Appunti quality and functionality of milk and dairy products

Properties of milk and dairy products

There is a difference between functional and technological properties of food components:

• Functional: Ability to provide benefit to one or more functions of the body, in addition to adequate nutritional effects, in a manner relevant to either improved health and well-being or reduced risk of disease.
• Technological: Viscosity, color contribution, flavor, emulsifier, foaming, and structuring properties, etc.

Functional foods

According to the ILSI (International Life Science Institute), they can be defined as "any food or ingredient that has been sufficiently proved to be capable of benefiting one or more functions of the body, in addition to its appropriate nutritional effects, in a manner relevant to either the improvement of health and well-being or the reduction of disease risk".

It should be noted that the definition says 'has sufficiently demonstrated' to mean that its effects must be based on scientific evidence and must therefore pass through EFSA's opinion. It is also important to communicate this information to the consumer through advertising or labeling (regulation n.1924/2006 of "health claims").

Definitions

• Bioavailability: A fraction or compound of an ingested foodstuff that is available for the exercise of physical functions. Bioavailability is the key to nutritional efficiency. A lower amount of the total intake is assimilated and used for storage and metabolic functions (for example, it reaches the circulatory system or a target organ).
• Bioaccessibility: The amount of a compound released upon gastric digestion that is absorbed by the intestine (epithelial tissue).
• Bioactivity: The specific effect (e.g., anti-inflammatory, antioxidant, etc.) arising from the exposure to a compound. This includes how to reach the target organ, interact with other biomolecules, the generation of biomarkers, and physiological responses.

Bioactive components of milk

These are compounds benefiting one or more functions of the body, in addition to appropriate nutritional effects. These are:

• Whey and milk protein hydrolysates: MPH and WPH. The latter are peptides < 20 amino acids, able to improve digestibility and reduce allergenicity (used for example in artificial milks, protein bars, or matcha latte).
• Milk and whey peptides: Casein-phosphopeptides (CPP) and glycomacropeptide (GMP).
• Milk and whey protein fractions: Lactoferrin, lactoperoxidase, colostrum, immunoglobulins (IgG), α-lactalbumin, and β-lactoglobulin. Lactoferrin is antioxidant, anti-inflammatory, antibacterial, promotes bone growth, regulates iron absorption, and promotes the development of the intestine (used for skincare, oral hygiene, and as a food supplement, in artificial milks and yogurt). α-lactalbumin, on the other hand, improves bowel growth, provides greater amounts of tryptophan, is a source of essential amino acids, and increases the speed of the immune response (used in artificial milks, sports bars, and to combat sleep disorders and depression).
• Lactose derivatives: They have a prebiotic effect (promote the growth of bifidobacteria), increase calcium absorption, promote gastrointestinal health, and give positive effects on the immunomodulatory response.
• Other bioactive peptides: β-casein, serum albumin (BSA), osteopontin, membrane fatty acid globules (MFGMs), and phospholipids.

The bioactive components of milk are found in many products, which are exploited for different commercial strategies. For example, infant formula (humanized milk). They are mainly used for performance, endurance (energy generation), and recovery (sports nutrition); they are also exploited for the digestion of proteins, the prevention of diseases, and treatments (clinical nutrition). Finally, the bioactive components can be studied to obtain natural ingredients with health benefits to be used in different products (food supplements and functional foods).

The goal of the market is to promote healthy growth and achieve a healthy state of health in the world (bone, heart, immune system health, fight obesity and overweight, muscle and intestinal health, counteract aging, and increased energy).

Effects of bioactive components in the market

There are both positive and negative effects related to the bioactive components of milk in the market.

The positive effects:

• Health benefits, therefore, increased consumer demand
• New technological processes that have lower costs
• Competitiveness due to product differentiation

The negative effects:

• Large raw material volume to be processed for small output
• Difficulty in obtaining a real economy of scale
• High research and development costs
• High-cost ingredients
• Complications in the union of raw materials and bioactive components on a large scale

MPH and WPH are the ones with the highest added value.

Bioactive peptides

They result from the selective hydrolysis of proteins:

• Fermentations/maturation by protease. In this case, proteolysis can be primary and, therefore, by endogenous proteases or coagulants, or secondary by laboratory starter cultures.
• During digestion (by gastrointestinal enzymes).

Not all foods produce bioactive peptides, but the sequence of these must already be present in the original proteins. The proteins in which they are most present are those of milk. They are defined as bioactive because they act on the system with effects:

• Effect on the cardiovascular system: antihypertensive and antithrombotic
• Effect on the nervous system: opioid agonists or antagonists (effect on the brain)
• Effect on the digestive system (e.g., absorption): casein-phosphopeptides (CPP) and glycomacropeptide (GMP). The latter has an effect on this system as it is devoid of an amino acid responsible for a series of pathologies (phenylalanine).
• Effect on the immune system.

Bioactive peptides can then be:

• Natives, such as minor whey proteins (GMP, lactoferrin, and lactoperoxidase).
• Latent, i.e., activated with proteolysis (opioids, hypotensives, antithrombotics, metal ligands, immunomodulators, antimicrobials, and satiating).

Opioid peptides

They are called opioids because they have a function similar to what is produced by the brain in response to pain, for example (in response to the control of certain symptoms). These opioids include endorphins (e.g., morphine), which are normally released to block pain’s neurotransmitters. There are some peptides that have activity similar to that of endorphins, which means that they block the release of neurotransmitters associated with the sensation of pain and promote the release of dopamine (neurotransmitter associated with positive sensations). The structure of opioid peptides is, in fact, very similar to that of endorphins. For example, sleep following a cup of milk is due to the action of these opioid peptides that aid relaxation.

Among the opioid peptides, there are also enkephalins (always endorphin family) that are always needed to regulate the sensation of pain (they are therefore endogenous opioids). Enkephalins consist of tyrosine-glycine-glycine-phenylalanine-leucine\methionine (structure found in many opioid peptides, some of which deriving from gluten). Their production is linked to the type of action proteolytic (it makes sense to look for them in a seasoned product; they increase significantly because of a proteolytic action).

Opioid receptor types (μ, κ, δ and k) are found in the endocrine nervous system and intestinal tract of mammals and interact with endogenous and exogenous opioid peptides. The first are represented by endorphin, enkephalin, and norphine derived from specific precursors.

Exogenous opioid peptides can be:

• Agonists: involved in the treatment of pain, binding to specific CNS receptors (e.g., β-casomorphins, α-lactoalbumin like lactarphine, serum albumins like serorphine, and β-lactoglobulin).
• Antagonists: bind to CNS receptors for the treatment of pain but do not activate them, thus blocking the effect of agonists (e.g., lactoferrin and k-casein).

Both exogenous and endogenous opioid peptides (apart from those derived from casein) are characterized by an N-terminal tyrosine residue in the presence of an aromatic amino acid in the third or fourth position, such as phenylalanine or tyrosine. This characteristic of the N-terminal is essential for binding to the opioid receptor and thus for opioid activity itself.

β-casomorphins

The most important are those that arise from the sequences 60-63 \60-64 \ 60-65 \ 60-66 \ 60-70 of β-casein. Of these opioids, the most effective are β-casomorphine 5 and 7. In fact, β-caseins have different sequences depending on the genotype of the breed: milk contains 2 variants of β-casein called A1 and A2. However, there are some breeds of "primordial cows" that produce milk with only A2 beta casein (hence the name A2 milk).

The difference between the two variants A1 and A2 is a single amino acid (in A2 it is a proline while in A1 it is a histidine) and therefore a different peptide is formed during digestion. If there is histidine, the peptide β-casomorphine 7 (BCM-7) is formed. BCM-7 has been shown to be responsible for type 1 diabetes, cardiovascular disease, intestinal inflammation, and autism. However, these effects are not confirmed by the literature.

The release of BCM7 through gastrointestinal enzyme digestion of bovine β-casein is dictated by different amino acid sequences of this protein, which vary genetically between cattle breeds. The amino acid present at position 67 of the β-casein sequence seems to be critical for the release of this BCM7: in the A2 variant of β-casein, there is a proline in position 67, while in the A1 and B variants of β-casein, a histidine residue is found at this position. In the case of variants containing proline, the enzymatic hydrolysis of the bond (and therefore the formation of BCM-7) does not occur or occurs at a very low rate; unlike if there is histidine at position 67, hydrolysis occurs and the casomorphine in question is formed.

In men, there is evidence, from a limited number of studies, that the consumption of A1 is also associated with intestinal transit delay and consistency loss of stool. In addition, digestive problems are related to the consumption of casein A1 and not A2 (consider that the opioid receptors μ to which β-casomorphins bind are found in the intestine).

EFSA monitoring the functional properties of BCM-7 peptide: EFSA says that the estimation of food peptide and risk does not seem recommendable; that, based on this review of the available scientific literature, a cause-and-effect relationship between oral intake of BCM7 or related peptides and the etiology or course of any suggested non-communicable diseases cannot be established (reduced amount of data supporting cause-and-effect relationship and reduced studies on β-casomorphins following intestinal absorption).

A2 Milk is milk in which the content of a natural variant of a beta casein protein A2 has been increased. This is done by selectively milking only cows that naturally produce milk containing A2. In this way, it is believed that the health benefits of drinking milk can be further realized. Such inclusion would ensure that any potential or perceived benefit associated with the consumption of A2 is maximized and thus increases its value for the most demanding consumers. Numerous scientific studies have been undertaken on a range of potential benefits associated with this protein.

As a result of extensive research carried out on the link between the consumption of this protein and the purported health benefits, A2 Milk can be considered a premium, value-added product with the potential to benefit the dairy industry and consumers. In fact, there are commercial products, like Granarolo milk "taste of the past" 100% Italian: it is produced by cows exclusively selected and controlled in farms and which have preserved the genetic heritage of cows of the past; in fact, they produce a milk whose β-casein is only A2, as it was originally.

To add is that β-casomorphins can permeate through the intestinal mucosa of newborns by passive transport: consequently, the breast tissue of pregnant or breastfeeding women is also permeable to these.

Possible role of exorphins in the development of psychiatric problems: there is a possible relationship between schizophrenia/autism and casein and/or gluten intake (considering the release of opioids such as β-casomorphins and exorphins during digestion). Both casomorphine and gluten exorphins (A5: Gly, Tyr, Pro, Thr; B5: Tyr, Gly, Trp, Leu; C: Tyr, Pro, Ile, Ser, Leu) could be absorbed by the gastrointestinal mucosa, especially if inflamed as in celiac disease, triggering psychotic disorders. The observation of a rapid regression of psychiatric symptoms after a period of "casein and gluten-free" diet seems to support the hypothesis of a precise pathogenetic role of casein and gluten in the genesis of schizophrenic disorders.

Hypotensive peptides

Inhibitory compounds of the angiotensin-converting enzyme (ACE), a peptide hormone that is part of a blood pressure regulatory cascade.

In the increase in blood pressure, there is a conversion of angiotensin I to angiotensin II, which is converted in turn to modified angiotensin (1-7), leading to an increase in blood pressure. These peptides block the activity of the enzyme that converts angiotensin I to II, thereby regulating blood pressure. While blocking the conversion, another enzyme is activated that converts a peptide to produce bradykinin, which is a vasodilator (opposite effect to angiotensin, which is a constrictor vessel, as it blocks this production of bradykinin and favors that of aldosterone; aldosterone reduces renal excretion and increases water retention). Consequently, the preventive aspect of these ACE inhibitors is precisely to avoid an increase in blood pressure (some drugs are, in fact, ACE inhibitors).

These peptides are ACE inhibitors because they have the function of intervening by chelating zinc: it is, in fact, a conversion by enzymes that require zinc for their activity. The ACE inhibitor, chelating the zinc, subtracts it from these enzymes, which no longer work.

Several peptides with this activity are present in the primary sequence of casein α, casokinins and β, and ks1caseins. Several peptides capable of inhibiting ACE have been found in cheese, i.e., their formation is influenced by proteolytic phenomena that occur during maturation. Obviously, the type of proteolytic activity (coagulant, ripening conditions) influences the amount and bioactivity of the hypotensive peptide present in the cheese. The maximum level is usually found after an average ripening period of 4-8 months), after which the bioactivity decreases. Ripening is the best time to take more ACE inhibitors with cheese (proteolytic activities that generate peptides). The curve at a certain point decreases because for each cheese there is a different curing time.

ACE-inhibitor peptides in several cheese samples: several peptides represent competitive substrates for ACE, and their inhibiting activity is mainly modulated by a specific C-terminal sequence, which is required for inhibitors to bind to ACE. In this regard, ACE-inhibitor (ACE-I) peptides having Arg, Trp, Tyr, Phe, or Pro residues in the three C-terminal positions have been reported as the most effective in enhancing binding to ACE.

In this study, the samples were Cheddar, Gorgonzola, Maasdam, and Grana Padano (subjected to in vitro α gastrointestinal digestion): it was shown that during ripening there is proteolysis of β-caseins and αs1-caseins that can generate ACE-inhibitor peptides (usually of 2-12 amino acids). In fact, ACE-I peptides from casein (CN) have hydrophobic or positively charged amino acid residues, such as Pro, Lys, or Arg, at the three C-terminal positions. Among ACE-I peptides identified in cheese and derived from bovine CNs, VPP and IPP have been the most studied (they prevailed in almost all cheese). The highest level of ACE-I peptides was recorded in Cheddar cheese. This sample contained all the searched peptides and, in particular, it showed the highest contents of VPP and IPP amongst the sampled cheeses. It also showed the maximal ACE-I activity in cheese after 6 months of ripening. During ripening, the CZE pattern evidenced the disappearance of both α-casein and αs-I-casein. This phenomenon and the contemporary action of plasmin on β-CN could explain the formation of substrates potentially degradable by intracellular peptidase to form VPP and IPP. The same study showed that the concentrations of the two tripeptides can vary to a large extent within Emmental cheeses differing in age (longer ripening means more substrates degradation and, so, more ACE-inhibitor peptides).

Lastly, the ACE-I potential of cheese can vary after gastrointestinal proteolysis: an increase at gastric level, followed by a decrease at intestinal level, of ACE-inhibitor peptide contents was demonstrated in Cheddar and other cheese samples. This study provides evidence that the degree of proteolysis itself cannot be regarded as a promoting or hindering factor for ACE-I peptide release during cheese digestion. At the same time, the results evidence that the ACE-I potential of cheese cannot be inferred based on the type and amount of ACE-I peptides present in undigested samples.

ACE-inhibitor peptides in Parmigiano Reggiano

The entire cheese-making process is rigorously regulated; for instance, only natural whey starter resulting from previous cheese-making can be added to starting raw milk. These starter lactic acid bacteria along with nonstarter lactic acid bacteria (NSLAB) naturally present in raw milk strongly contribute to CN breakdown as proteolysis represents the most intense biochemical process during PR maturation, which lasts not less than one year.

To establish any potential in vivo antihypertensive effect, the fate of ACE-I peptides during gastrointestinal digestion (GID) as well as their fate and absorption through the intestinal epithelium have to be preliminarily evaluated: In general, the content of ACE-I peptides in the PR samples was low in comparison with levels usually found in short- to medium-ripened undigested samples.