Nutritional compositions comprising milk-derived peptides and uses thereof
阅读说明：本技术 包含乳衍生肽的营养组合物及其用途 (Nutritional compositions comprising milk-derived peptides and uses thereof ) 是由 D·巴纳瓦拉 J·B·拉巴特 S·D·玛莉亚 S·C·菲利普斯 于 2019-01-04 设计创作，主要内容包括：提供了具有蛋白组分的营养组合物,所述蛋白组分包括某些肽和/或生物活性肽。还公开了具有蛋白来源的营养组合物,所述蛋白来源包括完整蛋白、富含β-酪蛋白的酪蛋白水解物和/或分子量为约500Da至约1,999Da的肽。所公开的营养组合物适合于施用于儿科受试者,例如婴儿。(Nutritional compositions having protein components that include certain peptides and/or bioactive peptides are provided. Also disclosed are nutritional compositions having a protein source comprising intact protein, a beta-casein-rich casein hydrolysate, and/or peptides having a molecular weight of about 500Da to about 1,999 Da. The disclosed nutritional compositions are suitable for administration to pediatric subjects, such as infants.)
1. A staged feeding regimen for infants comprising the steps of:
A) administering to a newborn infant a first nutritional composition comprising from about 5% to about 15% by weight of a protein source or protein equivalent source having peptides with a molecular weight of from about 500Da to about 1,999 Da;
B) administering to the middle-age infant a second nutritional composition comprising from about 6% to about 7% by weight of a protein or protein equivalent source having peptides with a molecular weight of from about 500Da to about 1,999 Da;
C) administering to the late stage infant a third nutritional composition comprising from about 4% to about 6% of a protein or protein equivalent source having peptides with a molecular weight of from about 500Da to about 1,999 Da.
2. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises a casein hydrolysate enriched in beta-casein.
3. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises intact proteins.
4. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises an amino acid.
5. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises lactoferrin.
6. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises at least one prebiotic.
7. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises at least one probiotic.
8. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises inositol.
9. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises at least one long chain polyunsaturated fatty acid, wherein the long chain polyunsaturated fatty acid is selected from the group consisting of docosahexaenoic acid, arachidonic acid, and combinations thereof.
10. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises lactoferrin.
11. The staged feeding regimen of claim 1, wherein at least one of the first, second, or third nutritional compositions further comprises a culture supernatant from a later exponential growth phase of a probiotic batch culture process.
12. A nutritional composition comprising:
a source of carbohydrates;
a fat or lipid source; and
a protein source, wherein the protein source comprises from about 4% to about 10% peptides having a molecular weight of from about 500Da to about 1,999Da, a casein hydrolysate enriched in β -casein and intact proteins.
13. The composition of claim 12, wherein the protein source further comprises amino acids.
14. The composition of claim 12, wherein the nutritional composition further comprises inositol.
15. The composition of claim 12, wherein the nutritional composition further comprises a probiotic.
16. The composition of claim 12, wherein the nutritional composition further comprises a prebiotic.
17. The composition according to claim 12, wherein the nutritional composition further comprises one or more long chain polyunsaturated fatty acids.
18. The composition of claim 17, wherein the one or more long chain polyunsaturated fatty acids comprise docosahexaenoic acid, arachidonic acid, and combinations thereof.
19. The composition of claim 12, wherein the nutritional composition further comprises beta-glucan.
20. The composition of claim 12, wherein the nutritional composition further comprises a culture supernatant from a late exponential phase of a probiotic batch culture process.
Technical Field
The present disclosure generally relates to nutritional compositions comprising proteins and/or peptides such that different protein molecular weight distributions are administered to pediatric subjects of different age stages. Also disclosed are nutritional compositions comprising protein hydrolysates produced using trypsin or chymotrypsin. The disclosed nutritional compositions may provide additive and/or synergistic beneficial health effects.
Background
Human milk contains different proteins and protein fragments, characterized by their molecular weight characteristics. Applicants have found that the molecular weight distribution of the protein content of human milk may change throughout the first year of lactation. Therefore, there is a need to prepare nutritional compositions with protein characteristics that vary with the age of the infant, in order to more closely mimic human breast milk.
In view of the need to provide nutritional compositions, such as infant formulas, that provide to pediatric subjects an amount and composition of a protein source that more closely mimics human breast milk, provided herein are nutritional compositions, i.e., infant formulas, that include protein hydrolysates having a peptide composition similar to that found in human milk. In some embodiments, infant formulas are provided that include a blend of intact protein, a casein hydrolysate enriched in beta-casein, and a blend of free amino acids. In some embodiments, nutritional compositions are provided that include hydrolysates produced by processes that utilize certain trypsin or chymotrypsin enzymes.
Brief summary
Briefly, in one embodiment, the present disclosure is directed to nutritional compositions comprising a protein component or protein source that includes certain bioactive peptides. In some embodiments, the protein component or protein source comprises intact protein, casein hydrolysate enriched in beta-casein, amino acids, and combinations thereof. In certain other embodiments, the nutritional composition may comprise a protein component comprising at least one of trypsin or chymotrypsin and intact protein. In some embodiments, the nutritional composition comprises a protein hydrolysate produced using trypsin or chymotrypsin.
In some embodiments, the nutritional compositions include a protein component described herein along with long chain polyunsaturated fatty acids (such as docosahexaenoic acid and/or arachidonic acid), one or more probiotics (e.g., lactobacillus rhamnosus GG), lactoferrin, beta-glucan, Phosphatidylethanolamine (PE), sphingomyelin, inositol, vitamin D, and combinations thereof.
It is to be understood that both the foregoing general description and the following detailed description present embodiments of the disclosure, and are intended to provide an overview or framework for understanding the nature and character of the disclosure as it is claimed. This description is made for the purpose of explaining the principles and operation of the claimed subject matter. Other and further features and advantages of the present disclosure will be readily apparent to those skilled in the art upon reading the following disclosure.
Brief description of the drawings
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.
Figure 1a shows the average molecular weight distribution of 15 human milk samples (5 samples from each of three geographically distinct regions-usa, mexico and china).
Fig. 1b shows the change in molecular weight distribution of the 1,999Da to 50Da portion of human milk in the first year of lactation for the same human milk sample as provided in fig. 1 a.
Figure 2 shows a pie chart of the parent protein of the human lactopepset. Endogenous pepsets of human milk were determined by LC-MS/MS based pepsets (n = 27). The most abundant peptides are derived from casein, especially beta-casein.
Fig. 3 shows the enzyme predictions for the total human milk peptide group. The endogenous peptide group of human milk was determined by LC-MS/MS-based peptide histology (n =27) and proteases were predicted using the enzepredictor software.
Detailed Description
Reference now will be made in detail to embodiments of the disclosure, one or more examples of which are set forth below. Each example is provided by way of explanation of the nutritional compositions of the present disclosure, and not by way of limitation. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the teachings of the disclosure without departing from the scope thereof. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Thus, it is intended that the present disclosure cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present disclosure are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present disclosure.
The present disclosure generally relates to nutritional compositions having a protein component or protein source that includes an amount of a biologically active peptide. In some embodiments, the protein component comprises a beta-casein rich hydrolysate. In some embodiments, the protein component may include intact protein along with trypsin or chymotrypsin. In still other embodiments, the protein component may include peptides produced by a process using trypsin or chymotrypsin.
"nutritional composition" refers to a substance or formulation that meets at least a portion of a subject's nutritional needs. The terms "nutrient," "nutritional formula," "enteral nutrient," and "nutritional supplement" are used throughout this disclosure as non-limiting examples of nutritional compositions. Furthermore, "nutritional composition" may refer to a liquid, powder, gel, paste, solid, tablet, capsule, concentrate, suspension or ready-to-use form of an enteral formula, an oral formula, an infant formula, a pediatric subject formula, a pediatric formula, a growing-up milk and/or an adult formula.
By "pediatric subject" is meant a human less than 13 years of age. In some embodiments, a pediatric subject refers to a human subject born to 8 years of age. In other embodiments, a pediatric subject refers to a human subject between 1 and 6 years of age. In still further embodiments, a pediatric subject refers to a human subject between 6 and 12 years of age. As described below, the term "pediatric subject" may refer to an infant (preterm or term infant) and/or a child.
"infant" refers to human subjects ranging in age from birth to no more than one year of age, and includes infants aged 0 to 12 months corrected. The term "corrected age" refers to the real age of the infant minus the amount of time the infant is born early. Thus, the corrected age is the age of the infant if it has been pregnant to term. The term infant includes low birth weight infants, very low birth weight infants and premature infants. By "preterm infant" is meant an infant born before the end of week 37 of gestation. By "term infant" is meant an infant born after the end of the 37 th week of gestation.
"child" refers to a subject ranging in age from 12 months to about 13 years. In some embodiments, the child is a subject between 1 and 12 years of age. In other embodiments, the term "child" refers to a subject that is 1 to about 6 years old, or about 7 to about 12 years old. In other embodiments, the term "child" refers to any age range from 12 months of age to about 13 years of age.
By "infant formula" is meant a composition that meets at least a portion of the nutritional needs of an infant. In the united states, the contents of infant formula are regulated by federal regulations set forth in
The term "medical food" refers to an enteral composition formulated or intended for the dietary management of a disease or condition. The medical food may be a food for oral ingestion or tube feeding (nasogastric tube), may be labeled for dietary management of a particular medical disorder, disease or condition with unique nutritional requirements, and may be intended for use under medical supervision.
The term "protein component" as described herein is used interchangeably with "protein source" and generally refers to various protein sources that may be used in a nutritional composition. Indeed, the use of the term "protein" is not limited to intact proteins, but also includes, but is not limited to, intact proteins, hydrolysed proteins, peptides and free amino acids. The term "protein equivalent source" may also be used interchangeably with "protein source" or "protein component".
The term "peptide" as used herein describes a linear molecular chain of amino acids, including single-chain molecules or fragments thereof. The peptides described herein comprise a total of no more than 50 amino acids. The peptide may further form an oligomer or multimer consisting of at least two molecules, which may be the same or different. Furthermore, the term "peptide" also includes peptidomimetics of such peptides in which amino acids and/or peptide bonds have been replaced by functional analogs. Such functional analogs may include, but are not limited to, all known amino acids other than the 20 gene-encoded amino acids, such as selenocysteine.
The term "peptide" may also refer to naturally modified peptides, wherein the modification is effected, for example, by glycosylation, acetylation, phosphorylation and similar modifications as are well known in the art. In some embodiments, the peptide component is different from the protein source also disclosed herein. Furthermore, the peptides may be produced, for example, recombinantly, semisynthetically, synthetically, or obtained from natural sources, e.g., after hydrolyzing proteins (including but not limited to casein), all according to methods known in the art.
The term "molar mass distribution" when used in relation to a hydrolysed protein or protein hydrolysate relates to the molar mass of each peptide present in the protein hydrolysate. For example, a protein hydrolysate having a molar mass distribution of greater than 500 daltons means that each peptide comprised in the protein hydrolysate has a molar mass of at least 500 daltons. To produce a protein hydrolysate having a molar mass distribution of greater than 500 daltons, the protein hydrolysate may be subjected to certain filtration procedures or any other procedures known in the art to remove peptides, amino acids and/or other protein material having a molar mass of less than 500 daltons. For the purposes of this disclosure, any method known in the art can be used to produce a protein hydrolysate having a molar mass distribution of greater than 500 daltons.
The term "protein equivalent" or "protein equivalent source" includes any protein source, such as soy, egg, whey or casein, as well as non-protein sources, such as peptides or amino acids. Furthermore, the protein equivalent source may be any protein equivalent source used in the art, such as skim milk, whey protein, casein, soy protein, hydrolyzed protein, peptides, amino acids, and the like. Sources of milk protein that may be used in the practice of the present disclosure include, but are not limited to, milk protein powder, milk protein concentrate, milk protein isolate, skim milk solids, skim milk powder, whey protein isolate, whey protein concentrate, sweet whey, acid whey, casein, acid casein, caseinate (e.g., sodium caseinate, sodium calcium caseinate, calcium caseinate), soy protein, and any combination thereof. In some embodiments, the protein equivalent source may comprise hydrolyzed proteins, including partially hydrolyzed proteins and extensively hydrolyzed proteins. In some embodiments, the protein equivalent source may comprise an intact protein.
The term "protein equivalent source" also encompasses free amino acids. In some embodiments, amino acids may include, but are not limited to, histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, carnitine, taurine, and mixtures thereof. In some embodiments, the amino acid may be a branched chain amino acid. In certain other embodiments, small amino acid peptides may be included as a protein component of the nutritional composition. Such small amino acid peptides may be naturally occurring or synthetic.
"milk fat globule membrane" includes components found in milk fat globule membranes, including, but not limited to, milk fat globule membrane proteins such as
The term "growing-up milk" refers to a broad category of nutritional compositions intended for use as part of a diverse diet to support the normal growth and development of children aged from about 1 to about 6 years.
"milk" refers to a component that has been extracted or extracted from the mammary gland of a mammal. In some embodiments, the nutritional composition comprises a milk component derived from a domesticated ungulate, ruminant, or other mammal, or any combination thereof.
By "nutritionally complete" is meant a composition that can be used as the sole source of nutrition, providing essentially all of the required daily amounts of vitamins, minerals, and/or trace elements in combination with protein, carbohydrate, and lipids. In fact, "nutritionally complete" describes a nutritional composition that provides sufficient amounts of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy to support normal growth and development in a subject.
By definition, a nutritional composition that is "nutritionally complete" for a term infant will qualitatively and quantitatively provide sufficient amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals and energy required for growth of the term infant.
By definition, a nutritional composition that is "nutritionally complete" for a child will qualitatively and quantitatively provide a sufficient amount of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the child.
"probiotic" refers to a microorganism of low or no pathogenicity that exerts beneficial effects on the health of the host.
The term "inactive probiotic" refers to a probiotic in which the metabolic activity or reproductive ability of the probiotic in question has been reduced or destroyed. More specifically, "inactive" or "inactive probiotic" refers to non-living probiotic microorganisms, cellular components thereof, and/or metabolites thereof. Such inactive probiotics may have been heat inactivated or otherwise inactivated. However, "inactive probiotics" still retain their cellular structure or other structures associated with the cell, such as exopolysaccharides and at least a portion of their biological diol-protein and DNA/RNA structures, at the cellular level, thus retaining the ability to favorably influence the health of the host. Conversely, the term "active" refers to a living microorganism. The term "inactive" as used herein is synonymous with "inactivated".
By "prebiotic" is meant a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the gut, which can improve the health of the host.
"phospholipid" refers to an organic molecule containing diglycerides, phosphate groups, and simple organic molecules. Examples of phospholipids include, but are not limited to, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol phosphates, phosphatidylinositol diphosphates and phosphatidylinositol triphosphates, ceramide phosphorylcholine, ceramide phosphorylethanolamine, and ceramide phosphorylglycerol. Also included within this definition are sphingolipids, such as sphingomyelin. Glycosphingolipids are quantitative minor components of MFGM and consist of cerebrosides (neutral glycosphingolipids containing uncharged sugars) and gangliosides. Gangliosides are acidic glycosphingolipids that contain sialic acid (N-acetylneuraminic acid (NANA)) as part of their carbohydrate moieties. There are various types of gangliosides derived from different synthetic pathways, including GM3, GM2, GM1a, GD1a, GD3, GD2, GD1b, GT1b, and GQ1b (Fujiwara et al, 2012). The major gangliosides in milk are GM3 and GD3(Pan & Izumi, 1999). The different types of gangliosides differ in the nature and length of their carbohydrate side chains and the number of sialic acids attached to the molecule.
The nutritional compositions of the present disclosure may be substantially free of any optional or selected ingredients described herein, provided that the remaining nutritional composition still contains all of the desired ingredients or features described herein. Herein, and unless otherwise indicated, the term "substantially free" means that the selected composition may contain less than a functional amount of optional ingredients, typically less than 0.1 wt%, and also contains 0 wt% of such optional or selected ingredients.
All percentages, parts and ratios used herein are by weight of the total composition, unless otherwise specified.
All references to singular features or limitations of the present disclosure shall include the corresponding plural features or limitations, and vice versa, unless otherwise indicated herein or clearly implied to the contrary by the context in which such references are made.
All combinations of method or process steps as used herein can be performed in any order, unless otherwise indicated herein or otherwise clearly contradicted by context in which the combination is referred to.
The methods and compositions of the present disclosure, including components thereof, may comprise, consist of, or consist essentially of: the essential elements and limitations of the embodiments described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in nutritional compositions.
The term "about" as used herein should be interpreted to refer to two numbers that are specified as the endpoints of any range. Any reference to a range should be considered as providing support for any subset of the ranges.
Indeed, the protein composition of human milk varies with time, and especially in the first year of lactation the amount of small proteins or peptides with a certain molecular weight decreases. However, these adjustments are not made in infant formula. Thus, formula-fed infants may experience reduced nutrition compared to breast-fed infants. Furthermore, human milk consumption is affected by changes throughout the lactation period, reflecting the changing needs of growing infants. However, in the united states, conventional infant formulas are sold to all term infants throughout the first year of life, which may include meeting the nutritional needs of the first twelve (12) months of life of the infant. Despite some adjustments to infant formula, there are still differences in protein digestibility, amino acid intake, and the presence of specific bioactive peptides in infant formula.
Human milk comprises about 80-85% intact protein, 5-10% peptides and about 3-5% free amino acids based on total weight of protein. The major proteins in human milk include whey and casein, i.e., beta-casein, kappa-casein, alpha-lactalbumin, lysozyme and lactoferrin. These proteins can be used as a source of biologically active peptides, as during digestion, the gastrointestinal tract of infants may break down these intact proteins to produce certain peptides with a variety of biological activities. Indeed, peptides derived from milk proteins may have a positive impact on physiological and metabolic functions, which have beneficial effects on infant health.
Applicants have found that the molecular weight distribution of human milk is different at different stages of lactation. Indeed, although the majority (more than 80%) of the total protein content in breast milk includes intact proteins with a molecular weight greater than 5000Da, it has been found that the molecular weight of proteins with a molecular weight between 1,999Da and 500Da (i.e. peptides typically ranging from about four (4) amino acids to twenty-four (24) amino acids) varies the most, significantly decreasing during lactation, from more than about 9% at
TABLE 1 percent molecular weight distribution of human breast milk
This higher level of a specific protein fraction with a molecular weight of 500Da to 1,999Da during the first three months of lactation is indicative of an important effect on growing infants. Indeed, a pepset analysis of human milk from the first month of lactation determined 328 peptides, 10-40 amino acids in length, with an average length of about 22 amino acids. The overall average peptide molecular weight was about 2383 Da. Indeed, certain peptides included in the range of 500Da to 1,999Da may have antimicrobial effects.
Thus, provided herein is a staged feeding regimen for infants in the first year of life, wherein the amount of certain peptides having a molecular weight of about 500Da to 1,999Da decreases as the infant ages. Methods of staged infant feeding regimens to promote healthy development and growth of formula-fed infants are also provided. In certain embodiments, the feeding regimen comprises feeding the infant with the first nutritional composition, the second nutritional composition, and the third nutritional composition as the infant ages.
Indeed, while conventional "unified fit" infant formulas may provide adequate nutrition for formula-fed infants, such formulas do not account for the changing needs in development. It would therefore be beneficial to provide an infant feeding regimen that includes a nutritional composition tailored to provide a combination of nutrients aimed at promoting healthy development and growth at each stage.
"infant" refers to a person from birth to an age of no more than 12 months, wherein a "newborn infant" is an infant from birth to 3 months of age, a "middle infant" is an infant from 3 months of age to 6 months of age, and a "late infant" is an infant from 6 months of age to 12 months of age or 1 year of age.
Thus, in certain embodiments, the first nutritional composition is fed to an infant that is a newborn infant, i.e. an infant born to 3 months of age, the second nutritional composition is fed to an intermediate infant, i.e. an
In certain embodiments, the feeding regimen of the present disclosure comprises:
A) a first nutritional composition comprising:
i) from about 7% to about 10% by weight of a protein source or protein equivalent source having peptides with a molecular weight of from about 500Da to about 1,999 Da;
B) a second nutritional composition comprising:
i) from about 6% to about 7% by weight of a protein or protein equivalent source having a peptide with a molecular weight of from about 500Da to about 1,999 Da; and
C) a third nutritional composition comprising:
i) from about 4% to about 6% of a protein or protein equivalent source having a peptide with a molecular weight of from about 500Da to about 1,999 Da.
In certain embodiments, the feeding regimen of the present disclosure comprises:
A) a first nutritional composition comprising:
i) from about 5% to about 15% by weight of a protein source or protein equivalent source having peptides with a molecular weight of from about 500Da to about 1,999 Da;
B) a second nutritional composition comprising:
i) from about 6% to about 7% by weight of a protein or protein equivalent source having a peptide with a molecular weight of from about 500Da to about 1,999 Da; and
C) a third nutritional composition comprising:
i) from about 4% to about 6% of a protein or protein equivalent source having a peptide with a molecular weight of from about 500Da to about 1,999 Da.
In certain embodiments, as the amount of peptides or proteins having a molecular weight of about 500Da to about 1,999Da present in the infant formula decreases, the amount of intact proteins may increase during the staging of the infant formula.
In certain embodiments, the feeding regimen of the present disclosure comprises:
A) a first nutritional composition comprising:
i) from about 5% to about 10% of a beta-casein rich hydrolysate;
B) a second nutritional composition comprising:
i) from about 2% to about 5% of a beta-casein rich hydrolysate; and
C) a third nutritional composition comprising:
i) from about 0.1% to about 2% of a beta-casein rich hydrolysate.
In certain embodiments, the feeding regimen of the present disclosure comprises:
A) a first nutritional composition comprising:
i) from about 10% to about 20% of a beta-casein rich hydrolysate;
B) a second nutritional composition comprising:
i) from about 2% to about 5% of a beta-casein rich hydrolysate; and
C) a third nutritional composition comprising:
i) from about 0.1% to about 2% of a beta-casein rich hydrolysate.
In certain embodiments, as the amount of beta-casein rich hydrolysate present in the infant formula decreases, the amount of intact protein may increase during the staged infant formula.
In addition, a portion of the non-protein nitrogen portion of human milk comprises a peptide sequence and free amino acids. The exact concentration of these nutrients will vary based on several factors, including the labor and lactation period. However, recent findings from human milk molecular weight and peptide composition (fig. 2 and 3) indicate that hydrolysis in the mammary gland appears to be specific, rather than a random event, suggesting that human milk peptides may have effects beyond providing amino acids to infants in overall human milk function.
Furthermore, specific Bio-IT analysis indicates that the human milk peptide group appears to be dominated by casein (mainly β -casein, see fig. 2) derived sequences, whereas specific proteases (including trypsin, chymotrypsin and plasmin) (see fig. 2) may lead to hydrolysis of casein in human milk. Furthermore, homology to known active peptide sequences suggests an overall role for the peptide group in overall human milk function.
Thus, in some embodiments herein, nutritional compositions include certain food-derived bioactive peptides that are short amino acid chains having a known sequence that may have one or more biological activities. Functionally or biologically active peptides may be derived from milk proteins and may exhibit a positive effect on physiological and metabolic functions. In fact, many of these bioactive peptides are released in the gastrointestinal tract after consumption of intact proteins by certain proteolytic enzymes. The functions of these bioactive peptides include antihypertensive, antioxidant, antimicrobial, immunomodulatory, opioid and opioid-antagonist properties. In addition, certain bioactive peptides can inhibit oxidative processes, help control or maintain healthy body weight, affect mineral binding activity, and improve the sensory value of certain foods (e.g., infant formula). Tolerogenic peptides in cow's milk may also lead to reduced allergenicity. Certain peptides present in casein hydrolysates may provide additional digestive benefits compared to intact proteins.
Milk proteins are a rich source of bioactive peptides and major milk proteins (i.e., α -S1-casein, α -S2-casein, β -casein, κ -casein, β -lactoglobulin, α -lactalbumin, bovine serum albumin, and lactoferrin), and can be used as precursors to bioactive peptides. Some functional peptides derived from bovine milk include casomorphin, casomokinin, immunopeptide and lactoferricin. Many of the bioactive peptides found in human breast milk are provided by enzymes present in the mammary gland. Human breast contains more of these enzymes than bovine breast, which provides an increased amount of biologically active peptides in human breast milk compared to bovine milk. In addition, the peptide distribution of these biologically active peptides is altered during lactation.
Thus, protein sources or protein equivalent sources provided herein include casein hydrolysates that are rich in β -casein. In certain embodiments, the protein source comprises a combination of intact protein, casein hydrolysate enriched in beta-casein, and free amino acids. Indeed, in certain embodiments, provided are beta-casein and beta-casein-enriched casein hydrolysates that deliver a mixture of beta-casein-derived bioactive peptides.
Indeed, from the dairy technology point of view, it is feasible to enrich for beta-casein or alpha-casein, and bovine caseinate enriched with beta-casein is commercially available. Thus, provided herein in some embodiments are enriched beta-casein, which is hydrolyzed by a particular protease, e.g., trypsin, chymotrypsin, and/or plasmin, resulting in a beta-casein enriched casein hydrolysate, which can be included in the protein sources disclosed herein.
In embodiments where the protein source comprises a beta-casein rich casein hydrolysate, the hydrolysate may be prepared by any method known in the art. The methods of producing beta-casein-enriched casein hydrolysates disclosed herein may be directed in part to the preparation of beta-, alpha-or kappa-enriched casein, acid casein or caseinate hydrolysates, for example for use in nutritional formulations. Casein refers to a family of related phosphoproteins, including β -casein, α -casein, and κ -casein. Bovine casein is commercially available from a variety of sources. In certain embodiments, casein enriched with β -, α -, or κ -casein is used. Methods for enriching beta-casein (see, e.g., U.S. patent publication No. 20070104847) and alpha-casein and kappa-casein (see, e.g., WO2003003847) are known in the art. Acid caseins or caseinates rich in beta-, alpha-or kappa-casein may also be used (e.g. sodium caseinate, sodium calcium caseinate, calcium caseinate). Caseinate is typically formed from the reaction of acid casein with a base.
In some embodiments, the beta-casein hydrolysate may comprise at least one or a combination of the following peptides: AVPYPQR, VLPVPQK, YQEPLGPVRGPFPI, YQEPLGPVRPGPIIV, PGPIPN and/or YPVEP.
It is known that the casein composition of human and bovine milk differs significantly. In human milk, the casein fraction is almost entirely composed of beta-casein. In the case of bovine milk, beta-casein is present in small amounts, the major component being alphasCasein, and thus there is a need for products enriched with beta-casein, i.e. products with a higher beta-casein content compared to other types of casein, for use in nutritional compositions, such as infant formula. Thus, beta-casein may be isolated or purified from any suitable milk source, including bovine milk from any known process.
Methods for preparing beta-casein-rich products are known in the art and are described, for example, in PCT publication No. WO1994/003606, the disclosure of which is incorporated herein by reference for all purposes. The methods disclosed herein further involve preparing a hydrolysate of polymeric immunoglobulin receptor (PIGR), osteopontin, bile salt activated lipase, and/or clusterin with any one or more of the proteases described herein.
As described herein, one or more proteases may be used to prepare the hydrolysate. Suitable proteases include trypsin, chymotrypsin, plasmin, pepsin or any combination thereof. In certain embodiments, trypsin, chymotrypsin, and plasmin are used. In certain embodiments, trypsin and chymotrypsin are used. In certain embodiments, trypsin and plasmin are used. In certain embodiments, chymotrypsin and plasmin are used. In certain embodiments, trypsin, chymotrypsin, plasmin, and pepsin are used. In certain embodiments, trypsin, chymotrypsin, and pepsin are used. In certain embodiments, trypsin, plasmin, and pepsin are used. In certain embodiments, chymotrypsin, plasmin and pepsin are used. In certain embodiments, cathepsin D is also used (e.g., using trypsin, chymotrypsin, plasmin, and cathepsin D; using trypsin, chymotrypsin, and cathepsin D; using trypsin, plasmin, and cathepsin D; using trypsin, chymotrypsin, plasmin, pepsin, and cathepsin D; using trypsin, chymotrypsin, pepsin, and cathepsin D; using trypsin, plasmin, pepsin, and cathepsin D; using chymotrypsin, plasmin, pepsin, and cathepsin D). In certain embodiments, an exonuclease is used. Proteases are known in the art and are available from a number of manufacturers, including, for example, from SigmaAldrich, St. Louis, MO and Worthington Biochemical Corporation, Lakewood, N.J..
Methods for preparing casein hydrolysates are known in the art and are described, for example, in japanese patent application No. JP2006010357 and new zealand patent application No. NZ619383, the disclosures of which are incorporated herein by reference for all purposes.
In certain embodiments, to prepare the hydrolysate, a protein (e.g., beta-casein-rich casein) is dissolved or dispersed in a solvent, such as water (e.g., distilled water), which may include an acid or base or a salt thereof. The concentration of the solution may be between about 1% and about 75% by weight, about 1% and about 50% by weight, about 1% and about 40% by weight, about 1% and about 30% by weight, about 1% and about 20% by weight, about 1% and about 15% by weight, about 1 and about 10% by weight, about 5% and about 15% by weight, about 5% and about 10% by weight.
The pH of the solution is then adjusted to be within the operable range of the protease or proteases used. The substrate concentration, enzyme concentration, reaction temperature, reaction time, etc. of the specific protease used are determined. The reaction conditions for a given enzyme are known in the art and are generally provided by the manufacturer of the enzyme. For example, the pH range may be adjusted between
The progress of the reaction can be monitored, for example, by collecting samples of the reaction solution at different time intervals, measuring the extent of protein degradation, and optionally measuring the molecular weight distribution of the protein hydrolysate.
The reaction may be terminated by any method known in the art, for example by addition of a hydrochloride solution and/or heat inactivation treatment. The heat-inactivation treatment conditions (heating temperature, heating time, etc.) can be determined depending on the thermostability of the enzyme used. The treatment may also be combined with other techniques, such as filtration, microfiltration, ultrafiltration or nanofiltration, to reduce and inactivate enzyme proteins.
After termination of the enzymatic reaction, one or more of the following may be used: filtering, micro-filtering, membrane separation (such as ultrafiltration membrane), resin adsorption separation, and purifying the obtained hydrolysate by column chromatography. The membrane separation process may be performed using any equipment known in the art. For example, microfiltration and ultrafiltration modules may be used to filter the hydrolysate, which is obtained as a permeate fraction of the membrane. Resin adsorption separation can be performed in any manner known in the art, for example, using resins, ion exchange resins, chelating resins, affinity adsorbent resins, synthetic adsorbents, and high performance liquid chromatography resins.
The properties of the peptide hydrolysate can be tested and evaluated by, for example, mass spectrometry and/or standard nitrogen and degree of hydrolysis measurements. An exemplary mass spectrometer suitable for use in the methods described herein is a high performance liquid chromatography triple quadrupole mass spectrometer (LC/MS, Waters TQD). The hydrolysate can be separated by gradient analysis using chromatography, e.g., a reversed phase ODS column, as a separation column and 0.1% aqueous formic acid and 0.1% formic acid containing acetonitrile as eluents prior to measurement by a mass spectrometer. The specific peptide content can be determined using a calibration curve using synthetic peptides as standards and/or labeled peptide standards.
In certain embodiments where the protein source comprises beta-casein rich casein hydrolysate, the beta-casein rich casein hydrolysate is present in the nutritional composition in an amount of from about 0.036g/100Kcal to about 3g/100Kcal, or from about 0.042 g/100Kcal to about 2.5 g/100Kcal, from about 0.042 g/100Kcal to 1.5 g/100Kcal, from about 0.042 g/100Kcal to about 1 g/100Kcal, or from about 0.042 g/100Kcal to about 0.5 g/100Kcal of the nutritional composition. In some embodiments, the beta-casein rich casein hydrolysate is present in the nutritional composition in an amount of from about 0.05g/100kcal to about 0.2g/100kcal of the nutritional composition.
In some embodiments, the casein hydrolysate enriched in beta-casein is provided in an amount of about 5 wt% to about 15 wt% based on the total weight of protein included in the nutritional composition.
In certain embodiments, the beta-casein rich casein hydrolysate peptides provide about 25% to about 60% (e.g., about 30% to about 50%, about 35% to about 45%) of the total peptides in the nutritional composition. In certain embodiments, the alpha-casein peptide provides from about 5% to about 25% (e.g., from about 10% to about 20%, from about 12% to about 18%) of the total peptides in the nutritional composition. In certain embodiments, the PIGR peptide provides from about 5% to about 25% (e.g., from about 10% to about 20%, from about 12% to about 18%) of the total peptides in the nutritional composition. In certain embodiments, the osteopontin peptide provides from about 1% to about 15% (e.g., from about 5% to about 10%, from about 6% to about 8%) of the total peptides in the nutritional composition. In certain embodiments, the kappa-casein peptides provide from about 1% to about 10% (e.g., from about 2% to about 8%, from about 3% to about 5%) of the total peptides in the nutritional composition. In certain embodiments, the bile salt activated lipase peptide provides from about 1% to about 10% (e.g., from about 2% to about 8%, from about 3% to about 5%) of the total peptides in the nutritional composition. In certain embodiments, the clusterin peptides provide from about 0.5% to about 5% (e.g., from about 1% to about 3%, about 2%) of the total peptides in the nutritional composition.
Non-limiting examples of hydrolysis methods are disclosed herein. In some embodiments, the method may be used for a β -casein-rich product to obtain β -casein-rich protein hydrolysates and peptides of the present disclosure. The protein is hydrolyzed using proteolytic enzyme protease N. Protease N "Amano" is commercially available from Amano Enzyme u.s.a. co., ltd., Elgin, Ill. Protease N is a proteolytic enzyme preparation derived from the bacterial species Bacillus subtilis. The protease powder is defined as "not less than 150,000 units/g", which means that one unit of protease N is an amount of enzyme that produces amino acids equivalent to 100 micrograms of tyrosine at pH of 7.0 for 60 minutes. To produce the infant formulas of the present disclosure, protease N may be used at a level of about 0.5% to about 1.0% by weight of total protein hydrolyzed.
The N-hydrolysis of proteins by proteases is typically carried out at a temperature of about 50 ℃ to about 60 ℃. The hydrolysis occurs for a period of time to achieve a degree of hydrolysis of about 4% to 10%. In particular embodiments, the hydrolysis occurs for a period of time to achieve a degree of hydrolysis of about 6% to 9%. In another embodiment, the hydrolysis occurs for a period of time to achieve a degree of hydrolysis of about 7.5%. Such hydrolysis levels may take from about half an hour to about 3 hours.
A constant pH should be maintained during the hydrolysis. In the process of the present disclosure, the pH is adjusted to and maintained at about 6.5 to 8. In particular embodiments, the pH is maintained at about 7.0.
To maintain the optimum pH of the solution of whey protein, casein, water and protease N, the pH during hydrolysis may be adjusted using caustic solutions of sodium hydroxide and/or potassium hydroxide. If sodium hydroxide is used to adjust the pH, the amount of sodium hydroxide added to the solution should be controlled to a level where it comprises less than about 0.3% of the total solids in the finished protein hydrolysate. To maintain the optimum pH, the pH of the solution can also be adjusted to the desired value using a 10% potassium hydroxide solution before the addition of the enzyme or during hydrolysis.
The amount of caustic solution added to the solution during proteolysis can be controlled by pH-stat or by continuous and proportional addition of caustic solution. The hydrolysate can be made by standard batch or continuous processes.
To better ensure consistent quality of the protein partial hydrolysate, the hydrolysate was subjected to enzymatic inactivation to terminate the hydrolysis process. The enzyme inactivation step may comprise heat treatment at a temperature of about 82 ℃ for about 10 minutes. Alternatively, the enzyme may be inactivated by heating the solution to a temperature of about 92 ℃ for about 5 seconds. After the enzyme deactivation is complete, the hydrolysate can be stored in liquid form at a temperature below 10 ℃.
In some embodiments, the protein source comprises a whole protein source. The source of intact protein may be any protein source used in the art, such as skim milk, whey protein, casein, soy protein, hydrolyzed protein, amino acids, and the like. Sources of milk protein that may be used in the practice of the present disclosure include, but are not limited to, milk protein powder, milk protein concentrate, milk protein isolate, skim milk solids, skim milk powder, whey protein isolate, whey protein concentrate, sweet whey, acid whey, casein, acid casein, caseinate (e.g., sodium caseinate, sodium calcium caseinate, calcium caseinate), and any combination thereof.
In one embodiment, the proteins of the nutritional composition are provided as intact proteins. In other embodiments, the protein is provided as a combination of both intact and partially hydrolyzed protein, and the degree of hydrolysis is about 4% to 10%. In certain other embodiments, the protein is more completely hydrolyzed. In yet other embodiments, the protein source comprises amino acids. In yet another embodiment, the protein source may be supplemented with a glutamine-containing peptide.
In a particular embodiment of the nutritional composition, the ratio of whey from which the protein is derived: the casein ratio is similar to that found in human breast milk. In one embodiment, the protein source comprises from about 40% to about 80% whey protein and from about 20% to about 60% casein.
In some embodiments, the protein source may include a combination of milk powder and whey protein powder. In some embodiments, the protein source comprises from about 5 wt% to about 30 wt% skim milk powder based on the total weight of the nutritional composition and from about 2 wt% to about 20 wt% whey protein concentrate based on the total weight of the nutritional composition. In yet another certain embodiment, the protein source comprises from about 10 wt% to about 20% skim milk powder based on the total weight of the nutritional composition and from about 5 wt% to about 15 wt% whey protein concentrate based on the total weight of the nutritional composition.
In some embodiments, the nutritional composition comprises from about 0.8g/100Kcal to about 3g/100Kcal of intact protein. In some embodiments, the nutritional composition comprises from about 1g/100Kcal to about 2.5g/100Kcal of intact protein. In still other embodiments, the nutritional composition comprises from about 1.3g/100Kcal to about 2.1g/100Kcal of intact protein.
In some embodiments, the protein source or protein equivalent source comprises amino acids. In this embodiment, amino acids may include, but are not limited to, histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, carnitine, taurine, and blends thereof. In some embodiments, the amino acid may be a branched chain amino acid. In other embodiments, small amino acid peptides may be included as a protein component of a nutritional composition. Such small amino acid peptides may be naturally occurring or synthetic. In one embodiment, 100% of the free amino acids have a molecular weight of less than 500 daltons.
In some embodiments, the nutritional composition comprises glutamic acid or glutamine in an amount of about 1mg/100Kcal to about 70mg/100 Kcal. In some embodiments, the nutritional composition comprises glutamic acid or glutamine in an amount of about 20mg/100Kcal to about 40mg/100 Kcal. In some embodiments, the nutritional composition comprises taurine in an amount of about 0.05mg/100Kcal to about 15mg/100 Kcal. In some embodiments, the nutritional composition comprises taurine in an amount of about 3mg/100Kcal to about 8mg/100 Kcal. In some embodiments, the nutritional composition comprises alanine in an amount from about 0.05mg/100Kcal to about 8mg/100 Kcal. In some embodiments, the nutritional composition comprises alanine in an amount from about 2mg/100Kcal to about 4mg/100 Kcal. In some embodiments, the nutritional composition comprises serine in an amount of about 0.05mg/100Kcal to about 5mg/100 Kcal. In some embodiments, the nutritional composition comprises serine in an amount of about 1mg/100Kcal to about 3mg/100 Kcal. In some embodiments, the nutritional composition comprises glycine in an amount of about 0.02mg/100Kcal to about 4mg/100 Kcal. In some embodiments, the nutritional composition comprises glycine in an amount of about 0.5mg/100Kcal to about 2mg/100 Kcal. In some embodiments, the nutritional composition comprises about 20mg/100Kcal of glutamic acid or glutamine, 6mg/100Kcal of taurine, and 3mg/100Kcal of alanine.
Proteases for protein digestion and absorption along the human gastrointestinal tract consist mainly of pepsin in the stomach, enzymes from the pancreas and small intestine brush border (di-and tri-) peptidases. For neonates and infants, the protein digestive system has not yet fully developed. For example, pepsin in the stomach is produced from hydrochloric acid activated pepsinogen (pro-pepsin) which has limited proteolytic function due to its low secretion and high post-prandial gastric pH compared to children and adults. Thus, the main source of infant protein digestive enzymes is the pancreas, mainly comprising trypsin, chymotrypsin, elastase, carboxypeptidase a and carboxypeptidase B. In addition, these pancreatic proteases are secreted as zymogens which are activated by trypsin, which itself is derived from an enterokinase-activated trypsinogen. Among them, trypsin and chymotrypsin are believed to play an important role in the digestion of proteins, since elastase and carboxypeptidase are less active in newborn children than in older children. Furthermore, even though trypsin activity in term infants is not significantly affected by age, chymotrypsin activity at birth is only about 60% of its activity in older children. Indeed, it has been found that the ratio of trypsin to chymotrypsin in term infants is in the range of 1: 2 to 2: 1, in the above range.
Accordingly, provided herein are nutritional compositions that include a suitable protein source in addition to a protease or other suitable enzyme, such as trypsin or chymotrypsin. Such nutritional compositions, including a suitable protein source and trypsin or chymotrypsin, may further mimic the digestion of major proteins present in the small intestine and provide for some selected hydrolysis to occur in the mammary gland. Indeed, while the use of certain protein hydrolysates may be generally known in the art, these hydrolysates are often prepared enzymatically or by a method other than trypsin or chymotrypsin. Trypsin and chymotrypsin provide a pre-digestion process that has physiological similarities to digestion that occurs in the gastrointestinal tract. Hydrolysis with trypsin or chymotrypsin may provide better absorption of certain peptides compared to free amino acids or intact proteins, especially in infant or pediatric subjects with digestive problems. In addition, the inclusion of intact proteins with trypsin or chymotrypsin can provide peptides with certain biological functions such as inducing a reduction in the allergenicity of intact proteins, enrichment of tolerogenic peptides, antimicrobial properties, antioxidant properties and promoting maturation of the enteric nervous system. Furthermore, the inclusion of a suitable source of intact protein in addition to trypsin and/or chymotrypsin can result in a hydrolysate with better organoleptic properties than extensively hydrolysed proteins or hydrolysates comprising a large number of free amino acids.
Thus, in some embodiments, the nutritional composition may include a suitable protein source in addition to trypsin or chymotrypsin. Suitable protein sources for inclusion in the nutritional composition may include any mammalian animal milk protein or vegetable protein, as well as portions or combinations thereof. Suitable protein sources include cow's milk, goat's milk, whey protein, casein, soy protein, rice protein, pea protein, peanut protein, egg protein, sesame protein, fish protein, wheat protein, and combinations thereof.
In some embodiments, the nutritional composition includes a suitable protein source, trypsin and/or chymotrypsin, and beta-enriched casein hydrolysate. In certain embodiments, the nutritional composition may include a protein component that includes intact proteins, amino acids, trypsin and/or chymotrypsin, beta-enriched casein hydrolysate, and any combination thereof. Other suitable protein sources include lactoferrin, immunoglobulins, components rich in milk fat globule membrane (i.e. MFGM10 whey protein from Ara FoodIngredients), casein rich in beta-casein, casein micelles, alpha-lactalbumin-rich whey or plant proteins, including rice, soy, maize, wheat, sorghum, barley, pea, hemp, sage, quinoa, spirulina, sesame, flax, almond, walnut, cashew, algae, fungi, yeast and/or bacteria.
Suitable sources of trypsin or chymotrypsin include, but are not limited to, any available source, such as those made from animal sources by extraction of pancreatic or microbial sources. Other suitable enzyme sources include commercially available enzymes from mammalian pancreas, such as Pancrematic Trypsin Novo 6.0S (PTN 6.0S or PTN) from Novozymes A/S, Denmark; corolase PP from AB Enzymes GmbH, Germany; pancreatin from porcine pancreas supplied by Sigma chemical company, USA; trypsin from bovine and porcine pancreas supplied by Sigma Chemical Company, USA; chymotrypsin from bovine pancreas from Sigma Chemical Company, USA; bovine ENZECO chymotrypsin 1:1 available from Enzyme Development Corporation, USA; trypsin 250, Pancreatin 1XNF and Pancreatin 4ANF supplied by Biocatalysts Limited, UK. In addition, microbial enzymes such as trypsin-like endopeptidases from Fusarium strains and chymotrypsin-like endopeptidases from Novozyme' S A/S from Denmark may also be supplied. Generally, both of these microbial-based enzymes are Generally Recognized As Safe (GRAS) to use as direct food ingredients.
In some embodiments, the nutritional composition may comprise a protein hydrolysate that has been hydrolyzed using trypsin or chymotrypsin. Indeed, if a combination of trypsin and chymotrypsin is used, in certain embodiments the trypsin and chymotrypsin are used in a ratio of from 20:1 to 1:20, preferably from about 10:1 to 1:10, more preferably from about 5:1 to 1:5, most preferably from 1:2 to 2: 1.
Example 1
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