WO2023221994A1

WO2023221994A1 - Milk protein composition, and preparation method therefor and use thereof

Info

Publication number: WO2023221994A1
Application number: PCT/CN2023/094598
Authority: WO
Inventors: 骆滨; 廖丽; 王松伟; 苏世源; 屠均亮; 杨秀华; 吴蓉; 许昱
Original assignee: 上海昌进生物科技有限公司
Priority date: 2022-05-17
Filing date: 2023-05-16
Publication date: 2023-11-23

Abstract

A milk protein composition, and a preparation method therefor and the use thereof, wherein the milk protein composition comprises a lactoglobulin peptide, and the lactoglobulin peptide is generated by means of degrading the recombinant β-lactoglobulin when expressed in a host cell. Also provided is a food composition or a feed composition comprising the above-mentioned milk protein composition.

Description

A milk protein composition and its preparation method and application

This application claims the priority of the Chinese patent application (application number: 2022105409902, invention title: a milk protein composition and its preparation method and application) with the filing date of May 19, 2022. The full text of this Chinese patent application is by reference The method is incorporated into this application in its entirety, and the specific expression forms are uniformly and uniformly proofread.

Technical field

This application belongs to the field of dairy product processing, and specifically relates to a milk protein composition and its preparation method and application.

Background technique

Milk is a universal source of nutrients, rich in protein, fat, lactose, minerals, vitamins and growth factors such as immunoglobulins and lactoferrin. There are two main types of milk proteins: whey protein (20%) and casein (80%). Whey protein is divided into α-lactalbumin and β-lactoglobulin; casein mainly exists 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 newborn babies. Secondly, casein can increase the absorption of calcium and phosphorus by newborn babies. Casein can release a variety of bioactive peptides after the action of proteases in the gastrointestinal tract of newborn babies. These active peptides regulate digestion, nutrition and immunity of pups. Kappa-casein can also stimulate the growth of beneficial bacteria in the gastrointestinal tract of newborn pups. β-Lactoglobulin is a high-quality protein in milk with a good amino acid ratio and high branched-chain amino acids. It also has the unique ability of the lipocalin family to bind hydrophobic ligands, such as fat-soluble nutrients. The combination of beta-carotene, retinol, unsaturated fatty acids and vitamin E can serve as a carrier or solvent for these fat-soluble nutrients, thereby reducing the intake of fat. Compared with casein, whey protein is more soluble and contains higher quality essential amino acids. Whey protein has a high content of sulfur-containing amino acids, which is an important component of the biosynthesis of glutathione. Glutathione is a tripeptide related to antioxidant, anti-cancer and improving the body's immunity. Whey protein is also the highest natural source of branched-chain amino acids, which are thought to stimulate muscle protein synthesis.

However, cow's milk is also one of the eight major allergens announced by the Food and Agriculture Organization of the United Nations. Its main allergens include casein, bovine serum albumin, β-lactoglobulin and α-lactalbumin, etc., affecting approximately 2%-6% of infants and young children. Milk allergy affects 0.1%-0.5% of adults. 82% of patients with milk allergy are allergic to β-lactoglobulin (β-Lg). This is mainly because breast milk does not contain β-Lg, so β-Lg is the first protein that infants and young children encounter. It is a foreign protein that is not easily hydrolyzed by chymotrypsin and pepsin. It can maintain its immune properties after being digested and absorbed by the human body. Therefore, β-Lg is considered to be the most important allergen in milk. Lactoglobulin is found in the whey of most mammals, but not in the whey of rodents, rabbits, and humans, which is one of the reasons why lactoglobulin is the main allergen in cow's milk.

Based on the above problems, there is an urgent need to develop a milk protein or composition that has the same or similar protein composition as natural milk protein and has lower allergenicity.

Contents of the invention

The purpose of this application is to provide a milk protein composition and its preparation method and application. The milk protein composition has an amino acid composition similar to that of natural milk protein and has at least lower allergenicity.

In order to solve the above technical problems, this application proposes the following technical solutions:

A first aspect of the present application provides a milk protein composition, which contains lactoglobulin peptides, which are produced by degradation of recombinantly expressed β-lactoglobulin when expressed in host cells.

The second aspect of this application provides a method for preparing the milk protein composition described in the first aspect of this application, which includes expressing the recombinantly expressed β-lactoglobulin in a host cell and recovering it from the host cell. The milk protein composition.

The third aspect of the present application provides a food composition or feed composition, which contains the milk protein composition described in the first aspect of the present application.

The beneficial effects of this application include:

(1) By exogenously expressing β-lactoglobulin in host cells, this application unexpectedly obtained a milk protein composition containing lactoglobulin peptide, which has lower allergenicity than natural milk protein.

(2) The milk protein composition provided by this application contains lactoglobulin peptides produced by the degradation of β-lactoglobulin. The fragments are smaller, and the solubility and in vitro digestibility are significantly better than that of natural β-lactoglobulin, thus having Nutritional value similar to or better than natural milk protein.

(3) What is even more surprising is that there are a variety of functional peptides in the lactoglobulin peptides produced by degradation when β-lactoglobulin is expressed in host cells, such as peptides with angiotensin-converting enzyme (ACE) inhibitory activity. , providing the milk protein composition with a blood pressure lowering effect.

(4) The milk protein composition of the present application is obtained through heterologous expression, which can eliminate environmental pollution and animal welfare problems caused by natural milk production.

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

Description of the drawings

Figure 1 is the fermentation broth supernatant of the Kluyveromyces marxianus expression strain at different times in Example 1 SDS-PAGE result chart;

Figure 2A is a physical diagram of the milk protein composition obtained in Example 2;

Figure 2B is an SDS-PAGE result diagram of the milk protein composition produced by the Kluyveromyces marxianus expression strain in Example 2;

Figure 3 is an SDS-PAGE result diagram of the milk protein composition in the fermentation broth supernatant of the Kluyveromyces marxianus expression strain at different times in Example 3;

Figure 4 is an SDS-PAGE result diagram of the milk protein composition in the fermentation broth supernatant of the Kluyveromyces marxianus expression strain at different times in Example 4;

Figure 5 is a diagram of SDS-PAGE results of milk protein compositions prepared from different Kluyveromyces marxianus expression strains in Example 5;

Figure 6 is an SDS-PAGE result diagram of the milk protein composition obtained by fermentation of Kluyveromyces lactis in Example 6;

Figure 7 is a SDS-PAGE result diagram of the milk protein composition obtained in the comparative example;

Figure 8 is a high performance liquid chromatogram of β-lactoglobulin standard;

Figure 9 is a high performance liquid chromatogram of the fermentation sample obtained in Example 2.

Detailed ways

The terminology and descriptions used herein are for the purpose of describing particular embodiments only and are not intended to limit the application. Unless otherwise defined, 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 otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

definition

Throughout this application, the terms "a" and "an" as well as "the" and similar referents refer to the singular and the plural unless otherwise indicated herein or otherwise clearly contradicted by context.

As used herein, the terms "about" and "similar to" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range may depend in part on how the value is measured or determined, or Depends on the limitations of the measurement system.

As used herein, "milk protein" refers to a protein found in milk produced by a mammal or having a sequence that is at least 50% (e.g., at least 50%, at least 55%, at least 60%, Proteins with 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 sequences. Non-limiting examples of milk proteins include beta-lactoglobulin, alpha-lactalbumin, kappa-casein, beta-casein, lactoferrin, alphaS1-casein, alphaS2-casein, and osteopontin, in addition to milk proteins. Proteins are known in the art.

"Peptide" refers to the polymerized form of amino acids, that is, two or more amino acids connected by peptide bonds. multimer. "Milk protein peptides" in this application particularly refers to a combination of peptides with different molecular weights produced by the degradation of one or more milk proteins. The milk protein peptides can be produced by the degradation of one milk protein or by multiple milk proteins. Produced by the degradation of milk proteins.

The term "recombination" is a term known in the art. When referring to a nucleic acid (e.g., a gene), the term may 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 related in whole or in part to naturally adjacent or proximal nucleic acids, a nucleic acid that is not linked to a naturally occurring nucleic acid. Nucleic acid is an operably linked nucleic acid, or a nucleic acid that does not occur naturally. When "recombinant" is used to describe a protein, it may refer, for example, to a protein that is produced in a different species or type of cell than the species or type of cell in which the protein is produced in nature.

"Host cell" refers to a specific recipient cell and the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may actually differ from the parent cell but still be included within the scope of the term "host cell" as used herein. In this application, the "host cell" particularly refers to a cell used for heterologous expression of the recombinant milk protein.

In this application, "degradation" has its general meaning, which refers to the process in which proteins are degraded into polypeptides and amino acids through the action of protein degrading enzymes. In this application, it especially refers to milk proteins expressed in host cells, which are unique in host cells. Under the action of protein degrading enzymes, it is degraded into a mixture of polypeptides with different molecular weights (ie, the milk protein peptides of the present application).

"Filamentous fungi" refers to filamentous forms of organisms from the phyla Eumycota and Oomycota. Filamentous fungi differ from yeast by their hyphal elongation during vegetative growth.

"Yeast" refers to an organism of the order Saccharomycetales. Vegetative growth of yeast is by budding/bubbing of single-cell thalli, and carbon catabolism may be fermentative.

"Bacteria culture" refers to the proliferation of bacterial cells under appropriate conditions. Non-limiting examples of suitable conditions include suitable culture media (e.g., having suitable nutrient content [e.g., suitable carbon content, suitable nitrogen content, suitable phosphorus content], suitable supplement content, suitable trace metal content, appropriate pH value of the culture medium), appropriate temperature, appropriate feed rate, appropriate pressure, appropriate oxygenation level, appropriate culture duration, appropriate culture volume (i.e., culture containing recombinant host cells base volume) and suitable culture vessels.

"Expression" refers to the process by which cells convert genetic information stored in DNA sequences into biologically active protein molecules through transcription and translation.

The term "vector" as used in this application means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Illustratively, one type of vector is a "plasmid," which generally refers to a circular double-stranded DNA loop into which additional DNA segments (foreign genes) can be ligated, and may also include linear double-stranded molecules, such as those obtained from Amplification by polymerase chain reaction (PCR) or treatment of circular plasmids with restriction enzymes yields linear double-stranded molecules. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., have complexes that function in the host cell). vector for making starting points). Other vectors can be introduced into a host cell, integrated into the host cell's genome, and thus replicated with the host genome. In addition, certain preferred vectors are capable of directing the expression of foreign genes to which they are linked. In this application, the vector to which the foreign gene is connected is called a "recombinant expression vector" (or simply "expression vector").

A first aspect of the present application provides a milk protein composition, which contains lactoglobulin peptides, which are produced by degradation of recombinantly expressed β-lactoglobulin when expressed in host cells. The inventor unexpectedly found in the study that when recombinantly expressed β-lactoglobulin is expressed in host cells, degradation will occur, thereby producing corresponding lactoglobulin peptides. The milk protein composition containing lactoglobulin peptides of the present application , less allergenic than natural milk protein.

In some embodiments of the present application, the milk protein composition further comprises recombinantly expressed β-lactoglobulin.

In some embodiments of the present application, the milk protein composition is directly expressed in the host cell.

In some embodiments of the present application, the milk protein composition is obtained by isolating and purifying the lactoglobulin peptide in a host cell.

In some embodiments of the present application, the milk protein composition contains more than 5% by weight, more than 10% by weight, more than 20% by weight, more than 30% by weight, more than 40% by weight, more than 50% by weight, more than 60% by weight , 70% by weight or more, 80% by weight or more, 90% by weight or more or 95% by weight or more of the lactoglobulin peptide.

In some embodiments of the present application, the milk protein composition contains less than 90% by weight, less than 80% by weight, less than 70% by weight, less than 60% by weight, less than 50% by weight, less than 40% by weight, and less than 30% by weight. below, 20% by weight below, 10% by weight below or below 5% by weight of the milk protein peptide.

In some embodiments of the present application, based on the total mass of the milk protein composition, the content of the recombinantly expressed β-lactoglobulin is 0-95%; the content of the lactoglobulin peptide is 5%-100% ; Preferably, the content of the recombinantly expressed β-lactoglobulin is 10%-90%; the content of the lactoglobulin peptide is 10%-90%; Preferably, the recombinantly expressed β-lactoglobulin The content of the lactoglobulin peptide is 20%-80%; preferably, the content of the recombinantly expressed β-lactoglobulin is 30%-70%; the lactoglobulin peptide The content of the recombinantly expressed β-lactoglobulin is 30%-70%; preferably, the content of the recombinantly expressed β-lactoglobulin is 40%-60%; the content of the lactoglobulin peptide is 40%-60%.

In some embodiments of the present application, the milk protein composition also includes other recombinantly expressed milk proteins, and the other recombinantly expressed milk proteins may be recombinantly expressed α-lactalbumin, recombinantly expressed κ-casein, recombinantly expressed β - at least one of casein, recombinantly expressed lactoferrin, recombinantly expressed αS1-casein, recombinantly expressed αS2-casein and recombinantly expressed osteopontin. For example, it can be: recombinant expression of β-lactoglobulin and recombinant expression of α-lactalbumin, recombinant expression of β-lactoglobulin and recombinant expression of κ-casein, recombinant expression of β-lactoglobulin Protein and recombinant expression of β-casein, recombinant expression of β-lactoglobulin and recombinant expression of lactoferrin, recombinant expression of β-lactoglobulin and recombinant expression of αS1-casein, recombinant expression of β-lactoglobulin and recombinant expression of αS2- A combination of casein, recombinantly expressed β-lactoglobulin and recombinantly expressed osteopontin, or the combination of the above two recombinantly expressed milk proteins also includes other recombinantly expressed milk proteins, for example, it can be a recombinantly expressed β-lactoglobulin. Lactoglobulin, combination of recombinant expression of lactoferrin and recombinant expression of α-lactalbumin, recombinant expression of β-lactoglobulin, combination of recombinant expression of lactoferrin and recombinant expression of κ-casein, recombinant expression of β-lactoglobulin, The combination of recombinant expression of lactoferrin and recombinant expression of β-casein, the combination of recombinant expression of β-lactoglobulin, the combination of recombinant expression of lactoferrin and the recombinant expression of αS1-casein, etc.

In some embodiments of the present application, the other recombinant expression milk proteins can be directly expressed in the host cells to obtain the milk protein composition; they can also be added to the milk protein composition through exogenous addition. among things.

In some embodiments of the present application, the milk protein composition 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 present application, the other milk protein peptides can be obtained by degradation when expressing the other recombinantly expressed milk proteins in the host cells, or can be added to the milk protein combination through exogenous addition. among things.

In some embodiments of the present application, the β-lactoglobulin can be cow, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, Mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, Wolf, fox, lion, tiger, echidna or mammoth beta-lactoglobulin. β-Lactoglobulin is a major allergen in milk produced by mammals (such as cow’s milk). The inventor found that the milk protein composition of the present application contains lactoglobulin peptides produced by the degradation of β-lactoglobulin, which can significantly reduce Sensitization by beta-lactoglobulin.

In some embodiments of the present application, the kappa-casein can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee , mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig , wolf, fox, lion, tiger, echidna or mammoth kappa-casein. The recombinant kappa-casein and the kappa-casein peptide produced by degradation when expressed in the host cell are beneficial to increase the solubility of the milk protein composition, making it easier to be absorbed by the organism.

In some embodiments of the present application, the alpha-lactalbumin can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee , mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig , wolf, fox, lion tiger, echidna or mammoth alpha-lactalbumin. The lactalbumin peptides produced by degradation of α-lactalbumin when expressed in the host cells are beneficial to further reducing the allergenicity of the milk protein composition.

In some embodiments of the present application, the β-casein can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee , mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig , wolf, fox, lion, tiger, echidna or mammoth beta-casein. The recombinant β-casein and the β-casein peptide produced by degradation when expressed in the host cell are beneficial to increase the solubility of the milk protein composition, making it easier to be absorbed by the organism.

In some embodiments of the present application, the lactoferrin can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, Mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, Wolf, fox, lion, tiger, echidna or mammoth lactoferrin. Recombinant lactoferrin and/or the lactoferrin peptide produced by degradation when expressed in the host cell has the effects of promoting the body's absorption of iron, inhibiting bacteria, improving intestinal flora, etc., making it Suitable for use as antibacterial and antiviral agents in food products such as infant formulas, functional dairy products and dietary supplements.

In some embodiments of the present application, the αS1-casein can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee , mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig , wolf, fox, lion, tiger, echidna or mammoth αS1-casein. The recombinant αS1-casein and the αS1-casein peptide produced by degradation when expressed in the host cell are beneficial to increase the solubility of the milk protein composition, making it easier to be absorbed by the organism.

In some embodiments of the present application, the αS2-casein can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee , mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig , wolf, fox, lion, tiger, echidna or mammoth αS2-casein. The recombinant αS2-casein and the αS2-casein peptide produced by degradation when expressed in the host cell are beneficial to increase the solubility of the milk protein composition, making it easier to be absorbed by the organism.

In some embodiments of the present application, the osteopontin can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, Mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, Opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna or mammoth osteopontin. Research shows that osteopontin (OPN active protein) activity can directly reach the intestines, enhance the intestinal protective barrier function, achieve systemic immune protection, and reduce biological functions such as fever and discomfort by 50%. The content of osteopontin in the umbilical cord blood plasma of newborns and the plasma of 3-month-old infants is very high, about 7-10 times that of adults, suggesting that it is closely related to the growth and development of infants and young children in early life.

In some embodiments of the present application, at least one of the recombinantly expressed milk proteins comprises a protein related to cow milk protein, sheep milk protein, horse milk protein, goat milk protein, human milk protein, buffalo milk protein, camel milk protein, yak milk protein, etc. The amino acid sequence of the milk protein, dog milk protein, elephant milk protein, whale milk protein, bear milk protein, lion milk protein or tiger milk protein is at least 80%, at least 90% or at least 95% identical.

In some embodiments of the present application, the recombinantly expressed β-lactoglobulin comprises a combination with bovine β-lactoglobulin, buffalo β-lactoglobulin, yak β-lactoglobulin, sheep β-lactoglobulin, goat β-lactoglobulin, -Lactoglobulin, camel β-lactoglobulin, horse β-lactoglobulin, dog β-lactoglobulin, elephant β-lactoglobulin, whale β-lactoglobulin, bear β-lactoglobulin, lion β- The amino acid sequence of lactoglobulin or tiger beta-lactoglobulin has an amino acid sequence that is at least 80%, at least 90% or at least 95% identical.

In some embodiments of the present application, the recombinantly expressed lactoferrin comprises a combination with bovine lactoferrin, human lactoferrin, buffalo lactoferrin, yak lactoferrin, sheep lactoferrin, goat lactoferrin, camel lactoferrin protein, equine lactoferrin, dog lactoferrin, elephant lactoferrin, whale lactoferrin, bear lactoferrin, lion lactoferrin or tiger lactoferrin having an amino acid sequence of at least 80%, at least 90%, or at least 95 % identity of the amino acid sequence.

In some embodiments of the present application, the recombinantly expressed α-lactalbumin includes a combination with bovine α-lactalbumin, human α-lactalbumin, buffalo α-lactalbumin, yak α-lactalbumin, sheep α-lactalbumin, goat α-lactalbumin, α-lactalbumin, camel α-lactalbumin, horse α-lactalbumin, dog α-lactalbumin, elephant α-lactalbumin, whale α-lactalbumin, bear α-lactalbumin, lion α-lactalbumin or tiger α-lactalbumin The amino acid sequence of lactalbumin has an amino acid sequence that is at least 80%, at least 90% or at least 95% identical.

In some embodiments of the present application, the recombinantly expressed kappa-casein comprises a combination with bovine kappa-casein, human kappa-casein, buffalo kappa-casein, yak kappa-casein, sheep kappa-casein, goat κ-casein, camel κ-casein, horse κ-casein, dog κ-casein, elephant κ-casein, whale κ-casein, bear κ-casein, lion κ-casein or tiger κ-casein The amino acid sequence of casein has an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical.

In some embodiments of the present application, the recombinantly expressed β-casein comprises a combination with bovine β-casein, human β-casein, buffalo β-casein, yak β-casein, sheep β-casein, goat Beta-casein, camel beta-casein, horse beta-casein, dog beta-casein, elephant beta-casein, whale beta-casein, bear beta-casein, lion beta-casein or tiger beta-casein The amino acid sequence of casein has an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical.

In some embodiments of the present application, the recombinantly expressed αS1-casein comprises a combination with bovine αS1-casein, human αS1-casein, buffalo αS1-casein, yak αS1-casein, sheep αS1-casein, goat αS1-casein, camel αS1-casein, horse αS1-casein, dog αS1-casein, elephant αS1-casein, whale αS1-casein, bear αS1-casein, lion αS1-casein or tiger αS1-casein The amino acid sequence of casein has an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical.

In some embodiments of the present application, the recombinantly expressed αS2-casein comprises a combination with bovine αS2-casein, human αS2-casein, buffalo αS2-casein, yak αS2-casein, sheep αS2-casein, goat αS2-casein, camel αS2-casein, horse αS2-casein, dog αS2-casein, elephant αS2-casein, whale αS2-casein, bear αS2-casein, lion αS2-casein or tiger αS2- The amino acid sequence of casein has an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical.

In some embodiments of the present application, the recombinantly expressed osteopontin comprises a combination with bovine osteopontin, human osteopontin, buffalo osteopontin, yak osteopontin, sheep osteopontin, goat osteopontin, camel osteopontin protein, horse osteopontin, dog osteopontin, elephant osteopontin, whale osteopontin, bear osteopontin, lion osteopontin or tiger osteopontin has an amino acid sequence of at least 80%, at least 90%, or at least 95 % identity of the amino acid sequence.

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

In some embodiments of the present application, the fungal cells include, but are not limited to, Ascomycotas, Basidiomycota, Zygomycota, Chythridiomycota, Oomycota ) and organisms of the phylum Glomeromycota, exemplarily, the fungus is selected from yeast or filamentous fungi.

In some embodiments of the present application, the yeast includes, but is not limited to, Candida, Cladosporium, Cryptococcus, Debaromyces, Endosporium Endomyces, Endomycopsis, Eremothecium, Hansenula, Kluyveromyces, Lipomyces, Pichia (Pichia), Rhodosporidium, Rhodotorula, Saccharomyces, Sporobolomyces, Sporidiobolus, Trichosporon , at least one species of Xanthophyllomyces, Yarrowia, and Zygosaccharomyces.

In some embodiments of the present application, the Kluyveromyces genus includes, but is not limited to, Kluyveromyces marxianus, Kluyveromyces marxianus, Kluyveromyces lactis, At least one of Kluyveromyces hubeiensis, Kluyveromyces wickerhamii and Kluyveromyces thermotolerans.

In some embodiments of the present application, the filamentous fungal cells include, but are not limited to, Acremonium spp., Aspergillus spp., Ceratospora spp., Neosartoria spp., Aureobasidium spp., Canariomyces, Trichosporon spp. Chaetomidium, Chaetomidium, Corynespora, Chrysosporium, Echinacea, Collarspora, Fusarium, Gibberellus, Humicola, Hypocrea, Lentinus, Oryzae oryzae Malbranchium, Melanocarpus, Mortierella, Mucor, Myceliophthora, Lactobacillus, Neocanthellae, Neurospora, Paecilomyces, Penicillium, Phenerochaete, Erythromyces Pythium, Ruminochytrid, Pythium, Rhizopus, Schizophyllum, Acremonium, Sporothrix, Pleurosporium, Talaromycetes, Thermoascomycota, Thermophilic Fungi , at least one species of the genus Thielavia, Trichoderma, and Trichoderma.

In some embodiments of the present application, bacteria include, but are not limited to, Firmicutes, Cyanobacteria (blue-green algae), Oscillatoriophcideae, Bacillales, Lactobacilli Members of the order Lactobacillales, Oscillatoriales, Bacillaceae, Lactobacillaceae, and any of the following genera, and their derivatives and hybrids: Acinetobacter, Acetobacter (Acetobacter) (e.g., Acetobacter suboxydans, Acetobacter xylinum), Actinoplane (e.g., Actinoplane missouriensis), Arthrospira Arthrospira (e.g., Arthrospiraplatensis, Arthrospira maxima), Bacillus (e.g., Bacillus cereus, Bacillus coagulans) coagulans), Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis), Escherichia (e.g., Escherichia coli), Lactobacillus, (e.g., Lactobacillus acidophilus, Lactobacillus bulgaricus), Lactococcus genus (e.g., Lactococcus lactis, Lactococcus lactis, Lancefield group N, Lactobacillus lactis) Lactobacillus reuteri, Leuconostoc (e.g., Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostoc mesenteroides) ), Micrococcus (e.g., Micrococcus lysodeikticus), Rhodococcus (e.g., Rhodococcus opacus, Rhodococcus opacus strain PD630), Spirulina, Streptomyces Streptococcus (e.g., Streptococcus cremoris, Streptococcus lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilus), Streptomyces (Streptomyces) (such as Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivaceus ( Streptomyces olivochromogenes), Streptomyces rubiginosus), and Xanthomonas (e.g., Xanthomonas campestris) (Xanthomonas campestris)).

“Protozoa” in this application refers to organisms of the phylum Protozoa, which are lower single-celled eukaryotes. For example, it may be Tetrahymena thermophila, Tetrahymena hegewischi, Tetrahymena hyperangularis, Tetrahymena malaccensis, Tetrahymena colourspot, Tetrahymena pyriformis or Tetrahymena bulimia.

In some embodiments of the present application, the fungal cell is selected from at least one of Kluyveromyces, Pichia, Aspergillus, and Fusarium; the bacterial cell is selected from Lactobacillus or Bacillus At least one species of the genus.

In some embodiments of the present application, the Bacillus genus includes Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus, and At least one species of Bacillus subtilis.

In some embodiments of the present application, the Lactobacillus genus includes at least one of Lactobacillus acidophilus and Lactobacillus bulgaricus.

In some embodiments of the present application, the host cell is selected from at least one of Kluyveromyces marxianus or Kluyveromyces lactis. The inventor found that expressing recombinant β-lactoglobulin in Kluyveromyces marxianus or Kluyveromyces lactis can obtain more degradation products—lactoglobulin peptides. The inventors also found that there are more active peptides in the lactoglobulin peptides obtained using the host cells.

The active peptides mentioned in this application refer to peptide fragments with biological functions, such as peptide fragments with ACE inhibitory activity.

In some embodiments of the present application, the milk protein composition further includes host cell proteins.

In some embodiments of the present application, the milk protein composition contains more than 5% by weight, more than 10% by weight, more than 20% by weight, more than 30% by weight, more than 40% by weight, more than 50% by weight, more than 60% by weight , 70% by weight or more, 80% by weight or more or 90% by weight or more of the host cell protein.

In some embodiments of the present application, the milk protein composition contains less than 90% by weight, less than 80% by weight, less than 70% by weight, less than 60% by weight, less than 50% by weight, less than 40% by weight, and less than 30% by weight. below, 20 wt% below, 10 wt% below, 5 wt% below, or 1 wt% below the host cell protein.

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

In some embodiments of the present application, the host cell protein is at least one of the extracellular secreted proteins of Kluyveromyces marxianus or Kluyveromyces lactis. The extracellular secreted protein is biosafe, edible, and can be used as a nutritional component supplemented into the milk protein composition.

In some embodiments of the present application, based on the total weight of the milk protein composition, the milk protein composition includes 1-40% host cell protein, 5-90% lactoglobulin peptide and 5-90% recombinant β-lactoglobulin. Preferably, the milk protein composition contains 1-20% host cell protein, 40-60% lactoglobulin peptide and 20-55% recombinantly expressed β-lactoglobulin.

Specifically, the preparation method of the milk protein composition described in the first aspect of the application provided in this application may include:

The expression vector capable of expressing the β-lactoglobulin is introduced into the host cell using known methods, such as lipofection, electrotransfection, transformation, etc.; in a cell suitable for the expression of the β-lactoglobulin Culturing the host cell under conditions to allow the host cell to express the β-lactoglobulin; using methods known in the art, such as size exclusion chromatography, ultrafiltration through a membrane or density centrifugation based on its molecular weight, Isolate proteins or peptides. Proteins or peptides can also be separated based on their surface charge by isoelectric precipitation, anion/cation exchange chromatography. Proteins or peptides can also be separated by ammonium sulfate precipitation based on their solubility. Hydrophobic interaction chromatography can also be used to separate proteins or peptides by their affinity to another molecule. Affinity chromatography can also exploit the binding properties of the stationary phase to separate proteins or peptides. The expressed β-lactoglobulin and/or the lactoglobulin peptides produced by its degradation are recovered from the host cells, thereby obtaining the milk protein composition of the present application.

In some embodiments of the present application, the recovery of the milk protein composition from the host cell further includes a separation and purification step. The separation and purification can be a method known in the art, such as size exclusion chromatography, membrane filtration. method, isoelectric point precipitation method, anion/cation exchange chromatography, ammonium sulfate precipitation method, affinity chromatography, etc., this application is not limited here. The separation and purification step can obtain lactoglobulin peptide with higher purity.

It should be noted that due to different expression vectors or host cells used, those skilled in the art can select conditions suitable for the expression of β-lactoglobulin according to specific circumstances, and this application is not limited here.

In some embodiments of the present application, the fungal cell is selected from at least one member of the genus Kluyveromyces, Pichia, Aspergillus, and Fusarium.

In some embodiments of the present application, the Kluyveromyces species comprise Max. Kluyveromyces marxianus, Kluyveromyces marxianus var., Kluyveromyces lactis, Kluyveromyces hubeiensis, Kluyveromyces wickerhamii and resistant At least one species of Kluyveromyces thermotolerans.

In some embodiments of the present application, the milk protein composition can be expressed in Kluyveromyces marxianus or Kluyveromyces lactis. The inventor unexpectedly found that using the host cell Expressing the β-lactoglobulin can obtain a higher yield of lactoglobulin peptides, which contain more functional peptides. In addition, the host cell protein is biosafe, edible, and can Supplemented into the milk protein composition as a nutritional component.

In some embodiments of the present application, the time for expressing the one or more recombinantly expressed milk proteins in the host cell is 12-168 hours, preferably 36-84 hours. The inventor found in the research that in the milk protein composition of the present application, the content of milk protein peptides is related to the expression time. The longer the expression time, the higher the content of milk protein peptides produced; if the expression time is too long, the host cells are prone to Aging reduces the production efficiency of milk protein compositions.

In some embodiments of the present application, the temperature at which the one or more recombinant expression milk proteins are expressed in the host cell is 20-45°C, preferably 25-40°C. The inventor found that within the temperature range, it is beneficial for the host cells to efficiently produce the milk protein composition.

Strains used in this application include:

(1) Kluyveromyces marxianus (K.marxianus) CJA0001, deposited in the China Type Culture Collection Center, the deposit number is CCTCC No: M20211601, the deposit date is December 13, 2021, and the deposit address is Wuhan University, Wuhan, China , the classification is named Kluyveromyces marxianus CJA0001.

(2) Kluyveromyces marxianus (K.marxianus) CJA0002, deposited in the China Type Culture Collection Center, the deposit number is CCTCC No: M20211602, the deposit date is December 13, 2021, and the deposit address is Wuhan University, Wuhan, China , the classification is named Kluyveromyces marxianus CJA0002.

(3) Kluyveromyces marxianus (K.marxianus) CJA0003, deposited in the China Type Culture Collection Center, the deposit number is CCTCC No: M20211603, the deposit date is December 13, 2021, and the deposit address is Wuhan University, Wuhan, China , classified as Kluyveromyces marxianus CJA0003.

(4) Pichia pastoris, Pichia pastoris collected by the applicant himself, the collection place is Shangri-La City, Yunnan Province, and the collection time is December 2021.

(5) Kluyveromyces marxianus was purchased from the China Industrial Microbial Culture Collection Center (CICC), numbered CICC 1953, and purchased in September 2020.

(6) Rubybacter lactis was purchased from the China Industrial Microbial Culture Collection Center (CICC), with the number CICC 32428, and the purchase time was May 2021.

The milk protein composition and its preparation method of the present application will be described below through specific examples.

Example 1 Preparation of milk protein composition

This example uses K. marxianus CJA0001 (China Type Culture Collection Center, CCTCC No: M20211601) to express β-lactoglobulin. The steps are as follows:

1: The bovine β-lactoglobulin gene fragment (NCBI Gene ID: 113901792) was synthesized at GenScript Biotechnology Co., Ltd. and named β-Lg. Using β-Lg as a template, obtain the target gene through PCR amplification (upstream primer F1: 5'-GGCTGAAGCTTTGATCGTTACCCAAACTATG-3'SEQ ID No: 1, 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'-GTAACGATCAAAGCTTCAGCCTCTTTTCTC-3'SEQ ID No: 4). The target gene is connected to the commercial pKLAC1 vector through seamless assembly to obtain the expression vector pKLAC1-β-Lg.

2: Use the fast endonuclease Sac II of New England Biolabs, Inc. (NEB) to digest plasmid pKLAC1-β-Lg to obtain the homologous recombination fragment of linearized pKLAC1-β-Lg, and use electroporation (0.1cm electroporation cup, 1.5-2.5kV voltage, time selection is ~5ms) method to transform linearized pKLAC1-β-Lg into Kluyveromyces marxianus CJA0001 (deposit number CCTCC No: M20211601) to construct K.marxianus::pKLAC1-β-Lg expression strain, At the same time, the control strain K.marxianus::pKLAC1 was constructed in a medium with acetamide as the only nitrogen source (1.17g of yeast basic carbon source, 3mL of 1mol/L KH ₂ PO ₄ -K ₂ HPO ₄ buffer with pH=7.0 (final concentration 30mmol/L), 2g agar powder, add acetamide after sterilization to a final concentration of 5mmol/L) for screening verification. Cultivate the above expression strain and control strain respectively in fermentation medium (peptone 20g/L, yeast powder 10g/L, glucose 10g/L, galactose 10g/L); adjust the pH of the medium to 5.5-6.5, and ferment at 30°C At 72h, galactose was added during the process to induce protein expression. The fermentation broth was taken at 0, 12, 24, 36, 48 and 60 hours respectively.

3: Centrifuge each fermentation broth at 12000g, collect the supernatant, select a separation gel concentration of 15% according to the size of the target protein, and prepare it using the SDS-PAGE Denaturing Acrylamide Gel Rapid Preparation Kit (C631100) (Sangon Bioengineering ( Shanghai) Co., Ltd.), please see the instructions on the attached page for the detailed formula. Pick Put 40 μL of the supernatant sample into a centrifuge tube, add 10 μL of 5× protein loading buffer, mix well, and heat with boiling water for 10 minutes to denature the protein sample. Add 10 μL of sample and 5 μL of Maker to each gel well. The electrophoresis voltage is constant voltage 110V and the time is 80-90 minutes. After electrophoresis, the protein gel was stained and destained. The protein staining results of the supernatant of the expression strain are shown in Figure 1, which shows the production of exogenous proteins (i.e., β-lactoglobulin) and degradation products (lactoglobulin peptides); the proportions were analyzed by Image J. As the fermentation time increases, , the ratio of β-lactoglobulin to β-lactoglobulin peptide content in the fermentation broth was reduced from 9:1 to 2:3. It can also be seen from Figure 1 that after 24 hours, the ratio of β-lactoglobulin to β-lactoglobulin peptide is about 3:2. After that, β-lactoglobulin continues to be generated and degraded, until about 60 hours , the ratio of β-lactoglobulin and β-lactoglobulin peptide is roughly stable at 2:3. There was no exogenous protein or degradation product production in the control strain, and the protein staining results are not shown.

4: Extraction of milk protein composition: Use membrane filtration method to purify the fermentation broth supernatant. Filtration conditions, first 2-stage filter element: precision filter element + NF nanofiltration membrane; NF nanofiltration membrane specification: NF-4040; filtration precision: 0.001μm; molecular cutoff value: ≤200D; water inlet pressure: 0.2-0.3Mpa; working pressure :0.5-0.7Mpa. During the concentration process, the nanofiltration membrane is backwashed every 10 minutes. After the concentration is completed, the concentrated liquid is recovered, weighed and recorded to obtain the milk protein composition of the present application, which is stored in a 4°C cold storage for later use.

Example 2: Expanding fermentation system to prepare milk protein composition

The 50L fermentation process is as follows:

1: Shake flask seed culture, take 1.5 mL of the K.marxianus::pKLAC1-β-Lg bacterial liquid cultured for 24 hours in Example 1 and inoculate it into 100 mL of liquid seed culture medium (peptone 20g/L, yeast powder 10g/L , glucose 10g/L, galactose 10g/L) in a 250mL Erlenmeyer flask, culture at 30°C, 220rpm for 12h to obtain the seed liquid.

2: Secondary seed culture, prepare 200mL seed liquid and connect it to the secondary seed culture medium (5L tank), 30℃, 220rpm, culture for 6-8h, OD reaches about 10-12, and obtain the secondary seed liquid.

3: Fermentation culture, add the secondary seed liquid into 50L of batch fermentation medium (20g/L peptone, 10g/L yeast powder, 10g/L glucose, 10g/L galactose) according to the inoculum amount of 10%. Batch culture is carried out in the fermentation tank with an initial liquid volume of 22-25L. The conditions are temperature 30°C, pH=5.5, initial rotation speed 300r/min, air flow ^2-3m3 /h, and tank pressure 0.04-0.055MPa.

4: Feeding process, sugar supplementation: starting from the bottom sugar consumption, add glucose (60%) at a rate of 6-15g/L/h until the end of fermentation; at the same time, add galactose to induce protein expression; supplement ammonium sulfate for 8 hours Afterwards, continuous feeding is carried out. The ammonia concentration is controlled between 0.1% and 0.2% based on the process ammonia measurement results. The addition is stopped 1 hour before the tank is placed.

5: Harvest the product. In the fermentation tank culture experiment, samples were taken every 4 hours to detect OD, and the samples were retained. After entering the stable period, the fermentation was completed at 60 hours and placed in the tank to obtain the fermentation liquid. The fermentation broth supernatant was purified using membrane filtration method. Filtration conditions, first 2-stage filter element: precision filter element + NF nanofiltration membrane; NF nanofiltration membrane specification: NF-4040; filtration precision: 0.001μm; molecular cutoff value: ≤200D; water inlet pressure: 0.2-0.3Mpa; working pressure :0.5-0.7Mpa. During the concentration process, the nanofiltration membrane is backwashed every 10 minutes. After the concentration is completed, Recover, weigh and record the quality of the concentrated liquid to obtain the milk protein composition of the present application, as shown in Figure 2A, and then store it in a 4°C cold storage for later use.

6: Protein detection. The milk protein composition was tested by SDS-PAGE. The results are shown in Figure 2B. It can be seen that under 50L culture conditions, β-lactoglobulin was partially hydrolyzed to produce lactoglobulin peptides. After purification by AKTA ion chromatography, hydrolyzed lactoglobulin peptides can also be seen.

Example 3: Expanding fermentation system to prepare milk protein composition

The 1T fermentation process is as follows:

1: First-level shake flask seed culture: 2 mL of K.marxianus::pKLAC1-β-Lg glycerol bacteria from Example 1 was added to 200 mL of LYPD medium (500 mL shake flask), 30°C, 220 rpm, cultured for 16 hours, and the OD reached about 12. Obtain first-level seed liquid.

2: Secondary seed shake flask culture: 7mL of primary seed liquid is connected to 700mL of secondary shake flask culture medium (20g/L peptone, 10g/L yeast powder, 10g/L glucose, 10g/L galactose), 30°C. 220rpm, culture for 16h, OD reaches about 12, and obtain the secondary seed liquid.

3: Seed tank (150L) culture: 2.8L secondary seed liquid is connected to 40L fermentation medium (40L/150L seed tank (g), peptone 20g/L, yeast powder 10g/L, glucose 10g/L, galactose 10g /L), 30°C, initial rotation speed 300 rpm, pH = 5.5, air volume 40L/min, dissolved oxygen and rotation speed are linked, DO is controlled at 30%, culture for 6-8 hours, OD reaches about 12, and the seed liquid is obtained.

4: Fermentation culture: 40L seed liquid is connected to 400L fermentation medium (1000L fermentation tank), 30°C, initial speed 300rpm, pH=5.5, air volume 400L/min, dissolved oxygen and speed are linked, and DO is controlled at 30%. Add sugar according to pH feedback before 16 hours; add sugar and galactose for induction according to DO feedback after 16 hours; culture for 64 hours to obtain fermentation broth. If the dissolved oxygen supply is insufficient, the subsequent air volume can be increased to 1000L/min.

5: Harvest the product and use membrane filtration method to purify the fermentation broth supernatant. Filtration conditions, first 2-stage filter element: precision filter element + NF nanofiltration membrane; NF nanofiltration membrane specification: NF-4040; filtration precision: 0.001μm; molecular cutoff value: ≤200D; water inlet pressure: 0.2-0.3Mpa; working pressure :0.5-0.7Mpa. During the concentration process, the nanofiltration membrane is backwashed every 10 minutes. After the concentration is completed, the concentrated liquid is recovered, weighed and recorded to obtain a milk protein composition, which is stored in a 4°C cold storage for later use.

6: Protein detection, the milk protein composition was tested by SDS-PAGE. The results are shown in Figure 3. It can be seen that under 1T culture conditions, β-lactoglobulin and milk protein hydrolyzate product milk protein peptide can also be expressed in large quantities.

Example 4: Prolonging fermentation time to prepare milk protein composition

Except that the fermentation culture time in step 4 was extended to 108 hours, the rest was the same as in Example 3. The supernatant of the fermentation broth at 12h, 36h, 60h, 72h, 84h, 96h, and 108h was used to prepare milk protein compositions using the same method as Example 3. The SDS-PAGE results are shown in Figure 4. From the figure, β- Lactoglobulin and milk Production of globin peptides.

Example 5: Different K. marxianus expressed milk protein compositions

Referring to the method of Example 1, linearized pKLAC1-β-Lg was transformed into Kluyveromyces marxianus of different origins: strain 1 K. marxianus CJA0002 (CCTCC No: M20211602), strain 2 K. marxianus CJA0003 (CCTCC No: M20211603), strain 2 3K. marxianus (CICC 1953), obtain an expression strain, and use the same conditions as in Example 1 for fermentation, detection, and purification of the fermentation product to obtain a milk protein composition.

The SDS-PAGE detection results of the milk protein compositions in the fermentation broth of different expression strains are shown in Figure 5. The results show that β-lactoglobulin expressed in different K. marxianus can be hydrolyzed to produce lactoglobulin peptides.

Example 6: Preparation of milk protein composition using Kluyveromyces lactis

Referring to the method of Example 1, linearized pKLAC1-β-Lg was transformed into Kluyveromyces lactis (Kluyveromyces lactis, deposit number CICC 32428), K.lactis::pKLAC1-β-Lg was constructed, and the control strain K.lactis was constructed at the same time: :pKLAC1. Cultivate Kluyveromyces lactis in a fermentation medium (peptone 20g/L, yeast powder 10g/L, glucose 10g/L, galactose 10g/L); adjust the pH of the medium to 5.5-6.5, after 25-30 The fermentation was carried out for 72 hours at ℃, and galactose was added during the fermentation process to induce protein expression, and the fermentation broth was obtained.

The fermentation broth was purified and detected by SDS-PAGE using the same method as in Example 1. The SDS-PAGE results of the milk protein compositions of the control strain and the transformed strain are shown in Figure 6. It can be seen that lactoglobulin peptides can also be obtained by using Kluyveromyces lactis to exogenously express β-lactoglobulin.

Comparative Example 1: Preparation of milk protein composition using Pichia pastoris

Using the β-Lg of Example 1 as a template, PCR amplified the lactoglobulin fragment (F1: 5'-AAAGAGAGGCTGAAGCTT ACTTGATCGTTACCCAAACTAT-3'SEQ ID No: 5, R1: 5'-AGGCGAATTAATTCGCGGCCTCAAATGTGACATTGTTCTT-3'SEQ ID No: 6) , the PCR product was ligated with the pPIC9k plasmid digested by SnaBI/NotI-HF to construct the Pichia pastoris expression plasmid pPIC9k-β-Lg. The pPIC9k-β-Lg plasmid linearized by SalI enzyme was transformed into Pichia pastoris GS115 to construct P.pastoris::pPIC9k-β-Lg. At the same time, the control strain P.pastoris::pPIC9k was constructed. The strains obtained above were successively used by BMGY The culture medium (Brand: Solarbio; Product No.: LA5250) and BMMY culture medium (Brand: Solarbio; Product No.: LA5250) are used for fermentation, and different volumes of methanol are added every day to keep the final content of methanol in the fermentation broth at 1%, and the fermentation is continued for 5 sky. Then, the supernatant was collected by centrifugation, concentrated by centrifugation, and quantitatively analyzed using SDS-PAGE.

The fermentation broth was purified and detected by SDS-PAGE using the same method as in Example 1. The results are shown in Figure 7. Lane 1 in the figure is the control strain, and lanes 2-9 are the constructed expression strains. It was found that using Pichia pastoris Lactoglobulin peptide cannot be obtained through fermentation. In order to verify this conclusion, this example was repeated many times, and the experimental results were consistent.

Effect verification:

Experimental Example 1: Identification of composition ingredients

In this experimental example, the HPLC-MS protein gel sequencing method was used to identify the components of the milk protein peptides in the milk protein compositions of Examples 1, 5, and 6.

1: Protein gel preparation

The fermentation broth supernatant was subjected to SDS-PAGE analysis, and gel strips of different molecular weight sizes were pretreated. First, the gel pieces were immersed in a buffer of 50 mmNH ₄ HCO ₃ and 50% (v/v) acetonitrile (ACN) until Decolorize; incubate with 100% ACN for 5 minutes and then remove ACN to complete the first dehydration, and add 10mM dithiothreitol (DTT) and incubate at 5°C for 60 minutes for the first hydration; the hydrated gel The strip was dehydrated again using 100% ACN for the second time, and incubated with 55mM iodoacetamide (IAA) at room temperature for 45 minutes in the dark to perform the second hydration; the hydrated strip was again dehydrated using 50mm NH ₄ HCO ₃ and 100 %ACN performs the third dehydration.

2: Protein enzymatic hydrolysis

(1) Glue cutting: Cut the above-mentioned rubber strips into rubber particles of about 1mm;

(2) Decolorization: Add 1 mL of decolorization solution, vortex for 10 seconds, decolorize at 37°C for 30 minutes, centrifuge briefly, blot dry, and repeat decolorization several times until the blue color is removed;

(3) Clean the gel particles: Wash the gel 3 times, add 1mL of ultrapure water each time, and vortex for 1 minute;

(4) Dehydration: Add 500 μL acetonitrile to dehydrate until the micelle completely turns white, repeat once;

(5) Thiol reduction: Aspirate off the acetonitrile, open the lid of the centrifuge tube and place it in a clean bench for 10 minutes to air-dry the acetonitrile. Add 10mM DTT until the liquid is submerged above the colloidal particles (twice the volume of colloidal particles), and bathe in a 56°C water bath for 1 hour;

(6) Thiol sealing: After cooling to room temperature, blot dry, quickly add 55mM IAM until the liquid is above the colloidal particles (twice the volume of colloidal particles), and place in a dark room for 45 minutes;

(7) Cleaning: Aspirate the liquid, wash twice with 500 μL decolorizing solution and once with pure water;

(8) Dehydration: Aspirate the liquid, add 500 μL acetonitrile, vortex for 5 minutes, aspirate the acetonitrile, open the centrifuge tube cover and place it on a clean bench for 10 minutes to air dry thoroughly;

(9) Add enzyme: Dilute the enzyme solution (already packed 1 μg/μL Trypsin) to 0.01 μg/μL with 25mM NH ₄ HCO ₃ , and use the enzyme solution to completely cover the gel particles;

(10) Colloidal particle absorption: rest at 4°C or on ice for 30 minutes;

(11) Add buffer solution: After the colloidal particles have swelled, add the corresponding buffer solution until the colloidal particles are submerged;

(12) Incubate at 37°C overnight;

(13) Gradient elution of peptides: add 5 times the volume of 50% ACN, vortex for 5 minutes, centrifuge at 5,000g for 1 minute, transfer the supernatant to a new centrifuge tube with the same number; add 5 times the volume of 100% ACN, vortex After vortexing for 5 minutes, centrifuge at 5,000g for 1 minute, and transfer the supernatant to a new centrifuge tube with the same number. Then, centrifuge the obtained supernatant at 25,000g for 5 minutes and take the supernatant;

(14) Drainage: Freeze and drain the extracted peptide solution.

3: LC-MS/MS: Re-dissolve the dried peptide sample with mobile phase A (2% ACN, 0.1% FA (formic acid)), centrifuge at 20,000g for 10 minutes, and take the supernatant for injection. Separation was performed by Thermo UltiMate 3000UHPLC. The sample is first enriched and desalted in the trapping column, and then enters the self-packing C18 column (internal diameter 75 μm, column size 3 μm, column length 25 cm), and is separated at a flow rate of 300 nL/min through the following effective gradient: 0 to 5 min, 5% Mobile phase B (98% ACN, 0.1% FA); 5 to 45 minutes, mobile phase B increases linearly from 5% to 25%; 45 to 50 minutes, mobile phase B increases from 25% to 35%; 50 to 52 minutes, mobile phase B increased from 35% to 80%; 52-54min, 80% mobile phase B; 54-60min, 5% mobile phase B. The nanoliquid phase separation end is directly connected to the mass spectrometer.

Peptides separated by liquid chromatography were ionized by a nanoESI source and then detected by a tandem mass spectrometer Q-Exactive HF X (Thermo Fisher Scientific, San Jose, CA) in DDA (data-dependent acquisition) mode. Main parameter settings: ion source voltage is set to 1.9kV, MS1 scanning range is 350~1,500m/z; resolution is set to 60,000; MS2 starting m/z is fixed at 100; resolution is 15,000. MS2 fragmentation ion screening conditions: the first 30 precursor ions with a charge of 2 ⁺ to 6 ⁺ and a peak intensity exceeding 10,000. The ion fragmentation mode was HCD, and fragment ions were detected in Orbitrap. Dynamic exclusion time is set to 30 seconds. AGC settings are: MS13E6, MS21E5.

The mass spectrometry peptide identification results from the β-lactoglobulin gel segment and lactoglobulin peptide gel segment in SDS-PAGE analysis are shown in Table 1 and Table 2 respectively.

Table 1: Mass spectrometry identification of β-lactoglobulin

Table 2: List of lactoglobulin peptides identified by mass spectrometry 2

Table 1 shows that in the β-lactoglobulin gel segment, the amino acid sequences of the polypeptides with high ion scores among the detected polypeptides are shown in SEQ ID No: 7 to 10, among which SEQ ID No: 7 has been queried the most times. . Table 2 shows that in the lactoglobulin peptide gel segment, the amino acid sequences of the polypeptides with high ion scores among the detected polypeptides are shown in SEQ ID Nos: 7, 10 and 11 respectively, and SEQ ID Nos: 7 to 11 are all It comes from β-lactoglobulin, not bacterial protein, or other metabolite proteins. Therefore, it can be seen that the composition ratio of each polypeptide fragment contained in the lactoglobulin peptide decomposed during the expression process is compared with the complete form that existed before decomposition. There are significant differences in expression, making the two show unexpected differences in various activity assays.

In addition, the components of milk protein peptides in the milk protein compositions of Example 1 and Example 5 were compared respectively. The results showed that different Kluyveromyces marxianus expressed exogenous β-lactoglobulin and were able to hydrolyze it to produce milk protein peptides. , the lactoglobulin peptide composition is close, indicating that different host cells of the present application can produce the lactoglobulin peptide of the present application.

In addition, it can be seen from the test results that, in addition to the main polypeptides with high ion scores, the milk protein compositions in the examples of the present application also contain a certain amount of host cell proteins.

When the applicant conducted activity analysis on the milk protein composition, unexpectedly, the applicant found that the milk protein composition of the present application has ACE inhibitory activity. It can be judged from this that the milk protein composition of the present application has higher nutritional value.

Experimental Example 2: Antigenicity Analysis of Milk Protein Composition

The antigenicity of β-LG in the 12-hour, 36-hour, and 72-hour fermentation broth samples of Example 1 was evaluated using an ELISA detection kit (product number: ml036565, manufacturer: Shanghai Enzyme Biotechnology Co., Ltd.).

Sample Preparation:

a: Hitrap Capto Q anion exchange chromatography separation and purification of β-lactoglobulin

a1: Take 10 mL of fermentation broth supernatant, centrifuge at 12000 rpm/min for 5 min, take the supernatant, adjust the pH to 7.5/8.5 with NaOH, and filter with a 0.45 μm sterile needle filter.

a2: The AKTAPure 150 protein purification system was tested on the machine. Use ultrapure water to flush the entire flow path for 5 column volumes at a flow rate of 5mL/min; balance 5 column volumes with buffer A at a flow rate of 5mL/min; before loading the sample. Buffer A cleans the loop, and the sample loading flow rate is 3mL/min; use buffers containing 100mM, 200mM, 300mM, 400mM, and 500mM NaCl to perform staged elution, with a flow rate of 5mL/min. Collect the elution peaks at each stage and use SDS-PAGE detects the molecular weight and purity of the collected proteins; wash 5 column volumes with ultrapure water, and then wash 5 column volumes with 20% ethanol at a flow rate of 5 mL/min. After optimization, the loading volume of Hitrap Capto Q 5mL anion exchange chromatography column to purify the protein was 2mL, and buffers containing 100-600mM NaCl were used for elution. Among them, 300mM NaCl was eluted to obtain β-lactoglobulin.

b: Membrane filtration separation and purification of β-lactoglobulin peptide

b1 Ultrafiltration: Take 500 mL of the fermentation broth of K.marxianus::pKLAC1 and K.marxianus::pKLAC1-β-Lg in Example 1, centrifuge at 12000rpm/min for 5 minutes, and take the supernatant. Start the ultrafiltration machine and use clean water to flush the ultrafiltration machine forward and back until the water is clear and has no peculiar smell. After pre-rinsing the ultrafiltration machine, add the above supernatant. Start the ultrafiltration machine and booster pump. After normal liquid discharge, adjust the opening of the concentrated water return valve to control the concentrated water return pressure at 0.04-0.08Mpar. During the concentration process, the ultrafiltration membrane is backwashed every 10 minutes (backwash time is set to 40s). Ultrafiltration membrane precision: 0.01μm-0.1μm; molecular cutoff value: ≤10000DA. After the concentration is completed, recover, weigh and record the quality of the concentrate and permeate, and store them in a 4°C cold storage for later use.

b2 Nanofiltration: Take the permeate of K.marxianus::pKLAC1 and K.marxianus::pKLAC1-β-Lg collected in b1 above and perform nanofiltration purification. Inject an appropriate amount of pure water into the concentrated water tank, start the nanofiltration machine, and use clean water to flush the nanofiltration machine forward and back until the water is clear and has no peculiar smell. After pre-rinsing the nanofiltration machine, add b1 permeate. Start the nanofiltration machine and booster pump. After normal liquid discharge, adjust the opening of the concentrated water return valve to control the concentrated water return pressure at 0.04-0.08Mpar. During the concentration process, the nanofiltration membrane is backwashed every 10 minutes. Nanofiltration membrane precision: 0.001μm; molecular cutoff value: ≤200DA. After the concentration is completed, recover and weigh the concentrated liquid to record the quality, and store it in a 4°C cold storage for later use.

b3: Subtract K from the concentration of K.marxianus::pKLAC1-β-Lg nanofiltration concentrate. marxianus::pKLAC1 nanofiltration concentrate concentration as K. marxianus expressing β-lactoglobulin peptide produced by hydrolysis of β-lactoglobulin.

c: Membrane filtration separation and purification of β-lactoglobulin and β-lactoglobulin peptide composition

c1 Ultrafiltration: Take 500 mL of the fermentation broth of K.marxianus::pKLAC1 and K.marxianus::pKLAC1-β-Lg from Example 1, centrifuge at 12,000 rpm/min for 5 min, and take the supernatant. Start the ultrafiltration machine and use clean water to flush the ultrafiltration machine forward and back until the water is clear and has no peculiar smell. After pre-rinsing the ultrafiltration machine, add the above supernatant. Start the ultrafiltration machine and booster pump. After normal liquid discharge, adjust the opening of the concentrated water return valve to control the concentrated water return pressure at 0.04-0.08Mpar. During the concentration process, the ultrafiltration membrane is backwashed every 10 minutes (backwash time is set to 40s). Ultrafiltration membrane precision: 0.01μm-0.1μm; molecular cutoff value: ≤20000DA. After the concentration is completed, recover, weigh and record the quality of the concentrate and permeate, and store them in a 4°C cold storage for later use.

c2 Nanofiltration: Take the K.marxianus::pKLAC1 and K.marxianus::pKLAC1-β-Lg permeate collected in c1 above and perform nanofiltration purification. Inject an appropriate amount of pure water into the concentrated water tank, start the nanofiltration machine, and use clean water to flush the nanofiltration machine forward and back until the water is clear and has no peculiar smell. After pre-rinsing the nanofiltration machine, add b1 permeate. Start the nanofiltration machine and booster pump. After normal liquid discharge, adjust the opening of the concentrated water return valve to control the concentrated water return pressure at 0.04-0.08Mpar. During the concentration process, the nanofiltration membrane is backwashed every 10 minutes. Nanofiltration membrane precision: 0.001μm; molecular cutoff value: ≤200DA. After the concentration is completed, recover and weigh the concentrated solution, record the quality, and store it in a 4°C cold storage spare.

c3: The sum of the concentrations of K.marxianus::pKLAC1-β-Lg ultrafiltration and nanofiltration concentrates minus the sum of the concentrations of K.marxianus::pKLAC1 ultrafiltration and nanofiltration concentrates is used as K.marxianus expression β-lactosphere Proteolytically produced beta-lactoglobulin and beta-lactoglobulin compositions.

d: Membrane filtration separation and purification of milk protein composition

d1: Use membrane filtration method to purify the fermentation supernatant. Nanofiltration conditions, first 2-stage filter element: precision filter element + NF nanofiltration membrane; NF nanofiltration membrane specification: NF-4040; filtration precision: 0.001μm; molecular cutoff value: ≤200D; water inlet pressure: 0.2-0.3Mpa; working Pressure: 0.5-0.7Mpa. During the concentration process, the nanofiltration membrane is backwashed every 10 minutes. After the concentration is completed, the concentrated solution is recovered, weighed and recorded, and stored in a 4°C cold storage for later use.

The milk protein composition prepared on d1 includes β-lactoglobulin, β-lactoglobulin peptide and K. marxianus extracellular secreted protein.

After separation and purification, β-lactoglobulin (A) fermented for 72 hours in Example 1; β-lactoglobulin peptide (B); β-lactoglobulin and β-lactoglobulin peptide composition (C); milk protein Composition (D, comprising 35% by weight of recombinant β-lactoglobulin, 60% by weight of β-lactoglobulin peptide and 5% by weight of Kluyveromyces marxianus extracellular fraction Secreted protein); Example 1 Milk protein composition fermented for 36 hours (G, comprising 50% by weight of recombinant β-lactoglobulin, 45% by weight of β-lactoglobulin peptide and 5% by weight of Kluyveromyces marxianus Extracellular secreted protein), the milk protein composition (H) fermented for 12 hours in Example 1, including 85% by weight of recombinant β-lactoglobulin, 10% by weight of β-lactoglobulin peptide and 5% by weight of Kluyveromyces marxianus Extracellular secreted protein); the milk protein composition of Comparative Example 1 (F); commercial β-lactoglobulin (E) (manufacturer: Sigma-Aldrich, product number: L0130).

Antigenicity analysis

The analysis method is: take the fermentation sample obtained in the above Example 2, prepare it to a concentration of 1g/L, and use commercial β-lactoglobulin (E) as a standard. Then Kjeldahl nitrogen determination, HPLC (high performance liquid chromatography) and Elisa (enzyme-linked immunosorbent) were used to detect the protein content in the two groups of samples. Based on the different principles of different detection methods, comparing the protein concentrations detected by different methods can Compare the allergenicity of each group of samples. The specific method is as follows:

a: Elisa detection

a1: Use Elisa kit (product number: ml036565, manufacturer: Shanghai Enzyme Biotechnology Co., Ltd.), in which the wells of the microtiter plate are pre-coated with bovine β-LG-specific antibodies. Different samples obtained using the above 4 methods were added to the wells and combined with specific antibodies and horseradish peroxidase (HRP)-conjugated specific antibodies.

a2: The antibody-antigen-HRP-conjugated antibody complex and 3,3',5,5'-tetramethylenebenzidine (TMB) substrate solution will turn blue after adding the stop solution, and then turn to yellow . Optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm. The OD value is proportional to antigenicity.

b: HPLC detection

Chromatographic conditions are: phase A pure water (containing 0.1% trifluoroacetic acid), phase C acetonitrile (containing 0.1% trifluoroacetic acid), column temperature 50°C, flow rate 1mL/min, sample loading 20μL, detection wavelength 214nm, collection time 9min .

The mobile phase gradient is as follows:

The liquid chromatogram corresponding to the standard product is shown in Figure 8, and the liquid chromatogram of the fermentation sample is shown in Figure 9.

c: Kjeldahl nitrogen test

The principle of Kjeldahl nitrogen detection is: decompose the protein, then combine the ammonia generated by the decomposition with sulfuric acid to form ammonium sulfate, then alkaline distillation to free the ammonia, absorb it with boric acid, and then titrate it with a sulfuric acid or hydrochloric acid standard solution. According to the acid consumption The amount multiplied by the conversion factor is the protein content.

The test results of the above four methods are as follows:

As can be seen from the above table, the protein content in the 1g/L sample to be tested prepared by Kjeldahl determination was 0.97g/L and 0.98g/L respectively. β-lactoglobulin was detected by HPLC. The fermentation sample composition The detection result was 0.89g/L, indicating that the sample was partially hydrolyzed. The Elisa detection result was 0.84g/L, indicating that the allergenicity of the fermented sample composition was reduced after hydrolysis.

Solubility analysis

Dry the fermentation sample to obtain sample powder. Weigh 1g of the fermentation sample powder and β-lactoglobulin standard respectively, and use the Kjeldahl nitrogen method to detect the total amount of protein contained in 1g of the fermentation sample powder and β-lactoglobulin standard. , then, dissolve the fermentation sample powder and β-lactoglobulin standard in distilled water to prepare a 1% (w/v) aqueous solution, shake it on a constant temperature shaker at 25°C for 1 hour and then centrifuge, take the supernatant and record it. The quality of the supernatant was determined by Kjeldahl nitrogen determination. The above-mentioned detection of each sample was repeated in three parallel groups.

Calculate protein solubility according to the following formula:

Protein solubility = protein content in supernatant/protein content per 1g of sample. The test results are as follows:

Digestibility test

The detection steps are as follows:

a: The total protein content in the unit sample (fermentation sample obtained in Example 2 and β-lactoglobulin standard) was determined using the Kjeldahl nitrogen method.

b: In vitro simulated gastric digestion: Use distilled water to prepare an emulsion with a protein mass fraction of 2%. Preheat the emulsion in a 37°C water bath for 10 minutes. Use 1 mol/L HCl to adjust the emulsion to pH=3. Add 0.04g of pepsin and 0.06g of chymosin to 100 mL of emulsion, digest and hydrolyze on a constant temperature shaker at 37°C for 1 hour, and then adjust the emulsion to pH=7 with 1 mol/L NaOH. Inactivate the enzyme and determine the gastric digestibility of the protein.

c: In vitro simulated intestinal digestion: prepare the same amount of sample as the above a with distilled water to obtain a protein mass fraction. Preheat the 2% emulsion in a 37°C water bath for 10 minutes, and adjust the emulsion to pH=3 with 1 mol/L HCl. Add 0.04g of pepsin and 0.06g of chymosin to 100 mL of milk sample, digest and hydrolyze it on a constant temperature shaker at 37°C for 1 hour, and then adjust the emulsion to pH=7 with 1 mol/L NaOH. Take another 100 ml milk sample and add 0.1 g of trypsin, digest and hydrolyze it on a constant temperature shaker at 37°C for 2 hours, then inactivate it in a boiling water bath for 5 minutes, and measure the total digestibility of the protein.

d: Determination of protein digestibility in vitro: Take 10 mL of the digested sample in step b or step c, add an equal volume of 24% trichloroacetic acid solution to precipitate the protein, centrifuge at 12000 r/min for 20 min, collect the supernatant, and use CaCl The nitrogen content was determined by the Nitrogen determination method, and then the digestibility was calculated using formula (1), in which distilled water was used to replace the milk sample digestive fluid in the blank test.

In the formula: m1-the nitrogen content in the sample supernatant, g; m0-the nitrogen content in the blank supernatant, g; m2-the protein content in the sample, g. The calculation results are as follows:

ACE activity inhibition ability test

(1) Preparation of solution

Prepare a 0.1 mol/L sodium borate buffer (pH=8.3) containing 0.3 mol/L sodium chloride. Lyophilize the fermentation sample obtained in Example 2 and then redissolve it in the buffer to the original volume before lyophilization. Hippuryl-histidyl-leucine (HHL) and ACE were dissolved in the buffer to 5 mmol/L and 0.1 U/mL respectively.

(2) Sample preparation

Mix the above 35 μL fermentation sample solution with 200 μL HHL solution and pre-incubate at 37°C for 5 min; add 20 μL ACE solution, incubate at 37°C for 30 min, and add 250 μL 1mol/L hydrochloric acid to terminate the reaction. In the control group, the purchased β-lactoglobulin reconstituted solution was used instead of the sample solution, and hydrochloric acid was added before pre-incubation.

(3)Instrument conditions

The chromatographic column is C18 (4.6mm×250mm), the mobile phase A is water containing 0.1% (v/v) trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.1% (v/v) trifluoroacetic acid. Use gradient elution method. The elution gradient is 30% to 60% B, 0 to 10 minutes; 60% to 30% B, 10 to 12 minutes; 30% B, hold for 2 minutes. The detector is an ultraviolet (UV) detector, the detection wavelength is 228nm, the sample volume is 20 μL, the flow rate is 1mL/min, the column temperature is 30°C, and each sample is measured three times in parallel.

(4) Determination of linear range

Take hippuric acid standard (HPLC, >98%) and prepare hippuric acid standard solutions with concentrations of 10 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, and 100 μg/mL (using 0.1 mol/ L of boric acid buffer), and filtered with a 0.45 μm micromembrane for HPLC analysis to determine that the standard concentration and detection peak area satisfy a linear relationship.

The IC ₅₀ result showed that the IC ₅₀ of the fermentation sample obtained in Example 2 was 11.34 μg/mL, which was higher than the 8.85 μg/mL of the purchased β-lactoglobulin standard.

Claims

A milk protein composition comprising lactoglobulin peptides, which are produced by degradation of recombinantly expressed β-lactoglobulin when expressed in host cells.
The milk protein composition according to claim 1, wherein the host cell is selected from at least one of Kluyveromyces marxianus or Kluyveromyces lactis.
The milk protein composition according to claim 1, further comprising recombinantly expressed β-lactoglobulin.
The milk protein composition according to claim 3, which is obtained by direct expression in the host cell.
The milk protein composition according to claim 3, which contains 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more. More than 80% by weight, more than 90% by weight or more than 95% by weight of the lactoglobulin peptide.
The milk protein composition according to claim 3, wherein the content of the recombinantly expressed β-lactoglobulin is 0-95% based on the total mass of the milk protein composition; the content of the lactoglobulin peptide is 5%-100%; preferably, the content of the recombinantly expressed β-lactoglobulin is 10%-90%; the content of the lactoglobulin peptide is 10%-90%; preferably, the recombinantly expressed β-lactoglobulin content is 10%-90%. The content of β-lactoglobulin is 20%-80%; the content of the lactoglobulin peptide is 20%-80%; preferably, the content of the recombinantly expressed β-lactoglobulin is 30%-70%; The content of the lactoglobulin peptide is 30%-70%; preferably, the content of the recombinantly expressed β-lactoglobulin is 40%-60%; the content of the lactoglobulin peptide is 40%-60% .
The milk protein composition according to claim 1, wherein the recombinantly expressed β-lactoglobulin comprises a combination with bovine β-lactoglobulin, sheep β-lactoglobulin, buffalo β-lactoglobulin or yak β-lactoglobulin. The amino acid sequence of a globulin has an amino acid sequence that is at least 80%, at least 90%, at least 95% or 100% identical.
The milk protein composition according to any one of claims 1 to 7, further comprising a host cell protein.
The milk protein composition according to claim 8, which contains 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, 20% by weight or less, 10% by weight or less. % by weight or less, 5% by weight or less, or 1% by weight or less of the protein of the host cell.
A method for preparing the milk protein composition according to any one of claims 1 to 9, which includes expressing the recombinant expression β-lactoglobulin in a host cell, and recovering the said recombinant expression β-lactoglobulin from the host cell. Milk protein composition.
The preparation method according to claim 10, further comprising a separation and purification step.
The preparation method according to claim 10, wherein the host cell is selected from Maxx At least one kind of Kluyveromyces marxianus or Kluyveromyces lactis.
The preparation method according to claim 10, wherein the time for expressing the recombinantly expressed β-lactoglobulin in the host cell is 12-168 hours, preferably 36-84 hours.
The preparation method according to claim 10, wherein the temperature at which the recombinant β-lactoglobulin is expressed in the host cell is 20-45°C, preferably 25-40°C.
A food composition or feed composition comprising the milk protein composition according to any one of claims 1-9.