, gibbon, gorilla, mountain snake (mandril), pig, wolf, fox, lion, needle mole, or rago like kappa-casein. Recombinant expression of kappa-casein and kappa-casein peptides produced by its degradation when expressed in the host cell contributes to increasing the solubility of the milk protein composition making it more readily absorbable by organisms.

In some embodiments of the application, the alpha-lactalbumin may be bovine, human, ovine, caprine, buffalo, camel, horse, donkey, marmoset, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, north american wild goat (mountain goat), monkey, ape, cat, dog, sand bag mouse (wallby), rat, mouse, elephant, negative mouse, rabbit, whale, baboon, gibbon ape, gorilla, mountain (mandril), pig, wolf, fox, lion, needle mole, or rague alpha-lactalbumin. The degradation of the alpha-lactalbumin peptide produced when expressed in the host cell facilitates a further reduction in the sensitization of the milk protein composition.

In some embodiments of the application, the β -casein may be bovine, human, ovine, caprine, buffalo, camel, horse, donkey, marmoset, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, north american wild goat (mountain goat), monkey, ape, cat, dog, sand bag mouse (wallby), rat, mouse, elephant, negative mouse, rabbit, whale, baboon, gibbon ape, gorilla, mountain (mandril), pig, wolf, fox, lion, needle mole, or branchlet like β -casein. Recombinant beta-casein and beta-casein peptides produced by its degradation when expressed in the host cell are advantageous for increasing the solubility of the milk protein composition, making it more easily absorbable by organisms.

In some embodiments of the application, the lactoferrin may be bovine, human, sheep, goat, buffalo, camel, horse, donkey, marmoset, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee (mountain goat), monkey, ape, cat, dog, sand bag mouse (wallby), rat, mouse, elephant, bell mouse, rabbit, whale, baboon, gibbon, gorilla, mountain (mandril), pig, wolf, fox, lion, needle mole, or rago lactoferrin. Recombinant lactoferrin and/or lactoferrin peptides produced by the degradation of recombinant lactoferrin when expressed in the host cells have the effects of promoting iron absorption by organisms, inhibiting bacteria, improving intestinal flora, etc., making it suitable for use as an antibacterial and antiviral agent in food products such as infant formulas, functional dairy products, and dietary supplements.

In some embodiments of the application, the αs 1-casein may be bovine, human, ovine, caprine, buffalo, camel, horse, donkey, marmoset, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, north american wild goat (mountain goat), monkey, ape, cat, dog, sand bag mouse (wallby), rat, mouse, elephant, negative mouse, rabbit, whale, baboon, gibbon ape, gorilla, mountain, pig, wolf, fox, lion, needle-mole, or ragon αs 1-casein. Recombinant expression of αs 1-casein and αs 1-casein peptides resulting from its degradation upon expression in the host cell is advantageous for increasing the solubility of the milk protein composition, making it more readily absorbable by an organism.

In some embodiments of the application, the αs 2-casein may be bovine, human, ovine, caprine, buffalo, camel, horse, donkey, marmoset, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, north american wild goat (mountain goat), monkey, ape, cat, dog, sand bag mouse (wallby), rat, mouse, elephant, negative mouse, rabbit, whale, baboon, gibbon ape, gorilla, mountain, pig, wolf, fox, lion, needle-mole, or ragon αs 2-casein. Recombinant expression of αs2-casein and αs2-casein peptides resulting from its degradation upon expression in the host cell is advantageous for increasing the solubility of the milk protein composition, making it more readily absorbable by organisms.

In some embodiments of the application, the osteopontin may be bovine, human, ovine, caprine, buffalo, camel, equine, donkey, marmoset, guinea pig, squirrel, bear, macaque, gorilla, northern wild goat (mountain goat), monkey, ape, cat, dog, sand bag mouse (walleby), rat, mouse, elephant, negative mouse, rabbit, whale, baboon, gibbon, gorilla, mountain, mangril, pig, wolf, fox, lion, pin mole, or rago osteopontin. Research shows that the activity of osteopontin (OPN active protein) can directly reach intestinal tract, strengthen the protective barrier function of the intestinal tract, realize the whole body immune protection, reduce 50% of biological functions such as fever discomfort, and the like. The content of osteopontin in the umbilical cord blood plasma of newborns and the blood plasma of infants of 3 months old is very high and is about 7-10 times of that of adults, which indicates that the osteopontin is closely related to the growth and development of the infants in early life.

In some embodiments of the application, at least one of the one or more recombinantly expressed milk proteins comprises an amino acid sequence that is at least 80%, at least 90%, or at least 98% identical to an amino acid sequence of a bovine milk protein, ovine milk protein, equine milk protein, caprine milk protein, human milk protein, buffalo milk protein, camel milk protein, yak milk protein, canine milk protein, a milk-like protein, whale milk protein, xiong Ru protein, or lion milk protein.

In some embodiments of the application, the recombinantly expressed β -lactoglobulin comprises an amino acid sequence having at least 80%, at least 90%, or at least 98% identity to the amino acid sequence of bovine β -lactoglobulin, buffalo β -lactoglobulin, yak β -lactoglobulin, sheep β -lactoglobulin, goat β -lactoglobulin, camel β -lactoglobulin, ma-lactoglobulin, dog β -lactoglobulin, like β -lactoglobulin, whale β -lactoglobulin, xiong-lactoglobulin, or lion β -lactoglobulin.

In some embodiments of the application, the recombinant expressed lactoferrin comprises an amino acid sequence that is at least 80%, at least 90% or at least 98% identical to the amino acid sequence of bovine lactoferrin, human lactoferrin, buffalo lactoferrin, yak lactoferrin, sheep lactoferrin, goat lactoferrin, camel lactoferrin, horse lactoferrin, dog lactoferrin, like lactoferrin, whale lactoferrin, xiong Rutie protein, or lion lactoferrin.

In some embodiments of the application, the recombinantly expressed α -lactalbumin comprises an amino acid sequence that is at least 80%, at least 90%, or at least 98% identical to the amino acid sequence of bovine α -lactalbumin, human α -lactalbumin, buffalo α -lactalbumin, yak α -lactalbumin, sheep α -lactalbumin, goat α -lactalbumin, camel α -lactalbumin, ma-lactalbumin, dog α -lactalbumin, like α -lactalbumin, whale α -lactalbumin, xiong-lactalbumin, or lion α -lactalbumin.

In some embodiments of the application, the recombinantly expressed kappa-casein comprises an amino acid sequence having at least 80%, at least 90% or at least 98% identity to the amino acid sequence of bovine kappa-casein, human kappa-casein, buffalo kappa-casein, yak kappa-casein, sheep kappa-casein, goat kappa-casein, camel kappa-casein, ma-casein, dog kappa-casein, like kappa-casein, whale kappa-casein, xiong-casein, or lion kappa-casein.

In some embodiments of the application, the recombinantly expressed β -casein comprises an amino acid sequence having at least 80%, at least 90%, or at least 98% identity to the amino acid sequence of bovine β -casein, human β -casein, buffalo β -casein, yak β -casein, sheep β -casein, goat β -casein, camel β -casein, ma-casein, dog β -casein, like β -casein, whale β -casein, xiong-casein, or lion β -casein.

In some embodiments of the application, the recombinantly expressed αs1-casein comprises an amino acid sequence having at least 80%, at least 90%, or at least 98% identity to an amino acid sequence of bovine αs1-casein, human αs1-casein, buffalo αs1-casein, yak αs1-casein, ovine αs1-casein, caprine αs1-casein, camel αs1-casein, ma S1-casein, canine αs1-casein, like αs1-casein, whale αs1-casein, xiong S1-casein, or lion αs1-casein.

In some embodiments of the application, the recombinantly expressed αs2_casein comprises an amino acid sequence having at least 80%, at least 90%, or at least 98% identity to an amino acid sequence of bovine αs2_casein, human αs2_casein, buffalo αs2_casein, yak αs2_casein, ovine αs2_casein, caprine αs2_casein, camel αs2_casein, ma S2_casein, canine αs2_casein, like αs2_casein, whale αs2_casein, xiong S2_casein, or lion αs2_casein.

In some embodiments of the application, the recombinantly expressed osteopontin comprises an amino acid sequence having at least 80%, at least 90% or at least 98% identity to the amino acid sequence of bovine osteopontin, human osteopontin, buffalo-bone osteopontin, yak-bone osteopontin, sheep-bone osteopontin, goat-bone osteopontin, camel-bone osteopontin, equine-bone osteopontin, dog-bone-bridge protein, like osteopontin, whale-bone-bridge protein, bear-bone-bridge protein, or lion-bone-bridge protein.

In some embodiments of the application, the host cell is selected from a fungal cell, a bacterial cell or a protozoan cell.

In some embodiments of the application, the fungal cells include, but are not limited to, organisms of the phylum ascomycota (ascomycota), basidiomycota (Basidiomycota), zygomycota (Zygomycota), chytriomycota (chytriomycota), oomycota (oomyceta), and sacculus mycota (glycomycota), illustratively the fungus is selected from the group consisting of yeast or filamentous fungi.

In some embodiments of the application, the yeasts include, but are not limited to, candida (Candida), amycola (Cladosporium), cryptococcus (Cryptococcus), debaromyces (Debaromyces), endomyces (Endomyces), candida (Eremothecium), hansenula (Hansenula), kluyveromyces (Kluyveromyces), olea (Lipomyces), pichia (Pichia), rhodosporidium (Rhodosporidium), rhodotorula (Rhodosporidium), rhodosporidium (rhodosporum), yarrowia (Rhodosporidium), yarrowia (Trichosporon), yarrowia (Rhodosporidium), yarrowia (Yarrowia) and at least one species of Zosterum.

In some embodiments of the application, the Kluyveromyces (Kluyveromyces) includes, but is not limited to, at least one of Kluyveromyces marxianus (Kluyveromyces marxianus), kluyveromyces marxianus variant, kluyveromyces lactis (Kluyveromyces lactis), kluyveromyces Hupehensis (Kluyveromyces hubeiensis), kluyveromyces weissei (Kluyveromyces wickerhamii), and Kluyveromyces thermotolerans (Kluyveromyces thermotolerans).

In some embodiments of the present application, the filamentous fungal cell includes, but is not limited to, at least one of Acremonium, aspergillus, paeonia, new Saprolegnia, geobacillus, aureobasidium, canariomyces, chaetomium, cyanomyces, vermilium, paeonia, fusarium, gibberella, humicola, hymenomyces, hymenochaete, lentinus, pyricularia, malbranchium, melanocarpus, mortierella, mucor, myceliophthora, rhizopus, new Mesona, neurospora, paecilomyces, penicillium, phenerochaete, neurospora, rumex, pythium, rhizopus, schizophyllum, courospora, cyanomyces, sporothecium, talaromyces, thermomyces, thiomyces, trichoderma, and Trichoderma.

In some embodiments of the application, bacteria include, but are not limited to, firmicutes, cyanobacteria (blue-green algae), rhodobacter (oscillozophediae), bacillus (Bacillales), lactobacillus (Lactobacillales), rhodobacter (oscilloriales), bacillus (bacilus) and lactobacillus (Lactobacillaceae) and members of any of the following genera and derivatives and hybrids thereof: acinetobacter (Acinetobacter), acetobacter (Acinetobacter) (e.g., acetobacter oxydans (Acetobacter suboxydans), acetobacter xylinum (Acetobacter xylinum)), actinoplanes (e.g., actinoplane (Actinoplane missouriensis)), arthrospira (e.g., arthrospira platensis (Arthrospira platensis), arthrospira maxima (Arthrospira maxima)), bacillus (e.g., bacillus cereus (Bacillus cereus), bacillus coagulans (Bacillus coagulans), bacillus licheniformis (Bacillus licheniformis), bacillus stearothermophilus (Bacillus stearothermophilus), bacillus subtilis (Bacillus subtilis)), escherichia (e.g., escherichia coli), lactobacillus, (Lactobacillus (Lactobacillus acidophilus), lactobacillus delbrueckii (Bulgaria (Lactobacillus bulgaricus, lactobacillus bulgaricus), lactobacillus casei (Lactobacillus casei subspecies), lactobacillus reus (Lactobacillus reuteri), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus crispatus (53), lactobacillus salivarius (5275), lactobacillus (Lactobacillus plantarum (Bacillus coagulans), lactobacillus salivarius (Lactobacillus plantarum (5275), lactobacillus plantarum (Bacillus subtilis), and the like Leuconostoc (Leuconostoc) (e.g., leuconostoc (Leuconostoc citrovorum), leuconostoc dextran (Leuconostoc dextranicum), leuconostoc mesenteroides (Leuconostoc mesenteroides)), micrococcus (e.g., micrococcus lywall (Micrococcus lysodeikticus)), rhodococcus (e.g., rhodococcus turbidi (Rhodococcus opacus), rhodococcus turbidi PD 630), spirulina (Spirulina), streptococcus (Streptomyces) (e.g., streptococcus cremoris (Streptococcus cremoris), streptococcus lactis (Streptococcus lactis), streptococcus lactis diacetyl subspecies (Streptococcus lactis subspecies diacetylactis), streptococcus thermophilus (Streptococcus thermophilus)), streptomyces (Streptomyces) (e.g., streptomyces calilight (Streptomyces chattanoogensis), streptomyces griseus (Streptomyces griseus), streptomyces naeus (Streptomyces natalensis), streptomyces olivaceus (Streptomyces olive), streptomyces olivaceus (Streptomyces olivochromogenes), streptomyces rust (Streptomyces rubiginosus)), xanthomonas (Xanthomonas) (e.g., xanthomonas campestris (Xanthomonas campestris)).

"Protozoa" in the present application means organisms of the phylum Protozoa (Protozoa), which are lower single-cell eukaryotes. For example, it may be tetrahymena thermophila, tetrahymena hegewischi, tetrahymena hyperangularis, tetrahymena malcicens, tetrahymena pigmentosum or tetrahymena bulimi.

In some embodiments of the application, the fungal cell is selected from at least one of the genera kluyveromyces, saccharomyces, pichia, aspergillus, fusarium; the bacterial cell is selected from at least one of the genus Lactobacillus or Bacillus.

In some embodiments of the application, the Bacillus comprises at least one of Bacillus cereus (Bacillus cereus), bacillus coagulans (Bacillus coagulans), bacillus licheniformis (Bacillus licheniformis), bacillus stearothermophilus (Bacillus stearothermophilus), and Bacillus subtilis (Bacillus subtilis).

In some embodiments of the application, the lactobacillus comprises at least one of lactobacillus acidophilus (Lactobacillus acidophilus) and lactobacillus bulgaricus (Lactobacillus bulgaricus).

In some embodiments of the application, the host cell protein is selected from at least one of kluyveromyces marxianus (Kluyveromyces marxianus) or kluyveromyces lactis (Kluyveromyces lactis). The inventors found that expression of recombinant milk proteins in kluyveromyces marxianus or kluyveromyces lactis can yield more degradation products, milk protein peptides. The inventors have also found that more active peptides are present in the milk protein peptides obtained using the host cells.

The term "active peptide" as used herein means a peptide having a biological function, for example, a peptide having ACE inhibitory activity.

In some embodiments of the application, the milk protein composition further comprises a host cell protein.

In some embodiments of the application, the milk protein composition comprises 80 wt% or less, 70 wt% or less, 60 wt% or less, 50 wt% or less, 40 wt% or less, 30 wt% or less, 20 wt% or less, 10 wt% or less, 5 wt% or less, or 1 wt% or less of the protein of the host cell.

In some embodiments of the application, the host cell protein content is 1-40%, preferably 1-20% based on the total mass of the milk protein composition.

In some embodiments of the application, the host cell protein is at least one of kluyveromyces marxianus (Kluyveromyces marxianus) or kluyveromyces lactis (Kluyveromyces lactis) extracellular secreted protein. The extracellular secreted protein is biosafety, edible, and may be supplemented as a nutritional ingredient into the milk protein composition.

In some embodiments of the application, the milk protein composition comprises 1-40% host cell protein, 5-90% milk protein peptide and 5-90% recombinantly expressed milk protein, preferably the milk protein composition comprises 1-20% host cell protein, 40-60% milk protein peptide and 20-55% recombinantly expressed milk protein, based on the total weight of the milk protein composition.

Specifically, the preparation method of the milk protein composition according to the first aspect of the present application may include:

introducing an expression vector capable of expressing the one or more recombinant expressed milk proteins into a host cell by known methods, such as liposome transfection, electrotransfection, transformation, and the like; culturing the host cell under conditions suitable for expression of the one or more recombinantly expressed milk proteins such that the host cell expresses the one or more recombinantly expressed milk proteins; proteins or peptides are isolated using methods well known in the art, for example, by size exclusion chromatography, ultrafiltration through a membrane, or density centrifugation, based on their molecular weight. Proteins or peptides may also be separated by isoelectric precipitation, anion/cation exchange chromatography based on their surface charge. Proteins or peptides may also be isolated by ammonium sulphate precipitation based on their solubility. The proteins or peptides may also be separated by their affinity to another molecule using hydrophobic interaction chromatography. Affinity chromatography can also utilize the binding properties of the stationary phase to separate proteins or peptides. Recovering the expressed one or more recombinant expressed milk proteins and milk protein peptides from the host cell to obtain the milk protein composition of the application.

In some embodiments of the application, the recovering the milk protein composition from the host cell further comprises a separation and purification step, which may be a method well known in the art, such as size exclusion chromatography, membrane filtration, isoelectric precipitation, anion/cation exchange chromatography, ammonium sulfate precipitation, affinity chromatography, and the like, and the application is not limited herein. The separation and purification steps can obtain the milk protein peptide with higher purity.

In some embodiments of the application, different recombinant expressed milk proteins may be expressed simultaneously or sequentially in the same host cell from which the expressed plurality of recombinant expressed milk proteins and/or milk protein peptides are recovered to obtain the milk protein composition of the application.

In other embodiments of the present application, different recombinant expressed milk proteins may be expressed in different host cells, and the expressed different recombinant expressed milk proteins and/or milk protein peptides may be recovered from the different host cells, respectively, and mixed to obtain the milk protein composition of the present application. In some embodiments of the present application, the milk protein composition may be obtained by directly extracting and purifying from a host cell, or the recombinant expressed milk protein and the milk protein peptide may be obtained by separately extracting and purifying from a host cell and then mixing them to obtain the milk protein composition of the present application.

It should be noted that, due to the difference of the expression vector or host cell used, the skilled person can choose the conditions suitable for the expression of the one or more recombinant expressed milk proteins according to the specific circumstances, and the present application is not limited herein.

In some embodiments of the application, the fungal cell is selected from at least one of the genera kluyveromyces, saccharomyces, pichia, aspergillus, fusarium.

In some embodiments of the application, the Kluyveromyces (Kluyveromyces) comprises at least one of Kluyveromyces marxianus (Kluyveromyces marxianus), kluyveromyces marxianus variant, kluyveromyces lactis (Kluyveromyces lactis), kluyveromyces hupehensis (Kluyveromyces hubeiensis), kluyveromyces weissei (Kluyveromyces wickerhamii), and Kluyveromyces thermotolerans (Kluyveromyces thermotolerans).

In some embodiments of the application, the milk protein composition may be obtained by expression in kluyveromyces marxianus (Kluyveromyces marxianus), and the inventors have unexpectedly found that milk protein compositions obtained using kluyveromyces marxianus (Kluyveromyces marxianus) have lower allergenicity.

In some embodiments of the application, the time for expressing the one or more recombinant expressed milk proteins in the host cell is 12-168 hours, preferably 36-84 hours. The inventors found in the study that in the milk protein composition of the present application, the content of milk protein peptide is related to the expression time, and the longer the expression time, the higher the content of milk protein peptide is produced; the expression time is too long, the host cells are easy to age, and the production efficiency of the milk protein composition is reduced.

In some embodiments of the application, the temperature at which the one or more recombinant expressed milk proteins are expressed in the host cell is 20-45 ℃, preferably 25-40 ℃. The inventors have found that within this temperature range efficient production of the milk protein composition by the host cell is facilitated.

The strains used in the present application include:

(1) Kluyveromyces marxianus (K.marxianus) CJA0001, which is preserved in China center for type culture Collection, with the preservation number of CCTCC No: m20211601, the preservation date is 2021, 12 and 13, the preservation address is university of Wuhan in Wuhan, china, and the classification name is Kluyveromyces marxianus CJA0001.

(2) Kluyveromyces marxianus (K.marxianus) CJA0002 is preserved in China center for type culture Collection, and the preservation number is CCTCC No: m20211602, the preservation date is 2021, 12 and 13, the preservation address is Kluyveromyces marxianus CJA0002, classified and named as university of Wuhan in Wuhan, china.

(3) Kluyveromyces marxianus (K.marxianus) CJA0003, which is preserved in China center for type culture Collection, with the preservation number of CCTCC No: m20211603, the preservation date is 2021, 12 and 13, the preservation address is Kluyveromyces marxianus CJA0003.

(4) Pichia pastoris, which is collected by the applicant, is collected in Shanglira city of Yunnan province, and the collection time is 2021 and 12 months.

(5) Kluyveromyces marxianus is purchased from China industry microbiological culture Collection center (CICC), and is numbered CICC 1953, and the purchase time is 09 months 2020.

(6) Lactic acid Lu Weijun, purchased from China center for type culture Collection (CICC), designated CICC 32428, was purchased for 2021, month 05.

The milk protein composition of the present application and the method for preparing the same are described below by way of specific examples.

Example 1 preparation of milk protein composition

This example uses Kluyveromyces marxianus (K.marxianus) CJA0001 China center for type culture collection, CCTCC No: m20211601) the expression of β -lactoglobulin is as follows:

1: bovine-derived beta-lactoglobulin Gene fragment (NCBI Gene ID: 113901792) was synthesized at Kirschner Biotechnology Inc., and designated as beta-Lg. The beta-Lg is used as a template, and a target gene is obtained through PCR amplification (an upstream primer F1:5'-GGCTGAAGCTTTGATCGTTACCCAAACTATG-3' SEQ ID No:1 and a downstream primer R1:5'-GGAGATCTTAAATGTGACATTGTTCTTCC-3' SEQ ID No: 2); the commercial vector pKLAC1 was amplified by PCR primers (upstream primer F2:5'-CAATGTCACATTTAAGATCTCCTAGGGGTACC-3' SEQ ID No:3, R2:5'-GTAACGATCAAAGCTTCAGCCTCTCTTTTCTC-3' SEQ ID No: 4). The target gene is connected with a commercial pKLAC1 vector in a seamless assembly mode to obtain an expression vector pKLAC 1-beta-Lg.

2: the plasmid pKLAC 1-beta-Lg was digested with the rapid endonuclease Sac II of New England Biolabs, inc. (NEB) to obtain a homologous recombinant fragment of linearized pKLAC 1-beta-Lg, which was transformed into Kluyveromyces marxianus CJA0001 (accession number CCTCC No: M20211601) by electrotransformation (0.1 cm electrotransformation cup, 1.5-2.5kV voltage, time-wise (-5 ms)) to construct K.marxianus:: pKLAC 1-beta-Lg expression strain, while constructing the control strain K.marxianus: pKLAC1, all in medium with acetamide as the sole nitrogen source (yeast base carbon source 1.17g, pH=1.0 mol/L KH) ₂ PO ₄ -K ₂ HPO ₄ 3mL of buffer (final concentration: 30 mmol/L), 2g of agar powder, and 5mmol/L of acetamide was added after sterilization. Culturing the above expression strain and control strain in fermentation medium (peptone 20g/L, yeast powder 10g/L, glucose 10g/L, galactose 10 g/L), respectively; regulating pH of the culture medium to 5.5-6.5, fermenting at 30deg.C for 72 hr, and feeding galactose to induce protein expression. Fermentation broths were taken at 0, 12, 24, 36, 48 and 60 hours, respectively.

3:12000g of each fermentation broth was centrifuged separately, the supernatant was collected, and 15% of the concentration of the separation gel was selected according to the size of the target protein, and the preparation was carried out using SDS-PAGE denaturing acrylamide gel rapid preparation kit (C631100) (biological engineering (Shanghai) Co., ltd.) and the detailed formulation was shown in the specification of the attached PAGEs. 40. Mu.L of supernatant was placed in a centrifuge tube, 10. Mu.L of 5 Xprotein loading buffer was added, and after mixing, the protein sample was denatured by heating in boiling water for 10 min. Each gel well is added with 10 mu L of sample and 5 mu L of Maker, and the electrophoresis voltage is constant voltage of 110V for 80-90min. After electrophoresis, the albumin glue is dyed and decolorized. The result of protein staining of the supernatant of the expression strain is shown in figure 1, and the generation of the external protein (namely beta-lactoglobulin) and degradation products (lactoglobulin peptides) can be seen; image J analysis ratio shows that the ratio of beta-lactoglobulin to beta-lactoglobulin peptide content in fermentation liquor is from 9:1 to 2:3. it can also be seen from fig. 1 that after 24 hours the ratio of beta-lactoglobulin to beta-lactoglobulin peptide was about 3:2, and then the beta-lactoglobulin is continuously generated and is continuously degraded until about 60 hours, wherein the proportion of the beta-lactoglobulin to the beta-lactoglobulin peptide is approximately stabilized at 2:3. no foreign proteins or degradation products were produced in the control strain and the protein staining results are not shown.

4: milk protein composition extraction: and purifying the supernatant of the fermentation broth by adopting a membrane filtration method. Filtering conditions, the first 2-stage filter element: a precise filter element and an NF nanofiltration membrane; NF nanofiltration membrane specification: NF-4040; and (3) filtering precision: 0.001 μm; molecular cut-off value: less than or equal to 200D; inlet water pressure: 0.2-0.3 Mpa; working pressure: 0.5-0.7 Mpa. In the concentration process, back flushing the nanofiltration membrane once every 10 minutes, recovering and weighing and recording the mass of the concentrated solution after the concentration is finished, so as to obtain the milk protein composition, and placing the milk protein composition into a refrigerator at 4 ℃ for storage for later use.

Example 2: preparation of milk protein composition by enlarging fermentation system

The 50L fermentation process is as follows:

1: shake flask seed culture, 1.5mL of pKLAC 1-. Beta. -Lg bacterial liquid cultured for 24 hours in example 1 was inoculated into a 250mL triangular flask containing 100mL of a liquid seed medium (peptone 20g/L, yeast powder 10g/L, glucose 10g/L, galactose 10 g/L), and cultured at 30℃for 12 hours at 220rpm to obtain a seed liquid.

2: and culturing the secondary seeds, preparing 200mL of seed liquid, inoculating the seed liquid into a secondary seed culture medium (5L tank), culturing at 30 ℃ and 220rpm for 6-8h, and obtaining the secondary seed liquid with the OD of about 10-12.

3: fermenting, inoculating the secondary seed solution into 50L fermenter containing 20L batch fermentation medium (peptone 20g/L, yeast powder 10g/L, glucose 10g/L, galactose 10 g/L) according to 10% inoculum size, performing batch culture, and culturing at initial liquid loading of 22-25L under the conditions of 30deg.C, pH=5.5, initial rotation speed of 300r/min, and air flow of 2-3m ³ And/h, the tank pressure is 0.04-0.055MPa.

4: and (3) a material supplementing process, namely supplementing sugar: feeding glucose (60%) according to the ratio of 6-15g/L/h from the end of bottom sugar consumption until fermentation is finished; meanwhile, galactose is added to induce protein expression; and (3) adding ammonium sulfate continuously after 8 hours, controlling the ammonia concentration to be between 0.1 and 0.2 percent according to the ammonia measurement result of the process, and stopping adding 1 hour before tank discharge.

5: and (3) harvesting a product, sampling and detecting OD every 4 hours in a fermentation tank culture experiment, reserving the sample, and after entering a stabilization period, fermenting until 60 hours, and discharging the fermentation tank to obtain fermentation liquor. And purifying the supernatant of the fermentation broth by adopting a membrane filtration method. Filtering conditions, the first 2-stage filter element: a precise filter element and an NF nanofiltration membrane; NF nanofiltration membrane specification: NF-4040; and (3) filtering precision: 0.001 μm; molecular cut-off value: less than or equal to 200D; inlet water pressure: 0.2-0.3 Mpa; working pressure: 0.5-0.7 Mpa. In the concentration process, back flushing the nanofiltration membrane once every 10 minutes, recovering and weighing and recording the mass of the concentrated solution after the concentration is finished to obtain the milk protein composition, as shown in figure 2A, and then placing the milk protein composition in a cold storage at 4 ℃ for storage for later use.

6: protein detection, the milk protein composition was subjected to SDS-PAGE, and the results are shown in FIG. 2B, and it can be seen that under 50L culture conditions, beta-lactoglobulin is partially hydrolyzed to produce lactoglobulin peptides. Hydrolyzed lactoglobulin peptides were also seen after purification by AKTA ion chromatography.

Example 3: preparation of milk protein composition by enlarging fermentation system

The 1T fermentation flow is as follows:

1: first-stage shake flask seed culture: 2mL of K.marxinus of example 1, pKLAC 1-beta-Lg glycerol was inoculated into 200mL of YPD medium (500 mL shake flask), cultured at 30℃and 220rpm for 16h with OD to about 12 to obtain a first seed solution.

2: shake flask culture of secondary seeds: 7mL of the primary seed solution is inoculated into 700mL of a secondary shaking flask culture medium (20 g/L of peptone, 10g/L of yeast powder, 10g/L of glucose and 10g/L of galactose), and the secondary seed solution is obtained after 16h of culture at 30 ℃ and 220rpm and OD is about 12.

3: seed pot (150L) culture: 2.8L of secondary seed liquid is inoculated into 40L of fermentation culture medium (40L/150L of seed tank (g), 20g/L of peptone, 10g/L of yeast powder, 10g/L of glucose and 10g/L of galactose), the initial rotating speed is 300rpm at 30 ℃, the pH value is=5.5, the air quantity is 40L/min, dissolved oxygen is linked with the rotating speed, DO is controlled at 30%, and the culture is carried out for 6-8h, and the OD is about 12, so that the seed liquid is obtained.

4: fermentation culture: 40L of seed liquid is inoculated into 400L of fermentation medium (1000L of fermentation tank), the temperature is 30 ℃, the initial rotating speed is 300rpm, the pH=5.5, the air quantity is 400L/min, dissolved oxygen and the rotating speed are linked, and DO is controlled at 30%. Sugar is added according to pH feedback before 16 h; adding sugar and galactose according to DO feedback after 16h for induction; culturing for 64h to obtain fermentation liquor. The subsequent air quantity can be increased to 1000L/min without supplying dissolved oxygen.

5: the product was harvested and the supernatant of the fermentation broth was purified by membrane filtration. Filtering conditions, the first 2-stage filter element: a precise filter element and an NF nanofiltration membrane; NF nanofiltration membrane specification: NF-4040; and (3) filtering precision: 0.001 μm; molecular cut-off value: less than or equal to 200D; inlet water pressure: 0.2-0.3 Mpa; working pressure: 0.5-0.7 Mpa. In the concentration process, back flushing the nanofiltration membrane once every 10 minutes, recovering and weighing and recording the mass of the concentrated solution after the concentration is finished to obtain the milk protein composition, and placing the milk protein composition into a refrigerator at 4 ℃ for storage for later use.

6: protein detection, the results of SDS-PAGE detection of the milk protein composition are shown in FIG. 3, and it can be seen that the beta-lactoglobulin and milk protein hydrolysate milk protein peptide can be expressed in a large amount under the 1T culture condition.

Example 4: preparation of milk protein composition by prolonging fermentation time

The procedure was as in example 3 except that the fermentation time in step 4 was prolonged to 108 hours. 12h, 36h, 60h, 72h, 84h, 96h, 108h of the supernatant of the fermentation broth were used to prepare a milk protein composition in the same manner as in example 3, and the SDS-PAGE results are shown in FIG. 4, from which the production of beta-lactoglobulin and lactoglobulin peptide were observed.

Example 5: different k.marxinus expressed milk protein compositions

Referring to the method of example 1, linearized pKLAC1- β -Lg was transformed into different sources of kluyveromyces marxianus, respectively: bacterium 1K.marxianus CJA0002 (CCTCC No: M20211602), bacterium 2K.marxianus CJA0003 (CCTCC No: M20211603), bacterium 3K. Marxinus (CICC 1953) were obtained as expression strains, and fermentation and detection and purification of the fermentation product were performed under the same conditions as in example 1 to obtain a milk protein composition.

SDS-PAGE results of milk protein compositions in fermentation broths of different expression strains are shown in FIG. 5, and the results show that beta-lactoglobulin can be hydrolyzed to produce lactoglobulin peptides by expressing the beta-lactoglobulin in different K.marxinus.

Example 6: preparation of milk protein composition by using kluyveromyces lactis

Referring to the procedure of example 1, linearized pKLAC 1-. Beta. -Lg was transformed into Kluyveromyces lactis (Kluyveromyces lactis, accession number CICC 32428), K.lactis:: pKLAC 1-. Beta. -Lg, and a control strain K.lactis:: pKLAC1 was constructed. Culturing a Kluyveromyces lactis strain in a fermentation medium (20 g/L peptone, 10g/L yeast powder, 10g/L glucose and 10g/L galactose); regulating pH of the culture medium to 5.5-6.5, fermenting at 25-30deg.C for 72 hr, and feeding galactose to induce protein expression during fermentation to obtain fermentation liquid.

The fermentation broth was subjected to purification and SDS-PAGE detection in the same manner as in example 1, and the SDS-PAGE results of the milk protein compositions of the control strain and the transformed strain are shown in FIG. 6. Thus, the lactoglobulin peptide can be obtained by exogenous expression of beta-lactoglobulin by using the Kluyveromyces lactis.

Comparative example 1: preparation of milk protein composition by pichia pastoris

Using the β -Lg of example 1 as a template, lactoglobulin fragments (F1: 5'-AAAGAGAGGCTGAAGCTT ACTTGATCGTTACCCAAACTAT-3' SEQ ID No:5, R1:5'-AGGCGAATTAATTCGCGGCCTCAAATGTGAC ATTGTTCTT-3' SEQ ID No: 6) were PCR amplified, and the PCR products ligated with SnaBI/NotI-HF digested pPIC9k plasmid to construct Pichia pastoris expression plasmid pPIC9k- β -Lg. The pPIC9 k-beta-Lg plasmid linearized by SalI enzyme is transformed into Pichia pastoris GS115, P.pastoris is constructed, a control strain P.pastoris is constructed, the obtained strain is fermented by using BMGY culture medium (brand: solarbio; product number: LA 5250) and BMMY culture medium (brand: solarbio; product number: LA 5250) successively, methanol with different volumes is added every day, and the final content of methanol in the fermentation liquid is kept to be 1%, and the fermentation is continued for 5 days. The supernatant was then collected by centrifugation, concentrated by centrifugation, and quantitatively analyzed by SDS-PAGE.

The fermentation broth was purified and SDS-PAGE tested in the same manner as in example 1, and the results are shown in FIG. 7, wherein lane 1 is a control strain and lanes 2 to 9 are constructed expression strains, and it was found that lactoglobulin peptides could not be obtained by fermentation using Pichia pastoris, and the experimental results were consistent in a number of replicates for verifying the conclusion.

And (3) effect verification:

experimental example 1: composition component identification

In this experimental example, the components of the milk protein peptides in the milk protein compositions of example 1, example 5 and example 6 were identified by using an HPLC-MS protein gel sequencing method.

1: preparation of protein glue

SDS-PAGE analysis of the supernatant of the fermentation broth, pretreatment of the gel strips of different molecular weight sizes, first immersing the gel block in 50mm NH ₄ HCO ₃ And 50% (v/v) Acetonitrile (ACN) in a buffer until decolorized; after 5 minutes incubation with 100% ACN, ACN was removed to complete the first dehydration, and 10mM Dithiothreitol (DTT) was added for 60 minutes incubation at 5 ℃ for the first hydration; the hydrated adhesive tape is dehydrated for the second time by using 100% ACN again, and is incubated for 45 minutes by using 55mM Iodoacetamide (IAA) at room temperature and in a dark place for the second hydration; the hydrated adhesive tape is reused with 50mm NH ₄ HCO ₃ And a third dehydration with 100% ACN.

2: proteolysis

(1) Cutting glue: cutting the adhesive tape into colloidal particles with the diameter of about 1 mm;

(2) Decoloring: adding 1mL of decolorization solution, vortex oscillating for 10s, decolorizing for 30min at 37 ℃, centrifuging briefly, sucking to dry, and repeating decolorizing for multiple times until blue is completely removed;

(3) Cleaning colloidal particles: washing the gel for 3 times, adding 1mL of ultrapure water each time, and carrying out vortex oscillation for 1min;

(4) Dehydrating: adding 500 mu L acetonitrile for dehydration until colloidal particles become white completely, and repeating the steps once;

(5) Thiol reduction: sucking acetonitrile, opening a centrifugal tube cover, placing in an ultra-clean bench for 10min, air-drying the acetonitrile, adding 10mM DTT until the liquid is over colloidal particles (colloidal particles with double volume), and carrying out water bath at 56 ℃ for 1h;

(6) Sulfhydryl blocking: after cooling to room temperature, the mixture was blotted dry, and then 55mM IAM was rapidly added until the liquid was above the colloidal particles (twice the volume of the colloidal particles), and the mixture was placed in a dark room for 45min;

(7) Cleaning: sucking the liquid, washing twice with 500 mu L of decolorizing liquid and washing once with pure water;

(8) Dehydrating: sucking off the liquid, adding 500 mu L of acetonitrile, vortex oscillating for 5min, sucking off the acetonitrile, opening a centrifugal tube cover, and placing the centrifugal tube cover on an ultra-clean bench for 10min for thorough air drying;

(9) Adding enzyme: with 25mM NH ₄ HCO ₃ Diluting the enzyme solution (1. Mu.g/. Mu.L of Trypsin split-packed) to 0.01. Mu.g/. Mu.L, and completely covering the colloidal particles with the enzyme solution;

(10) And (3) sucking and expanding colloidal particles: standing at 4deg.C or on ice for 30min;

(11) And (3) adding buffer solution: after the colloidal particles are swelled, adding corresponding buffer solution until the colloidal particles are soaked;

(12) Incubating overnight at 37 ℃;

(13) Gradient elution peptide: adding 5 times of 50% ACN by volume, vortex shaking for 5min, centrifuging for 1min at 5,000g, and transferring the supernatant to a new centrifuge tube with the same number; adding 5 times of 100% ACN by volume, vortex oscillating for 5min, centrifuging for 1min at 5,000g, transferring the supernatant to a new centrifuge tube with the same number, centrifuging for 5min at 25,000g, and collecting supernatant;

(14) And (5) pumping: and freezing and drying the extracted peptide solution.

3: LC-MS/MS: the dried peptide samples were reconstituted with mobile phase A (2% ACN,0.1% FA (formic acid)) and centrifuged at 20,000g for 10min, and the supernatant was taken for injection. The separation was performed by Thermo UltiMate 3000 UHPLC. The sample was first enriched and desalted in a trapping column and then fed into a self-packed C18 column (inner diameter 75 μm, column size 3 μm, column length 25 cm) and separated by the following effective gradient at a flow rate of 300 nL/min: 0-5 min,5% mobile phase B (98% ACN,0.1% FA); 5-45 min, the mobile phase B is linearly increased from 5% to 25%; 45-50 min, the mobile phase B is increased from 25% to 35%; 50-52 min, the mobile phase B rises from 35% to 80%; 52-54 min,80% mobile phase B; 54-60 min,5% mobile phase B. The nanoliter liquid phase separating end is directly connected with the mass spectrometer.

Peptides isolated by liquid chromatography were ionized by a nanoESI source and then subjected to DDA (data dependent acquisition) mode detection by a tandem mass spectrometer Q-exact HF X (Thermo Fisher Scientific, san Jose, CA). Principal ginsengSetting the number: the voltage of the ion source is set to be 1.9kV, and the scanning range of the MS1 is 350-1,500 m/z; resolution was set to 60,000; MS2 initial m/z fixed to 100; the resolution is 15,000. Ion screening conditions for MS2 fragmentation: charge 2 ⁺ ～6 ⁺ The first 30 parent ions with peak intensities exceeding 10000. The ion fragment mode is HCD, and fragment ions are detected in Orbitrap. The dynamic exclusion time was set to 30 seconds. The AGC is set to: MS1, 3e6, MS2, 1e5.

Mass spectrum polypeptide identification results from the beta-lactoglobulin gel section and the lactoglobulin peptide gel section in SDS-PAGE analysis are shown in table 1 and table 2, respectively.

Table 1: mass spectrum identification of beta-lactoglobulin

Table 2: mass spectrum identification of lactoglobulin peptides

Table 1 shows that in the beta-lactoglobulin gel section, the amino acid sequences of the polypeptides with high ion scores in the detected polypeptides are shown as SEQ ID No. 7-10, respectively, wherein the number of times of inquiry of SEQ ID No. 7 is the greatest. In the lactoglobulin peptide gel section, the amino acid sequences of the polypeptides with high ion scores in the detected polypeptides are shown as SEQ ID No. 7, 10 and 11 respectively, and SEQ ID No. 7-11 are all from beta-lactoglobulin, but not mycoprotein or other metabolite protein, so that the composition ratio of each polypeptide fragment contained in the lactoglobulin peptide decomposed in the expression process is obviously different from the expression of the complete form existing before decomposition, and the two show unexpected differences in each activity detection.

Further, the results of comparing the components of the lactoprotein peptides in the milk protein compositions of example 1 and example 5, respectively, show that different kluyveromyces marxianus expressing exogenous beta-lactoglobulin can be hydrolyzed to produce lactoprotein peptides, and the components of the lactoprotein peptides are close, which indicates that different host cells of the application can produce lactoglobulin peptides of the application.

In addition, it can be seen from the detection results that the milk protein composition in the examples of the present application contains a certain amount of host cell proteins in addition to the main polypeptides having high ion scores.

Applicants have unexpectedly found that the milk protein compositions of the present application have ACE inhibiting activity when assayed for activity. From this, it can be judged that the milk protein composition of the present application has a higher nutritive value.

Experimental example 2: antigenic analysis of milk protein compositions

The antigenicity of beta-LG in 12, 36 and 72 hours samples of the fermentation broth of example 1 was evaluated by ELISA detection kit (product number: ml036565, manufacturer: shanghai enzyme-linked Biotechnology Co., ltd.).

Sample preparation:

a: hitrap Capto Q anion exchange chromatography separation and purification of beta-lactoglobulin

a1: the supernatant of the fermentation broth was centrifuged at 12000rpm/min for 5min and pH was adjusted to 7.5/8.5 with NaOH and filtered through a 0.45 μm sterile needle filter.

a2: the AKTA Pure 150 protein purification system is used for on-machine detection, the whole flow path is flushed with ultrapure water for 5 column volumes, and the flow rate is 5mL/min; equilibrate 5 column volumes with buffer a at a flow rate of 5mL/min; before loading, cleaning the loop ring by using a buffer solution A, wherein the loading flow rate is 3mL/min; performing stage elution with buffer solution containing 100mM, 200mM, 300mM, 400mM and 500mM NaCl respectively at flow rate of 5mL/min, collecting eluting peaks at each stage, and detecting and collecting molecular weight and purity of protein by SDS-PAGE; the column was washed with ultrapure water for 5 column volumes and then with 20% ethanol for 5 column volumes at a flow rate of 5mL/min. After the optimization, the protein is purified by using a Hitrap Capto Q5 mL anion exchange chromatography column, the loading amount of the protein is 2mL, and the protein is eluted by using a buffer solution containing 100-600 mM NaCl respectively, wherein 300mM NaCl is eluted to obtain beta-lactoglobulin.

b: membrane filtration separation and purification of beta-lactoglobulin peptide

b1 ultrafiltration: 500mL of the fermentation solutions K.marxiannius:: pKLAC1 and K.marxiannius:: pKLAC 1-. Beta. -Lg in example 1 were centrifuged at 12000rpm for 5min, and the supernatants were collected. And starting the ultrafilter, and flushing the ultrafilter with clear water until the effluent is clear and has no peculiar smell. After pre-flushing the ultrafilter, the supernatant was added. And after the ultrafilter and the booster pump are started and normally discharged, the opening of a concentrated water reflux valve is regulated, and the concentrated water reflux pressure is controlled to be 0.04-0.08 Mpar. During the concentration process, ultrafiltration membrane back flushing is performed once every 10 minutes (back flushing time is set to 40 s), and ultrafiltration membrane precision is: 0.01-0.1 mu m; molecular cut-off value: less than or equal to 10000DA. After the concentration is finished, the concentrated solution and the permeate liquid are respectively recovered, weighed and recorded, and put into a refrigerator at 4 ℃ for storage for standby.

b2 nanofiltration: collecting the K.marxiannius, pKLAC1 and K.marxiannius, pKLAC 1-beta-Lg permeate, and performing nanofiltration purification. And injecting a proper amount of pure water into the concentrated water tank, starting the nanofiltration machine, and using clear water to wash the nanofiltration machine forward and backward until the effluent is clear and has no peculiar smell. After the nanofiltration machine is pre-washed, the b1 permeate is added. And after the nanofiltration machine and the booster pump are started and the liquid is discharged normally, the opening of a concentrated water reflux valve is regulated, and the concentrated water reflux pressure is controlled to be 0.04-0.08 Mpar. During the concentration process, every 10 minutes, the nanofiltration membrane is backwashed once, and the nanofiltration membrane precision is as follows: 0.001 μm; molecular cut-off value: and is less than or equal to 200DA. After the concentration is finished, the quality of the concentrated solution is recovered, weighed and recorded, and the concentrated solution is put into a refrigeration house at 4 ℃ for storage for standby.

b3: k.marxianius is used for expressing beta-lactoglobulin peptide generated by beta-lactoglobulin hydrolysis by subtracting K.marxianius from the concentration of the K.pKLAC 1-beta-Lg nanofiltration concentrated solution.

c: membrane filtration separation and purification of beta-lactoglobulin and beta-lactoglobulin peptide composition

c1 ultrafiltration: the fermentation broths of K.marxians:: pKLAC1 and K.marxians:: pKLAC 1-. Beta. -Lg of example 1 were centrifuged at 12000rpm for 5min, respectively, and the supernatants were collected. And starting the ultrafilter, and flushing the ultrafilter with clear water until the effluent is clear and has no peculiar smell. After pre-flushing the ultrafilter, the supernatant was added. And after the ultrafilter and the booster pump are started and normally discharged, the opening of a concentrated water reflux valve is regulated, and the concentrated water reflux pressure is controlled to be 0.04-0.08 Mpar. During the concentration process, ultrafiltration membrane back flushing is performed once every 10 minutes (back flushing time is set to 40 s), and ultrafiltration membrane precision is: 0.01-0.1 mu m; molecular cut-off value: and 20000DA or less. After the concentration is finished, the concentrated solution and the permeate liquid are recovered, weighed and recorded, and put into a refrigerator at 4 ℃ for storage for standby.

c2 nanofiltration: collecting K.marxiannius, pKLAC1 and K.marxiannius, pKLAC 1-beta-Lg permeate collected in the above step c1, and performing nanofiltration purification. And injecting a proper amount of pure water into the concentrated water tank, starting the nanofiltration machine, and using clear water to wash the nanofiltration machine forward and backward until the effluent is clear and has no peculiar smell. After the nanofiltration machine is pre-washed, the b1 permeate is added. And (3) starting the nanofiltration machine and the booster pump, and after normal liquid outlet, adjusting the opening of a concentrated water reflux valve to control the concentrated water reflux pressure to be 0.04-0.08Mpar. During the concentration process, every 10 minutes, the nanofiltration membrane is backwashed once, and the nanofiltration membrane precision is as follows: 0.001 μm; molecular cut-off value: after concentration of less than or equal to 200DA, recovering, weighing and recording the mass of the concentrated solution, and storing in a refrigerator at 4 ℃ for later use.

c3: k.marxianius is subtracted from the sum of the concentration of the ultrafiltration and nanofiltration concentrated solution of the pKLAC 1-beta-Lg to obtain the K.marxianius which expresses beta-lactoglobulin and beta-lactoglobulin composition produced by beta-lactoglobulin hydrolysis.

d: membrane filtration separation and purification of milk protein composition

d1: the fermentation supernatant was purified by membrane filtration. Nanofiltration conditions, the first 2-stage filter element: a precise filter element and an NF nanofiltration membrane; NF nanofiltration membrane specification: NF-4040; and (3) filtering precision: 0.001 μm; molecular cut-off value: less than or equal to 200D; inlet water pressure: 0.2-0.3 Mpa; working pressure: 0.5-0.7 Mpa. In the concentration process, back flushing the nanofiltration membrane once every 10 minutes, recovering and weighing the concentrated solution after the concentration is finished, and placing the concentrated solution into a cold storage at 4 ℃ for storage for later use.

d1 comprises beta-lactoglobulin, beta-lactoglobulin peptide and K.marxiannius extracellular secretion protein.

The beta-lactoglobulin (A) of example 1 was obtained by separation and purification for 72 hours of fermentation; beta-lactoglobulin peptide (B); beta-lactoglobulin and beta-lactoglobulin peptide composition (C); a milk protein composition (D comprising 35 wt.% recombinant β -lactoglobulin, 60 wt.% β -lactoglobulin peptide, and 5 wt.% kluyveromyces marxianus exocrine protein); example 1 milk protein composition fermented for 36 hours (G, comprising 50 wt% recombinant β -lactoglobulin, 45 wt% β -lactoglobulin peptide with 5 wt% kluyveromyces marxianus exocrine protein), milk protein composition fermented for 12 hours (H, comprising 85 wt% recombinant β -lactoglobulin, 10 wt% β -lactoglobulin peptide with 5 wt% kluyveromyces marxianus exocrine protein); commercial beta-lactoglobulin (F) (manufacturer: sigma-Aldrich, cat# L0130).

Antigenic analysis

The analysis method comprises the following steps: the fermentation sample obtained in example 2 was prepared at a concentration of 1g/L, and commercial beta-lactoglobulin (F) was used as a standard. Then, respectively adopting Kai-nitrogen determination, HPLC (high performance liquid chromatography) and Elisa (enzyme-linked immunosorbent assay) to detect the protein content in the two groups of samples, comparing the protein concentration detected by different methods based on different detection methods, and comparing the sensitization of the samples of each group, wherein the specific method is as follows:

a: elisa assay

a1: an Elisa kit (product number: ml036565, manufacturer: shanghai enzyme-linked biotechnology Co., ltd.) was used, in which wells of the microtiter plates were pre-coated with antibodies specific for bovine beta-LG. Different samples obtained using the 4 methods described above were added to wells and bound to specific antibodies and horseradish peroxidase (HRP) -conjugated specific antibodies.

a2: the antibody-antigen-HRP-conjugated antibody complex and 3,3', 5' -tetramethylene benzidine (TMB) substrate solution became blue and then yellow after addition of the stop solution. Optical Density (OD) was measured spectrophotometrically at a wavelength of 450 nm. OD values are proportional to antigenicity.

b: HPLC detection

The chromatographic conditions are as follows: phase A pure water (containing 0.1% trifluoroacetic acid), phase C acetonitrile (containing 0.1% trifluoroacetic acid), column temperature 50 ℃, flow rate 1mL/min, loading 20 μl, detection wavelength 214nm, and acquisition time 9min.

The mobile phase gradient is as follows:

time min	C phase proportion%
		0～2.5	40 to 70 liters
2.5～8	70 liter to 100
		8～11	100
11～14	100 to 40
		14～19	40

The liquid chromatogram corresponding to the standard is shown in FIG. 8, and the liquid chromatogram of the fermented sample is shown in FIG. 9.

c: kai-type nitrogen determination detection

The Kai-type nitrogen determination detection principle is as follows: decomposing protein, combining ammonia generated by decomposition with sulfuric acid to generate ammonium sulfate, then performing alkalization distillation to enable ammonia to be free, absorbing with boric acid, then titrating with sulfuric acid or hydrochloric acid standard solution, and multiplying the consumption of acid by a conversion coefficient to obtain the protein content.

The detection results of the four methods are as follows:

	kai-type nitrogen determination	HPLC	Elisa
				Fermentation sample (D)	0.97	0.89	0.82
Purchase of beta-lactoglobulin	0.98	0.93	0.92

As can be seen from the above table, the protein content in the 1g/L sample to be detected prepared by Kjeldahl determination is 0.97g/L and 0.98g/L respectively, and the beta-lactoglobulin is detected by HPLC, wherein the detection of the fermented sample composition is 0.89g/L, which indicates that the sample is partially hydrolyzed, and the detection result of Elisa is 0.84g/L, which indicates that the detection sensitization is reduced after the hydrolysis of the fermented sample composition.

Solubility analysis

Drying the fermented sample of example 2 to obtain sample powder, weighing 1g of the fermented sample powder and 1g of the beta-lactoglobulin standard substance respectively, detecting the total amount of protein contained in each of 1g of the fermented sample powder and the beta-lactoglobulin standard substance by a Kjeldahl method, dissolving the fermented sample powder and the beta-lactoglobulin standard substance in distilled water to prepare 1% (w/v) aqueous solution respectively, shaking the mixture for 1h at a constant temperature of 25 ℃, centrifuging the mixture, taking the supernatant, recording the quality of the supernatant, and measuring the protein content of the supernatant by Kjeldahl nitrogen, wherein each sample detection is repeated in three groups.

Protein solubility was calculated according to the following formula:

protein solubility = protein content in supernatant/protein content in per 1g sample, the test results are as follows:

Numbering device	Sample of	Protein solubility%
			1	Fermentation sample	92
2	Purchase of beta-lactoglobulin	87

Digestibility detection

The detection steps are as follows:

a: the total protein content in the unit samples (the fermented sample obtained in example 2 and the beta-lactoglobulin standard) was determined by the Kaplan nitrogen determination method.

b: in vitro simulated gastric digestion: the medium sample in a is prepared into an emulsion with the mass fraction of protein of 2% by using distilled water, the emulsion is preheated for 10min in a water bath with the temperature of 37 ℃, and the emulsion is adjusted to pH=3 by using HCl with the concentration of 1 mol/L. To 100mL of the emulsion was added pepsin 0.04g, chymosin 0.06g, digested and hydrolyzed on a constant temperature shaker at 37 ℃ for 1h, then the emulsion was adjusted to ph=7 with NaOH 1 mol/L. Inactivating the enzyme and determining the gastric digestibility of the protein therein.

c: in vitro simulated intestinal digestion: the medium sample in a is prepared into an emulsion with the mass fraction of protein of 2% by using distilled water, the emulsion is preheated for 10min in a water bath with the temperature of 37 ℃, and the emulsion is adjusted to pH=3 by using HCl with the concentration of 1 mol/L. To 100mL of the milk sample, 0.04g of pepsin and 0.06g of chymosin were added, and the mixture was digested and hydrolyzed on a constant temperature shaker at 37℃for 1 hour, and then the emulsion was adjusted to pH=7 with 1mol/L NaOH. From this, 100ml of milk sample was taken, 0.1g of trypsin was added, digested and hydrolyzed on a shaking table at a constant temperature of 37℃for 2 hours, and then inactivated in a boiling water bath for 5 minutes, to determine the total digestibility of the protein.

d: determination of in vitro digestibility of proteins: taking 10mL of the digested sample in the step b or the step c, adding an equal volume of 24% trichloroacetic acid solution to precipitate protein, centrifuging for 20min at 12000r/min, collecting supernatant, measuring the nitrogen content in the supernatant by a Kjeldahl method, and calculating the digestibility by a formula (1), wherein the blank test uses distilled water to replace the milk-like digestive juice.

Wherein: m 1-nitrogen content in the supernatant of the sample, g; m 0-nitrogen content in the supernatant, g; m 2-protein content in sample, g. The calculation results are as follows:

numbering device	Sample of	Protein digestibility (%)
			1	Fermented sample composition	82
2	Purchase of beta-lactoglobulin	75

ACE activity inhibition capability detection

(1) Preparation of the solution

A sodium borate buffer (ph=8.3) containing 0.3mol/L sodium chloride was prepared at 0.1mol/L, and the fermented sample of example 2 was lyophilized and reconstituted in the buffer to the original volume before lyophilization. The hippocampal-histidyl-leucine (HIL) and ACE were dissolved in the buffer to 5mmol/L and 0.1U/mL, respectively.

(2) Preparation of samples

Mixing 35. Mu.L of the fermentation sample solution obtained in example 2 above with 200. Mu.L of HIL solution, and pre-incubating at 37℃for 5min; 20. Mu.L of ACE solution was added and incubated at 37℃for 30min, and the reaction was quenched by the addition of 250. Mu.L of 1mol/L hydrochloric acid. The control group was prepared as a control sample solution from a purchased beta-lactoglobulin reconstituted solution and hydrochloric acid was added prior to pre-incubation.

(3) Instrument conditions

The column was C18 (4.6 mm. Times.250 mm), mobile phase A was water containing 0.1% (v/v) trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% (v/v) trifluoroacetic acid. Adopts a gradient elution mode. The elution gradient is 30 to 60 percent of B, and the time is 0 to 10 minutes; 60 to 30 percent of B and 10 to 12 minutes; 30% B, for 2min. The detector is an Ultraviolet (UV) detector with a detection wavelength of 228nm, a loading amount of 20 mu L, a flow rate of 1mL/min, a column temperature of 30 ℃, and each sample is measured in parallel for 3 times.

(4) Determination of the Linear Range

A hippuric acid standard (HPLC, > 98%) was prepared as a hippuric acid standard solution (with 0.1mol/L boric acid buffer) having a concentration of 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 60. Mu.g/mL, 80. Mu.g/mL, 100. Mu.g/mL, and the solution was filtered through a 0.45 μm microfilm and analyzed by HPLC to confirm that the concentration of the standard and the area of the detection peak satisfy a linear relationship.

Result IC ₅₀ The IC of the fermented sample obtained in example 2 is shown ₅₀ 11.34 μg/mL, higher than 8.85 μg/mL of the beta-lactoglobulin standard purchased.

	Fermented sample composition	Purchase of beta-lactoglobulin
			IC ₅₀	11.34μg/mL	8.85μg/mL

Claims

1. A milk protein composition comprising one or more recombinantly expressed milk proteins, and a milk protein peptide, wherein the milk protein peptide is produced by degradation of the recombinantly expressed milk protein when expressed in a host cell.

2. The milk protein composition of claim 1, wherein the recombinantly expressed milk protein is selected from at least one of recombinantly expressed β -lactoglobulin, recombinantly expressed α -lactalbumin, recombinantly expressed kappa-casein, recombinantly expressed β -casein, recombinantly expressed lactoferrin, recombinantly expressed αs1-casein, recombinantly expressed αs2-casein, or recombinantly expressed osteopontin.

3. The milk protein composition of claim 1, wherein the one or more recombinantly expressed milk proteins are:

i. recombinant expression of beta-lactoglobulin,

recombinant expression of beta-lactoglobulin and recombinant expression of alpha-lactalbumin,

recombinant expression of beta-lactoglobulin and recombinant expression of kappa-casein,

recombinant expression of beta-lactoglobulin and recombinant expression of beta-casein, or

iv recombinant expression of beta-lactoglobulin and recombinant expression of lactoferrin.

4. The milk protein composition of claim 1, wherein at least one of the one or more recombinant milk expression proteins comprises an amino acid sequence that is at least 80%, at least 90%, or at least 98% identical to an amino acid sequence of a cow milk protein, sheep milk protein, horse milk protein, goat milk protein, human milk protein, buffalo milk protein, camel milk protein, yak milk protein, dog milk protein, milk-like protein, whale milk protein, xiong Ru protein, lion milk protein, or tiger milk protein.

5. The milk protein composition of claim 1, wherein the host cell is selected from at least one of a fungal cell, a bacterial cell, or a protozoan cell;

preferably, the fungal cell is selected from at least one of the genera kluyveromyces, saccharomyces, candida, pichia, aspergillus, or fusarium; the bacterial cell is selected from at least one of Lactobacillus or Bacillus;

preferably, the Kluyveromyces (Kluyveromyces) comprises at least one of Kluyveromyces marxianus (Kluyveromyces marxianus), kluyveromyces marxianus variant, kluyveromyces lactis (Kluyveromyces lactis), kluyveromyces hupehensis (Kluyveromyces hubeiensis), kluyveromyces weissei (Kluyveromyces wickerhamii) or Kluyveromyces thermotolerans (Kluyveromyces thermotolerans);

preferably, the Bacillus comprises at least one of Bacillus cereus (Bacillus cereus), bacillus coagulans (Bacillus coagulans), bacillus licheniformis (Bacillus licheniformis), bacillus stearothermophilus (Bacillus stearothermophilus) or Bacillus subtilis (Bacillus subtilis);

preferably, the lactobacillus comprises at least one of lactobacillus delbrueckii bulgaricus (Lactobacillus bulgaricus), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus casei subspecies casei (Lactobacillus casei subsp. Casei), lactobacillus reuteri (Lactobacillus reuteri), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus fermentum (Lactobacillus fermentum), lactobacillus grignard (Lactobacillus gasseri), lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus plantarum (Lactobacillus plantarum) or lactobacillus salivarius (Lactobacillus salivarius).

6. The milk protein composition of claim 1, comprising 5 wt.% or more, 10 wt.% or more, 20 wt.% or more, 30 wt.% or more, 40 wt.% or more, 50 wt.% or more, 60 wt.% or more, 70 wt.% or more, 80 wt.% or more, 90 wt.% or more, or 95 wt.% or more of the milk protein peptide.

7. The milk protein composition of claim 1, comprising 90 wt.% or less, 80 wt.% or less, 70 wt.% or less, 60 wt.% or less, 50 wt.% or less, 40 wt.% or less, 30 wt.% or less, 20 wt.% or less, 10 wt.% or 5 wt.% or less of the milk protein peptide.

8. The milk protein composition of claim 1, wherein the weight ratio of the recombinantly expressed milk protein to the milk protein peptide is 100:1 to 1:100; preferably 10:1-1:10; more preferably 5:1-1:5, a step of; and more preferably 3:2 to 2:3.

9. The milk protein composition of any one of claims 1-8, further comprising a protein of a host cell.

10. The milk protein composition of claim 9, comprising 80 wt.% or less, 70 wt.% or less, 60 wt.% or less, 50 wt.% or less, 40 wt.% or less, 30 wt.% or less, 20 wt.% or less, 10 wt.% or less, 5 wt.% or 1 wt.% or less of the protein of the host cell.

11. The milk protein composition of claim 9, comprising 5 wt.% or more, 10 wt.% or more, 20 wt.% or more, 30 wt.% or more, 40 wt.% or more, 50 wt.% or more, 60 wt.% or more, 70 wt.% or more, 80 wt.% or more, 90 wt.% or more, or 95 wt.% or more of the milk protein peptide.

12. A method of preparing a milk protein composition according to any one of claims 1-11, comprising expressing the one or more recombinantly expressed milk proteins in a host cell and recovering the milk protein composition from the host cell.

13. The method of claim 12, wherein the host cell is selected from at least one of a fungal cell, a bacterial cell, or a protozoan cell;

preferably, the fungal cell is selected from at least one of the genera kluyveromyces, saccharomyces, candida, pichia, aspergillus, fusarium; the bacterial cell is selected from at least one of Lactobacillus or Bacillus;

14. The method of claim 12, wherein the time for expressing the one or more recombinantly expressed milk proteins in the host cell is 12-168 hours, preferably 36-84 hours.

15. The method of claim 12, wherein the temperature at which the one or more recombinantly expressed milk proteins are expressed in the host cell is 20-45 ℃, preferably 25-40 ℃.

16. A food or feed composition comprising the milk protein composition of any one of claims 1-15.