WO2023221994A1 - Composition de protéine de lait, son procédé de préparation et son utilisation - Google Patents

Composition de protéine de lait, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2023221994A1
WO2023221994A1 PCT/CN2023/094598 CN2023094598W WO2023221994A1 WO 2023221994 A1 WO2023221994 A1 WO 2023221994A1 CN 2023094598 W CN2023094598 W CN 2023094598W WO 2023221994 A1 WO2023221994 A1 WO 2023221994A1
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WIPO (PCT)
Prior art keywords
lactoglobulin
milk protein
weight
protein composition
casein
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PCT/CN2023/094598
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English (en)
Chinese (zh)
Inventor
骆滨
廖丽
王松伟
苏世源
屠均亮
杨秀华
吴蓉
许昱
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上海昌进生物科技有限公司
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Publication of WO2023221994A1 publication Critical patent/WO2023221994A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • This application belongs to the field of dairy product processing, and specifically relates to a milk protein composition and its preparation method and application.
  • Milk is a universal source of nutrients, rich in protein, fat, lactose, minerals, vitamins and growth factors such as immunoglobulins and lactoferrin.
  • Whey protein is divided into ⁇ -lactalbumin and ⁇ -lactoglobulin; casein mainly exists in the form of micelles, consisting of ⁇ S1 - Casein, ⁇ S2-casein, ⁇ -casein and ⁇ -casein (4:1:4:1).
  • Casein contains almost all essential amino acids and is the most nutritionally valuable protein for newborn babies.
  • 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.
  • ⁇ -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.
  • 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.
  • 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, ⁇ -lactoglobulin and ⁇ -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 ⁇ -lactoglobulin ( ⁇ -Lg). This is mainly because breast milk does not contain ⁇ -Lg, so ⁇ -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.
  • ⁇ -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.
  • 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.
  • a first aspect of the present application provides a milk protein composition, which contains lactoglobulin peptides, which are produced by degradation of recombinantly expressed ⁇ -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 ⁇ -lactoglobulin in a host cell and recovering it from the host cell.
  • the milk protein composition includes expressing the recombinantly expressed ⁇ -lactoglobulin in a host cell and recovering it from the host cell.
  • 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 milk protein composition provided by this application contains lactoglobulin peptides produced by the degradation of ⁇ -lactoglobulin.
  • the fragments are smaller, and the solubility and in vitro digestibility are significantly better than that of natural ⁇ -lactoglobulin, thus having Nutritional value similar to or better than natural milk protein.
  • 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.
  • 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.
  • 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 ⁇ -lactoglobulin standard
  • Figure 9 is a high performance liquid chromatogram of the fermentation sample obtained in Example 2.
  • 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.
  • 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.
  • Protein 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • vector means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • plasmid 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.
  • PCR polymerase chain reaction
  • 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 ⁇ -lactoglobulin when expressed in host cells.
  • the inventor unexpectedly found in the study that when recombinantly expressed ⁇ -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.
  • the milk protein composition further comprises recombinantly expressed ⁇ -lactoglobulin.
  • the milk protein composition is directly expressed in the host cell.
  • the milk protein composition is obtained by isolating and purifying the lactoglobulin peptide in a host cell.
  • 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.
  • 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.
  • the content of the recombinantly expressed ⁇ -lactoglobulin is 0-95%; the content of the lactoglobulin peptide is 5%-100% ; Preferably, the content of the recombinantly expressed ⁇ -lactoglobulin is 10%-90%; the content of the lactoglobulin peptide is 10%-90%; Preferably, the recombinantly expressed ⁇ -lactoglobulin The content of the lactoglobulin peptide is 20%-80%; preferably, the content of the recombinantly expressed ⁇ -lactoglobulin is 30%-70%; the lactoglobulin peptide The content of the recombinantly expressed ⁇ -lactoglobulin is 30%-70%; preferably, the content of the recombinantly expressed ⁇ -lactoglobulin is 40%-60%; the content of the lactoglobulin peptide is 40%-60%.
  • the milk protein composition also includes other recombinantly expressed milk proteins, and the other recombinantly expressed milk proteins may be recombinantly expressed ⁇ -lactalbumin, recombinantly expressed ⁇ -casein, recombinantly expressed ⁇ - at least one of casein, recombinantly expressed lactoferrin, recombinantly expressed ⁇ S1-casein, recombinantly expressed ⁇ S2-casein and recombinantly expressed osteopontin.
  • the other recombinantly expressed milk proteins may be recombinantly expressed ⁇ -lactalbumin, recombinantly expressed ⁇ -casein, recombinantly expressed ⁇ - at least one of casein, recombinantly expressed lactoferrin, recombinantly expressed ⁇ S1-casein, recombinantly expressed ⁇ S2-casein and recombinantly expressed osteopontin.
  • it can be: recombinant expression of ⁇ -lactoglobulin and recombinant expression of ⁇ -lactalbumin, recombinant expression of ⁇ -lactoglobulin and recombinant expression of ⁇ -casein, recombinant expression of ⁇ -lactoglobulin Protein and recombinant expression of ⁇ -casein, recombinant expression of ⁇ -lactoglobulin and recombinant expression of lactoferrin, recombinant expression of ⁇ -lactoglobulin and recombinant expression of ⁇ S1-casein, recombinant expression of ⁇ -lactoglobulin and recombinant expression of ⁇ S2-
  • a combination of casein, recombinantly expressed ⁇ -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
  • Lactoglobulin combination of recombinant expression of lactoferrin and recombinant expression of ⁇ -lactalbumin, recombinant expression of ⁇ -lactoglobulin, combination of recombinant expression of lactoferrin and recombinant expression of ⁇ -casein, recombinant expression of ⁇ -lactoglobulin, The combination of recombinant expression of lactoferrin and recombinant expression of ⁇ -casein, the combination of recombinant expression of ⁇ -lactoglobulin, the combination of recombinant expression of lactoferrin and the recombinant expression of ⁇ S1-casein, etc.
  • 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.
  • 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.
  • 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.
  • the ⁇ -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.
  • ⁇ -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 ⁇ -lactoglobulin, which can significantly reduce Sensitization by beta-lactoglobulin.
  • 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.
  • 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 ⁇ -lactalbumin when expressed in the host cells are beneficial to further reducing the allergenicity of the milk protein composition.
  • the ⁇ -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 ⁇ -casein and the ⁇ -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.
  • 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.
  • the ⁇ S1-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 ⁇ S1-casein.
  • the recombinant ⁇ S1-casein and the ⁇ S1-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.
  • the ⁇ S2-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 ⁇ S2-casein.
  • the recombinant ⁇ S2-casein and the ⁇ S2-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.
  • 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.
  • 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.
  • 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.
  • the recombinantly expressed ⁇ -lactoglobulin comprises a combination with bovine ⁇ -lactoglobulin, buffalo ⁇ -lactoglobulin, yak ⁇ -lactoglobulin, sheep ⁇ -lactoglobulin, goat ⁇ -lactoglobulin, -Lactoglobulin, camel ⁇ -lactoglobulin, horse ⁇ -lactoglobulin, dog ⁇ -lactoglobulin, elephant ⁇ -lactoglobulin, whale ⁇ -lactoglobulin, bear ⁇ -lactoglobulin, lion ⁇ -
  • 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.
  • 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.
  • the recombinantly expressed ⁇ -lactalbumin includes a combination with bovine ⁇ -lactalbumin, human ⁇ -lactalbumin, buffalo ⁇ -lactalbumin, yak ⁇ -lactalbumin, sheep ⁇ -lactalbumin, goat ⁇ -lactalbumin, ⁇ -lactalbumin, camel ⁇ -lactalbumin, horse ⁇ -lactalbumin, dog ⁇ -lactalbumin, elephant ⁇ -lactalbumin, whale ⁇ -lactalbumin, bear ⁇ -lactalbumin, lion ⁇ -lactalbumin or tiger ⁇ -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.
  • 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 ⁇ -casein, camel ⁇ -casein, horse ⁇ -casein, dog ⁇ -casein, elephant ⁇ -casein, whale ⁇ -casein, bear ⁇ -casein, lion ⁇ -casein or tiger ⁇ -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.
  • the recombinantly expressed ⁇ -casein comprises a combination with bovine ⁇ -casein, human ⁇ -casein, buffalo ⁇ -casein, yak ⁇ -casein, sheep ⁇ -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.
  • the recombinantly expressed ⁇ S1-casein comprises a combination with bovine ⁇ S1-casein, human ⁇ S1-casein, buffalo ⁇ S1-casein, yak ⁇ S1-casein, sheep ⁇ S1-casein, goat ⁇ S1-casein, camel ⁇ S1-casein, horse ⁇ S1-casein, dog ⁇ S1-casein, elephant ⁇ S1-casein, whale ⁇ S1-casein, bear ⁇ S1-casein, lion ⁇ S1-casein or tiger ⁇ S1-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.
  • the recombinantly expressed ⁇ S2-casein comprises a combination with bovine ⁇ S2-casein, human ⁇ S2-casein, buffalo ⁇ S2-casein, yak ⁇ S2-casein, sheep ⁇ S2-casein, goat ⁇ S2-casein, camel ⁇ S2-casein, horse ⁇ S2-casein, dog ⁇ S2-casein, elephant ⁇ S2-casein, whale ⁇ S2-casein, bear ⁇ S2-casein, lion ⁇ S2-casein or tiger ⁇ S2-
  • the amino acid sequence of casein has an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical.
  • 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.
  • the host cell is selected from fungal cells, bacterial cells, or protozoan cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Acinetobacter Aceto
  • 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.
  • 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.
  • the Bacillus genus includes Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus, and At least one species of Bacillus subtilis.
  • the Lactobacillus genus includes at least one of Lactobacillus acidophilus and Lactobacillus bulgaricus.
  • the host cell is selected from at least one of Kluyveromyces marxianus or Kluyveromyces lactis.
  • the inventor found that expressing recombinant ⁇ -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.
  • the milk protein composition further includes host cell proteins.
  • 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.
  • 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.
  • the protein content of the host cells is 1-40%, preferably 1-20%, based on the total mass of the milk protein composition.
  • 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.
  • the milk protein composition includes 1-40% host cell protein, 5-90% lactoglobulin peptide and 5-90% recombinant ⁇ -lactoglobulin.
  • the milk protein composition contains 1-20% host cell protein, 40-60% lactoglobulin peptide and 20-55% recombinantly expressed ⁇ -lactoglobulin.
  • 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 ⁇ -lactoglobulin in a host cell and recovering it from the host cell.
  • the milk protein composition includes expressing the recombinantly expressed ⁇ -lactoglobulin in a host cell and recovering it from the host cell.
  • 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 ⁇ -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 ⁇ -lactoglobulin Culturing the host cell under conditions to allow the host cell to express the ⁇ -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 ⁇ -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.
  • 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.
  • the host cell is selected from fungal cells, bacterial cells, or protozoan cells.
  • the fungal cell is selected from at least one member of the genus Kluyveromyces, Pichia, Aspergillus, and Fusarium.
  • 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.
  • the milk protein composition can be expressed in Kluyveromyces marxianus or Kluyveromyces lactis.
  • the inventor unexpectedly found that using the host cell Expressing the ⁇ -lactoglobulin can obtain a higher yield of lactoglobulin peptides, which contain more functional peptides.
  • the host cell protein is biosafe, edible, and can Supplemented into the milk protein composition as a nutritional component.
  • 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 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 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.
  • Strains used in this application include:
  • 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.
  • 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.
  • 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.
  • Kluyveromyces marxianus was purchased from the China Industrial Microbial Culture Collection Center (CICC), numbered CICC 1953, and purchased in September 2020.
  • CICC China Industrial Microbial Culture Collection Center
  • 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.
  • CICC China Industrial Microbial Culture Collection Center
  • the milk protein composition and its preparation method of the present application will be described below through specific examples.
  • the bovine ⁇ -lactoglobulin gene fragment (NCBI Gene ID: 113901792) was synthesized at GenScript Biotechnology Co., Ltd. and named ⁇ -Lg.
  • ⁇ -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- ⁇ -Lg.
  • ⁇ -lactoglobulin continues to be generated and degraded, until about 60 hours , the ratio of ⁇ -lactoglobulin and ⁇ -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.
  • 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: 0.001 ⁇ m; molecular cutoff value: ⁇ 200D; water inlet pressure: 0.2-0.3Mpa; working pressure :0.5-0.7Mpa.
  • 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:
  • Example 1 Shake flask seed culture, take 1.5 mL of the K.marxianus::pKLAC1- ⁇ -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.
  • liquid seed culture medium peptone 20g/L, yeast powder 10g/L , glucose 10g/L, galactose 10g/L
  • Secondary seed culture prepare 200mL seed liquid and connect it to the secondary seed culture medium (5L tank), 30°C, 220rpm, culture for 6-8h, OD reaches about 10-12, and obtain the secondary seed liquid.
  • 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.
  • Example 3 Expanding fermentation system to prepare milk protein composition
  • the 1T fermentation process is as follows:
  • First-level shake flask seed culture 2 mL of K.marxianus::pKLAC1- ⁇ -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.
  • 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.
  • 40L/150L seed tank (g) 40L/150L seed tank (g)
  • peptone 20g/L peptone 20g/L
  • yeast powder 10g/L glucose 10g/L
  • galactose 10g /L galactose 10g /L
  • air volume 40L/min dissolved oxygen and rotation speed
  • Example 3 Except that the fermentation culture time in step 4 was extended to 108 hours, the rest was the same as in Example 3.
  • the SDS-PAGE results are shown in Figure 4. From the figure, ⁇ - Lactoglobulin and milk Production of globin peptides.
  • linearized pKLAC1- ⁇ -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.
  • linearized pKLAC1- ⁇ -Lg was transformed into Kluyveromyces lactis (Kluyveromyces lactis, deposit number CICC 32428), K.lactis::pKLAC1- ⁇ -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 °C, 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.
  • 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 ⁇ -lactoglobulin.
  • Example 1 Using the ⁇ -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- ⁇ -Lg.
  • the pPIC9k- ⁇ -Lg plasmid linearized by SalI enzyme was transformed into Pichia pastoris GS115 to construct P.pastoris::pPIC9k- ⁇ -Lg.
  • 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.
  • 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.
  • 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.
  • the fermentation broth supernatant was subjected to SDS-PAGE analysis, and gel strips of different molecular weight sizes were pretreated.
  • 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.
  • ACN acetonitrile
  • 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 ⁇ m, column size 3 ⁇ m, 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.
  • Table 2 List of lactoglobulin peptides identified by mass spectrometry 2
  • Table 1 shows that in the ⁇ -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 ⁇ -lactoglobulin, not bacterial protein, or other metabolite proteins.
  • 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.
  • Example 1 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 ⁇ -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.
  • the milk protein compositions in the examples of the present application also contain a certain amount of host cell proteins.
  • 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.
  • the antigenicity of ⁇ -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.).
  • 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 ⁇ m sterile needle filter.
  • 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.
  • Nanofiltration Take the permeate of K.marxianus::pKLAC1 and K.marxianus::pKLAC1- ⁇ -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: 0.001 ⁇ m; 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.
  • Nanofiltration Take the K.marxianus::pKLAC1 and K.marxianus::pKLAC1- ⁇ -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: 0.001 ⁇ m; 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- ⁇ -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 ⁇ -lactosphere Proteolytically produced beta-lactoglobulin and beta-lactoglobulin compositions.
  • Nanofiltration conditions First 2-stage filter element: precision filter element + NF nanofiltration membrane; NF nanofiltration membrane specification: NF-4040; filtration precision: 0.001 ⁇ m; molecular cutoff value: ⁇ 200D; water inlet pressure: 0.2-0.3Mpa; working Pressure: 0.5-0.7Mpa.
  • 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 ⁇ -lactoglobulin, ⁇ -lactoglobulin peptide and K. marxianus extracellular secreted protein.
  • Example 1 Milk protein composition fermented for 36 hours (G, comprising 50% by weight of recombinant ⁇ -lactoglobulin, 45% by weight of ⁇ -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 ⁇ -lactoglobulin, 10% by weight of ⁇ -lactoglobulin peptid
  • 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 ⁇ -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:
  • 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 ⁇ -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.
  • HRP horseradish peroxidase
  • 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.
  • 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 20 ⁇ L, detection wavelength 214nm, collection time 9min .
  • the mobile phase gradient is as follows:
  • 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 protein content in the 1g/L sample to be tested prepared by Kjeldahl determination was 0.97g/L and 0.98g/L respectively.
  • ⁇ -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.
  • Protein solubility protein content in supernatant/protein content per 1g of sample. The test results are as follows:
  • the detection steps are as follows:
  • step 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.
  • 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 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 ⁇ L, the flow rate is 1mL/min, the column temperature is 30°C, and each sample is measured three times in parallel.
  • UV ultraviolet
  • hippuric acid standard (HPLC, >98%) and prepare hippuric acid standard solutions with concentrations of 10 ⁇ g/mL, 20 ⁇ g/mL, 40 ⁇ g/mL, 60 ⁇ g/mL, 80 ⁇ g/mL, and 100 ⁇ g/mL (using 0.1 mol/ L of boric acid buffer), and filtered with a 0.45 ⁇ m 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 ⁇ g/mL, which was higher than the 8.85 ⁇ g/mL of the purchased ⁇ -lactoglobulin standard.

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Abstract

L'invention concerne une composition de protéine de lait, son procédé de préparation et son utilisation, la composition de protéine de lait comprenant un peptide de lactoglobuline, et le peptide de lactoglobuline étant obtenu par dégradation de la β-lactoglobuline recombinante lorsqu'il est exprimé dans une cellule hôte. L'invention concerne également une composition alimentaire ou une composition alimentaire comprenant la composition de protéine de lait mentionnée ci-dessus.
PCT/CN2023/094598 2022-05-17 2023-05-16 Composition de protéine de lait, son procédé de préparation et son utilisation WO2023221994A1 (fr)

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