Milk protein hydrolysates, content in cow’s milk, hydrolysis process used and their related bioactivity.
Milk is nature’s most complete food. While milk clearly provides basic nutritional requirements, bioactive components within milk also impart a wide range of additional health benefits to both the neonate and the adult. However, human milk is compositionally different from cow’s milk, and certain protein components of cow’s milk can act as allergens to susceptible humans. One way of extracting the benefits of cow’s milk proteins, while eliminating the risk of allergenicity in humans, is to hydrolyse the milk proteins. Hydrolysis of milk proteins generates smaller peptide sequences from their parent protein that can be biologically active when released. At an industrial scale, hydrolysis of milk proteins can be achieved through either enzymatic hydrolysis or fermentation. An alternative process of generating similar sized peptides is by in silico synthesis. These compounds can subsequently be developed as fortifying food agents.
- milk hydrolysates
- gut health
- gut microbiota
- gut homeostasis
1. Overall composition of milk
The overall composition of milk depends on a range of factors including genetics (species and breed), physiological state (age and stage of lactation) and environment (food and climate) [1–5]. While water is the main constituent of milk, comprising ~87% of the total volume, the remainder is composed of carbohydrates, fats and proteins in varying volumes across different species [6–8]. Among the numerous nutritional benefits of milk, milk proteins have gathered enormous attention for being a ‘complete’ protein as they provide all nine essential amino acids (leucine, isoleucine, valine, phenylalanine, tryptophan, histidine, threonine, methionine, lysine) required by humans . The proteins in milk are categorised into major proteins that include casein and whey fractions  and minor proteins that include lactoferrin, lactoperoxidases, lipases, lactase [6, 10] and miscellaneous proteins (cytokines, immunoglobulins, etc.) .
2. Milk protein hydrolysates
The process of breaking down milk proteins to shorter peptide sequences is termed ‘hydrolysis’. This process happens naturally in the gastrointestinal tract and can be simulated in the laboratory or on an industrial scale. During the normal transit through the gastrointestinal tract, milk proteins are exposed to proteinases such as pepsin, trypsin and chymotrypsin which break them down into smaller peptides. These peptides are further digested by brush border peptidases present at the surface of intestinal epithelial cells where they produce amino acids; however, some oligopeptides still remain intact . In laboratory or at an industrial scale, milk hydrolysates are released either by treatment of milk proteins with food grade enzymes or through fermentation with bacteria, which is described in detail in the following sections.
The shorter peptide sequences often possess bioactive properties beyond their nutritional contribution along with eradicating any protein-specific allergenicity [13, 14]. Processing and enriching for food grade bioactive peptides is a goal for the functional food industry. A functional food can be described as:
Once the hydrolysates are released, they can potentially have bioactive properties which can exert their effects in receptive cells, including those present in the gastrointestinal tract . The bioactivities of the resulting hydrolysates are variable depending on a range of factors, including the enzyme used, the processing conditions and the final size of the peptide sequence following hydrolysis . The degree of hydrolysis (DH) is defined as the percentage of cleaved peptide bonds, i.e. the number of hydrolysed bonds per total number of peptide bonds in the protein . This affects the size and amino acid composition of the peptides, which subsequently determines the biological activity of the peptide. Hence, DH is an important consideration from the perspective of functional food research .
2.1. Enzymatic hydrolysis
The enzymatic hydrolysis process is conducted under mild conditions (pH 6–8, temperature 40–60°C) to minimise side reactions and to retain the amino acid composition similar to the starting material . Enzymatic hydrolysis improves the solubility and heat stability of peptides, which is of benefit to the food industry. However, consumption of certain enzymes leads to allergic or toxic responses; hence, consumer safety is an important factor and requires the regulation of enzymes used for hydrolysis . Enzymes that obtain ‘generally recognised as safe’ (GRAS) status and special approval of ‘food grade’ quality are legally considered as safe . The food grade enzymes generally used to hydrolyse milk proteins into hydrolysates include pepsin, trypsin and chymotrypsin [21, 22]. In addition, food grade proteolytic enzymes, derived from microorganisms, can also be used to generate hydrolysates . Proteolytic enzymes are of two types, depending upon their hydrolysing mechanism: endopeptidases which hydrolyse peptide bonds within protein molecules and exoproteases which hydrolyse N or C terminal peptide bonds. Post enzymatic hydrolysis, the hydrolysates usually need an additional treatment. The most common procedures include ultrafiltration, heat treatment and/or activated carbon treatment to control molecular size and elimination of bitterness in the hydrolysates .
2.2. Hydrolysis through microbial fermentation
Fermentation of milk proteins with proteolytic starter culture is another method of bulk production of hydrolysates. Safety measures should be considered with regard to toxicity and pathogenicity associated with the microorganisms used for fermentation. Food grade microorganisms with no related toxigenic and pathogenic response in humans are widely used. During microbial fermentation, milk proteins are subjected to ‘splitting’ as they are broken down by the proteolytic system of microorganisms . Bacterial cultures of
2.3. Peptide synthesis
3. Models used for bioactivity evaluation and challenges
After the generation of milk hydrolysates, their bioactivity profile needs to be determined. In laboratory or at an industrial scale, the primary screening for the bioactivity is performed on
Although cell- and tissue-based model systems are an alternative to animal experiments, they do not reflect the
4. Functionality of bioactive hydrolysates
To date, the bioactivities of a wide variety of hydrolysates have been characterised using
|Immunomodulation||[37, 38, 92, 93, 99]|
|↑IgG, ↑IgA||[86, 87]|
|↑IgG, ↑IgA||[86, 87]|
|↑IgG, ↑IgA||[86, 87]|
|EH||Immunomodulation||[94, 100, 101]|
lysozyme, proteose-peptone, glycomacropeptide
|~3%||EH||Bifidogenic||[55, 56, 58]|
|EH, PS||Antimicrobial||[67, 68, 69]|
|EH, PS||↑IgG, ↑IgA, ↑IgM|||
4.1. Prebioitc activity of milk hydrolysates
The World Health Organisation now recommends breastfeeding for up to 6 months, as breast milk has a major positive impact on the health and growth of the infant . One of the most important benefits of breastfeeding the newborn is the colonisation of the gut by ‘healthy’ microbiota. ‘Healthy’ gut microbiota confers nutritive, metabolic and protective functions that affect intestinal physiology, immunity and whole-body metabolism. The establishment of a ‘healthy’ microflora in the gut during early life is crucial for the healthy development of a balanced immune regulatory network in the gut, a feature which affects the overall health of the individual . Beneficial gut microorganisms aid gut health by releasing growth substrates from milk , improving vaccine responses  and decreasing gut permeability [45, 46]. After birth, the gut is colonised with bacteria from four main phyla namely
Milk hydrolysates show bifidogenic activity, i.e. they support the growth of Gram-positive anaerobic bacteria namely
4.2. Antimicrobial activity of milk hydrolysates
Antimicrobial milk peptides prevent attachment and invasion of pathogens by either directly interacting with the pathogen and killing them or changing the host environment, leading to the inhibition of growth of microorganisms [59, 60]. The direct interaction of antimicrobial milk hydrolysates with microorganisms is specific, as they show affinity towards polarised bacterial membranes rather than dipolar membranes of eukaryotic cells . There is growing evidence that the antimicrobial property of milk hydrolysates is related to the formation of α-helical structure of the peptides. The modifications of peptide’s secondary and tertiary structures by phosphorylation of specific amino acid or chemical modification of C or N terminal dramatically affects the antimicrobial activity [50, 61]. Another mode of action for antimicrobial peptides is by aggregating in the cytoplasmic membrane, disrupting the membrane permeability of bacteria, and causing cell death [13, 30]. On the contrary, the indirect antimicrobial activity of milk hydrolysates is achieved by decreasing the host intestinal pH and thus limiting the growth of pathogenic microorganisms. This mechanism is also known as ‘colonisation resistance’ .
The antimicrobial effects of milk hydrolysates have been listed in Table 1. Casein is a major source of antimicrobial peptides, and hydrolysates of α-S1 casein exert protective effects against
4.3. Milk hydrolysates preserve gastrointestinal mucosal integrity
The intestinal epithelial cell layer of the gastrointestinal tract lies at the border between the gut-associated lymphoid tissue (GALT), which is the most abundant accumulation of lymphocytes in the body, and the intestinal lumen which contains a high number of dietary antigens and a varied commensal microbiota . The intestinal epithelial cell layer is covered by a mucus gel, which functions as a protective layer for the gastrointestinal system. This barrier function includes the prevention of entry of pathogenic microorganisms, toxins and allergens. The mucus gel is composed of glycoproteins called mucins, with up to 20 mucin genes identified. Mucin genes are expressed by specific cells (goblet cells and enterocytes) and categorised as gel-forming secretory mucins (
Milk hydrolysates can influence the expression and secretion of mucins, as outlined in Table 1. The modulation of mucin production by milk hydrolysates may assist in the development of dietary strategies to enhance and protect the mucus layer. In rats, jejunal
4.4. Milk hydrolysates can modulate the gastrointestinal immune system
The intestinal mucosa exists in a non-pathological state of continuous ‘physiological inflammation’. This low level of inflammation is required to prime the GALT for potential pathogenic bacteria . The mucosal immune system features immune cells including neutrophils, monocyte/macrophages, dendritic cells, mast cells, B and T cells. The crosstalk between intestinal epithelial cells, gut microbiota and local immune cells is essential to maintain intestinal homeostasis, whereas, dysregulation leads to chronic intestinal inflammation . Much of the experimental data come from model organisms such as mice and rats; however, a number of studies have been carried out in humans. Several examples of anti-inflammatory activity exhibited by a variety of milk hydrolysates across a range of experimental models are listed elsewhere [59, 60, 82, 84].
Several milk protein hydrolysates enhance immune cell function by increasing secretion of immunoglobulins, as outlined in Table 1. Immunoglobulins are glycoprotein molecules that specifically recognise antigens from bacteria or viruses and aid in their destruction through a highly complex and specific immune response . Hydrolysates of αs1-casein, αs2-casein and β-casein stimulated the immune system through the enhancement of immunoglobulin G (IgG) and IgA concentrations [86, 87]. Casein hydrolysates conferred protective effects against pathogenic microorganisms in mice challenged with bacterial endotoxin, LPS, by increasing intestinal and faecal IgA and anti-LPS IgA levels . Similar modulation of immune response was recorded in mice against
Immune cells, such as monocytes and macrophages, play an important role in inflammatory responses and tissue repair and remodelling by either interacting directly with microorganisms during infections and/or secretion of cytokines that mediate biological effects . Milk hydrolysates can modulate the gastrointestinal immune system by modulating proliferation and maturation of localised immune cells; the immunomodulatory activities of milk hydrolysates are outlined in Table 1. Casein peptides induced innate host immune responses in humans, by stimulating the proliferation of lymphocytes and macrophages,  and in mice, by activating monocytes and macrophages . On the contrary, rennin-digested κ-casein fragments inhibited the proliferation of mouse spleen lymphocyte and rabbit Peyer’s patch cells . The mechanisms of this κ-casein fragment include acting either as an anti-IL-1 antibody or suppressing IL-2 receptor expression on CD4+ T-cells . Functionally, the phagocytic activity of inflamed murine macrophages was increased by
Particular milk hydrolysates modulate the MAP kinase and NF-κβ pathways that consequently control the secretion of several cytokines that can induce inflammatory responses and strengthen the host defence mechanisms . Mice supplemented with
5. Future developments
The potential health benefits of milk hydrolysates are a subject of growing commercial interest from a health-promoting functional-food perspective. Several commercial products are currently available in the market, and this trend is likely to continue. There are three major areas where developments can be made. The generation of milk hydrolysates is the first area of development. The generation and processing of food grade milk hydrolysates should be carefully designed to yield hydrolysates with diverse bioactivities. Novel technologies can be developed, focussing on the process of enrichment of the hydrolysates with active peptides from milk proteins. The second area of development is the research technologies used to evaluate the bioactivity of milk hydrolysates. The investigation of biochemical properties using newly developed modern analytical technologies is required to understand the cross reactivity between milk hydrolysates and the carrier food matrix. Third, robust platforms should be developed to study the molecular mechanisms by which the bioactives exert their activities. This area is the most challenging research area as the outcome from these studies forms the basis of tailored dietary formulations.